New Approach to the Treatment of CoV-2 Infection by Means of Immune-modulators and Non-Steroid Anti- Inflammatory Drugs

Historical Background of the “COVID-19” Pandemic

The first known case of coronavirus was described as “severe acute respiratory syndrome” (SARS), which occurred on November 16, 2002, in Foshan, a city about 20 km from Guangzhou in China’s Guangdong province. Since November 2002, an unknown infectious agent had caused outbreaks of an atypical pneumonia that spread throughout Guangdong province in southern China. The disease usually started with high fever and mild respiratory symptoms, but rapidly progressed to pneumonia and within a few days new cases emerged in mainland China, so that by February 2003 more than 300 cases had been reported, about one-third of which involved health care workers [1]. Persons who became infected and subsequently traveled spread the outbreak to Hong Kong [2] and from there to Vietnam, Canada, and several other countries [3]. By the end of February 2003, the disease had spread to neighboring regions and countries, was severe, could be transmitted from person to person, and appeared to cause significant outbreaks in health care workers [3,4]. On March 13, 2003, WHO issued a global alert on the disease that it termed “severe acute respiratory syndrome” (SARS) [5], and a remarkable global effort led to the identification of the SARS coronavirus (SARS-CoV). In early April of the same year [4,6], 6 outbreaks occurred in Southeast Asia, North America and Europe and led to the first pandemic of the 21st century. In July 2003 and after a total of 8,096 reported cases, including 774 deaths in 27 countries [7], no further infections were detected and the SARS pandemic was declared to be over. Five additional cases of SARS, as a result of zoonosis, occurred between December 2003 and January 2004 [8], but no further human cases of SARS have been detected since then. Infection control measures, rather than medical interventions, then put an end to the first SARS-CoV pandemic of the 21st century. However, the possibility of transmission in a variety of ways was noted. It was later shown that certain viruses, similar to SARS-CoV found in bats, could infect human cells without prior adaptation [9,10], indicating that SARS could re-emerge [11]. Indeed, 10 years after the first occurrence of SARS-CoV, a man in Saudi Arabia died of “acute respiratory syndrome” and in his serum the “coronavirus” had been isolated, this syndrome was called “Middle East Respiratory Syndrome coronavirus” (MERS) because of its place of origin. In April 2012, several cases of “severe respiratory illness” had already occurred in a hospital in Jordan [12], these cases were retrospectively diagnosed, and considered to be human-to-human transmitted, furthermore in the United Kingdom, 3 cases of MERS were reported in September 2012 [13].

In May 2015, a single person, returning from the Middle East, initiated a nosocomial MERS outbreak in South Korea that affected 16 hospitals and 186 patients [14]. By April 26, 2016, 1,728 MERS cases, including 624 deaths, had been confirmed in 27 countries [15,16]. (Figure 1) In the accompanying figure, published in 2016 (copied from review paper: de Wit E, Doremalen VN, Falzarano D, et al. SARS and MERS: recent insights into emerging coronaviruses. Nat Rev Microbiol. (2016) 14: 523-34.doi: 10.1038/nrmicro.2016.81) shows as different ways of coronavirus transmission. Bats could have been the main reservoir of the coronavirus 30 years before passing to humans, due to “cross-species transmission” between bats and camels; these animals, through continuous contact with humans, could have produced the direct zoonosis that gave rise to MERS-CoV. Moreover, the detection of the virus in “palm civets” (Chinese species) and in a “raccoon dog” (Japanese raccoon), as well as the detection of antibodies to the virus in the Chinese ferretbadger (also known as small-toothed ferret- badger) observed at a live animal market in Shenzhen, China [17] alerted researchers to the possible transmission of the virus to humans. However, these animals were only incidental hosts, as there was no evidence of SARS-CoV-like virus circulation in “palm civets,” both in the wild and in breeding facilities [18]. Thus, the search for the MERSCoV reservoir initially focused on bats, but a serological study in dromedaries from Oman and the Canary Islands showed a high prevalence of MERS-CoV neutralizing antibodies in these animals [19]. In addition, MERS-CoV RNA was detected in swabs collected from dromedaries on a farm in Qatar that was associated with two human cases of MERS, and infectious virus was isolated from dromedaries in Saudi Arabia and Qatar [20-23], and serological tests also detected circulation of a MERS-CoV-like virus in dromedaries in the Middle East, East Africa, and North Africa. Dromedaries in Saudi Arabia harbor several viral genetic lineages [24], including those that have caused outbreaks in humans. Taken together, these data pointed to the role of dromedaries as a reservoir of MERS-CoV. The ubiquity of infected dromedaries near humans and the resulting zoonosis may explain why MERS-CoV continues to cause human infections, whereas SARS-CoV, without the continued presence of an infected intermediate host and with relatively infrequent human-bat interactions, had not caused further human infections.

Figure 1.

Person-to-person transmission of SARS-CoV and MERS-CoV occurred primarily through nosocomial transmission. Between 43.5 and 100% of MERS cases in individual outbreaks were hospital-related, and very similar observations were made for some of the SARS clusters [25-26]. Transmission among family members occurred in only 13 to 21% of MERS cases and 22 to 39% of SARS cases. Patient-to- patient transmission of MERSCoV was the most common route of infection (62-79% of cases), whereas for SARS-CoV, infection of health care workers by infected patients was very common (33-42%) [25]. The predominance of nosocomial transmission is probably due to the fact that substantial virus shedding occurs only after symptom onset [27-28], when most patients are already seeking medical care [29]. An analysis of hospital surfaces after treatment of patients with MERS showed the ubiquitous presence of viral RNA in the environment for several days after patients stopped testing positive [30]. In addition, many SARS or MERS patients were infected through “superpropagators” [25-27,31-33]. As of 2016, it had already been provided, in various publications, that the key features of these viruses are: the predominance of nosocomial transmission, pathogenesis driven by a combination of viral replication in the lower respiratory tract and an aberrant host immune response, and several potential treatments for SARS and MERS in animal models and “in vitro” had also been suggested, including small-molecule protease inhibitors, neutralizing antibodies and inhibitors of the host immune response.

Current Pandemic COVID-19

In December 2019, a new coronavirus (“nCoV”) emerged in Wuhan, Hubei province in China. Attention was focused on the Huanan food market, where in addition to fish, livestock animals were also traded. However, analysis of the first 41 hospitalized patients showed that the Wuhan seafood market might not be the main source for the spread of a new virus [34]. Nevertheless, an epidemic of severe pneumonia of unknown cause soon appeared [35], and genomic sequencing of viral isolates from five pneumonia patients hospitalized from December 18 to 29, 2019, indicated the presence of a previously unknown “b- CoV” strain in patients [36]. This “new” coronavirus (nCoV) subsequently spread from the original outbreak site in China and was designated as “SARS-CoV-2” by the World Health Organization (WHO) on January 12, 2020 and the disease as “COVID-19” on February 11, 2020 [37] and this virus was confirmed to have 75-80% similarity to the coronavirus that caused severe acute respiratory syndrome (SARS-CoV) [38]From February 2020 to April 2020, the disease “COVID-19” affected 188 countries worldwide. [38]and up to July 14, 2020 the cumulative number of confirmed cases was 13.1 million people and at least 572,426 people died from SARS-CoV-2 infection [39], the incidence of deaths ranged from less than 1% to 3.7% among the different countries [40], these figures were compared with the rate of deaths from influenza which was less than 0.1% [35].

After the first pandemic period, the incidence of COVID-2 infected cases declined during the summer months and then rose again significantly from September/October 2020 to date (31 January 2021), the increase in incidence is statistically shown as a “wave”, with 3/4 “waves” with “peaks”, “plateaus” and “valleys” in different countries; Most European Union countries, including Spain, have experienced high levels of incidence, but the highest number of infections has been observed in Great Britain, the USA, Brazil and India, up to this point. As of January 30, 2021, the number of cases in the world since the pandemic began at the end of 2019 has been: 102,000,000, and the number of deaths: 2,210,000.

The Acute Inflammatory Process

From the clinical point of view, the disease caused by CoV-2 presents 3 fundamental stages: in the first stage the patient shows signs and symptoms similar to infection by other viruses and/ or bacteria of the respiratory tract (e.g., Influenza), in this stage the symptoms are shown to a lesser degree and the patient may even be asymptomatic. In the second stage the patient feels worse and the signs and symptoms are more evident (fever, tiredness, general malaise, anosmia, hypoacusis, etc.); this stage is definitive for the patient, who may improve in the following days until cured or worsen until reaching the third stage, which may worsen to the point that the patient has to be admitted to the ICU, where intubation and assisted respiration may even be necessary; this moment is crucial for the patient since the feared “cytokine storm” may occur. From the immunological point of view, infections by bacteria and/or viruses, accidental or provoked trauma (e.g. surgical interventions), allograft rejection and the development of neoplasms have a common point: inflammation. Inflammation is the result of multiple interactions of the systems involved in the homeostasis of the organism, mainly the immune system, which have as their first objective the localization of the process and the elimination of the aggressor agent. When the infection is aggravated by a huge excess of antigen (due to the unstoppable and rapid replication of the virus), the inflammation reaches its climax and becomes a systemic process that affects the whole organism, it is called “systemic inflammatory response syndrome” (SIRS), and in the case of COVID-19, since the respiratory system is the main system affected, it is called “SARS-CoV-2” (“systemic acute respiratory syndrome”), the response of the immune system overflows and the “cytokine storm” appears, which can lead to “multiorgan failure” (MOF) and death of the patient. In fact, from a biological point of view, tissue injury and its sequelae are involved in most medical problems and the response of living tissues to aggression is the basis and foundation of the immune response. [41-45].

In addition to cytokine storming, COVID-19 viral particles can also directly induce multiple organ dysfunctions. In this regard viral particles from COVID-19 infection have been identified in bronchial and alveolar type 2 epithelial cells, and in fecal and urine samples [46,47]. Therefore, it is suggested that multiple organ dysfunction in patients with severe COVID-19 may also be caused by a direct attack by the virus. Many authors think that the synergistic effects of both effects contribute to the “multi-organ” failure of patients with severe COVID-19 however, we and some authors believe that in fatal COVID-19 cases, severe dysfunction of the immune response is responsible to a greater degree than the direct damaging effect of the virus itself [42,47]. (Figure 2) When macrophages or any other “antigen presenting cell” (APC) are stimulated, the “proinflammatory” cytokines par excellence are released: IL-1, IL-6, IL-8, IL-15, IL-17, IL-18, TNFs, IFN□ and PAF (platelet-activating factor). These cytokines play a relevant role in the inflammatory process and, in turn, can give rise to the so-called “cytokine storm”, the consequence of which is “systemic inflammatory response syndrome” (SIRS) and finally “multi-organ failure” (MOF), leading to death. On the other hand, as Niels Jerne (1974) said: “any stimulus capable of producing an immune response provokes a reaction comparable to the transmission of the ripples that can be observed in a pond when a stone is thrown, so that in the immune system the variation at the site of the stimulus receptor is transmitted everywhere”. In “SARS” this allegory reaches a dramatic expression and encompasses not only the network of signals, which cross and intersect within the immune system, but between the different systems (coagulation, fibrinolytics, cyanins, arachidonic acid, leukotrienes and thromboxanes, the immune system itself (complement system, circulating immune complexes ICC, ADCC, NK cells, adaptive immune response: CTL and cytokines) (Figure 2).

Figure 2: Navarro-Zorraquino M Immunologic response in shock and multiorgan failure.
In: Navarro-Zorraquino M, editor. Immunological aspects of surgery. Zaragoza:
Prensas Universitarias de Zaragoza; 1997. p. 261-300.

For this reason, the lack of control of the servomechanisms that maintain homeostasis in any of the mentioned systems can cause an unstoppable situation of mediator release leading irremediably to tissue damage [42]. From the pathophysiological point of view, inflammation is the result of multiple interactions between the various systems of the organism, which have as their first objective the localization of the process and the elimination of the aggressor agent; this is followed by a repair process. The main physical-chemical events that occur during inflammation are: increased blood supply to the site of the attack, increased capillary permeability – which allows larger molecules than usual, such as antibodies and fractions of the complement system and other enzyme systems, to pass through the vascular endothelium – and the activation of leukocytes: initially neutrophils and macrophages, then lymphocytes. The development of the inflammatory reaction is controlled by cytokines, which are the intercellular messengers of the “immunocompetent” molecules, the products of the plasma enzyme systems, the coagulation, fibrinolytic, cyanin and complement systems, vasoactive mediators released from mast cells, basophils and platelets, and endothelial adhesion molecules. Since CoV-2 exhibits tropism to the lung, the initiation of the immune response against coronavirus begins with direct infection of the bronchus and bronchiole epithelium. First, antigen-independent innate immunity provides the first line of defense of leukocytes against microorganisms. The “innate immune response involves several cell types, including leukocytes, neutrophils, eosinophils, eosinophils, basophils, monocytes, macrophages, lung epithelial cells, mast cells, and NK cells. After initial CoV-2 infection, dendritic cells (DCs) residing in the lungs become activated and change to “antigen presenting cells” (APCs).

Figure 3.

In the lung, DCs reside within and beneath the airway epithelium, alveolar septa, pulmonary capillaries and airway spaces. Activated APCs” cells ingest and process the antigens and migrate to the lymph nodes, in the lymph nodes the “APC cells” present the antigen in the form of MHC/peptide complex to the “virgin circulating T helper cells” (Th0), inducing the immune response. Following activation of the Th0 receptor by the MHC/peptide complex, Th2 cells are activated, proliferate and differentiate into CD4+ (Th lymphocytes) and CD8+ (cytotoxic T lymphocytes) cells. Subsequently, Th lymphocytes further differentiate into Th1 and Th2 cells, which are capable of releasing different cytokine profiles: Th1 cells drive cell-mediated immunity and release pro- inflammatory cytokines such as IFN-γ, IL-1β, IL-12 and pro-inflammatory factors such as TNFs, IFNs, PAF, GM-CSF, MCSF; Th2 cells activate the production of antibody-producing B cells and release anti- inflammatory cytokines such as TGF-β, IL-4, IL-5, IL-9, IL-10 and IL-13 [42,47]. In the immune response of healthy adults with CoV-2 infection there is a balance between Th1 and Th2 lymphocyte activity. The inflammatory reaction initiated by the immune system, through the Th1 activation pathway and with the participation of Th17 cells and various cytokines, is regulated by the immune response itself through a “regulatory servo-mechanism” involving mainly Th2 cells (considered as the main pathway of the “anti-inflammatory response”), through sub-populations of Th2 cells, called “regulatory cells”: Treg (CD4+25+FOXp3 and CD8+25+FOXp3) and Th-17 cells (Figure 3). Th17 cells” regulate the response by increasing the release of “pro-inflammatory” cytokines and Treg cells” regulate the response towards the release of anti-inflammatory cytokines. (Figure 3) Navarro-Zorraquino M. Immunologic response in shock and multiorgan failure. In: Navarro-Zorraquino M, editor. Immunological aspects of surgery. Zaragoza: Prensas Universitarias de Zaragoza; 1997. p. 261- 300. We wish to emphasize here that the “regulatory pathway” exerts its role by responding to the needs of the immune response, at a given time, against the corresponding antigen, by increasing the inflammatory activity of the Th1 pathway, mainly by means of Th17 cells and IL-17A, or by increasing the anti-inflammatory activity of the Th2 pathway, mainly by means of transforming growth factor β (TGF-β). Since these 2 cytokines are going to be the key in the design of our research project, we will insist on them later.

Systems of the Human Organism Affected by the “Cytokine Storm”

It is important to remember here the influence and consequences that the immune response has on the most important systems of the human organism, especially when it overflows producing the “cytokine storm”. If we look at Fig. 2, we can see that this response is related to the release of histamine, activation of the coagulation, fibrinolysis and “kinins” systems, release of arachidonic acid metabolites, neuroendocrine response, release of free radicals and release of prostacyclins and prostaglandins [42]. When the complement system is activated, the different fractions are released (activation by the classical pathway begins with the C1 fraction, and activation by the alternative pathway begins with the C3 fraction), but the most important for their pathophysiological actions are the C3a and C5a fractions (called anaphylatoxins), which increase capillary permeability and produce smooth muscle contractionboth at the level of the bronchial tree and the gastrointestinal tract; the C3a fraction is capable of producing tachycardia, impairing cardiac function and inducing coronary vasoconstriction. The C3a and C5a fractions stimulate basophils and mast cells to release histamine, whose main action is to increase vascular permeability and smooth muscle contraction. When aggression to the organism occurs, activation of the enzymatic cascades of the complement system, kinins, coagulation and fibrinolysis occurs rapidly, as well as cell activation of PMN leukocytes, macrophages, endothelial cells and platelets. Tissue damage produced by viruses (the case of CoV- 2) induces platelet aggregation and adhesiveness on subendothelial collagen when the vascular endothelium is damaged, thus initiating an activation, by means of the so-called intrinsic pathway, through the activation of factor XII (Hageman’s factor), which gives rise to FXIIa [42] which is an active protease; this is a key factor that directly relates the coagulation system to the so-called “kinin system”, “kinins” or “kinins” (Figure 2). kinins” or “kinins” (Figure 2). FXIIa activates pre-kallikrein which becomes kallikrein and this, in turn, becomes kininogen, a high molecular weight substance, which together with factor XII and pre-kallikrein binds directly to sub- endothelial collagen, as do platelets through the mediation of Willebrand factor (Figure 2).

At the same time that activation of the coagulation system by the intrinsic pathway occurs, activation of the so-called “extrinsic pathway” can also occur, by means of tissue thromboplastin released by damaged cells; tissue thromboplastin activates the extrinsic pathway in collaboration with factor VIIa (FVIIa) causing factor X (FX) to also become an active protease -FXa-. The result of the activation of the coagulation system by both pathways is the conversion of prothrombin to thrombin, which increases platelet aggregation and induces the release of arachidonic acid metabolites, especially thromboxane A2 (TxA2) (Figure 2).This activation of the coagulation system would be implicated in the immune response to CoV-2 and the production of clots in patients with COVID-19, especially in the most severe stage of the disease, as well as in the finding of clots in the necropsies of deceased patients. The hypothalamic-pituitary-adrenal axis responds to stimuli that represent the release of mediators and the organism’s own aggressor agent in a given situation. At the present time there are numerous studies that attempt to relate different hormones, whose synthesis and release is regulated by the neuroendocrine system, with the immune response in various situations. We will refer here only to what seems to us most relevant in relation to the inflammatory response and in particular to the pathophysiology of the “cytokine response”[42].

Cortisol is the most important glucocorticoid secreted by the adrenal cortex in response to ACTH and corticotropin-releasing hormone (CRH). Cortisol plays a very important role in many aspects of the inflammatory response and shock (it increases the effect of catecholamines, increases protein catabolism at the muscular level, has action (together with epinephrine and norepinephrine) on vascular smooth muscle, on lipolysis and on neoglycogenesis). But here we try to emphasize that cortisol inhibits the release of “kinins” and that it is closely connected with the release of other mediators and with the systems of coagulation, fibrinolysis and the complement system in the inflammatory response. In addition, cortisol considerably reduces the number of lymphocytes, especially the number of T- lymphocytes, in patients with sepsis. in this regard, it is very noticeable that the majority of patients affected by COVID-19 show lymphopenia. Nitric oxide (NO) is synthesized in the body from L-arginine by an enzyme: nitric oxide synthase. There are two types of this enzyme: one is a constituent of the cytoplasm and is Ca++ and calmodulin dependent for NO release; the other enzyme is also a cytoplasmic component, but is Ca++ independent, however it requires tetrahydro-biopterin and other cofactors for its activation and is inhibited by glucocorticoids. Following the studies of Furchgott and Zawadzki, et al. [47-50] there is no doubt that NO is a very important neurotransmitter. The enzyme nitric oxide synthase is found in brain neurons, but is not present in glia; in the pituitary it is found in brain neurons, but is not present in glia; in the pituitary it is found mainly in neurons located in the posterior lobe (which are the neurons that synthesize and release vasopressin and oxytocin), it is also found in the adrenal medulla in neurons that stimulate the cells that release adrenaline or epinephrine), in the intestine it is found in the mesenteric plexuses, regulating peristaltic movements. In addition, nitric oxide synthase is present in numerous tissues, but especially in the cells of the endothelial layer of blood vessels, where it seems to play an important role in vasomotor phenomena, but also as a “messenger” molecule closely connected to the immune system. In all these tissues NO release by nitric oxide synthase appears to be Ca++ and calmodulin dependent (as described above), constituting the “physiological NO production pathway”.

The point of view that most interests us here is the relationship of NO with the immune response, not only because it is able to stimulate macrophages, endothelial and dendritic cells against bacteria, but also against viruses and rikettsias, and because it is actively involved in the inflammatory process. Its excess production may contribute to a high degree to the pathophysiology of SARS and multiorgan failure. Macrophages produce detectable levels of NO about 6 hours after activation by IFN-g, reaching the maximum level at 24h. However, there is a “servo-control mechanism” by which NO can regulate its own synthesis, inhibiting IFN-g production from Th1 cells and also that of nitric oxide synthase. In addition, some cytokines, including IL-4 and IL-10 and TGF-b, also have an inhibitory effect (apparently “dose-dependent”) on NO production. In this respect, antagonists of cytokine and NO production could be a therapeutic measure in the treatment of COVID-19, as evidenced by some in vitro studies.

Risk Factors Associated with COVID-19 Infection

Diseases associated with COVID-19 infection, mainly severe heart disease, chronic kidney disease, chronic obstructive pulmonary disease (COPD), cancer (patients undergoing active treatment), immunosuppression due to solid organ transplantation, obesity and type 2 diabetes mellitus, together with advanced age of the patients, can result in “immune dysregulation” leading to failure of the “system regulatory pathway” and “anti-inflammatory pathway” with an exaggerated shift towards the “inflammatory pathway”, can result in “immune dysregulation”, leading to failure of the “system regulatory pathway” and the “anti-inflammatory pathway” with an exaggerated shift to the “inflammatory pathway” which can develop into a huge release of cytokines and inflammatory factors called “cytokine storm”.

Advanced age is perhaps the most important factor in our century, since there are populations of people living in the world at very advanced ages of life (even people > 100 years), especially in developed countries. Overall, published work with respect to patient age shows that the COVID-19 pandemic is causing a large increase in mortality in the elderly population, relative to the mortality rate observed in patients under 70 years of age.” The mortality rate is dramatically alarming in the case of patients older than 80 years, about 30% compared to the total population of COVID-19 infected patients [44]. Some currently published statistical data show that the probability of death from COVID-19, compared with the age group of infected patients aged 18-29 years, can be summarized as follows: persons aged 30-39 years (2 times higher), 40-49 years (3 times higher), 50-64 years (4 times higher), and 50-64 years (4 times higher). higher). 65-74 years (5 times higher), 74-84 years (8 times higher), > 85 years (13 times higher) [51]. All of the above shows that the COVID-19 pandemic is causing a large increase in mortality in the elderly population, compared to the mortality rate observed in patients younger than 70 years of age. The mortality rate is dramatically alarming in the case of patients over 80 years of age, about 30% compared to the total population of CoV-2 infected patients.

Important Characteristics of Aging

Chronic inflammation in aging, described as “inflammatory aging, may occur in elderly patients, and may also be associated with other related disorders. with inflammation: diabetes mellitus, obesity, arthrosis, etc. Consequently, the increased generation of pro-inflammatory markers in “inflammatory aging” may have an impact on the severe inflammatory process that occurs in patients with COVID-19 and increased risk of mortality. Several factors, including altered ACE2 receptor expression, excess reactive oxygen species (ROS) production, senescent adipocyte activity, altered autophagy and mitophagy, “immunosenescence”, as well as severe vitamin D deficiency (VD) may be associated with “inflammatory aging” and contribute to the cytokine storm in elderly patients suffering from COVID-19 [52,53].

Alteration Of Ace2 Receptor Expression

SARS-CoV-2” uses the same receptor “angiotensin-converting enzyme 2” (ACE2) as “SARS-CoV” (the coronavirus associated with the SARS outbreak in 2003). The “renin-angiotensin system” (RAS) is an important regulator of several physiological events, including cardiovascular and blood volume, natriuresis, diabetes, chronic kidney disease and liver fibrosis. The study by Xudong and colleagues in 2006 observed in the rat lung that ACE2 expression is significantly reduced with aging; these authors suggest that ACE2, which is higher in young adults compared to older age groups, may contribute to the prevalence of SARS episodes in this age group. On the other hand, Chen and colleagues, in 2020, found a markedly higher expression of ACE2 in Asian women compared with men; they also found an age-dependent decrease in ACE2 expression, and a highly significant decrease in type II diabetic patients, and established a negative correlation between ACE2 expression and death from COVID-19 [54].

Excess Production of Reactive Oxygen Species (ROS)

The effects of reactive oxygen species (ROS) on cellular metabolism have been well documented in a wide variety of species. These include not only roles in programmed cell death and necrosis, but also positive effects, such as induction of defense genes and mobilization of ion transport systems. It is also frequently implicated in “redox signaling” or “oxidative signaling” functions. In particular, platelets involved in wound repair and blood homeostasis release reactive oxygen species to recruit more platelets to sites of injury. They also provide a link to [immune system] adaptation through white blood cell recruitment. Reactive oxygen species are involved in cellular activity in a variety of inflammatory responses including cardiovascular disease. They may also be involved in cochlear damage induced by elevated sound levels, ototoxicity of drugs such as cisplatin, and in congenital deafness in animals and humans. Redox signaling is also involved in mediating apoptosis or programmed cell death and in ischemic injury. Specific examples are strokes and heart attacks. Garrido, et al. [55] identified that immune cells from prematurely aging mice had lower values of antioxidant defenses and higher values of ROS and pro-inflammatory cytokines, thus suggesting that excessive ROS production during aging may activate the inflammatory response and subsequently increased release of pro-inflammatory cytokines, which include TNF-α, IL-1β, IL-2 and IL-6 and adhesion molecules. Therefore, excessive ROS production and inflammation are closely related, as they are involved in the pathogenesis of chronic inflammation and “inflammatory aging” in older adults.

Autophagy and Age

Autophagy is a conserved catabolic turnover pathway in eukaryotic cells by which cellular material is delivered to lysosomes for degradation. The autophagy process is related to the maintenance of cellular homeostasis, and its dysregulation could lead to the development of several pathophysiological diseases related to aging [56]. It has been shown that the autophagy process decreases during aging and leads to the accumulation of damaged macromolecules and organelles. Decreased autophagy during aging may also lead to dysfunctions in mitochondria and consequently to increased ROS production [57] (since mitochondria are the main source of ROS. On the other hand, mitophagy, which is characterized by autophagic degradation of mitochondria, decreases in aging the decrease in mitophagy, together with the decrease in antioxidant capacity during aging [58], may increase the levels of ROS in the human organism and also to the increased secretion of proinflammatory cytokines during aging [59-62].

Senescent Adipocytes and Age

Some studies on aging highlight the importance of adipose tissue inflammation in aged animals by elevated release of IL- 6, IL-8, IL-1β, and TNF-α. [63-65] Adipose tissue is a dynamic structure that plays an important role in modulating metabolism and inflammation. It is very likely that adipose tissue dysfunction (e.g., obesity during aging) is associated with chronic inflammation in elderly subjects [66]. The mortality rate of obese elderly patients with COVID-19 is approximately 14%. Covarrubias ,et al. [67] found that during aging senescent cells accumulate significantly in visceral adipose tissue and that “inflammatory cytokines” are found in the supernatant of senescent cells, Alicka et al. in 2020 found that “stem cells” derived from adipose tissue of old horses (older than 5 years) exhibited increased gene expression of pro-inflammatory and miRNA genes (such as IL-8, IL-1β, TNF-α, miR-203b-5p and miR-16-5p) and markers of apoptosis (such as p21, p53, caspase-3, caspase-9) [68]. Therefore, it is possible that elevated release of pro-inflammatory cytokines by senescing adipocytes carries an elevated risk of the “cytokine storm” in obese elderly patients with COVID-19.

Age and Immunosenescence

Immunological senescence” is characterized by alterations in both humoral and cell-mediated immune response. Dysregulation of the response severely impacts the pro-inflammatory/antiinflammatory balance when the organism is attacked by an infectious agent. It is known that NK cells and macrophages link the innate and cell-mediated immune systems. Some authors have described an increase in the number of circulating NK cells during aging [69]. One of the important cytokines for the cytotoxic activity of NK cells is IL-2, which increases the killing properties and proliferation of NK cells. In a young healthy individual, IL-2 can induce IFNg secretion by NK cells, but this effect is diminished in the elderly [70]. On the other hand, it has been observed that T cell numbers do not decrease during aging, but the T cell pool shows significant age-related alterations, including impaired responses to T cell stimulation by mitogens, an inverted CD4+/CD8+ T cell ratio, a reduced proportion of Th0 cells, and an increased proportion of “memory cells,” in animals and humans [71-73]. In addition, aging is associated with overproduction of pro-inflammatory cytokines by T cells, leading to immune pathology [74]. The proportion of Th17 cells increases during aging, resulting in an “inflammatory aging” state in adults [75]. The “Th17 regulatory” cells have the “pro-inflammatory” phenotype and are in balance with “antiinflammatory Th-reg cells.” Both cells are derived from a common precursor: Th0 cells [76]. During aging, the generation of several macrophage-induced factors, including fibroblast growth factor, vascular endothelial growth factor, epithelial growth factor, transforming growth factor (TGFβ), is reduced. TGFβ is one of the most important “cytokines” released by “anti-inflammatory regulatory cells”. Therefore, it is thought that the fragile and mildly overactive immune system in older adults cannot turn off proinflammatory response in COVID- infection. 19. The clinical findings in severe patients with COVID-19 infection are consistent with the literature mentioned above. In 2019, Schouten et al. identified that the increase in “pro-inflammatory cytokines” during aging also correlated with SARS severity and could explain, at least in part, the difference in COVID-19 severity between young adult patients and elderly patients [77].

Age and Vitamin D Deficiency

Older adults are at risk for vitamin D deficiency due to several factors, including decreased pre-vitamin D production, poor skin integrity, decreased dietary intake of vitamin D, increased adiposity, obesity, decreased kidney function, as well as less time outdoors [78].Vitamin D deficiency has been linked to various inflammatory diseases related to aging, such as rheumatoid arthritis, asthma, inflammatory bowel disease, multiple sclerosis, cardiovascular disease, hypertension, diabetes mellitus, and cancer [79].

Vitamin D together with the vitamin D receptor (VDR) have an important anti-inflammatory function, acting as “immunomodulators” by decreasing the release by Th1 cells of “proinflammatory cytokines” and increasing the release by Th2 cells of “anti-inflammatory cytokines”. Furthermore, vitamin D deficiency in elderly subjects is associated with the pro-inflammatory phenotype of immune cells, which probably increases the risk of “inflammatory aging” in older adults [80], and this chronic inflammatory condition could contribute to the “cytokine storm” in elderly patients with COVID-19. However, patients with renal failure or granulomatous disease are at high risk for side effects and should be excluded from being treated with vitamin D supplementation. Upcoming vitamin D supplementation trials will provide more clarity on the in vivo effects and the opportunities and possible limitations of vitamin D as an immuno-regulatory agent. In this regard, recent work by Murai, et. al [81] shows that high-dose vitamin D3 shows no significant difference among hospitalized patients with COVID-19, nor does it significantly reduce the length of hospital stay. These findings do not support the use of high- dose vitamin D3 for the treatment of moderate to severe COVID-19.

Influence of Sex

The higher COVID-19 case fatality rate and greater disease severity in men compared to women are likely due to a combination of behavioral/lifestyle risk factors, prevalence of comorbidities, aging, and underlying biological sex differences. However, the underlying biological sex differences and their effects on COVID-19 outcomes have received less attention. The recent review conducted by Haitao Tu, Vermunt JV et al. of the Mayo Clinic (October 2020) [82] summarizes the available literature regarding proposed molecular and cellular markers in COVID-19 infection, their associations with health outcomes, and any reported modifications by sex.

Biological sex differences characterized by such biomarkers exist within healthy populations and also differ with age- and sex-specific conditions, such as pregnancy and menopause. In the context of COVID-19, descriptive biomarker levels are often reported by sex, but data regarding the effect of patient sex on the relationship between biomarkers and COVID-19 disease severity/outcome are scarce. Such biomarkers may offer plausible explanations for the worse COVID- 19 outcomes observed in men. Larger studies with sex-specific reporting and robust analyses are needed to elucidate how sex modifies the cellular and molecular pathways associated with SARS-CoV-2. This would improve biomarker interpretation and clinical management of patients with COVID-19 by facilitating a personalized medical approach to risk stratification, prevention, and treatment. Several comorbidities, which occur disproportionately in men, likely contribute to worse COVID-19 outcomes, it is thought that perhaps ACE inhibitors are involved or that angiotensin receptor blockers may exert adverse effects on COVID-19. Experimental and epidemiological evidence is conflicting as to whether the use of ACE inhibitors and angiotensin receptor blockers upregulate ACE2 expression and affect susceptibility to infection and/or disease severity. Ongoing randomized clinical trials could inform whether this differs by sex and recommendations on the use of such therapy in patients with COVID-19.

Immunologically

It appears that women have a stronger immune response overall; however, men are more likely to develop the “cytokine storm associated with poor outcomes against COVID-19. Further research on immuno-modulation by sex hormones, age and X-linked gene expression could help explain the poorer survival of men and identify sex-specific risk factors for SARS-CoV-2 infection and the course, outcome and prognosis of COVID.

Current Treatment of COVID-19

Despite advances in the deterioration of the COVID-19 patient population, there is no approved drug that has considerable beneficial effects in the medical treatment of COVID-19 patients. Hydroxychloroquine was the first drug of choice for the treatment of the disease, but today it is being rejected because of its ineffectiveness and because in some cases it has aggravated the condition of the treated patient. At present, umifenovir, remdesivir and favipiravir are thought to be the most promising antiviral agents for improving the health of infected patients. Dexamethasone is being considered as the first known steroid drug that can save the lives of critically ill patients, as it was shown in a randomized clinical trial in the UK to reduce the death rate in patients with COVID-19. However, despite its increased use worldwide it is not a truly effective treatment over the current high mortality rate in severe cases.

Based on the evidence, the US Food and Drug Administration (FDA) approved some drugs that had already been used in the treatment of SARC-CoV and MERC-COV. The primary treatment chosen for COVID-19, lopinavir, is an antiretroviral (ARV) drug used for the treatment of HIV-1 and has been used for COVID- 19 in combination with ritonavir (potent anti-HIV drug). Currently, 64 clinical trials are underway with lopinavir-ritonavir along with other drug implications, and most of them are in the early stage of progress. The latest evidence for the management of COVID-19 will be uncovered shortly. No single drug may be superior or inferior, however, the use of a single drug may not be effective enough to control this deadly virus, considering PK and drug metabolism, the use of a combination of antivirals with different mechanisms of action may be more effective [83].

Antiviral Agents Used to Date

Remdesivir

Remdesivir (GS-5734) was developed by Gilead Sciences (Foster City, CA, USA). It is an adenosine triphosphate analog and has been used to treat coronavirus and Ebola virus. Remdesivir stops viral replication by inhibiting essential replication enzymes (RNA-dependent RNA polymerase). Currently, more than 24 clinical trials are underway in patients with COVID-19 [84].

Favipiravir

Favipiravir directly inhibits viral transcription by inhibiting RNA polymerase. Currently, 18 clinical trials in various stages of development are underway for the treatment of COVID-A Phase 3 clinical trial has recently been initiated in India, and full study results are expected to be published soon. Clearance for the clinical trial phase evaluation for the safety and efficacy of favipiravir in tablet form has been granted to Appili Therapeutics to monitor COVID-19 in long-term care services [85].

Lopinavir/ritonavir

Lopinavir (Kaletra) is a potent anti-HIV drug used to treat HIV infection in combination with ritonavir. Ritonavir inhibits the pharmacological metabolism of lopinavir to improve PK (half-life) and activity. The Infectious Diseases Society of America (IDSA) recommended ritonavir-boosted combination therapy for HCV patients as first-line therapy. Lopinavir / ritonavir have shown anti-SARS-CoV-2 activity “in vitro” by inhibiting protease in Vero E6 cells [86]. In addition, SARS patients revealed that lopinavirritonavir plays an important role in explaining clinical outcomes and in combination with IFN improved clinical outcomes in some MERS patients [87]. In India, the EMR division has recommended the dosing schedule of this drug combination for the clinical management of COVID-19.

Ribavirin

Ribavirin is a broad-spectrum antiviral drug developed by Bausch Health Companies (Bridgewater Township, New Jersey, USA). It is a guanosine analog used to treat several viral diseases. It showed a lower risk of death in ARDS (acute respiratory distress syndrome) infection in combination with lopinavir- ritonavir. In recent “in vitro” studies, ribavirin showed high efficacy against COVID-19; however, in other studies rivavirin showed an unexpected adverse effect, which was very detrimental to some patients with SARS. [88-89].

Umifenovir

Umifenovir, also known as Arbidolâ , is a broad-spectrum antiviral agent developed by the Russian Institute of Chemical and Pharmaceutical Research. Lopinavir-ritonavir and umifenovir were previously used to treat acute SARC-CoV in clinical practice; however, their efficacy remains debated. The clinical safety and efficacy of umifenovir monotherapy were analyzed in patients with COVID-19 and compared with lopinavir-ritonavir therapy. Umifenovir was found to be better than lopinavir-ritonavir for the treatment of COVID-19 [90]. This drug has obtained approval to proceed with the phase III clinical trial of umifenovir. This randomized, double-blind, placebo-controlled trial will test the efficacy, safety and tolerability of umifenovir. Results are expected to be reported soon [83].

Nitazoxanide

Nitazoxanide inhibits viral infection by potentiating the hostspecific mechanism. Although the “in vitro” activity of nitazoxanide against SARC-CoV-2 suggests that it is effective, more clinical data are needed to estimate efficacy and safety against CO-VID-19 [91]. Currently, many clinical trials of nitazoxanide are underway with various doses to treat patients with COVID-19. 969Although the results are not encouraging or available yet, the FDA has given approval to Azidus Brazil for nitazoxanide to continue with the Phase II clinical trial.

Ivermectin

Ivermectin, an FDA-approved antiparasitic agent as effective as Albendazoleâ, has shown activity against many viruses. Recently, an in vitro study has shown that ivermectin inhibits COVID-19 replication. Its antiviral activity may play a key role and be a potential candidate to treat COVID-19. Finally, the FDA announced a statement for the administration of ivermectin in patients with COVID-19 [92].

Interferons

Interferon (IFN) is a broad-spectrum antiviral agent that inhibits viral replication by interacting with the toll-like receptor (TLR Type III IFNs (IFN-λs) were identified in 2003 and were independently used to elicit antiviral resistances in cells. One member of this family (IFN-λ) [93] was shown to be effective in 2013. IFNs of this type have been used to treat patients critically ill with chronic hepatitis C virus and have also been effective in treating people infected with hepatitis B virus, so they are believed to have the ability to protect patients during outbreaks of other viruses. IFN-λ has also been shown to be more efficacious compared to IFNα-based therapies, also leading to less increase in inflammation and tissue damage, and potentially restricted viral spread from the nasal epithelium to the upper respiratory tract. Moreover, IFNα and β exhibited activity against SARS-CoV “in vitro”. IFNβ also showed potential action to decrease MERS-CoV replication. For the most part, type I IFN showed a rapid decrease in viral load in patients with mild or moderate COVID-19. In severe COVID- 19 infection, IFN showed an antiviral response, but with elevated pulmonary cytokine levels, and weakened T-cell response and acute clinical relapse [94].

Dexamethasone

The main synthetic glucocorticoids: dexamethasone, triancinolone and prednisone are used as immunosuppressants, but their therapeutic indications also include their anti-inflammatory action, and because of their qualities as anti-lymphocyte cytostatics they are used in oncology and in the treatment of allergic diseases. The immunological effects of these drugs are multiple and differ between experimental animals (rodents) and man. In man there is, within a few hours of administration, an increase in neutrophils and a decrease in all other white blood cells in peripheral blood, this decrease being more pronounced for B and T lymphocytes. Although a single dose of glucocorticoids has little effect on B lymphocytes, treatment for several days (3 to 10) may result in a decrease in IgG, IgA and IgM. The FDA approved dexamethasone as a spectrum immunosuppressant in 1958. It is 30 times more potent and longer lasting than cortisone and reduces the ability of B cells to synthesize antibodies [95]. However, a clinical trial showed that dexamethasone saved the lives of severely ill COVID-19-infected patients in the United Kingdom [96]. The UK government declared that dexamethasone was allowed as an immediate treatment option for hospitalized patients who were critically ill and on ventilators. WHO added dexamethasone to the list of life-saving drugs that are readily available at low cost. In the U.S., guidance was issued to recommend dexamethasone as a treatment option for patients infected with CO- VID-19. However, clinical evidence does not support the use of corticosteroids in COVID-19 infection [96]. Dexamethasone may regulate, to some extent, the damaging effects of cytokines by limiting their release, but it has not been shown to be able to inhibit the “cytokine storm”, when the antigen overwhelms the regulatory capacity of the immune response. In addition, dexamethasone prevents macrophages and NK cells from eliminating nosocomial pathogens associated with “coronavirus”.

Tetracyclines

Tetracycline can be used as a possible treatment option for patients with COVID-19 because of its known activity to decrease the level of inflammatory cytokines such as IL-1b and IL-6 [97]. Both IL-1b and IL-6 levels increase significantly in the body of patients during COVID-19 infection. Tetracycline has also been shown to decrease inflammatory factors in the circulation through activation of protein kinase C and induction of programmed cell death [98].

Tocilizumab

Tocilizumab (called Actemra) is a recombinant monoclonal antibody developed by Roche Pharmaceuticals (Basel, Switzerland). Tocilizumab is basically used to treat rheumatoid arthritis. It was designed as an IL-6 receptor blocker to inhibit the binding of IL-6 to its receptor, thus alleviating the “cytokine release” syndrome.

IL-6 is significantly increased in the body of patients when exposed to COVID-19 infection. This is why tocilizumab is used as a therapeutic option for the treatment of patients with COVID-19 [99]. In COVID- 19 infected patients, T lymphocytes and macrophages produce IL-6 and and help the “cytokine storm” and severe inflammatory responses in the lungs and other tissues. Tocilizumab has binding affinity for the IL- 6 receptor and renders the receptor unable to bind IL-6, decreasing the inflammatory response and ultimately decreasing the IL-6 signal transduction pathway [100]. Consequently, it may be essentially an effective therapeutic drug for the treatment of patients with severe COVID-19 infection [101]. The FDA has given Genentech approval to proceed with the Phase III clinical trial of intravenous tocilizumab to evaluate its safety and efficacy in adult patients infected with COVID- 19.

Itolizumab

Itolizumab (called Alzumab) is a recombinant monoclonal antibody against CD6 (IgG1 (Immunoglobulin G1) differentiation group. It was developed for the treatment of psoriatic patients [102]. It showed reduction of IL-6 in critically ill patients. Itolizumab has been shown to have the effect of regulating downstream activation pathways and reduction of inflammatory cytokines, such as IFN-γ, TNF- α and IL-6 [103]. Based on the mode of action, it could be used as a treatment option for COVID-19 infection [103].

Teicoplanin

Teicoplanin (called Targocid) was developed by Sanofi Pharmaceuticals (Paris, France). It is an antiviral drug that can inhibit replication and transcription of the competent virus. It also works against MERS and SARS [104]. Mechanistic investigations revealed that teicoplanin specifically inhibits the activity of host cell cathepsin L and cathepsin B; these proteins are responsible for cleaving the viral glycoprotein, allowing contact of the receptorbinding domain of its core genome and subsequent release into the host cell cytoplasm [105-106]. Since COVID-19 is also “virusdependent” on cathepsin L, some studies suggested that teicoplanin could be used as a therapeutic option to treat COVID-19. According to Ceccarelli, et al. [107], teicoplanin would have a possible therapeutic effect in COVID-19 infected subjects. At present, an in vivo study using teicoplanin in subjects affected by COVID-19 has already been performed for the first time and the results seem quite acceptable compared to a previous report from the same geographical area. Teicoplanin is now thought to be a promising option for the treatment of COVID-19 although more safety data in humans are still required.

Meplazumab

Meplazumab is a humanized monoclonal antibody that acts against the CD147 spike protein. In in vitro studies, it has been shown to effectively inhibit virus replication in Vero E6 cells [108]. Based on this evidence, a study has been conducted to determine the clinical outcomes with the use of meplazumab in treating patients infected with COVID-19. Meplazumab was previously reported to exhibit activity against “Chauge-Strauss syndrome” (characterized by eosinophilic vasculitis, pulmonary infiltration, sinusitis, neuropathy and asthma) [109].

The Phase I clinical trial (NCT0436369586) in healthy volunteers with maplazumab injection is currently being completed to find the safety, efficacy, tolerability, pharmacokinetic characteristics and dosing regimen for the Phase II clinical trial. In the U.S., an openlabel Phase I and Phase II clinical trial is underway to determine the safety and efficacy of meplazumab injection in patients infected with COVID- 19 (NCT04275245). Meplazumab could be used as a therapeutic option to treat patients with COVID-19.

Eculizumab

Eculizumab (Soliris, Alexion Pharma International, Zürich, Switzerland), a human monoclonal antibody, is a highly selective and effective C5-binding protein of the complement system with high affinity. It prevents cleavage to C5a and C5b and inhibits the production of the membrane attack complex (MAC) C5b- 9 to lyse cells. Interestingly, blockade of C5 reveals an indirect “immunoprotective” action by preserving early components of the complement system [110]. Consequently, eculizumab could function as an emergency therapy to treat patients with CO-VID-19 associated with SARS. Some studies have supported the use of eculizumab as a treatment for severe COVID-19. In addition, more clinical trials are approved, some already completed, studying the action of eculizumab in combination with ruxolitinib for efficacy in patients with severe COVID-19 [111].

AMY101

AMY101 is a highly selective inhibitor of the C3 fraction of the complement system that was developed by Amyndas Pharmaceuticals). AMY101 has successfully completed clinical phase I with acceptable safety and tolerability and is now in phase II clinical trial (NCT04395456) AMY101 could be a unique therapeutic option to overcome the complement-mediated inflammatory response in patients with COVID-19 [112-113].

ARDS-003

Cannabinoid (CBD) is also a potential treatment for patients with severe COVID-19. It was designed as an injectable form to treat a severe case of coronavirus with “acute respiratory distress syndrome” It may have the advantage of affecting several proinflammatory signaling pathways by enhancing the effectiveness of the drug to rapidly dampen cytokine release and prevent acute ARDS outcomes [114]. The cannabinoid drug named “ARDS-003” has been approved for a Phase I clinical trial, which is still being conducted by Tetra Bio-Pharma. Initially, the FDA emphasized that the results of the non-clinical studies were appropriate to begin the study in COVID-19 infected patients.

LCB1

CB1 has been shown to be the “SARS-CoV-2 neutralizing antibody”. It is a computer-engineered mini- protein that has been synthesized by researchers at the University of Washington School of Medicine. It binds tightly to SARS-CoV-2 spike proteins and prevents it from infecting cells. “LCB1” was shown to protect “Vero E6” cells from SARS-CoV-2 infection. Synthetic antiviral candidates were designed to stop infection by interfering with the mechanism used by the coronavirus to penetrate and enter cells. LCB1 is currently being evaluated in rodents [115]. These “hyperstable mini-agglutinants” provide a starting point for the most novel COVID-19) therapeutics.

Convalescent plasma

Convalescent plasma therapy has potential to cure COVID-19 (145). Clinical data are very limited to date, but suggest that it is safe, clinically effective, and reduces mortality. However, there is an urgent need for “multicenter clinical trial” studies to establish its efficacy in patients with COVID-19. The U.S. FDA has issued an “emergency use” clearance for “convalescent plasma”, currently under investigation, for the treatment of patients with COVID-19. In addition, polyclonal antibodies from convalescent individuals and immunoglobulin concentrates (human and bovine) may also be of interest in the treatment of COVID-19, at this moment a Spanish company is working on it.

Vaccine development

Coronaviruses are a family of single-stranded RNA viruses that infect many animal species, including bats and humans. Prior to 2003, only twelve animal or human coronaviruses were identified. In the last eighteen years, three new and deadly strains have spread to humans. In 2003, the severe acute respiratory syndrome coronavirus (SARS-CoV) had an official number of 8096 cases and 774 deaths, with people with pre- existing conditions suffering the highest mortality. The overall effect of COVID-19 vaccine development has been a massive invigoration of the field of pandemic vaccine development. The current vaccines are realizing the theoretical promise of antigen sequence-only platforms, such as mRNA and vector-based platforms, and have massively accelerated their development toward rapid “Phase 3 vaccination against COVID-19” evaluation in a timeframe never seen before for vaccines. However, it is important to note that, despite their rapid manufacturing timeline, these platforms encode an antigen that was developed over a timeline of many years through basic research on coronavirus biology and protein engineering. Largescale investment and unprecedented mobilization of the research community have generated insights into the design, manufacture, formulation, and deployment of candidate vaccines that may pay dividends in the future when society must cope with the next inevitable infectious disease outbreak [116-117,83].

Herbal Medicines

In China, during the COVID-19 outbreak, some traditional medicines were used, such as Astragali Radix (Huangqi), Saposhnikoviae Radix (Fangfeng), Glycyrrhizae Radix et Rhizoma (Gancao), Atractylodis Macrocephalae Rhizoma (Baizhu) [118]. Some cannabinoid products were also used [119]. As a treatment option to control the inflammatory response medicinal plants with proven antiviral effects and related beneficial effects could be considered as an alternative approach to prevent high-risk population from COVID-19. However, there are no randomized studies to know the true efficacy and side effects of these natural products obtained from plants. Currently, other researchers, and ourselves, are focusing our attention on the acute and systemic inflammatory process that leads to the activation of “damageassociated molecular patterns” (DAMPs), as well as the study of substances capable of preventing or decreasing cell damage by “suppressing/inhibiting DAMPs” (SAMPs), leading to the resolution of the “inflammatory disease”.

Authors such as Land WG (Laboratory of Excellence Transplantex, University of Strasbourg, Strasbourg, France and German Academy for Transplantation Medicine), think that current or future therapeutics will include the inhibition of “DAMPs” in hyper-inflammatory processes, e.g., “systemic inflammatory response syndrome” (SIRS), which is currently observed in Covid-19, as well as the application of “SAMPs” in chronic inflammatory diseases, in “hyperresolution” processes, systemic inflammatory response syndrome” (SIRS), currently observed in Covid-19, as well as the application of “SAMPs” in chronic inflammatory diseases, in “hyper-resolving” processes (e.g. compensatory anti-inflammatory response syndrome) and in the administration of “SAMPs” in the treatment of chronic inflammatory diseases. We are in full agreement with this author that controlled production of “DAMPs” and “SAMPs” is necessary to achieve complete homeostatic restoration and repair of tissue injury and tissue damage. On the other hand, we fully agree with this author that a controlled production of “DAMPs” and “SAMPs” is necessary to achieve complete homeostatic restoration and repair of tissue injury, and also with the need to identify and define “a priori” a context-dependent “homeostatic DAMPs/ SAMPs ratio” in each case and a “homeostatic window” of DAMP, and SAMP concentrations, to ensure a safe treatment modality in patients [120]. In this aspect, our research group has recently published the work: “Implant of mesenchymal cells decreases acute cellular rejection in small bowel transplantation”[121]in which the inhibition of acute cell-mediated rejection is observed in an experimental model of allogeneic small intestine transplantation, through the implantation of mesenchymal cells and the activation of the “immune-regulatory response”, with an increase in the percentage of Treg cells, a significant increase in TGFb-1 and a decrease in IL-17. This finding will serve as the basis for the project: “Treatment of coronavirus-19 infection using nonsteroidal immunomodulators”, where the DAMPs will be the proinflammatory cytokines and the SAMPs will be the “anti-IL17” and “TGF-β1” molecules.

TGF- β1

As we have described above, the “pathway of regulation of the immune response” exerts its role by responding to the needs of the immune response, at a given moment, against the corresponding antigen, increasing the inflammatory activity of the Th1 pathway, mainly by means of Th17 cells and IL-17A, or increasing the antiinflammatory activity of the Th2 pathway, mainly by means of the “transforming growth factor β” (TGF-β). In the scheme shown on page 7 of this paper, we can see that this factor already acts from the “immune-regulatory pathway” and is part of the “anti-inflammatory pathway”, tipping the balance towards this second pathway. In our previously cited work on the inhibition of acute cell-mediated rejection in intestinal transplantation, “TGFb-1” is shown to be the most important factor, in relation to the other cytokines studied, acting as an inhibitor of the inflammatory immune response in rejection. In 2000, the Canadian researchers Prud’homme GJ and Piccirillo CA already pointed out that the importance of the factor “TGF-β” in “immuno-regulation” and tolerance had been recognized once again [122].

Like us, the authors propose that there are regulatory T-cell (T-reg) populations, some called T-helper type 3 (Th3), exert their action mainly by secreting this cytokine, and furthermore these authors emphasize the following concepts: 1) TGF-β1 has multiple suppressive actions on T cells, B cells, macrophages and other cells, and increased TGF-β1 production correlates with protection and/ or recovery from autoimmune diseases; 2) TGF-β1 and CTLA-4 are molecules that work together to terminate immune responses; 3) Th0, Th1 and Th2 clones can secrete TGF-β1 following CTLA-4 cross-linking; 4) TGF-β1 may play a role in the switch from effector T cells to memory T cells; 5) TGF-β1 acts with some other inhibitory molecules to maintain a state of tolerance, which is most evident in immunologically privileged sites, but may also be important in other organs; 6) TGF-β1 is produced by many cell types, is always present in plasma (in its latent form) and permeates all organs, binding to matrix components and creating a reservoir of this immunosuppressive molecule; and 7) TGF-β1 has beneficial effects in several autoimmune diseases and shows that it can be effectively administered by a somatic gene therapy approach, resulting in depressed inflammatory cytokine production and increased production of endogenous regulatory cytokines.

Currently, March 2021, Aydemir MN, et al. [123] have published an interesting paper. The authors believe that despite the information obtained on the structure of the SARS-CoV-2 viral genome, many aspects of virus-host interactions during infection are still unknown; their purpose in this study has been to identify the “microRNAs” (“miRNAs”) encoded by SARS-CoV- 2” and their cellular targets. The authors have employed for this purpose a computational method to predict SARS-CoV-2-encoded miRNAs along with their putative targets in humans. The predicted miRNA targets were grouped into clusters according to their biological processes, molecular function and cellular compartments. Aydemir MN, et al. note that the “TGF-β1 pathway” has important functions in many cellular processes, and that it is often manipulated by viruses, as it is a simple pathway. The authors expose that proteins that play a crucial role in almost all steps of this pathway are targeted by SARS-CoV-2 miRNAs and demonstrate that the SARS-CoV “nucleocapsid protein” (“N”) inhibits the formation of the “SMAD” complex (family of inducing genes of this pathway), resulting in blocking TGF-β1-induced apoptosis of Cov-2-infected cells and, conversely, tissue fibrosis in SARS-CoV-infected “host cells” [124]. Finally, these authors performed an integrative pathway network analysis with target genes and identified 40 SARS-CoV-2 miRNAs and their regulated targets, the analysis shows that the targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C , JARID2 plays an important role in NFKB, JAK/STAT and TGFB signaling pathways as well as epigenetic regulatory pathways in cells and they believe that their results may help to understand the virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. Since there is currently no drug or effective treatment available for COVID19, it may also help to develop new treatment strategies.

Monoclonal Antibody Against Il-17

COVID-19 is caused by SARS-CoV-2, a “beta-coronavirus” closely related to MERS-CoV and SARS- CoV, the causative agents of “Middle East respiratory syndrome (MERS)” and “severe acute respiratory syndrome” (SARS), respectively. COVID-19 appears to follow a similar pattern, with 81% of fatal cases diagnosed with SARS (2). In consideration of this, a recent publication in The Lancet [125] suggests that all patients with COVID-19 should be evaluated for “hyper-inflammation” in order to identify those who would benefit from targeted immunosuppression or immunomodulation to prevent acute lung disease. (“ALI”) (acute lung injury) [126]. IL-17 (formally IL-17A) is the best known member of a family of multifunctional cytokines. Its predominant role seems to depend on where the cytokine is expressed (gut, lung or skin) and what the trigger is. These two factors appear to influence whether the predominant effect of its expression is protective or whether it leads to a detrimental hyper-inflammatory state. For MERS-CoV, SARS-CoV and SARS-CoV-2, disease severity was shown to correlate positively with levels of IL-17 and other T helper 17 (Th17) cellrelated pro-inflammatory cytokines, such as IL-1, IL-6, IL-15, TNF and IFNγ. (see page 7 of this paper) Increased IL-17 levels in LPS-induced “ALI” (“acute lung injury”) mice correlated with increased lung injury scores, increased protein-rich inflammatory lung infiltration and decreased overall survival. Furthermore, the addition of exogenous IL- 17 further exacerbated LPS-induced production of TNF, IL-1β, IL-6 and CXCL2, revealing the role of IL- 17 as a key principal modulator of the inflammatory pathway. In the same study, mice genetically deficient in IL-17 or those that received anti-IL-17 antibodies demonstrated improved survival, less pulmonary infiltration, and improved lung pathology scores after LPS exposure [127] Taken together, analyses of patients with coronavirus-induced lung disease suggest that IL-17 may serve as a biomarker (“DAMP”) of disease severity and a potential target for therapy to mitigate SARS-CoV-2 damage, particularly in the lung. Of note, COVID-19 mortality is also associated with myocarditis in the context of SARS.

Zhao Y, et al. [128] in a very recent paper (February 2021) propose a model to understand the underlying mechanisms involved in lung pathology by investigating the role of the lungspecific immune response. The authors obtain immune cells in bronchoalveolar lavage fluid and in blood drawn from patients with COVID-19 with severe disease and patients with bacterial pneumonia not associated with viral infection. By tracing T-cell clones across tissues, they identify Th17 cells similar to clonally expanded “memory T cells” resident in lung tissue, which they term “Trm17 cells” and which reside in the lungs even after viral clearance. Analysis of the lung suggests that Trm17 cells may interact with lung macrophages and Tc/s (CD8+ cytotoxic) cells, and is associated with disease severity and lung damage. Ultimately, elevated serum IL-17A and GM-CSF protein levels in patients with COVID- 19 are associated with a more severe clinical course. Zhao Y, et al. suggest that lung Trm17 cells are a potential orchestrator of hyperinflammation in severe COVID-19. On the other hand, Trm17 cells become activated or reactivated as part of the ongoing cytokine storm, during which they may begin to produce pro-inflammatory cytokines such as GM-CSF. This could lead to increased activation of macrophages and cytotoxic CD8+ cells, which other authors have linked to disease severity and ultimately mediate lethal lung damage [42,45].

As related by Zhao Y, et al. To date there have been 2 small pilot studies that have indicated that targeting GM-CSF in patients with severe lung disease by COVID-19 using anti-GM-CSF receptor monoclonal antibodies mavrilimumab or lenzilumab, respectively, may be a strategy to improve clinical outcomes [3,4], although larger controlled clinical trials would be needed to determine the efficacy and biological impact of such approaches. This network of tissue-resident cells may persist in the lungs even after the initiating event, e.g., a viral infection, has been eliminated, contributing to chronic lung pathology. There are three commercially available options: secuquinumab (human monoclonal antibody against IL- 17), ixeki-zumab (humanized monoclonal antibody against IL-17) and brodalumab (human monoclonal antibody against the IL-17 receptor). Both secukinumab and ixekizumab are approved for psoriasis, psoriatic arthritis and ankylosing spondylitis; brodalumab is approved for the treatment of psoriasis alone. All three of these drugs come with warnings about an increased risk of infections. Compared to placebo, clinical trials showed a moderate increase in upper respiratory tract infections (“URIs”) for patients treated with secukinumab and a similar number of URIs for patients treated with ixekizumab, while treatment with brodalumab resulted in a lower rate of “URIs.” The risk of serious infections is unchanged or low in the short term. Therefore, the use of these drugs in the acute setting of COVID- 19 should not lead to an increased risk of secondary infections.

“NSAIDs” (Non-Steroidal Anti-Inflammatory Drugs)

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a group of often chemically unrelated compounds that have potent antiinflammatory, analgesic and antipyretic activity and are among the most widely used drugs worldwide. It is generally thought that one of their main mechanisms of action is the inhibition of cyclooxygenase (COX), the enzyme responsible for the biosynthesis of prostaglandins (PGs) and thromboxane. NSAIDs are also associated with an increased risk of gastrointestinal, renal and cardiovascular adverse effects.

The review paper by Bacchi S et al., [129] describes the clinical pharmacology of “NSAIDs, their classification, molecular mechanisms of action and adverse effects, including their possible contribution to “neuro-inflammation” and carcinogenesis, as well as some recent developments aimed at designing effective antiinflammatory agents with improved safety and tolerability profiles. In the late 1980s, it was discovered that COX has two isoforms, each produced by a different gene. The COX-1 gene is located on chromosome 9 and functions as an internal gene that regulates numerous cellular functions, including the complex series of processes responsible for protecting the gastrointestinal mucosa from ulceration. The COX-2 gene, located on chromosome 1, is an early and immediately activated gene and is rapidly deregulated in response to a variety of inflammatory cytokines and cellular injury.

The COX-1 enzyme produces the prostaglandins responsible for gastrointestinal cytoprotection and platelet function, while the COX-2 enzyme produces the reactions responsible for pain perception and inflammation. Thus, COX-1 and CoX-2 enzymes can produce both beneficial and adverse effects due to inhibition of prostanoids, derived from arachidonic acid (AA), which is converted to prostaglandin G2 (PGG2) and H2 (PGH2) as a result of cyclooxygenase (COX) activity, and PGH2 is subsequently metabolized by terminal synthases into biologically active prostanoids. COX-2 expression is greatly restricted under basal conditions, but is greatly increased at inflammatory sites in response to cytokines such as interferon-gTNFa, IL-1, hormones, growth factors, and hypoxia. The pharmacological effects of NSAIDs are due to blockade of COX and consequent reduction of PG synthesis, leading to a decrease in inflammation, pain and fever. The anti-inflammatory action of “NSAIDs” is due to the decrease of vasodilator PGs (PGE2, PGI2), which indirectly reduces edema.

Within the group of non-steroidal anti-inflammatory molecules, the group of pyrazolones stands out and among them metamizole (dipyrone). In 2014, the research group of Jasiecka A et al, [130] published a paper on the pharmacological characteristics of “metamizole”: a popular, non-opioid analgesic drug commonly used in human and veterinary medicine. In some cases, this agent is still incorrectly classified as a non-steroidal anti-inflammatory drug. Metamizole is a “pro-drug” that spontaneously breaks down after oral administration into structurally related pyrazolone compounds. In addition to its analgesic effect, the drug is an antipyretic and spasmolytic agent. The mechanism responsible for the analgesic effect is complex and most likely based on inhibition of a central cyclooxygenase-3 and activation of the opioidergic and cannabinoid systems. Metamizole can block both PG-dependent and PG-independent LPS-induced fever pathways, suggesting that this drug has a distinctly different antipyretic action profile than the other NSAIDs. The mechanism responsible for the spasmolytic effect of metamizole is associated with inhibition of intracellular Ca2 + as a result of reduced inositol phosphate synthesis [130]. Metamizole is predominantly applied in the therapy of pain of different etiology, spastic conditions, especially affecting the digestive tract, and fever refractory to other treatments. Coadministration of morphine and metamizole produces super-additive anti-nociceptive effects [131]. On the other hand, metamizole is a relatively safe pharmaceutical preparation, although it is not completely free of undesirable effects. Among these side effects, the most serious and most controversial is the myelotoxic effect; however, it seems that in the past the risk of metamizole-induced agranulocytosis was exaggerated [132]. Today it is considered that the side effects of metamizole appear only in long periods of treatment of chronic inflammatory diseases. Our research team has studied the effects of “magnesium metamizole” marketed under the name of Nolotilâ and currently produced by Boehringer Laboratories (Germany).

The active ingredient of this drug is the “methylated oxyquinazine” molecule, whose chemical structure is included here: The complete structure of this synthetic drug corresponds to a phenyl-dimethyl pyrazolone derivative whose “R” root is magnesium methylene sulfonate. Its complete formula is dimethyl oxyquinazine methylene methylamine magnesium sulfonate. Since 1975, we have used this drug in the surgical clinic as an analgesic in the first days of the immediate postoperative period, due to its high analgesic power and also to its anti-adhesiveness and anti-platelet aggregation properties. These findings were obtained in “in vivo” and “in vitro” studies carried out by our research team in the 70’s of the last century. [132-134]. Our results have now been ratified by Pfrepper C et al. in 2019 [135]. It is very important to emphasize here that increased platelet adhesiveness and aggregation leads to initial thrombus formation and eventually to thrombosis. Methylated oxyquinazine by its anti- aggregating and antiadhesive action on platelets may contribute to prevent vascular thrombosis. When “CoV-2 antigens” stimulate macrophages or any other “antigen presenting cell” the “pro- inflammatory” cytokines par excellence are released: IL-1, IL-6, IL-8, IL-15, IL-17, IL-18, TNFs, IFNg and PAF (platelet activating factor) (and source of PF4). Through the release of PAF, the immune response acts on the coagulation and fibrinolytic systems, giving rise to signals that cross and intersect between the immune response and the different systems: coagulation, fibrinolytics, cyanins, arachidonic acid, leukotrienes and thromboxanes, etc. Thus, we believe that methylated oxyquinazine could contribute to inhibit or palliate the immune response overwhelmed by COVID-19 in the most severe stage of Cov-2 disease [42]. (see Figure 2) page 5 of this paper). It has recently been published those vaccines using adenovirus vectors can produce thrombosis by activation of PAF and PF4, resulting in increased platelet aggregation and adhesiveness accompanied by thrombocytopenia [136-143]. In this aspect, methylated oxyquinazine (metamizole) could also serve as a prophylaxis of thrombus formation when these vaccines are given (Figure 4).

Figure 4: The complete structure of this synthetic drug corresponds to a phenyl-dimethyl pyrazolone derivative whose “R” root is magnesium methylene sulfonate. Its complete formula is: dimethyl oxyquinazine methylene methylamine magnesium sulfonate [134-136].

Our research project is aimed at avoiding the “cytokine storm” by means of molecules that do not inhibit the response to the virus but can avoid the excessive inflammatory response, by activating the “immuno-regulatory pathway” of the immune system. The treatments applied to date fail when patients, especially the elderly or “immuno-compromised” people (suffering from severe heart disease, chronic kidney disease, chronic obstructive pulmonary disease, cancer –patients undergoing active treatment–, immunosuppression by transplantation of solid organs, obesity or type 2 diabetes mellitus, or elderly people who also suffer from any of these diseases, reach the most serious stage of the disease, and their body is not capable of avoiding the “cytokine storm” and the multi-organic failure that leads inexorably to death.

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How COVID-19 Pandemic Indirectly Affected Orthopedic Patients: A Case Report of a Rescue Treatment For a Proximal Humerus Nonunion

Introduction

Humeral fractures account for 5% to 8% of all fractures, whereas proximal humerus fractures represent the seventh most frequent fractures in adults [1,2]. Nonunion is a complication that occurs in 15% of all the humeral fractures [3] and its incidence increases in case of proximal humerus fracture [4]. Risk factors are advanced age, osteoporosis, obesity, smoking, alcoholism, and infection. Comminution and impaction of fractures and loss of fixation also contribute to the develop of nonunion [5,6]. This condition results in pain and loss of shoulder function. The management of proximal humerus nonunion is challenging and often the results are disappointing. Treatment of these kind of complications include open reduction and internal fixation with bone grafting but often it is an unsuccessful treatment resulting in bad clinical outcomes and further surgery is required. Other options are fixation with tension wires or with intramedullary nail [7].

These are optimal options for bone of good quality such as in young patients and with no signs of gleno-humeral arthritis. Shoulder arthroplasty is a reasonable option in case of proximal humerus nonunion associated with a rotator cuff damage and osteoporosis [8]. This case-report describes a proximal humeral nonunion in a 69-year-old woman who was first treated with an external fixation before the advent of COVID-19 pandemic. After removing the external fixator (EF), she was lost at follow-up because of the closure of our department during the Italian lockdown. She came back after seven months with pain and functional limitation. X-Ray reported a nonunion of proximal humerus. In the end she underwent to reverse shoulder arthroplasty, recovering with a good result.

Case Report

A 69-year-old woman came to ER of Poliambulanza of Brescia in October 2019. X-rays were obtained. Fracture involved proximal humerus of the right, dominant, upper arm. The fracture was 11C3.1 according to AO classification. At first, the fracture was treated with an external. fixation using a Galaxy EF in the first twenty-four hours. The EF was removed after one month because of the loss of reduction with displacement of the fracture. Physio- kinesitherapy was indicated but she was unable to underwent to treatment. She was lost at follow -up for several months due to COVID-19 pandemic and the social limitations that resulted. After 7 months, Xrays showed a dislocated nonunion of proximal humerus with necrosis of the head. She complained shoulder pain with passive elevation of 40° and scapular dyskinesia. In March 2021 she was listed for a reverse shoulder arthroplasty. During the surgery, Synovasure test and white blood cell count were performed: both tested negatives. A cemented trauma stem “Equinoxe” by Exactech number 8 mm was applied with a standard baseplate fixed with three screws of 26, 18, 18 mm. External rotators were reinserted, and range of motion (ROM) was good at three months follow-up.

Discussion

The impact of COVID-19-related restrictions has resulted in changes in patients’ healthcare and follow-up. During the pandemic, injured patients have experienced difficulties in receiving medical assistance, due to the lack of healthcare personnel and fear of contagion. Lombardy was the most affected region of Italy and orthopedic surgeons were involved in the emergency as other specialists [9,10]. Our level-2-trauma center in Brescia (Lombardy) went on admitting injured patients unceasingly, since several domestic accidents happened during that time, despite reports out of Italy noted a 65% reduction in trauma services provided for shoulder and elbow injuries during the time residents were asked to stay in the home [11]. One of the effects of the pandemic was the loss of follow-up of outpatients [12]. It is hypothesized that the main causes of this issue were the isolation and the fear of contagion in hospital environment [10] (Figure 1).

Figure 1: First radiograph showing comminuted and dislocated fracture.

Figure 2: First treatment with EF.

Figure 3: Partial loss of reduction after the removal of the EF.

Figure 4: Nonunion and dislocation at 7 months after trauma.

Figure 5: Final treatment.

Figure 6: Range of motion at 3 months after arthroplasty.

Radiographic checks showed a partial loss of reduction and physio-kinesitherapy was indicated but she was unable to underwent to treatment because of the new social restrictions. Further radiographs showed gradual loss of reduction. Therefore, we started contemplating a definitive treatment by performing a ORIF with bone graft or a shoulder arthroplasty, but at that moment, the patient was lost at follow-up. Given the poor bone quality, after the removal of the EF, we would probably have implanted a hemiarthroplasty or a reverse prosthesis with a press-fit primary humeral stem fixation, considered an optimal choice because of the possible easier revision, decreased operative time, healing time, and resolution of the symptoms [16]. After 7 months, the patient came back to our department, suffering from pain and severe functional limitation, compounded by a preternatural movement of the joint. Radiographs showed evident dislocated nonunion with reabsorption of tuberosities and metaphysis.

Therefore, our choice has been to implant a reverse shoulder prosthesis with a cemented trauma stem “Equinoxe” by Exactech number 8 mm. This choice involved several compromises like technical difficulties due to the severe bone loss and higher risks of dislocations, infections, nerve injuries and thromboembolism due to the use of cement, compared to an arthroplasty with a pressfit stem [17-20]. At three-month follow-up, the patient showed no pain and a sufficient function of the joint. Since the exact amount of loss to follow-up is not valuable, there is a chance that cases of nonunion in longstanding fractures like this could increase in the near future. Our experience shows that cemented stem fixation can be an important choice of treatment for these patients. Other strategies, like telemedicine, should be considered and eventually implemented to prevent this kind of consequences resulting from the pandemic [21-23].

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Environmental Impacts and Control of Duststorms

Mini Review

Duststorms and sandstorms are natural meteorological phenomena and severe weather condition frequently occurring in arid and semi-arid regions mainly during summer season when these regions are subjected to strong winds; and driven by different factors: availability and nature of source sediments, vegetation cover density, prevailing climatic conditions, and the textural characteristics of the surface deposits, environmental, geomorphological and relief variation factors. One of the major terrestrial sources of moving sand worldwide is the Arabian peninsula and Sahara desert, while minor sources come from Iran, Pakistan, and India which deposit dust in the Arabian sea, and from China depositing dust in the Pacific. According to [1], the recent surface deposits are the major source of duststorms in Kuwait which are potentially originated from:
1) Dry sabkhas muddy sediments in the lower Mesopotamian flood plain;
2) Old sandstone, limestone and dolostone sediments exposed in the western desert of Iraq;
3) Dibdibba Formation Paraconglomeratic sediments exposed in southern Iraq and northern Kuwait; and
4) Air locally picked up particles from playa, sabkhas, and finegrained mobile sand.
Duststorms winds have variant local nomenclature. In Sahara desert they are named as Simoom, in some Africa Arabian countries like Egypt, Libya, Sudan, Morocco, and Tunisia, they are named Khamasine, Ghibli, Haboob, Sahel and Chili, respectively, while in Asian areas like India and the Arabian Gulf region they are named Loo and Shamal or Toze, respectively [2]. A duststorm is distinguished from a sandstorm on the basis of particle size. Dust storms are made up of a multitude of very fine particles while sandstorms have larger particle sizes that range from .08mm to 1mm [3]. The fine “dust” particles may be elevated as high as 3km or more while the “sand” particles are confined to the lowest 3.5m, rarely rise more than 15m above the ground. The term sandstorm is oftenly used in desert sandstorms context, especially in the Sahara Desert, or places where sand is a more prevalent soil type than dirt or rock, when, in addition to fine particles obscuring visibility, a considerable amount of larger sand particles are blown closer to the surface.
The term duststorm is more likely to be used when finer particles are blown long distances, especially when the duststorm affects urban areas. A sandstorm can transport and carry large volumes of sand unexpectedly. Dust storms can carry large amounts of dust, with the leading edge being composed of a wall of thick dust as much as 1.6 km (0.99 mi) high. In desert areas, dust and sand storms are most commonly caused by either thunderstorm outflows, or by strong pressure gradients which cause an increase in wind velocity over a wide area. Drought and wind contribute to the emergence of dust storms, as do poor farming (e.g., dryland farming techniques) and grazing practices by exposing the dust and sand to the wind. In addition to the environmental factors including: wind speed, atmospheric stability, source region surface characteristics, surface heating, soil moisture, soil type and surface vegetation.
Moreover, in Kuwait human activities in the desert contribute to duststorms occurrence including: extensive motor car movements, extensive urban development, environmentally uncontrolled quarrying activities, and overgrazing by cattle throughout the year. In Kuwait duststorms are more frequent during the Spring and Summer due to:
1) The dry fresh (15-24 m/s) northwesterly winds blowing from Iraq and local lands [4];
2) The deserts surrounding Kuwait: Iraqi desert from N-NW and Saudi Arabian from W-S;
3) The loose sediments covering most of the surface area [5]. There are 3 types of dust in Kuwait [6,7]. Duststorms (wind speed ≈18 knots (33.336 km/h), horizontal visibility is <1 km (if < 200 m it is called a severe duststorm)); 2) Rising dust (wind speed is moderate, horizontal visibility is ≥ 1 km); and 3) Suspended dust (horizontal visibility is < 1 km but with moderate wind speed (6-14 m/s) it is in the range of 1-5 km). Dust and sandstorms may have impacts in different aspects; physical, environmental, economic, social, human health … etc.
As physical and environmental sandstorm can possess a huge power that it can move whole sand dunes; Duststorms and suspended dust can reduce visibility to < 200 m; dust may block roads, damage materials and equipment and affect transportation and severely pollute the air. Dust particles can reflect and absorb solar radiation causing radioactive effect as they are tropospheric aerosols a significant component of the earth’s climatic system changing climate by their direct radiative scattering and absorption [8], and indirectly by their radiative effects through affecting on clouds microphysics [9] and affecting the processes of atmospheric chemistry. Dust can remarkably affect the soil characteristics, ocean productivity, and air chemistry by influencing the nutrient dynamics and biogeochemical cycling of ecosystems. Economically, duststorms lead to soil loss, which, in turn, will remove the organic matter and nutrient-rich particles reducing the soil fertility and by abrasion they damage the young crop plants and reduce the crop productivity.
Moreover, duststorms reduce visibility affecting aircrafts and road transportation, that would have consequences of financial and human lives loss. Duststorms reduce the amount of sunlight that reaches the surface, and hence cause critical complications on plants photosynthesis and productivity and reduce the livestock forage. Increased clouds of dust and sandstorms can affect the ecosystem stability by increasing the heat blanket effect. Socially wise, by reduction of livestock forage, ecosystem biodiversity and increase hunger, water availability and farmland yields, the land resources will be lost which result in turn will spread poverty, the spread of poverty and hunger will increase, which eventually will result in migration in search of food and relief, and increasing the environmental refugees number that poses pressure on neighborhood areas and leading to enormous social problems. In relation to public health, duststorms have adverse shorttime impacts on the public health including immediate increased symptoms and worsening of the lung function in individuals with asthma, increased mortality and morbidity long-transported duststorm particles adversely affect the circulatory system. Prolonged and unprotected exposure of the respiratory system in a dust storm can also cause silicosis, which, if left untreated, will lead to asphyxiation; silicosis is an incurable condition that may also lead to lung cancer. It was found by [10] that the concentrations of all pollutants (including Particulate Matter (PM10)) in the ambient air of Kuwait in the residential areas is dependent on the meteorological conditions (PM10 and NOx). It was indicated by [11] that in Kuwait duststorms and fossil fuel combustion strongly contribute to the air pollutants (especially (PM)) which play a significant role in determination the symptoms of Rheumatoid Arthritis (RA) disease and worsening it on overall.
It was stated by [12] that the chronic and long-term exposure to calcite and quartz particles (the major constituents of dustfallout in Kuwait) may produce alkalosis and hypercalcemia and can have potentially serious respiratory effects. There is also the danger of keratoconjunctivitis sicca (“dry eyes”) which, in severe cases without immediate and proper treatment, can lead to blindness. There are short-term approaches for dust and sand storm control (e.g., forecasting and early warning) and others are long-term (e.g., source area rehabilitation). In general, dust and sand storms can be controlled by applying different kinds of dust suppressants or wind breakers. Such suppressants may include physical covers, e.g., vegetation, aggregate, mulches or paving; and chemical compounds, e.g., water, either fresh, sea water or even reclaimed, especially on construction sites and unpaved roads; calcium and magnesium chloride and petroleum-based chemicals, which can stabilize the soil by absorbing the moisture from the atmosphere.
This will change the soil surface physical properties as by applying the suppressant the soil particles will be coated and aggregated together becoming heavy to be airborne particles hence unsusceptible for wind erosion. Controlling the movement and sand encroachment by wind can be done by creating tree windbreakers, reducing ground level wind velocity by inserting straw bundles into the sand in a checkerboard pattern, or using creeping plants. Sand or dust encroachment can also be controlled by rehabilitating and improving the land surface by reducing barren land through reforesting and planting degraded land, and improving the environmental capacity of the soil by introducing water-saving and water management techniques for the efficient use of water and application of farm animal manure.

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Keratoacanthoma and Well Differentiated Squamous Cell Carcinoma Have a Distinct Prognosis Running Head: Prognosis of Keratoacanthoma

Introduction

Keratoacanthoma (KA) is a rapidly growing skin tumor thought to originate from the hair follicle [1]. The exact classification of the tumor is still a matter of debate. Due to its ability to spontaneously regress some consider KA a benign lesion [2]. However, KA can also display perineural as well as venous invasion [3,4] and cases of metastatic KA were also reported [5,6] suggesting the classification as a subtype of well differentiated SCC is more appropriate. On a molecular level, the etiology of KA and Squamous Cell Carcinoma (SCC) seems to differ, since deletion of polarity proteins in mouse models can rescue SCC formation but promote KA formation [7]. Expression of tumor suppressors and promoters is also different in KA compared to SCC with a gain of 11q and subsequent amplification of the cyclin D1 locus being the most frequent aberration in KA [8]. Mutated p53 is more frequently found in SCC compared to KA and the cell cycle inhibitor p16 is expressed in KA but downregulated in SCC [9]. Additionally, the tumor microenvironment of KA and SCC is different providing a possible explanation for the ability of KA to regress [10].

The therapy of KA usually consists of complete excision since it is impossible to predict whether the tumor will regress or progress to invade the underlying tissue and even metastasize [5,6]. Since current guidelines on SCC do not take the KA histological subtype as a prognostic factor into account the question whether KA has a distinct prognosis compared SCC remains open. This question is particularly relevant since KA often have an increased tumor thickness due to rapid growth and may unnecessarily fall into the category of high risk SCC thereby burdening the financial system with frequent follow-up of these patients. In this study, we compared the prognosis of KA with that of well differentiated SCC without KA histology (wSCC) and found that KA histology favorably impacts metastasis-free survival but not local relapse-free survival.

Patients and Methods

A retrospective analysis of medical records of the Department of Dermatology, University Hospital of Cologne identified 403 KA and 905 wSCC. Tumors with an incomplete excision were excluded. The histopathologic criteria for KA were a crateriform tumor surrounding a central keratin plug showing epithelial lipping [1,11]. Solar elastosis was defined as a fibrillary basophilic material in the upper dermis [12]. Immunosuppression was defined as use of chemotherapy, other malignancies than non-melanoma skin cancer and use of immunosuppressive drugs. Diabetes was not considered immunosuppressive. Statistical evaluations were performed with the statistical software package IBM SPSS, version 20.0. Graphs were made using GraphPad Prism, version 5.0. Student’s t test was used with continuous variables, while the X2 test was used for categorical variables. Survival rates were calculated by the Kaplan– Meier method and compared using log-rank tests. A P-value of <0.05 was considered significant.

Results

A total of 403 KA and 905 wSCC were retrospectively analyzed. The median follow-up was 23 months. 8 Patients with KA (2%) developed metastasis after a median of 7 months (range 1-49) and 35 patients with wSCC (3.9%) developed metastasis after a median 8 of months (range 1-37). Except for 2 patients with wSCC, who developed lung metastasis all patients developed metastasis of skin and/or lymph nodes. The metastasis-free survival was significantly decreased in the wSCC groups compared to the KA group (p=0.042) (Figure 1). 9 KA (2.2%) recurred locally following complete excision after a median of 32 months, while after a median of 8 months 24 wSCC (2.7%) recurred locally. The local recurrence-free survival was not significantly different in the KA group compared to the wSCC group (p=0.426) (Figure 1).

Figure 1:

a) Metastasis-free survival in keratoacanthoma compared to well differentiated squamous cell carcinoma (SCC) (p=0.042).
b) Local recurrence-free survival in keratoacanthoma compared to well differentiated SCC (p=0.436).

Patients with KA were more likely to be female (40.2% versus 27%, p<0.0001) and to have thicker lesions compared to patients with wSCC (tumor thickness 3.2 vs 2.7, p=0.012) (Table 1). Age was also significantly decreased in KA compared to wSCC (p<0.0001). Furthermore the patterns of tumor localization were different in KA compared to wSCC, with KA occurring more frequently on the lower extremity and to a lesser extent in the head and neck region. Since the difference in tumor location could be explained by sun exposure, we analyzed the presence of solar elastosis in the periphery of tumors and found that solar elastosis occurred more frequently in wSCC compared to KA (82% vs 60.7%, p<0.0001). In contrast, the presence of immunosuppression and tumor diameter was not significantly different in the KA group compared to the wSCC group (Table 1).

Table 1: Clinical data keratoacanthoma compared to well differentiated SCC.

Note: SCC- squamous cell carcinoma; STDEV-standard deviation.

Discussion

Currently, no specific guideline for the follow-up of patients with KA exists and it is unclear whether the guidelines regarding the follow-up of SCC [13] can also be applied to KA. To our knowledge, this the first study comparing the prognosis of KA with wSCC. We found that the metastasis-free survival was significantly increased in KA compared to wSCC. 2% of KA developed metastasis in the skin and regional lymph node indicating that similar to SCC ultrasound of the regional lymph nodes is also important in patients with KA. Since local recurrence-free survival was not different in KA compared to wSCC we propose to use the same frequency of physical examinations of the skin in KA as in SCC [13]. In line with the hypothesis that SCC and KA have different etiologies our study could show that clinical and pathologic characteristics were also significantly different in the wSCC group compared to KA. As expected from a rapidly growing tumor, the tumor thickness of KA was significantly increased compared to wSCC. Interestingly, patients with KA were more likely to be female and were significantly younger compared to patients with wSCC further underlining the different etiology of the two tumors.
In accordance with a previous study [14] we could show that the predominant distribution of KA is on the lower extremity while SCC were mostly localized in the head and neck region. Moreover, the presence of solar elastosis was significantly increased in the KA group suggesting sun exposure plays a more minor role in KA. Consistent with the hypothesis that UV plays a less important role in the etiology of KA the mutation burden of KA is lower compared to cutaneous SCC [15]. Further supporting a different etiology of KA and SCC the rate of HPV DNA detection was higher in KA compared to wSCC suggesting a possible viral etiology in the pathogenesis of KA [16]. This would then imply that immunosuppression would play a more important role in the etiology of KA compared to SCC, however the incidence of immunosuppression was not different in KA compared to the wSCC group in our study, suggesting other etiologic factors such as the tissue microenvironment could play a role.

Conclusion

We could show that KA histology favorably impacts metastasisfree survival but does not influence local relapse-free survival.

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Comparison of Three Treatment Methods of Ⅰ-Ⅲ Degree Hemorrhoids: A Meta Analysis

Introduction

Hemorrhoids is a common clinical disease and its pathogenesis is not yet clear. The theory of anal cushion displacement is widely accepted at present, that is, supporting tissue degeneration, including fibrous tissue fragmentation, elastic connective tissue and submucosal muscle fiber weakening, which may be related to lack of dietary fiber, constipation, improper defecation habits and lifestyle. The main clinical manifestations of hemorrhoids are bleeding during defecation, pain, anal prolapse, swelling, pruritus and perianal secretions [1]. Depending on the location of the disease, hemorrhoids can be divided into internal hemorrhoids, external hemorrhoids and mixed (internal and external) hemorrhoids. Internal hemorrhoids are formed by tissue covered by columnar epithelium, and located above the dentate line; External hemorrhoids are formed by tissue covered by squamous epithelium, and located below the dentate [2]. Mixed hemorrhoids appear at the top and bottom of the dentate line. The staging of internal hemorrhoids is not completely unified in the world. Goligher’s classification is more commonly used, which divides internal hemorrhoids into gradeⅠ-Ⅳ.

i. Grade Ⅰ: Bleeding during defecation, but not prolapse, most patients have no obvious symptoms.
ii. Grade Ⅱ: The hemorrhoids protrude out of the anus during defecation, and the prolapse can be returned by itself.
iii. Grade III: Prolapse out of the anus during defecation, fatigue, long walking, or coughing. After prolapse, internal hemorrhoids cannot be repaid by themselves, and they need to be repaid by hand.
iv. Grade Ⅳ: With external hemorrhoids, the hemorrhoids remain outside the anus for a long time and cannot be repaid or prolapse immediately after being repaid.
The treatment methods of internal hemorrhoids include non-surgical treatment and surgical treatment. Most internal hemorrhoids of grade I-III can be alleviated by non-surgical treatment such as drug treatment, RBL, IS, and IRC; grade IV internal hemorrhoids, internal hemorrhoids that fail or have complications should undergo surgical treatment [3]. RBL is a simple, quick and effective method for treating patients with grade I-II and part of grade III internal hemorrhoids. The method is to ligate with ligator above the dentate line, and the internal hemorrhoids are necrotic due to blood flow blockade and fall off automatically [4]. IS is an endoscopic or anal endoscopic injection of sclerosis agent into the submucosal layer of internal hemorrhoids, forming fibrosis and scars. IRC is to coagulate the hemorrhoid blood vessels or cause fibrosis of the hemorrhoid submucosa through the instant high heat generated by infrared rays, to fix the anal cushion. IRC is mainly used for the treatment of internal hemorrhoids of grade I – III [2]. The purpose of this study was to compare the efficacy and safety of RBL, IS and IRC in the treatment of internal hemorrhoids. We evaluated the evidence from the random control trial (RCT) and the research data from the systematic review of the RCT, and performed a meta-analysis, which is reported below.

Materials and Methods

Search strategy

Use keywords such as “Hemorrhoids”, “Hemorrhoid sclerotherapy”, “Injection sclerotherapy”, “Rubber band ligation”, “Hemorrhoid ligation”, “Infrared coagulation”, “Hemorrhoid infrared coagulation”. Searched the literatures from PubMed, Cochrane Library, and Embase database up to 2021. The research method limited the RCT to improve the sensitivity and did not limit the language, and the related references in the included literature were manually searched, and consistent standards were used to determine the included and excluded literature.

Inclusion and Exclusion Criteria

• Participants of the study included patients ≥18 years of age who met the Goligher’s grading classification criteria and patients with grade I-Ⅲ internal hemorrhoids; patients diagnosed with grade IV hemorrhoids and patients with other anorectal diseases other than the study disease were excluded.
• The original text is publicly published literature; it is limited to RCT, in which patients are randomly assigned to two or more treatment groups, clinical results are recorded, and follow-up time is at least 3 months.
• The original literature provides corresponding indicators of effectiveness and safety: including effective rate, recurrence rate, complication rate, etc.
• The type of literature is limited to treatises, and literatures with incomplete original data are excluded. reviews, conference reports, reviews, case reports, etc. are excluded.
• The included RCTs are all evaluated by the Cochrane handbook to evaluate the quality of the literature.

Data Extraction

General data of the literature were extracted: including the first author, year of publication, number of patients included, observation data, etc. The extraction of all data was done independently by two researchers. For the controversial data, the third researcher participated in the discussion and decided.

Outcome Indicators

Main Outcomes: Effective Rate, Recurrence Rate.
Secondary Outcome: Complications.

Statistical Analysis

Meta-analysis was conducted with Review Manager 5.4 software to study the effective rate, recurrence rate and complication rate of RBL, IS and IRC in the treatment of patients with internal hemorrhoids. As a dichotomous variable analysis, odds ratio (OR) was used as the effect index, and 95% confidence intervals (95%CI) is calculated. The Cochrane Q test was used to evaluate the heterogeneity among studies, and the magnitude of heterogeneity among studies was determined by combining the I2 value. Moderate to high heterogeneity was considered when I2 was greater than 50%, and the test level was α=0.1. The data were combined and analyzed for heterogeneity. If there was no heterogeneity (I2≤50% and P≥0.1), the fixed-effect model was selected for analysis; If there was heterogeneity (I2>50% and/or P<0.1), then analyze its sources and discuss, choose random effects model analysis. Meta analysis test level was α=0.05. For highly heterogeneous variables, the single study deletion method was used to conduct sensitivity analysis to find the source of heterogeneity. If the heterogeneity decreased after deleting the document, the document was the source of heterogeneity, and the forest figure was finally made. Regarding the analysis of publication bias, by observing whether the two sides of the funnel chart were symmetrical, if the two sides were symmetrical, there was no obvious publication bias, and if the two sides were asymmetric, there may be publication bias.

Meta Analysis Results

A total of 379 relevant literatures were retrieved, 159 duplicate literatures were excluded, 155 were excluded after reading the title, 39 were excluded after reading the abstract, and 14 were excluded after reading the full text. A total of 12 literatures were screened according to the inclusion criteria (Figure 1). A total of 1438 patients were included, including 592 patients in the RBL group, 350 patients in the IS group, and 496 patients in the IRC group. The detailed data of the included literatures were shown in Table 1 (Figure 2).

Figure 1: Literature retrieval process and results Table 1 Features of the included literatures.

Figure 2: Risk assessment of inclusion literature.

Effective Rate

a. RBL vs IRC: A total of 782 cases are included in 6 literatures, and heterogeneity analysis indicates no heterogeneity (P=0.60, I2=0%). The fixed-effect model analysis is carried out, and the results shows that there is no significant difference in the effective rate between the RBL group and the IRC group (OR=1.36, 95%CI:0.89-2.09, P=0.16), indicating that the treatment effect of the two groups is similar, as shown in Figure 3.

Figure 3: RBL vs IRC effective rate.

b. RBL vs IS8: A total of 480 patients are included in 5 literatures. Fixed effect model analysis is used, and the heterogeneity analysis indicates that there is heterogeneity (P=0.06, I2=55%). By eliminating literatures one by one, it is found that the heterogeneity decreases after eliminating literatures Greca.1981, with statistical difference (OR = 2.56, 95% CI: 1.53-4.27, I2 = 46%, P = 0.0003). It suggests that the effective rate of RBL group is better than that of IS group, as shown in Figure 4.

Figure 4: RBL vs IS effective rate.

c. IS vs IRC: A total of 267 patients are included in 2 literatures. Heterogeneity analysis shows that there is no heterogeneity (P = 0.77, I2 = 0%). Fixed effect model analysis is carried out. The results shows that there is no significant difference in the effective rate between IS group and IRC group (OR = 0.66, 95% CI: 0.37-1.19, P = 0.17), indicating that the treatment effect of the two groups is similar, as shown in Figure 5.

Figure 5: IS vs IRC effective rate.

The Recurrence Rate

a. RBL vs IRC: A total of 612 patients are included in 4 literatures. Fixed effect model analysis is used and the heterogeneity analysis indicates that there is heterogeneity (P=0.004, I2=77%). By eliminating literatures one by one, it is found that the heterogeneity decreases after eliminating literatures Walker.1990, with statistical difference.(OR=0.40, 95%CI:0.24-0.67, I2=44%, P=0.0003), suggesting that the recurrence rate of the RBL group is lower than that of the IRC group, as shown in Figure 6.

Figure 6: RBL vs IRC recurrence rate.

b. RBL vs IS: A total of 480 patients are included in 5 literatures. Fixed effect model analysis is used and the heterogeneity analysis indicates that there is heterogeneity (P=0.02, I2=67%). By eliminating literatures one by one, it is found that the heterogeneity decreases after eliminating literatures Walker.1990, with statistical difference (OR=0.34, 95%CI:0.19-0.61, I2=264%, P=0.0003), suggesting that the recurrence rate of the RBL group is lower than that of the IS group, as shown in Figure 7.

Figure 7: RBL vs IS recurrence rate.

c. IS vs IRC: A total of 267 patients are included in 2 literatures, and the heterogeneity analysis indicates that there is heterogeneity (P=0.04, I2=76%). Since there are only 2 literatures, random effect model analysis is carried out, and the results showed that there is no significant statistical difference in the recurrence rate between IS group and IRC group(OR=0.59,95%CI:0.14- 2.39,P=0.46), suggesting that the recurrence rate of IS group and IRC group is similar, as shown in Figure 8.

Figure 8: IS vs IRC recurrence.

Incidence of Complications

a. RBL vs IRC: A total of 782 patients are included in 6 literatures. Fixed-effects model is used and the heterogeneity analysis indicates that there is heterogeneity (P=0.03, I2=59%). By eliminating literatures one by one, it is found that the heterogeneity decreases after eliminating literatures Walker.1990, with statistical difference (OR=1.91, 95%CI: 1.15~3.16, I2=19%, P=0.01), suggesting that the incidence of complications in the RBL group is lower than that in the IRC group, as shown in Figure 9.

Figure 9: RBL vs IRC complication rate.

b. RBL vs IS: A total of 554 patients are included in 6 articles and heterogeneity analysis indicates no heterogeneity (P=0.49, I2=0%). The fixed-effect model analysis is carried, and the results shows that there is no significant difference in the incidence of complications between the RBL group and the IS group (OR =0.02, 95%CI: -0.03~0.07, P=0.42), as shown in Figure 10.

Figure 10: RBL vs IS complication rate.

Discussion

Hemorrhoid is a soft venous mass produced by the dilation and flexion of the submucosal and cutaneous inferior venous plexus at the end of the rectum, which contains normal spongy tissue structures such as minute arteriovenous anastomosis, connective tissue, and nerve tissue, thus playing an important role in assisting and controlling defecation. Hemorrhoids are the most common anorectal diseases in adults, accounting for 89.25% of all anorectal diseases, and treatment needs are huge. The main clinical manifestations of internal hemorrhoids include bleeding, prolapse, pain and perianal itching, and in several cases, it can be complicated with thrombosis, incarceration, strangulation and difficulty in defecation, which significantly affects the patient’s quality of life. The treatment of internal hemorrhoids focuses on the elimination of symptoms caused by internal hemorrhoids. The commonly used treatment methods for internal hemorrhoids include non-surgical treatment and surgical treatment, among which non-surgical treatment includes RBL, IS, and IRC. The national guidelines of the United States, Japan, France, and China countries recommend that the above methods are mainly used for the treatment of I-III degree internal hemorrhoids [1,5,6], but the efficacy and safety of various methods are still controversial. In this article, the efficacy, recurrence rate and complication rate of the above three methods for the treatment of internal hemorrhoids are analyzed and compared in order to comprehensively and objectively evaluate the efficacy and safety of RBL, IS and IRC in the treatment of internal hemorrhoids. The results of this study show that the efficiency of RBL group is better than that of IS group, which is consistent with the research results of Jacobs, D et al. it is mentioned in the research report that the long-term effective rate of RBL group is about 90% among patients with internal hemorrhoids of grade I-III, while the long-term remission rate of only one-third of the patients treated with IS, indicating that the long-term effective rate of RBL is superior to that of the IS group [7]. Similarly, the research results of MacRae HM et al. also show that for I-III degree internal hemorrhoids, it is recommended to use RBL as the first-line treatment, and its curative effect is better than IS. Compared with patients receiving IS or IRC treatment, the need for retreatment in RBL group is obviously reduced [8]. This study has shown that the efficacy of the RBL group was comparable to that of the IRC group. The research by Ricci MP et al. also reported that the success rate of 4 weeks after RBL was not different from that of IRC, that is, the short-term clinical efficacy of RBL was comparable to that of IRC [9].
This study shows that the efficiency of IS group is equivalent to that of IRC group, which is similar to the research results of MacRae, MD et al. . It is mentioned in the research report that there is no difference in any outcome index between IS group and IRC group [10]. IS is the most effective for I-II degree internal hemorrhoids, and postoperative bleeding is rare. For patients with high risk of bleeding, such as patients receiving anticoagulant therapy, this method should be considered [7]. This study suggests that the incidence of complications in RBL group is lower than that in IRC group. However, the research of Johanson JF et al. Showed that RBL has better long-term efficiency, but the incidence of pain after treatment is higher. In contrast, the complications of IRC are few and not serious [11]. The reason for the analysis may be that the current guidelines classify pain as a type of complication. This article does not analyze pain alone, but classifies it as a complication for analysis. This study shows that the recurrence rate of RBL group is lower than that of IS group and IRC group. A C Poen et al. reported that 18% of the patients receiving RBL treatment had symptomatic recurrence, while 20% of the patients receiving IRC treatment had symptomatic recurrence to the level before treatment [12], and the longer the follow-up time was, the higher the symptom recurrence rate was [13]. Meta-analysis of non-surgical treatment showed that the recurrence rate of Ⅰ-II internal hemorrhoids patients after IS was relatively high, while the discomfort caused by RBL was relatively high [14-24].

The Limitations of this Study

1) Although the research documents included in the systematic review are all RCTs, the follow-up time varies, and there is a lack of multi-center, large-scale, long-term follow-up RCT research results.
2) Some studies have a small sample size, and some evaluation indicators only have 2-3 literatures for effect combination, and the outcome indicators of the analysis are not fully mentioned, such as: surgical recovery time, cost-benefit ratio, patient satisfaction, etc. To systematically evaluate the three methods, there is still a lack of high-quality RCTs research.
3) The cases of internal hemorrhoids in some literatures are not classified.
In conclusion, for the treatment of grade I – III internal hemorrhoids, the safety of RBL group is better than IS group and IRC group, and the efficacy is better than IS group or equivalent to IRC group. Therefore, RBL can be the first choice among the three treatment methods, but it still needs to be verified by multi-center, large-sample and high-quality RCTs.

Table 1.

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Optimization of Ovulation Induction in Clomifene Resistant Patient with Infertility

Introduction

It has been proven that the induction of ovulation is the main method of treatment in infertile women with PCOS [1-3]. According to the WHO, from 10 to 15% of married couples suffer from infertility. In the conditions of Central Asia, where large families are common and this is traditionally encouraged, childlessness is considered a great misfortune and often leads to family disintegration [4,5]. Up to 20-25% of women with PCOS are resistant to clomiphene citrate [6,7].

Materials and Research Methods

Our randomized trials were carried out in the central polyclinic of Urgench from 2018 to 2020. It included 40 clomiphene-resistant women with PCOS. In group I (n = 20) women, we used clomiphene citrate 100mg + recombinant follicle-stimulating hormone p FSH 37.5 IU / day. Group II (n = 20) received only p FSH 37.5 using a low dose escalating protocol. Informed written consent was obtained from all patients. Women were considered clomiphene resistant if ovulation did not occur when taking CC at a dose of 150 mg / day. PCOS was diagnosed based on the Rotterdam criteria, in which at least 2 of the following three criteria were met:
1) Oligo menorrhea (a cycle lasting 35 or more days) and / or amenorrhea (absence of menstruation for 6 or more months);
2) Hyperandrogenism (defined as a Ferriman-Gallvi index of more than 8) which is clinically manifested by acne / hirsutism and / or biochemical – the determination of testosterone in the blood serum of more than 0.7ng / mg;
3) Sonographic manifestations of polycystic ovary: if the ovary contains 12 or more follicles with a diameter of 2 to 9 mm and / or the volume of the ovaries is more than 10 ml.
Inclusion criteria are, clomiphene citrate resistant women with PCOS aged 20 to 38 years, BMI, no previous ovulation induction, partners with normal sperm counts according to WHO standards, opening of the fallopian tubes (confirmed by hysterosalpingography in the previous 6 months), without presence operations on the genitals. The exclusion criterion is the presence of any factors of infertility, except for CV-resistant women with PCOS. The study also included the measurement of blood pressure, abdominal circumference, hormonal study of the serum of patients such as basal FSH, LH / FSH ratio, free testosterone (T), insulin, progesterone, AMG on the 3rd day of the menstrual cycle. HDL High Density Lipoproteins, serum estradiol was determined on the day of ovulation trigger administration. Insulin resistance (HOMA-IR) was determined as follows: HOMA-IR = fasting insulin (IU / ml) x fasting glucose (mol / l) / 22.5. Ultrasound of the ovaries with a transvaginal sensor on the 2nd – 3rd day of the menstrual cycle to assess the number of antral follicles with a diameter of 2 to 9 mm (in an amount of 12 or more is considered polycystic) and an assessment of the volume of the ovary, which is determined by measuring three perpendicularly directed ovarian diameters and applying the formula: D1xD2xD3x0.5236.

Results of the Study

(Table 1) shows the results of clinical and laboratory studies of both groups, which reflects the average age of a woman, type of infertility, BMI, abdominal circumference, ovarian volume, type of menstruation disorder, hormonal and biochemical studies (Table 1). As our study showed, group I (CC + rFSH) received a lower dose of rFSH (532.5 ± 315) and the duration of stimulation days (12.34 ± 4.5) was less than in group II (18.42 ± 6.2 days of stimulation). The number of growth of the middle and dominant follicle, the thickness of the endometrium, the number of ovulations and the frequency of pregnancy are shown in (Table 2). The study showed that the dose of gonadotropin preparations for obtaining ovulation can be reduced by the simultaneous administration of CC + rFSH.

Table 1: Results of clinical and laboratory studies.

Table 2: Induction cycle indicators with results.

Conclusion

The combined administration of CC + rFSH in clomipheneresistant women with PCOS compared to the use of rFSH alone, gives higher ovulation rates and lower financial costs. The use of this protocol enables monofollicular growth and a decrease in the risk of multiple pregnancies and, in turn, is the prevention of ovarian hyper stimulation.

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Coats Disease in Young Patient with Congenital Cataracts History

Case Report

He is 14-year-old boy, attended at the Ophthalmology Unit of High Specialty at Hospital Civil de Guadalajara Fray Antonio Alcalde since 2013. He had an ophthalmologic precedent of bilateral congenital cataract, treated with cataract faco aspiration and capsular bag implantation of intraocular lens (IOL) of both eyes in the same year, as well as correction of air lenses after the surgical procedure and visual therapy, with adequate evolution. Nevertheless, the patient loses his follow up as of 2018. Later, in May 2021 he requests a new assessment due to progressive visual loss of the right eye of 5 months of evolution, with no other association. Left eye reports no symptomatology. Other personal precedents without significant data.

Ophthalmologic Exploration

Best corrected VA OD 20/400, OS 20/30. No alterations in OU eyelids and annexes, eucromic conjunctiva, clear cornea, formed anterior chamber, isochoric pupils, nomoreflectic, Pseudofaquia, intraocular lens in capsular bag (Figures 1 and 2).

Figure 1: Clinical picture of IOL in situ, opacity of posterior capsule is observed, as well as fibrosis with free visual axis right eye (A), left eye (B).

Figure 2: fundus image: right eye (A), left eye (B).

Fundoscopy

OD clear vitreous, isochoric pupil, normochromic, retina with exudation at inferior and superior temporal arcade level with presence of vascular tortuosity, macular exudates. OS without alterations (Figures 3-5). In ultrasound of OD, we observe normal ocular globe contour, with intraocular lens of posterior chamber, vitreous with small mobile condensations of low reflection, thickened retina due to important cystic edema with macular involvement, thickened choroid secondary edema, irregular papilla. OS is reported without alterations (Figure 4). Macular spectral domain ocular coherence tomography of OD, where 242-micron central foveal thickness is observed, in presence of intra-retinal cysts and sub-retinal fluid.

Figure 3: Mode B ultrasound, right eye (A), left eye (B).

Figure 4: Macular SD- OCT.

OS with central foveal thickness of 286-micron, within normal parameters (Figure 4). In the fluorescein angiography (FA) of OD we observe retinal vessels with dilation and leaks, with telangiectasias in retina surrounded by hard exudates, in peripheric retina with hyperfluorescent zones corresponding with areas of capillary closure. FA of the OS is observed without alterations (Figure 5) In accordance with clinical characteristics of the patient, as well as findings in complementary studies, Coats disease is diagnosed. Treatment with scheme of intra vitreous anti-VEGF (Aflibercept) of OD, and with previous informed acceptance from his parents, first dose was applied on Aug 05, 2021. After three weeks he is evaluated, and patient refers recovery of visual quality. Nevertheless, BCVA of OD didn´t improve further than 20/400. A second dose of anti-VEGF was applied in September 2021. He was evaluated one week later presenting vision improvement, with a BCVA 20/200 and reduction of retinal exudation as shown in Figure 6.

Figure 5: Fundus images, right eye (A1), left eye (B1). Red-free images of the retina, right eye (A2), left eye (B2). Flouoresceine angiography of right eye (A3- A6), left eye (B3- B6).

Figure 6: Fundus image A) and macular SD OCT (B) of the right eye.

Discussion

Coats Disease is a retinal vascular disease, characterized by telangiectasias and vascular leaks that lead to exudation. It is typically found in young males, between the first and second decade of life, with a peak of incidence between 5 and 11 years old. In 85- 90% of patients, it is a unilateral affectation. In cases of bilateral disease, the other eye shows no symptoms with slight telangiectasic changes in the periphery. There is no preference for race, and is a sporadic non inherited condition, without systemic association; the gold standard for the diagnosis of this disease is the eye fundus clinical exploration through direct ophthalmoscopy [1,2] and as our patient fitted in the above-mentioned characteristics, the diagnosis of Coats has reached. However, there are very few reports regarding the association of this pathology with the presentation of Congenital cataracts, even less in a bilateral way, as is the case of our patient [3,4].
The etiology of Coats disease is not completely defined; however, it is well known that retinal vascular leak is one of the main pathologic processes found in this disease. Several studies have been recently found that where an increase of cytokines is evidenced in the aqueous humor of patients with Coats disease, mainly the vascular endothelial growth factor (VEGF) of the aqueous humor was strongly elevated in this entity and had correlation with the extension of the retinal exudation. Tingy Liang et al. analyzed the aqueous humor of two groups of patients. One of them had 36 patients with Coats disease and another one with 15 patients as a control group with congenital cataract. Concentrations of 22 different cytokines, of which, concentrations of 8 cytokines (VEGF, IL-6, IL-8, MCP-1, MIP-1α, IP-10, VCAM-1 e ICAM-1) were significantly higher in the group of Coats disease, however, significant differences were observed in bFGF, TNF-a and IFN-y between the group of Coats disease and the control group, being this of importance for our patient since he presented both diseases and thus the use of anti-VEGF drugs for his treatment is sustained [5].
Broadly speaking, the objective of the treatment for slight and moderate disease is preservation of the vision and prevention of progression of the disease as retinal detachment and all the other complications already mentioned. That is why treatment is focused in ablation of the actual abnormal vasculature of the retina by means of photocoagulation with laser and cryotherapy. However, the innovative therapy is the use of intra vitreous VEGF, which is also recommended as a contributory manner with the traditional therapies already mentioned, since it seems to reduce macular edema and exudates, stabilize visual acuity and enhance regression of abnormal vessels, as observed in our patient, with a vision improvement and reduction of posterior retinal exudation to the first dose of intra vitreous anti-VEGF. Even though a complete regression of the disease is not expected, our objective is to stop progression and avoid appearance of future complications. [2,6,7]. Even though association of the appearance of cataracts is reported, after presentation of Coats disease, as mentioned by Daruich A. et al. [7], inverse association (that means, late presentation of Coats disease with bilateral congenic cataracts background) without a syndromic association is very rare.

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Overview of Carbapenem-Resistant Enterobacteriaceae

Introduction

Respiratory infection is one of the most common diseases in the world, with high incidence and mortality. Enterobacteriaceae is the most clinically important gram-negative pathogenic bacteria, which are increasingly being reported worldwide. Antimicrobial resistance is globally recognized as one of the greatest threats to public health. For years, carbapenems have been used successfully to treat infections due to resistant Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae. However, recently Carbapenem-resistant Enterobacteriaceae have emerged, which confer broad resistance to most ß-lactam antibiotics including lastline carbapenems. Carbapenem-resistant Enterobacteriaceae refers to Enterobacteriaceae that are resistant to any drug of ertapenem, Doripenem, imipenem, meropenem, or enterobacteriaceae that produce carbapenemase. Infection with Carbapenem-resistant Enterobacteriaceae is emerging as an important challenge in healthcare settings and a growing concern worldwide, it is very easy to spread in patients with long-term hospitalization or low immunity, leading to nosocomial infection, and may even cause a small- or large-scale outbreak [1-3]. Most Enterobacteriaceae belong to the normal flora in the intestinal tract and can become opportunistic pathogens once the body’s immunity declines. Enterobacteriaceae obtain genetic material mainly by horizontal gene transfer mediated by plasmids and transposons [4] Carbapenem-resistant Enterobacteriaceae can cause a number of serious infection types (such as pneumonia, abdominal cavity infection, urinary tract infections, Bloodstream infection, skin and soft tissue infection, central nervous system infection, and device-associated infections) or asymptomatic colonization, among which Ventilator-associated pneumonia (VAP) was the most common [5]. Severe pneumonia has always been a common respiratory disease, which can endanger life. Statistics [6] show that infectious diseases account for 30% of all deaths worldwide, with severe pneumonia leading the way. Carbapenem-resistant Enterobacteriaceae infection was reported in 68.8% of patients with hospital acquired bacterial pneumonia [7]. Consistent mortality rates of 40-50% are observed among inpatients with infections caused by CRE in hospitals worldwide, while the mortality rate from CRE infection in pneumonia patients is as high as 60% [8]. Carbapenem-resistant Enterobacteriaceae infection is a very difficult problem in clinical practice.

Risk Factors of Acquisition of CRE Infection

There are a number of factors that predispose persons to infections by CRE. Exposure to these resistant organisms can cause serious infections in patients with the following reported risk factors: immune-suppression, advanced age, admission to intensive care unit (ICU), mechanical ventilation, previous exposure to antimicrobials, organ or stem-cell transplantation and prolonged hospital stays. Healthcare associated infections caused by CRE, mainly Klebsiella pneumoniae, have been encountered most commonly in ventilator-associated pneumonia, bacteremia, urinary tract and surgical site infections. Growing evidence suggests early detection of CRE-colonized patients on admission to long-term care facilities may help to prevent institutional outbreaks and limit regional spread of this emerging public health threat. Respiratory disease is one of the risk factors for CRE infection, probably because a variety of bacteria grow in the respiratory tract and maintain a dynamic balance in the body. Elderly patients with respiratory diseases have low immunity and are vulnerable to infection. Moreover, most patients with respiratory diseases have a history of invasive operation. When tracheotomy or endotracheal intubation is performed, the respiratory mucosa will be damaged, resulting in a variety of complications. Some bacteria are easy to form biofilm in the open airway, which leads to abnormal expression of outer membrane pore protein and bacterial drug resistance. In particular, frequent aerosol inhalation and other operations change the airway environment, requiring frequent contact with patients by medical staff, which makes patients susceptible to colonization and infection by multi-resistant bacteria.

Prevention and Control measures for CRE Infections

2.1 There are reports which suggest that overuse of carbapenems is closely related to the incidence of CRE infection, and unreasonable use of carbapenems can easily induce bacterial resistance and spread [9]. One of the chief difficulties in the treatment of CRE is the excessive use of antibiotics, not only those acquired by the community but also in hospitals. On the one hand, the use of broad-spectrum antibiotics can kill the sensitive bacteria, and the resistant bacteria can survive and become the dominant growth, thus increasing the probability of CRE infection. On the other hand, drug resistance may occur due to the change of drug binding sites after drug use, resulting in carbapenemases and other drug resistance mechanisms [10]. Growing evidence suggests that carbapenem-resistant Gram-negative bacteria are sufficient to develop in the intestinal flora of intensive care patients just a few days of application of carbapenems antibiotics [11]. Therefore, it is necessary to strengthen the supervision and management of clinical application of carbapenems, and strictly implement the classification of antibiotics and the management of doctors’ prescribing rights, limiting the over-use and abuse of antibiotics in humans and agriculture.

Standardized Collection and Correct Interpretation of Microbial Test Reports

Specimens should be collected before antibiotic treatment, sterile site specimens should be collected as far as possible and microbial reports should be correctly interpreted, eliminating contamination and colonization and avoiding unnecessary use of antibiotics. Therefore, clinical microbiology laboratories at all levels of medical institutions should establish the ability to receive and process microbial specimens within 24 hours. There are reports which suggest that that patient carrying other multidrug-resistant bacteria are mostly in serious and complex conditions, with low immunity and relatively long stay in intensive care units, leading to the development of CRE. Therefore, it is necessary to strengthen the contact and isolation of MDR-resistant bacteria to avoid the spread of MDR-resistant bacteria in hospitals. Hand hygiene is the simplest, most effective, most convenient and economical method to control the infection in hospitals, which can significantly reduce the incidence of CRE infection. Mobile water sinks, non-contact faucets, hand sanitizers, hand drying facilities, quick-drying hand disinfectants, and related charts can reduce the colonization rate of CRE [12].
Studies show that the wash basin and its surrounding environment are seriously polluted, which is an important source of CRE pollution. Therefore, medical institutions should pay attention to the cleaning, disinfection and management of the location of the sink in the diagnosis and treatment area, taking anti-splash measures. Symptomatic colonized patients can become potential sources of infection. The significance of active screening lies in early identification of CRE colonized patients so that timely isolation measures can be taken to reduce the risk of transmission. Stool is the best specimen for active screening, and if not readily available, a rectal swab is taken. If the patient has a definite history of CRE infection, specimens from the infected site should be screened again. Patients with positive initial screening and hospitalized for less than 30 days do not need further screening, while patients hospitalized for more than 30 days were screened once a month. Those who have been screened negative for the first time should be screened regularly, either once a week or every two weeks, or twice a week, depending on the severity of the outbreak. Health care facilities should implement isolation of all CRE infected/colonized persons. Isolation refers not only to the establishment of physical spatial barriers, but also to the strict enforcement of isolation measures.

Treatment Options for CRE Infections

There are numerous different types of carbapenemase enzymes, each conferring varying spectrums of resistance. In general, the presence of a carbapenemase confers broad resistance to most ß-lactam antibiotics including penicillins, cephalosporins, and the monobactam aztreonam (excluding metallo-β-lactamases [MBLs] and oxacillinases [OXAs]) [13]. At present, the main drugs for the treatment of CRE in the world are polymyxins, Tigecycline, fosfomycin, Ceftazidime-Avibatam and aminoglycoside antibiotics. Polymyxins and tigecycline were highly sensitive to CRE in vitro and were not affected by the type of carbapenemases produced by bacteria. Due to heterogeneous drug resistance and positive correlation between dose and renal toxicity, polymyxins are often used in combination with other antibacterial agents. The conventional dose of tigecycline is difficult to reach sufficient concentration in the areas including the blood flow and alveolar lining fluid, so it is often necessary to increase the dose and use it in combination with other drugs. Ceftazidime-avibactam lacks effective antibacterial activity against metalloenzyme-producing CRE, so it may be an important choice for the treatment of nonmetalloenzyme- producing CRE infection. The most common adverse drug reactions of Ceftazidime-avibactam in trials were vomiting, nausea, constipation, and anxiety [14].
Combination therapy for CRE infections may decrease mortality compared with monotherapy. Benefits of combination therapy include reduction of initial inappropriate antimicrobial therapy, potential synergistic effects, and suppression of emerging resistance [15]. For patients who are critically ill or with deep-seated infections, consider empiric and antibiogram-directed combination therapy with 3 drugs, basing on antimicrobial sensitivity results. Polymyxins may be most effective as part of a combination for serious CRE infections [15,16].

Conclusion

In summary, the burden of carbapenem-resistant Enterobacteriaceae is increasing rapidly worldwide. CRE is widely spread and is now a major factor in morbidity and mortality in health-care settings. The results at present are still not good, especially in elderly patients with a history of CRE infection. The extremely high mortality rates of patients with CRE infections have driven efforts to prevent the acquisition and spread of these bacteria in hospitals. Although the above measures are simple, they can prevent the spread of CRE to some extent. However, continued research is desperately needed to determine the most appropriate treatment for serious CRE infections.

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Oscillatory Diffusion and Chemical Vortices in Multicomponent Plasma

Introduction

Often laboratory and astrophysical plasmas are multicomponent, and diffusion of elements is important for some phenomena. For instance, diffusion processes can lead to chemical inhomogeneities and, as a result, to change the emission, heat transport, and conductivity [1-3]. In thermonuclear fusion, the source of impurities is usually the chamber walls, and diffusion determines the distribution impurities [4-6]. Even a small number of heavy ions influences the rate of radiative losses in plasma and changes its thermal properties. In astrophysical conditions, chemical inhomogeneities have been detected in the surface layers of many stars of various spectral types. It is widely accepted that these inhomogeneities are determined by diffusion processes but, however, the mechanisms resulting information of chemical spots is still uncertain. Diffusion in plasma can differ qualitatively from that in neutral gases because of the presence of charged particles – electrons. This particularly concerns hydrogen plasma [7]. In such plasma, the influence of electrons on diffusion of heavy ions is especially pronounced.
Chemical inhomogeneities can appear because of various reasons, for instance, it is often thought that chemical spots occur due to the presence of magnetic fields. The magnetic field B can magnetize electrons and produce a non-uniform distribution of ions. Anisotropy of transport is characterized by the Hall parameter, xe =ωBeτ where is the gyrofrequency of electrons and τe is their relaxation time. In hydrogen plasma, where n and T are the number density and temperature of electrons, respectively, L is the Coulomb logarithm. The current-driven diffusion can lead to a formation of chemical inhomogeneities like other diffusion processes. Using a simple model, we show that the interaction of the current and field with impurities leads to their diffusion in the direction perpendicular to both. This type of diffusion can contribute to formation of chemical spots even in a relatively weak magnetic field with xe << 1. We also show that such diffusion can be accompanied by a particular type of waves in which only the impurity number density oscillates.

Formulation of the Problem and Basic Equations

Consider a cylindrical magnetic configuration with the field in a z-direction, are cylindrical coordinates and the corresponding unit vectors. Then, the electric current is given by

If jϕ → 0 at large s then B→ B0 =const at s→∞. Note that such magnetic configurations can be unstable for some dependences B(s) [9-11] but we assume that the instability does not occur for chosen dependencies B(s). Plasma is assumed to be fully ionized and consists of electrons e, protons p, and small amount of heavy ions i. A small admixture of heavy ions does not influence the dynamics of a background hydrogen plasma and these ions can be considered as test particles. The partial momentum equations in ionized plasma have been discussed by [7,12]. The paper [12] considers transport processes in the hydrogen-helium plasma but the derived equations describe the hydrogen plasma with a small admixture of heavy test ions. Then, the partial momentum equation for ions i has the form

where is the velocity, Zi is the charge of the species i, pi and ni are the partial pressure and number density, respectively, and is the electric field; is the external force on species i. We neglect the external force in our model since its influence is often insignificant. The forces and are caused by the interaction of ions i with electrons and protons, respectively. The forces and are internal and their sum over all components is zero.

The velocity can be represented as a sum of and where is the hydrodynamic velocity of plasma and ~V i is a diffusive velocity of ions i relative plasma. Since in our model ni is small compared to np, the hydrodynamic velocity is approximately equal to the mean velocity of protons, . In this paper, we assume that background plasma is in hydrostatic equilibrium. Then, = 0 and, hence, . If the temperature is uniform, the friction force has only a component proportional to the relative velocity of ions i and protons, . This force can be easily calculated if Ai = mi /mp >>1. Since the velocity of a background plasma is zero, can be represented as

[12], where is the characteristic time of scattering between protons and heavy ions; L is assumed to be the same for all types of scattering.

The friction force on the l.h.s of Eq. (2), , can be estimated as and the first term in this equation is where Dt is the timescale of diffusion processes. Therefore, this term is much smaller than the friction force . The second term on the l.h.s. of Eq. (2) is of the order of where L is the characteristic length scale. Since L ~ Vi Δt , this term can also be neglected if . Then, Eq. (2) transforms into

If ni << np , then, is given by [12], where is the force acting on the electrons. Since ni<< n, is determined by scattering of electrons on protons but scattering of electrons on heavy ions gives a small contribution. Therefore, one can use for the expression obtained by [7] for one-component plasma. In the case of a cylindrical isothermal configuration, this force reads

where is the velocity of electric current; ; coefficients α ⊥ and α ∧ have been calculated by Braginskii [7]. The force (5) is caused a friction due to a relative motion of the electron and proton gases. Then, Eq.(1) yields for the current velocity

Consider the current-driven diffusion in a magnetic field with xe <<1 . Substituting into Eq.(5) and using coefficients α ⊥, ∧ we obtain with the accuracy in linear terms in xe

The momentum equation for the species i (Eq.(4)) depends on s E and Eϕ components of the electric field. The momentum equations for e and p, containing these electric fields, read

Using Eq. (5), we obtain

Substituting Eqs.(6), (3), and (9) into the s- and ϕ -components of Eq.(4), we arrive to the expression for a diffusion velocity, ,

Vni is the standard diffusion velocity and VB is the velocity of diffusion caused by the current. The corresponding diffusion coefficients are

where .

Distribution of Ions Under the Influence of Electric Currents

The condition of hydrostatic equilibrium reads in our model

where p and r are the total pressure and density of plasma, respectively. In hydrogen plasma, we have p ≈ 2nkB T where kB is the Boltzmann constant. Integrating Eq. (14) and assuming the temperature to be constant, we obtain

where are the values of (p, n, T, b) at s → ∞.

Consider the distribution of elements in equilibrium. Since Vis = 0 in equilibrium, Eq.(10) yields

The r.h.s. is responsible for the influence of currents on the distribution of impurities. One has from Eq. (16)

Taking into account Eq. (16) and integrating Eq.(17), we have

where μ = − 2Zi (0.21Zi− 0.71) and ni0 is ni at s →∞. If the local abundance of i is γi = ni/ n , then taking into account Eq. (17), obtain

where γi0 = ni0 /n Local abundances depend essentially on the field strength. These dependences are particular sensitive to B in the case of heavy ions with large charge numbers. If other diffusion mechanisms are negligible, then the exponent (μ − 1) reaches large negative values for large Zi and, as a result, strong abundance anormalies can be produced in this case. For instance, (μ − 1) is equal 1.16, -0.52, and -2.04 for Zi =2, 3, and 4, respectively. Note that (μ − 1) changes its sign with an increase of Zi : (μ − 1) > 0 if Zi = 2 but (μ − 1) < 0 for Zi ≥ 3. Therefore, elements with Zi ≥ 3 are in deficit (γi <γi0 ) in the region with a weak magnetic field (B < B0) but these elements should be overabundant in the region with a stronger field, B > B0.

Eq. (20) describes the distribution of impurities in diffusive equilibrium. The characteristic timescale to reach this equilibrium, tB, can be estimated as

where L is the length scale of the magnetic field, L = |d In B/ds|-1 . The characteristic timescale of baro-diffusion is given by the wellknown expression

Hence, the current-driven diffusion operates on a shorter timescale if DB > D or

where cs is the sound speed, . Therefore, the current-driven diffusion is more efficient if the magnetic pressure exceeds the gas pressure.

Chemical Vortices

In our simplified model of cylindrical magnetic configuration and velocity (10), the continuity equation for ions i has the form

Consider the behaviour of small perturbations of ni by making use of a linear analysis of Eq. (23). In the unperturbed state, we assume that plasma is in a diffusive equilibrium and, therefore„ the unperturbed impurity number density satisfies Eq. (18). For the sake of simplicity, we assume that small perturnations are independent of z. Denoting disturbances of the impurity number density by i δ n and linearizing Eq.(23), we obtain the equation that describes the evolution of such small perturbations,

Consider only disturbances with a short radial wavelength. If this wavelength is shorter than the length scale of unperturbed quantities, then we can use the so-called local approximation. Then small disturbances are α exp(−iks)where k is the radial wavevector, ks >>1. Since the basic state does not depend on time and ϕ , perturbations can be represented in the form where m is the azimuthal wavenumber and ω should be calculated from the dispersion equation. Such disturbances have a shape of cylindrical vortices of a chemical composition. Substituting i δ n into Eq. (24), we derive the dispersion equation for such perturbations in the form

This dispersion equation describes a special type of magnetohydrodynamic waves where only the number density of impurities oscillates. Note that there are many different sorts of waves in magnetized plasma that can produce oscillations of a chemical composition. Such oscillations, for example, can be generated by standard magnetohydrodynamic waves [12] but, usually, fluctuations of impurities in such waves are accompanied by fluctuations of other hydrodynamic quantities. Indeed, the electric field has actively to take part in such waves and interact more efficiently with heavily charged ions than with protons. As a result, MHD waves can produce fluctuations of a chemical composition. However, these fluctuations are typically very small because the period of MHD waves is much shorter than the diffusion timescale. Chemical fluctuations become significant only if the period of a wave is comparable to the diffusion timescale.
The waves considered in this paper are caused by diffusion processes and their period is generally comparable to the diffusion timescale. Therefore, fluctuations of a chemical composition can reach a significant value in such waves. The quantity ωR is responsible for decay of these waves with the timescale typical for standard diffusion. The frequencies ωI and ωH describe oscillations of the impurities caused by the combined action of electric current and the Hall effect. The considered waves are oscillatory if | ωI + ωH|> ωR. The latter condition is equivalent to

Therefore, the waves are oscillatory if the magnetic pressure is substantially greater than the gas pressure. The frequency of diffusion waves is higher in the region where the magnetic field has strong gradients. The order of estimate of ωI is

Where li = ciτi Note that different impurities in such waves oscillate with different frequencies.

Conclusion

Diffusion of heavy ions in plasma under the influence of the current-driven mechanism has a number of features. Generally, the diffusion velocity can be comparable to (or even greater than) that caused by other diffusion mechanisms. The currentdriven mechanism can lead to a formation of chemical spot even if the magnetic field is relatively weak. Note that other diffusion mechanisms usually require a much stronger magnetic field. The current-driven 83 diffusion is caused by the Hall effect and electric current and, therefore, it leads to diffusion of heavy ions in the direction perpendicular to both the magnetic field and current. Therefore, a distribution of elements is determined by a geometry of the magnetic fields and electric currents. Chemical inhomogeneities can manifest themself, for instance, by a nonuniform distribution of plasma temperature and emission. Note that this type of diffusion can be important also in some conductive fluids if the magnetic field is sufficiently strong. Our consideration shows that a special type of magnetohydrodynamic waves may exist in multicomponent plasma.

These waves can be periodic if the magnetic field is sufficiently strong, and they are characterized by oscillations of the impurity number density alone. The frequency of such waves is rather low and is determined by a characteristic diffusion time. The order of magnitude estimates of this frequency yields Lλ where λ = 2π / k is the wavelength. In stellar conditions, such waves can manifest themselves in pecular magnetic stars where the magnetic field is ∼ 104 G and the number density and temperature are ∼ 1014 cm−3 and ∼ 104 K, respectively. If the length scale, L, and λ are of the same order of magnitude (for instance, ∼ 1011 cm), then the period of such compositional waves is ∼ 3 × 103 yrs. This is much shorter than the magnetic timescale and generation of such waves should lead to short-term spectral variability. Such waves can exist in laboratory plasmas as well, but their frequency is much higher in this case. If B ∼ 105 G, n ∼ 1015 cm−3, T ∼ 106K, and L ∼ l ∼ 102 cm, then the period of compositional waves is ∼ 10−8 s. Note that frequencies of waves with various impurities can differ essentially since they depend on the sort of heavy ions.

In terrestrial conditions, the compositional waves also can manifest themselves by oscillations in spectra. In astrophysical conditions, the magnetic field has usually more complex topology than that considered in our model. In some cases, however, cylindrical magnetic configurations can mimic the magnetic field in certain regions. For example, the field near the magnetic poles in stars has approximately cylindrical symmetry [13] and our results describe qualitatively diffusion in the region near the poles. Therefore, the current-driven mechanism in combination with other diffusion processes contributes to a formation of chemical spots in various types of stars. For example, this mechanism can contribute to formation of element spots in Ap-stars where the magnetic fields have been detected. Also, many neutron stars have strong magnetic fields and topology of these fields is rather complex with spot-like structures at the surface. Such magnetic configurations can be responsible for formation of a spot-like chemical distribution at the surface. For example, evolution of neutron stars is very complicated, particularly, in binary systems [14] and, therefore, their surface chemistry can be complicated as well. Diffusion processes may play an important role in this chemistry.

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Palatability Evaluation, DNA Analyses, and Functional Processing of Rice

Evaluation of Palatability of Cooked Rice

Abstract

Rice is one of the most important crops in the world. It is useful not only for the supply of energy, but also for giving palatability and good health to the people. The rice quality depends on various aspects such as consumer safety, nutrition, appearance, price level, and palatability. Sensory test and physico-chemical measurements are important measures for quality assay of rice grains. Sensory test is a fundamental method that requires large amounts of samples, many panel members, and long hours. Physico-chemical measurements estimate the eating quality based on the results of various kinds of measurements, such as chemical composition analyses, cooking quality test, gelatinization property test, and the measurements of physical properties of cooked rice. Novel method to evaluate the quality of the cooked rice is necessary to breed high-quality rice cultivars and to select the suitable rice for each consumer and each utilization purpose. It is necessary to develop the novel method to evaluate the rice quality using various kinds of novel measurements, such as Texture Analyzer, Tensipresser, RVA, NIR, and spectrophotometer. Simple, rapid, and accurate method to assay the quality of rice grains would be very valuable.

Introduction

Rice, wheat, and maize are the most important crops in the world. Rice consumers need not only enough amount of rice but also high-quality rice grains. The many rice cultivars grown in the world vary markedly in their cooking, sensory, and processing quality [1]. Consumers are heterogeneous with respect to their perceived differentiation of rice quality among regions, countries, cities, and urbanization levels [2] The rice quality depends on various aspects such as consumer safety, nutrition, appearance, price level, and palatability. Because rice is eaten every day, it is indispensable that it should be safe to eat and highly nutritious. Furthermore, because rice provides income for farmers and is prepared, milled, and sold by wholesalers and retailers, the rice yield and price are extremely important to the farmers. Consumers have been asking for more palatable rice because they have recently become more affluent. Calingacion et al engaged local experts across the world and they showed that there are at least 18 different quality types of rice that are favored around the world and this complexity immediately reveals the extent of the specificity of consumer preference [3]. As described by Juliano, rice quality evaluation includes eating quality assays for sensory evaluation and aroma testing, as well as physical property measurements using Instron, Texturometer, or Tensipresser etc., which were highlighted as indirect methods, in addition to amylose content, starch gelatinization temperature, gel consistency, amylography, and protein content determination [4]. Ohtsubo et al. reported on the quality assay of rice using traditional and novel tools, such as the measurements of amylose, physical properties and pasting properties [5].

Starch comprises the most abundant component in rice grain and con-sists of am- ylose (linear ƒ¿-1,4-glucan) and amylopectin (highly branched molecule with ƒ¿-1,4 bonds and ƒ¿-1,6 bonds). Apparent amylose content (AAC) is measured by the iodine colorimetric method [6] and high-AAC rice becomes harder and non-sticky on cooking [1,6,7]. Lian et al. investigated on the identification of the main retrogradation-related properties of rice starch and reported that retrogradation rates of different rice starch showed significant positive correlation with proportion of the chains (DP>10) in amy- lopectin [8]. Protein content is another extremely important factor that determines rice quality [1]. Protein contents are measured by Kieldahl method, combustion method or NIR method. As Juliano [8] and Bhattacharya [9] pointed out, the physical properties of cooked rice grains are important determinants of the eating quality of rice. The pasting properties of rice are useful indicators in the quality assay of cooked rice, rice cake, rice bread, rice extrudate, etc. In several countries, such as Australia, China, Japan, and the United States, the RVA has become the standard method with which the rice processing industry and breeding programs determine rice pasting properties [1]. A rapid, simple, and accurate method is required to evaluate rice. Therefore, if a such novel method could be developed, it would become a great help to the producers and the consumers of rice all over the world. Sensory testing is the fundamental method that evaluates the eating quality of rice grains. The members of a trained panel taste cooked rice samples and give scores based on appearance, flavor, taste, hardness, stickiness, and overall sensory evaluation.

Sensory Test and Physicochemical Measurement [10]

Sensory Test: Sensory testing is the fundamental method that evaluates the eating quality of rice grains. The members of a trained panel taste cooked rice samples and give scores based on appearance, flavor, taste, hardness, stickiness, and overall sensory evaluation. The test results can be expressed as numerical values and treated statistically; there are disadvantages to using this test. The results differ depending on the preference of panel members, and the results cannot be directly compared if the time or country differs.

Physicochemical Evaluations: Physicochemical evaluation is only indirect evaluation of rice palatability, its results are common all over the world if we use the same method and apparatus. Physicochemical measurements include component analysis, such as protein, amylose, and fibers, pasting properties of starch, texture measurements of the cooked rice grains, etc. For the sake of estimating the total palatability, statistical treatment is adopted using the results of each measurement as the variables. Multiple regression analysis, principal com-ponent analysis, and PLS analysis are very useful to clarify the characteristics of the rice samples. Recently, spectrophotometric analyses, such as NIR or visible-light spectrometer, are utilized to estimate the chemical components and to develop the estimation formulae for palatability. “Taste Analyzer” is an example of the NIR system as a non-destructive estimation apparatus for palatability of rice grains. Examples of sensory test and physico-chemical evaluation of rice palatability are shown in Table 1.

Table 1: Evaluation of rice palatability.

Amylose Content and Amylopectin Molecular Structure: Amylose content is the most important factor that determines rice quality because starch shares approximately 85% (w/w, dry basis) of the milled rice grains. Because North- east Asian consumers prefer soft and sticky cooked rice, low amylose rice is the dominant rice sold in the rice market. Amylose content is generally measured by the iodine colorimetric method of Juliano [6]. Li, et al. [10] reported that stickiness of cooked rice is affected not only by AAC but also the proportion of short amylopectin chain. They found that the autoclave cooking, which is used to produce sticky “convenience rice”, effects on sensory properties and increase leached amylopectin [11].

Protein Content: Protein content is another extremely important factor that determines the eating quality of rice. Cooked rice with high protein content tends to be hard and non-sticky. Protein content is measured by the Kieldahl or the combustion method. It can be measured by NIR method (Near Infrared Reflectance) easily and non-destructively. Recently, not only protein content, but also molecular compositions of protein, such as glutelin or prolamin, is reported to affect the quality of rice [12,13].

Gelatinization Properties Using an RVA: The RVA (Figure 1) provides various information, for examples, as indicators in cerealbased products, on apparent viscosity of cake batters and quality of flour for cake making, about interactions between starch and other compounds, on extruded products and the measurement of the degree of cook, as quality indicators for hydrocolloids and fibers, for simulation and monitoring of processes, and on enzymatic reactions [14]. Comejo and Rosell used RVA in the research on the influence of germination time of brown rice in relation to flour and gluten free bread quality [15]. Although the Brabender Viscoamylograph has been used to assess rice pasting properties [1], Blakeney, et al. [16] and Champagne, et al. [17] showed that the RVA is useful in determining the “degree of cook” after processing of rice into precooked and extruded products. Yao et al. reported that the high temperature during ripening of rice decreased setback and trough viscosities and increased breakdown, implying that the pasting properties were slightly better [18]. Zhu et al. investigated the effect of soaking and cooking on structure formation of cooked rice through thermal properties, dynamic viscoelasticity, and enzyme activity, in which they used RVA for the measurements of enzyme activity [19].

Figure 1: Rapid visco analyzer (RVA).

Nakamura et al. developed a novel estimation formula for AAC and resistant starch (RS) based on the pasting properties measured by an RVA [20], enabling evaluation of the starch properties and processing suitability of material rice flours. Furthermore, we developed novel estimation formulae for oleic and linoleic acid contents based on the pasting properties of brown rice flours using an RVA, making it possible to predict easily and rapidly the nutritive and bio-functional characteristics of material rice [21]. To evaluate the pasting properties, the behavior of rice starch at temperatures higher than 100 ‹C should be examined to elucidate the changes in rice quality after cooking and processing, such as extrusion cooking [22] or super-heated moisture treatment [23]. Recently, Nakamura et al. reported about the comparison of the three different programs and estimating the hardness and retrogradation of cooked rice based on its pasting properties using a novel high-temperaturetype programs [24]. They have made improvements to analyzing pasting properties in a new program using RVA 4800 (Figure 2), and novel estimation formulae were developed in this paper for estimates of the retrograded hardness of cooked rice H1(R) and the degree of retrogradation H1(RD), which has led to an easy and rapid evaluation of the cooking qualities of various rice samples. The following are the main features;
1. Among the three kinds of RVA programs, Program 2 (120‹C) showed the highest correlations with starch microstructure (Amax), RS, physical properties, and degree of retrogradation of the cooked rice grains.
2. The novel Program 2 (120 ‹C) showed high determination coefficients for hard-ness and the degree of retrogradation of cooked rice grains.
3. The novel RVA Program 2 enables us to estimate easily and rapidly the cooking and processing characteristics of various kinds of rice cultivars. Commercial rice of Japonica rice cultivars Consistency (93°C) Consistency (120°C) Consistency (140°C).

Figure 2: Comparison of “Consistency” among the three programs using an RVA 4800.

Fat Acidity: Fat acidity is a good index to measure the quality deterioration of rice grains during storage. In Japan, newlyharvested rice is preferred to aged rice because consumers like soft and sticky cooked rice. Ohtsubo proposed a new colorimetric method to accurately measure fat acidity [25].

Cooking Quality Test [26]: A cooking quality test was developed by the researchers of USDA in 1950s. An expanded volume and water uptake ratios are measured after cooking rice samples in excess amounts of water. Dissolved amylose is measured by color generation by combining iodine and amylose.

Physical Properties of Cooked Rice Grains: Physical properties of cooked rice grains, such as hardness and stickiness, are measured by a Texturometer, Tensipresser, Texture analyzer, or Rheolograph-micro. Okabe reported the texture measurement of cooked rice and its relationship to eating quality using a Texturometer [27]. Low-compression and high-compression test using a Tensipresser [28] is shown in Figure 3. Recently, Texture Analyser has been used for measuring the physical properties of the cooked rice [29] or the parboiled rice [30]. And dynamic viscoelasticity gives valuable information for the elucidation of the relationship between the texture and the molecular structure of starch, which showed that gconsistency coefficient h affected hardness and tanƒÂ affected stickiness of cooked rice grains [31].

Figure 3: Tensipresser used for texture measurement of cooked rice grains.

Palatability Estimation Formula: Chikubu et al. developed a new equation to estimate the palatability of rice grains using a multiple regression analysis based on physico-chemical measurements, such as an amylograph viscosity profile, protein assay, and iodine blue value measurements [32]. The formula showed a high correlation between the results of a sensory test (R = 0.84) and the rice samples that will be harvested next year.

Application of Near Infra-Red Spectroscopy to Evaluate Rice Quality: Near Infra-red spectroscopy is a rapid, non-destructive, and extremely promising technique to evaluate qualities of the rice grains. In Japan, NIR spectroscopy was initially used to determine the protein and moisture content of the rice flours. Satake Co. Ltd subsequently combined NIR and palatability estimation formula and developed a new system called “Taste Analyser”.

Protein Content: Iwamoto et al. reported that NIR was equally sensitive to both rice and wheat proteins. A standard error of estimation (SEP) was decreased for calibrations using a derivative absorbance. However, the derivative procedure may possibly make an increment bias in cases that predict unknown sam-ples [33]. Inatsu utilized NIR spectroscopy to select palatable rice cultivars by measuring the protein content of rice because low protein rice is preferred in Japan because of their palatability [34].

Estimation of Rice Amylose Contents by NIR [35]: Japanese rice breeders have tried to develop palatable rice cultivars by selecting low amylose rice; however, the amylose contents among Japanese rice grains are not significantly different (15% – 20%). NIR spectroscopy can easily compare protein and moisture; however, it does not easily differentiate amylose from amylopectin. Villareal and Delwiche had already developed excel-lent calibrations to detect amylose in various rice samples through NIR spectroscopy [36,37]. Japanese consumers request high-performance calibration for the measurement of narrow range amylose of Japanese rice cultivars. Ap-parent amylose content (AAC) analysis is a rapid and simple technique uses NIT spectroscopy. It was developed based on the near infrared transmission spectra and the reference value of AAC determined by the iodine colorimetric method. The wide AAC range (0%-35.3%) PLS model was inadequate for the accurate prediction of the extremely narrow range of the AAC (13.2% 20.7%) of Japanese milled rice samples. The statistics performed on the 11 factor PLS model for the extremely narrow range of AAC analyses was 0.78, 0.74, and 2.01 in SECv, r2, and RPD, respectively. The performance of this model was 1.25 and 0.49 for SEP and r2, respectively, on the validation set. The previous models developed a wide range of AAC samples, which were difficult to apply to the narrow range of AAC rice. The present AAC analysis technique is based on NIT spectroscopy, which can discriminate between the extremely narrow differences in AAC of Japanese milled rice in the same sub family.

Eating Quality Evaluation System by NIR: Satake Co. Ltd. Developed a taste analyser in the 1980s that combined the palatability estimation formula that was based on the physicochemical measurements and NIR. The taste analyzer principle is based on the multiple regression analysis that uses NIR data (protein, moisture, amylose and fat acidity) against the sensory test results. A previous study by the Japanese As-sociation of Milling Companies evaluated 3 different types of palatability evaluation system using NIR. The results showed that the taste scores significantly correlated significantly with the results of the sensory test (r = 0.54 to 0.63), but the scores were affected by the moisture contents and milling yield [38]. There were 8 Japanese 2 companies that developed the characteristic systems to evaluate the palatability of rice 3 grains based on the spectroscopic technique. More than 1000 agricultural cooperatives, 4 wholesalers, and retailers introduced these NIR systems to evaluate the quality of their rice 5 grains (Figure 4). In 1996, the Japanese Food Agency conducted a survey regarding the palatability evaluation system, and the results showed that 67% users were satisfied with the performance of the system. The protein and moisture data were extremely reliable, but 8 some users requested an improvement in the taste score and amylase accuracy.

Figure 4: Taste Analyzer using NIR technology (Satake Co.).

Proposal as Novel Physicochemical Measurement, Iodine Colorimetric Analysis, for Rice Palatability Evaluation [39]

Introduction: As described in section 3, physicochemical test is very important because it is time- saving and labor-saving objective test for rice palatability. Main component of rice grains is starch, therefore, amylose content affects eating quality [6]. Low-amylose rice generally be-comes soft and sticky after cooking, whereas high-amylose rice be-comes hard with fluffy separated grains [40]. The most widely used method for amylose determination is a colorimetric assay where io-dine binds with amylose to produce a blue-purple color, which is measured spectrophotometrically at a single wavelength (620nm) [6]. Nakamura et al. characterized the starch of various rice cultivars; evaluated the relationship between their iodine absorption curve and apparent amylose contents, amylopectin fractions, and resistant starch. They improved the iodine colorimetric method, and developed the novel estimation formulae against the amylose contents, resistant starch or certain fractions of definite chain-length amylopectin. This novel method would lead to an easy and low-cost spectroscopic method for analyses of starch characteristics.

Figure 5: Estimation of apparent amylose contents based on iodine colorimetric analyses.

Iodine Scanning Colorimetric Measurement: Nakamura et al. used Japonica rice, Iindica rice, Indi-ca-Japonica hybrid rice, ae mutant rice, and waxy rice produced in Japan and China. They found that the iodine absorption curve differed between the Indica rice, Japonica rice, Japonica-Indica hybrid rice, ae mutant rice cultivars, and glutinous rice cultivars. And they proposed a novel index, “new max” based on iodine absorption curve, which shows higher correlation with amylose, resistant starch or CD of amylopectin than ordinary max. Furthermore, they developed the novel estimation formulae for AAC, RS contents, amylopectin chain lengths (Fa; DP<12, Fb3; DP>37), and fraction of DP10 (short chain glucans of amylopectin) and fraction of DP22 (medium chain glucans of amylopectin) based on the iodine absorption curve. These formulae would lead to an easy and low-cost spectroscopic method for the starch characteristics [39] (Figure 5).

Examples of Physicochemical Measurements of Rice Palatability

Comparison of Physicochemical Properties between Japanese and Chinese Rice Cultivars: As described in section 3, in the case of physicochemical evaluation, we can get the common results for the rice samples produced in the different countries if we use the same method. As an example, we report the results of physicochemical measurements of rice pal- atability using rice samples produced in Japan and China. China is the largest riceproducing country, accounting for 32% of the global production from 20% of the global rice-growing area. China produces Indica subspecies mainly in the southern region and Japonica subspecies mainly in the northern region (Heilongjiang, Liaoning), and eastern or southern region (Jilin, Jiangsu, Zhejiang, and Yunnan), whereas the other three countries, India, Indonesia, and Bangradesh, primarily grow Indica rice. In China, consumers are now choosing Japonica rice based on its shape and color as well as its texture and taste. Zhang et al. performed a sensory test of Chinese Japonica rice cultivars [41], in which a Chinese panel mainly determined the overall eating quality based on the stickiness and hardness. In the present section, we conducted physicochemical evaluations of some Chinese and Japanese Japonica rice cultivars using traditional and novel indicators based on the iodine absorption curve and RVA.

Recently, Nakamura et al. evaluated 16 Japanese and Chinese rice cultivars in terms of their main chemical components, iodine absorption curve, apparent amylose content (AAC), pasting property, resistant starch content, texture of cooked rice grains, and SDS PAGE of proteins [42]. Based on these quality evaluations, we can conclude that Chinese rice cultivars are characterized by their high protein content. The hardness of the surface layer (H1) and overall layer (H2) were higher in the Chinese rice cultivars than the Japanese rice cultivars, whereas the stickiness of the surface layer, and the balance degree of the surface layer were lower in the Chinese rice cultivars than the Japanese rice cultivars, although the stickiness of the overall layer |H2 was higher in the Chinese rice cultivars than the Japanese rice cultivars. In addition, rate of staling of cooked rice was faster for Chinese rice than Japanese one. In a previous study, they developed a novel formula for estimating AAC based on the iodine absorption curve [39]. The validation test showed a determination coefficient of 0.996 for estimating AAC of these Chinese rice cultivars as unknown samples as shown in Figure 6. Therefore, it became possible to estimate AAC, using iodine scanning colorimetric method [39], or the RVA method [20], easily and rapidly not only for Japanese rice but also various japonica rice cultivars produced in all over the world.

Figure 6: Validation test of the formula for estimating AAC in Chinese rice cultivars.

Summary of Evaluation of Rice Palatability

There are various kinds of rice cultivars of which qualities are diversified, such as hard Indica rice and soft Japonica rice in the world. Consumers in the Southern Asia prefer to hard rice grains and people in the North-eastern Asia like soft and sticky rice grains. Novel method to evaluate the quality of the cooked rice is necessary to breed high-quality rice cultivars and to select the suitable rice for each consumer and each purpose. Many researchers are trying to develop the novel method to evaluate the rice quality using various kinds of apparatus, such as Tensipresser, RVA, NIR, and spectrophotometer, etc. Simple, rapid, and accurate method to evaluate the quality of rice grains is very valuable.

Cultivar Identification of Rice (Oryza Sativa L.) by PCR Method and its Application to Processed Rice Products

Abstract

As the cultivars of rice affect markedly eating quality, processing suit-ability and price, identification or differentiation of rice cultivar is very important. The present authors developed suitable STS (sequence-tagged-site) primers for PCR (Polymerase Chainm Reaction) and it became possible to identify rice cultivar using template DNA extracted and purified from rice grains. Multiplex primer set was shown to be useful to differentiate effectively rice cultivars produced in various countries by PCR. Two kinds of multiplex kit for identification of “Koshihikari”, dominant cultivar in Japan, have been developed. Application of cultivar identification method by PCR method to processed rice products was investigated. The present authors developed “enzyme treatment method”, in which the gelatinized starch is decomposed by the heat stable alpha-amylase at 80°C, followed by hydrolysis of proteins by proteinase K with SDS and purification of extracted DNAs by PCI (Phenol/Chloroform/iso-amyl alcohol). It became possible to identify the material rice cultivars of the processed rice products, such as cooked rice and rice cake, by PCR method using template DNA prepared by the “enzyme treatment method” [43].

Introduction

Rice grains of the famous cultivars are traded or distributed at higher price as “Premium rice” than the ordinary rice because of their high palatability, processing suitability or special aroma, etc. As those premium rice grains sell at high price, some dishonest rice wholesalers or retailers blend low quality cheap rice to high quality premium rice and mislabel to be “high-quality premium rice”. Recently, rice consumers are tend to demand more information, such as cultivar name, location of production, year of production, etc., about rice which they purchase. For example, it is obliged to display the name of rice cultivar on its package under the JAS LAW (Law Concerning Standardization and Proper Labeling of Agricultural and Forestry Products in Japan). Therefore, technology to identify rice cultivar is very important for breeders, farmers, inspectors, wholesalers, retailers, food industries and consumers. Rice cultivar has been identified based on morphological characteristics of rice plant or grains, ripening ratio after crossing, or the isozyme patterns, etc. Cultivated rice (Oryza sativa L.) is one of the most polymorphic crop species in the world.

On the contrary, many high-quality rice or premium rice is closely related because inbred lines are often used for their development. Therefore, it is necessary to develop a time-saving technology to differentiate rice cultivars clearly and precisely. DNA fingerprinting was developed in 1985 [44] and is used for criminal investigation and trial at court. Recently, novel cultivar identification method based on DNA polymorphism has been developed accompanied with the progress in molecular biology. DNA-based markers have the obvious advantage of sampling the genome directly and RFLP analysis has been widely used for assessing variation of plants [45,46].

RFLP analysis has been used to distinguish between species of Oryza [47] and particularly between indica and japonica type of Oryza sativa [48]. Recently, PCR-based marker system has been developed by Williams et al [49]. In this RAPD (Random Amplified Polymorphic DNA) method, short oligo-nucleotides of arbitrary sequence are used to support the amplification of regions of the test plant genome and amplified DNAs are separated by gel electrophoresis. There are some reports on RAPD analysis of rice germplasm including indica and japonica types, to identify suitable parents for linkage map construction, and for gene tagging for drought resistance [50,51]. RAPD analysis was revealed to be reproducible and amenable for identification of each single plant line of F1 hybrid rice in China [52] and Australian rice cultivars [53]. Diversified rice cultivars were classified into separate groups by PCR using RAPD markers, but many primers were needed to resolve closely related japonica cultivars [54-56]. In case of RAPD markers, many other DNA bands than the target DNA for cultivar differentiation appear in the electrophoregram after PCR. Therefore, it is recommended to develop the STS (sequence tagged site) markers or SCAR (sequence characterized amplified region) markers based on RAPD markers. In addition to RAPD markers, microsatellites or simple sequence repeats (SSRs), which are DNA sequences with repeat lengths of a few base pairs and variation in the number of nucleotide repeats, can be detected with PCR by selecting the conserved DNA sequences flanking the SSR as primers. In 1993, SSR markers were developed for rice [57,58].

SSR markers are useful not only characterize the relationship between heterosis and marker genotype heterozygosity but also to identify chromosome segments that may have significant effects on yield and its component traits in rice [59]. Genetic diversity among Indian elite rice varieties was evaluated using three different types of DNA markers and parentage analysis [60]. SSLP was reported to be more reliable than AFLP for identify rice cultivars [61]. RAPD, RFLP, nuclear SSLP and chloroplast SSLP analyses were carried out to clarify the phylogenetic relationships among A-genome species of rice [62]. SSR was reported to provide useful genetic information on weedy rice [63]. Development of genome-wide DNA polymorphism database for map-based cloning of rice genes was investigated using SNP markers [64]. And SNPs were used for the discussion on the sequence variations between the rice cultivars [65]. Although SSR and SNPs are very useful DNA markers, it is impossible to use several PCR primers simultaneously, which is “multiplex PCR”, to simplify the PCR and electrophoresis and save time.

Furthermore, it was not reported to identify or differentiate the material rice cultivars by PCR method using the processed rice products, such as boiled rice or the rice cake. The present authors have investigated about the cultivar identification of rice by PCR method [66-70]. In the present study, application of PCR technology to cultivar identification of rice grains was investigated. SCAR markers and multiplex primer sets were developed based on RAPD marker analyses to differentiate closely related rice cultivars clearly and efficiently by PCR. The efficient method to prepare the template DNA for PCR from the processed rice products, such as cooked rice or rice cake, was investigated. The identification or the differentiation of material rice cultivars by the multiplex PCR was carried out using the processed rice products as samples.

Materials and Methods

Materials: Fifteen various kinds of rices were collected or purchased. Sample rice grains used are as follows; Koganemochi (Japan), Hinohikari (Japan), Akitakomachi (Japan), Kirara397 (Japan), Ilpum (Korea), Calmochi101 (USA), Medium grain (USA), Fobidden rice (USA), Pelde (Australia), Kyeema (Australia), Paellea (USA), Long grain (USA), Doongara (Australia), Nanjing 11 (China), IR2061 (Philippines). Thirty three original Koshihikari rice seeds, produced in 1999, 2000 and 2001, were used for DNA extraction and purification. All the rice samples including other 49 different cultivar rice samples than Koshihikari were kindly provided by National Food Agency, Japan. Forty nine cultivars are Hitomebore, Hinohikari, Akitakomachi, Kirara397, Kinuhikari, Hosh-inoyume, Haenuki, Mutsuhomare, Niopponbare, Sasanishiki, Tsuga- ruroman, Hanaechizen, Yumetsukushi, Hatsushimo, Asanohikari, Tsukinohikari, Aichinokaori, Matsuribare, Akiho, Yukimaru, Mutsukaori, Mana-musume, Kakehashi, Kiyonishiki, Domannaka, Koshijiwase, Yukinosei, Hohohonoho, Yumeakari, Notohikari, Akitsuho, Akebono, Asahi, Yama-houshi, Yamahikari, Koganenishiki, Koganemasari, Reiho, Mineasahi, Fusaotome, Karinomai, Dontokoi, Akinishiki, Naganohomare, Fukuhikari, Goropikari, Hatsuboshi, Nakateshinsenbon and Morinokumasan. Paddy rice samples were hulled by an experimental huller (Ketto Science Laboratory, Tokyo) and milled to the yield of 90% by an experimental rice polisher (Pearlest, Ketto Science Laboratory, Tokyo). Milled rice flours were prepared by a coffee mill (Millser IFM- 100, Iwatani, Tokyo).

Physical and Chemical Properties of the 20 Rice Cultivars: Amylose contents were measured by the iodine-colorimetric method using the milled rice flours by Juliano [6]. Protein contents were calculated by the multiplication of nitrogen- protein conversion coefficient (5.95 for rice) to the nitrogen contents measured by the Kjeldahl method [71]. Physical properties of the boiled rice grains were measured by the method of Okadome [28] using a Tensipresser.

Preparation of Boiled Rice and Rice Cake: White rice prepared by the experimental rice polisher (Ketto Science Laboratory) was boiled by an electric rice cooker (RC183, Toshiba, Tokyo) after soaking for 1 hr and each single boiled rice grain was subjected to the extraction of template DNA for PCR. Milled waxy rice was pulverized using experimental impact mill (Udy cyclone Mill, Udy Corporation, Fort Collins, USA). Thirty five gram of water was added to 50g of the rice powder and kneaded manually. Rice cake was prepared using electric rice cake maker for home-use (SMK-1800, Tiger Co. Ltd, Tokyo). Rice cake was lyophilized and pulverized using a coffee-mill (IFM-100) and subjected to DNA extraction.

Extraction and Purification of Template DNA from Milled Rice Flours: According to the CTAB method [64,65], DNAs of the milled rice flours were extracted. Milled rice flours (0.4g) were placed in the micro-centrifugal tube (2 ml) and DNAs were extracted into the 0.6ml of 2 x CTAB (Cetyl tri-methylammonium bromide, 2% CTAB, 20 mM EDTA (ethylenediamine-N, N, N’, N’- tetraacetic acid), 1.4 M NaCl, 0.1 M tris-hydroxyl aminomethane/ HCl buffer, pH 8.0) solution and 0.2 ml distilled water for 30 min at 65 °C. The solution of chloroform and isoamyl alcohol (24:1, v/v) (0.8 ml) was added and stirred gently for 15 min using a rotator. Thereafter, the solution was centrifuged (8,000 g, 15 min) by a refrigerated centrifuge (hi-mac CR21F, Hitachi, Hitachi) and the upper layer was transferred to another micro-tube. CTAB solution (10%, 0.08 ml) and chloroform/isoamyl alcohol (24:1, v/v) were added to the solution and was stirred gently for 15 min followed by centrifugation (8,000 g, 15 min). The upper layer was transferred to another tube and was stood for 5 min in the freezer (-80°C) after the addition of 2.5 times volumes of the precipitation buffer (50mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% CTAB). The precipitate was collected by the centrifugation (6,000 g, 15 min) and dissolved in the 0.5 ml of Tris-EDTA buffer (TE) and was added with the same volume of isopropyl alcohol. After the gentle stirring by a rotator for 15 min, the precipitate was collected by the centrifugation (6,000 g, 15 min). The precipitate was dissolved in 0.2 ml of TE followed by the decomposition of RNAs by the addition of 1 μl of RNase (RNase A, bovine pancreas, 10 mg/ml, Nippon-gene Inc. Tokyo) and incubation for 1 hr at 55°C. Thereafter, neutral phenol solution was added and upper layer was transferred to another tube after the centrifugation (8,000 g, 15 min). The same volume of the solution of phenol/chloroform (1:1, v/v) was added to the solution followed by the cen-trifugation (8,000 g, 15 min) and the upper layer was transferred to another tube. The solution was added with 0.2 M NaCl and two times volume of cold ethanol to generate the precipitate of DNAs. The DNAs were washed by the 50 μl of 70% ethyl alcohol and dissolved in 30 μl of 0.1 TE and were subjected to PCR.

Preparation of DNA in Case of Processed Rice Products: In case of processed rice products, such as boiled rice grains or rice cake, different DNA extraction/purification method was developed [57,58]. Processed rice product, such as a cooked rice grain, was subjected to the de-composition of starch by heat-stable alphaamylase (790 unit/mg solid, 30 mg/ml, from Batillus richenoformis, Sigma, USA) for 60 min at 80°C. For the starch digestion, 20 μl of above mentioned alpha-amylase (1mg/1ml) was added to the 1.5 ml of the sample solution (0.1 g of single boiled rice grain in the solution of 50 mM Tris-HCL buffer, pH 8.0) and digestion was performed for 1 hr at 80°C. Thereafter, the protein hydrolysis was carried out by pro-teinase K (Takara-bio, Otsu, Japan) 55°C or 2 hrs at 37°C. For the protein digestion, 10 μl of proteinase K solution (27.7 u/mg, 20 mg/ml) was added to the starch digested solution with 1% of sodium- dodecylsulphate (SDS). The protein digestion was carried out for 1 hr at 55°C. Thereafter, DNAs were extracted by the same amount of Tris-EDTA saturated phenol solution and purified by the phenol/chloroform/iso-amyl alcohol (PCI, 25/24/1, v/v/v) and 70% ethyl alcohol. For the control, commercial DNA extraction kit (Isoplant, Nippongene, Tokyo) was used.

Polymerase Chain Reaction (PCR): DNAs were proliferated by the PCR method using 600 commercial random primers (10 mers or 12 mers) as primers [56]. Taq-DNA polymerase (Takarabio Inc., Otsu, Japan) was used for amplification of DNAs. Each DNA was denatured for 1 min at 94°C, annealed for 1 min at 62°C and elongated for 2 min at 72°C. This procedure was repeated for 40 times. As a PCR apparatus, Thermal Cycler MP (Takara-bio, Otsu, Japan) was adopted.

Electrophoreses of the Amplified DNAs: Proliferated DNAs were subjected to the electrophoresis for 30 min through the agarose gel (2%) using a Mupid-2 electrophoresis system (Advance, Tokyo, Japan) at the charge of direct current of 100 V. After the electrophoresis, the DNAs were stained by the ethydium bromide and detected by the irradiation of UV light.

Development of SCAR Markers and Multiplex Primer Sets: In the present study, SCAR markers were developed based on RAPD analysis to differentiate rice cultivars by PCR (25-28). DNAs were extracted from the agarose gel after the electrophoresis of the PCR products using “Easy trapＴＭ” (Takara-bio, Otsu, Japan). DNA cloning was carried out using “TOPO XL PCR Cloning KitＴ Ｍ” (Invitorogen Corporation, Carlsbad, USA). DNA sequence was determined using a commercial DNA preparation kit (QIAprep Spin Miniprep kit, Qiagen, K.K, Tokyo) and DNA proriferation kit (Big Dye Terminator Cycle sequencing kit, V1-1, Applied Biosystems Japan) and automatic DNA sequencing system (DNA sequenser ABI PRISM Genetic Analyzer 310, Applied Biosystems Japan, Tokyo). SCAR markers were designed based on the DNA sequences with from 20 mers to 29 mers so that the transition temperatures are around 62°C. The combinations of these SCAR markers, “multiplex primer set” were developed to identify Koshihikari, most dominant rice cultivar in Japan.

Results and Discussion

Table 2: Sequences of SCAR markers.

Note: F: forward, R: reverse.

Development of SCAR Markers: Example for the development of SCAR markers for identification or differentiation of rice cultivars is shown in Table 2. The sequence of the differential DNA, which was extracted from the electrophoresis agarose gel after PCR of RAPD method, was determined and the suitable forward and reverse primers were designated as shown in Table 2 [59,60]. As shown in Figure 7, SCAR markers are more useful than RAPD markers [59,60,66,67] because it is clearer in electrophoregram. Furthermore, it is possible to use several SCAR markers simultaneously in PCR, which leads to saving of time and labor. As shown in Table 2, several kinds of SCAR markers were developed. The length of markers was adjusted so that the transition temperatures of the markers are around same temperature, 62°C. Therefore, several SCAR markers can be used simultaneously in “multiplex PCR.”

Figure 7: Comparison of RAPD and SCAR markers in PCR and electrophoresis.

Properties of Rice Samples: Various rice samples were collected from different district in the world and their chemical and physical properties were measured [60]. The results are shown in Table 3. As shown in Table 3, Indica subspecies, such as IR2061 and Nanjing 11, showed high amylose content and less adhesiveness, on the contrary, Japonica sub-species, such as Hinohikari and Akitakomachi, revealed low amylose content and more adhesiveness. In case of trade con-tract, inspection or grading or the survey of traceability of rice grains, PCR method must be applied using rice grains as samples. Shoot or leaves are not available as sample specimen. Therefore, milled rice flour, single kernel of polished rice or even boiled rice is used as materials for PCR in the inspection carried out in post-harvest inspection.

Rice is markedly diversified from the viewpoint of genetics, morphology and properties. On the contrary, high-quality rice is closely related by inbred breeding to attain high-palatability, high processing suitability or characteristic aroma, etc. Rice grains of the famous cultivars are traded or distributed at higher price as “Premium rice” because of their high palatability, processing suitability or special aroma, etc. As those premium rice grains sell at high price [1]. In the present study, various rice samples were collected from different district in the world and their chemical and physical properties were measured. As shown in Table 3, the grain properties between Indica subspecies, such as IR2061, and Japonica sub-species, such as Hinohikari, were revealed to be very different, but those among the same sub-species are not different.

Table 3: Proximate components and physical properties of various cultivar rice.

Note: Adhesion was measured by a Tensipresser and the unit is mm.
Consistency was measured by a Rapid Visco Analyser and the unit is RVU.

Application of PCR to Various Rice Samples: CTAB method can be applied to milled rice flour for extraction and purification of template DNA for PCR [72,73]. The result of PCR using 15 rice cultivars from various countries, such as USA, India, Thailand or Japan, as samples was successful. The primer set used for PCR was our commercial “Koshihikari Positive Kit” (Takara-bio). Four DNA bands appeared clearly in case of Hitomebore and three ones appeared clearly in case of Koshihikari, which were revealed to be useful to differentiate various rice cultivars by a single time PCR and electrophoresis. As shown in Figure 8, the multiplex primer set for PCR, “Koshihikari Positive Kit” is very useful to differentiate various rice cultivars from the different district in the world. It was shown that RAPD analysis is a useful tool in determining the genetic relationships among rice cultivars [51-56].

Figure 8: Example of the PCR using the primer set for the differentiation of various kinds of rice cultivars

Nevertheless, it is necessary to perform many PCRs and electrophoreses to distinguish rice cultivars by RAPD or SSR markers because those markers are difficult to use simultaneously in the same experiment. It became possible to save experiment time and cost using multiplex primer set based on SCAR markers. Figure 8 is an example that the multiplex SCAR marker set is useful to differentiate various rice cultivars by a single time PCR and electrophoresis. Three DNA bands appeared clearly in case of Koganemochi, premium Japonica waxy rice, and other three ones appeared clearly in case of Hinohikari, premium non-glutinous Japonica rice. In case of Nanjing 11, typical Indica non-glutinous rice, showed the different three bands, which are useful to differentiate with the other Indica rice, IR2061.

Primer Set for “Koshihikari” Identification: In Japan, leading variety Koshihikari shares more than one thirds of total cultivation area of rice because it is palatable and traded at higher price than other rice cultivars. It is obliged, in Japan, that the name of rice cultivar, location of cultivation, and year of rice production are labeled on the package of rice by the Japan Agricultural Standard (JAS) Act. Therefore, it was necessary to develop the technology to identify Koshihikari cultivar by DNA analysis. As shown in Figure 9A, Koshihikari can be discriminated from any other cultivars using this “primer set for identification” showing three specific DNA bands [69]. There is no other cultivar rice which shows the same three band pattern with Koshihikari among 50 dominant non-glutinous rice cultivars in Japan. All the Koshihikari from the different districts showed the same pattern of three DNA bands, which correspond to the same grouping under Japanese Seeds and Seedling Law based on representative characteristics of rice plant (Figure 9B). On the contrary, no cultivars other than Koshihikari showed the same DNA patterns with Koshihikari even Hitomebore or Hinohiari, which are descendant cultivars of Koshihikari as shown in Figure 9A [69].

Figure 9: Primer set for identification of Koshihikari. A: 50 different rice cultivars (No1 is Koshihikari)
B: Koshihikari of 34 different prefectures.

Primer Set for Differentiation of Other Rice with Koshihikari: As the premium rice Koshihikari sells at high price, mislabeling “Koshihikari 100%” occurs by the dishonest rice retailers in Japan. For the purpose of detection of rice grains of other cultivars which were dishonestly blended into Koshihikari, “Detection-kit” was developed. In case of detection-kit, no DNA is proliferated in case of all Koshihikari rice grains of various prefectures (Figure 10A), and more than one DNA bands never fail to in-crease by PCR in case of rice grains of other cultivars (Figure 10B). This “primer set for detection” can be used to detect the illegal blending of other rice to Koshihikari labeled as “Koshihikari 100% premium rice.” [69] SSR markers are useful not only characterize the relationship between heterosis and marker genotype heterozygosity but also to identify chromosome segments that may have significant effects on yield and its component traits in rice [57-62]. And SNPs were used for the discussion on the sequence variations between the rice cultivars [64,65]. Nevertheless, “multiplex primer sets” using SCAR markers were revealed to be more useful and practical for efficient and precise cultivar differentiation.

Figure 10: Primer set for detection of other cultivar rice grains to Koshihikari. A: 50 different rice cultivars
B: Koshihikari of 34 different prefectures

Novel DNA Extraction/Purification Method from Processed Rice Products and Differentiation of Material Rice Cultivars of Boiled Rice by PCR: In case of boiled white rice grain, gelatinized rice starch and heat de-natured proteins inhibit the extraction of DNA. Therefore, it is necessary to remove these starch and proteins without the damage or decomposition of DNAs themselves. Comparison of template DNAs prepared by the three methods, commercial kit method, CTAB method and “enzyme treatment method” was carried out [67]. “Enzyme treatment method” was shown to be profitable from the viewpoint of quality and quantity. High temperature of 80 degrees during the starch decomposition by the heat stable alpha-amylase and coexistence of SDS during the protein digestion are meaningful to inhibit the activity of endogenous DNase. Although commercial DNA extraction kit is very useful to prepare the template DNAs from rice leaves, it is not suitable for boiled rice. No amplified DNA bands appeared after PCR in case of template DNA prepared by the commercial kit [67].

In case of CTAB method, it is possible to prepare purified DNAs, but it was inferior to “enzyme treatment method” in terms of quantity of DNAs. Lysozyme is sometimes used for DNA preparation [58]. But lysozyme decomposes cell wall mainly, and it is difficult to digest the gelatinized starch and denatured proteins. It became possible to proliferate specific DNAs by PCR using template DNA prepared by the “enzyme treatment method” and the multiplex primer set is very useful to differentiate various rice cultivars by a single PCR and electrophoresis as shown in Figure 11 [67]. In case of boiled white rice grain, as shown in Figure 11, it became possible to differentiate Hitomebore or Hinohikari from Koshihikari, their parental cultivar by PCR using the template DNA prepared from each single grain of boiled rice by the “enzyme treatment method”, which was de-scribed in Materials and Methods [67].

Figure 11: Differentiation of Japanese rice cultivars by PCR using template DNA extracted from a single boiled rice grain by the “enzyme treatment method”

Differentiation of Material Rice Cultivars of Rice Cake by PCR: The results of PCR using template DNAs prepared from each rice cake by “enzyme treatment method” were same with those using template DNAs from Multiplex PCR for identification of material rice of rice cake. ”Ezymetreatment method” is also useful for preparation of template DNAs from rice cake. Solubility of rice cake in boiling water and expansion on heating varies depending on cultivar and producing area of the material glutinous rice [69]. Therefore, identification of cultivars of the material glutinous rice using rice cake as a sample is very important. The results of PCR using template DNAs prepared from each rice cake by “enzyme treatment method” were same with those using template DNAs from material raw rice flour directly by CTAB method as shown in Figure 12. It became possible to differentiate each material waxy rice cultivar by the single PCR and electrophoresis by the development of primer set for “multiplex PCR” to identify waxy rice cultivars [70]. material rice flour directly by CTAB method as shown in Figure 12 [68]. In conclusion, it became possible to identify or differentiate rice cultivars by PCR. Practical primer set for Koshihikari, dominant cultivar in Japan, was developed. It became possible to identify or differentiate the dominant cultivar by a single PCR without timeconsuming RAPD analysis or elaborate elec-trophoresis for SSR analysis. Furthermore, the results were same with ones based on the plant characteristics [1,69]. It became possible to use not only raw rice grains but also processed rice products, such as boiled rice or rice cake, as materials for cultivar identification by PCR method by the development of “enzyme treatment method.” The interference of DNA extraction by the ge-latinized starch and denatured proteins of the processed rice products is re-moved by the decomposition of starch and proteins. Heat-stable alpha-amylase was very useful because the high temperature of 80 degrees inhibits the DNase activities during the starch decomposition and SDS is useful to prohibit the DNase during the protein digestion [67]. Single boiled rice grain or rice cake can be used as a sample for identification or differentiation of material rice cultivars by PCR method [67,68].

Figure 12: Differentiation of Japanese rice cultivars by PCR using template DNA extracted from the raw rice flours or the rice
cake using “enzyme treatment method”
A: Raw Rice Flours
B: Rice Cake

Recent Progress of Rice Authentication Technology: Hayashi et al. assessed the utility of single-nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphisms (InDels) as DNA markers in genetic analysis and breeding of rice [74]. They surveyed SNPs and InDels in the chromosomal region containing the Piz and Piz-t rice blast resistance genes and developed PCRbased markers for typing the SNPs. Their findings suggest that, because of its ability to generate numerous markers within a target region and its simplicity in assaying genotypes, SNP genotyping with allele-specific PCR is a valuable tool for gene mapping, mapbased cloning, and marker-assisted selection in crops, especially rice. Satake Corporation developed and commercialized “Cultivar Identification System for Rice” in 2005 based on DNA tip method. It is an automatic system and it became possible to identify or differentiate rapidly and simply about 98% of Rice cultivars in Japan. Paddy, brown rice, polished rice, or the pre-washed rice samples are available for the system. It takes only 8 hours for 6 samples although it took about 3 days before (Figure 13). Starch characteristics determine the quality of various products of rice, e.g., eating, cooking and processing qualities. Bao et al. reported microsatel-lite polymorphism in the Waxy (Wx) gene, soluble starch synthase I gene (SS1) and starch branching enzyme 1 gene (SBE1), single nucleotide poly-morphism (SNP) in Wx and starch branching enzyme 3 gene (SBE3), and a sequence tagged site (STS) in starch branching enzyme 1 gene (SBE1) among 499 nonwaxy rice samples and their relationships with starch physicochemical properties [75].

Figure 13: Cultivar Identification System for Rice (Satake Inc.).

Analysis of the starch physicochemical properties of the samples withdifferent microsatellites, SNPs and STS groups indicated that these molecular markers can differentiate almost all the physicochemical properties examined, e.g., apparent amylose content (AAC), pasting viscosity characteristics, etc. These SSRs, SNPs and STS are useful in marker-assisted breeding for the improvement of starch quality of rice. To identify the chromosomal regions controlling the eating quality of cooked rice, Takeuchi et al. performed a quantitative trait locus (QTL) analysis using 93 backcross inbred lines (BILs) and 39 chromosome segment substitution lines (CSSLs) derived from crosses between a japonica rice cultivar Koshihikari (glossier appearance, tasty, sticky and soft eating quality of rice when cooked) and an indica cultivar Kasalath (less glossy appearance, less sticky and hard eating quality of rice when cooked) [76]. They evaluated the eating quality of rice including overall evaluation (OE), glossiness (GL), taste (TA), stickiness (ST) and hardness (HA) in each line based on the sensory test of cooked rice. They also mapped the QTLs for chemical properties, such as amylose content (AC) and protein content (PC), which affected the eating quality.

Consequently, QTL was mapped in the interval between the SSR markers RM1332 and RM6676 in the middle region of the short arm of chromosome 3 by fine mapping of three sub-CSSLs. Five QTLs, qOE6, qGL6, qTA6, qST6 and qHA6, seemed to be associated with the Waxy (Wx) gene located on chromosome 6. Evaluation of eating quality in early breeding generations of rice is critical to developing varieties with better palatability. Lestari et al. reported about DNA markers associated with eating quality of temperate japonica rice and an evaluation method aided by multiple regression analysis [77]. A total of 30 markers comprising STSs, SNPs, and SSRs were tested for their association with palatability using 22 temperate japonica varieties with different palatability values. Eating quality-related traits of the 22 varieties were also measured. Of the 30 markers, 18 were found to be significantly associated with palatability and, consequently, a model regression equation with an R2 value of 0.99 was formulated to estimate the palatability by the marker data set. Validation of the model equation using selected breeding lines indicated that the marker set and the equation are highly applicable to evaluation of the palatability of cooked rice in temperate japonica varieties.

X. Zhou et al. reviewed about the utilization of next generation sequencing (NGS) on direct identification of candidate genes [78]. The genome-wide association analysis (GWAS) has become a commonly used approach toward identifying the genetic loci and candidate genes for several traits that are closely associated with grain yield. The Multi- parent Advanced Generation Inter-Cross population (MAGIC) is introduced to discuss potential applications for mapping QTLs for rice varietal development. Their strategies broaden the capacity of functional gene identification and its application as a complementary method to insert mutants that comprise T-DNA and transposons. Moreover, accurate genome sequence information enables genome editing for the utilization of key recessive genes that control important agronomic traits. Their review summarized how NGS accelerates rice genetic improvements through the identification and utilization of key functional genes that regulate agronomic traits.

Bio-Functionality of Super-Hard Rice with Long- Chain Amylopectin- Multiple Prevention Against Diabetes and Dementia

Abstract

Diabetes and dementia are life-style related diseases, and patients have been increased all over the world. We tried to prevent diabetes using the high-amylose rice cultivars. It was shown that the postprandial BGL of high-amylose rice was lower than that of low-amylose rice. “Super hard rice”, of which starch contains longchain amylopectin, was developed in Kyushu University, Japan, and we clarified its characteristics by the cooperative re-search. It was very hard after cooking, and rich in resistant starch. “Super hard rice” lowered postprandial BGL not only as cooked rice grains but also as rice noodle and rice bread. We prepared super-hard rice bread blended with 5% of black rice bran (SRBBB), which contained much amylose and showed inhibitory activity against β- secretase after heating. Aged mice, which were fed SRBBB diet for 4 weeks, showed lower amyloid β40 in the blood than the control. We investigated the effects of the blend of ordinary brown rice and black rice cooked after a high-pressure treatment (HPTKO). A randomized, single-blind, crossover-designed study was conducted using 15 subjects, and BGLs at 90 and 120 min after ingesting the cooked HPTKO were significantly lower than that for cooked ordinary polished rice. Furthermore, postprandial blood amyloid β40 did not increased markedly compared with the control.

Introduction

According to WHO report, globally, an estimated 422 million adults were living with diabetes in 2014, and diabetes of all types can lead to complications in many parts of the body and increase the overall risk of dying prematurely [79]. Therefore, there is an urgent need to implement population-based interventions that prevent diabetes, enhance its early detection, and use lifestyle and pharmacological interventions to prevent or delay its progression to complications [80]. The WHO and Food and Agriculture Organization of the United Nations recommend foods with low glycemic index (GI) to prevent diabetes [81]. The concept of GI was introduced by Jenkins, et al. [82]. as a ranking system for carbohydrates based on their immediate impact on blood glucose levels, and low GI and glycemic load diets have more recently been widely recommended for the prevention of chronic diseases including diabetes, obesity, cancer and heart disease [83], because the re- sulting glycemic index classification of foods provided a numeric physiologic classification of relevant carbohydrate foods in the prevention and treatment of diseases such as diabetes [84]. Alzheimer’s disease affects more than 25 million people worldwide and is the most common form of dementia [85]. β-amyloid precursor protein (APP) is processed to generate β-amyloid (Aβ) by β- and γ-secretase, in a highly regulated process. Many drugs have been approved for the treatment of Alzheimer’s disease, at different stages of the disease, although they all have limited efficacy. Recent epidemiological studies have suggested a link between Alzheimer’s disease and type 2 diabetes mellitus associated with insulin resistance [86-88].

Diabetes is a lifestyle disease, and its prevention and treatment are extremely important. Low glycemic index (GI) foods inhibit rapid increases in blood glucose and insulin secretion after meals. The β-amyloid precursor protein (APP) generates the amyloid β peptide (Aβ) via β- and γ- secretase in a highly regulated process. Because working memory is impaired by AD, the disease has spawned dynamic research investigating the influence of gammaaminobutyric acid (GABA) on working memory performance in AD sufferers [89]. It is well-recognized that the prevalence of dementia is higher in diabetic patients than in non- diabetic subjects [90], and Tokutake, et al. [91] showed that there is a molecular link between AD and insulin signaling. Cereal grains are indispensable for the people all over the world. Intake of calories, 1st functions, satisfaction of sensory preference, 2nd functions, and the maintenance and promotion of health, 3rd functions, could be possible by the intake of various grains. For example, brown rice contains vitamin Bs, vitamin E, dietary fibers, gamma- oryzanol, ferulic acid, phytic acid, and gamma-Aminobutyric acid (GABA), various minerals, and polyphenols. Colored grains, such as red rice, purple wheat, purple barley, and purple corn, contain much amount of anti-oxidative polyphenolic substances.

Several studies have reported the development of highly resistant starch rice [92,93] as well as high-amylose and high-dietary fiber rice [94] via physical or chemical mutation. The hydrolysis of starch is a key factor in controlling the GI of foods. Functional foods that have α-glucosidase inhibi-tory activities have proven effective in controlling blood sugar levels in people at risk of developing diabetes [95]. Furthermore, epidemiological studies suggest that the low incidences of certain chronic diseases in rice-consuming regions of the world may be attributable to the antioxidant compounds found in rice. The molecules with antioxidant activity contained in rice include phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, γ-oryzanol, and phytic acid. Rice bran also contains various functional substances, such as γ-oryzanol, ferulic acid, sterol, wax, ceramide, phytin, inositol, and protein [96].

Rice bran oil, the only domestic edible vegetable oil made from the rice bran produced in Japan, is known to have high oxidative stability and serum cholesterol- lowering activity [97,98]. You et al. showed that in mice fed a ferulic acid-enriched diet, exercise endurance capacity was enhanced and fatigue was reduced by elevating antioxidative potentials [99]. Matsuzaki et al. showed that bran rice and γ-oryzanol reduced hypothalamic endoplasmic reticulum stress and attenuated the preference for dietary fat in mice [100]. Matsuoka et al. showed that plant sterols/stanols decreased blood cholesterol levels through the inhibition of cholesterol absorption in the intestines [101]. Intake of fermented brown rice could minimize insulin secretion, thus attenuating any subsequent rise in the levels of blood sugar [102]. Abe et al. showed that rice components including inositol hexaphosphate significantly inhibited Aβ production in neuroblastoma cells without causing cytotoxicity, suggesting such foods may prevent Alzheimer’s disease [103]. Pigmented rice contains naturally occurring colored substances that be-long to the flavonoid group called anthocyanins. Positive health effects of these pigments present in the bran layer of rice have been reported [104].

Ling et al. showed that red and black rice decreases atherosclerotic plaque formation and increase antioxidant status in rabbits due to their enhanced serum high-density lipoprotein (HDL) cholesterol and apolipoprotein A1 concentrations [105]. Recently, food technology to prevent the decrease of those biofunctional components during the cooking and increase their functions by the germination, high- pressure treatment, or the coextrusion cooking have been reported. High-pressure treatment (HPT), a technological process that limits the negative effects of food preparation on hydro-soluble vitamins, is recognized as being very useful in preserving nutritional quality in foods [106,107]. Yamakura, et al. [108] showed that subjecting rice to HPT before cooking results in more free amino acids and stickier cooked rice. The consumption of a western diet, which is characterized by a high in-take of red meats and high-fat dairy products, may contribute to obesity and metabolic syndrome, as well as increase the risk of developing type 2 diabetes and cardiovascular disease. In contrast, the traditional Asian diet, which is rich in soy and fish but low in animal protein and fat, may help reduce the frequency of severe chronic diseases [109]. There is also a significant association between a Mediterranean diet and reduced risk of major chronic de- generative diseases, including Alzheimer’s disease. The optimal diet for the prevention of cardiovascular and other major chronic diseases has rapidly evolved [110].

Development of Specialty Rice Cultivars (High-Amylose Rice, Black Rice)

Super rice research project of Japan started in 1989. It was supported by Ministry of Agriculture, Forestry and Fisheries, Japan, to enhance rice consumption. Low-amylose, high-amylose, giant embryo, aromatic, pigmented and high-prolamin rice cultivars were bred and utilized (Figure 14). We prepared wheat/rice bread blending three kinds of specialty rice, high-amylose, sugary, and purple rice cultivars. The bread showed good taste and maintained softness even after 4 days [111].

Utilization of High-Amylose Rice (Prevention of Diabetes)

Diabetes patients are more than 8.2 million and 18.7 million including those to be diabetes in near future in Japan. It is indispensable to prevent diabetes for the reduction of the increase of the medical costs. It has been reported by the large-scale medical tests that the inhibition of drastic increase of the blood glucose after meals reduce the rate of diabetes initiation [81-84,93]. Cristiana reviewed about the rice compounds with impact on diabetic control [112]. They pointed out that rice macro nutrients, such as starch, proteins, lipids, and dietary fiber, and rice bran compounds, such as gam-ma-oryzanol, phytic acid, ferulic acid, and gamma-aminobutyric acid (GABA)and vitamin E, and elucidated the metabolic mechanisms and bioactive towards diabetes [112]. We aimed to evaluate the palatability of the high-amylose rice of which eating qualities are inferior to ordinary Japonica rice. Another objective of this research was to clarify the mechanism to pre-vent diabetes initiation by the high-amylose rice by the feeding test of rats and diet test by the human beings. Proximate components and gelatinization properties of the high-amylose rice were clarified and texture and eating qualities of the cooked rice from high-amylose rice were reported [113]. In the case of highamylose rice, drastic increase of blood glucose and insulin after meals were inhibited (Figure 15) [113]. By serving the retrograded rice samples (2hrs after cooking), increase of blood insulin was more inhibited for high-amylose rice than for low-amylose one in animal feeding test [113].

Figure 14: Specialty rice cultivars and rice bread in Niigata Prefectural Agricultural Research Institute

Figure 15: Change in digestion degree between high-amylose rice and low- amylose one

Super-Hard Rice, Amylopectin Long Chain Rice (Prevention of Diabetes)

Super-hard rice (ae mutant rice) was developed through chemical mutation method (MNU method) by H. Sato et al. of Kyushu University Japan (Figure 16) [114]. Its starch is characteristic because its apparent amylose content is markedly high because its amylopectin has much amount of middle and long glucan chains [114]. Although its cooked rice is unpalatable due to hard texture, it contains high amount of resistant starch [115-118]. Therefore, super-hard rice is promising as a material to prevent obesity and diabetes. Nakamura and Ohtsubo [119], and Maeda, et al. [120] tried to improve the texture of cooked rice from super-hard rice (CRSH), and they showed that the high-pressure treatments improve the texture of CRSH and maintained the effect to inhibit abrupt increase of postprandial blood glucose level.

Figure 16: Super-hard rice and its molecular structure of starch amylopectin.

Diabetes/Dementia Multi-Prevention Rice Product by the Combination of Specialty Rice and High- Pressure Treatment

Bio-Chemical Test for Prevention of Life-Style Diseases

Type-2 diabetes and Alzheimer’s disease are very serious disease and the former has been suggested to be one of the causes of the latter [86-88]. Low glycemic index foods inhibit rapid increases in blood glucose and insulin secretion after meals [93,94]. Antioxidative capacity of brown rice and rice oil may be useful for the prevention of dementia [96,98,103]. In this study, we investigated the palatability of boiled rice and inhibition of an abrupt increase in blood glucose level (BGL) and amyloid β peptide production after eating blend of ordinary brown rice, “Koshihikari” and anthocyaninrich black-rice, “Okunomurasaki” unpolished rice cooked after a high-pressure treatment (HPT KO). “Okunomurasaki” showed a high antioxidant capacity and high inhibitory activity against β-secretase even after HPT and cooking [121].

Animal Feeding Test

Multiple prevention of type-2 diabetes and Alzheimer’s disease by the rice products would be very important objective for us [86,88,93,94]. Therefore, we prepared super- hard rice bread blended with black rice bran (SRBBB), which showed strong inhibitory activities against β-secretase and acetyl-cholinesterase and contained high amount of resistant starch even after heating. Black rice bran showed greater β-secretase inhibitory activity (3.6- fold) than Koshihikari rice [121]. The bran contained more oleic acid and anthocyanin, meaning that it is potentially a bio-functional food with a high antioxidant capacity. Furthermore, aged mice, which were fed a SRBBB diet for 4 weeks, showed lower amyloid β 40 peptide in the blood than mice fed a commercial diet (p < 0.01) [121] (Figure 17B). Additionally, their initial blood glucose levels after 12 weeks of being fed SRBBB were significantly lower than those in the control group (Figure 17A). Taken together, our results indicate that SRBBB seems to be promising for inhibiting not only amyloid β production but also abrupt in-creases in post prandial BGL.

Figure 17: Change in blood glucose and amyloid β40 in mice.

Human Intervention Test (Single-Dose Test)

It seemed to be necessary to conduct a human intervention test of the promising rice product in terms of inhibitory activity against the increase of BGL and amyloid β peptide production to prove the probability to prevent type-2 diabetes and Alzheimer’s disease. Therefore, we prepared heat moisture treatment cooked brown rice (HMT), and high- pressure treatment cooked brown rice (HPT), which contained higher amounts of resistant starch, antioxidant capacity, and β-secretase inhibitory activity than cooked ordinary white rice (Table 4) [122]. A randomized, single-blind, crossoverdesigned study was conducted using 15 subjects with a normal BGL. It was reported that GI varies depending on the subjects (e.g., young men and women), so we divided the 15 subjects into two groups; a high BGL subclass (seven subjects) and a low BGL subclass (eight subjects). BGL at 90, and 120 min after eating cooked HPT KO unpolished rice for 15 subjects showed a significantly lower BGL compared with that after eating cooked Koshihikari polished rice (p < 0.05). BGL at 90 min and 120 min after eating cooked HPT KO unpolished rice for high BGL subjects (n = 7) showed a significantly lower BGL compared with that after eating cooked Koshihikari polished rice (p < 0.05). AUC was significantly lower compared with cooked Koshihikari white rice 120 min after eating (p < 0.05) [122]. Furthermore, the increase in the amyloid β40 peptide in the blood 120 min after eating HPT KO (unpolished rice, blend of Koshihikari, ordinary rice, and Okunomurasaki, black rice (ratio was 6:4)) tended to be lower than that of cooked Koshihikari polished rice in human intervention test (single-dose test) [123].

Table 4: Bio-functional properties of raw rice and cooked samples.

Note: HPT, high pressure treatment; KO unpolished rice, blend Koshihikari and Okunomurasaki(6:4) unpolished rice, Significant
difference(p<0.05) among the two cooked rice and the three raw rice is shown by a, b and c.

Long-Term Human Intervention Test (12-Week Test)

Furthermore, we conducted a long-term human intervention test using the HPSHRB (high-pressure treated super-hard rice (40%) blended with black rice (40%)) in terms of inhibitory activity against the abrupt increase of BGL (Figure 18) and amyloid β peptide production for the probability to prevent type-2 diabetes and Alzheimer’s disease. Tokutake et al. pointed out that amyloidβ secretion and hyperphosphorylation of tau are closely related [91], and we had ascertained it by the cell-culture test. By the long-term in- tervention test, we aimed to confirm the effects to inhibit both of abrupt in-crease of post- prandial blood glucose and amyloid β peptide production. And we could ascertain the safety and acceptability of our multi-preventive rice product by this long-term test. Test was performed under the permission of ethical committee for the human test. As a result, significant reduction of abrupt increase of blood glucose (Figure 18) and inhibition of decrease in amyloid β ratio (amyloid β42/amyloid β40, Figure 19), bio-marker of Alz- heimer’s disease [124], were shown after the 12-week human test [125]. And all 12 subjects accepted the rice sample and finished test with no adverse effects.

Figure 18: Change in IAUCg120 in subjects after eating cooked rice HPT KO (after 12 weeks).

Figure 19: Difference in blood amyloid β42/ amyloid β40 after 6 and 12 weeks in human test

Summary

Diabetes and dementia are life-style related diseases, and patients have been increased all over the world. Rice is a staple food for Japanese and they eat rice at least once or twice every day. Rice does not only supply calories and give tasty dishes but also maintains healthy life for consumers and prevents some diseases. Since 1989, specialty ricecultivars, such as hard rice, soft rice, colored rice, aromatic rice, giant-embryo rice, etc. have been developed in Japan. We tried to prevent diabetes using the highamylose rice cultivars. It was shown that the postprandial BGL of high-amylose rice was lower than that of ordinary rice. It was shown that not only high-amylose rice but also pre-germinated giantembryo rice have the effects to lower the postprandial BGL and to lower the bloodpressure compared with the ordinary rice. “Super hard rice”, of which starch contains long- chain amylopectin, was developed in Kyushu University and we showed its characteristics by the cooperative research. It was very hard after cooking, and rich in resistant starch. “Super hard rice” lowered postprandial BGL not only as cooked rice grains but also as rice noodle and rice bread. High pressure treatment was introduced to food processing by Hayashi in 1980’s. We showed that the super rice becomes softer and sticker by the high-pressure treatment preserving high amount of resistant starch.

We prepared super-hard rice bread blended with 5% of black rice bran (SRBBB), which contained a high amount of resistant starch that showed strong inhibitory activities against β-secretase after heating. Aged mice, which were fed SRBBB diet for 4 weeks, showed lower amyloid β 40 in the blood than the control. We investigated the effects of the blend of ordinary brown rice, super-hard rice, and black rice (2:4:4, w/w) cooked after a high-pressure treatment (HPT KO). A randomized,single-blind, crossover-designed study was conducted using 15 subjects with a normal BGL, and BGLs at 90 and 120 min after ingesting the cooked HPT KO were significantly lower than that for cooked ordinary polished rice (p < 0.05). Furthermore, we conducted a long-term test using the HPSHRB in terms of inhibitory activity against the abrupt increase of BGL and amyloid β peptide production for the probability to prevent type-2 diabetes and Alzheimer’s disease. As a result, significant reduction of abrupt increase of blood glucose and change in amyloid β ratio were shown after the 12-week human test. And all 12 subjects accepted HPSHRB and finished test with no adverse effects. Furthermore, the ratio ofamyloid β42 to amyloid β40, an indicator for the proceeding of Altzheimer disease, did not increase markedly compared with the control.

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