Association of Body Weight Misperception and Hand Grip Strength(HGS) in Korean Older Adults: KNHANES VII-1

ABSTRACT

This study aims to investigate the association of body weight misperception and handgrip strength (HGS) in elderly Koreans. A total of 1,199(male=572, female=627) elderly subjects (age ≥ 65 years), who participated in the Seventh Korea National Health and Nutrition Examination Survey (KNHANES VII-1, 2016) were analyzed. Participant’s hand grip strength, Body Mass Index (BMI), and perceived body weight were measured. By comparing participants’ perceived bodyweight category with their actual bodyweight category, participants were divided into three groups: correct estimate, underestimate, and overestimate. In results, the odds of sarcopenic HGS tended to increase with underweight perception but tended to decrease with normal weight perception(ORs: 2.54, 95% C.I: 1.527-4.230). Compared with the Obese group by BMI categorization, the underweight group, BMI under 18.5, increased ORs 4 times of sarcopenic HGS. Compared with correct estimated weight status, underestimation and overestimation of weight status were high odds of sarcopenic HGS (ORs: 3.21, 95% CI: 1.230-8.381 and ORs: 3.954, 95% C.I: 1.479-10.542). In conclusion, this study supports the hypothesis that weight misperception and the accuracy of the perceived weight are related to HGS.

Abbreviations: HGS: Handgrip Strength; BMI: Body Mass Index; ADLs: Activities of Daily Livings; ORs: Odds Ratio; CIs: Confidence Intervals

Introduction

Research on body weight perception in the adult population suggests that women have body shape ideals significantly smaller in size than their perceived current body shapes, whereas men are equally divided between those who want to be bigger than their perceived current shape. Research on the relationship between body weight perception in young adults suggests that regular participation in exercise can bring about positive changes in body image and self-concept. Handgrip strength (HGS) has been used to indicate muscle strength and as an important marker of frailty [1]. The reduction of dominant HGS in the older population undermines the Activities of Daily Livings (ADLs) such as washing, dressing, using the toilet, showering, defecating, and mobility. Further, HGS is largely proven as an explanator of overall strength, fractures, falls, cognitive impairment [2], and Metabolic syndrome [3].
To avoid weak muscle strength status and alleviate all risks above, we need to understand related factors and the relation between HGS and weight control in this population. Previous research, however, shows inconsistent results. Long-term overweight and obese status have been associated with lower HGS among the older population [4]. Others, however, reported that being underweight [5], higher fat mass, and weight loss are associated with weak muscle strength [6]. Meanwhile, the association between weight misperception perceived weight status, and handgrip strength is unclear. Recently, Lee [7] reported that fatter weight perception, correct weight estimation, and overestimated body weight was associated with a lower risk of decreased HGS in Korean adults. However, this association between HGS and bodyweight misperception in the senior population is unclear. Therefore, the present study was designed to examine the association between body weight misperception and handgrip strength in older adults.

Method

Study Population

This study is the second analysis of the data acquired in the KNHANES VII-1, 2016. The KNHANES has been examined since 1998 to assess the Korean population’s health and nutritional status. This survey utilized a multistage, complex, stratified, probability cluster survey of a representative sample of the noninstitutionalized population in South Korea. The annual sample of the survey is around 10,000-12,000 persons; 4600 households are selected and surveyed from a panel. The KNHANES VII-I consists of the nutrition survey, the health exam, and the health interview survey. Among a total of 8,150 persons age 19 and older who completed KNHANES VII-I, we used data 1,199 whose aged over 65 years and who had handgrip strength (HGS), BMI, body perception survey data (Figure 1).

Figure 1: Flow diagram for selection of study subjects.

Ethics Statement and Data Access

Access to the KNHANES data was acquired after getting approval from the Korea Center for Disease Control and Prevention. This study is a secondary analysis that used and analyzed the data from 2016 KNHANES data collected; therefore, approval from IRB was exempted and not required.

Data Collection

The data for this study comprises participants in the KNHANES 2016. Sociodemographic variables are age, gender, level of education, household income, marital status, regular exercise performance. Education level was classified as below elementary level, middle school graduate (<9 years of school), High school graduate (10-12 years of school), and college or above (>13 years of education). Monthly household income was divided by the number of family members and classified into quantifies for household income. For marital status, those with a spouse were defined as ‘with the spouse,’ singles after divorced as ‘divorced,’ singles of the widow, and widower as ‘widow/widower.’ Regular resistance exercise was defined as performing.

Assessment of Hand Grip Strength (HGS), BMI, Body Perception Parameter

Handgrip strength was measured three times in each hand with a grip strength dynamometer (TKK5401; Takei Scientific Instruments, Co., Ltd., Tokyo, Japan). Trained technicians instructed subjects to hold measures with the distal interphalangeal finger joints of the hand at 90 degrees to the handle and squeeze the handle as hard as possible. We analyzed data of max HGS for this study. Sarcopenic handgrip strength (HGS) was defined as the mean value of HGS <27kg for men and <16kg for women Cruz-Jentoft, et al. [8]. Bodyweight and height were measured, and BMI was calculated as weight divided by height squared (kg/m2). Based on the Korean Society for the Study of Obesity practice guidelines Korean Society for the Study of Obesity [9], we classified BMI into four groups according to BMI: underweight (BMI<18.4kg/m2), normal weight(18.5≤BMI<23.0kg/m2), overweight (23≤BMI<25kg/m2), moderate obesity (25≤BMI<30kg/m2) and severe obesity(30kg/ m2≥BMI). BMI classification was collapsed into a three-tier variable: in each BMI category, ‘underweight’ was labeled as ‘underweight,’ ‘normal’ and ‘overweight’ were re-labeled as ‘normal,’ and ‘moderate obesity and severe obesity were re-labeled as’ obesity.’
Body perception was asked to each participant as rate his or her body weight as very underweight, underweight, normal, overweight, and very overweight. The responses to body weight were re-categorized into a three-tier variable: In each perception of body weight, ‘very underweight’ and ‘underweight’ were considered as ‘underweight,’ ‘normal’ was considered as ‘normal,’ ‘overweight’ and ‘very overweight’ were considered as ‘overweight.’ By comparing participants’ perceived bodyweight category with their actual bodyweight category, participants were divided into three groups: correct estimate (the group with the agreement between BMI category and self-recognized body weight), underestimate (group recognized as lighter than BMI criteria), and overestimate (group recognized as heavier than BMI criteria).

Statistical Analysis

A Chi-square test or t-test was conducted to compare characteristics between participants with normal HGS and those with sarcopenic HGS. In all participants, multiple logistic regression analyses were conducted to estimate the odds ratio (ORs) and 95% confidence intervals (CIs) between the gap of body perception and grip strength, as well as between BMI categories and selfperceptions of the body using IBM SPSS version 22.0(IBM CO, Armonk, NY, USA). We also conducted a chi-square test to analyze differences in variables from baseline characteristics. The covariate included were age, education, household income, and resistance training.

Results

Baseline Characteristics

The general characteristics of subjects are shown in Table 1. The study population included 572 men (mean age 72.18 years) and 627 women (mean age 72.15 years) from KNHANES VI-1. There was no difference in mean age between men and women. While 58.6% of the men had more than middle school education, only 27.1% among women (F=141.19, p<.001). While 88.2% of men have a spouse, it was only 54.3% among women, and widowhood was more prevalent in women than men (42.0% vs. 7.6% among men, F=186.38, p<.001). As for household income, 41.3% of men and 52.6% of women had the lowest quartile (F=16.26, p<.001). When classified by BMI category, 33.0% and 42.1% of the men and women were classified as overweight/obese, while 3.0% and 2.2% were underweight, respectively (F=10.61, p<.01). Among 1199 subjects, 8.9% of subjects(n=107) had underestimated, that was, seniors’ perceived body weight was lighter than their actual body weight. 61.6% of seniors (n=739) had accurately estimated, that was, perceived their body weight was consistent to their actual body weight; 29.4% of subjects (n=353) had overestimated, that was, they perceived their body weight heavier than their actual body weight. More women (10.5% vs. 7.2%) underestimate their actual body weight compared with objective measures (F=11.01, p<.01).

Table 1: Baseline characteristics of participants (n=1199).

Note: BMI: Body Mass Index.

In our subjects, 12.8% had sarcopenic HGS. Compared with participants with normal HGS, those with sarcopenic HGS were more likely to be older, be women, perceive themselves as underweight, be categorized by underweight based on BMI, be overestimated their weight, perform less regular resistance exercise, have lower household income, and be less educated (Table 2). Table 3 shows the associations between perceived weight misperception and sarcopenic Handgrip strength (HGS) according to adjustment levels of confounding factors. In fully controlled models, the odds of sarcopenic HGS tended to increase with underweight perception but tended to decrease with normal weight perception (ORs: 2.54, 95% C.I: 1.527-4.230). Compared with the Obese group by BMI categorization, the underweight group, BMI under 18.5, increased ORs 4 times of sarcopenic HGS. Compared with correct estimated weight status, underestimation and overestimation of weight status were high odds of sarcopenic HGS (ORs: 3.21, 95% CI: 1.230-8.381 and ORs: 3.954, 95% C.I: 1.479-10.542).

Table 2: Summarizes the comparison of the actual weight status, sociodemographic, health, lifestyle factors between an individual with normal and sarcopenic handgrip strength (HGS).

Table 3: Summarizes the comparison of the actual weight status, sociodemographic, health, lifestyle factors between an individual with normal and sarcopenic handgrip strength (HGS).

Note: Values represent Odd Ratio(ORs) (95% confidence interval) for sarcopenic handgrip strength using logistic regression after controlling for age and gender (model 1), for age, gender, education, household income, marital status (Model 2), for age, gender, education, household income, marital status, regular resistance exercise (Model 3) p for trend using linear regression analysis after adjusting for same confounding factors in each model.

Discussion

This study examined the association between body weight misperception and handgrip strength in older adults, using extensive nationally representative population data. Results showed that underweight perception, objectively measured underweight status, incorrect estimation of weight were associated with a higher risk for sarcopenic HGS after controlling for sociodemographic factors and health behavior. These findings were consistent across both genders and weight statuses. According to the previous research targeting the older population, weight tends to be underestimated or overestimated[10]. Findings from a study of Japanese seniors [11] reported that the increase in the risk for underestimation was more obvious than that for overestimation in women, which is comparable with our results that more elders overestimated their body weight (29.4%) than underestimation (8.9%).
Given that women rarely overestimated their weight, the overestimation found in our study may indicate an increasing rate of attention toward healthy habits and lifestyles changes in the older population. Because Korean National Health Insurance Services provide medical exams every two years free of charge to make most of the elder’s attention to their body shape and condition. This study reported the association between weight misperception and low grip strength in older adults. Although mechanisms for the results are unclear, given that grip strength is critical for independent living in later life, this result should be attending. One potential factor could be obesity or sarcopenia. In a previous study, percent body fat among older adults was associated with a higher volume of muscle mass and muscle strength when examined knee extension strength [12]. Therefore, seniors who believe their weight to be lighter or heavier than their actual weight status affect their lifestyles such as exercise and diet habits. Seniors who underestimate their weight may be less motivated to make efforts to lose weight.
In contrast, elders who overestimate their weight may be less motivated to make efforts to gain weight. This study (to the best of our knowledge) reported the relationships of weigh-perception with HGS based on a Nationally representative sample of older Korean populations. However, this study has limitations such as uncontrolled factors and potential causality, a possible bias of measurement errors of HGS to generalize the results to other populations. In conclusion, this study supports the hypothesis that weight misperception and the accuracy of the perceived weight are related to HGS. The results indicate that the programs for seniors make them aware of their actual body weight and health education for seniors to motivate them for healthy lifestyles based on their body weight and disease status to keep their independent lives.

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Role of Diffusion Weighted Magnetic Resonance Imaging in Diabetic Foot and Ankle Disease

Diabetes mellitus (DM) is one of the most common chronic diseases in the United States (US) and worldwide. The National Diabetes Statistics Report 2020 by the Center of Disease prevention and Control (CDC) estimates approximately 34.2 million people in the US alone to be affected with DM (10.5% of total US population), of which 34.1 million will be more than 18 years of age (13% of all US adults) [1]. An overwhelming 88 million adults 18 years or older have prediabetes (34.5% of the adult US population), adding substantially to the overall burden of the disease [1]. The estimated total economic burden of diagnosed DM in 2017 was $327 billion and 1 in 7 healthcare dollars is spent on treating diabetes and its complications [2]. DM is the leading cause of atraumatic lower extremity amputations, which accounted for 130,000 hospital discharges in 2016 (5.6 per 1,000 adults with diabetes) [1].
Diabetic foot and ankle disease include several pathologies that result from a combination of peripheral neuropathy and peripheral arterial disease (PAD) [3]. The wide spectrum of diabetic foot and ankle disease ranges from superficial soft tissues pathologies like edema, cellulitis, Diabetic foot ulcer (DFU), and inter-fascial abscess to deeper soft tissue infections like necrotizing fasciitis, intermuscular abscess, dry and wet gangrene, infected tenosynovitis and infected bursitis, diabetic myonecrosis and bone infections, including osteomyelitis (DFO) and intraosseous abscess. Underlying Charcot’s neuropathy or neuroarthropathy is commonly associated with such lesions. In addition, charcot neuroarthropathy (CNA) and DFO often coexist, rendering clinical diagnosis challenging. PAD is common in patients with DM. Arterial hypoperfusion can lead to ulceration, delayed wound healing, limb ischemia and gangrene, and may ultimately necessitate amputation. Early and accurate diagnosis of diabetic foot complications can reduce patient morbidity, health care costs, and limb amputations while potentially improving wound healing and quality of life [4].
Imaging, in conjunction with clinical examination, plays a vital role in the diagnosis and follow-up of DFD. The diagnostic evaluation often includes a variety of imaging modalities including radiographs, ultrasound (US), CT, nuclear medicine scintigraphy, and MRI, each with their own advantages and limitations [4]. Plain radiographs are cost-effective for screening fractures, advanced neuroarthropathy, soft tissue swelling and gas, and cortical erosions of DFO. US assists in identifying and draining soft tissue fluid collections/abscesses. CT shows fluid collections, gas in necrotizing fasciitis and cortical erosion / sequestrum. MRI provides accurate information of both soft tissue and bone pathology and, in conjunction with a plain radiograph, is usually the imaging modality of choice for evaluating the extent of musculoskeletal infection. MRI also renders early diagnosis of CNA and helps differentiate CNA and DFO from other conditions, such as Gout [5]. Intravenous gadolinium is often needed to detect soft tissue abscess in the mound of edema and differentiate simple effusions from synovitis as well as to characterize sinus tracts [6].
Diffusion weighted imaging (DWI) is being increasingly used in musculoskeletal imaging in conjunction with traditional MRI sequences for tumor and infection imaging [7-9]. Hydrogen proton diffusion refers to the random Brownian motion of the water molecules, which in the human body live in a complex milieu divided between extracellular and intracellular compartments. Different tissues of the human body have different proportions of intra and extracellular compartments, and hence have characteristic diffusion properties [10]. DWI also exploits the differences in diffusion between the normal and abnormal tissues. Typically, more cellular, protein or pus containing structures exhibit restricted diffusion, and high intensity on DWI with correspondingly low intensity on apparent diffusion coefficient (ADC) images. Different ADC values are seen in various tissues and pathologies, assisting the the MRI diagnosis of different pathologies in the setting of DFD. DWI use is still limited to only a few centers despite added value in conventional MRI [7,8]. This review article highlights the optimal technical considerations for DWI and discusses its role as a problem-solving tool in differentiating the wide spectrum of DFD pathologies. The reader will be able to use these principles and apply DWI for DFD for the benefit of their patient population.

Diffusion Weighted Imaging

The detailed physics behind the acquisition of DWI is beyond the scope of this article. Briefly, a single-shot echo planar imaging (SS-EPI) technique is used to acquire such images [11]. Two diffusion gradients are applied on either side of the 180-degree refocusing pulse to interrogate information about tissue diffusion. Strength of diffusion gradients is characterized as ‘b’ (diffusion moment) parameter and higher b-values correspond to stronger moments. At least two images, one with no diffusion gradient (b=0 s/mm2) and a second with a diffusion gradient (e.g. b=50-1000 s/ mm2) are needed to mathematically compute an ADC value which quantifies the amount of diffusion at each pixel (9). Bright signal areas on high b-value DWI images with corresponding low ADC values indicate restricted diffusion while a bright area on DWI and high ADC indicates T2 shine through effect [12]. ADC is calculated on a pixel-by-pixel basis, and minimum, maximum, and mean values can be measured, usually expressed as square millimeters per second (mm2/sec) using a complex mathematical equation [11]. For musculoskeletal MRI, b values of 50, 400 and 800 s/ mm2 are commonly acquired. For best technical performance, echo times are kept at minimum, echo spacing should be below 0.7ms, fat suppression is best kept as adiabatic inversion recovery to reduce ghosting artifacts, axial plane of imaging provides the most distortion free imaging, and motion of extremity should be minimized with foot padding or patient comfort during scanning. Typical parameters of DWI are (TR= 6500-8000ms, TE= 56-65ms, slice= 4mm, matrix 128×128, fat suppression= adiabatic inversion recovery, SS-EPI sequence). ROI (region of interest) is placed on the ADC images to evaluate mean and minimum ADC within the lesion for a quantitative assessment. In authors practice, mean ADC is used in almost all circumstances. The range of mean ADC values (x10-3 mm2/s) for the spectrum of MSK infections as described by Kumar et al. are illustrated in Table 1 [8].

Table 1: Range of ADC values (x10-3 mm2/s) in the spectrum of musculoskeletal infections.

Role of DWI in Diabetic Foot and Ankle Diseases

Soft Tissue Pathologies

Almost all diabetic foot infections begin with a foot ulcer [13]. Subcutaneous noninfectious edema is commonly seen in the diabetic patient population either due to diabetic vascular insufficiency or lymphedema. It is a noninfectious inflammatory condition of the superficial soft tissues, clinically presenting as soft tissue swelling and pain. Cellulitis is a non-necrotizing superficial soft tissue infection usually caused by a breach in the skin surface/ulcer. Staphylococcus aureus and Streptococcus pyogenes are common pathogens responsible for cellulitis [6]. t also clinically presents with soft tissue selling, pain, redness, warmth, and erythema of the superficial soft tissues with accompanied systemic manifestations of fever and malaise. The diagnosis of these conditions is usually clinical, but imaging is often obtained to rule out deeper extent of the infection, which may necessitate intravenous or prolonged course of antibiotics. Conventional MRI shows overlapping features between cellulitis and noninfectious edema with both showing T2 bright superficial soft tissues and skin thickening. Cellulitis shows enhancement of the soft tissues and skin ulceration in addition [14]. On DWI, both entities show T2 shine through effect, less so in cellulitis (ADC= 1.2-2.0) versus noninfectious superficial edema (ADC = 2.0-3.0) (Figure 1). This differentiation helps clinically, as cellulitis, if not adequately managed, may result in further complications such as necrotizing fasciitis, abscess, and gangrene.

Inter-fascial and intermuscular abscess refers to loculated pocket of fluid collection within the fascial plane or in-between muscles. These are fluid like-T2 hyperintense collections with peripheral rim enhancement on post contrast images. In the setting of the DFD with cellulitis, small pockets of fluid collections may be challenging to identify on conventional MRI from the background diffuse mound of hyperintense soft tissue edema. DWI images are particularly helpful in accurately finding these small pockets of abscesses as they typically show restricted diffusion (ADC= 0.6- 1.2). Figure 2 Diffusion in an abscess is extremely slowed due to the presence of inflammatory cells, cellular debris, bacteria, and proteins. DWI helps to identify the deeper extent of a superficial abscess guiding appropriate drainage and management. A study by Harish et al. showed that DWI, in conjunction with other unenhanced MR imaging sequences of the area of interest, lead to similar confidence levels of readers as the post-contrast images in diagnosing abscess [15]. In another study by Unal et al. DWI showed a sensitivity and specificity for detecting soft tissue abscesses of 92% and 80% respectively [16]. DWI also helps differentiate abscess from adventitial bursitis adjacent to DFU or simply necrotic tissue without pus (both showing T2 shine through effect).

Figure 1: Cellulitis and myositis. Middle aged man with known type II DM presenting with skin ulcer, skin thickening (short arrow), plantar soft tissue swelling (medium arrow) and myositis (long arrow). Axial T1W (A), FS T2 (B), DWI (C), ADC (D), and color map (E). Myositis and cellulitis demonstrate increased signal on DWI (C) and associated enhancement on ADC and color map (D and E) with ADC values 1.6 for myositis and 1.7 x 10-3 mm2/s for cellulitis.

Figure 2: 54-year-old man with proximal phalanx osteomyelitis and abscess. Sagittal T1 (A), T2 FS (B), T1 FS post (C), STIR (D), T1 FS post contrast (E), DWI (F), ADC (G) and color map (H). Osteomyelitis (long arrow) is demonstrated by low T1 signal, edema and enhancement after contrast. Associated osseous enhancement on ADC and color map (green compared to blue in normal marrow). Subcutaneous fluid collection (short arrow) demonstrates restricted diffusion (blue) with ADC of 0.5 x 10-3 mm2/s indicative of abscess (E-H).

Infectious tenosynovitis is characterized by infection of the closed synovial sheath of the tendon [17]. Infected fluid/pus accumulates within the tendon sheath. DWI images help distinguish it from mechanical or reactive tenosynovitis as infectious tenosynovitis will show restricted diffusion. Adjacent joints should be evaluated to exclude associated septic arthritis. Septic bursitis is also within the same spectrum of closed synovial space infection, most commonly affecting superficial bursae likely secondary to direct inoculation. Again, DWI images help differentiate septic bursitis from reactive bursitis due to restricted diffusion with infection. Identification of these two entities is necessary as early treatment with appropriate antibiotics and surgery or drainage of the infected bursa, if necessary, can be performed in a timely fashion [17]. In authors’ experience, accumulation of pus in a soft tissue abscess, tendon sheath or a bursa, all will have ADC values ranging from 0.6- 1.1 (x10-3 mm2/s). In an abscess with a communicating ulcer or a draining fistula, the restriction on DWI is more often localized to the edges of the abscess as opposed to a closed infected cavity.
Necrotizing fasciitis (NF) is a medical emergency characterized by a rapidly progressing, potentially fatal soft tissue infectious process. It is usually polymicrobial in etiology. Soft tissue infection dissecting along the fascial planes without a penetrating injury or iatrogenic cause is pathognomonic [18]. Historically, presence of gas in the fascial planes as observed on plain radiographs or CT is described as a classic imaging finding. However, absence of air along the fascial planes does not rule out necrotizing fasciitis in the setting of high clinical suspicion [19]. It is commonly seen in patients with diabetes and early surgical debridement can be lifesaving in these cases. MRI is often not performed given the urgency of these cases and the time taken to perform a complete MRI. However, MRI has been shown to diagnose NF in earlier stages where subcutaneous emphysema has not yet developed [20]. In a study by Kim et al. patients with necrotizing fasciitis showed thick (≥3 mm) fascial hyperintensity on fat-suppressed T2-weighted images, or extensive involvement of the deep fascia with low signal intensity on fat-suppressed T2-weighted images, a focal or diffuse non-enhancing portion in the deep fascia, and involvement of three or more compartments in one extremity [21]. DWI can help identify small inter-fascial or intramuscular abscesses (pyomyositis) in such setting. Foci of soft tissue air are seen as signal void on all pulse sequences and exhibit blooming artifact on gradient echo sequences like DWI [22,23].
Gangrene refers to necrosis of the soft tissues, almost exclusively seen in DM or PAD patients adjacent to a DFU [24]. It is characterized as devitalized soft tissue with characteristic nonenhancement on post contrast imaging (Figure 3). Gangrene is of two types: dry and wet, with dry, usually without infection, and wet indicating superimposed infection. Wet gangrene can also have soft tissue emphysema. Air is again seen as blooming artifact on DWI. It has been proposed that gas associated with wet gangrene is far more extensive when compared to a penetrating ulcer [25]. Wet gangrene unless associated with an abscess exhibits T2 shine through effect. Also, as noted by Ledermann et al., areas of abscess can be masked within the region of necrotic tissue on routine non-contrast MRI [24]. DWI can easily find abscess within the devitalized soft tissues.

Figure 3: Devitalized tissue and muscle denervation. Sagittal STIR (A), T1 (B), T1 FS post contrast (C), DWI (D) and color map (E). Patchy edema in plantar subcutaneous fat (A and B, arrows) from tissue ischemia with mild enhancement (C, arrow) due to cellulitis. Associated increased signal on DWI (D, arrow) and enhancement (red) on the color map (E, arrow) due to surrounding cellulitis.

Muscle Pathologies

Pyomyositis exhibits T2 hyperintense muscle and peri-epimysial edema. Small intramuscular abscesses may be difficult to recognize without contrast MRI. DWI helps identify localized abscess within the area of myositis. Formation of intramuscular abscess is a hallmark of pyomyositis [23]. DWI also helps in differentiating pyomyositis from diabetic myonecrosis described below, the latter shows only T2 shine through effect [8].

Diabetic myonecrosis is a unique pathologic entity seen in diabetic patients with unknown exact etiology, possibly a result of microangiopathy. Clinical presentation includes acute onset pain, induration, swelling and elevated serum lactate and CPK levels. MRI shows areas of heterogeneous T2 hyper intensity without an intramuscular abscess, as opposed to pyomyositis [26] (Figure 5). DWI assists in recognizing abscess in pyomyositis. Diabetic myonecrosis shows T2 shine through effect with elevated ADC>1.5 (normal muscle ADC= 1.3-1.5). It is prudent to differentiate these two conditions as diabetic myonecrosis is treated conservatively with bed rest, glycemic control and nonsteroidal anti-inflammatory agents whereas pyomyositis may require surgical debridement [27,28].

Figure 4: Charcot arthropathy. Lateral foot radiograph (A), sagittal T1 (B), STIR (C), T1 FS post contrast (D), DWI/ADC/color map (E). Osseous destruction and debris in the hindfoot (A and B) with associated osseous edema (B) and enhancement after contrast (C). Fluid collection (short arrow) does not demonstrate restricted diffusion (ADC 2.5 and red on color map) consistent with sterile collection. Corresponding enhancement on ADC and color map imaging (E).

Figure 5: 46-year-old diabetic man with muscle denervation and ischemia, elevated LDH, CPK and serum lactate levels. Axial T1 (A), FS T2 (B), DWI (C), ADC (D) and color map (E). Muscle edema in the medial gastrocnemius (small arrow) readily apparent on the FS T2 sequence; however, DWI and ADC with color map demonstrate enhancement in the anterior compartment (long arrow) indicative of denervation that is not appreciated on the T1 and FS T2 imaging.

Osseous Abnormalities

Diabetic Foot Osteomyelitis (DFO) is one of the most common complications in diabetics with a foot ulcer. DFO is usually due to non-healing ulcer and is associated with higher risk of limb amputation, longer duration of hospital stay, prolonged need of intravenous antibiotics and delayed wound healing [29]. DFO can affect any bone but most frequently affects the forefoot and midfoot [30]. Transtibial amputation is more frequently performed with DFO involving the hindfoot when compared to the forefoot, which can be salvaged with below ankle amputation [31]. Early diagnosis of DFO, i.e. before the development of exposed bone, extensive ischemia or soft tissue necrosis, is critical for the success of conservative surgery and can obviate the need for local or highlevel amputation [32].
MRI is the imaging modality of choice to diagnose osteomyelitis. The easiest way to diagnose osteomyelitis is to identify the superficial ulcer and its sinus tract extending to the bone with confluent marrow signal abnormality [33]. Reactive marrow edema is identified as T2 hyperintensity without corresponding T1 hypo intensity while DFO shows confluent T1 hypo intensity as well. DWI signal is low in the normal bone marrow (ADC= 0.2- 0.4 x 10-3 mm2/s), which becomes hyperintense in the settings of reactive marrow edema and intraosseous or subperiosteal abscess. In authors experience, ADC of reactive marrow edema falls within 1.4-1.9 x 10-3 mm2/s while in osteomyelitis, there is relatively less ADC enhancement (0.6-1.3 x 10-3 mm2/s) (Figures 2,6,7). Eren et al. showed that DWI supplements conventional MRI without added intravenous contrast in diagnosing DFO (34). They found the ADC values were significantly lower in patients with osteomyelitis (0.75 x 10-3 mm2/s) compared to those without osteomyelitis (0.90 x 10-3 mm2/s). While ADC values may vary depending upon the scanner strength and signal of the image, the presence of intra-osseous abscess reduces the ADC values to below 0.8 x 10-3 mm2/s versus its absence leads to higher ADC values up to 1.3 x 10-3 mm2/s in authors’ experience. ADC can also be used to assess treatment response, i.e. partially treated osteomyelitis will have slightly higher ADC ranging from 1.4-1.9 x 10-3 mm2/s and these values increase with resolution of abscess and decrease later on with the return of fatty marrow.

Figure 6: 41-year-old diabetic man with osteomyelitis. Coronal T1 (A), FS T2 (B), DWI (C), ADC (D) and color map (E). Subcutaneous edema and fluid collection (short arrow) with subjacent osteomyelitis of the calcaneus (long arrow) demonstrated by cortical erosion, T1 hypointense signal and enhancement on post contrast imaging. Associated enhancement noted on ADC and color map with an ADC of 1.4 x 10-3 mm2/s (green).

Figure 7: 34-year-old woman with trauma and superimposed osteomyelitis. Coronal T1 (A), FS T2 (B), T1 FS post contrast (C), DWI (D), color map (E). T1 hypointense signal at the metatarsal bases (A, arrow) with cortical edema (B, arrow) and enhancement on post contrast imaging (C, arrow). Cortical hyperintense outline also noted on DWI (D, arrow) and color map with an ADC of 1.5 x 10-3 mm2/s (E, arrow, green), blue represents first and fifth metatarsals as control for normal marrow (E).

Diabetes mellitus is currently the most common cause of neuropathic osteoarthropathy (Figure 4), which most frequently affects the foot and ankle [35]. Diabetic CNA is a devastating and disabling complication of diabetic neuropathy. It is characterized by acute excessive inflammatory response leading to local osteoporosis and devastating fractures and deformity of the foot [36]. A severe deformity, in addition to the above described mechanisms, will result in a secondary ulceration, infection, and amputation [37]. It is secondary to a combination of peripheral neuropathy and PAD resulting in decreased proprioception causing repetitive trauma, ischemia, poor healing, joint instability, cartilage loss, deformity, and increased new bone formation [38]. CNA often begins in the midfoot, and subluxation usually starts at the second tarsometatarsal joint [39].
MRI is very sensitive in detection of early changes of CNA as offloading the extremity can halt or delay the progression of this devastating disease [40]. Early signs on MRI include bone marrow edema and soft tissue edema, joint effusion, and eventually subchondral microfractures [40,41]. Disruption of the Lisfranc ligament, plantar calcaneo-navicular ligament, and plantar fascia is seen in early stages, which can result in midfoot collapse and malalignments [42].
Distinguishing between DFO and CNA is challenging in patients with diabetes. Ahmadi et al concluded that the presence of a sinus tract, replacement of soft-tissue fat, fluid collection(s) and extensive marrow abnormality indicate superimposed infection [41]. Neuroarthropathy commonly affects the tarsometatarsal and metatarsophalangeal joints whereas osteomyelitis occurs distal to the tarsometatarsal joint and in the calcaneus. DFO develops, almost exclusively, by the contiguous spread of infection from skin ulceration at predictable sites, whereas CNA is primarily articular, bilateral, and presents as subchondral cysts [33]. CNA with superimposed infection further complicates the landscape. DWI and ADC can play a vital role in differentiating these entities. A prospective study by Razek et. al. found that the mean ADC of acute diabetic osteoarthropathy was 1.27 ×10-3 mm2/s and that for diabetic osteomyelitis was 0.86 ×10-3 mm2/s. They established a cut-off ADC value of approximately 1.0 ×10-3 mm2/s to differentiate acute diabetic osteoarthropathy and osteomyelitis with approximately 94% accuracy [43].

Peripheral Nerve Pathologies

The four prime mechanisms causing hyperglycemic nerve damage are oxidative stress from the polyol pathway, increased hexosamine pathway flux, elevated levels of intracellular advanced glycated end products (AGE) and activation of protein kinase [44-46]. Excessive oxidative stress and vasoconstriction lead to ischemia of the nerve cells, nerve cell injury and death [47]. Neuropathy in diabetic patients is manifested in motor, autonomic and sensory divisions of the nervous system [48]. Damage to motor nerves leads to atrophy and edema-like T2 signal of the foot muscles leading to foot deformities and eventually CNA. Autonomic disturbances lead to dry skin of the foot, predisposing to fissuring [49]. Sensory neuropathy in extremities lead to loss of sensitivity to pain, temperature and proprioception resulting in repeated foot injuries, ulcer formation and/or secondary infection.
Diabetic neuropathy is a well-recognized complication of longstanding diabetes. Up until recently, nerve conduction studies and biopsy were the only available options to diagnose peripheral neuropathy in diabetic patients, demonstrating a loss of myelin in peroneal and sural nerves [50]. MRI can recognize denervation changes in the muscles, seen as high signal on DWI and ADC maps (Figures 5&8). Dedicated peripheral nerve imaging, MR neurography (MRN) present another avenue to recognize this complication [51-54]. Anatomic MRN is typically performed using 3D heavily T2W imaging evaluation of peripheral nerves and regional muscles [55]. More recently, DWI and diffusion tensor imaging (DTI) neurography have been extensively studied and used in the assessment of peripheral nerves [56-58]. While the pathologic nerves exhibit increased T2 signal alteration with or without enlargement in the setting of neuropathy, DTI in addition exhibits elevated ADC and low fractional anisotropy (FA) values. DWI and DTI also depict abnormal muscles with increased conspicuity as compared to conventional MR images. The recommended maximum b value for DWI neurography range from 600 to 800 sec/ mm2 to enable adequate visualization of peripheral nerves with an acceptable signal to noise ratio (SNR) (50). Further research is ongoing with respect to the use of DTI in diabetic neuropathy to further refine its clinical and diagnostic utility.
Table 2 illustrates a reference guide for interpretation of DWI and ADC signal intensities in the spectrum of diabetic foot and ankle diseases.

Figure 8: Type II DM with muscle denervation. Axial T1 (A), STIR (B), DWI (C), ADC (D), color map (E). Diffuse muscle fatty replacement demonstrating relative enhancement on DWI (C, arrow), ADC (D, arrow) and color map (E, arrow).

Table 2: Interpretation of diffusion and ADC signal intensities.

Limitations of DWI

It is easier to perform DWI on a 1.5T strength MRI magnet compared to a 3T MRI. Although a 3T MRI has higher SNR, there are usually more susceptibility artifacts due to the increased B0 [11]. SS-EPI sequence is used for DWI, which is also particularly sensitive to susceptibility artifacts induced at tissue boundaries like those occurring at the fat, water, and bone interfaces in the musculoskeletal system [11]. Use of parallel imaging, autoshimming, correction algorithms, and modified radiofrequency pulses (monopolar or bipolar) can mitigate these effects. Use of multishot EPI technique reduces susceptibility artifact, but at the expense of longer acquisition time [59]. Another artifact common to DWI is Eddy currents, seen as contraction or dilation of the image, and overall shift and shear of the image [60]. This occurs due to the rapidly switching on and off, of the radiofrequency gradients. The resulting image distortion can lead to incorrect quantification of ADC values. Newer techniques using rectangular field of view, motion correction and multi-segmented read-out, etc. have improved the image quality with distortion-free acquisition possible in sagittal and coronal planes.

Pitfalls

A common interpretation pitfall of DWI is misidentifying T2 shine through as true restricted diffusion. This can be easily mitigated by simultaneous assessment of the high b value DWI and ADC images. Many malignancies, soft tissue or intraosseous, show restricted diffusion due to increased cellularity and can be misdiagnosed as infection/abscess or vice versa. A thorough understanding of the indication and clinical presentation sometimes may be the only clue to make an accurate diagnosis, as infections can have a very aggressive appearance on imaging. Another common pitfall is the presence of a hematoma. Hematoma in varying stages of evolution may show restricted diffusion and can be confused with underlying infection or tumor (Figure 9). A study by Oka et al, however, did show that the mean ADC value of chronic expanding hematoma (1.55 x10-3 mm2/s) was significantly higher than that of malignant soft tissue tumors (0.92 x10-3 mm2/s) with no overlap in the minimum ADC values [61]. A knowledge of clinical presentation with recent history of trauma or procedure may help to make an accurate assessment.

Figure 9: 77-year-old with swelling, subcutaneous hematoma as DWI pitfall. Axial T1 (A), STIR (B), DWI (C), ADC (D), color map (E). T1 intermediate to hyperintense fluid collection (long arrow) that demonstrates restricted diffusion.

The guidelines for choosing the ROI are also not well established, but the darker region on ADC map with corresponding brighter area on DWI map is used by most since theoretically these areas correspond to the regions of most cellularity/infection/abscess [9].

Conclusion

Diabetes-related foot complications are common problems associated with high morbidity and mortality. There is a considerable overlap in the clinical and imaging findings of soft tissue and bone infections in patients with diabetes and neuroarthropathy. Treatment also varies greatly for different complications and relies heavily on an accurate diagnosis. The decision of oral versus systemic antibiotics versus surgical intervention (debridement versus amputation) is based on correctly differentiating DFO from other soft tissue infections [62]. MRI plays a central role in determining the diagnosis and extent of these complications. DWI complements and supplements the conventional MRI evaluation, especially where post contrast imaging is not possible. DWI should always be interpreted in conjunction with conventional MRI and in the light of pertinent clinical information to avoid imaging pitfalls. Knowledge of DWI utility for DFD, can help guide appropriate and timely treatment.

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Is Obstructive Sleep Apnea A Risk Factor for Severe Acute Respiratory Syndrome Coronavirus 2 Infection?

Introduction

Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV- 2) has emerged as public health crises first in the city of Wuhan, Hubei province in China in December 2019, and has subsequently turned out to be a global problem. Coronavirus Disease 2019 (COVID-19) may present in a wide spectrum of clinical forms ranging from mild symptoms such as fever, cough, or fatigue to severe pneumonia, septic shock, organ failure, or death. Understanding risk factors for disease susceptibility and severity is essential to prioritize target populations and patients that are at most risk. Which factors predict the susceptibility to COVID-19 and the severity of the infection have been studied earlier. Diabetes mellitus (DM), hypertension, respiratory and cardiovascular disorders are among the factors with high consistency of association to lifethreatening outcomes [1]. COVID-19 and obstructive sleep apnea (OSA) share many demographic characteristics and comorbidities such as advanced age, male gender, obesity, hypertension, cardiac complications, and DM. Both OSA and COVID 19 are associated with pro-inflammatory mediators. Coronavirus-2 enters the cell with the help of the Angiotensin-Converting Enzyme-2 (ACE-2) receptor. The number of ACE-2 receptors in adipose tissue increases in obesity [2]. Hypoxemia in OSA may affect the coagulation cascade and enhance the tendency to coagulation caused by COVID 19 [3]. Taken together, sharing the mentioned putative risk factors in common, is OSA risk and poor prognostic factor for COVID-19 infection?
There are several studies focused on the frequency of OSA in COVID-19 patients and the effect of OSA on the prognosis of COVID-19 [4,5]. However, there are not enough data in the literature regarding the prevalence of COVID-19 in the population with OSA. In our study, we aimed to evaluate the prevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in patients diagnosed with OSA and the effect of OSA on the severity of the infection in these patients.

Material and Methods

We conducted a retrospective observational study in the OSA population diagnosed by polysomnography (PSG) in our clinic. The records of consecutive patients who underwent PSG between March 2015 and March 2020 in our clinic were reviewed. OSA was diagnosed using overnight PSG. The standard overnight PSG included electroencephalography, electrooculography, submental and bilateral leg electromyography, and electrocardiography recordings. We measured airflow with a nasal pressure transducer and an oronasal thermistor, respiratory effort via respiratory inductance plethysmography, and arterial oxyhemoglobin saturation via a finger pulse oximeter. Experienced technicians collected and digitalized all signals using computerized PSG systems (Comet Grass: Astro-Med, Inc., West Warwick, Rhode Island, United States, and Viasys Cephalo-Pro, SomnoStar: VIASYS Healthcare, Hochberg, Germany) following established standards [6]. Certified sleep specialists, experienced in sleep medicine, scored sleep stages using the American Academy of Sleep Medicine (AASM) scoring system [7,8]. Grading of the apnea-hypopnea index (AHI) followed AASM’s 1999 criteria as follows: an AHI less than 5 was normal, an AHI higher than 5 but less than 15 was mild, an AHI higher than 15 but less than 30 was moderate and an AHI higher than 30 was severe [9].
The electronic medical records of the Public Health Management System were queried for the results of the SARS-CoV-2 polymerasechain- reaction (PCR) tests for all these subjects with PSG confirmed sleep disorders. Whether the subjects have been tested for SARSCoV- 2 with PCR and the test results were recorded. For patients with positive tests, the demographic data, results, and diagnoses of the sleep study were recorded. Comorbidities were ascertained by ICD-10-CM coding and medical record data. A case of Covid-19 was defined by a positive result on a PCR assay of a specimen collected on a nasopharyngeal swab. The clinical findings, laboratory and radiological data, outpatient/inpatient treatment status, and the course of the COVID-19 of the patients with positive PCR tests were recorded.

The Study Protocol was Approved by the Local Ethics Committee

All statistical analyses were performed using SPSS software (version 17.0). For baseline characteristics, mean (standard deviation) for continuous variables and number and percentages for categorical variables were calculated. Given that this is a descriptive study, no analysis for statistical significance was performed.

Results

Our analysis included 1317 OSA patients diagnosed by PSG. A review of the medical records demonstrated that 51 patients have been tested for SARS-CoV-2 with PCR. The reasons for testing were suspicion of infection, contact tracing, scanning before hospital admission or interventional procedures, or screening for travel. We identified 14 patients with positive PCR results for SARSCoV- 2 (Figure 1). The mean age of the 14 patients was 48.9 ± 12.1 years. The majority of the patients were male (n=13, 93%). The mean BMI was 29.7 ± 2.4 kg/m2. The polysomnographic data is demonstrated in Table 1. Eight (57%) cases had mild OSA, three (21%) had moderate OSA, and three (21%) had severe OSA. Three cases were asymptomatic. Main complaints were chest pain (n=6, 43%), fever (n=5, 36%), fatigue (n=3, 21%), cough (n=3, 21%), shortness of breath (n=3, 21%), loss of taste and smell (n=2, 14%), and diarrhea (n=1, 7 %). Two patients (14%) had DM and two (14%) had hypertension. Two patients (14%) did not need radiological evaluation. Others underwent computed tomography (CT) scanning; normal CT findings was observed in six cases (43%); involvement was unilateral in three cases (21%) and bilateral in three (21%) cases. The mean percentage of oxygen saturation was 97.4±3.0(90-99) on initial evaluation. The laboratory data of COVID-19 patients is demonstrated in Table 2. All the patients underwent outpatient treatment and no hospital or intensive care unit (ICU) admission, progression to respiratory failure or mortality was observed.

Figure 1.

Table 1: The polysomnographic data of OSA patients with COVID-19.

saturation

AHI: apnea–hypopnea index (events per hour); AI: apnea index (events per hour); ODI: oxygen desaturation index (events per hour); spO2: Arterial oxygen three(21%) had moderate OSA, and three(21%) had severe OSA.

Table 2: Genetic mutation in GIST.

spO2, Arterial oxygen saturation

Discussion

We have observed that the prevalence of COVID-19, the need for hospitalization, and progression to respiratory failure, namely severe infection did not seem to increase in OSA patients. In our large OSA population, no hospital admission or death occurred due to COVID-19. Recognition of conditions substantially associated with significant morbidity and mortality is essential to offer prudent preventive measures to vulnerable populations. Theoretically, OSA patients should have increased susceptibility and severity for SARS-CoV-2 infection as they share essentially identical risk factors. Due to overlapping predisposing factors, OSA patients are thought to show a heightened risk of poor outcomes in the case of COVID-19. Our findings are contradictory to this fact. Several studies including a small population of severe COVID-19 patients have shown that 21-28% of patients had OSA (10,11) A recent study on the relationship between OSA and risk of COVID-19 infection has revealed that the risk for COVID-19 infection was about 8-fold greater in OSA patients. The authors stated that the risk of hospitalization and respiratory failure increased, as well [12]. Obesity predisposes to OSA [13]. Links between obesity and COVID-19 have been investigated. In a recent analysis, obesity has been reported as an independent risk factor for invasive mechanical ventilation in COVID-19 patients [14].
Up through January 22, 2021, a total of 28.195.901 tests have been applied and 2.418.472 people had tested positive for the new SARS-CoV-2 coronavirus in Turkey. The total number of deaths is 24789 [15]. These data reveal that about 3% of the Turkish population has been infected with SARS-CoV-2. Concerning these data, the prevalence of COVID-19 in our selected population does not seem to be higher than the general population. One possible explanation is the fact that this specific cohort, as well as their families are aware of the increased risk of morbidity and mortality from Covid-19, due to their comorbid medical conditions, such as obesity, hypertension, diabetes, and thus, they were extra cautious about exposures. One major problem with treatment in OSA is nonadherence to CPAP treatment. CPAP adherence has been shown to improve significantly during the COVID-19 lockdown [16]. Staying at home, travel restrictions, and the fear of having a poor prognosis, and the probability of being hospitalized might have been motivating factors in better CPAP adherence [16]. This may be considered as a protective factor for OSA patients. The current study has several limitations. Coding and recording of data may be imprecise and missing. Still, the administrative data we based our investigation on is very reliable. Our data reflect OSA as it is diagnosed by PSG. However, OSA is widely underdiagnosed, therefore the true prevalence is probably higher. It may be argued that some OSA patients might have PCR negative COVID-19 infection, on the other hand, the same argument may be true for all the population. In the population we reviewed, all OSA patients who had negative PCR results have been on basis of screening. They had no infection symptoms or signs.
In contrary to previous reports suggesting an increased risk of COVID-19 in OSA patients, our study represents novel data on the incidence of COVID-19 in population with confirmed OSA. To our knowledge, this is the first study to claim that susceptibility, severity, and mortality are not increased in COVID-19 patients with sleep disorders.

Conclusion

Our results provide some initial data regarding COVID-19 risk in a large OSA population. We demonstrated that OSA cannot be considered as one of the underlying medical conditions predisposing to increased risk or poor outcome in COVID-19. Poor COVID-19 related prognosis, if exists, may be attributed to other risk factors or comorbidities accompanying OSA. We have observed that the prevalence of COVID-19, the need for hospitalization, and progression to respiratory failure, namely severe infection did not seem to increase in OSA patients. In our large OSA population, no hospital admission or death occurred due to COVID-19. Recognition of conditions substantially associated with significant morbidity and mortality is essential to offer prudent preventive measures to vulnerable populations. Theoretically, OSA patients should have increased susceptibility and severity for SARS-CoV-2 infection as they share essentially identical risk factors. Due to overlapping predisposing factors, OSA patients are thought to show a heightened risk of poor outcomes in the case of COVID-19. Our findings are contradictory to this fact. Several studies including a small population of severe COVID-19 patients have shown that 21- 28% of patients had OSA [10,11] A recent study on the relationship between OSA and risk of COVID-19 infection has revealed that the risk for COVID-19 infection was about 8-fold greater in OSA patients.
The authors stated that the risk of hospitalization and respiratory failure increased, as well [12]. Obesity predisposes to OSA [13]. Links between obesity and COVID-19 have been investigated. In a recent analysis, obesity has been reported as an independent risk factor for invasive mechanical ventilation in COVID-19 patients [14]. Up through January 22, 2021, a total of 28.195.901 tests have been applied and 2.418.472 people had tested positive for the new SARS-CoV-2 coronavirus in Turkey. The total number of deaths is 24789 [15]. These data reveal that about 3% of the Turkish population has been infected with SARS-CoV-2. Concerning these data, the prevalence of COVID-19 in our selected population does not seem to be higher than the general population. One major problem with treatment in OSA is nonadherence to CPAP treatment. CPAP adherence has been shown to improve significantly during the COVID-19 lockdown [16]. Staying at home, travel restrictions, and the fear of having a poor prognosis, and the probability of being hospitalized might have been motivating factors in better CPAP adherence [16]. This may be considered as a protective factor for OSA patients. The current study has several limitations. The study does not include a control group to determine the prevalence of hospitalization or severe disease in a cohort without OSA. Still, we have the prevalances from the total population to compare the prevalence of the cohort. Coding and recording of data may be imprecise and missing. Still, the administrative data we based our investigation on is very reliable. Our data reflect OSA as it is diagnosed by PSG. However, OSA is widely underdiagnosed, therefore the true prevalence is probably higher. It may be argued that some OSA patients might have PCR negative COVID-19 infection, on the other hand, the same argument may be true for all the population. In the population we reviewed, all OSA patients who had negative PCR results have been on basis of screening. They had no infection symptoms or signs.
In contrary to previous reports suggesting an increased risk of COVID-19 in OSA patients, our study represents novel data on the incidence of COVID-19 in population with confirmed OSA. To our knowledge, this is the first study to claim that susceptibility, severity, and mortality are not increased in COVID-19 patients with sleep disorders. In conclusion, our results provide some initial data regarding COVID-19 risk in a large OSA population. We demonstrated that OSA cannot be considered as one of the underlying medical conditions predisposing to increased risk or poor outcome in COVID-19. Poor COVID-19 related prognosis, if exists, may be attributed to other risk factors or comorbidities accompanying OSA.

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Use of Eichhornia Crassipes, Lemna sp and Salvinia Minima Plant Scrubbers in the Decontamination of Wastewater of Livestock Origin, in the Province of Imbabura

Introduction

Livestock activities are the basis of economic development at the rural level and constitute food sources for the urban sector. In Ecuador, the production of cattle and pigs has increased notably, so in the reports of the last National Agricultural Census of the year 2000, it is seen that with respect to the Census of 1976 the increase of these species has been 76, 80%, this has increased the economic movement, but has affected the good quality of the water resource, since contamination by livestock activity is frequent. However, if an adequate treatment of these wastes is carried out, the negative impact that is generated can be minimized and contribute positively to rural development with the obtaining of other benefits [5].
Several investigations have been carried out in the province of Imbabura, with optimal results. The use of phyto-scrubbers is encouraging as part of Environmental Management in caring for the water resource. In this way, knowing the potential for treating wastewater of the plant species Eichhornia crassipes and Lemna sp, they were used at La Pradera Farm to include them in a productive decontamination system, continuing with research to treat wastewater. Salvinia minima was also included due to its accelerated growth and because it is found abundantly in coexistence with Eichhornia crassipes; for this reason, it is considered important to be evaluated as a phyto-scrubber.

Methodology

In the research, which was carried out at La Pradera Farm, located in the province of Imbabura, Antonio Ante canton, San José de Chaltura parish, a biodigester, 12 mini ponds, 7.5 kg of Eichhornia crassipes, 1.5 kg of Lemna sp. and 3 kg Salvinia minima. The Completely Random Design was used, with four treatments and three repetitions. When finding a significant difference between treatments, the functional analysis was performed with the 5% Tukey test. The variables that were evaluated in the laboratory and the methods used were: pH (potentiometric), Conductivity (conductimetric), Hardness (volumetric), Total Solids (gravimetric), anions and cations (atomic absorption), BOD (APHA 5210B), COD (5520 D), total coliforms (EPA 40 CFR). Percentage of dry matter, protein and fat. The average flow was 3 L / s. The biodigester was built, located at the outlet of the effluent with wastewater, at the outlet of the biodigester a system of pipes led the water to the ponds 0.50 m wide x 1.00 m long and 0.30 m deep. depth, which contained the three investigated species.

Results Evaluation and Discussion

Table 1 Overall result of the evaluated variables and their reference parameters

Variables pH and Electrical Conductivity (ds / m)

Graph 1 Eichhornia crassipes, Lemna sp and Salvinia minima decrease the pH levels in the wastewater, Eichhornia crassipes lowers the pH levels by 24.05% with respect to the control, this makes the water reach the optimal levels to be used in irrigation with Regarding this parameter, Lemna sp and Salvinia minima also decrease the pH values by 16.53% and 1.15% respectively. These results are consistent with the research carried out by Valderrama (1996) [6], in which he states that E. crassipes stabilizes the pH and contributes to producing values closer to the neutrality of the water. Eichhornia crassipes with 65.11%, followed by Salvinia minima with 20.9%, and finally Lemna sp with 27.9% decrease the concentrations of electrical conductivity. Eichhornia crassipes absorbs a large number of metals present in the water, tending to decrease the conductivity parameters, with this, what is stated by Valderrama (2005) is corroborated.

Graph 1: Average values of pH and electrical conductivity of the evaluated treatments.

Variables Cations (Ca, Mg, Na, K)

Graph 2 Eichhornia crassipes reduces Ca concentrations by 33.3%, this phyto-scrubber is very easy to absorb this type of minerals due to its root and foliar structure, Lemna sp slightly decreases the calcium content of the water by 1.33%, but surprisingly Salvinia Minima contributes a greater amount of calcium to the water since it increased its concentration by 21.33%, probably due to the fact that this ion accumulates in its roots, the type of tangled roots that this species has and they save this mineral from the area collection at Lake San Pablo.

Eichhornia crassipes, decreases the concentration of Mg with respect to the wastewater of origin is 65.5%, Lemna sp and Salvinia minima also lower the levels of concentration of Mg by 22.8% and 18.71% respectively, this is evidenced in the absorption produced by these phytodepurative species. Eichhornia crassipes decreased the Na content in the water by 80.88%, Salvinia minima decreased by 61.1% and finally Lemna sp decreased by 53.1%, this confirms the research carried out by García et, in which it indicates that the aquatic species like E crassipes, they have a high affinity for adsorption and complexation with organic matter, cations and anions, assimilating them through the root. Eichhornia crassipes tends to decrease the amount of K significantly, the reduction percentage of it was 73.9%, Lemna sp and Salvinia minima also decreased this parameter, the percentage decreases were 65.2% and 45.4% respectively, with this it is verified what Valderrama (2005) [6] states in terms of the absorption of nutrients by aquatic macrophyte species is highly efficient in wastewater, being able to exceed 50% of their removal.

Graph 2: Average cation values of the evaluated treatments.

Variables Anions (HCO3, SO4, NO3, B, P, Cl)

Graph 3 Eichhronia crassipes achieved the highest percentage of HCO3 removal with 57.2%, followed by Lemna sp with a decrease of 21.4% and Salvinia minima had a reduction of this ion of 11.9%, this is consistent with Orozco, Saimonds (2006), who point out that floating macrophytes are capable of eliminating various substances and ions dissolved in water by adsorption and absorption. Eichhornia crassipes notably decreases the levels of SO4 in the wastewater, decreasing 98.7% of it, Salvinia minima decreasing by 38.5% and Lemna sp by 25%, the three phytodepurating species lower the levels of SO4 in a significant way. S. minima decreased the highest amount of NO3 with 59.7%, E. crassipes and Lemna sp did not decrease 50% of this anion. García (2012) [7] in his research states that nitrates are not eliminated by ion exchange due to their negative charge, rather they are transported as part of the residual water, being easily assimilated to new plant tissues and eliminated through the denitrification process by microorganisms present in the middle, which happened in the investigation although not significantly. Eichhornia crassipes significantly decreases the amount of B in the water, thus its decrease is 84.6%, Lemna sp and Salvinia minima also show a great decrease in 69.2% and 46.15% respectively, which shows that the phytodepurating species they highly absorb this nutrient.
Eichhornia crassipes, Salvinia minima and Lemna sp decreased 100% of the phosphorus present in the water. Rodríguez (2001) [8] in his study of Hydrology and Groundwater uses Eichhornia crassipes to reduce phosphorus levels, resulting in a 40-60% decrease, in this research these values were exceeded with the use of the three phyto-scrubbers. Eichhornia crassipes, Lemna sp and Salvinia minima decreased chlorine concentrations, the removal percentage was 4.2% for E. Crassipes and 3.6% for Lemna and S. minima respectively.

Graph 3: Average values of anions of the evaluated treatments.

Variables RAS, Hardness, Total Dissolved Solids

Graph 4 The Sodium Adsorption Ratio is decisive in the quality of the water, so that the waters that contain less than 1 are excellent for agriculture, from 1 to 2 are good waters, from 2 to 4 are considered regular waters, from 4 At 8 bad waters and more than 15 inappropriate waters, the values obtained through the phyto-scrubbers show the decrease of the RAS and as they fit into good evaluations, Eichhornia crassipes reached 0.91 RAS, making the water excellent, since it decreased 70.6% of This value, Lemna sp Salvinia minima and Lemna sp also lowered the RAS levels by 61.3% and 48.3% respectively. Eichhornia crassipes the best aquatic species to reduce the hardness of the water, with 54.2%, Lemna sp followed with a decrease of 15.3% and in the end the smallest decrease was had by Salvinia minima with 4.7%, E. crassipes is the only species plant that converts the average water hardness of the source water to soft, which makes this water more useful to be used in irrigation. Eichhornia crassipes decreased 80.2% of the amount of STD dissolved in wastewater, Salvinia minima had a reduction of 63.6% and Lemna sp decreased 54.2%, E. crassipes was the most effective species to decrease the amount of STD in water irrigation, this coincides with the research carried out by Valderrama (1996), in which he uses E. crassipes for the treatment of wastewater of agro-industrial origin and determines that this species is capable of eliminating more than 50% of STD of the water. Likewise, the percentages of decrease of E. crassipes coincide with those obtained by Camacho and Ordóñez (2008), who through their investigation of evaluation of recovery of wastewater systems with Eichhornia crassipes, determine that this species was able to decrease 83.69%.

Graph 4: Average values of Total Hardness of the evaluated treatments.

Variables BOD5, COD and Total Coliforms

Graph 5 Eichhornia crassipes decreased by 75% of BOD5, Lemna sp decreased by 52.5% and Salvinima minima decreased this value by 30%, the values obtained in this investigation agree with other investigations carried out, especially Eichhornia crassipes that has been more studied. that Obando (2006) [8] through his research, achieves reductions of 89.3% of BOD5 with Eichhornia crassipes, 76.6% through Salvinia minima and 70.7% with Lemna sp; Rodríguez (2006) [9] with Eichhornia crassipes decreases the concentration of BOD5 in a range of 80-90%. Camacho and Ordóñez (2008), in their research, found that E. crassipes was highly effective in reducing BOD5 values, decreasing 56.84%. With the chemical oxygen demand (COD) Eichhornia crassipes achieves the highest removal of the three species with 78%, followed by Lemna sp with 71.5% and Salvinia minima decreases in 65.3%. Eichhornia crassipes, Lemna sp and Salvinia minima, which decreased Total Coliform Colony Forming Units from 4000 to less than 10.

Graph 5: Average values of residual chlorine, nitrates and phosphates of the evaluated treatments.

Variable Percentage of Dry Matter, Protein and Fat

Graph 6 Eichhornia crassipes presented an accumulation of 9.42% of dry matter, 12.13% of protein and 1.14% of fat, being the species with the best bromatological characteristics in these principles, since Lemna sp weighed 9.36% of dry matter, 3.67% protein and 0.89% fat, finally Salvinia minima had 8.39% dry matter, 0.41% protein and 1% fat.

Graph 6: Values in percentage of dry matter, protein and fat of the phyto-scrubbers.

Conclusions and Future Work

With the use of the phytodepurators Eichhornia crassipes, Lemna sp and Salvinia minima, it was possible to reduce the concentrations of the evaluated parameters Ca, Mg, Na, K, HCO3, Cl, SO4, B, RAS, hardness, STD, BOD5, COD and Total coliforms, being able to reuse the residual water in irrigation. The best phytodepuration species is Eichhornia crassipes since it reduces the values of the essential parameters to determine the quality of irrigation water: pH, electrical conductivity, Ca, Mg, Na, K, HCO3, Cl, SO4, B, RAS, hardness, STD, BOD5, COD and total coliforms evaluated with greater efficiency, compared to the other phytodepurating species Lemna sp and Salvinia minima, it also has a better adaptation in the field and can be used as an additional feed source for the livestock of the farm (previous research), due to its nutritional properties and their acceptability, for this reason the Wastewater Productive Decontamination System of La Pradera Farm was implemented with this species [10-12].
This research is the preamble to many other investigations, since the efficiency of Eichhornia crassipes as a phyto-scrubber has been determined, but there are still many other parameters to be evaluated, and thus prove that phyto-scrubbers are excellent allies in the care and maintenance of the environment [13-60].

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Negative Emotions Damage the Heart

The heart (from the Latin cor) is the main muscular organ of the circulatory system. In humans, it is a hollow muscle located in the thoracic cavity with a slight inclination to the left whose function is to pump blood throughout the body through the blood vessels. The heart muscle is myogenic, that is, it excites itself. Rhythmic contractions occur spontaneously, as well as their frequency, which can be affected by the response of our body to different situations that may arise in the course of life, including:
1. Surprise: a fleeting and unexpected emotion.
2. Perception of a danger.
3. A dislike at work.
4. Jealousy.
5. An illness.
6. The infidelity of the partner or spouse.
7. The culmination of a loving or fraternal relationship.
8. The loss of a loved one.
9. An energetic discussion with someone.
10. Social exclusion.
Among the most recurrent emotional symptoms of people who have suffered from such ailments, we could mention:
1. Suffering
2. Principles of despair and insanity.
3. Loss of meaning towards life and daily tasks.
4. Moods such as: sadness, melancholy, depression.
5. Negative emotional arousal (irritation, anger, impatience, anxiety)
6. Very strong stress
All this can affect the normal functioning of our body causing physical symptoms such as: tachycardia, chest pain, fatigue, asthma, diarrhea, eczema and other skin conditions, lack of sleep, physical exhaustion, lack of appetite, or due to on the contrary, a voracious appetite, muscle spasms, among others, but they also directly, and sometimes seriously, affect the heart. There are studies that address the effects on the heart after receiving bad news and suffering severe emotional stress, one of them is temporary heart failure commonly called broken heart syndrome or Tako-tsubo cardiomyopathy, (this is the name of a vessel, domed and narrow neck traditionally used by Japanese fishermen to catch octopus) which was first described in the 1990s in Japan. It is a stressinduced cardiomyopathy in which there is a sudden temporary weakening of the myocardium. Chest pain is one of the common signs of this disease. From research carried out, it has been possible to determine that many of the cardiovascular problems that occur are mainly caused by depression [1-3].

What Happens in our Body When We are Depressed?

The body of a depressed person sets in motion a series of substances from structures and glands that regulate their functioning. Among them are the hypothalamus in the brain, which issues orders for the pituitary to order the thyroid to produce thyroid hormones, these streamline all functions and, in turn, act on the adrenal glands, where adrenaline is produced, High doses of this hormone have a strong impact on blood pressure, heart rate and the size of the arteries of the heart. Also, the levels of serotonin (a neurotransmitter that helps calm and produces a feeling of wellbeing) fall; If this situation is recurrent, inflammatory responses can occur, a tendency to arrhythmias and a decrease in cardiac flow; which can cause heart attacks and sudden death. Therefore, scientists support the theory that people who cannot adapt and overcome emotional pain, are those who must go through a higher level of physical pain. Many people manage to adapt to these situations, but many others do not reach those levels of resilience (the ability to be reborn after adversity).All this without taking into account also that depressed people tend to adopt unhealthy lifestyles, evidenced in bad eating habits, the tendency to smoke, sedentary lifestyle and even alcoholism, therefore, there is a dangerous increase in risk factors coronary.
We are not exempt from encountering situations that push us to our limits, what is truly important is that the rational part enables us better or worse to face these changes. Under new conditions or any change that requires a biological adaptation process, man can respond in an exaggerated way for fear of punishment, not finding a reasonable way out, he ends up acting effusively. Negative emotions adversely affect our health, they can contribute to the development of diseases and interfere with recovery, the way you react to them is extremely important. Avoid disorders and learn from what has been lived. Episodes of sadness should help us learn, take new directions and emerge stronger [4-6]. A sad brain produces less serotonin; If we cannot get out of this state by making new decisions and assuming what happened, in the long term this deficit in serotonin can cause us to suffer diseases such as depression, compulsive obsessions and / or violent outbursts. But we have to be strong and find in those moments of meditation, reflection and look for new resources with which to get ahead. If sadness is of any use to us, it is to learn from what we have experienced, we all know that existence is not a flat easy road to travel, there are stones to overcome and new paths to find, realities from which we must learn. This is how we will be stronger and more capable.

Conclusion

In life no one is exempt from the difficult situations that can arise. We must be prepared to face them, no matter how difficult it may seem. Do not seek solutions that further harm our health such as frequent ingestion of alcoholic beverages, addiction to cigarettes, resorting to diets that alter blood pressure, assuming a sedentary life, staying in unhealthy environments, among others. Learning to overcome and remedy our emotional and physical pain with favorable solutions for our body will allow us to contribute to “heal the hearts” of others.

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The Green Economy is A Must of Our Society

Introduction

Given the increasing global level of waste and pollution and its detrimental effects on air, water and the human health, The safeness environment became a mast for worldwide consumers together with wellbeing and longevity [1,2]. It is to underline in fact that according to the World Health Organization (WHO),every year occurs 4.2 million deaths as a result to outdoor pollution (Figure1) [3], also if it isn’t to be forgotten the indoor nanoparticulates represented from 10.000 to 240.000 nanoparticles/ml air [4]. Thus, it has been estimated that in 2015,diseases caused by air pollution had a cost of USD 3.8 trillion in 176 Countries .Moreover, the COVID-19 pandemic ,ravaging the World and attacking societies at their core by an high impact on health and the economy never ever imagined until today, has further increased the need to adopt a Green Circular Economy [5,6]. Therefore, It has became a painful evidence the necessity to shift healthcare system away from profiting, giving a much bigger role to wellness and wellbeing, thus changing the actual way of living [7]. However, our future survival will depend on a new alignment between healthcare/wealth and the preservation of both Environment and Biodiversity [3,8].
The materials and energy actually used to make consumer and commercial goods ,in fact, produce a large share of greenhouse gas (GHG) emissions impacting the environment by global warming and climate changing [9,10]. Therefore, the urgent necessity to produce and consume biodegradable goods that, decomposed or deconstructed into different materials, can easily be recycled and used without impacting the environment [10]. In conclusion the Linear Economy, based on the taking, making and producing waste, has to be changed by the Circular Economy of Reducing, Reusing and Recycling (Figure 2). At this purpose many research studies have been dedicated to emulate the productive strategies of nature for making bio-based products from renewable feedstocks, without waste and by a low consume of energy [5,6]. Thus, for example, both cosmetic ingredients and packaging materials could be realized by the use of biopolymers, involving the bacteria machinery to make these products at zero waste, safe and inexpensive [11]. Consequently the new products of the circular economy might be realized by the use of renewable sources and sustainable technologies, possibly obtained from agroforestry or food waste, such as chitin and lignin [12,13], preferably used in their nanosize, such as chitin Nanofibrils (CN ) and Nanolignin (LG).

Figure 1: Air pollution and its risk on health (by courtesy of WHO [3]).

Figure 2: Differences between Linear and Circular Economy.

Chitin And Lignin for Innovative Tissues

Both the natural polymers chitin and lignin, easily obtainable from food and agro-forestry waste at low cost, may be used as interesting biocompatible carriers to make smart particles and innovative biodegradable non-woven tissues for medical [14- 16] and cosmetic use [17-19], as well as to realize biodegradable films and nanocomposites for food packagings and cosmetic containers [20-22]. But how chitin and lignin can be used? Due to the fact that chitin is an electropositive polymer, while lignin has an electronegative backbone, they have been used to realize block polymeric particles by the gelation method (Figure 3). Successively, embedding the particles into a bio polymeric gel it has been possible to make tissues by the electrospinning technology or films by the casting technology. It is interesting to underline that these particles, more effective when in their nanosize, are able to encapsulate various active ingredients necessary to characterize the activity of the respective tissues and films [5,14-20] Thus, encapsulating nanoparticles made by nanostructured silver bound to fibers of chitin nanofibrils-chitosan ,it has been realized an innovative tissue that has shown an interesting anti-inflammatory and skin repairing activity both in vitro and in vivo [23-25].

Figure 3: CN-LG particles at the Scanning Electron Microscopy(SEM)

On the other hand, encapsulating other active ingredients such as vitamin A and E and nicotinamide and other compounds into the same tissue, innovative cosmeceuticals have been realized, effective to slow down the formation of fine lines and wrinkles and to repair the damaged hair, always respecting the environment [26-28]. However, it results important to underline that : polymer quality and purity ,method and size adopted to produce particles and complexes, quality and dose of the active ingredients selected and encapsulated, result fundamental to obtain effectiveness and safeness of the designed final product. Just to understand the importance of the polymer size it is to remember, for example, that small chitin (<40 micrograms) has shown an antiinflammatory activity, having on the other hand a pro-inflammatory activity with a size of 40-70 micrograms [29]. Moreover, the tissues and films possibilities to enter in contact with the right cells releasing the active ingredients at level of the different skin layers, depend not only to the polymeric fibers size, the electric charge of their surfaces and the pH of the environment, but first of all to the inter- fibrillar spaces necessary to permit the cell adhesion, proliferation and differentiation (Figure 4) [12,30]. However, the non-woven tissue, results effective to regenerate skin and other tissues when made by natural polymeric fibers reproducing the structure of the natural extra cellular matrix (ECM) [31].

Figure 4: Skin penetrability of Nanoparticles depends from their size, electrical charge and type of polymer but also from the tissue structure that has to be similar to native extra cellular matrix (ECM).

Conclusion

In a world where waste, pollution and carbon dioxide became a serious global threat to human health and the environment provoking many worldwide disasters, it results necessary to produce goods at zero waste by the use of bio-natural polymers and micro-nanoparticles, finally adopting the Circular Economy [13].The reported engineered micro-nano particles and tissues, in fact, could provide numerous advantages as green compounds characterized for their availability, reproducibility, biodegradability, renewability biocompatibility and non-toxicity. However due to their multi-functionality, these innovative biomaterials and carriers could represent active matrices effective to regenerate wounded, burned and aged skin as well as to make biotextiles for producing sportswear, biodegradable medical masks and other hygienic biomaterials or film-packagings to preserve food from bacterial contamination [5-7,12-28]. In any way, It is to underline that chitin, lignin and their complexes may be considered active carriers, being metabolized by the human enzymes to produce glucose, glucosamine, acetyl glucosamine and polyphenol compounds used from the cells as food or energy.

Additionally, it seems possible to stop the ocean’ mcroplastics waste, utilizing these biopolymers to produce the many plastic goods actually made by petrol-derived compounds. So doing it will be possible to avoid many toxic compounds dangerous for the algae, fish, sea mammals and birds as well as for the human health, because of their activity acting as endocrine disruptors for its content of toxic bis phenols [32]. For all these reasons the worldwide consumers, including Z Millennials (aged 18-34), Generation X (aged 35-50 ) and Baby boomers (51-69), request to prohibit the actual deforestation, eliminate air and water pollution and the natural resources depletion for the necessity to produce and consume, for example, recyclable or compostable packagings necessary to stop the climate change, as for our proposal. (Figure 5& 6) [33,34]. In conclusion ,natural climate solutions, that avoid GHG emissions and soil sequestration ,offer a way to limit warming and disasters by the worldwide introduction of the Green Economy.

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Possible Use of CRISPR-Cas13 Technology in the Pathogenesis of the SARS-CoV-2 Virus

In order to start thinking about this alternative, it is important to know the mechanism of action of viruses in order to identify the attack sites of the technique. It is important to know that the infection begins in the host cell, when the virus binds to a receptor on the cell surface. The SARS-CoV-2 virus is bound by its protein (S) and the receptor for angiotensin converting enzyme 2 (ACE2). This binding directs the north of the virus in the target cell and its specificity [1]. ACE2 contributes to the regulation of blood pressure by converting angiotensin I into angiotensin [2]. The ACE2 receptor is expressed in many tissues such as the lower respiratory tract, heart, kidney, stomach, bladder, esophagus, and intestine [2]. In the lung, it is expressed in the alveoli [3]; and in the oral cavity, in epithelial cells of the tongue [4]. The SARS-CoV-2 protein (S) has two subunits (S1 and S2). The S1 subunit is the one that interacts and binds to the ACE2 receptor through the receptor-binding domain (RBD), while the S2 subunit determines the fusion of the virus membrane with that of the host cell [5].
The virus completes its entry into the cell when the S protein is cut by the protease enzyme (TMPRRS2). This generates the separation of the RBD union of the S1 subunit with the ACE2 receptor and its fusion with the membranes, and in this way the virus is left with the door of entry open to the cell by endocytosis [6]. Once inside the cell, the translation of the viral genome and the transcription of the SARS-CoV-2 proteins begin. When it enters the cytoplasm, the nucleocapsid of the virus is released and the viral genomic RNA is released. This RNA acts as an mRNA so the viral replicase gene can be directly transcribed [1,7]. To generate the necessary proteins of the replicase transcriptase complex (RTC), which is assembled in the endoplasmic reticulum (ER) ([1,5,7-11]. Finally, the complex (RTC) replicates and synthesizes a set of sub genomic mRNA (sgRNA) [8-11] that code for the elaboration of the main structural proteins (S), (M), (E), (N) and accessory proteins [1-5].
So, this is how RNA replication, Protein Assembly and SARSCoV- 2 Egress from the Host Cell are built. In SARS-CoV-2 replication, single-stranded RNA (+ ssRNA) serves as a template to initially synthesize a copy of single-stranded RNA (-ssRNA) [12]. And from this copy of -ssRNA, polyproteins will be produced, which will make up the RTC complex [1,8]. The RTC complex again creates a copy of the original virus genome + ssRNA from the -ssRNA template. This associates with the protein (N) forming the nucleocapsid. The structural proteins (S), (M) and (E); and accessory proteins from the endoplasmic reticulum (ER) and then, in the Golgi complex, are assembled together with the nucleocapsid to produce new viral particles, which are produced towards the plasma membrane for the release of the virus [5]. According to this final mechanism, it is where the grouped and regularly spaced short palindromic repeats (CRISPR) emerge as a therapeutic alternative, together with the endonuclease Cas, form the CRISPR / Cas complex.
This system was discovered as an immune defense mechanism present in bacteria and archaea, which incorporate DNA from pathogens, such as bacteriophages, between repeated palindromic sequences and subsequently generate an RNA called “crRNA” when transcribed. Due to its activity as an endonuclease and recognition capacity in specific sequences, the CRISPR / Cas system has been exploited in genetic engineering to activate genes, repress them, induce point mutations and change sequences through homologous recombination. CRISPR has also been used to generate murine models of human diseases and to evaluate cell physiology through the simultaneous activation or repression of various genes [13,14]. This mechanism is made up of two factors, an endonuclease and a complementary recognition sequence. Thus: an RNA from the CRISPR sequence, called “crRNA”, and the Cas endonuclease. The crRNA is in charge of directing Cas towards its complementary sequence, where Cas makes the cut.
The CRISPR sequence is composed of a leader or promoter and different spacer sequences of more or less than 40 nucleotides on average and side and side repeated sequences called palindros with an extension of approximately 32 nucleotides Figure 1 [15]. When this mechanism enters into action in the cytoplasm, the cell recognizes a sequence known as a motif adjacent to the PAM protospacer and incorporates the nucleotides adjacent to the PAM. Figure 1. Subsequently, the crRNA is transcribed. The Cas then associates with the mature crRNA and forms the CRISPR / Cas complex. The crRNA will be the one who guides the complex towards its target through the recognition of the complementary sequence. The cuts produced by the explained mechanism are repaired by non-homologous end joining (NHEJ) and homology directed repair (HDR). As NHEJ can cause unwanted effects of unexpected mutations, it is preferred to choose HDR for this method. This CRISPR / Cas complex is a good alternative for targeted genomic editing.

Figure 1: Model of the CRSPR / Cas9 system. Mar Benito, MSc https://www.linkedin.com/in/marbenito/.

The system has made it possible to insert, eliminate or generate mutations in the sequences, which leads it to target a specific sequence and induce a cut in both DNA strands [13,14]. However, this system would not be very relevant for SarsCov2, since it is an RNA virus, but within the range of possibilities and versatility of the CRISPR system, a subsystem of this complex, known as CRISPR-Cas13, has been identified. CRISPR-Cas13 are widely distributed among Leptotrichia species. Estimating the occurrence, composition, and diversity of CRISPR-Cas13 systems in the genus Leptotrichia is particularly challenging because complete information on the entire genome is limited [16]. Cas13 is a crRNAguided RNA-targeted effector, which has two distinct RNase sites [17]. Not only does it highlight its unique RNA-directed ssRNA lysis activity, but Cas13a becomes a promiscuous RNase that can nonsequentially specifically cleave host cell RNA, leading to host cell or cell death latency [18,19].
Taking this information into account, this work aimed to propose an anti-COVID-19 therapeutic alternative based on the CRISPR-Cas13 system, and generate mutations in the virus, specifically in the sequence of the SARS-Cov2 protein S RBD gene that It has the function of binding to the cellular ACE2 protein and that it is the entry site of the virus for its infectivity.

Methods

First, an exhaustive review of the reported sequences of Sars- Cov2 was made, in the databases: NCBI, Google Scholar, Scopus, GeneBank, NIH. With descriptors COVID-19, CRISPR-Cas13, Gen SARS-CoV-2. The gene sequences and spike protein sequences for SARS-CoV-2 were downloaded to FASTA from GeneBank NCBI Reference Sequence: NC_045512.2. To determine the binding points of the virus to the ACE2 receptor, the sequence reported by Walls A, et al. [20] in Cell was taken as a model. With the MEGA software [21]. The sequences were compared, and mutational analysis was carried out. In addition, mutations were made in silico that allowed the generation of stop codons that would prevent the configuration of the virus RNA and make it unfeasible for its duplication and subsequent infection. Multiple couplings of the RBD site and the ACE2 receptor were made to find binding to it and whether or not it was diminished. This was run in PyMol software [22].

Results and Discussion

SARS-CoV-2 is an RNA virus, so the best technology to attack it inside the cell is when it deposits its genetic material in the cell. The appropriate technique presented is CRISPR-Cas13, which attacks RNA and not DNA [23,24]. Based on this, the following strategy was proposed here using its CRISPR technology: This is the sequence of the Virus genome, downloaded in FASTA from GeneBank: NCBI Reference Sequence: NC_045512.2. the genome corresponding to Surface Glycoprotein S. Below, highlighted in yellow is the genome of the virus and the proteins of the virus [25].

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Therapeutic Strategies of Ischemic Stroke Based on Proton-Activated Chloride Channel

Ischemic stroke is a multifactorial disease with high rate of morbidity, mortality and disability rate worldwide [1]. When acute ischemia occurs, it leads to excessive oxidative stress, causing irreversible damage to nervous tissue [2,3]. However, current treatment strategies of ischemic stroke are relatively limited, actually, a large number of neuroprotective agents that have proved ineffective in clinical trials [4]. PAC, also known as ASOR (acid-sensitive outwardly rectifying anion channel) [5] or PAORAC (proton-activated outwardly rectifying anion channel) [6], which was first observed in rat Sertoli cells in 2003[7], has shown encouraging results in providing partial neuroprotection against tissue acidosis caused by ischemic stroke.

Pac Channel and its Therapeutic Potential

Under normal physiological condition, the pH of extracellular fluid, including the blood plasma, is normally tightly regulated between 7.32 and 7.42 by the chemical buffers, the respiratory system, and the renal system [8]. However, in cerebral acidosis caused by ischemic stroke, the pH of the ischemic core may be as low as 6.0 [9]. Ischemic tissue acidosis is a sensitive metabolic indicator for the progression of cerebral ischemic injury. PAC was reported to be activated by extracellular acidity as well as its sensitivity to temperature and pH [5]. The threshold pH for PAC activation is relatively low, around pH 5.5 at room temperature and around pH 6.0 at 37℃ [10]. As a highly conserved channel ubiquitously expressed in mammals, the core protein of PAC (TMEM206) has the highest molecular expression in the cerebral cortex of human [11]. By cooperating with other ion transporters and channels such as V-ATPase, acid-sensing ion channel 1a (ASIC1a), and Na+/ H+ exchanger (NHE), PAC is activated by extracellular acidity and mediates Cl- influx into the cell, cytotoxic edema and cell necrosis ensue [12-14]. The simultaneous increase in intracellular Ca2+ promotes cell death cascade associated with intracellular Ca2+ accumulation [15].
Several studies have reported that inhibition of PAC provides partial neuroprotection against tissue acidosis after ischemic stroke:
1. Neurons undergoing massive necrosis 1h after exposure to acidic solution were not only protected by PAC channel blockers but also by cooling down to 25 ℃ [10],
2. Knocking down the core component of PAC (TMEM206) almost abolished proton activated Cl- current in rat neurons and reduced neuronal cell death caused by acid treatment [16],
3. Application of the chloride channel blocker 4,4′-Diisothiocyano- 2,2′-stilbenedisulfonic acid (DIDS) in vivo attenuated cell injury induced by ischemia-reperfusion in hippocampal CA1 neurons [17],
4. Knockout of mouse PAC abolished proton-activated Cl- current in neurons and attenuated brain damage after ischemic stroke [11]. Based on above lines of evidence, PAC is a promising therapeutic target to protect neurons in cerebral ischemia.

Therapeutic Strategies of Ischemic Stroke Targeting PAC Channels

Metabolic acidosis occurring in ischemic stroke is a sensitive metabolic indicator capable of targeting drugs to the salvageable ischemic penumbra with high specificity. Targeting ischemic brain tissue using pH sensitive marker polymers or nanoparticles has enabled medical imaging to accurately distinguish ischemic tissues from normal ones [18,19]. The development of bio-responsive materials that undergo conformational or solubility changes under acidic environments offers great promise for the development of smart targeted drug delivery nano-systems [20], such as the hydrophobically modified chitosan nanoparticles (Chit NPs) with C6-side chains were released the Ca2+ channel blocker nimodipine (NIMO) drug at the pH of ischemic tissue (=6.0), while at normal pH (=7.4) the drug molecules was remained closed in polymer shell [21]. pH directing effect and bio-responsive nanomaterials have been applied in solid tumor therapy and inspired the application of nanotechnology in the treatment of ischemic stroke [22,23]. Selective delivery of PAC specific blockers to the ischemic penumbra via a pH responsive smart nanosystem holds promise for the treatment of injury sites without adverse nontargeted side effects. However, currently there are only several universal drugs available to target chloride channels, such as DIDS, niflumic acid (NFA), and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), specific PAC blocker has not yet been reported [11,12].
Therapeutic hypothermia has been regarded as one of the most effective neuroprotective strategies since 1987 [24,25]. Over the past few decades, the neuroprotective effects of hypothermia in cardiac arrest [26] and resuscitation from neonatal hypoxicischemic encephalopathy [27] have been confirmed by multiple clinical trials. Numerous preclinical studies based on vascular recanalization models have shown that hypothermia exerts neuroprotective effects mainly by reducing the cerebral metabolic rate [28], but also involves cell death, inflammation and white matter integrity [29], and the temperature sensitivity of PAC also provides potential mechanisms [16]. Intra-arterial selective cooling infusion (IA-SCI), as a novel therapeutic method of hypothermia, utilizes the characteristics of high blood flow to the brain to directly perfuse hypothermic fluid into the cerebral arteries for rapid and selective cooling [30]. IA-SCI can precisely achieve regional cooling of brain tissue with a much faster cooling compared with traditional superficial cooling and systemic transvenous cooling and can be combined with mechanical thrombectomy to avoid the side effects brought by systemic cooling [31,32], which has been clinically validated in rodent models [33], large animal models [34] and ischemic stroke patients [35]. Precise cooling of the acidotic brain tissue in ischemic stroke by IA-SCI can elevate opening threshold of PAC, thereby shutting the channel, thereby reducing the influx of Cl-, improving cytotoxic edema, and achieving neuroprotection [10,12].

Conclusion

Targeted delivery of PAC blockers by smart nano-systems is highly specific and efficient and enables the gradual release of drugs to extend the time of drug exposure, reducing the risk of adverse side effects or off target toxicity. Compared with the simultaneous closure / blockade of multiple cationic channels associated with cellular edema (such as ASICs and NHEs, etc.), the blockade of a single and unique anion channel (PAC) by hypothermia has higher applicability and feasibility experimentally or clinically. Although there is currently a lack of sufficient experiments to validate the above two therapeutic strategies, smart drug targeting nanosystems and hypothermia therapy that inhibit PAC also pose an promising opportunity for translational trials to protect brain tissue.

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The Current State of Target Therapy for Subtypes of Gastrointestinal Stromal Tumors

Introduction

Gastrointestinal stromal tumors (GISTs) are the most common primary mesenchymal tumor of the gastrointestinal tract, although with low incidence. They are connective tissue tumor arising from the interstitial cells of Cajal (ICC) cells. The most common tumor location is the stomach (50%-70%), followed by the small intestine (25%-35%), rectum (5%-10%), and esophagus (<5%) [1]. The signs and symptoms of GISTs, which grow slowly and generally occur in 50 to 70 years old, are gastrointestinal hemorrhage, trouble in swallowing and metastases [2]. About 30% of GISTs can be defined as evil and led to metastasize occurring drug resistance during the treatment because of gene mutuation [3]. Some study show that the patients of GIST have mutations in PDGFR (PDGFRα or PDGFRA) but not in the wild-type GISTs without c-kit or PDGFRA mutations were accounted for 10-15% of the total number of GISTs (Table 1) [4] According to researchs, wild-type GISTs might lack succinate dehydrogenase (SDH) leading by mutations of SDH genes.The occurrence of GISTs are related with KIT/PDGFRA gene mutations, which c-kit mutations were accounted for 80~90% and PDGFRA mutations for 7% [5].
The radical surgery may be the only chance of cure, but the treatments of traditional radiation and chemotherapy are ineffective for advanced GIST (recurrent, inoperable or distant metastasis). In recent years, the targeted therapy of GISTs has obtained significant efficacy with the development of precision medical. Tyrosine kinase inhibitor (TKI) is the first-line treatment drug of GISTs, such as imatinib mesylate (IM). The locus secondary mutations of KIT/PDGFRA genes were key factors for prognosis. At the same time, the application of targeted drugs to achieve the maximum therapeutic effect has become the focus of research. The latest NIH classification system for GISTs declare that Mid- to Late GISTs are applicative for the adjuvant therapy, which preoperative IM is widely available. This article aims to make a summary of different genotype, mutation and molecular mechanism of GISTs, and the progress of targeted drugs.

Table 1: Genetic mutation in GIST.

Genetic Mutation in GISTs

KIT Mutation

C-kit gene is considered at the homologue of HZ4 feline sarcoma Virus KITs oncogene. C-kit proto-oncogene, which consisting of 21 coding exons in III type of protein tyrosine kinase receptor superfamily members, is located in chromosome 4q11-12. It is a kind of Kit protein receptor, which consist of extra-cellular region, trans-membrane region, near-membrane region and 2 tyrosine kinase (TK) region. Its ligand is stem cell growth factor (SCF). The most common mutations are exon11 mutations (70%), exon9 mutations (5%~10%), and exon13 mutations (1%) [6]. Exon14, 17, 18 mutations are relatively rare [7]. The main mutation types are deletion mutation, point mutation, mixed mutation and insert mutation [8].
When c-kit mutated, CD117 protein expresses and forms a dimer autonomously without the participation of the ligand. Therefore, it cannot precisely regulate the differentiation, proliferation, and programmed cell death. Finally, c-kit mutations can cause more cells enter the neoplastic hyperplasia stage from the quiescent stage, which may be one of the key mechanisms causing malignant transformation of GIST. Studies have found that tumors with c-kit exon11 deletion are more likely to appear in the patients who are older than 50 years, with a tumor diameter of 5~10cm and the NIH grade as high risk. Moreover, the secondary and high-risk GISTs are susceptible to secondary mutations that cause recurrence and metastasis because of the deletion of exon11 of c-kit gene [9], which has potential value for predicting poor prognosis of patients.

PDGFRA Mutation

PDGFRA gene stretches approximately 65 kb on human chromosome 4 (4, q11-13) and consists of 23 exons in III type of protein tyrosine kinase receptor superfamily members, including exon3-10 coding region exocellular five immunoglobulin sample, exon11 coding intracellular membrane area, near exon13-15 and exon17-21 coding intracellular kinase 2 cheese ammonia acid (tyrosine kinase, TK) area. Similar to c-kit, PDGFRA gene mutation is also a function-acquired mutation and most of them occur in CD117 negative GISTs [10]. In the absence of ligand binding, the autonomous dimerization leads to cell division and proliferation by promoting DNA synthesis through downstream signaling pathways, thus the growth, proliferation, attachment, metastasis, differentiation, and apoptosis of cells are regulated. The mutations of PDGFRA essentially clustered in three regions and the most common of these is exon18 that accounting for 82.5%, the most common site is D842V [11-13]. The process of GISTs formation by PDGFRA mutation is similar to c-Kit, the tyrosine kinase receptor encoded by PDGFRA located on the cell membrane is abnormally and continuously activated, resulting in the loss of autoinhibition function.

Wild-Type GIST

The morphology of wild type GIST is in line with the GIST, CD117 positive or negative expression, at the same time cannot detect c-kit and PDGFRA gene mutations, but often coupled with abnormalities in the structure or expression of other genes, such as, subunits of succinate dehydrogenase (SDH) complex, Rapidly accelerated fubrosarcoma B (BRAF) gene, neurofibromatosistype1 (NF1) gene mutation, multiple gene fusion and other abnormalities. The most common type in the wild type is the SDH expression deficient or decreased, which can be accompanied by other neoplastic diseases, including paraganglioma, pulmonary chondroma, pheochromocytoma, etc. At first, the disease was recognized as Carney triad and Carney Stratakis syndrome [14]. The high expression of insulin-like growth factor 1 receptor (IGF-1R) receptor RNA or protein in the GISTs due to SDH gene defects is detected，IGF-1R binds to ligand and is activated by autophosphorylation, leading to the activation of mitogen-activated protein kinase (MAPK) and phosphatidlinositol 3-kinase (PI3K) cascades [15].
IGF signaling can inhibit IGF-1R-induced apoptosis in SDHdeficient GIST cells and inhibit the signaling of Akt and MAPK pathways in GIST cells [16]. For BRAF gene mutant GIST, most of the mutation sites are V600E of exon 15, which can affect the function of PI3K [17], BRAF mutations have been found in a small number of patients with imatinib (IM) resistance, suggesting that BRAF mutations may be the cause of secondary resistance [18]. KRAS mutations are also one of the wild-type GIST, some scholars analyzed the gene sequence of 578 cases of GIST and found no KRAS mutation, So KRAS mutations may be extremely rare [19].

Other Biological Markers

Many research experiments show that c-kit mutations represent a poor prognosis in high-risk GISTS, PDGFRA mutations associated with less malignant GIST [20]. However, these alone are not absolute and require more biological indicators related to prognosis and efficacy. In addition, the proliferation index Ki-67 is a very effective marker for predicting the aggressive behavior and malignant potential of GIST and can be employed as an independent predictor of GIST [21]. P16 expression is also related to the malignant of GIST [22]. Moreover, there are evidence from 19 studies showing p53 have the predictive value in the risk of GIST [23].

TKI Therapy in GIST

Complete surgical resection is the only chance for cure for the disease, but there is still a possibility of recurrence and metastasis. For advanced (unresectable or recurrent, metastatic) GISTs, molecular targeted therapy is the preferred treatment [24].

First-Line Imatinib Therapy for Advanced GIST

Imatinib is a derivative of 2-phenylaminopyrimidine, which is a kind of small molecule selectively activated tyrosine enzyme inhibition agent to inhibit the BCR-ABL protein, ABL, KIT and PDGFRs. Though selective inhibition of tyrosine kinase lives sex, blocking the phosphate group on tyrosine residues. In 2002, the FDA approved STI571 for the treatment of unresected or metastatic GIST. Demitri et al conducted a trial, 147 patients with advanced GIST were randomly assigned to receive 400mg and 600mg of imatinib per day. The results showed that imatinib has achieved a good objective response in patients with advanced GIST, and there were no significant differences in the safety of treatment dose [25]. Subsequent phase 2 and phase 3 trials of metastatic GIST also confirmed the efficacy of imatinib in advanced GIST (Table 2). Treatments which provided the sites to inhibit tumor cell proliferation were widely applied advanced and metastatic tumor, and the preoperative and postoperative adjuvant therapy.
However, an effective dose of imatinib cannot inhibit other open sites in the tyrosine kinase pathway except for c-kit or PDGFRA gene mutations, allowing cell proliferation signals to bypass cell proliferation inhibition caused by imatinib, c -kit exon11 mutant GIST is generally sensitive to imatinib and not sensitive to exon9 and PDGFRA mutation types [26]. Further research found that the relationship between kinase genotype and treatment outcome, Kit exon11 genotype mutation has better benefit than other genotype mutations [27]. Our previous meta-analysis estimated the imatinib treatment for different genotypes of GIST and found that personalized treatment makes patients with exon 11 mutation more profitable [28]. To optimize the treatment of imatinib, a metaanalysis of 1,640 patients with advanced disease showed that the patient could get a small PFS advantage of 2 times the standard dose (800mg/d) of imatinib, but no significant difference in OS between the dosages, especially in kit exon9 mutation.
Compared with exon11 mutation, exon9 mutation and wild mutation have a worse prognosis [29]. Moreover, Asian Consensus Guidelines agreed that a higher dose may also be beneficial in Asian patients with KIT exon 9 mutation [30]. To assess longterm survival with two doses of imatinib, one long-term result of a randomized trial showed the 800mg/d group had a better 10- year PFS rate and an average OS rate than the 400mg/d group [31]. Whether patients with advanced GIST benefit significantly in terms of long-term survival and will they differ due to the differences of mutations? An analysis of Phase 3 SWOG Intergroup Trial S0033 showed that imatinib as a first-line drug in advanced GIST has resulted in long-term survival of 10 years for a significant number of patients, especially those with KIT exon11 mutations or KIT/ PDGFRA mutations lacking (mainly succinic dehydrogenase mutant tumors) [32]. Most people can benefit from imatinib, but this result does not lasting. Many patients will initially be resistant even if they were recommended to extend the duration of medication from one year to three years [20].
GIST resistance of imatinib can be divided into primary and secondary, primary resistance is no response to imatinib treatment, while secondary resistance occurs 6 months after the initial treatment is effective. The secondary gene mutation of KIT/ PDGFRA is considered closely related to secondary resistance. The secondary mutation of kit occurred in exons 13, 17, 14 [33] and occurred in exon 18 of PDGFRA [34]. Further research found that heterogeneity of KIT secondary mutations is the principal mechanism of tumour progression to KIT inhibitors in imatinibresistant GIST patients [35]. The sensitivity of PDGFRA mutant GISTs to targeted drugs is obviously worse than that of C-KIT, while the exon 18 D842V mutation of PDGFRA mutant GISTs is also primary resistance to imatinib, so it is difficult to achieve satisfactory efficacy even if the dose of imatinib is increased [36]. Many resistance mechanisms are still being researched due to the diversity of mutation sites, heterogeneous multitarget inhibitors and precise personal treatment are more worthy of promotion.

Table 2: Some important trials about TKIs.

Second-line Sunitinib for Advanced GIST

Secondary drug resistance occurred in more than 50% of patients after treatment with imatinib, thus the second-line treatment drugs such as sunitinib, an oral multitarget tyrosinase inhibitor, emerged. The growth, proliferation and metastasis of malignant tumors were affected by blocking the signaling pathway by inhibiting tyrosine kinases such as VEGFR, PDGFR, KIT, and RET. Sunitinib is considered as a second line TKI because of its considerable benefit in patients with advanced imatinib resistance and intolerance. A randomized, blank-control phase 2 clinical trial of random taking sunitinib 50mg / d and placebo in the blank control group. The results of this experiment showed that progressionfree survivals were 27.3 weeks and 6.4 weeks, respectively. Overall survival was 24.1 weeks and 6 weeks, respectively (Table 2) [37]. However, Lile Wu et al. conducted a meta-analysis clinical efficacy of seconed-generation TKI in imatinib-resisitant GISTS showed that sunitinib are effective for improving PFS but not OS in patients with imatinib-resistant GIST [38].

One of our previous pooled analysis showed that sunitinib treatment after imatinib resistance varied according to the genetic subtypes of GIST. Compared with PDGFRA, the mutation of KIT gene showed better clinical rate of cure, especially the mutation of KIT exon 9, 11. Furthermore, the mutation cure rate of KIT exon 9 was better than that of exon 11 [39]. Other second-line TKI drugs such as nilotinib, a tyrosine kinase activity of ABL/BCR, and KIT, PDGFRs, significantly improve PFS [40]. In a phase III study of nilotinib versus imatinib as first-line therapy for unresectable or metastatic GIST, Blay JY et al. divided 324 advanced patients on nilotinib 400 mg twice daily and 320 advanced patients on imatinib 400 mg daily. The results showed that the 2-year progression-free survival rate was better in the imatinib group (59.2%) than in the nilotinib group (51.6%). There was no difference between the two groups for progression-free survival of KIT exon 11 mutation, but imatinib group was better than nilotinib of the exon 9 mutation. Therefore, in the future, first-line drugs need to be determined based on subtype analysis [41].

Third Line Regorafeniband Ripretinib for Advanced GIST

Regorafenib, a multikinase inhibitor was candidated as a third-line treatment option for patients with advanced GIST, which is recommended after failure of both high-dose imatinib and sunitinib. In a randomized phaseIII trial of regorafenib GRID, the results showed that median progression-free survival was 4.8 months for regorafenib and 0.9 months for placebo, with no significant difference in overall survival [42]. Another study of regorafenib showed the efficacy and safety of manageable profile in Japanese were consistent with the overall GRID study population of patients with advanced GIST. As a third line TKI, the effectiveness of regorafenib has been proven in GIRD, however, frequent dose reductions were required of the administration plan. In a study involving 25 patients who failed treatment with imatinib and sunitinib, low-dose continuous treatment with regorafenib showed that the disease control rate for at least 3 months in 64% of patients and had a median progression-free survival of 7.3 months. Maybe this method of administration has become a better choice [43,44].
In addition, regorafenib prolonged progression-free survival of patients with advanced mutations in exon17 [45-48]. Another threeline targeted therapy, ripretinib is a Ⅱ type switchpocket inhibitor that binds to the switchpocket and acts as a structural substitute for an inhibitory switch, preventing the activation loop from entering the switchpocket, thus locking the kinase in an inactive state and inhibiting downstream signaling. Ripretinib has strong inhibitory effects on different secondary drug resistance mutations because it is acting on the final link of the kinase pathway [49-50]. However, clinical data on ripretinib therapy and genotyping have not been reported yet, so there may be differences in the efficacy of ripretinib in GIST with different genetic mutation types.

Other TKIs

Other TKIs identified in clinical trials, such as sorafenib, as a third line / four-line TKI. A Korean clinical trial used two or more TKIs to treat unresectable or distant metastic, 36% of patients had more than six months of disease control after using sorafenib [51]. Moreover, including vatalanib (PTK789), masatinib (AB1010), pazopanib (PAZOGIST), dasatinib (NCT0056875) (Table 2) [52-54].

Conclusion

Despite the many benefits of targeted drugs for advanced GIST, drug resistance presents a new challenge. Through the understanding of the mutation sites of GIST and the collection of information about current treatment regimens, we can see that the efficacy of targeted drugs in different genotypes still needs a large number of standardized clinical open trials. Similarly, to leverage the strength of targeted drugs to make up for the shortcomings, personalized treatment and evaluation are essential. New therapeutic ideas should be applied to more actions, such as the combination of targeted and chemotherapy drugs, new targets to be discovered, updated TKIs and combined immunotherapy. Further studies on drug resistance mechanisms and new molecular markers are the breakthrough points for the treatment of advanced GIST to optimize the treatment of advanced GIST.

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High Resolution Melting Curve for the Rapid and Efficient Detection of SARS-Cov-2 Gene Variation in the Greek Population

As of May 6, 2021, approximately 153 million cases of Coronavirus disease 19 (COVID-19) have been reported worldwide [1]. Due to the rapid spread of the causative SARS-CoV-2 corona virus, the development of a quick and accurate detection assay is considered vital aiming to control the possible sources of infection, in order to design effective measures to prevent further transmission. Routine laboratory confirmation of SARS-CoV-2 infection is based on nucleic acid amplification tests (NAAT), such as the real-time reverse transcriptase-PCR (RT-PCR), which is adopted as a simple qualitative assay that combines a relatively high sensitivity [2] with high specificity for the detection of the virus [3,4]. The most commonly used targets for primer/probe development derive from the conserved viral genome, including the ORF1ab, RNA dependent RNA polymerase (RdRp), nucleopcapsid (N), envelope (E) and spike protein (S) genes [5-7]. Although SARS-CoV-2 quasispecies variants can be most accurately identified through whole genome sequencing or alternatively by the means of Sanger or next generation sequencing amplicon-based sequencing of selected parts of the viral genome [1], these methods are regarded both as time consuming and expensive.

Thus, a simple and more rapid screening approach is needed to be developed, for the key SARS-CoV-2 mutations that define variant strains. High Resolution Melting (HRM) is a novel, homogeneous, close-tube, post-PCR method, enabling genomic researchers to analyze genetic variations either as single nucleotide polymorphisms (SNPs), point mutations, or methylation degree in PCR amplicons. This assay prevails the power of a classical melting curve analysis by enabling a detailed study in the thermal denaturation of a double-stranded DNA, providing us finally sufficient information. In this study we tested the possibility that a Real-Time PCR combined with HRM assay will serve as useful and efficient diagnostic technique for the detection of SARS-CoV -2 variables in 4 viral genome locations, namely RdRp, N1, E and S2 genes. The High-Resolution Melting Curve was performed in a post RT-PCR assay, and amplified a fragment of these genes, in order the generic diversity of the SARS-CoV -2 can appropriately be examined. Our hypothesis was that this method can be adopted as a valuable tool for the rapid screening of large numbers of patient samples for the tested variants, providing an early warning for the emergence and spread of these strains of concern.

Materials and Methods

Clinical Samples

A total of 620 clinical specimens collected in the municipal area of West Attica and sampling was accomplished to our premises with all the required precautions. All volunteers were suspicious COVID-19 cases, according to World Health Organization criteria (World Health Organization, 2021c). We only collected nasopharyngeal swabs, which subsequently were placed in 2 ml of transport medium with neutralizing agent. We used Disposable Virus Sampling Tube (Zybio; Inc; China) which adopts efficient virus inactivation technology and special flocked swab. It can be used for the collection and storage of clinical novel coronavirus, influenza, avian influenza (such as H7N9), hand-foot-mouth virus, measles and other virus specimens, as well as for chlamydia, mycoplasma, and ureaplasma. Specimen processing was performed in a class II biological safety cabinet using biosafety level three (BSL3) work practices.

RNA Extraction

Nucleic acids were recovered from clinical specimens using an automatic extractor (MagDEA DNA / RNA 200 virus), The RNA samples were separated in two aliquots.

qRT PCR

The first aliquot was used for the qRT-PCR using the Mutaplex SARS-CoV-2 commercial kit (Immundiagnostik AG). Specific primers were used for highly conserved regions and double-labeled probes to enhance and differentiate RNA SARS-CoV-2 and other beta Coronaviridae such as MERS. Detection of SARS-CoV-2 was visualized at the FAM / GREEN channel. Beta-coronaviruses (SARSCoV- 1 and SARS-CoV-2) are detected at Cy5 / RED channel. Internal Process Control (IPC), which was added during RNA extraction, was detected in the same reaction at HEX/ YELLOW. Detection of RNA Polymerase (human gene) allows RT-PCR detection of inhibitors confirming in addition viral RNA was isolated from specimen.

cDNA Synthesis

The second aliquot was used for the cDNA synthesis following a qRT-PCR with a post High Resolution Melting Curve. cDNA was synthesized using Luna Script® RT SuperMix Kit (New England Biolabs). A 5 μL aliquot of purified RNA was added to 4 μL of the Luna Script® RT SuperMix Kit. The reaction was performed in a total volume of 20 μl.

qRT-PCR and HRM Assay

5 μL cDNA was added to 20 μL of six different reaction mixtures containing 500 nM each primer. The primer sequences used for RNAdependent RNA polymerase gene detection were FW 5’AGA-ATA-GAGCTC- GCA-CCG-TA3’and REV 5’ CTC-CTC-TAG-TGG-CGG-CTA-TT-3’ giving an amplified product of 101bp. The primer sequences used for E gene detection were FW 5’TTCGGAAGAGACAGGTACGTTA-3’ REV 5’AGCAGTACGCACACAATCG-3’ giving an amplified product of 116bp. The primer sequences used for N1 gene detection were FW 5’CAATGCTGCAATCGTGCTAC-3’ and REV 5’GTTGCGACTACGTGATGAGG-3’ giving an amplified product of 117bp. The primer sequences used S2 gene detection were FW 5’GCTGGTGCTGCAGCTTATTA-3’ and REV 5’AGGGTCAAGTGCACAGTCTAA-3’ giving an amplified product of 107bp. 25-μL reaction was setup that contained 5 μL of cDNA, 12.5 μL 12.5μL Melt Doctor master mix, which includes HRM dye (Melt Doctor Applied Biosystem).

PCR Cycling for HRM Curve Acquisition was Run Under the Following Conditions

One cycle at 95°C for 10 min; 40 cycles at 95°C for 15 s, 60°C for 40 s, and 72°C for 30 s; Then the fragment was melted by raising the temperature from 60°C to 95°C, with an increment of 0.11°C/s, in order to obtain information on melting profiles. Melting-curve analysis was performed using the HRM software of Applied Biosystems This software analyzes the HRM curve data to identify changes in the shape of the curve that indicate sequence polymorphisms.

PCR Test for cDNA Quality

As an optional step, a cDNA quality test was performed after cDNA synthesis to verify the appropriate synthesis of the cDNA from each sample. GAPDH genes of Homo sapiens with primer set FORW 5’ CAA-TGA-CCC-CTT-CAT-TGA-CC.3’ and REV 5’ TTG-ATTTTG- GAG-GGA-TCT-CG was used for the human IPC. In this PCR protocol, 5 μL of cDNA was used with the following PCR cycling conditions: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 60 °C for 40 s, and 72 °C for 1 min, with the final elongation step at 72 °C for 5 min. Each primer was used at a concentration of 500 nM in 2× PCR premix reagent (Promega Hot Start Green Master Mix). The amplicons were subjected to electrophoresis in a 2% agarose gel at 130 V for 20 min and visualized under UV light giving an amplicon with 159bp.

Standard Curve and Limit of Detection (LOD)

The real-time PCR with Melt Doctor standard curve was generated by serial 10-fold dilutions of synthetic positive controls in RdRp, E, N1 and S2 genes with known copy numbers (10.000, 1.000, 100, 10 and 1 copies/μL). These dilutions were tested using 10 replicates and they were used as quantification standards to construct the standard curve by plotting the copy number against the corresponding threshold cycle values (Ct). To verify the specificity of the reaction, the melting curve analysis and electrophoresis on agarose gel were carried out for the products of the real-time PCR reaction. Five microliters of the amplicons were electrophoresed in 2% (w/v) agarose gel stained with ethidium bromide in 1x (TBE) buffer and visualized by ultravioltet (UV) light in order to check All specimens were aliquoted at reception and those not used in the assays were stored at -800C for later confirmation of PCR results. Positive results were considered valid when the PCR results matched in two different aliquots and analyzed with the commercial kit and the real-time PCR with HRM assay.

Biomedical Ethics Issues

The collection of clinical data from volunteers, were correlated with the laboratory research results and were conducted in such a way as to fully guarantee the patients’ anonymity and personal data confidentiality.

Statistical Analysis

Statistical analysis. Standard statistical analyses (average, standard deviation, correlation coefficient) and graphing were performed using Microsoft excel (ver 2102) for Windows.

Results

The linearity and efficiency of the real-time PCR were determined by generating a standard curve in which serial 10-fold dilutions of positive control were tested. The standard curve was generated by plotting the real-time PCR threshold cycle numbers (Ct) of each dilution against the known copy numbers of positive control. The resulting slope showed a linear relationship over 5 orders of magnitude, ranging from 10.000 to 1 copies/μL with a correlation coefficient R2>0.99. The detection rate was 100 % for up to 2,5 copies/μL having 10/10 replicates positive for E and N genes and 5 copies/μL having 10/10 replicates positive for S and RdRp genes. Strong linear correlations (r² ≥0.99) were obtained between C_T values and transcript quantity. Assay reproducibility and repeatability was tested by using replicate 10-fold serial dilutions of the RNA transcripts evaluated for each dilution point in triplicate on three different days. At the lower copy detection limit for SARS-CoV-2 and assay reproducibility exceeded 95%. Over the linear range of the assay, the coefficient of variation of the mean C_T values within and between runs was 0.46%–2.54% and 0.64%– 2.39%, respectively.

The methods have thus been shown to be highly capable of detecting the novel SARS-CoV-2 with 100 % specificity. The specificity (100%) of the reactions was confirmed by a melting temperature for positive control dilutions, indicating the formation of a single PCR product with no artefacts, such as nonspecific amplification products or primer dimers (results not shown). Furthermore, amplification products were also checked on agarose gel stained with ethidium bromide in standard TBE buffer and clear and well-defined specific bands with the expected sizes for all replicates of positive control dilutions. All 620 RNA samples tested, were positive for the human gene (GADPH) which was included as internal control to evaluate the quality of clinical specimens (nasopharyngeal swabs) and nucleic acid extraction. From the 620 RNA specimens, 60 were tested positive for SARS-CoV-2 indicating a prevalence of 9,7% by both methods. The Ct Value ranged from 19 to 36 that corresponded to 10.000- 1 copy numbers. The qRT-PCR and the real-time PCR with HRM assay displayed 100% sensitivity.

A 50% of the specimens tested comprised 10.000 to 1.000 copy numbers of E and RdRp amplicons. Sixty and 66,7% of the specimens tested, contracted 10.000 to 1000 copy numbers of the S2 and N1 genes, respectively. A range of 1000 to 100 copy numbers was also identified in 33.3% of the specimens for the RdRp gene. A range of 100 to 10 copy numbers was further identified in 40 and 33.3% of the specimens for the S2 and N1 genes, respectively. Finally, a range of 10 to 1 copy numbers was identified in 50 and 16.7% of the specimens of the E and RdRp genes. All the sixty positive for the virus-RNA samples were synthesized to cDNA following a qRTPCR with a post High-Resolution Melting Curve. The HRM diversity assay was used to analyze the regions of the four genes. As shown in Tables 1-4 and Figure 1, all SARS-CoV-2 positive samples produced measurable Tm values. The E gene demonstrated the highest variation with three clusters, with 66.7% belonging to cluster I. N1 gene amplicons identified two clusters with 50% of the specimens in each cluster. The S2 gene did not show any variation, forming only one cluster. Similar results were shown for RdRp gene, where 95% of amplicon belonged to one cluster (Table 5).

Figure 1: High Resolution Melt Curve This is an aligned melt curve. The plot demonstrates the sharp decrease in fluorescence when the double-stranded DNA melts into its single-stranded form. Diversity of positive specimens of gene Rdp.

Table 1: Diversity of positive specimens of gene E.

Table 2: Diversity of positive specimens of gene Rdp.

Table 3: Diversity of positive specimens of gene S2.

Table 4: Diversity of positive specimens of gene N1.

Table 5: Quantification of positive samples.

Discussion

SARS-CoV-2 is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory disease, named Covid-19, which threatens human health and public safety. Little is known about the genetics of this virus but the health communities have to respond to any new viral genetic variants, pursuing immediate countermeasures. However, it is inevitable that in a limited number of individuals a new virus population during transmission will be established. Thus, remains crucial the concerning variants to be early detected by the means of rapid and effective assays and therefore we need ascertained genomic epidemiology projects for large scale monitoring of the SARS-CoV-2 evolution. High Resolution Melting Analysis is a molecular technique based on RT-PCR that has been largely used to rapidly detect other pathogen strains, resistant to treatment. We developed the first homemade RT PCR combined with high resolution melting curve for the detection and screening of Sars-CoV-2 variants.

Although detection of the viral nucleic acid using an RT-PCR assay has become a standard and formative assessment for the diagnosis of COVID-19 [8,9] the main concern in RT PCR are the false negative results usually attributed to the low quality of the specimen and to the inappropriate sample handling. In order to be ensured that our samples had a suitable genetic material for RTPCR and adequate portion for HRM would be then extracted, we used Internal Positive Control primer sets of GADPH genes of Homo Sapiens [10,11]. All of the 620 samples studied were positive for GADPH gene, confirming therefore the quality of the extracted RNA and the converted cDNA portions. The quantification of SARS-CoV-2 RNA in clinical specimens by reporting Ct values and copy numbers of RT-PCR is generally limited [12], but this is due to the current needs of diagnosis. Efficacy of RT-PCR in the diagnosis of SARSCoV- 2 infection is greatly dependent on the pre-analytical phase, including the patient selection and material collection.

Even more the extraction method of RNA and the performance of RT-PCR test kit [12] interfere significantly with the results. Although Ct values are affected by a number of factors, they may be still able to provide important clinical data for decision making by providing an indication of viral load [13]. A positive correlation has been demonstrated between the viral load and either the severity of COVID-19, or the intensity of hypoxemia, the risk of death, or with various other hematological, biochemical, and inflammatory alterations [14,15]. As we were not aware of the complete medical history for the majority of the volunteers, the detected low Ct numbers in the 50% of the examined specimens. This probably corresponds to a high viral load of the sample which was taken 3-6 days following the symptoms onset from individuals residing in an area with high COVID-19 prevalence. Most of our study participants did not suffer from any underlying diseases and they received home-based treatment, while none them was hospitalized for any reason. In specimens that were taken 15 days later, it was shown to have higher or negative Ct values, in comparison to samples taken in the beginning of the infection (data non shown), indicating somehow a recovery status.

Although reverse transcription PCR remains the most sensitive and accurate method for the detection of the new coronavirus, the use and the sufficient supply of commercial kits for this purpose may not considered as a cost-effective solution [16]. Currently RT PCR using different sets of primers and dyers, is applied for the detection of variants [13,11,12]. However, HRM assay seems to be a promising tool, when it is combined with RT-PCR, even without any other previous testing. Even more HRM technique can be noted as a rapid, low-cost, and, high-throughput method for quantifying genetic diversity. Using thermal denaturation of double-stranded DNA, the technique extracts significant details and is capable of finding SNPs. With this method we are able to identify smaller differences in PCR amplicons, down to the single base level and therefore this method is accepted as an ideal procedure for single nucleotide polymorphism genotyping, species identification, sequence matching and mutation scanning, without the need for any further separation and additional processing following a PCR. Several HRM assays have already been successfully conducted in the past and have been applied for the detection and genotyping of viruses such as HIV[17], astroviruses [18], polyomaviruses [19], noroviruses [20] and influenza A viruses [21].

To our knowledge, no study has been conducted as yet, using high-resolution melting analysis (HRM) technique, for the rapid detection of variations within S, N, E and RdRp genes of SARS-CoV-2. In our study the highest variation with three clusters was identified in the gene E which encodes the Envelope protein E proteins, that help in the assembly and release of the virions [22]. The E protein plays important roles in viral morphogenesis, replication, and pathogenesis [23] and is conserved in coronaviruses [24]. Among the structural proteins of the SARS-CoV-2, E protein is considered as a potential drug target. However, according to the GISAID database (as of 25th May 2020), more than 40 amino acid mutations of the E gene were found from 4085 SARS-CoV-2 genomes. The E gene of SARS-CoV-2 seemed to have a high mutation rate and thus it would be difficult to synthesize an effective antiviral molecule aiming to E gene expression which may be carrying a diverse population of different strains.

Nucleocapsid proteins (N) play an important role in the packaging of viral RNA and mediate viral assembly by interacting with the viral genome and M protein, which are helpful in the augmentation of viral RNA transcription and replication [25]. Based on the high sequence similarity of N protein within the coronavirus family, it may be suggested that antibodies against the N protein of SARS-CoV would likely recognize the N protein of SARS-CoV-2. Although our results indicate variations divided in 2 distinct clusters, N gene show highly conserved regions and therefore N proteins are also considered as potential drug targets. Nevertheless, the clinical relevance of N2 negativity is considered to relate with asymptomatic or subclinical disease course as was the case of our volunteers [26]. S gene encodes the spike (S) protein which interacts with angiotensin-convertin enzyme 2 (ACE2). The inhibition of this association is a possible target for the development of novel therapeutic approaches [27]. The S2 subunit mediates the viral cell membrane fusion and the detected gene stability in our study is encouraging the idea that inhibiting the gene expression, would serve as a potent therapeutic intervention with constant effect for the prevention of disease transmissibility and pathogenesis.

The lack of any S2 variability seen in our study is providing confidence that we will not face a potentially compromised vaccine effectiveness in Greece for the near future, since S protein serve as the major viral antigen for the current vaccines. RNA-dependent RNA polymerase (RdRp), is considered a promising but challenging drug target for inhibiting replication and hence, the growth of various RNA-viruses. The widely used antiviral drug Remdesivir has an anti-RdRp activity and various under development other potent prospective drug candidates against the SARS-CoV-2 are targeting RdRp proteins. The absence of any variations in our results in regard of RdRp gene empower the steady effect of this therapeutic approach in mitigating the disastrous global effects of the COVID-19 pandemic. The emergence of SARS-CoV-2 variants which may be proved potential for increased transmission, disease severity, and resistance to vaccine induced immunity is of grave concern. A simple screening assay to monitor the emergence and spread of these variants may be helpful for epidemiological studies. With our study it was demonstrated that our assay using the highthroughput PCR assay platform is a simple, rapid, and sensitive and specific tool for detecting variant-identifying mutations.

Our HRM assay may reinforce the further investigation of the novel coronavirus diversity and the detection of newly emerging virus variants. High-Resolution Melting Assay will also possibly lead to a reduction in the need of sequencing techniques by the exclusion of the samples with the same HRM curves. Therefore, it could be then adopted as an inexpensive molecular tool for public health screening studies in order to pursue SARS-Cov-2 variants. Probably the most important limitation of the method is that it may not reveal all the sequence variations in a cDNA fragment. The limitations regarding the detection method of our home set up RTPCR was regarded similar to the commercial kit used in primary diagnosis of SARS-CoV-2 2 infection in the routine laboratory. Another limitation of the study is the rather small sample study, but the need for the prompt circulation of all relevant information regarding the SARS-CoV-2, convinced us to submit our preliminary results. Our data may then facilitate the design of a universal admission screening course.

Conclusion

A home-made RT-PCR, determining separately the expression of four gene targets, is useful for the current needs of SARS-CοV-2 laboratory diagnosis. Post RT-PCR, High Resolution Melting curves (HRM) could be considered as a rapid screening method for the identification of variants and strains of concern. Improvements of this method and further research are required in order to monitor effectively the virus evolution and the corresponding host immunity.

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