The Implementation of Disaster Management Life Cycle During the Earthquake 2005 in Kashmir Pakistan and the Disaster Response of EMS Services

Organizations involved:
1. Earthquake Reconstruction & Rehabilitation Authority Pakistan (ERRA since 2005)
2. NDMA (National Disaster management authority Pakistan (Since 2010)
3. US Marine and Army helicopters from Afghanistan, Pakistan Army and retired.
4. UN, WHO, EU, OIC, UNDP, UNESCO, UNICEF, Oxfam, ICRC-red crescent, JEN-Japan Others.

Management Cycle

(Figures 1 & 2) [2].

Figure 1: Management cycle (Google Disaster life cycle, 2021).

Figure 2: Management cycle (Google Disaster life cycle, 2021).

Kashmir Earthquake 2005 [3]

The 2005 Kashmir earthquake occurred at 08:50 am Pakistan Standard (Wikipedia, 2021) Time on 8 October in Pakistani Azad Kashmir. It registered a moment magnitude of 7.6 and had a maximum Mercalli intensity of VIII (Severe) or XI (Extreme). The earthquake also affected countries in the surrounding region where tremors were felt in Afghanistan, Tajikistan, India and the China Xinjiang region. The severity of the damage caused by the earthquake is attributed to severe upthrust. Over 86,000 people died a similar 100,000 number were injured and millions were displaced. It is considered the deadliest earthquake to hit South Asia (Table 1).

Table 1: The magnitude of Disaster because of Earthquake 2005 in Kashmir Pakistan and disaster lifecycle.

Preparation [4]

Disaster preparedness: 5 key components to effective emergency [4] management were used in the 2005 Kashmir earthquake.
1. Clear communication.
2. Comprehensive training.
3. Knowledge of assets.
4. Technology fail-safes and protocol.
5. Healthcare leadership involvement. Disaster response in the early phase of [5] earthquake relief is complex with local facilities often overwhelmed and damaged. Coordinated effort is required for success with lessons learnt to improve future disaster management.

Financial Assistance and Aid [6]

In late 2006 a staggering $20 billion USD development scheme was mooted [6] by Pakistan for reconstruction and rehabilitation of the earthquake hit zones in Azad Kashmir. A land use plan for Muzaffarabad city had been prepared by Japan International Cooperation Agency. Countries of Asia, Africa, EU, Americas, Oceana, Multinational organizations, NGOS – On November 19, 2005, it was estimated that the international community as a whole pledged about US$5.8 billion. (Wikipedia 2005 Kashmir earthquake, 2021) [7] (Figures 3-5).

Figure 3: (Wikipedia 2005 Kashmir earthquake, 2021).

Figure 4: (Wikipedia 2005 Kashmir earthquake, 2021).

Figure 5: (Wikipedia International response, 2021).

Mitigation [8]

Risk mitigation refers to the process of planning and developing [8] methods and options to reduce threats or risks to project objectives. The following five strategies can be used in risk mitigation planning and monitoring.
1. Assume and accept risk.
2. Avoidance of risk.
3. Controlling risk.
4. Transference of risk.
5. Watch and monitor risk.
Although susceptibility zoning maps represent [9] a powerful tool in natural hazard management caution is needed when developing and using such maps. The October 2005 earthquake triggered several thousand landslides in the Lesser Himalaya of Kashmir in northern Pakistan and India. Preliminary results from repeat photographs from 2005 and 2006 after the snowmelt season reveal that much of the ongoing land sliding occurred along rivers and roads, and the extensive earthquake-induced fissuring. Although the susceptibility zoning success rate for 2001 was low many of the co post seismic land sliding in 2005 occurred in areas that had been defined as being potentially dangerous on the 2001 map [9]. Within a designated study area of 2250 km2 the number of landslides increased from 369 in 2001 to 2252 in October 2005.

Response [10]

Disaster response is the assistance and intervention [10] during or immediately after an emergency or disaster. Focus is on saving lives and protecting community assets (buildings, roads, animals, crops, infrastructure). Usually measured in hours, days or weeks. Immediately after the earthquake occurred the largest rescue and relief [11] operation was launched in the history of Pakistan. The Pakistani Army was directed to extend help to the civilian population in the quake affected areas and all civilian and military hospitals were directed to deal with the situation on an emergency basis. Many countries international organizations, and nongovernmental organizations offered relief aid to the region in the form of donations as well as relief supplies including food, medical supplies, tents, and blankets. International rescue and relief workers brought rescue equipment including helicopters and rescue dogs.

Recovery [12]

During the recovery period, restoration efforts occur concurrently with regular operations and activities. Preventing or reducing stress related illnesses and excessive financial burdens. Rebuilding damaged structures and reducing vulnerability to future disasters. It posed unique challenges and efforts on a massive scale for [13] reconstruction. For residential buildings the Pakistan government adopted a house owner driven approach. The reconstruction policy stated that the government and other agencies would provide equal technical assistance and subsidy to each family without differentiating between who lost what. To increase capacity in earthquake resistant construction large scale training of artisan’s technicians, engineers, and community mobilisers has been conducted. Campaigns to “build back better” have raised awareness in the communities. Local Housing Reconstruction Centers have been established for training advice and [13] dissemination of earthquake resistant technology. This decentralized approach has helped in achieving reconstruction smoothly [14].

Conclusion

To conclude, the importance of disaster medicine and disaster management is well recognized in last decades. The role of disaster management cycle with steps, preparation, mitigation, response and recovery with detail efforts enables the EMS of the countries to help during disaster prevention and recovery.

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Perception of Quality of Life of People with Kidney Transplantation and Transplant Candidates in Merida, Yucatan, Mexico

Introduction

Chronic kidney disease (CKD) affects around 11% of the population over the age of 20 globally, with an increase in incidence in recent years [1]. Peritoneal dialysis, hemodialysis and kidney transplantation are treatments that have been effective in increasing the life expectancy of people with CKD [1,2]. In the last three decades, the analysis of quality of life has been integrated as an indicator of the evolution of health status in patients with CKD to see beyond the number of years of survival. Quality of life is, according to the WHO, “the perception that an individual has of his place in existence, in the context of the culture and value system in which he lives and in relation to his objectives, his expectations, his norms, his concerns. It is a concept that is influenced by the physical health of the subject, their psychological state, their level of independence, their social relationships, as well as their relationship with the environment.” This concept encompasses objective and subjective aspects that reflect the degree of physical, emotional, social and economic well-being of each individual. The analysis of the quality of life in people with CKD allows us to understand the impact of the disease and its treatment, to know more about patients, how they evolve and how they adapt to organic alteration [3,4]. Currently, the analysis of quality of life in people with CKD seeks to generate evidence, qualitative and quantitative, to facilitate: the process of assessing human needs and the implementation of quality interventions in the care sectors [5]. In the health sciences, phenomenological research, and those with a qualitative approach in general, generate evidence that serves as a guide to practice sensitive to the realities of the people to whom care is directed, to their cultural diversity and to the contexts in which their lives unfold [6,7].
In studies related to quality of life in transplanted people and candidates for kidney transplantation, participants manifest as the main human responses: recurrent hospitalizations, uncertainty about the work situation, deterioration of body image, deterioration of sexual functionality, dependence on third parties, stress and guilt [2,8-12]. Specifically, people who are candidates for kidney transplantation manifest as the main human responses: anxiety and depression [13,14]. Transplanted individuals report acute rejections, medication side effects, and emotional instability; [12-15,16] immediately, after transplantation, they may perceive release with respect to dependence on renal replacement therapy, but as time goes by, they have to face various adaptation problems: side effects of medications, medical and social complications, among the latter the return to work, social and family life [12,16,17]. The analysis of quality of life, with its respective components and human responses in patients with a history of CKD is recent. Therefore, the needs inherent in the nursing care process may go unnoticed when directing care for people with these characteristics. Although there are numerous studies that quantitatively address health-related quality of life, [4,18,19] qualitative studies such as the present one provides particular evidence to integrate it into the holistic process of the nursing-patient relationship at different levels of care [13,20]. Therefore, the objective of this study is to analyze the perception of quality of life of people with kidney transplantation and kidney transplant candidates treated at the High Specialty Medical Unit of Mérida, to identify the related human responses through an interpretive phenomenological approach.

Methodology

Design

A qualitative study was conducted with an interpretative phenomenological approach. From this design it is possible to reach the understanding of the experiences and the articulation of similarities and differences in the meanings and human experiences of people with kidney transplantation and candidates for kidney transplantation. Although it is not possible to make generalizations of the results of this study, particular data are reached with transferability to other populations with similar characteristics [6,7,14]. This article followed the COREQ [Consolidated criteria for reporting qualitative research] criteria to enhance its quality and clarity [21].

Study and Sampling Population

An intentional sampling was carried out, obtaining a final sample consisted of 11 people with a history of ERD: 7 candidates to receive kidney transplant and 4 transplanted, who received health services in the High Specialty Medical Unit of Mérida [UMAE] of the Mexican Institute of Social Security [IMSS]during the period from November 2019 to February 2020.

Data Collection

Data were collected through semi-structured interviews conducted during their follow-up consultations. Interviews lasted 30 to 40 minutes, were recorded in audio format, and field notes were taken. Table 1 presents the questions asked during the semistructured interviews.

Table 1: Semi-structured interview questions.

Ethical Considerations

The study respects ethical principles: beneficence, nonmaleficence, justice and autonomy. The research study protocol, with folio R-2018-785-129, was approved by the ethics committee of the High Specialty Medical Unit of the Mexican Social Security Institute. The testimonies presented herein are referenced with codes to safeguard the identity of the participants.

Information Processing

Semi-structured interviews were transcribed verbatim and then analyzed using content analysis. This analysis process consisted of:
1. Encoding the data and establishing a data index.
2. Categorize the content of the data into meaningful categories; and
3. Determine the topics related, in this case human responses, to the previously defined categories [7,22]. In the results section, tables are presented that allow to visualize the categories of analysis delimited in Table 2 from Urzúa and Caqueo [23], the human responses within the categories and, finally, testimonies of the participants; all of the above accompanied by interpretive narrative.

Table 2: Number, crude and age-standardized rate per 100,000 by sex and time during 2005-2014 in the Nghe An province.

Note: * Categories of the concept of quality of life from Urzúa and Caqueo.

Quality Criteria

Once the transcript of the interviews was completed, the 11 participants were asked to verify that the information interpreted was correct. Also the protocolization related to the organization of the data, the detailed and meticulous description of the selection of the sample and the context in which the study is carried out, facilitate the possibility of transfer and reproducibility of the same in similar conditions, thus providing another criterion of qualitative quality.

Results

Characteristics of the participants Years of age were a median of 37 [mean 39]and SD=13 in the 11 participants. In people who were candidates for RT, the median was 37 [mean 41]and in those with RT it was 35.7 years [mean 41), respectively. In the latter group two people were 6 months or less old after receiving RT, one was 1 year old, and one person was 10 years old. Table 3 shows that the majority of the total sample was made up of men who worked as employees.

Table 3: Sociodemographic characteristics of the 11 participants included in the study.

Quality of Life: Perception in Kidney Transplant Candidates

Table 4 shows the interpretations related to the categories: concept of quality of life with their respective domains: physical, economic, family, and social, then the identified human responses are presented. Most of the participants said that quality of life is to be well physically, mentally, and emotionally, as well as to have all the basic services and not depend on renal replacement treatments: dialysis or hemodialysis. In the physical domain, people highlight discomfort, pain and discomfort related to the procedures of renal replacement therapies or the body itself: chronic or bone pain, for example, these human responses largely condition the inability to enter the labor field. In the economic domain, the participants report that they are unable to carry out the activities of any employment due to physical disability, and therefore, consider that their monetary income from a trade or employment is limited, scarce or null. In addition, they stressed that the economic resources are focused on financing the management of the health itself: laboratory tests, transportation, extraordinary treatments, appointments, and medical consultations, among others; these efforts are complicated precisely by the lack of monetary inputs.

Table 4: Quality of life: perception of kidney transplant candidates.

Note: *Categories of the concept of quality of life from Urzúa and Caqueo.

In the family domain, people identify the importance of the support, care, and understanding they receive, received, and expect to receive from their family in the ups and downs related to their state of health and well-being. In this regard, some express feelings of feeling a burden for their relatives for the extra activities that the latter perform in health management, which generates tension and uncertainty. However, the interviewees expressed the motivation generated by their family environment: mothers, children and grandchildren, among other ties, drive the desire to want to get out of their problem and be patients waiting for the transplant. In the emotional domain, each of the people interviewed expressed their affectation at different points that leads them to present low self-esteem: fear, frustration, depression, sadness and uncertainty are some of the emotions they expressed among their testimonies. Participants follow a continuous coping process, because not every day they feel with all the energy and motivation to continue with everyday life. The emotional perception of the interviewees was reflected in their features during the interviews, they touched points that led them to cry, they expressed how difficult it is to live with a dysfunctional organ, the uncertainty before the latent complications that can even make them lose their lives.

Quality of life: Perception in People with Kidney Transplantation

Table 5 shows that most participants consider that quality of life involves physical, environmental and personal well-being as components. For one of the interviewees, it means no longer relying on external factors to sustain life; another considered that the longer he can extend his life is better for the quality of it, considered that discomforts are companions of life. In the physical domain, the interviewees expressed the freedom to perform various activities and eat food without affecting their quality of life. They expressed that they could move and travel without thinking about the need to carry too many supplies related to their treatment. They also stated that they can eat food without causing discomfort or altering their clinical parameters, especially water, which was previously restricted. In the economic domain, participants report that they have time and autonomy to build opportunities for insertion into trades, jobs and vocational or educational training. One case mentioned that the ability to acquire economic resources improves their quality of life, another participant reports that they can work freely without thinking about the times of some renal therapy, finally, a case refers that they returned to normal by fully taking these opportunities that they previously addressed discreetly.

Table 5: Quality of life: perception of kidney transplants.

Note: *Categories of the concept of quality of life from Urzúa and Caqueo.

In the family domain, the perception and feelings of being considered a burden on their families has decreased along with the amount of care related to renal replacement therapies from which transplanted participants are already exempt; people mentioned that despite the constant support of their relatives there was a physical distancing seeking to reduce the crossing of infections, a situation that in recent times has ended and they can share more time and experiences together. In the emotional domain, trust and emotional balance were interpreted in the participants. Two people mentioned that they feel they have a new opportunity before life, to restart it and have new experiences that they previously did not consider possible. Two people referred to the need to have confidence and know how to take the advice of health personnel: doctors and nurses. Finally, one participant described that he was overwhelmed by living a few days in isolation after his transplant, necessary to prevent infections, but at the same time accepts that it is necessary to improve his quality of life.

Discussion

The quality of life of people with a history of renal pathologies is affected since the first clinical manifestations, the QoL in this sector has shown deficiencies, low levels or areas of opportunity with respect to the rest of the population [24]. Physical, environmental and personal well-being are part of the conception of quality of life in people with renal pathologies, whether they have been transplanted or not. In the early stages of the disease there are a series of negative perceptions of the disease and its mediate and immediate quality of life that, ultimately, can influence their coping actions, these perceptions can trigger anxiety, depression, coping, autonomy, self-esteem and accelerated progression of the disease [25]. In the identification of human responses in patients with chronic kidney disease, the main physiological risks related to this pathology have been highlighted. Farias et. al. points out the overstating of biological and complication-related human responses by nursing staff providing care to patients with nephropathies in a renal center. Among 24 diagnostic labels identified, the most frequent were “risk of infection”, “excess fluid volume”, “hypothermia”, among others whose main domains were located in Safety / Protection and Activity / Rest, on the other hand, “low situational self-esteem” was ranked 16th in frequency [26] corresponding to the Self-perception domain in the NANDA-I [20]. The above shows what Spilogon et. al. points out as an area of opportunity in the nursing process because it has the flexibility and openness to consider the perceptions and preferences of the user, in this case of the patient with nephropathies [27].
In the emotional category, low self-esteem was detected in participants with CKD without transplantation, and that is that a patient with CKD has needs for recognition and esteem, so the people in charge of their care should promote favorable behaviors in coping with the pathology and attachment to treatment, avoiding judging and repressing the failures of our human condition [28]. In contrast, participants who had received a kidney transplant manifested confidence and emotional balance, something that could be considered normal after receiving the expected transplant according to Tucker, et. al. [29]. From a quantitative approach Rocha et. Al. point out that the higher the quality of life, the better the assessment of the self-esteem of people with chronic kidney disease after transplantation [30].
In the economic category, while people who had not received kidney transplantation conceived the inability to enter the workforce among their perception of quality of life, those who had received kidney transplantation indicated greater time and autonomy to build job and academic opportunities. Reports indicate that patients with chronic kidney disease face many barriers to staying or joining the workforce after starting dialysis: limited opportunities, lack of financial resources to invest, fatigue and other symptoms of kidney failure, potential loss of disability benefits or medical follow-up, dialysis scheduling, and employer bias. The societal perception that patients with CKD cannot work completes a vicious cycle of low employment expectations [25,31]. In the family category, the perception of “being a burden” for family members influences is an important component in the perception of the quality of life of people with transplantation and without kidney transplantation. Evidence indicates that family members of patients with a history of renal pathologies manifest sleep interruptions, depression, anxiety, among other disorders associated with unforeseen responsibilities related to the treatment and logistics of their relatives; they must also deal with insufficient information, medication regimen and be accompanied by periodic hospitalizations [32]. NANDA International classifies problems into plausible diagnostic labels of interventions focused on promoting the health of individuals, the family. and community, we can mention: Risk of fatigue of the role of caregiver, Fatigue of the role of the caregiver, Dysfunctional family processes, Willingness to improve family processes, among others [20].
In the physical category, participants without kidney transplantation are identified as a condition for quality of life, a common and often severe manifestation in various populations with CKD; with prevalence’s of 40% to 60% is a strong imperative to establish the management of chronic pain as a clinical and research priority [33]. In this regard, the labels acute and chronic pain are available in NANDA-I [20]. Although pain and physical limitation decreases after a kidney transplant, it is important to mention that the physical and nutritional autonomy indicated by the participants of the present can generate an excess of confidence and the acquisition of unhealthy practices. Physical training regulated by physiotherapy specialists appears to be safe in kidney transplant recipients and is associated with improved quality of life and exercise capacity [34]. With respect to diet, the Mediterranean and DASH (Dietary Approaches to Stop Hypertension) diets have been shown to be the most beneficial dietary patterns for the population after kidney transplantation by focusing on less meat and processed foods, while increasing intake of fresh foods and plant-based options. [35]. Knowledge and awareness in the renal transplant population should be a cornerstone of therapy and an integral part of nursing responsibilities. Therefore, nurses should educate patients about self-care behaviors and remind them of the dangerous complications of abandoning them [28].
In participants who had not received a kidney transplant, there was an expectation of receiving a kidney transplant to improve their quality of life and from it to improve their quality of life. In this aspect we can mention the benefits before the expectation of receiving a kidney transplant mentioned by Santos et. al. who in a group of people with Brazilian nephropathies detected that patient who were not waiting for transplantation were at risk of poor quality of life, mainly in the emotional and physical aspects; those who were not awaiting transplantation died more frequently in the next 12 months [36]. However, betting on kidney transplantation to improve the quality of life in patients with nephropathies is not entirely recommended, in this regard we can cite the studies of Schulz et. al. and Smith et. al. published in 2014 and 2019, [29,37] who reported that before transplantation patients can overestimate gains in quality of life without finding significant improvements in quality of life after being transplanted.
Kidney transplantation is not a guarantee of improvement in quality of life in all patients with nephropathies, in the present study, those people who had received the kidney transplant did not consider an absolute improvement in their quality of life. The literature notes that kidney transplants can provide dramatic improvements in quality of life and health status, however, the effects on improvement are not universal and patients live in constant uncertainty as they are aware of the likelihood of graft dysfunction [29]. There are samples that have indicated that the expectation about the functionality or rejection of the graft generates greater fear and uncertainty than death itself [38]. The results on the perception of quality of life in people receiving renal replacement therapy support the trend of the last decade focused on the analysis of this category beyond only assessing life expectancy [39]. The limitations of the present are the risk of bias due to the same interpretative approach and the inability to generalize the results to the study population. To compensate for the above, criteria of methodological rigor were followed and from a particular context the search for generalities was made, reinforcing the results with respect to other studies [21].

Conclusion

In transplant patients, a perception of absolute or discomfortfree quality of life is not achieved and human responses that require care and interventions to achieve the highest level of well-being are still manifested. The construction of the concept of quality of life includes physical, mental, personal and social elements feasible to document and in which to exercise interventions for the benefit of the people treated and their families, it is evident that human responses do not only obey physiological needs.

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Lower Sensitivity of CAT and ER to Melatonin May Lead to Poor Ooplasmic Maturation of Porcine Oocytes from Heat Stress

Introduction

High temperature not only reduces the oocytes maturation quality [1-4] but also has a tremendous adverse impact on the animal ovarian function and embryo attachment [5,6]. Heat stress (HS) damage of oocyte can inhibit cumulus cells expansion [7], which lead to the abnormal distribution of organelles [8,9], decreased antiapoptotic and estrogen receptor gene expressions, enhanced apoptosis gene expression [10], as well as increased Reactive Oxygen Species (ROS) concentration [11,12] in the oocytes. In fact, excessive ROS can induce DNA damage and lipid peroxidation, disrupt the mitochondrial function [13,14], and induce abnormal gene expression and protein synthesis [15]. Reportedly, mitochondrial maturation distribution is an important indicator of the oocyte quality. Oocytes with poor maturation quality have a non-uniform mitochondrial distribution, whereas mitochondria are uniformly distributed in the ooplasm [16]. In order to protect oocytes from HS damage and improve their maturation quality, several materials are supplemented in the maturation medium during in vitro oocyte maturation, such as insulin-like growth factor, β-mercaptoethanol, astaxanthin, anthocyanins, Melatonin (MT), and coagulated proteins [17-20].
It has been reported that MT is involved in regulating several different physiological processes; it can promote the expression of antioxidant-related genes and improve the oocyte maturation quality and embryo developmental potential [21]. In addition, MT concentration of 10-9 M has been proven to be effective in promoting porcine oocyte maturation and development [22]. Oocyte maturation involves several complex events that coordinates nuclear and cytoplasmic maturation processes. Cytoplasmic maturation events following meiotic maturation is much more difficult to assess microscopically, such as the abnormal distribution of the mitochondria, lipid droplets [23], and Glutathione (GSH) concentration detection. However, the nuclear maturation process involves the Germinal Vesicle (GV) breakdown, chromosomal arrangement, and completion of Metaphase 1 (MI) by extruding the first polar body into the perivitelline space of the oocytes, all of which were observed by stereomicroscopy in a previous study [24]. It has been reported that HS can increase the expression of proapoptotic genes, enhance the activity of caspase proteins, and trigger the apoptosis pathway [25,26]. The members of the BCL- 2 family play a key role in regulating apoptosis, among which the expressions of the proapoptotic gene BAX and antiapoptotic gene BCL-2 as well as the ratio of these two gene expression levels are generally deemed as indicators for predicting the oocyte maturation quality and the embryo developmental potential.
Past studies in cattle have reported that the expressions of genes related to oocyte maturation quality and developmental potential were greatly reduced after the Cumulus and Oocyte Complexes (COCs) were subjected to HS at 41℃ for 12h. The present study discusses the sensitivity of BCL-2, BAX, CAT, and ER to high temperature and MT and analyzes the relationship among their sensitive differences and porcine oocyte maturation quality and developmental potential in vitro. We first discovered that the low sensitivity of CAT and ER to MT possibly contributes to the strong relationship with the poor porcine oocyte maturation quality and the developmental capacity of the embryos in vitro. We believe that the present results would be helpful in enhancing oocytes utilization and would provide a practical guide toward improving pig fertility during the high-temperature season.

Materials & Methods

All reagents used in this experiment were purchased from Sigma Chemicals (St. Louis, MO, USA), unless otherwise specified.

Oocytes Collection and Culturing In Vitro

The ovaries were acquired from a slaughterhouse and dispatched to our laboratory within 2 h of collection in a thermos flask containing sterile saline at 35-37℃. The COCs were extracted from follicles (of 2-6-mm diameter) with a disposable syringe (10 mL; No. 18 needle), and only COCs with uniform ooplasm and compact cumulus cells were maturation cultured in an incubator with 5% CO2 and 95% humidified air atmosphere, as follows: some COCs were cultured at 38.5℃ for 42 h in a maturation medium (Control, No HS); some COCs were cultured at 41.5℃ for 4 h and then transferred for continuous culturing at 38.5℃ for 38 h in the maturation medium (HS group); and the other COCs were cultured at 41.5℃ for 4 h in a medium supplemented with 10-9 M MT, after which it was subjected to continuous cultured for 38 h in a maturation medium at 38.5℃ (HSMT group). The maturation medium consisted of TCM199 (with Earle’s Salts; Gibco, Grand Island, NY, USA) supplemented with 10% porcine follicular fluid (PFF), 0.1 mg/mL cysteine, 0.065 mg/mL penicillin, 10 ng/ mL epidermal growth factor (EGF), 10 IU/mL equine chorionic gonadotropin (eCG; Intervet Pty. Ltd., Boxrneer, Australia), and 10 IU/mL human chorionic gonadotrophin (hCG; Intervet Pty. Ltd.). All the experiments were repeated thrice.

Assessment of the First Polar Body Expulsion Rate

The COCs from different groups were respectively stripped off cumulus cells by gentle pipetting in Phosphate-Buffered Saline (PBS) supplemented with 0.1% hyaluronidase, and the first polar body expulsion rate was determined under a stereomicroscope. A total of 150 denuded oocytes from each group were used for determining the rate of the first polar body expulsion. All oocytes used in the subsequent experiments had their first polar body expulsed.

Ooplasmic ROS Detection

ROS was detected using the Reactive Oxygen Species Assay Kit (S0033; Beyotime®, Haimen, Jiangsu, China) as per the manufacturer’s instructions. Briefly, 50 matured oocytes from each group were rinsed in the PBS solution thrice, followed by dying with 10× M ROS dye in the dark for 10 min. Next, photographs were taken under a fluorescence microscope (TE2000-s; Nikon, Japan). The fluorescence intensity analysis was performed with the Image J (Version 1.8.0) software, and the experiment was repeated thrice.

Ooplasmic Mitochondrial Distribution Analysis

Mitochondrial staining was performed with the Mito-Tracker Red CMXROS (C1049; Beyotime®) as per the manufacturer’s instructions. Briefly, 50 oocytes from each group were stained with 200-nM mitochondrial dye in the PBS solution for 25 min at 37℃ in the dark after washing in PBS thrice. Then, the stained oocytes were observed under the fluorescence microscope. The mitochondrial distribution pattern of porcine oocytes was then characterized based on two main distribution features: uniform distribution throughout the ooplasm or non-uniform distribution throughout the ooplasm.

Parthenote Production and Culture In Vitro

A total of 100 matured oocytes from each group were transferred to the activation medium (composed of 1.0 mM CaCl2, 0.1 mM Mg Cl2, 0.3 M mannitol, and 0.5 mM HEPES). Matured oocytes were activated with two pulses of 120 V/mm DC for 60 ms with the Electro-Cell Manipulator BTX 2001 (BTX Inc., USA). After activation, the parthenotes were subsequently cultured in 2 mM 6-dymethylaminopurine (6-DAMP) for 6 h and then the parthenotes were transferred into the PZM-3 medium for 7 days in an incubator at 39℃ under 5% CO2 atmosphere in humidified air. The rates of cleavage and blastocyst transfer were observed respectively on days 2 and 7 after oocytes activation.

Blastocyst Cells Staining

A total of 10 blastocysts were randomly selected from each group and fixed in 4% paraformaldehyde prepared in PBS containing 0.1% polyvinyl alcohol (PVA) for 1 h after washing thrice, and the blastocysts were then permeabilized in PBS-0.1% PVA solution containing 0.3% Triton for 30 min. After washing, the blastocysts were transferred to a solution supplemented with 10 μg/mL DAPI dye for 1 min, and then the blastocysts were mounted on a slide and covered with a coverslip. The total blastocyst count in each group was counted under the fluorescence microscope.

Gene mRNA Expression

The expressions of BCL-2, BAX, CAT, and ER were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA of 100 oocytes from each group was extracted by using the Micro RNA Extraction Kit (160027349; Qiagen, CA, USA) as per the manufacturer’s instruction. After washing in PBS, the oocytes were transferred to a 200-μL centrifuge tube for pre-cooling and then stored at -80℃ for further processing. After total RNA extraction, cDNA synthesis was performed for 30 min at 55°C using the Omniscript Reverse Transcription Kit (Invitrogen) with oligodT primer. PCR was performed by using the Maxime PCR Premix with SYBR Green (TaKaRa Bio Inc., Otsu, Japan) supplemented with each primers and cDNA samples under the following conditions: predenaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 56°C for 30 s, elongation at 72°C for 30 s, and a final extension at 72°C for 5 min for 40 cycles using the Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). According to the mRNA sequences of Sus scrofa genes published on Gen Bank, we designed primers with the Primer 5.0 software and synthesized by Shanghai Bioengineering Co., Ltd. (Shanghai, China). The primers used in the present study were verified for their availability by RTPCR. Real-time quantitative PCR was performed by the comparative Ct (2 -△△Ct ) method, and the results obtained for each gene in each cDNA pool were normalized based on the GAPDH ratio. The primers and Genebank source accessions for each gene are listed in Table 1.

Table 1: RT-PCR primers and Genebank source.

Statistics

The percent values were subjected to log transformation before analysis, and the quantitative data were analyzed by least-square analysis of variance (ANOVA) using the General Linear Models (GLM) procedures of the Statistical Analysis System (SAS Institute, Cary, NC, USA). We corrected the real-time PCR data by using the GAPDH data as a covariate for the analysis of differences. All data were expressed as mean ± SEM, with P < 0.05 deemed as statistically significant. All experiments were repeated thrice.

Results

Assessment of the First Polar Body Expulsion Rates of Oocytes in Different Groups

The first polar body expulsion rates of porcine oocytes are given in Table 2. Although the first polar body expulsion rate greatly decreased in the HS group in comparison with that in the control group, the corresponding rate in the HSMT group significantly increased again but exhibited no significant difference with that in the control group (P > 0.05). The first polar body of porcine oocytes was observed under a stereomicroscope (Figure 1A), and the observation was confirmed under a fluorescence microscope (Figure 1B). Figure 1C represents oocytes without the expulsed first polar body, indicating that fluorescence occurred only at the nuclear sites observed under the fluorescence microscope.

Table 2: The porcine first polar body expulsion rates in different groups.

Figure 1: Porcine oocytes with or without the first polar body expulsed
A. The stereomicroscopic examination of the matured oocytes with the first polar body expulsed, as pointed by the arrows.
B. The matured oocytes were stained with fluorescent dye Hoechst 33342, where both the polar body and the nucleus exhibited fluorescence.
C. Oocytes with no polar body expulsed were stained with fluorescent dye Hoechst 33342, where only the nucleus exhibited fluorescence as pointed by the arrows. Bar=100 μm.

ROS Concentration in Oocytes of Different Groups

The ROS concentrations in the HS group was significantly increased relative to those in the control group. In addition, although the ROS concentration in the HSMT group decreased significantly, it remained significantly higher than that in the control group as showed in Table 3 (P < 0.05). The proportion of oocytes with uniform mitochondrial distribution in porcine oocytes of different groups. Figure 2 supports that the proportion of oocytes with a uniform mitochondrial distribution in the HS group was significantly lower than that in the control group (P < 0.05). Although the proportion of oocytes with uniform mitochondrial distribution was greatly enhanced in the HSMT group, it remained significantly lower than that in the control group (P < 0.05). Figure 3 represents the uniform and non-uniform mitochondrial maturation distribution in the ooplasm.

Figure 2: The proportion of oocytes with better mitochondrial maturation distribution in different groups. Different letters (a-c) over a bar means significant difference (P < 0.05). Each experiment was repeated three times.

Figure 3: The condition of mitochondrial maturation distribution in ooplasm Oocyts were fluorescent stained by Mito-Tracker Red, and A. Represents the oocyte of better maturation quality with even distribution of mitochondria in ooplasm. B. Represents the oocyte of poor maturation quality with uneven distribution of mitochondria in ooplasm. Bar = 100 μm.

Table 3: ROS contents of oocytes in different groups.

Assessment of Porcine Oocytes Developmental Potential In Vitro

As can be seen in Table 4, the cleavage rate, blastocyst rate, and the total number of blastocysts were significantly lower in the HS group than in the control group (P < 0.05). As compared with those in the HS group, the rates of cleavage, blastocysts, and the total number of blastocysts in the HSMT group were significantly increased (P < 0.05), with no significant difference in the cleavage rate relative to that in the control group (P > 0.05). Nevertheless, the blastocyst rate and the total number of blastocysts remained significantly lower than the respective control values (P < 0.05). The results of blastocyst cells staining are depicted in Figure 4.

The mRNA Expressions of CAT, BCL-2, BAX, and ER in Oocytes of Different Groups

As shown in Figure 5, the mRNA expressions of CAT, ER, and BCL- 2 were decreased, whereas the BAX expression was significantly increased in the HS group in comparison with those in the control group. However, in the HSMT group, the mRNA expression levels of BCL-2 and BAX were restored to the control levels, while the CAT and ER expressions remained significantly lower than the control values (P < 0.05). Moreover, no significant difference was noted in the mRNA expression of ER between the HS and HSMT groups (P > 0.05).

Figure 4: The fluorescence staining of blastocyst cells. The blastocyst cells was fluorescent stained with 10μg/ml DAPI dye and a number of 10 blastocysts randomly selected from each group.
A. The blastocyst comes from the Control
B. The blastocyst comes from the HS group
C. The blastocyst comes from the HSMT group. Bar = 100 μm.

Figure 5: The mRNA expressions of CAT, BCL-2, BAX and ER genes in oocytes of different groups. Different letters (a-c) over a bar means significant difference (P < 0.05). Each experiment was repeated three times.

Table 4: The porcine oocytes developmental potential in different groups.

Discussion

Previous studies have shown that the effects of MT exposure on protecting oocytes from HS damage varied among different animals. Negrón-Pérez reviewed that the cleavage rate and the total number of blastocysts in dairy cows increased when MT was supplemented in the HS system [27], while another bovine study demonstrated that MT has no significant effect on the cleavage rate and on the total number of blastocysts [28]. The present study on porcine suggested that, although 10-9 M concentration of MT could significantly improve the cleavage rate, the blastocyst rate and total cell of blastocysts remained significantly lower in the HSMT group than in the control group (P < 0.05). Indeed, neither previous bovine studies nor the present porcine study indicated whether high-concentration MT supplementation could increase the total cell of blastocysts. With regards to porcine oocyte maturation, the ooplasmic maturation is usually evaluated by some molecular events, bioreaction, or organelle distribution [29], and the nuclear maturation is estimated by the first polar body expulsed into the perivitelline space of oocytes [30].
The results of the present study indicated that the first polar body expulsion rate was greatly increased in the HSMT group in comparison with that in the HS group, exhibiting no significant difference relative to that in the control group. We thus speculated that supplementation with 10 IU/mL Equine Chorionic Gonadotropin (eCG) and 10 IU/mL human Chorionic Gonadotrophin (hCG) to the maturation medium was sufficient for porcine oocyte nuclear maturation in the present experiments, considering that gonadotropins are responsible for the resumption of oocyte meiosis and for the promotion of nuclear maturation by the cAMP/PKA/MAP kinase pathway [31]. Although MT supplementation could significantly increase the ratio of oocytes with a uniform mitochondrial distribution, it remained significantly lower in the HSMT group than in the control group. Several other researches have demonstrated that HS could influence abnormal mitochondrial distribution, which is detrimental to oocyte maturation and development in vitro [32-37]. The data from production also suggests that the ooplasmic mitochondrial distribution was poor during the summers, but it improved during the autumn season [38].
The poor mitochondrial maturation in the HSMT group oocytes may attribute to the higher intracellular ROS concentration, considering that ROS is one of the main factors that result in diverse damages to oocyte maturation and development [39-42]. The present experiments demonstrated that 10-9 M MT concentration is insufficient to eliminate the excessive ROS in the oocytes of the HSMT group. Our study is the first to indicate that the BAX and BCL-2 were sensitive to both high temperature and MT. As for the CAT, 10-9 M concentration of MT could not restore its expression to the control level, which may explain the higher ROS concentrations in the HSMT group. Estrogen receptor gene is closely associated with the fertility of female animals and follicular development and oocyte maturation [43-45]. In our study, we found that the ER was sensitive to high temperature, but extremely insensitive to MT exposure. The reduction in ER expression induced the lack of binding sites for estrogen, which lowers the fertility of female animals under hot conditions [46]. Recent data in production also confirmed that the fertility of sow was lower in the summer season than in other seasons [47]; moreover, past studies in cows have reported that subcutaneous implantation with 18 mg MT could only partially alleviate the adverse effects of HS on the reproductive performance of cows in the hot season [48-50].
Backed by our research, we propose that the reduced secretion of MT and the insensitivity of ER to MT is most probably related with the lower fertility of animals in high temperature season. However, until date, the quantity of MT required to counteract the adverse effects of HS on pig remain unreported. Based on our experiments, we believe that much more than the 10-9 M concentration of exogenous MT would be required. The present experimental results imply that the lower fertility of animals in high temperature season can be attributed to the sensitive differences in the expressions of BCL-2, BAX, CAT, and ER to high temperature and MT, which provides important insights to the application of MT toward improving the fertility of animals during the high temperature season.

Conclusion

BCL-2 and BAX were found to be sensitive to both high temperature and MT exposure; however, CAT and ER, especially the latter, were found to be sensitive only to high temperature and extremely insensitive to MT exposure. The sensitive differences in these genes contributed to poor ooplasmic transfer, lack of nuclear material transfer, and maturation, which further hampered the developmental capacity of porcine oocytes.

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Derived Biobased Catalyst from the three Agro Wastes Peel Powders for the Synthesis of Biodiesel from Luffa Cylindrical, Datura Stramonium, and Lagenaria Siceraria Oil Blend: Process Parameter Optimization

Introduction

Over-dependence on petroleum reserves for the supply of energy, increasing demand for energy, price volatility of fossil fuel, the monopoly in the crude oil market, energy crisis associated with technological advancement are indicators that the current quantity will not meet the mandate with time [1,2]. Therefore, countries (EU, Spain, North America, South America, China, Brazil, India, Argentina, Australia, Canada, Cuba, Colombia, France, Ghana, Kenya, Sweden, Singapore, USA, UK, Zimbabwe, Peru, Pakistan, Italy, Japan, Malaysia, Mali, Mexico, Iran, Ireland, Norway, Germany, etc.) around the globe have shifted attention to biodiesel due to its excellent environmental attributes, sustainability attributes, biodegradability, non-toxic, readily available, and reduction or elimination of over-dependence on fossil fuel [3-6]. Meanwhile, biodiesel potential feedstocks come from first generation biodiesel feedstocks, which associated with the use of edible vegetable oil (beniseed oil, soyabean oil, corn oil, canola oil, palm oil, sunflower oil, coconut oil, olive oil, linseed oil, peanut oil, corn oil, papaya oil, etc.), the second generation biodiesel feedstocks, which make use of non-edible vegetable oil and animal fat (Jatropha curcus oil, pongamia pinnata oil, waste cooking oil, yellow oleander oil, cotton oil, grease, tallow, rapeseed, castor oil, karanje oil, neem oil, fish fat, pig fat, rubber seed oil, etc.), and the third generation biodiesel feedstocks, which involve the use of microalgae, algae, fungi, bacteria, latexes [5,7-15]. Nevertheless, exploiting firstgeneration biodiesel feedstock leads to a major problem especially in the present world of food shortage [16]. On the other hand, the use of third generation biodiesel feedstock requires a large amount of water for algae productivity, significant fertilizer for algae growth, high production cost using current technology, the long time needed for conversion to biofuel, contenders with regional suitability issues, lack of energy-efficient product, variations in the biofuel quality, and monoculture issue. Therefore, non-edible feedstocks in second-generation biodiesel feedstock are the only secure and viable future for all through biofuel production.
Meanwhile, it has been reported that the use of mix/blend oil tends to improve the yield and the quality of biodiesel; hence, researchers have reported the use of different blend ratios of oil for biodiesel synthesis. Khalil, et al. [17] reportedly the used oil blend ratio of 40:60 for rubber seed oil and palm oil, with NaOH as a base catalyst. The study reported by Qiu, et al. [18] adopted a ratio of 50:50 for the mixture of soybean and rapeseed oil with NaOH as a base catalyst. Milano, et al. [19], combined cooking with Calophyllum inophyllum oil in the ratio of 75:25, with KOH f as a base catalyst, while Hadiyanto, et al. [20] combined waste cooking with castor oil in the ration of 1:0, 1:2, and 2:1, respectively. Falowo, et al. [21], reported a blend of Neem and rubber oil in a 60:40 ratio with a base catalyst developed from elephant-ear tree pod husk. The work recently reported by Falowo, et al. [22], adopted the ternary blend ratio for Honne-Rubber-Neem oil, with mixed catalyst from three agro wastes. Observation from the reports showed that only Falowo, et al. [21,22] used heterogeneous catalysts for the synthesis of biodiesel production via oil blend. This was due to the heterogeneous catalytic nature such as reusability, recyclability, less water usage, non-toxic, low cost, eco-friendly, and high purity of by-product over the use of homogeneous catalysts (NaOH/KOH) [16,23,24]. To the author’s awareness so far, no single report on the use of the API gravity ratio has been reportedly used for oil blend for biodiesel synthesis. Also, no report has ever derived a based catalyst from the mixture of three green wastes of Cucurbita pepo, Musa acuminate, and Citrullus lanatus unripe peels for catalytic application. Literature survey showed that the unripe Cucurbita pepo peels contain 27.85% calcium [25] while unripe Musa acuminate peels contained 57% calcium mineral [26], the calcium content found in Citrullus lanatus unripe peels was reported to be 43% [27]. Proper processing of the peels through drying, sieving, and calcination at a higher temperature above 550oC has been established as a way of improving the content of the calcium in the peels [28-30].
Hence, this work focusses on the synthesis of a mesoporous based catalyst from the mixture of Cucurbita pepo, Musa acuminate and Citrullus lanatus peels, applied it to transesterification of Luffa cylindrical – Datura stramonium – Lagenaria siceraria oil blend to biodiesel. Detail characterization of the catalysts developed was carried out using Scanning Electron Microscopy (SEM), Fourier Transforms Infrared Spectroscopy (FTIR), X-ray Diffraction Analysis (XRD), X-ray Diffractometer (XRD), and Brunauer-Emmett- Teller (BET-adsorption) to determine its catalytic potential. Process optimization of transesterification of oil to biodiesel was carried out via response surface methodology (RSM). The quality of biodiesel was determined, and the results were compared with ASTM D6751 and EN 14214 biodiesel recommended standard.

Materials and Methods

Materials

Matured, Luffa cylindrical, Datura stramonium, Lagenaria siceraria seeds were collected from the nearby location around the rural house, Omu-Aran, Kwara State with proper permission obtained from the landowner in Nigeria. The seeds were separated from the husks by sundried for two weeks (14 days), and then oven-dried to a constant weight. The husk Datura stramonium has started splitting even before oven dried. The separated dried seeds were further obtained purely by winnowing and then milled into powders of 0.30 mm particle sizes, kept in separate cleaned containers for further processing (oil extraction). Cucurbita pepo, Musa acuminate, and Citrullus lanatus peels were obtained from the fruits. The peels were washed with distilled water twice, sundried for five days (5 days), and then oven-dried in a DHG-9101- 02 oven at 80oC for 2 h to achieved constant weight. The dried peels were milled into powders, separated into small particle sizes using a mesh strainer (mesh size: 125 mm-20 μm) to aid calcination. The fine sieved powders Cucurbita pepo peels powder (CPPP), Musa acuminate peels powder (MAPP) and Citrullus lanatus peels powder (CLPP) were kept in crucibles for further processing (Calcination/ thermal treatment). The seeds were identified by the Adepoju T. F., and the sample of the seeds have been deposited in Chemical and Petrochemical Laboratory 2, Akwa Ibom State University, Nigeria with a deposition number LC2019 for Luffa cylindrical, DS2019 for Datura stramonium and LS2019 for Lagenaria siceraria. All chemicals used were of analytical grades and need no further purification.

Methods

Oil Extraction: Oil extractions from the powders were carried out using solvent extraction in 1 L soxhlet extractor apparatus. Since the heating mantle was designed to handle three-Soxhlet extractors at once, mass extraction was carried out simultaneously. The oils were extracted from Luffa cylindrical, Datura stramonium, Lagenaria siceraria powders using n-hexane as the solvent. The procedures were as follows: the powders were put in a muslin bag and inserted in a Soxhlet extractor condenser with n-hexane at the round bottom flask placed on the heating mantle. The reaction temperature was monitored at 70oC for a complete extraction period (1 h). At the end of the reaction, the residual cakes were kept as a supplement for animal feeds while excess n-hexane in oils was recovered using the rotary evaporator. The clean oils Luffa cylindrical oil (LCO), Datura stramonium oil (DSO), and Lagenaria siceraria (LSO) were collected and kept in separate containers for further treatment. Oil qualities were ascertained by determining the physical, chemical, and other properties of the oils through AOAC (1997) standard test methods.
Oil Blend: The blend is the acts of mixing two or more substances, either miscible or non-miscible. For oils proper mix, it is worthwhile to know that the action of oil always increased by mixing several oils, nevertheless, the order in which the oil must be mixed must be factor properly. Lighter oil with smaller molecules will produce less viscous oils with high volatility, but heavier oil and larger molecules produce high viscous oils with low volatility. Hence, to obtain a low viscous, low density, and high volatile oil, there is a need for oil mix in an accurate blend ratio to increase the synergistic effect within the blended oil. One must know the nature (heavy or light) of the oil before mixing. The extracted oil is defined with API gravity, API gravity greater than 10 indicated lighter oil and the oil floats on water, the value of API gravity lesser than 10 indicated heavier oil, and the oil sinks on water. The API gravity of oil is calculated using Eq. (1) [31].

Oil Blend via API Gravity Estimate: The API gravity of the oils was estimated based on the specific gravity of the oil. The total API gravity of the oils was obtained from the API gravity of the oils obtained, the mix ratio of oil was computed using the mathematically derived Eq. (2), and the oil was properly mixed by heating at 50 oC on a magnetic shaker for 30 min.

Catalyst’s Calcination and Characterization

The fine powders (CPPP, MAPP, CLPP), with mixed powder (30 g CPPP + 30 g MAPP + 30 g CLPP) (MP) were calcined at 650 oC for 3 h to obtain the powdered catalysts [6]. After cooling, the powdered were characterized using scanning electron microscopy (SEM), to examine the surface morphology of the catalysts, energy dispersive spectroscope (EDS) to determine the elemental analysis of the samples and the quantitative composition of the catalysts, X-ray diffractometer (XRD) equipped with Kά and Cu radiation source, accelerated at 20 mA and 30 kV, to establish the angular scanning electron performed in the range of 20o <2θ <80o at speed of 2oC min-1, Fourier transform infrared spectroscopy (FTIR), to check the presence of functional group and verify the presence of characteristic absorption bands of major elements present. The surface area and the basicity of the catalysts were examined using BET isothermal adsorption and the Hammett indicator method [32].

Synthesis of Biodiesel

The mixed oil (29:50:21) free fatty acid (FFA = 0.82<1.5) was within the moderate value for transesterification of oil to biodiesel [33]. Therefore, transesterification of mixed oil (MO) to biodiesel through methanolysis of the mixed powder (MP) was carried out using the procedure earlier reported by Adepoju, et al. [29] with few modifications. A three-necked-reactor was used to carry out biodiesel production, four factors with five-level were considered viz. reaction time, calcined mixed powder (CMP) amount, reaction temperature, and methanol/oil molar ratio (MeOH/OMR). The MO was preheated at 60oC for 30 min, a known CMP was added to 40 ml of methanol in a 250 ml flask, heated at 65oC for 20 min in a magnetic shaker, the insoluble methanolic-catalyst was transferred into the preheated oil in the reactor, and the reaction was monitored at a particular temperature until the reaction reach completion. At the end of the reaction, the solid phase catalyst was separated by decantation and the biodiesel phase was separated from the ethanol phase by separating funnel. The leached catalyst in the biodiesel was removed by washing with a solution (containing a mixture of 2.0 g of NaCO3 and 40 ml ethanol thermally heated for 2 h) under agitation. The mixture was filtered, washed with distilled water three times before the separation of biodiesel through gravity settling was carried out. The washed biodiesel was then dried over anhydrous Na2SO4 and then separated by filtration to obtain pure biodiesel. These processes were repeated based on experimental runs generated by response surface methodology experimental design.

Experimental Design for Biodiesel Synthesis and Its Statistical Analysis: The four-level-five-factors used for biodiesel experimental designed with the respective units are presented in Table 1. A central composite design (CCD) was used which contains 24 non-center and 6 center points with high and low center factors, the design is a full type with 2 alpha and 1 block. A total of 30 experimental runs were obtained with central composite design replication occurred for every categorical combination. For analysis through the statistical approach, the model for response surface (biodiesel) and the interaction (variable factors) was evaluated by mean of fit summary. The model order, significant effects, and desired terms were evaluated by model effects. ANOVA was used to analyze the chosen model and view results, while diagnostic, evaluate the model fit, and also the transformation choice with the graph to interpret and evaluates the model. Moreover, process optimization was established by determining the probability value (p-value), the f-value (factor value), the degree of freedom (df), and the variance inflation factor (VIF), respectively. Linear regression parameters were obtained through evaluation of the coefficient of determination the predicted coefficient of determination, the adjusted coefficient of determination, and the adequate precision (Adeq. Prec.) to confirm the model suitability. Meanwhile, to show the relationship between two input variables and one output, three dimensional and contour plots prove to be the best representative of the correlation (Adepoju et al., 2016). The second-order polynomial model equation that further explains the relationship between biodiesel yield and the independent variables is mathematically expressed as Eq. (3).

Where FAME is the response (biodiesel) in percentage, P_0 is the intercept, P_i is the linear coefficient, P_ii is the interaction coefficient, P_ij is the quadratic coefficient terms, X_i 〖 and X〗_j are the four factors and ϵ is the residual error.

Table 1: Experimental design for biodiesel production.

Biodiesel Quality Characterization: The effectiveness and industrial application of biodiesel produce depend solely on its quality such as moisture content, density, viscosity, mean molecular mass, peroxide value, iodine value, saponification value, cetane number, higher heating value, API gravity, and diesel index. These properties were determined using AOAC, (1997) standard methods; the results were compared with biodiesel recommended standards [34,35]. All methods were performed in accordance with the relevant guidelines and regulations governing institutional, national, and international guidelines and legislation including the collection of plant material, the Experimental research and field studies on plants/seeds.

Results and Discussion

Extracted Oils Qualities and It Mixed Ratio

Table 2 showed the results of the qualities of the oils and the mixed oil obtained. From the table, it was observed that the specific gravity of the LSO (0.95) was highest, followed by the LCO (0.910), and then DSO (0.820). The higher the specific gravity, the lower the API gravity of the oil, and such oil tends to be less light in nature. The extracted oils were light oils with low free fatty acid value and low moisture content. The iodine value and the viscosity of the LSO (98.02 g I2/100g oil; 4. 10 mm2/s) appear higher than the value of LCO (96.50 g I2/100g oil; 4.10 mm2/s) and DSO (72.32 g I2/100g oil; 2.12 mm2/s). However, the acid value of the LCO (4.50 mg KOH/g oil) is higher than the acid value of LSO (1.84 mg KOH/g oil) and DSO (1.43 mg KOH/g oil), these showed that the LCO is non-edible oil. The API gravity of DSO (50) is greater than the values obtained for LCO (29) and LSO (21) which accounted for an oil mixed ratio of 29:50:21 for LCO: DSO: LSO. This mixed ratio produced oil with moderate FFA and of low density used for biodiesel production (transesterification).

Catalyst Characterization and Elemental Analysis

Scanning Electron Microscopy (SEM): Figure 1a showed the result of SEM images of morphological characteristics of the calcined mixed powder (CMP) at a different magnification of 1000x and 500x performed in the range of 20o<2θ < 80o at speed of 2oC/ min. The decomposed images after calcination at a temperature of 650oC for 3 h indicated non-uniform sizes with diverse shapes and slightly rough surface with cracks. This observation implied thermal treatment degrades organic substances in the mixed powder (MP) and also transforms the calcium carbonate (CaCO3) to the calcium oxide (CaO), owing to the gaseous form of carbon dioxide (CO2) which appears more porous, brittle and easy to ground.

Table 2: Qualities of the oils and API gravity mixed ratio.

Figure 1:

a. SEM image of CMP at different magnifications.
b. FTIR analysis of CMP at 650 oC for 3h.

Fourier-Transform Infrared Spectroscopy (FTIR): Figure 1b shows the FTIR spectra of CMP, distinct peaks were noticed at 711.9, 872.2, 1080.9, 1401.5, 1640.0, 1796.6, and 2512.2, respectively. The band at 1401.5 cm-1 represents bending vibration of the O-Ca-O group while the band at 771.9 – 1080.9 represents a stretch of CO32- molecules to higher energy value [3,36]. The spectrum stretches from 1640.0-2512.2 cm-1 indicated the presence of functional groups such as O-H, C-H for sp3 carbon, C=O for sp2 carbon, CHO, and N-H bond. Nevertheless, the undistorted assembly of CMP catalyst transformed into a spongy like structure signifies that calcination of the mixed powder at a temperature of 650oC was appropriate for a complete transformation of CaCO3 to CaO [16]. This observation could be attributed to the presence of functional groups present in the fruits during the growth period which occurred as a result of carbon growth inhibition.
Brunauer-Emmett-Teller (BET): Table 3 represents the properties of the catalysts indicating the BET surface, total pore volume, basicity, and percentage of CaO converted through N2 adsorption-desorption isotherm Brunauer-Emmett-Teller (BET) analysis. Observation from the results shows the calcined mixed powder (CMP) has a high basic site than other calcined catalysts (CMAPP, CCLPP, and CCPPP) owing to the high CaO (75.65%) obtained during analysis in the calcined powder. The basic site density obtained for CMAPP (158. 00 μmole/m2) was higher than that of CCLPP (120.00 μmole/m2), but the value obtained for CCPPP (157.80 μ mole/m2) is approximately the same with CMAPP. However, the basic site density of the CMP (160.83 μ mole/m2) was the highest which produced the highest biodiesel yield during transesterification. The table also reflects the result of biodiesel yields, at the same process conditions, each catalyst was tested based on the CaO yield of the calcined powders (CCPPP: 62.83%; CMAPP: 65.50%; CCLPP: 58.67 and CMP: 75.65%), the yields of biodiesel based on the nature of catalyst showed the three calcined catalysts have a basic site for conversion of oil to biodiesel (83.50, 88.75, 80.32 (%wt.)), but the mixed calcined catalyst produced highest biodiesel yield (96.50% wt.), due to the percentage of CaO in the catalyst and high basic site. Hence, the catalysts could be an economic viable promising source for CaO catalyst production for industrial applications.

Table 3: BET-adsorption and XRD analysis of calcined catalysts at 650 oC for 3 h.

Optimization of Transesterification Mixed Oil to Biodiesel

Experimental Results Analysis: Table 4 shows the coded experimental conditions, the experimental biodiesel yield, the predicted, and the residual values of the 30 standard runs generated by CCD. The table showed the maximum experimental yield of 97.20 (% wt.) at runs 18, while the minimum yield was obtained at runs 19 with a value of 85.66 (% wt.). Based on statistical analysis, the results were transformed into a fit summary, quadratic model, analysis of variance evaluation, diagnostics and graphs modeling, the predicted biodiesel yield of 96.63 (% wt.) was obtained at the following condition: reaction time of 80 min, CMP amount of 3.53 (g), reaction temperature of 90 oC, and CH3OH/OMR of 9:1 (ml/ml), at the desirability of 95.10%. This value was validated in triplicate, an average mean value of 96.50 (% wt.) was obtained which was close to the predicted value. The result proved that the methanolysis of CMP for the transesterification of mixed oil was successful.

Table 4: Coded factors, experimental, predicted biodiesel yield, and residuals values.

ANOVA and Fits Statistic: Table 5 shows the analysis of variance (ANOVA) for the response surface quadratic model and the Fit statistics. Observation from the table shows that the Model F-value of 1765.55 with a degree of freedom (df) of 14, implies the model is significant with prob value >0.0001. There is only a 0.01% chance that a “Model F-value” this large could occur due to noise. Meanwhile, values of “Prob > F” less than 0.05 show variable terms are significant. In this case, X1, X2, X3, X4, X12, X22, X32, X42, X1X2, X1X3, X1X4, X2X3, X2X4, and X3X4 were remarkable significant variable terms. The coefficient of determination is the correlation coefficient, also known as R-square,which allows it to display the degree of linear correlation between two variables. The value obtained in this study is high (99.94%), indicate a high degree of correlation between the interacting variables. The “Pred. R-Squared” of 99.65% is in reasonable agreement with the “Adj R-Squared” of 99.88%. The “Adeq Precision”, which measures the signal to noise ratio. Usually, a ratio greater than 4 is desirable, the ratio of 188.58 obtained in the study specifies an adequate signal.The polynomial model quadratic equation that shows the relationship between the biodiesel yield and the four-variable factors is presented in Eq. (4).

The final equation in term of coded

Table 5: ANOVA and Parametric Data Fit.

Meanwhile, the positive and the negative coefficients in the equation are the direct measure of the influence of variables on the response value. In this equation, the variable X2 with a coefficient of 1.23, f-value = 4796.50, with p-value<0.0001 is the most significant variable among the second-order polynomial equation in Eqn. (4).
Graphical Plots: Furthermore, the relationship between the response variable (biodiesel) and the interactive variables (X1X2, X1X3, X1X4, X2X3, X2X4, and X3X4) can be represented in contour and the three-dimensional plots as displayed in Figures 2a-2f. The observation from the graph shows Figure 2e has the highest mutual interaction between CMP amount and MeOH/OMR on the response biodiesel yield. The mutual interactive effects between reaction time and MeOH/OMR (Figure 2f) is higher than that observed in the interactive effects noticed between reaction time and reaction temperature (Figure 2b) on the response, but the interactive effects in Figure2c (MeOH/OMR and reaction temperature) is lesser than the interactive effects showed by Figure 2b, but higher than the interaction between CMP amount and reaction temperature (Figure 2d). Meanwhile, the least interactive effects were noticed in Figure 2a, which was the interaction between CMP amount and reaction time on response biodiesel produced. In all, there exist perfect interaction among the variables, which confirmed the variable factors considered in this study play an important role in biodiesel yield. The relationship between the predicted biodiesel and the experimental biodiesel yields as well as power transformation boxcox were as illustrated in Figures 3a & 3b.

Catalyst Reusability Test

For catalyst purification and reusability analysis of the reaction mixture, a built-in heating system vacuum centrifuges operated at 3500 rpm was used, the recovered catalyst was washed with alcohol to remove the impurity at the surface of catalyst that occurred during the transesterification. The alcoholic washed catalyst was oven-dried at 100oC for 1 h and then cooled to room temperature before reused. Nevertheless, the reactor wall accumulates a 0.15 g catalyst during the reaction, which tends to reduce the surface area and lowered catalyst activity. Hence, the need for catalyst reusability tests on catalytic activities. Figure 4 shows that the catalytic activities-maintained stability from the 1st cycle to the 4th cycle with little decreased from 93.6 (% wt.) to 92.20 (% wt.). However, there was significant decrease observed in 5th and 6th cycles (89.7 and 88.7% wt.), this can be associated to the continuous intermediate products formed during the reaction, such as monoglyceride and diglyceride, which obstructed the catalyst holes as well as water to oxygen reaction that occurred at the catalyst surface, which reduce the catalyst sensitivity. Hence, catalyst reusability was stopped after the 4th cycle [37-39].

Figure 2: Contour and 3-D’s Plots.

Figure 3: Diagnostics graph.

Figure 4: Catalyst recyclability test plot.

Properties of the Mixed Oil and Biodiesel

Method of AOAC were adopted to examine the properties of the blended oil and the product, the obtained results of the biodiesel were compared with the ASTM D6751 and EN 14214 biodiesel recommended standard (Table 6). It was observed that the decreased in the moisture content, density, viscosity, acid value, the iodine value, saponification, and the peroxide value of the blended oil to biodiesel was due to process transesterification. This confirmed that the synthesized product is consistent with biodiesel and the translation of blended oil complete transesterification reaction to biodiesel was achieved with insignificant resistance to flow and lessen internal drag in the engine. Further observation showed that the cetane number, the higher heating value (HHV), the API gravity, and the diesel index increased as blended oil was converted to biodiesel, this could be attributed to energy formation from viscous oil to low viscous oil. The high biodiesel yield obtained in this study could be attributed to due to decrease base consumption for neutralization. Based on cetane number, the higher the peroxide value, the better the cetane number and the decrease in ignition time [31,40]. The value of 4.34 meq O2/kg oil, can be attributed to the cetane number of 57.52 obtained. The higher heating value (HHV) of biodiesel is greater than that of blended oil which signify high heat of vaporization of water in the combustion of biodiesel. The American petroleum institute (API) gravity usually used to determine the weight of oil/petroleum in comparison with water, the value of 33.03 and 49.91 obtained for blended oil and biodiesel exhibited light oils. Diesel index which denotes the efficiency of the biodiesel as well as the ignition properties, the value obtained in this study were well within the required recommendation standard for biodiesel that can be used in I.C engine [41-44].

Table 6: Quality of mixed oil and biodiesel.

Conclusion

The study concluded that the ratio of blended oil produced low viscous oil, and the derived catalyst from the mixture of Cucurbita pepo, Musa acuminate and Citrullus lanatus peels powders, calcined at a temperature of 650oC for 3 h, produced high CaO- base (75.65%). Transesterification of blended oil to biodiesel was successfully carried out with maximum biodiesel yield of 97.20 (%wt.). A statistical software with second order model analysis predicted biodiesel yield of 96.63 (% wt.) at the following reaction time of 80 min, the CMP amount of 3.53 (g), the reaction temperature of 90 oC, and the CH3OH/OMR of 9:1 (ml/ ml), respectively. To validate this value, three experimental runs were carried out, and the average mean was determined as of 96.50 (% wt.) was obtained which was close to the predicted value. Analysis of variance test confirmed the significant of variables with p-value<0.0001. Catalyst reusability test was immobile at the 4th cycle due to loss of basicity that occurred due to leaching as a result of several recyclability during the reaction. Hence, the produced biodiesel conformed to biodiesel recommended standard, and the CaO catalyst could serve as promising economical feedstock for industrial application.

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The Relationship of Chronic Rhinosinusitis with Nasal Polyps and PDL-1

Introduction

Chronic rhinosinusitis with nasal polyps (CRSwNP) are benign inflammatory and hyperplastic outgrowths of the sinonasal mucosa [1]. It is a disease that seriously affects the quality of life of patients by causing nasal congestion, snoring, postnasal discharge, nasal discharge, loss of sense of smell/taste. The prevalence of CRSwNP is 1% to 4% in the general population [2]. CRSwNP is observed more frequently in male patients between the 4th and 6th decades [3]. İn histologically, nasal polyposis is characterized by respiratory epithelium covering oedematous stroma infiltrated by inflammatory cells [2]. Eosinophilic infiltration is the most common finding in polyp histopathology. In addition, there is an infiltration of inflammatory cells such as lymphocytes, basophils and macrophages.
Mucosal changes, including squamous metaplasia, secretory hyperplasia and epithelial cell proliferation, are observed in polyp tissues with the effect of chemical mediators secreted from these cells. T-helper 2 (Th2) cell-directed eosinophilia develops as a result of elevated immunoglobulin-E (IgE) and interleukin-5 (IL-5) [4]. In Asians, the majority of patients with CRSwNP show features of neutrophilic inflammation in addition to eosinophilia [5,6]. PD-1 (B7-H1,CD 274) is a protein-structured receptor belonging to the immunoglobulin superfamily found on the surface of activated T cells and antigen presenting cells. By binding to the PDL-1 ligand, T cell inhibition occurs and escape from the immune system occurs. PD-L1 works like a stop sign, inactivating the immune system’s T cells. One of the ways in which sick cells escape from the immune system is to densely host the proteins called PD-L1 on the cell surface. Studies have shown that PD-1 plays an important role in immune tolerance and in the regulation of T cell function [7].
Genetic studies have found that PD-1 gene polymorphism is associated with autoimmune diseases such as systemic erythematosus and rheumatoid arthritis [8,9]. PD-1 and PDL-1 are expressed in many different tumors such as lung carcinoma, lymphoma, pancreatic carcinoma, and renal cell carcinoma. It has been reported that PD-1 expression is increased in T cells in patients with CRSwNP with using flow cytometry [10]. This checkpoint led to the development of immune checkpoint inhibitors, which are the next generation immunotherapy drugs. These drugs prevent the interaction of molecules such as PD-L1 and PD-1, thereby activating the immune system against diseased cells. Among the immune checkpoint inhibitors nivolumab and pembrolizumab act by inhibiting PD-1 receptors, while atezolizumab and durvalumab act by inhibiting PD-L1 receptors [11].
If the importance of pdl1 in nasal polyp becomes clear, these immune checkpoint inhibitors drugs can be used in the treatment. There are not enough studies in the literature regarding the presence of PDL-1 in nasal polyps. In our study, our aim is to explain the presence of pdl1 in nasal polyp by immunohistochemical method. Detecting of the PDL-1 changes in nasal polyp tissue may explain the development of chronic rhinosinusitis with nasal polyps and may help develop new methods in the treatment of nasal polyps.

Materials and Methods

In our study, 20 patients who underwent endoscopic sinus surgery due to nasal polyps and 20 patients who underwent surgery for functional and/or aesthetic reasons and whose septum and turbinate mucosa were removed were included retrospectively, in Alanya Alaaddin Keykubat University Medical Faculty Hospital between 2010-2020. Previous sinus surgery, age is under 18 and nasal malignancies were exclusion criteria. 4 micron thick sections were taken from paraffin blocks for applying immunohistochemical PDL-1 antibodies. These sections were processed with Ventana PDL-1 (SP 263) monoclonal antibody on the fully automated Ventana Ultra Benchmark XT instrument. In the evaluation of PDL- 1 antibody applied immunohistochemically, the percentage of cells showing membranous staining with PDL-1 antibody was calculated among 100 tumor cells under light microscope. IHC staining results in groups were evaluated with Mann Whitney test using SPSS software version 22 (SPSS Inc., Chicago, IL, USA). P <0.001 was considered significant. Ethics committee approval was obtained from Alanya Alaaddin Keykubat University (no:2021/09-06).

Results

There were 2 female and 18 male patients in the study group, and 4 female and 16 male patients in the control group. The mean age was 37.4±8.6 years in the study group and 35.4±8.8 years in the control group. PDL-1 positivity in nasal polyp tissues was significantly higher than normal nasal mucosa epithelium. (P=0.000) (Table 1) (Figures 1-3) The recurrence rate was higher in PDL-1 positive cases with nasal polyps, and the recurrence rate increased as the staining rate increased, and it was statistically significant. (P=0.003).

Figure 1: Epithelial cell of the polyp with 50% membranous staining.

Figure 2: Epithelial cell of the polyp with 10% membranous staining.

Figure 3: Control normal nasal mucosa epithelium not stained with PDL-1.

Table 1: Percentage of immunohistochemical staining of cells with PDL-1.

Discussion

There are some hypotheses exist for how CRSwNP develops but the aetiopathologenetic mechanism of it is still unclear [2,12]. The pathogenesis of it is thought to involve persistent inflammation of the nasal mucosa due to infiltration by eosinophils, basophils, lymphocytes, neutrophils and mast cells [2]. T lymphocytes act as the main actor in this inflammation process. PDL1 İs a member of the B7 family that regulate the T-cell response and a predictive biomarker. It is a cell-surface glycoprotein primarily expressed by antigen-presenting cells. Studies on PDL 1 in the literature are mostly about tumor cells. Different cut-off values have been reported in these studies [13-16]. However, variable cut-off levels create difficulties for researchers in making decisions during evaluation. Determining a standard cut-off value for researchers is important for the correct interpretation of the results and for the consistency between the results of the studies. In our study, we considered the presence of PDL-1 positive in staining of 1% or more. Different companies have antibodies and subclones for PDL- 1. In the literature, there are studies reporting different results with antibodies and clones of different companies in the same cell group [17,18].
We used the SP263 clone of Ventana, which is frequently used in studies in the literature, to increase the staining quality and the reliability of the results. There are studies explaining that the infectious process plays a role in the pathogenesis of CRSwNP [19,20]. İt is reported that IFN-γ secretion increases after incubation of lymphocytes with bacterial and fungal extracts [21,22]. IFN-γ is an important cytokine thas is a strong inducer of PDL-1 expression [23]. Before studies showed IFN-γ have been identified in nasal polyposis [24,25]. PDL-1 levels in other cell membranes increases by the IFN-γ charged by T-cells. Thus, it causes a negative feedback in nasal polyposis by causing down-regulation of T-cell responses. İn a study about mRNA expression of PDL-1 in patients with CRSwNP, resarchers found mRNA levels of PDL-1 was increased in nasal polypies epitel compared to healty nasal tissue epitel [26].
Although there are studies in the literature that measure PDL1 level in nasal polyp tissue and blood cells by PCR and flow cytometry, but there is not enough study in which the pDL1 level in nasal polyp tissue is analyzed immunohistochemically. In a small study in the literature using only 3 nasal polyp tissues and 3 nasal tissues as control, immunohistochemical evaluation was made and PD-1+ cells were indeed increased in the submucosa of NP tissue compared to control tissue [27]. İn our study we found that the pdl1 level in CRSwNP tissue was significantly higher than the pdl1 level in the control healthy nasal tissue, and it was was statistically significant. (p:0.01). For this reason, the use of monoclonal antibodies developed against PDL-1 as a target drug as a non-surgical drug therapy in nasal polyps is a matter of debate. Recurrence in surgical procedures is an undesirable situation for the patient and the surgeon. Different researchers used several markers for predicting nasal polyp recurrence.
Recurrence is frequently observed in nasal polyposis, and recurrence is higher in eosinophilic polyps than in noneosynphilic polyps [28]. Zhong et al. in their study, the relationship between peripheral eosinophil count and recurrence was examined and they found a significant correlation between high peripheral blood eosinophil count and recurrence [29]. In another study showed researchers that a tissue eosinophil proportion of >27% of total cells was a marker for recurrence within 2 years after endocopic sinus surgery in patients with CRSwNP [30]. In our study, recurrence rate was high, and it was found in 10 (50%) patients. The recurrence rate was higher in PDL-1 positive cases with nasal polyps, and the recurrence rate increased as the staining rate increased, and it was statistically significant. (P=0.003) These findings suggest that the presence of PDL-1 may be a predictive factor for recurrence.

Limitations

The most important limitation of our study is that it was a retrospective study, and we could not reach sufficient patient material, since the operated patients took the operation materials to an external center for histopathological consultation. It will be possible to make a clearer comment on the fact that it is a determining factor in the development of recurrence.

Conclusion

PDL-1 was significantly increased in our patients with CRSwNP compared to normal nasal mucosa tissue. Presence of PDl-1 is a predictive factor for recurrence in nasal polyposis. Since there are not many studies explaining the presence of CRSwNP and PDL- 1 in the literature, we think that our study will contribute to the literature. New studies with a larger number of patients are needed to determine the presence and importance of PDL-1 in nasal polyps.

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Silver Nanoparticles for Hydrogen Peroxide Sensors

Introduction

Silver particles have been used as antibacterial agents since the 19th century, and now their uses have diversified to include many new physical, chemical and biological ones [1-4]. AgNPs are considered to be one of the most widespread NPs, with about 500 tons of annual global production [5]. More recently, AgNPs have been incorporated into industrial and surgical device coatings,dental coatings, and automotive smoke filters and textiles due to their effective properties against microbes [6]. The mechanism on this topic is currently under discussion by researchers [7]. In this review, relevant techniques of synthesis of metal nanoparticles will be discussed. After that, a detailed study of the properties and applications of AgNPs for hydrogen peroxide sensors will be presented.

Bottom-Up and Top-Down Synthesis

As a historical background for metal nanoparticles (MNPs), it was reported that many exploited the strengthening of ceramic matrices, including natural asbestos nanofibers, more than 4500 years ago [8]. Lead-based chemistry was pioneered in ancient Egypt for cosmetic preparation over 4000 years ago. Here, we look at a hair dye recipe using lead salts described in the text since Greco-Roman times. We report direct evidence for the shape and distribution of PbS nanocrystals that form in the hair during darkening [9]. Likewise, “Egyptian blue” was the first synthetic pigment prepared and used by the Egyptians using a mixture of sintered nanometer-scale glass and quartz around the 3rd century BC [10]. Egyptian Blue represents a complex mixture of CaCuSi4O10 and SiO2 (both glass and quartz). In the ancient geographic regions of the Roman Empire, including Egypt, Mesopotamia, and Greece, Egyptian blue for decorative purposes has been observed during archaeological excavations.
Natural or synthetic ways can synthesize NPs by two basic approaches, including various sub-preparation methods. The first approach is called the “top to bottom” method, including breaking down bulk solid materials into smaller pieces by applying external energy from physical, chemical, and thermal techniques [11]. The second approach, called “from the bottom up, “brings together and combines atoms or molecules of gases or liquids. The top-down approach is expensive. However, it is impossible to obtain perfect surfaces and edges due to the cavities and roughness in NPs, while a bottom-up approach can get excellent results of nanoparticle synthesis. In addition, with the bottom-up approach, no waste is formed to be eliminated, and the smaller NPs can be obtained with better control of the sizes [12-16]. However, adapting the production of large quantities of powders on an industrial scale is not simple. A significant advantage of treatment in solution is the possibility of generating encapsulated NPs using surfactants as a protective shell, which makes it possible to obtain very homogeneous and well dispersed NPs [17].
The main bottom-up approaches are the supercritical fluid synthesis, the use of the template, the spinning, the synthesis by plasma or flame projection, the green synthesis, the sol-gel process, the laser pyrolysis, the aerosol-based process, the chemical vapor deposition, and the atomic or molecular condensation. The main top-down approaches are the mechanical milling, the etching (chemical), the electro-explosion (thermal/chemical), the spraying (kinetics), and the laser ablation (thermal). The green synthesis approach is based on biological methods (accommodating plants, algae, the mushrooms, yeasts, actinomycetes and bacteria).

Types and Synthesis of Silver Nanoparticles

Among the various types of NPs, Ag NPs have been widely developed to be utilized in various applications due to their outstanding properties. Typical applications of Ag NPs include clothing and textiles, medical devices, food storage, cosmetics, sunscreens, laundry detergents [18], bandages [19], and sensors [20]. Some studies have found that Ag NPs have cytotoxicity that can induce ROS formation in cells [21]. Therefore, many products such as detergents, toiletries, etc. In addition, their synthetic or in-use personal care, whether industrial or household, produces a release of NPs, which ultimately ends up in the sewer. This untreated wastewater affects aquatic ecosystems and thus microorganisms. Recently, Ag NPs had great concerns regarding aquatic toxicology due to the difficulty of tracking these particles in the environment and accessing their effects on living organisms [22]. The fate of NPs in the aquatic environment and their interactions, the interactions between NPs with biological and abiotic components, and their potential for damage are not well understood, and these uncertainties raise concerns about related risks. These molecules impose on humans on health and the environment [23]. Based on the Scopus database [24], the publications on Ag NPs increase with time, where it started in 1990 (two reports) and reached 7105 reports in 2020.
Colloidal Ag NPs are molecules with an average diameter of 20- 40 nm and comprise 80% silver atoms and 20% silver ions. They are the best-selling nanoparticles ahead of carbon nanotubes and titanium nanoparticles and are released into the environment. The demand for Ag NPs has increased due to their applicability in multiple fields. Over the years, various synthesis techniques have been developed, and procedures have been improved to prepare small and uniform Ag NPs. Ag NPs were prepared by a chemical reduction technique, where the silver ions are reduced by sodium citrate [25]. As referenced, various kinds of Ag NPs have been utilized in various applications [26]. Specifically, Ag NPs of differing sizes and shapes have been used in a broad scope of uses and clinical gear, such as electronic gadgets, coatings, cleansers, swathes, etc. [27]. Explicit physical a nd optical properties of Ag NPs are subsequently essential factors in advancing their use in these applications. In such a manner, the accompanying subtleties of the materials are imperative to consider in their combination: surface property, size dissemination, clear morphology, molecule composition, dissolution rate (i.e., reactivity in arrangement and effectiveness of particle delivery), and kinds of diminishing and capping specialists utilized. The blend techniques for metal NPs are partitioned into top-down and bottom-up approaches. In these approaches which also include chemical and green methods, the electron transfer initiates the bioreduction through nicotinamide adenine dinucleotide (NADH)-dependent reductase as an electron carrier to form NAD+. The resulting electrons are obtained by Ag+ ions, which are reduced to elemental Ag NPs.
The physical technique usually utilized to prepare Ag NPs is the evaporation-condensation method. It is commonly performed using a tube furnace at atmospheric pressure, synthesizing various sizes [28]. Several attempts have been made. For instance, Tsuji, et al. [29] proposed a new method for synthesizing Ag NPs by a laser ablation technique with focused and unfocused laser beam irradiation carried out at 12 and 900 mJ cm−2 intensities, respectively. The radiation wavelengths used were 355, 532, and 1064 nm. This study revealed that the surface plasmon wavelength of Ag NPs irradiated using 355, 532, and 1064 nm is ~400 nm for focused and unfocused beams. The famous chemical method for Ag NPs synthesis is a reduction by natural and inorganic reducing agents. By and large, unique reducing agents, for example, sodium citrate, ascorbate, sodium borohydride, essential hydrogen, polyol measure, Tollen’s reagent, N, N-dimethylformamide (DMF), and poly (ethylene glycol)-block copolymers are utilized for the reduction of silver particles (Ag+) in aqueous or non-aqueous arrangements. These reducing agents decrease Ag+ and lead to metallic silver (Ag0), trailed by agglomeration into oligomeric clusters. These clusters ultimately arrange the metallic colloidal silver particles [30]. It is critical to utilize defensive agents to stabilize dispersive NPs during metal nanoparticle planning and ensure that the NPs consumed or tied onto nanoparticle surfaces stay away from their agglomeration [31]. Green synthesis methodologies dependent on natural reducing agents rely on different reaction parameters such as solvent, temperature, pressing, and pH conditions (acidic, fundamental, or impartial). For the union of metal oxide nanoparticles, plant biodiversity has been extensively viewed as the accessibility of successful phytochemicals in different plant separates, particularly in leaves such as ketones and aldehydes flavones, amides, terpenoids, carboxylic acids, phenols, and ascorbic acids [32,33]. These parts are fit for decreasing metal salts into MNPs [34]. The fundamental focus of such nanomaterials has been researched for biomedical diagnostics, antimicrobials, catalysis, atomic detecting, optical imaging, and marking of natural frameworks.

Applications of Silver Nanoparticles

Over the last decades, the production of NPs has been increasing rapidly for applications in electronics, chemistry, biology, and almost all our daily life applications [35]. This is mainly due to their properties of being very small, close to the biomacromolecules and providing high surface area, rapid diffusion, and high reactivity in both liquid and gas phases [36,37]. Recently, Ag NPs have attracted attention in various applications such as biological, food, optoelectronics, electronic devices for energy conversion, electron field emission sources for emission displays, and surface-enhanced Raman properties [38-41]. Accordingly, utilizing Ag NPs in sensing, mainly for the excellent physico-chemical properties, will be reviewed here. Developing fast, sensitive and selective gas sensors has received great attention in environmental monitoring, national security, and food safety applications [42]. Typically, the total performance of gas sensors strongly depends on the specific area surface of the materials utilized for detection gas. Thus nano-scale sensing is predictable to display improved sensing performance [20]. Typically, semi-conductive nano metal oxides such as ZnO and SnO2 exhibit high sensitivity and fast responses to some gases [43]. A relatively higher temperature (150–600◦C) required for maximum response is a big problem that restricts the practical applications in most areas [44]. Therefore, developing suitable nanomaterials to enhance the sensor response is highly recommended. Noble metal nanostructures are the most promising in this field due to LSPR. The LSPR is an optical phenomenon observed when the electromagnetic radiation excites the surface conducting electrons of MNPs, resulting in a coherent resonance oscillation of the particles. The location of the extinction maximum is highly dependent on the reflective index and dielectric properties of the surrounding environment and the adsorption of the molecules on the metal surface [45,46]. Therefore, the peak wavelength shift in the extinction maximum of NPs is used to fast detect moleculeinduced changes surrounding the NPs. Thus, UV-Vis spectroscopy and the naked eye can observe the changes in the absorbance of the visible and near-infrared wavelength regions.
There are many published articles on the use of LSPR sensing applications in the liquid phase to detect organic phosphorous pesticides [47] and ammonia [48]. Generally, SPR absorption of noble metal NPs is strong in the visible to near-infrared (IR) region. Moreover, SPR is particularly sensitive to its size, shape, composition, distance, and surroundings. Therefore, it displays a promising ability for various sensors. However, Ag NPs have higher extinction coefficients than gold nanoparticles (Au NPs) of the same size [49]. Due to this feature and high specific surface area, high catalytic, high crystallinity, and their electrical and optical properties, Ag NPs have been widely investigated as gas sensor applications [41,50]. This section will present the recent progress on Ag NPs as gas sensors for ammonia, methane, and hydrogen peroxide. Despite the wide use in industry, such as fertilizers, animal feed production, and manufacturing of paper and plastics, ammonia is a toxic material with a harmful effect on the human body as it can harm tissues and the immune system [51]. Ammonia is largely produced in deteriorating food and fruitbodies by various micro/macrofungi [52]. Thus, monitoring the concentration of ammonia in air and liquid in the atmosphere is extremely important. Recently, Ag NPs have been widely used to sense organic gases such as methane and ethanol [20,53,54]. Cannilla, et al. [55] successfully prepared Ag NPs in a poly-methacrylic acid (PMA) matrix by a photo-induced reduction process followed by deposition on a ceramic substrate to sense ammonia gas in resistive base sensors. To improve the conductivity of the thin films to be suitable for sensing, the as-prepared Ag NPs/PMA were loaded with multi-walled carbon nanotubes (MWCNTs). The developed sensor shows the considerable ability to work at low temperature with a wide range of detection range and represent fast response/ recovery times. Kumar et al. prepared face-centered cubic polyvinylpyrrolidone (PVP) capped Ag NPs at room temperature and a chemical reduction method [41]. The prepared sample showed an average size of ~22 nm, showing a conductive and metallic nature. As ammonium gas sensing, the synthesized NPs thin films showed the maximum sensitivity towards ammonia gas.

Hydrogen Peroxide Sensing

In the last decade, hydrogen peroxide (H2O2) monitoring has gained importance due to its widely employed in many industrial, atomic power stations, medical sectors and its application as a disinfecting agent for water pools [56-58]. However, the high presence of H2O2 can cause various biological damages, leading to aging, neurodegeneration, and cancer [59,60]. Therefore, developing a high-response, cheap method is highly recommended for medical, pharmaceutical security, and environmental protection [59]. Various techniques, such as spectrophotometry [60,61], chemiluminescence [62], and electrochemistry [63], have been utilized to detect H2O2. These methods are categorized as enzymatic and non-enzymatic methods. In the case of enzymatic, the peroxidase is an illustrative enzyme for H2O2 exposure; however, both pH and temperature are limited.
As mentioned above, noble metal NPs display strong SPR absorption with extreme sensitivity to the size, shape, composition, and surroundings suitable for colorimetric sensors in the range of visible to near-infrared region. For colorimetric assays, Ag NPs have a high ability toward the decay of H2O2 [64]. This reaction can mainly be sensed by colorimetric principles, as the colloidal Ag NPs exhibit a characteristic color from the LSPR. The alteration in both particle size and shape results from the incorporation of colloidal Ag NPs with H2O2 can be identified by determining the change in the absorption spectral at the wavelength of LSPR. For instance, Zhang, et al. [65] investigated H2O2 via colorimetric detection using three different morphologies of Ag NPs (triangular, spherical, and cubic). The Ag NPs with various shapes reacted with H2O2, and the edges of Ag NPs had been etched. The change shape transformation induced visible color change, which was used for the quantitative determination of H2O2. The triangular Ag NPs display the highest sensitivity for a quite small level of H2O2 (5 mM), while the cubic Ag NPs display lower sensitivity. Recently, Srikhao, et al. [66] prepared Ag NPs with an average particle size of 16.9 nm by using phenolic compound extracted from sugarcane leaves as a reducing agent. The results showed that 90◦C and stirring for 20 min are the optimum conditions for phenolic compound extraction. The prepared Ag NPs were evaluated as ammonia and H2O2 solution sensing by UV-Vis spectrophotometer and the naked eye. It observed that the Ag NPs sensitively detect both H2O2 and ammonia even at low concentrations.
Moreover, the prepared Ag NPs could detect these toxic agents even after two weeks. However, measuring in spectrophotometric methods requires some standard solutions and tools such as standardized cuvettes, microwell plates, and spectroscopic equipment. To overcome these limitations, Yoshikawa et al. utilized an optical technique for detecting H2O2 in their work Ag NPs deposited on a glass plate/Au NPs [66]. The Ag NPs chip diffracts incident light, and the diffraction efficiency is correlated with the amount of Ag NPs. By applying a drop of H2O2 onto the chip, the diffraction strength debilitates due to the decay of Ag NPs. A movable measurement technique of the diffraction intensity changes is assembled, and the H2O2 detection in a concentration range of 6.7–668 mmol L−1 in about 2 min by dropping the H2O2 solution onto the substrate.
The electrochemical technique is promising to detect H2O2 due to its simplicity, lower cost, ease of operation, and high sensitivity and selectivity compared to the other techniques [62,63]. Many electrochemical sensors based on metals and metal oxides-based complexes have been developed to overcome the high overpotential. Due to the high electron transfer rates, high catalytic activity toward reducing H2O2, and significantly decreasing overpotential at oxidizing and reducing agents, Ag NPs are widely utilized to fabricate these sensors. Zhan, et al. [67] decorated Ag NPs on three-dimensional graphene (3DG) via the hydrothermal process as a sensing electrode for electrochemical detection of H2O2 in phosphate-buffered solutions. The electrochemical results approved that the Ag NPs-3DG based biosensor exhibits fast amperometric sensing, low LOD, wide linear responding range, and perfect selectivity for non-enzyme H2O2 detection. Recently, Maduraiveer, et al. [68] utilized an electrochemical sensor for H2O2 by using Ag NPs introduced in a silicate matrix (APS(SG). The prepared APS(SG)-Ag NPs were deposited on glassy carbon (GC) electrode. The electron transfer behavior of the APS(SG)-Ag NPs was investigated by potassium ferricyanide ([FeCN)6]3−), methyl viologen (MV2) and ruthenium hexamine ([Ru (NH3)6]3+). The GC/APS(SG) electrode displayed a twofold increase in the peak currents and fast electron transfer kinetics toward [Fe (CN)6]3− in comparison with the GC electrode. The GC electrode modified with APS(SG)-Ag NPs significantly improved electron transfer. The GC/ APS(SG)-Ag NP electrode as an electrocatalytic sensor against H2O2 offered a reduction of H2O2 at less negative potential and showed the experimental low detection limit of 25.0 μM with the sensitivity of 0.042 μA/μM. Additionally, an APS(SG)-Ag NP-based sensor showed a fast response, good stability, and reproducibility.

Conclusion

This article presented the synthesis, properties, and hydrogen peroxide applications of silver nanoparticles in a brief detail. Despite the great role of Ag NPs in sensing applications for detecting gases and vapors of some organic compounds and detecting hydrogen peroxide in industry and biosystems, the function of this effect needs more study to be fully understood. Furthermore, the longterm stability and sensitivity should be more developed by adjusting the preparation conditions, utilizing eco-friendly stabilizing agents. Moreover, recycling Ag NPs from their applications wastes is an important issue from both environmental and economic points of view. This would help in developing Ag NP based nanomaterials more safe, biocompatible, and efficient for some vital applications in our life.

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Education of a Child in the Ethics of Al-Ghazali

Of course, raising a child is not an easy task. However, the scientist emphasizes that raising children should not be the reason for not generosity and indifference of parents to others. The purpose of starting a family is to bring up a healthy generation. The upbringing of society begins in the family. Raising a child is a serious responsibility that every parent must be aware of and fulfill. Parents should be the support and guardians of their children and should do everything possible for their peace of mind, happiness and full development. Parents, raising their children, pay back their duty to their parents. Indeed, the precious heritage of parents for a child is a good upbringing. The ethical conclusions of Muhammad Al-Ghazali on the sphere of raising children are attractive, in which the ways and methods of upbringing are shown in detail. The scientist’s ethical teachings can be used as a guide by educators, psychologists and the general public.

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How to Facilitate Simulation in Telemedicine in the COVID Era

Health Care Simulation is widely used to train healthcare professionals as it contributes to long-term retention of acquired complex technical, clinical and teamwork skills when compared to traditional learning methods. In addition, translational science research shows that measured outcomes transfer to improved patient care practices, and improved patient and public health [1]. Health Care Simulation follows experiential learning theory that proposes that a two-dimension cycle is necessary for learning to occur. The first dimension represents perception, where the learner begins by either grasping a concrete experience or a concept. The second dimension represents processing, where the learner transforms the experience by reflecting on it and the concept by actively experimenting on it [2]. To complete this cycle, learners and educators usually get together on site to participate in highly interactive, high-fidelity clinical scenarios using mannequins, actors, task trainers or a combination of them, followed by debriefing [3]. But the rapid development of technology-assisted learning methods, which are on the rise since the coronavirus (COVID-19) pandemic in order to help reduce the transmission risk among professionals, opens up new alternatives for distance experiential learning [4].

The best approach to carrying out clinical simulation activities in this context remains to be elucidated. Several solutions are proposed to fit the pieces in a suitable way that would allow professionals to continue with their training activities and, at the same time, to serve the clinical safety of the participants, avoiding, as far as possible the spread of the disease. They include a variety of simulation based asynchronous (e.g. recorded videos, vignettes, serious games) and synchronous (e.g. webinars, team collaboration) methods. They recreate the clinical environment with a different level of fidelity and learner personal engagement in combination with reflection strategies in combination with reflection strategies [5]. Moreover, innovations in healthcare simulation technologies can now provide the learner with opportunities to practice increasingly complex motor, decision-making and communication skills using virtual patient simulation. They recreate dynamic health conditions in a variety of clinical settings that respond to user interventions and show improvements in learning satisfaction when compared to a case-based learning approach [6].

Where to Start

The basic principle is to generate a training tool that maintains the essence of the simulation as a clinical practice that allows participants not to be in the simulation room in person in order to guarantee their safety in the midst of the COVID 19 pandemic. We have developed in Spain a model of distance clinical simulation in Valdecila Virtual Hospital, suitable for emergency physicians and we have proved in staff and also in internal medicine resident’s program [7] obtaining promising perspectives. This model can achieve positive results using distance clinical simulation combining their traditional simulation technology to address the training needs of healthcare organizations.

The Goal

Using virtual patient simulation to provide simulation-based distance learning experiences that are extremely realistic and highly interactive for the learner remains a challenge. Communicating with patients, family members and healthcare workers, performing physical examination on patients, monitoring of physiological parameters, evaluating complementary examinations in an actual clinical environment are all constricted by mathematical algorithms and software capabilities. A mix of synchronous on-site/on-line distance learning methods may address aspects of the richness and complexity of a true clinical experience and facilitate reflection on action promoting participant engagement, while limiting exposure to infectious diseases during a pandemic. Distant learning combining on site simulation technology with teleconferencing software based on experiential learning principles is feasible and easy-to-implement, and is well-accepted by participants for acute care training in the emergency room. The promising results of this new approach of “taking care of patients from a distance” can be useful not only during a pandemic, but also in our daily work because surely this new model will be able to facilitate future learning courses in the health field.

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Turning the Circadian Clock Slow: A New Function of Gaseous Signal H₂S

Gaseous signal molecules, such as nitric oxide (NO), carbonic oxide (CO), and hydrogen sulfide (H₂S), widely exist in most tissues of the body, and exert important physiological functions. These molecules are endogenously synthesized and catabolized, can freely diffuse across the plasma membrane and regulate certain biological processes at physiological concentrations. H₂S is the latest discovered among the above three gaseous signal molecules. Endogenous H₂S is produced by the catalytic synthesis of L-cysteine hydrochloride by cystathionine beta synthase (CBS) or cystathionine gamma lyase (CSE). CBS and CSE belong to pyridoxal 5’-phosphate dependent enzymes, with differences in tissue distribution. CBS mainly distributes in the brain, especially in hippocampus and cerebellum. The H₂S concentration in rat serum was about 46μmol/L and was 50-160μmol/L in rat brain tissues. In vivo, endogenous H₂S mainly exists in two forms, i.e., free gas (about 1/3) and sodium hydrosulfide (NaHS) (about 2/3). The two forms keep a dynamic equilibrium. As a H₂S donor, NaHS mainly exists in peripheral tissues, and maintains the stabilities of free H₂S level and pH in vivo. Studies on endogenous NO and CO have demonstrated that both of them play important roles in the regulation of circadian rhythm in organisms. It has also been proved that H₂S can change the expression patterns of clock genes such as Clock, Per2, Bmal1, and Rev-erbα by influencing the content ratio of NAD/NADH and the expression of Sirt1 in mice hepatocytes [1]. However, it is still unclear whether H₂S regulate the central circadian clock In vivo. The present study aimed to test the hypothesis that H₂S plays an important role in the regulation of central circadian clock located in the suprachiasmatic nucleus (SCN) by affecting the expression patterns of clock genes and clock-controlled genes.

To test the hypothesis, we performed voluntary wheel running experiments in adult male C57BL6/J mice. Animals were first entrained to 12:12h light: dark (LD) circumstance for 7 days and then subjected to constant dark (DD) condition for experiments, with circadian time 12:00 (CT12) indicated as the beginning of subjective night. Data were analyzed using the ActiView Biological Rhythm Analysis software (MiniMeter Co., USA). To observe the effects of higher or lower H₂S on the circadian locomotor rhythm, intracerebroventricular (i.c.v.) injections of exogenous NaHS (50mmol/L, 4ml per injection) or amiono-oxyacetate (AOAA) (a CBS inhibitor) (50mmol/L, 4μl per injection) were performed, respectively. AOAA was dissolved in artificial cerebrospinal fluid (ACSF) composed of (in mmol/L) NaCl 119, NaHCO3 26.2, KCl 2.5, NaH2PO4 1.0, MgCl2 1.3, glucose 10.0, pH 7.4. To check whether the SCN showed a normal light entrainment, short-time (15min) light exposure (200 lux) was performed. Period length (tau) was determined by the ActiView periodogram analysis. Phase shifts were defined as the difference between the predicted activity onset times and observed activity onset times in response to NaHS or light pulses.

Figure 1: H₂S modulation on the circadian rhythm in mice.
A. Wheel running actograms showing that H₂S i.c.v. injection at CT14 induced phase delay and circadian period prolongation.
B. ACSF i.c.v. injection at CT14 (control).
C. H₂S i.c.v. injection at CT20 also induced phase delay and circadian period prolongation.
D. ACSF i.c.v. injection at CT20.
E. Actograms showing phase shift and circadian period prolongation induced by i.c.v. injection of AOAA at CT14 (red asterisk), light exposure at CT15 (blue asterisk) and AOAA plus light exposure (red asterisk plus blue asterisk).
F. Statistical phase shifts at different conditions.
G. Immunohistochemical stains of SCN showing the robust circadian expression pattern of CBS.
H. RT-qPCR showing the mRNA levels of Bmal1, Per1, Per2 and Sirt1 in the SCN at different conditions.

After wheel running experiments, animals were decapitated under dim red light (4 lux), and SCN tissues were immediately harvested, liquid nitrogen frozen and total RNA were extracted as we previously reported [2]. Transcriptions of circadian genes, including Bmal1, Per1, Per2 and Sirt1, were examined by realtime quantitative PCR (RT-qPCR). Some SCN tissues were routinely developed, sectioned, and the protein expression of CBS was visualized by immunohistochemistry. Data were expressed as mean ± standard deviation (SD). Student’s t test or analysis of variance was used to perform the statistics where necessary. The criterion for statistical significance was P < 0.05. Animal use protocol was in compliance with the guidelines for the Care and Use of Laboratory Animals (NIH, revised 2011). Results showed that bolus i.c.v. injection of NaHS at CT14 (early subjective night) induced significant phase delay of the circadian locomotor rhythm in mice by about 50 ± 8 min, and also obviously prolonged the circadian period length from 23.8h to 24.3h (Figure 1A), compared with the control ACSF injection at CT14 (Figure 1B). Bolus NaHS (i.c.v.) at CT20 (late subjective night) also caused a phase delay of 30 ± 10min (Figure 1C) and a prolongation of period length from 23.8h to 24h, compared with control ACSF injection at CT20 (Figure 1D). Above results showed that NaHS (H₂S donor) i.c.v. injection induced phase delay and prolongation of circadian period length, suggesting that H₂S could slow down the running of central circadian clock. We thus proposed that suppression of H₂S production by inhibiting CBS would lead to opposite changes of phase and period length. However, to our surprise, i.c.v. injection of AOAA at CT14 produced almost the same results as that induced by NaHS, i.e., phase delay (30 ± 10 min) and period length prolongation, but the extents of phase shift and period prolongation were obviously less than that induced by NaHS (Figures 1E & 1F).

To check whether NaHS i.c.v. injection would affect the light entrainment function of SCN, twelve days after bolus NaHS i.c.v. injection, we gave the mice a short-time (15min) light exposure at CT15. Results showed that short light exposure induced significant phase delay by about 120 ± 20min (Figures 1E & 1F). This result indicates that the SCN responded well to light signal and was not damaged by NaHS i.c.v. injection. A combination of AOAA at CT14 and light exposure (CT15) led to further phase delay (180 ± 15min) and prolongation of circadian period length (Figures 1E & 1F). As CBS is a H₂S synthetase, and H₂S donor NaHS affected the circadian rhythm as we observed, we thus examined whether CBS expression would show circadian rhythm, and whether inhibition of CBS would affect the expression of clock genes and clock-controlled gene Sirt1, in the SCN of mice. We detected the expression of CBS over 24 hours in mice SCN tissues by immunohistochemistry. SCN tissues were obtained at CT2, CT6, CT10, CT14, CT18 and CT22. CSB protein expression levels showed a robust circadian pattern with a cycle of nearly 24 hours (Figure 1G). The expression peak appeared at CT14, and the trough showed at CT2 (Figure 1G).

Because NaHS induced phase delay and period length prolongation as shown above, it is possible that NaHS may change the transcription of clock genes and clock-controlled gene Sirt1. We checked the mRNA levels of Bmal1, Per1, Per2 and Sirt1 before and 15min after NaHS i.c.v. injection at CT14 by qRT-PCR. Results showed NaHS significantly decreased the mRNA levels of Per2 by 0.69-fold but increased the mRNA level of Sirt1 by 1.5-fold, while NaHS did not affect the mRNA levels of Per1 and Bmal1 (Figure 1H). To clarify the major findings of the present study, bolus i.c.v. injections of NaHS at CT14 and CT20, a way thought to increase the H₂S level in the SCN, induced phase delay and period length prolongation of mice free-running locomotor rhythms; deactivating CBS by AOAA to reduce the H₂S level in the SCN exerted similar result as NaHS did; the used dosage of NaHS did not damage the light entrainment function of SCN; CBS expression in the SCN showed a pattern of circadian rhythm; NaHS i.c.v. injection at CT14 increased the protein expression of CBS and the transcription of Sirt1, decreased the transcription of Per2, but did not significantly affect the transcription of Bmal1 and Per1, in the SCN. These results suggest that H₂S slows down the running of circadian rhythm, or in other words, H₂S turns the central circadian clock slow. This study provides new evidences to support that H₂S takes a role in the regulation of central circadian clock. The circadian expression pattern of CBS in the SCN is at least partially associated with the regulatory function of H₂S on the central clock, and PER2 and SIRT1 may take major roles in the function of H₂S.

We noticed that either increasing the H₂S level by NaHS or decreasing the H₂S level by CBS inhibitor induced the same results, i.e., phase delay and period length prolongation, only that the extent of NaHS-induced phase delay was greater than that caused by AOAA. Similar contradictory phenomena have also been observed in other studies. For example, Luo, et al. [3] demonstrated that both overexpression and deletion of miR-279 disrupted the circadian rhythm in Drosophila. We recently also found that either elevation or reduction of the microRNA-17-5p level in mice SCN shortened the free-running period length [4]. This unconventional regulatory algorithm deserves further investigations both in mechanism and significance. H₂S has been indicated having an anti-aging effect. It is still unknown whether H₂S exerts the anti-aging effect through changing the circadian rhythm. Some studies have shown that the free-running periods are shortened in senescent C57BL/6 mice, DBA mice, rats and hamsters, although some contradictory results were also reported. We show here that H₂S induced phase delay and free-running period prolongation in mice, and decreased Per2 mRNA level but increased Sirt1 mRNA level in the SCN. It is possible that H₂S exerts the anti-aging effect by prolonging the endogenous cycles of circadian rhythm via promoting the expression of Sirt1, because Sirt1 is considered a gene of longevity, and H₂S can prevent cell senescence through SIRT1 activation. SIRT1 regulates the expression of clock genes by PER2 acetylation [5]. Higher SIRT1 accelerates the degradation of PER2 protein, thereby promoting the transcription of Clock/Bmal1. This regulatory signaling pathway may explain our finding that exogenous H₂S increased the expression of Sirt1 and decreased the expression of Per2.

A question may be raised as whether exogenous NaHS (i.c.v.) would damage the function of SCN. The physiological concentration of H₂S in mouse brain is about 50-160μmol/L, but the exact transient local H₂S levels in the SCN are unknown. The concentration of NaHS used in the present study was 50mmol/l (i.c.v. 4ml per injection). This NaHS concentration looked much higher than the H₂S level of natural brain, but the actual acting H₂S level in the SCN should be much lower than the used NaHS concentration if considering the dilution and diffusion of NaHS/ H₂S in the cerebrospinal fluid and brain tissues including SCN. We showed that the used NaHS concentration did not affect the light entrainment function of SCN, suggesting that SCN, or including other parts of mouse brain, is quite tolerant to NaHS/H₂S. This finding suggests safety of relatively higher H₂S in the treatment of related brain diseases. In conclusion, H₂S can induce phase delay and circadian period length prolongation by affecting the expression patterns of clock genes and clock-controlled genes at SCN. This study suggests H₂S is regulator of central circadian clock.

Acknowledgment

This work was supported by NSFC grants (31471126 and 82170523 to J.C.), Sichuan Science and Technology Program (2019YJ0472 to P.Z.), and Luzhou Science and Technology Program (2018LZXNYD-ZK44 to P.Z.).

Conflict of Interests

The authors declare that they have no conflict of interest in this work.

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The Osseous Dissipation- Otosclerosis

Preface

Otosclerosis is a primary osteodystrophy of the otic capsule or a disorder of bone remodelling incriminating the stapes footplate or bony labyrinth of the inner ear. Otosclerosis literally implies “abnormal hardening of body tissue of the ear”. The condition is confined to the middle ear and is associated with anomalous remodelling of bone. Normally dense endochondral layer of bony otic capsule situated within the labyrinth is substituted by irregular, spongy bone. Consequently, fixation of the stapes ensues. Otosclerosis was initially scripted by Antonio Maria Valsalva in 1735 and is additionally designated as otospongiosis. Otosclerosis alters functioning of the middle ear and inner ear and is a predominant contributor to deafness in adults. Otosclerosis may engender significant disability and morbidity due to hearing loss. Antecedent disease detection with cogent therapy is associated with superior outcomes.

Disease Characteristics

Typically, otosclerosis induces lucent alterations instead of sclerotic bony modifications. Temporal bones are frequently incriminated [1,2]. Otosclerosis appears as an antecedent, adult onset disease. Generally, otosclerosis commences within second decade or third decade although hearing loss commences following the fourth decade. Disease occurrence within children is exceptional. However, the progressive condition demonstrates gradually worsening clinical symptoms and it may be challenging to ascertain precise disease onset [1,2]. A female predominance is observed with a female to male proportion of ~2:1. Otosclerosis worsens in pregnancy [1,2]. Ethnic predilection demonstrates frequent disease emergence within the Caucasian population. Otosclerosis is exceptionally discerned in Asians or Black population. Familial disease incidence appears in an estimated 50% instances. Around 85% instances are bilateral [1,2]. Otosclerosis is associated with distinctive phases such as
• Early or active otospongiosis and
• Late or inactive otosclerosis.
Of obscure and multifactorial pathogenesis, otosclerosis may arise due to genetic, viral, inflammatory or autoimmune components [1,2]. Preliminary lesions are predominantly comprised of an admixture of histiocytes, osteoblasts and osteocytes wherein osteocytes are an active cellular component. Bony circumscription of pre-existing vascular articulations is resorbed with meliorated microcirculation. Eventually, osteoblasts configure irregular foci of nascent, spongy bone, designated as “blue mantles of Manasse” [1,2].

Disease Pathogenesis

Of obscure aetiology, otosclerosis is posited to denominate a multifactorial emergence such as
• Anatomical factors wherein fissula ante fenestram is incriminated along with persisting remnants of embryonic cartilage [3,4].
• Genetic factors of otosclerosis are associated with several loci situated upon chromosomes 6p, 9p, 1q, 3q, 6q, 7q, 15q, 16q and a contemporary locus upon chromosome 7q22.1. Additionally, diverse genes as type I collagen (COL1A1 gene), transforming growth factor-β1 (TGF-β1) with BMP2 and BMP4 genes, angiotensin II with AGT M235T and ACE I/D genes may induce otosclerosis [3,4]. Sex hormones, autoimmune reaction, human leucocyte antigen (HLA), inflammatory or regulatory cytokines, parathyroid hormone, parathyroid hormonerelated peptide receptors and oxidative stress may initiate otosclerosis [3,4].
• Hereditary factors wherein an estimated 50% subjects with otosclerosis demonstrate a family history. Individuals with hereditary otosclerosis are associated with an antecedent disease onset. Majority of instances depict an autosomal dominant mode of inheritance accompanied by reduced penetrance in nearly ~40% subjects and variable expression of disease [3,4].
• Viral infection is implicated in the emergence of otosclerosis. Ribonucleic acid (RNA) of measles virus may be discerned within footplate of stapes upon ultrastructural examination and immunohistochemistry. Vaccination for measles virus may appear as a safeguard for occurrence of otosclerosis [3,4]. Additionally, factors such as menopause, trauma, bone dyscrasia or major surgery may initiate or aggravate otosclerosis [3,4].

Clinical Elucidation

Otosclerosis is commonly confined to the temporal bone and engenders fixation of footplate of stapes within the oval window. Therefore, inability to transmit sound waves manifests as conductive hearing loss [5,6]. Generally, otosclerosis engenders conductive deafness and appears in association with a normal tympanic membrane. Contingent to focal incrimination of the bony labyrinth, otosclerosis can be asymptomatic or represent with neurosensory decimation [5,6]. Frequent clinical representation of otosclerosis is hearing loss accompanied by tinnitus and vertigo. Gradually progressive bilateral hearing loss is a common symptom which typically commences in one ear and progresses to contralateral ear [5,6]. Inability to adequately discern low-frequency sounds as a whisper can emerge as an initial clinical symptom. Hearing may be meliorated in a noisy environment, a feature denominated as “paracusis willisii” which is indicative of conductive deafness. Incriminated subjects adopt a low-volume, monotonous tone of voice.
Extensive otosclerosis is associated with worsening tinnitus. Mild dizziness appears and deteriorates with disease progression, simulating Meniere’s disease [5,6]. Otoscopy may be normal or depict minimal modifications. However, around 10% instances of active otosclerosis or cochlear otosclerosis are accompanied by enhanced vascularity of the promontory which can be discerned through the tympanic membrane and is designated as “Schwartze sign” [5,6]. Of indolent clinical course, otosclerosis manifests with frequently bilateral, conductive hearing loss although sensorineural or mixed deafness may ensue. Hearing loss can be exacerbated in pregnant subjects [5,6]. Otosclerosis is comprised of distinctive subtypes denominated as
• Fenestral or stapedial variant which constitutes of a majority (~80%) of instances and incriminates the oval window along with stapes footplate. Frequently, conductive hearing loss is associated with thickening and fixation of stapes [5,6].
• Retrofenestral or cochlear subtype which is composed of nearly 20% instances. Incriminated cochlea is associated with demineralization of cochlear capsule. Sensorineural hearing loss emerges, possibly due to uncertain mechanisms [5,6].
Retrofenestral otosclerosis is usually accompanied by fenestral variant and the conditions are contemplated to represent a disease continuum [5,6].

Histological Elucidation

Upon gross examination, foci of otosclerosis appear as chalky white, greyish or yellowish. Active centric zone and rapid disease progression is associated with reddish lesions due to enhanced vascularity. Upon macroscopic examination of dissected tissue, specimens of un-altered head and crura of stapes are obtained [5,6]. Microscopically, foci of initial bone resorption and circumscription of vascular articulations by cellular, fibrovascular tissue are observed. Subsequently, deposition of immature bone, perpetual bone resorption and remodelling is exemplified. Gradually, deposition of bone, enhanced collagen and decimated ground substance ensue with consequent emergence of densely sclerotic bone demonstrating prominent cement lines [5,6]. Dense, endochondral layer of otic capsule appears to be constituted of spongy bone. Immature, active lesions of otosclerosis are imbued with abundant bone marrow, vascular spaces and numerous osteoblasts admixed with osteoclasts. Significant quantities of cement substance are deposited within the lesion [5,6]. Mature foci of otosclerosis are mildly vascular and composed of abundant bone and fibrillar substance with minimal cementum. Localized foci of bone remodelling and bone resorption of the otic capsule are followed by subsequent bone deposition.
Otoclerosis predominantly arises due to replacement of normal bone with sclerotic or spongiotic bone [5,6]. Osteolytic osteocytes are configured upon growing perimeter of the lesion. Sheets of connective tissue displace the bone. Delayed stage of otosclerosis depicts dense, sclerotic bone associated with zones of previous bone resorption. Consequently, disorganized bone is articulated, osteocytes are quantifiably enhanced and expansive bone marrow spaces are incorporated with vascular articulations and connective tissue [5,6]. Bone marrow spaces are eventually substituted by dense, sclerotic bone imbued with narrow vascular articulations and minimal Haversian canal system [5,6]. Preliminary and delayed stage of otosclerosis occurring within a singular temporal bone engenders pleomorphism. Antecedent lesions may adjoin fissula ante fenestram and expand through abutting vascular channels [5,6]. Majority of lesions are confined to anterior oval window and appear associated with calcification of annular ligament or incrimination of stapes, thereby engendering conductive hearing loss [5,6] (Figures 1-8).

Figure 1: Otosclerosis exhibiting sclerotic bone of footplate of stapes and bony labyrinth of inner ear [9].

Figure 2: Otosclerosis enunciating spongiotic and sclerotic bones replacing foot plate of stapes [10].

Figure 3: Otosclerosis exemplifying bony growth within footplate of stapes and spongiotic bone at the junction of incus and stapes [11].

Figure 4: Otosclerosis exemplifying bony sclerosis at footplate of stapes with an admixture of spongiotic bone, osteocytes and minimal Haversian system [12].

Figure 5: Otosclerosis delineating bony sclerosis of footplate of stapes with configured spongiotic bone and associated conductive hearing loss [13].

Figure 6: Otosclerosis demonstrating bony sclerosis and spongiotic bone replacing footplate of stapes with intermingled osteocytes and fibro-connective tissue [14].

Figure 7: Otosclerosis displaying spongy and sclerotic bone replacing footplate of stapes intermingled with abundant osteocytes, osteoblasts and fibro-connective tissue [15].

Figure 8: Otosclerosis exhibiting bony sclerosis of the inner ear labyrinth with several osteocytes, decimated Haversian canal and fibroconnective tissue [16].

Differential Diagnosis

Otosclerosis requires a segregation from conditions such as
• Osteogenesis imperfecta where otic capsule demonstrates lucencies simulating otosclerosis. Osteogenesis imperfecta is associated with non deformed, fragile bones and blue sclera. Severe variants of osteogenesis imperfecta are devoid of an organized bone trabecular pattern. Crowding of osteocytes within bone occurs due to decimated collagen synthesis. Enlarged foci of woven bone are delineated. Minimally severe instances depict crowding of osteocytes associated with attenuated lamellar bone [2,4].
• Paget’s disease of bone predominantly occurs in elderly individuals and demonstrates bony expansion. Acute stage is primarily composed of woven bone admixed with focal, mosaic configuration of lamellar bone associated with irregular cement lines. Osteoclasts are preponderant upon the bone surface. Osteoclasts appearing within osteolytic phase may depict around ~100 nuclei [2,4]. Chronic stage is comprised of thickened bony trabeculae and thick bones which are imbued with finely fibrotic bone marrow [2,4].
• Osteoradionecrosis arises due to vascular injury with consequent emergence of bony ischemia. Osteoradionecrosis is associated with distinctive phases denominated as
• Pre-fibrotic phase demonstrating foci of chronic inflammatory cell exudate
• Phase of organized fibrosis predominantly displaying aberrant fibroblastic activity along with an inadequately organized bone matrix. Aforesaid regions appear adjacent to foci of aging fibroblasts disseminated within a minimally cellular, fibrotic, densely sclerotic matrix [2,4].
• Fibro-atrophic phase is composed of dense hyalinization and fibrosis along with absence of bone marrow cells. Focal, reactive stratified squamous epithelium may layer a fistulous tract. Patchy, secondary infiltration of chronic inflammatory cells may occur. Lesions may display necrotic or sclerotic bone along with empty osteocyte lacunae. Fibrosis of bone marrow is admixed with irregular bony trabeculae devoid of osteocytes within the lacuna. Foci of bone marrow necrosis may arise due to emergence of a preliminary stage of oedematous, fibromyxoid disease [2,4]. Additionally, otosclerosis requires a segregation from conditions which are associated with conductive deafness such as serous otitis media, adhesive otitis media, congenital fixation of the stapes, Meniere disease, tympanosclerosis, attic fixation of head of the malleus or ossicular discontinuity [2,4].

Investigative Assay

Upon otoscopy, features indicative of otosclerosis are minimal to absent. However, severe instances with cochlear incrimination can engender hyperaemia of the cochlear promontory, a feature designated as “Schwartze sign” [7,8]. Otosclerosis can be appropriately investigated with the tuning fork and demonstrates a negative Rinne’s test. Weber’s test appears lateralized to the ear and is indicative of a severe conductive hearing loss [7,8]. Pure tone audiometry demonstrates low frequency loss of air conduction with normal bone conduction. Pure tone audiometry exhibits a characteristic decimation of bone conduction, especially at higher frequencies [7,8]. Mixed hearing loss can be observed wherein preliminary otosclerosis is associated with normal tympanometry [7,8]. High resolution computerized tomography (CT) of temporal bone is optimal in discerning otosclerosis besides recognizing and segregating associated causes of deafness. An estimated 80% instances depict fenestral foci which are situated anterior to the oval window. Also, thickened stapedial footplate and incriminated round window can be discerned, features which regulate appropriate therapy [7,8]. Retrosternal focus emerges as a “double halo” sign, a feature encountered with cochlear otosclerosis. Otosclerosis can be appropriately graded with CT [7,8].
Pertinent imaging depicts representations such as • Fenestral otosclerosis which is the commonest variant and demonstrates site of incrimination just anterior to oval window while configuring a miniature cleft denominated as fissula ante fenestram. Besides, bony overgrowth may engender fixation of the stapes [7,8].
• Retrofenestral otosclerosis incriminates the niche of round window and usually accompanies fenestral disease. Nevertheless, isolated round window otosclerosis may be occasionally discerned. Predominant site of disease emergence appears as focal or circumferential bone circumscribing the cochlea. Circumferential bony implication engenders a “fourth turn” or “double ring” sign [7,8]. Imaging features are contingent to phase of the disease and appear as
• Otospongiotic phase composed of demineralization and configuration of spongy bone. Pertinent phase manifests as decimated attenuation or lucent zone within homogeneously dense perimeter of otic capsule [7,8].
• Otosclerotic phase is associated with enhanced attenuation within disease specific region. As it may be challenging to discern otosclerotic bone from encompassing normal bone, features such as thickness of otic capsule or aberrant convexity of contour of otic capsule cortex anterolateral to anterior margin of the oval window may aid the distinction [7,8]. Severe instances are associated with comprehensive impaction of oval window or round window with a dense, bony plate associated with complete fixation of stapes [7,8]. Thin-slice computerized tomography (CT) through the temporal bone is a preferred imaging modality for adequately exemplifying anatomy of inner ear and emergent, subtle, preliminary modifications of otosclerosis. Upon computerized tomography, otosclerosis is categorized as
• Grade I which is singularly comprised of fenestral, spongiotic or sclerotic lesions which appear as thickened footplate of stapes along with decalcified, narrowed or enlarged round window or oval window [7,8].
• Grade 2 is constituted of patchy, localized cochlear disease along with the presence or absence of fenestral otosclerosis and is subdivided into~grade 2A which implicates basal cochlear turn~grade 2B which implicates middle or apical cochlear turn~grade 2C which implicates basal turn and middle or apical cochlear turn [7,8].
• Grade 3 is comprised of diffuse, confluent involvement of cochlea and otic capsule along with the presence or absence of fenestral involvement [7,8]. Magnetic resonance imaging (MRI) of retrofenestral otosclerosis exhibits a distinctive pericochlear and perilabyrinthine soft tissue intensity upon T1 weighted imaging with contrast enhancement. Besides, enhanced signal intensity upon T2 weighted imaging may be observed [7,8].

Therapeutic Options

Medical management of otosclerosis is required to circumvent or arrest disease progression. However, optimal, efficacious medical therapy which alleviates otosclerosis remains lacking. Administration of sodium fluoride in order to decimate disease progression is debatable [7,8]. Bisphosphonates are antiresorptive and induce osteoclastic apoptosis. Contemporary bisphosphonates, commonly employed to treat otosclerosis, appear promising [7,8]. Bilateral hearing aids can be utilized singularly or in combination with diverse therapies [7,8]. Fenestral otosclerosis can be appropriately treated with stapedectomy along with the employment of stapes prosthesis. Optimal and recommended surgical treatment of otosclerosis is stapedotomy or stapedectomy or correction of fixation of foot plate of stapes. Surgical intervention is commonly followed by implantation of prosthesis [7,8]. Therapeutic outcomes of surgical intervention in otosclerosis appear to be superior, regardless of surgical manoeuver adopted [7,8]. Though surgical intervention is beneficial, hearing aids may be required in certain subjects following surgery [7,8]. Untreated otosclerosis may engender significant hearing loss although complete deafness is uncommon [7,8].

Although infrequent, surgical intervention for treating otosclerosis can induce comprehensive sensorineural deafness within the operated ear. Surgical manoeuvers may induce facial nerve injury or tinnitus. An unpleasant taste may arise for a brief duration [7,8]. Prosthetic implantation is associated with granulomatous inflammation and erosion or necrosis of the incus [7,8]. Following surgical intervention, an estimated 90% subjects demonstrate significant amelioration of hearing ability. Hearing remains unaltered or declines in occasional instances [7,8]. Laser techniques and vein grafting is associated with superior outcomes. Additionally, reoccurrence of conductive hearing loss may ensue due to displacement of prosthetic implant. Original position of prosthesis is altered on account of collagen contraction within the neo-membrane configured between prosthesis and bony labyrinth [7,8]. Revision surgery may exhibit ambiguous outcomes and is usually adopted for treating associated clinical symptoms as facial nerve palsy, persistent vertigo or failure of meliorated hearing [7,8]. Nevertheless, revision surgery may be accompanied by inferior outcomes [7,8]. Strict monitoring is required in subjects with a family history of otosclerosis. Hearing loss in pregnant subjects may appear due to otosclerosis [7-16].

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