Lymphocytic atypical was a finding present only in HTLV-1 carriers (p = 0.0001). There was no high frequency in the leukocyte counts of those infected by HTLV-1 not among them concerning a significant increase or decrease. It is concluded that HTLV-1 infection is prominent in women over 50 years old. The hematological profile of those infected shows a reduction of segmented cells, an increase of eosinophils, and the presence of atypical lymphocytes. The hematological profile of the HTLV-1 carrier should always be evaluated to identify early some diseases associated with the infection.The human T-lymphotropic virus type 1 (HTLV-1), isolated in 1980, causes T-cell leukemia/lymphoma in adulthood, a type of lymphoproliferative disease, and chronic HTLV-1-associated myelopathy, a disease that causes paralysis of the lower limbs, which occur in about 5% of cases in this viral infection. This study aimed to establish the hematological profile of patients with HTLV-1 infection in Belém do Pará, describing the hematological parameters under study, estimating the frequency of lymphocytic atypical, and associating the hematological profile with diseases and symptoms. Hematologic data from 202 individuals were analyzed, including 87 HTLV-1 infected individuals and 115 non-HTLV-1 infected individuals as a control group, composed, at a great part, of relatives of the infected. The seroprevalence of HTLV-1 infection was observed in 71.3% of female individuals, with predominance in the group older than 50 years (44.8%). The analysis of hematological parameters showed a significant difference in the counts of the segmented cells ( The human T-lymphotropic virus type 1 (HTLV-1), isolated in 1980, is a slow-progression virus, being considered of low morbidity, is related to some diseases with emphasis on adult T-cell Leukemia/Lymphoma (ATL) and chronic myelopathy, occurring in about 5% of cases of virus infection . HTLV-2 Numerous factors can influence the hematological picture of individuals. The data provided by the blood count are essential within the investigation of hematological diseases. The diversity of information that the blood count can provide, although it is generally quite non-specific, makes this examination one of the most requested in clinical and surgical practices.During the last decades, there has been a great technological evolution in the performance of the blood count, and manual techniques have been replaced by automated systems that present greater precision in the results and in a shorter time interval. The importance of hematology as a means of study for signs of certain diseases is very clear, helping professionals to establish possible diagnoses, establish prognoses, and monitor evolving patients.flower cells). The cytoplasm is often scarce and the nucleus is irregular , a unit of the Federal University of Pará (UFPA), develops different projects that benefit the population by offering free outpatient and laboratory care in the diagnosis of different tropical diseases.Chlamydia trachomatis (C. trachomatis), and HTLV.The Laboratory of Molecular and Cellular Biology (LMCB) of NTM/UFPA develops teaching, research, and extension projects, aiming at the training, and improvement of its human resources. The lines of research and extension are focused on the prevention and study of sexually transmitted infections/diseases (STIs/STD), more specifically Human Papillomavirus (HPV), In LMCB serological and molecular tests are performed for the detection and diagnosis of HTLV.The present study was submitted and approved (CAAE: 31014114.2.0000.5172) by the research ethics committee of the NMT/UFPA, in compliance with the norms and guidelines of the Federal University of Pará.This study is aimed at all people assisted by the Molecular Biology Laboratory and or the Center for Tropical Medicine of UFPA, carriers or not of the human T-lymphotropic virus, from September 2015 to August 2016. The study included 202 individuals, among them 87 people infected with HTLV-1 (Case Group) and 115 individuals negative for HTLV-1 (Control Group), mostly relatives of carriers of the virus, due to the work of active search of infected family members.Case Group: Individuals with HTLV-positive serology confirmed by the molecular biology method (PCR) for HTLV-1. Control Group: Individuals with HTLV-negative serology and PCR. Both groups: Individuals who agreed to participate in the study by signing the Free Informed Consent Term (FICT).Individuals with inadequate samples for laboratory analysis. Individuals with HTLV-positive serology and not confirmed by molecular biology or confirmed for HTLV-2 or cases undetermined in serology and unconfirmed by molecular biology.Before the interview and the completion of a questionnaire with epidemiological data, the individuals were informed about the study and invited to participate in it, signing the Free and Informed Consent Form (FICF) in the event of an agreement. Blood was then collected for serological, molecular, and hematological examinations.The blood collection was performed in 5 ml tubes containing the anticoagulant EDTA, previously identified with the names of the patients and their registration numbers corresponding to their care stipulated by the laboratory. The blood collection was obtained through venous puncture according to the safety recommendations of blood collection. After collection, the samples were sent to perform the blood count and later to perform other tests.the research of flower cells and other atypical lymphocytes. The hematological parameters analyzed for the study were as follows: the count of leukocytes, lymphocytes, segmented, eosinophils, monocytes, hematocrit, red blood cells, hemoglobin concentration, and platelet count.All blood samples collected from the patients were submitted to the blood count. These samples were previously homogenized and processed in the humacount30TS hematological automation equipment of 18 parameters with a three-part differential. The Hematological Counter is based on electrical and photocolorimetric impedance technology and consists of the following parameters: Erythrocyte count, hemoglobin dosage, hematocrit determination, mean corpuscular volume (MVC), mean corpuscular hemoglobin (HCM), mean corpuscular hemoglobin concentration (CHCM), and distribution amplitude of erythrocytes (RDW) that make up the erythrogram; leukocyte count, and leukocyte formula that the leukogram and platelet count. From all samples, two slides were made on smears and blushed for the differential counting of leukocytes and As a result of the different reference values cited in the literature for hematological parameters, in order to adapt all the variables in a single reference, reference values according to the literature were adopted in this study .The blood smear was used to differentiate between leukocytes, that is, to make a count of the number of neutrophils, lymphocytes, monocytes, and eosinophils reaching a percentage of each cell found. The confection was made with a small drop of blood placed on a glass slide where a smear was made, dragging the drop of blood with another blade to form a film. The blood was homogenized before the smear was made so that the cells were well distributed. The smear was cordoned with Leishman dye, a yellowish eosin compound, and methylene blue oxidation products. Hence the differential count of leukocytes, with the count of 100 leukocytes in the blood smear, differentiating them according to their varieties of color and morphology observed from the microscope with the 100× lens.flower cell and other atypical lymphocytes were researched. The research was carried out through the blade throughout its length, using on average 50 fields with the immersion lens (40×), and when necessary for better visualization also used the 100× lens  according to the manufacturer’s instructions at the Laboratory of Molecular and Cellular Biology of NMT/UFPA.For the identification of anti-HTLV-1/2 antibodies, qualitative and enzyme immunoassay, ELISA (Enzyme-Linked Immuno Sorbent Assay) was performed with the kit cut-off were analyzed in duplicate.This test is used for the detection of IgM, IgG, and IgA anti-HTLV-1 and HTLV-2 antibodies in human serum or plasma by the ELISA method. It uses recombinant antigens (gp46 and gp21) fixed to the microplate. The positive samples and those with a value close to that of Wizard Genomic DNA Purification  kit protocol.The blood samples from the individuals were submitted for DNA extraction. The method of extracting total DNA from mononucleated peripheral blood cells was used, according to the All extracted genomic DNA was submitted to amplification of the human β-globin gene with primers G73 (5’-GAAGAGCCAAAGGACAGGTAC-3’) and G74 (5’-CAACTTCATCCCTCACC-3’) generating a fragment of 268 bp, whose objective is to evaluate DNA integrity and exclude the presence of PCR inhibitors.® Green Master Mix Purification , 2.0 μL of autoclaved distilled water, 10 pmol/μL of each primer, and 1 μL of DNA were used for a final volume of 7 μL. The amplification protocol followed the denaturation temperature of 94°C for 5 min, followed by 30 cycles, 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s, followed by the final extension temperature of 72°C for 10 min and 10°C for 10 min. The amplification of the segments mentioned above was performed in the thermocycler of Biocycler MJ96 + /MJ96G   .Go Taq Green Master Mix , 2.0 μL of water, 1 μL (10 pmol) of each primer–HTLV_External F 5’-TTCCCAGGGTTTGGACGAAG-3’  and HTLV_External R 5’-GGGTAAGGACCTTGAGGGTC-3’ –and 1.0 μL of the DNA, for a final volume of 10 μL.Once the proviral DNA was amplified, a nested PCR was run, followed by enzymatic digestion for the confirmation of the HTLV infection and the differentiation of the virus types 1 and 2. For this, the pX region of the virus was amplified. This initial reaction was run in a solution containing 5.0 de In the second phase of the nested PCR, the same quantity of Go Taq Green Master Mix  was used, together with 1 μL (10 pmol) of the primers HTLV_internal F 5’CGGATACCCAGTCTACGTGTT3’  and HTLV_internal R 5’GAGCCGATAACGCGTCCATCG3’ , 2.5 μL of water and 0.5 μL of the product of the first PCR, producing a fragment of 159 bps.Positive (a sample known to be infected) and negative controls were used for each PCR reaction. The products were electrophoresed (60 min at 100 V) in 2% agarose gel, stained with ethidium bromide in 1 × TAE buffer , and visualized using an ultraviolet transilluminator .After the identification of cases with positive PCR results, using the 1% agarose gel, enzymatic digestion (RFLP—polymorphisms of the length of restriction fragments) of the amplified product was performed to identify the type of HTLV-1/2 present in the samples.pX gene product (159 bp) was performed by mixing 6.0 μL of the amplified product, 7 μL of H20, 1.5 μL of buffer E , and 0.5 μL of the restriction enzyme TaqI , with subsequent incubation at 65°C for 2 h.The RFLP reaction of this The presence of the restriction site (T/CGA) generates three fragments  in HTLV-2 and two fragments (21 and 138 bp) in HTLV-1.The products of enzymatic digestion were visualized in agarose gel of 3% to 100 V for 45 min in 1 × TAE buffer , using transilluminator in light with an ultra-violet light source .All data from hematological parameters as well as all information collected in the interview and medical records were inserted into a spreadsheet using the Microsoft Office Excel© 2010 program.The statistical tests were performed with the aid of the BioEstat version 5.0 program, to identify the frequencies and make the graphs and tables.p-value of 0.05< was considered statistically significant.Initially, descriptive statistical analyses were performed, i.e., through graphs and frequency tables of the variables analyzed, estimation of mean, standard deviation, maximum, and minimum of numerical variables. The significance level to reject the null hypothesis was 5%, i.e., a For the hematological parameters, the Z and Mann-Whitney tests were used to evaluate the relevant difference and evaluate the significance among the populations under study.2) was used to compare the significant factors between the frequencies of patients and non-carriers of HTLV-1.The Chi-square test  and 115 individuals negative for HTLV-1 (56.9%), between September 2015 and August 2016.p = 0.009).Among the study samples, 72 individuals were male (35.6%), while 130 were female (64.4%). When the frequency of individuals negative for HTLV-1 infection was observed, it was found that 40.9% of these were male individuals, while 59.1% were female. When only individuals positive for HTLV-1 infection were analyzed, the frequency of male infected individuals was 28.7%, corresponding to 25 cases, while the female individuals corresponded to 71.3%, corresponding to 62 cases , demonstThe mean age of HTLV-1-positive patients was 46.5 years with a standard deviation (SD) of 16.08 years varying ages between 17 and 81 years. In negative cases, the mean was 40.3 years, with a standard deviation of 17.60 ranging from 2 to 73 years of age.p = 0.0092) showed the increased eosinophilic ratio in HTLV-1 positive patients  in the study population . In thispatients  and alsoThe data show the presence of HTLV-1 positive individuals with a predominance of ages above 50 years. 3  presented a change in the number of leukocytes, taking into account the reference interval of 5,000–10,000/mm3 , being f3 . HTLV-1 p = 0.6191).n = 18) and none of the negative cases presented lymphocytic atypical  in HTLV-1 patients. Most of these patients had counts below 7% of eosinophils in their blood counts.It was found in the eosinophil count that eosinophilia above 15% was present at a frequency of 2.3% (n = 16) of patients below the cut-off reference (12–16 g/dL) and anemia was evidenced in these patients, and most individuals positive for HTLV-1 had a hemoglobin concentration above or equal to 12 g/dL.Regarding hemoglobin concentration, a percentage of 18.4%  of those infected with HTLV-1 had thrombocytopenia, with a value below 150.000/mm3.Platelet count between cases with and without HTLV-1 infection was within the acceptable standard with a minimum reference of 150,000/mmn = 87), 22 cases were studied with any sign, symptom, or clinical diagnosis related to infection , aged between 31 and 50 years (40.3%), characteristics similar to those found in a study conducted in Belém on the clinical-epidemiological profile of HTLV-1 infected patients , who shoThe existence of a higher tendency of infection in females may suggest that they are more susceptible to HTLV infection, due to a possible more efficient sexual transmission from man to woman, possibly associated with a greater amount of infected and viable lymphocytes released during male ejaculation as described by other authors . In chilThe results of the blood count parameters found in this study were presented in tables, showing the point values for each blood count parameter referring to the positive and negative population for HTLV-1 as well as its minimum, maximum, mean, and standard deviation values . ThroughThe increase in eosinophils may be related to the presence of co-infection, especially Strongyloides or another parasitosis, or the process of leukemogenesis itself. 3) or above this reference.Studies suggest that HTLV-1 infection is associated with immunosuppression, HTLV-1 is a slow-progression virus with low virulence . Thus maWhen we have a viral process, it is common for the number of lymphocytes to increase. However, HTLV-1 responds to infection preferably peripheral T cells, predominantly CD4+ T lymphocytes  and acticalled flower cells. Considering that ATL presents distinct clinical forms, Shimoyama (1991) classified it into four types, based on disease extension, evolution time, lymphocytic and biochemical changes. The acute form presents abnormal circulating lymphocytes and flower cells, with infiltration of lymph nodes and viscera, as well as tumor manifestations. Symptoms are usually non-specific and may appear in any lymphoproliferative disease.Lymphocytosis accompanied by leukocytosis requires greater attention and may have the presence of atypical lymphocytes and may correspond to more than 10% of circulating lymphocytes. Atypical lymphocytes are highly pleomorphic, many of them large with a diameter between 15 and 30 μm, with abundant cytoplasm, and intensely basophilic. Some have large central nucleolus, nuclei can be round, oval, reniform, lobulated, or occasionally clover-shaped. Lymphocyte morphology may be the first sign of the diagnosis of ATL . These cChronic and latent forms have a better prognosis, but may evolve to the acute form at any time and have no lymphocytosis. The HTLV-1 genome is randomly integrated into the cellular genetic material. In HTLV-1 infection there is little or no vírion in the plasma of the infected individual, reflecting the main viral replication pathway, viral synapse .Studies show that HTLV-1-infected CD4+ T lymphocytes persist for about 7 years in the same individual, suggesting that the virus participates in the process of transformation and immortalization of these lymphocytes. After HTLV-1 infection, the latency period is long (about 50 years), indicating that in the development of ATL multiple steps are required, accumulating genetic mutations in infected cells .The probability of an HTLV-1 carrier developing ATL in the course of prolonged latency shows that the process of cell transformation and leukemogenesis should involve many complex events within the host’s T-cells .Lymphocytes with different morphology, which can be found in the peripheral blood of healthy patients in a small percentage (1–4%) called atypical lymphocytes, were also found in HTLV-1-positive patients where 18 of them (20.7%) had at least one of these lymphocytes, and in two patients had a count above the reference range, one with 5% of atypical lymphocytes and the other with 7% of atypical lymphocytes, the negative population did not even present a count of these lymphocytes. No lymphocyte with the flower-shaped nucleus of positive patients was found.n = 11) of negative patients had eosinophilia between 7 and 14%, and among the positive 14.9% (n = 13) had eosinophilia between 7 and 14%, alteration sofa not related to either an allergic process or any parasitosis, and there was no difference in frequency between populations.About eosinophils, 9.5% .As for hemoglobin concentration, specifically, the data showed that, of the 87 HTLV-1-positive patients, 12 (13.8%) had anemia, with hemoglobin ranging from 10.0 to 11.9 g/dL, taking as a cutoff point 12.0 g/dL, especially 4 HTLV-1 patients (4.6%) presenting hemoglobin below 10 g/dL and generally a normal distribution among the population  below this profile for negative patients and 4.6% (n = 6) among the eight patients with lower-normal platelet concentration positive for HTLV-1, and two of them with a score below 100,000/mm3, patients without having problems with heavy bleeding. There was normality regarding the distribution of platelet concentrations among the population studied.Also, evaluating platelet count in the population, with a cut-off value of 150,000 platelets/mmThe human T-lymphotropic virus type 1 infection can lead to numerous changes from the immunological, oncological-hematological, neurological, rheumatological, infectious, and ophthalmological points of view. HTLV causes persistent infections throughout life and this virus is associated with serious diseases such as ATL. Most infected individuals remain asymptomatic throughout their lives, and about 5% develop these diseases . HTLV-1 3), hemoglobin (4.9 g/dL), and hematocrit (16.8%) values below the reference  by the research ethics committee of the NMT/UFPA, in compliance with the norms and guidelines of the Federal University of Pará. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


HHP is more relevant among pediatric physicians in the context of the COVID-19 pandemic since, children are more vulnerable to infection. Therefore, assessment of the COVID-19 impact on HHP could be useful in minimizing lethal virus transmission from pediatric physicians to patients and vice versa.The hand hygiene practice (HHP) is the most effective and simplest preventive measure The present cross-sectional, electronically self-administered supplement based survey study was conducted among different professional levels of pediatric physicians involving consultants, specialists, and residents. The supplement includes information related to demography, knowledge, awareness, preventive measures, demonstration and practice of HHP. The information was collected and summarized on a Microsoft excel sheet before being imported to SPSS for statistical analysis.(98.01%), after touching surrounding of patients (74.75%) may prevent germ transmission to patients whereas HHP after touching patients (98.27%), before clean/aseptic procedure (67.57%), after exposure to immediate surroundings of patients (97.02%) may prevent germ transmission to pediatric physicians. Rubbing hands is preferred before palpation of abdomen (74.25%), before giving injection (56.68%), after removing gloves (61.88%), after making a patient's bed (47.80%), while washing of hands preferred after emptying bedpan (67.82%) and after visible exposure to blood (84.40%), 92.57% believed gloves can't replace HHP, posters display at point of care as reminders (95.30%), received frequent HHP education (82.92%), 50.49% do not need HHP reminder, 51.73% preferred alcohol based sanitizer, 53.46% facilitate daily morning huddle, HHP >10 times per day before COVID-19 (24.62%) while in COIVID-19 (56.44%). HPP is the most effective way to prevent the spread (98.01%) of microbes because it kills germs (90.35%), health care associated infections is the major (38.06%) cause of germ transmission, 86.88% will be remains committed to HHP even after pandemic. In comparison to residents and specialists, consultants gave more importance (p = 0.02) to HHP and were more adherent during (p = 0.007) and even after (p = 0.001) COVID-19 pandemic.Of the total (N = 404) pediatric physicians, 56.68% male, 43.06% belongs to 25–35 years, 42.32% were consultants, 98.01% respondents were familiar with five moments of HHP. Further, HHP immediately before touching patients (99.26%), clean/aseptic procedure (95.04%), after body fluid exposure (72.28%), after touching patients Assessing knowledge of pediatric physician, awareness, and adherence to hand hygiene measures could be helpful to reduce the contact transmission of lethal viruses to patients and vice versa. Further increase in the awareness, knowledge and education of HHP are required in order to maximize its utilization. •to reduce the risk of infection.The hand hygiene practice (HHP) is the most effective and simplest preventive measure •Of the total (N = 404) pediatric physicians, 56.68% male, 43.06% belongs to 25–35 years, 42.32% were consultants, 98.01% respondents were familiar with five moments of HHP.•Assessing knowledge of pediatric physician, awareness, and adherence to hand hygiene measures could be helpful to reduce the contact transmission of lethal viruses to patients and vice versa. It can be performed by washing hands with an alcohol based hand rub or wash hands with either plain or antimicrobial soap or water [In December 2019, an outbreak of coronavirus disease (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV-2) was detected in Wuhan, China and then became unprecedented global health crisis. Healthcare workers (HCWs) are the frontline fighters during COVID-19 pandemic, so they are more likely to get infected with SARS-CoV-2 which appears to be transmitted primarily through respiratory droplets, from face-to-face contact and, to a lesser extent, through contaminated surfaces. Our hands are the most critical body part for transferring pathogens . Severalor water . In todaor water . Continuor water . Ataee eor water . Despiteor water . Moore eor water . Among aor water  since, cor water . Ministror water   or disagreed (no) with each of the five questions [The current multi-center, cross-sectional supplement-based survey study was conducted among pediatrics physician during the period of 17 September 2021 to 28 October 2021 to assess the impact of the COVID-19 pandemic on HHP in the Kingdom of Saudi Arabia. An electronically self-administered supplement was designed and modified based on a previously published study ,29,36. T2.1The statistical analysis was carried out by using SSPS software, windows version 21.0 .The data was presented using frequency, percentage, arithmetic mean, and standard deviation.p < 0.05 were considered as significant values.Chi-squared test/Fisher's exact test was used for the analysis of the impact of professional ranks on the importance, and adherence to HHP. The 3Of the total (N = 404) pediatric physicians invited to fill the questionnaire consisting 229 males (56.68%) and 175 females (43.32%). Most of the participants’ ages were under the age group of 25–35 years (43.06%). Majority of participants were consultants 171 (42.32%), followed by residents 127 (31.44%) and specialist 106 (26.24%). The most of respondents worked in general pediatrics department 166 (41.08%) followed by critical department 150 (37.13%) and another pediatric subspecialty 88 (21.78%) are shown in table-1. The provincial distribution data of respondents revealed that most of the respondents were from Riyadh followed by Makkah province . In comparison to residents and specialists, consultants gave more significance (p = 0.02) to HHP and were more adherent to HHP during (p = 0.007) and even after (p = 0.001) the pandemic (Table-2). A comparison of respondents' attitudes toward the use of alcohol and soap at various time intervals were presented in figure-2. Alcohol based HHP  at 20 s and soap at 60 s  were the most preferred . When comparison was made to find out the associative risk factors for the increased risk of COVID-19, we found Jewelry and finger nails ranked top . In Figure-4, demonstration of the duration of hand rubbing and washing in order to prevent viral transmission have shown that 20–40 s for hand rubbing and 40–60 s for hand washing were preferred.When data were analyzed for the five moments of HHP, we found 396 (98.01%) respondents were familiar with my five moments of HHP, 265 (65.60%) thought that hand hygiene affected during this pandemic, unfortunately, 98 (24.25%) had been infected with covid-19.HHP immediately before touching patients (99.26%), clean/aseptic procedure (95.04%), after body fluid exposure (72.28%), after touching patients (98.01%), after touching surrounding of patients (74.75%) may prevent germ transmission to patients whereas HHP after touching patients (98.27%) before clean/aseptic procedure (67.57%), after exposure to immediate surroundings of patients (97.02%) may prevent germ transmission to pediatric physicians.Rubbing hands is preferred before palpation of abdomen 300 (74.25%), before giving injection 229 (56.68%), after removing gloves 250 (61.88%), after making a patient's bed 193 (47.80%), while washing hands preferred after emptying bedpan 274 (67.82%) and after visible exposure to blood 341 (84.40%), 374 (92.57%) believed gloves can't replace HHP, posters display at point of care as reminders (95.30%), 332 (82.92%) received frequent HHP education, 204 (50.49%) do not need reminder for HHP, 209 (51.73%) preferred alcohol based sanitizer, 216 (53.46%) facilitate daily morning huddle, before COVID-19 only 98 (24.26%) adopting >10 times HHP while 228 (56.44%) since the start of the pandemic, 396 (98.01%) believe that HHP is the best to prevent spreading of COVID-19 infection, 365 (90.35%) HHP kills germ, 273 (67.57%) would be last longer than expected, 125 (30.94%) hand dryness forgetting is the major obstacle in HHP, 24.25% infected by COVID-19 of which 94.89% because of close contact, 147 (36.38%) may prevent COVID-19 outbreak, 351 (86.88%) will be committed to HHP even after this pandemic (Table-1).4The unprecedented COVID-19 outbreak and its high transmission rate become a major threat, globally. Despite the efforts of the entire world to comprehend COVID-19, understanding of this novel virus remains limited, and there is no medication therapy to cure COVID-19. Preventing spread is extremely important to reduce the overall burden of the disease and to remain safe. Healthcare professional are front warier therefore, they are at high risk of exposure and had the likelihood of acquiring this disease more. Currently, WHO recommends physical distances, appropriate use of all PPE, and HH practices to reduce the spread ,23. It ip = 0.02), adherent to HHP during (p = 0.007) and even after COVID-19 pandemic (p = 0.001) significantly as compared to residents and specialist (Table-2). Respondents from major cities, like Riyadh, indicating HHP is more important to them. . It has been suggested that knowledge of good HHP and compliance in hand hygiene as per WHO guidelines is essential for lowering COVID-19 infection [Our study demonstrates male respondents were higher than female and more than half of respondent's age was lying between 25 and 35 years, consultant and general pediatric physicians were rank top among the responders. The higher response rate from high-ranking experts of pediatrics departments may be useful in evaluating pediatrics physician approach for safe clinical practices during COVID-19 in Saudi Arabia. Our study have shown consultants prioritize the hand hygiene . In our study, the respondents preferred the application of alcohol for 20 s and soap for 60 s. Furthermore, respondents believed that hand rubbing for 20–40 s and hand washing for 40–60 s may prevent viral transmission . It has been recommended that regular and thorough hand washing with alcohol for 20 s and soap for at least 40 s can remove viruses from the hands. Similar findings were also reported in Indian population, 51.73% agreed that using alcohol based hand sanitizer is less time consuming and efficient than hand washing with water and soap. According to our findings, more than half of respondents believes frequent education on HHP, poster exhibition could serve as HHP may improve the knowledge, awareness and reminders, more than half believe that ABHS is less time consuming and efficient than washing hand with water and soap. Hand washing with soap and water or using an ABHS is a simple yet efficient strategy to reduce the transmission of COVID-19 in hospital settings ,34. The When the data was evaluated to compare hand hygiene compliance before and after the pandemic, we observed hand washing or rubbing >10 times increased from 24.26% to 56.44%. Our data showed, the increase in the level of awareness and importance of hand hygiene among respondents.However, we noticed that after one year of the pandemic, 32.42% of the pediatrics physicians became less strict about hand hygiene. One likely cause is hand dryness and forgetfulness, which prevent proper HHP in our study. Other than this, hand dryness and eczema can also influence adherence of hand hygiene. American contact dermatitis society published a recommendations to prevent hand dryness especially among health care workers during COVID-19 pandemic suggesting using less irritating agents such as alcohol based rub instead of frequent hand washing .Similar to this, a study conducted in nine hospitals of United States to observe the impact of COVID-19 on hand hygiene using electronic monitoring, results showed significant increase in awareness and adherence at the start of the pandemic. In a reassurance responses, we found 86.88% of the respondent will be committed to hand hygiene even after the pandemic. In order to review the study group perspective regarding what do they think for the most frequent source of germs transmission, 38.86% stated that it is the hospital environment such as surfaces, doors handles. However, in an Indian study, 34.05% of healthcare workers believe it's the germs existing on or inside the patient. Several other studies have shown that HCWs compliance with hand hygiene recommendations is generally poor, and evidence regarding the proper usage of PPE is few and underwhelming . The fac5Assessing knowledge of pediatric physician, awareness, and adherence to hand hygiene measures may help to reduce the contact transmission of lethal viruses to infants, children and HCW. Our data evidenced that proper hand hygiene implementation can significantly reduce risk of germ transmission from pediatric healthcare practitioners to patients or vice versa. Our study has certain limitations in terms of the self-reporting of HHP performance and volunteer bias. To support the current findings, a larger cross-sectional study with a bigger sample size is required to generalize the findings of the study.Ethical approval has been given by the Institutional Review Board (IRB) of our institution.NO FUNDING FOR THIS RESEARCH.Drafting of the manuscript: All authors contributed to the drafting of this manuscript,Critical revision of the manuscript for important intellectual content: All authors contributed to the critical revision of the manuscript.NONE.Abdullah AlGhobaishi, corresponding author of the manuscript, accept full responsibility for the work and the conduct of the study, had access to the data, and controlled the decision to publish.NO CONFLICTS OF INTREST.


Bryonopsis laciniosa (L.) Naud.  for treating sexual dysfunction, impaired fertility, and as a general virility-booster in both males and females. To investigate the scientific basis for such claims, the current study was designed for the chemical characterization of the super critical fluid extracted Shivlingi seed oil (SLSO), and subsequent evaluation of its reproductive fecundity in the zebrafish model of N-ethyl-N-nitrosourea induced infertility.The Ayurvedic system of medicine mentions the use of seeds of Linoleic and linolenic acids were the major fatty acids in the SLSO, with trace amounts of β-sitosterol and stigmasterol. Both male and female zebrafish, when fed orally with the SLSO infused pallets, showed a dose-dependent increase in fertility and fecundity rates. Microscopic observations revealed recovery in the gross ovarian anatomy and consequential improvement in egg production in infertile female zebrafish. Similarly, cytological studies exhibited increased sperm counts and motility in male zebrafish. SLSO exhibited effects similar to the human equivalent dose of Letrozole.Shivlingi seeds in Ayurvedic treatment for infertility in humans. Finally, but importantly, this study warrants further scientific investigations into different aspects of SLSO on human reproductive health.Taken together, these observations demonstrated the fertility-boosting potentials of SLSO comparable to the widely used infertility drugs. As a whole, this research work has provided scientific evidence for the rationale behind the use of  Fertility and problems associated with it are among one of the most complex issues in medical science. The World Health Organization (WHO) has classified infertility as a disability and a ‘disease of the reproductive system’. Infertility is estimated to affect approximately 186 million people worldwide, with 10% of women and 12% of men , 4. AmonBryonopsis laciniosa (Shivlingi) seeds (Cucurbitaceae family) for the treatment of infertility since ancient times [Rāja Nighaṇṭu (Guḍūcyādi-Varga) and Śāligrāma-nighaṇṭu (Guḍūcyādi-Varga) from the 14 and nineteenth centuries, respectively. It is an annual climber with bright red fruits and is used as a hypoglycemic herb in some parts of India. However, its most recognized use, as orally consumed whole seeds, is in the improvement of sexual behaviour and treating infertility [The Ayurvedic system of medicine advocates the use of nt times , 10. Beint times  Numerousertility , 13. HowShivlingi seeds for the treatment of fertility problems [2) promotes high-quality extraction with minimal thermal degradation compared to traditional extraction methods. This method of extraction also allows changing the process parameters for optimum recovery of specific compound(s) with minimum post-extraction purification steps [Traditional systems of medicine used crude preparations of problems . With adproblems , 16. Fooon steps . The useShivlingi seed extract on fertility.The overall ease of dosing zebrafish (just by water dissolution or through the feed), makes it an extremely attractive pharmacological and toxicological study model system for recapitulating several human diseases and disorders, including reproductive abnormalities. Zebrafish is a very relevant in vivo model for studying human reproductive problems, due to its short reproductive cycle and a high degree of similarity with human reproductive regulatory systems, at hormonal and neuronal levels . The useShivlingi seeds has been provided through this study. This study can be divided into two aspects: (1) production of SLSO through SCFE and its compositional investigation through Gas Chromatography-Mass spectrometry (GC–MS/MS) and High-Performance Thin Layer Chromatography (HPTLC), and (2) its functional validation as infertility remedial using the ENU mutagenic zebrafish model of the disorder. The potential of SLSO in restoring fertility was comparable to that of commonly used infertility drugs.Scientific validation for the Ayurvedic practise of treating fertility problems with Several batches of SLSO were extracted and the average yield was estimated. Oil from one such extraction was used to determine the phyto-chemical constituents and subsequent in vivo demonstration of the rescue of zebrafish with impaired fertility phenotype. The SLSO in this particular batch of extraction resulted in a yield of 65 gm oil from 1.5 kg powdered seed material (4.33% yield). This was also the yield from five different extractions. GC–MS/MS analysis of the oil using helium as a carrier led to the detection of five fatty acids; two major ones, namely, linoleic (29.13%) and linolenic (12.37%), being polyunsaturated and essential. The other fatty acids present in SLSO in lesser amounts are oleic (11.85%), palmitic (11.06%), and stearic (6.75%) acids .In the case of male zebrafish, they were subjected to 100 mM metronidazole for further 20 days post-ENU treatment. Metronidazole leads to testicular cell ablation and therefore, only partial treatment was used to obtain zebrafish impaired with reversible fertility. A total of 550 fish were subjected to the mutagenesis protocol and out of the 519 survivors, 450 males were termed as fertility impaired mutants as determined by the production of less than 20 embryos upon breeding with healthy wild-type females.Once the fertility impairment was induced, the fish were kept in groups of 24 individuals along with a healthy control group. The fertility impaired mutants were given a diet with either the reference drugs  or with the SLSO at different dosages Fig. .Fig. 3ScZebrafish with diets containing either the standard drugs or the SLSO did not show any mortality over the study period. The healthy control group and the fertility impaired group also did not have any mortality. This suggested that treatment with SLSO even at 100X (128 µg/kg) of the human equivalent dosage was non-toxic to the zebrafish model.The fertility rate in the female zebrafish study groups was calculated as the number of positive spawning events irrespective of the formation of confluent embryos to the total number of breeding pairs. The control group comprising of healthy females in a mating event with four healthy males showed a fertility rate of 100%, while the fertility impaired group had a rate of 83.3% Fig. . All thep < 0.0001) percentage of healthy eggs at just 27.21%  than that of the fertility impaired group. The SLSO at the lowest dosage  led to the production of only 32.5% healthy eggs but was still significantly higher (p < 0.01) when compared to the fertility impaired group. With increasing dosages of SLSO, a dose-dependent increase in the number of healthy eggs per spawning was observed as shown in Fig. With a 100% fertility rate, the number of healthy eggs per spawning was 92.13% in the healthy control group. In comparison, the fertility impaired model had a significantly lower  to only 16.8% in the fertility impaired group  conception rate at 43.57%. The human equivalent dosage (1.28 µg/kg/day) restored the conception rate to near-normal levels (94.43%), while the other three dosages of 5X, 10X, and 100X had similarly high rates of conception.In addition to the fertility rate, the rate of conception and the fecundity rate also showed a dose-dependent increase with increasing dosages of SLSO. The conception rate was defined as the percentage of mating events that resulted in the formation of viable embryos to the total number of mating events. The healthy control group had a conception rate of 99.54% while this was significantly reduced (p < 0.0001) but was still low at 42.47%. The other dosages improved the fecundity rate comparable to the healthy controls  number of follicles at each developmental stage as well as a lower number of total eggs in the ovarian reserve. The SLSO at the lowest dosage did not show a significant increase in the number of follicles at the PG, EV, and the MV stages but had a significantly higher (p < 0.01) number at the PV and FG stages. The total number of eggs in the ovarian reserve was also significantly higher (p < 0.0001) compared to the fertility impaired group. The human equivalent dosage had an egg reserve that was slightly higher than that in the group treated with letrozole. The follicles were predominantly in the PG and PV stages and were significantly higher (p < 0.0001) than that seen in the fertility impaired group. A similar distribution was also seen in the group treated with SLSO at 5X of human equivalent dose, along with an increase in the follicles at the Final Growth stage. The dosages that were 10X and 100X of the human equivalent dosage showed near-complete reversal of the fertility impairment. The total egg reserve was almost equal to that of the healthy control group and there was a good distribution of all the different developmental stages.The ovarian reserve of eggs is an important factor in assessing the fertility of zebrafish. The higher the reserve of eggs, the higher is the fertility rate. In the parallel experiment, the fish were terminated on the 15p < 0.0001) number of cysts  in the number of cysts compared to the fertility impaired group, but the mean percentage of cysts in this group was still about 18%. Although treatment with higher dosages showed a dose-dependent reduction in the percentage of cysts, treatment with the human equivalent dosage (1.28 µg/kg/day) still had about 11% cysts. Dosages at 10X and 100X showed maximum reversal of the phenotype with only 3% of the total ovarian follicles determined to be cysts.Cysts in the ovary are identified as irregularly shaped cells, dark in colour and filled with fluid rather than the yellowish yolk. The covering of the cyst is also very thin, which ruptures easily during handling. The ovary was carefully dissected and observed under the 40X objective of a stereomicroscope to quantify the number of cysts present in the different study groups. No cysts were identified in the healthy control group, while the fertility impaired model showed a significantly higher  sperm count compared to the healthy control  in the number of sperm compared to the fertility impaired group. However, individuals of the group showed a great degree of variability in the sperm count, similar to that seen in the fertility impaired group. Treatment with SLSO at dosages less than the human equivalent dosage showed a modest but non-significant increase in the sperm count compared to the fertility impaired group (Table p < 0.0001) in the sperm count compared to the fertility impaired group, but this number was only 16.5% of the sperm count in the healthy control group. Importantly, there was very little variation in the sperm count among the individual members of the group suggesting the robustness of the treatment response. Treatment with 10X and 100X human equivalent dosage restored the sperm count to levels that were almost equal to that of the healthy control group.The lower rate of fertility in the fertility impaired group could be explained by the significantly lower . Treatment with clomiphene increased sperm motility significantly (p < 0.0001) compared to the fertility impaired model; however, this increase was only 21%. The lowest dosage at 0.2X (0.25 µg/kg/day) significantly increased the motility and was comparable to that of the healthy reference group. A dose-dependent increase in sperm motility was observed with an increase in the dosage of SLSO. The human equivalent dosage showed 46% motility which was a much better response compared to clomiphene and this increase was statistically significant when (p < 0.0001) compared to both the fertility impaired group as well as the clomiphene treated group. The highest dosage of SLSO (128 µg/kg/day) at 100X of the human equivalent dosage reversed the sperm motility to about 73%, but still lower than the healthy control group. However, this reversal was highly significant when compared to the fertility impaired group.A drastic reduction in sperm motility was observed in the fertility impaired model compared to the healthy controls Fig.  and thisThe testis was dissected from the male zebrafish from all the different groups in the study and a cytological smear was made. The tissue was stained with hematoxylin–eosin and used to study the tissue architecture. The testis from the healthy control group was observed to have a good distribution of sperm covering all the different stages of maturation such as spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa Fig. . The nucShivlingi are used in the Ayurvedic system of medicine as a general virility booster and are often used to treat sexual dysfunction and impaired fertility. The current work was undertaken to evaluate the scientific basis for such claims by chemically analyzing the supercritical fluid extracted SLSO and evaluating its reproductive fecundity in a zebrafish model of N-ethyl-N-nitrosourea-induced infertility.The seeds of High quantities of linoleic (47.8%) and linolenic acid (28.9%) were found in the SLSO. Fertility impaired female and male zebrafish showed improvement in fertility rates when fed with a diet rich in SLSO. In female zebrafish models, a dose-dependent increase in the follicular reserve of eggs was observed, along with a significant decrease in the number of cysts in the ovary. Similarly, in male zebrafish models, a significant increase was observed not only in the sperm count but also in the sperm motility. Moreover, gross improvement in the reproductive organs was observed, both in terms of morphological as well as cytological features. Microscopic studies revealed improvement in ovarian morphology with increased dosage of SLSO. Accordingly, the number of cysts and inflammatory cells also reduced in fertility impaired zebrafish as is evident from cytological studies of the ovaries. Similarly, in zebrafish male models, the cytological studies of the testis confirmed improvement in testicular health with healthy spermatogonia at various stages of maturity. These results establish the role of SLSO-rich diet in reversing the phenotype associated with impaired fertility in both males and females.Polyunsaturated fatty acids such as linoleic and linolenic acids are known to enhance follicle growth in cattle . They alPimephales promelas) [The SLSO was also found to contain β-sitosterol and stigmasterol. Since stigmasterol is a precursor in the synthesis of β-sitosterol and shares structural similarity with cholesterol, it is quite possible that it may undergo oxidation by free radical mediated pathways such as those documented for cholesterol . Howeverromelas) . In factromelas) . Studiesromelas) . Moreoveromelas) . In addiromelas) .Shivlingi seed oil in fertility impaired zebrafish models. Apart from providing scientific validation to Ayurvedic treatment, a solid groundwork is established for further research in this area. Further studies to decipher the possible pathway(s) by which the phyto-constituents of Shivlingi seeds rescue impaired fertility are needed to develop this classical Ayurvedic treatment into a therapeutic for impaired fertility in humans.The fatty acid constituents of SLSO, linoleic and linolenic acids, along with the phyto-sterols, β-sitosterol and stigmasterol, have known effects on cholesterol metabolism, oxidative stress, and inflammation. In this study, the rescue of fertility impairment in ENU-mutagenized zebrafish models was observed. There was a significant increase in the fertility rate and fecundity of female zebrafish treated with the SLSO. A dramatic increase in the number of follicles at different developmental stages was also observed. These results are a reflection of the beneficial effects of SLSO on ovarian morphology and cytology. Similarly, fertility impaired males treated with SLSO showed a rescue of testicular anatomy evident through a significant increase in sperm count as well as sperm motility. Overall, this study demonstrated for the first time the fertility restoring effects of Super Critical Fluid extracted All the chemicals used in the study were of analytical grade (AR) and were procured from Sigma-Aldrich  unless specified otherwise. Commonly used reagents such as PBS pH 7.4 and embryo buffer were made in house according to accepted methods.Bryonopsis laciniosa (L.) Naud. (Shivlingi) were procured from Divya Pharmacy, Haridwar, India. These seeds were further evaluated by our in-house botanists Dr. Rajesh Mishra and Acharya Balkrishna and authenticated for purity. The seeds from five different batches were used for extraction and oil from one such batch (Batch number D4/CHM/SCFE15510919) was used for the chemical analyses as well as in vivo experiments in this study.Fully matured seeds of 2). Briefly, the seeds of Shivlingi were pulverized into a fine powder and 1.5 kg of this powder was used to extract the oil in a supercritical extractor SFE 5000 Bio-Botanical Extraction System . The extractor was equipped with a CO2 recycler and the backflow pressure was maintained at 450/80 and 55/4 Bar/°C and a CO2 flow rate of 70 g/min over a period of 480 min. The initial extraction was in the static phase followed by a dynamic phase extraction to get a yield of 65 gm oil. This puts the yield of oil from this batch at 4.33% and was also the average yield from several other batches of extraction.The extraction process was accomplished using Super Critical Carbon dioxide  was collected.The fatty acid content in SLSO was determined according to the accepted method  of the American Chemist’s Society . BrieflyGC–MS/MS  was used to analyse the fatty acid mixture in the FAME using helium as a carrier. A standard mix for C4-C24 FAME containing a mixture of 37 FAMEs  was used to ascertain the identities of the fatty acids present in the SLSO.Briefly, 500 mg sample was dissolved in 5 mL of chloroform. It was sonicated in a Fisherbrand™ Model 505 sonicator  for 20 min on ice to obtain a clear solution. The sample was centrifuged at 8000 rpm for 5 min to remove the insoluble material. The supernatant was transferred to a sterile volumetric flask and the volume made up to 10 mL with chloroform. A standard stock solution was prepared by dissolving 10 mg of β-sitosterol  in 1 mL methanol. Working solutions were prepared by further dilutions as required.254 . The different phyto-constituents were resolved on the plate using a mobile phase consisting of chloroform and methanol in a ratio of 9:1, which was finalized after many trials during the protocol development stage. After resolution, the bands were developed upon derivatization using anisaldehyde sulphuric acid reagent and photographed under white light. Densitometric scanning was performed using the derivatized TLC plate at 450 nm. The concentration of β-sitosterol in SLSO was estimated using a standard curve of known concentrations of the standard and compared with duplicate loadings of the SLSO sample.Standard β-sitosterol (1 µL) at different concentrations (800 to 1800 µg/mL) and SLSO sample (2 µL) were spotted on an aluminium backed TLC plate with silica gel 60 FHPTLC analysis was done for the identification and quantification of stigmasterol in SLSO. The SLSO sample solution for HPTLC was prepared as described above by dissolving 1 g of SLSO in a total of 10 mL chloroform. A standard stock solution of stigmasterol  was prepared at a concentration of 10 mg/mL in methanol. It was further diluted as appropriate to get standard working solutions of 100 to 1200 µg/mL. A TLC plate with aluminium backing coated with Silica gel 60 F254 was used to resolve 1 µL of standard stigmasterol (100 to 1200 µg/mL concentrations) and 2 µL of SLSO sample. The bands were resolved and derivatized as described above.The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, guidelines were followed throughout the study period. All animal handling was done ethically to minimize stress to the zebrafish and all the protocols were duly submitted and approved by the Institutional Animal Ethics committee of Patanjali Research Institute, Haridwar (vide protocol number: 216/Go092019/IAEC).Danio rerio) of AB strain, from a single breeding pair, were used in the study to generate fertility impaired mutants. The fish were reared in groups of 24 with 2 L of water per tank in sturdy polypropylene tanks that meet the CPCSEA guidelines. The water temperature was maintained at 27 °C ± 1 °C using automated temperature control and water circulation systems. The tanks were checked daily for left-over food particles that were not cleared by the filtration system to prevent unwanted growth in the tanks. Aged water (for de-chlorination) was used and pH was routinely checked and adjusted if needed. The water was replenished as needed and full water exchange was done every week.Wild-type zebrafish . The accepted mode of induction can cause a higher rate of mortality in the fish and therefore, the protocol was modified to suit the needs of the study , 30, 31.After the mutagenesis, the fish were allowed to recover and during this time, they were used in mating experiments with wild-type males  twice, under standard conditions. In our experience, this ratio provided the best rates of positive breeding and therefore, was used throughout the study. The number of embryos, irrespective of their viability, was determined for each breeding pair and only females that produced less than 20 embryos were termed as ‘fertility impaired females’ and were included in the study. Fish were randomly assigned into groups of 24 individuals each, while un-mutagenized fish from the same breeding pair was used as a healthy or normal control group.In addition to ENU mutagenesis, the male zebrafish were further treated with 100 mM metronidazole in the tank water for further 20 days. Metronidazole is known to cause testicular cell ablation and therefore, a complete treatment protocol, which generally results in sterile individuals, was not used , 33. SixOnce the study population was selected, groups with 24 individuals each were moved to tanks designated to hold them during the study period and were acclimatized for 7 days. Fish were fed with normal Tetrabit flakes , using a 24 h feeding cycle until the start of the experimental period.Shivlingi seeds per day. In our studies, the average yield of oil from the seeds is 4.33%. The equivalent dosage of oil extract for humans was calculated based on the amount of seeds to be consumed and the yield of oil and was determined to be 90 mg/day.The Ayurvedic texts recommend the consumption of 2 gm of The translational dosage for the zebrafish was calculated according to the accepted guidelines of USFDA , 35. In The test and reference compounds were mixed with the normal feed at different dosages based on the known amount of feed. The feed was finely ground using a mortar and pestle and mixed with the compounds before being extruded out as pellets of 4 mg each. A fixed number of pellets with the target dose was given to the fish per day to achieve the required dosing. The study groups were dosed with either the reference drug or the SLSO in case of the treatment groups, and with normal feed in case of control and the fertility impaired groups.th day, the different study end-points were analysed. Female fish in the different study groups were allowed to spawn with wild-type males  and the fertility rate, conception rate, and fecundity rate were assessed. The study groups were returned to their respective tanks and after 5 days (next ovulation cycle), the ovary was dissected and the follicle count for the ovarian reserve of eggs was estimated. In a parallel experiment, the same dosing protocol was followed and on the 15th day, the ovary was dissected to determine the general morphology and cytology of the ovary as well as for the determination of the number of cysts.The dosing was for 14 days and on the 15A positive spawning that resulted in the formation of embryos, irrespective of their viability was used to calculate the fertility rate. The fertility rate was calculated from the number of positive mating to the total number of breeding pairs (24 breeding pairs).Fertility Rate = (Number of Positive breeding/ Breeding pair setup) × 100.Conception affirms that fertilization has taken place and highly confluent viable embryos are present. The percentage of fertilized eggs per mating was calculated based on the number of highly confluent viable embryos to the total number of breeding pairs.Conception Rate =  × 100.Embryos collected from the breeding tank were transferred to a 30 mL embryo medium for allowing the embryos to mature into larvae. The number of larval survivors to the total number of viable embryos produced by each breeding pair was estimated as the fecundity rate.Fecundity Rate =  × 100.Euthanization was performed ethically and with minimal distress to the fish by using water at 2 to 4 °C , 37. TheThe cytology smears of the ovary and testis were stained with hematoxylin and eosin for 2 min each using standard procedures. Excess stain was washed multiple times in PBS at pH 7.4 and images were captured using the image viewer software under a 40X bright-field objective . Triplicate fields were randomly chosen and the cells per field were quantified using Qupath software .The ovary of an adult zebrafish is identified as a thick structure with extensive lobules. The developmental cycle of the oocyte is asynchronous containing eggs at various stages of development . The fulThe ovary was dissected gently to prevent any damage to the gonad and was washed twice in PBS at pH 7.4. The oocytes at different maturation phases were gently dispersed onto a counting slide. The slide was observed under the 40X objective of a stereo-microscope . The follicles were identified by the presence of yolk and the different stages were identified by their morphological features. The number of follicles at each stage of development were counted and estimated as the ovarian reserve of eggs.The presence of cysts in the ovary is a sign of immature follicles that do not form fertilizable eggs and therefore, increased infertility in female zebrafish. The cysts were identified as a dark mass of cells that lack yolk and are irregularly shaped under a 40X objective of a stereo-microscope .th day, individual fish were allowed to spawn with healthy females (1:1 ratio) to estimate the fertility rate. In addition, the sperm count and motility were also determined.Male zebrafish in the different treatment groups were treated with either SLSO or clomiphene as a reference drug. Treatments were similar to those for the female zebrafish for 14 days. On the 15The testis was isolated and the cytological smear was stained with hematoxylin–eosin to assess the distribution of the male gametes at different developmental stages to determine the recovery from fertility impairment.Fish were anaesthetized in water at 17 °C until the operculum movement came to a stop . Each fi8 sperm per µL of HBSS. If the sperm count was below the desired number, then the entire sample was re-suspended in 1 µL and used for the analysis. Following this, 1 µL of sperm was transferred to a clean glass slide and activated using 9 µL deionized water. Thereafter, the motility of the sperm was recorded using a 10X objective.The sperm was freshly collected as mentioned previously and the motility was assessed according to the protocol devised by Selvaraj et al. with modifications ,41. BrieThe number of moving objects were tracked using the wrMTrck plugin in the Z project . The scap-value less than 0.5 was considered significant.Data are expressed as mean ± Standard Deviation (SD) of observations. Statistical analysis was performed using GraphPad Prism 7.04 . One-way ANOVA followed by Tukey’s multiple comparisons test was used to detect the significance of the data. In the analysis, the fertility impaired group was compared to the healthy control group, while the reference standard and SLSO treatment groups were compared to the fertility impaired group.


Kallikrein‐bradykinin‐forming cascade is known to cause hereditary angioedema (HAE) acute angioedema (AE) attacks. Further research of HAE attacks is needed to explain disease heterogeneity, predict treatment response and identify biomarkers for monitoring HAE attacks. Differential expression of the microvascular endothelial cell‐surface receptors for example, g‐C1qR, cytokeratin‐1, and plasminogen‐activator‐urokinase‐receptor (PLAUR) were hypothesized as biomarkers of AE attacks.n = 11; F:M = 8:3) were compared. To understand the HAE variants, DEGs in skin biopsies from HAE with normal C1 inhibitor , and non‐HAE  patients were compared. Gene‐set enrichment analyses and regulator effects analysis of these DEGs identified biological pathways in HAE attacks and their regulators.To understand HAE attacks, the differentially expressed genes (DEGs) in RNAseq and mi‐RNAseq data of total RNA extracted from skin biopsies of lesional versus non‐lesional skin collected during and between attacks in Type‐1 HAE patients (p = 0.05). The 18 significant DEGs investigated between baseline and AE attack samples in Type1‐HAE were enriched in beta1/beta3‐integrin cell surface interactions and IL‐6‐mediated signaling. Regulator effects analysis suggests a role for IL‐1b in HAE flares. AKT2, the mRNA regulated by the differentially‐expressed miR‐184A, was also associated with HAE attacks.PLAUR gene, encoding urokinase‐type plasminogen activator (u‐PAR), was constitutively over‐expressed in HAE‐Type‐1 versus non‐HAE controls suggestive of overactive u‐PAR‐mediated signaling via binding to Factor‐XII. Baseline PLAUR expression was associated with severe AE (β1‐integrin signaling pathways causing endothelial destabilization, and avid binding of factor XII to u‐PAR are possible novel mechanisms for progression of the endothelial kinin‐bradykinin‐forming cascade in HAE attacks.Angiopoetin‐activated  Although more effective HAE therapies have evolved over the past 15 years, further research of disease mechanism(s) is needed to explain disease heterogeneity, predict treatment response and identify biomarkers for monitoring disease during and between attacks. Clinically, characterization of HAE swelling attacks is difficult to predict because of their heterogeneity within and between patients with respect to frequency, location, severity and triggers such as psychological and physical stimuli that induce an episode.The pathogenesis of HAE involves complex interaction of the complement, contact and fibrinolytic pathways. The primary mutations for HAE Type I and II involve the serine protease inhibitor G1 but more recently a number of genetic factors including factor XII (F12), plasminogen (PLG) and mutations in angiopoietin 1 have been found to result in increased bradykinin 2 receptor mediated signaling leading to increased bradykinin production. Additionally, ANGPT1 gene is known to perturb the cytoskeletal assembly of vascular endothelial cells.Because of the possible involvement of the endothelial cell microenvironment in the pathogenesis of HAE flares, it was hypothesized that differential expression of the microvascular endothelial cell‐surface receptors such as g‐C1qR, cytokeratin‐1, and plasminogen activator urokinase receptor (PLAUR) could be novel biomarkers for disease activity. These molecules exist in the endothelial cell membrane as the biomolecular complexes, gC1qR‐cytokeratin 1 and cytokeratin 1 – urokinase‐type plasminogen activator (u‐PAR), which primarily bind high molecular weight kininogen‐prekalikrein complex and F12, respectively.This study aimed to determine the expression profiles of endothelial cell surface receptors,  from skin biopsies of patients with HAE Type 1 and HAE with normal C1 inhibitor (HAEnCI) between and during swelling attacks. In addition, investigation of the upstream regulators and molecular networks for such differentially expressed genes (DEGs) was performed to understand mechanisms of acute angioedema attacks based on canonical pathways from existing medical knowledge. Finally, using the regulator effects analysis, we also predicted novel pathways from predicted upstream regulators to downstream molecular/biological functions based on their common association with the DEGs.22.1n = 11; F:M = 8:3) were collected during flares and during quiescent (asymptomatic) periods that were separated by at least 7 days after completion of the flare. These biopsies were collected from edematous skin at the site of the HAE flare and from an adjacent area of skin after complete resolution of the attack. Skin biopsies during and in between swelling episodes were also obtained from HAE patients with normal complement . The HAE normal complement subjects all endorsed a family history for angioedema and were tested negative for the F12 mutation. Screening for other known mutations was not performed. A single skin biopsy was obtained from non‐HAE patients  for comparison to HAE Type I samples.To understand the mechanisms of HAE Type 1 angioedema attacks (referred to as flares in this manuscript), skin biopsies from consented HAE Type 1 subjects (2.1.1(1) Ages 18–75 years, (2) confirmed HAE Type 1 (low C1INH), or HAEnCI for the comparison group, (3) patients using on‐demand therapy only, and (4) ability to provide informed consent. Non‐HAE subjects without other allergic disorders were enrolled as the control group.2.1.2(1) Pregnancy, (2) cognitive disability, (3) urticaria, (4) acquired angioedema (low C1q), (5) ACE Inhibitor‐induced angioedema, (6) clinical response to H1‐antihistamines, (7) evidence of chronic diseases involving the microvasculature for example, coronary artery disease, diabetes or chronic renal failure not stable per clinical judgment of the investigator, (8) on estrogen oral contraceptives, and (9) prophylactic HAE therapy.2.2See Appendix 2.3See Appendix 2.4Five ml of whole blood was collected in RNA stabilizing blood tubes and stored at −80°C until further processing  versus non‐HAE controls and within the HAE patients between samples collected during flares and quiescence. These analytical methods attempted to identify biomarkers that could predict flares and disease progression, and thereby identify potential therapeutic agents to control such events. The DEGs were queried within the Ingenuity knowledge base included in the Ingenuity Pathway Analysis®  software to perform the core analysis that permits interpretation of the biological relevance of expression changes in the omics data and to determine which metabolic and signaling canonical pathways were enriched in the active flare lesions in HAE Type 1 versus normal healthy skin from non‐HAE controls. The core and subsequent comparison analyses using this application are summarized below. Additionally, the regulator effects analysis, based on the comparison analysis, were also performed that explains the upstream regulators expected to affect the target molecules in the dataset.2.7Differential gene‐expression of PLAUR in skin biopsies from HAE subjects at baseline versus non‐HAE controls were regressed on the severity of angioedema attacks in HAE subjects using generalized linear regression assuming a log‐normal distribution of gene expression values. Severity of attacks were determined clinically by the treating HAE specialist.2.8p‐value of overlap calculated using the right‐tailed Fisher's Exact test with Benjamini‐Hochberg correction for multiple testing, and the z‐score, indicative of similar or dissimilar biological signatures between analysis for downstream effects or upstream regulators, were used for determining statistical significance.Gene expression between comparison groups was explored by IPA® using the expression analysis component of the core analysis to determine the canonical pathways that were enriched in the data. This analysis identified relationships, mechanisms, functions, and pathways relevant to the dataset. Subsequently, the significant upstream regulators affecting the molecules in the dataset were predicted. The biological functions significantly over‐represented were determined. Molecular networks biologically relevant for HAE were determined based on the predicted activation or inhibition of regulatory molecules or biological processes. The 2.9Comparison analysis was performed on the core analysis results from all the comparator groups to identify trends, similarities and differences between those results. Components of such comparison analyses include comparison of the canonical pathways, and the regulator effects analysis. Thus, the canonical pathways commonly represented in the core analysis between the comparison groups  were compared. Thereafter, upstream analysis compared the potential upstream regulators for the DEGs between the different comparison groups. Enriched biological and pathological functions based on these regulators and their target molecules were compared. Regulator effects analysis examined connections between upstream regulators, the dataset molecules and downstream functions in HAE pathogenesis, and determined if any specific therapeutic molecules could hypothetically minimize or prevent the differential gene expression thus preventing the pathogenesis of HAE flares.2.10The list of differentially expressed miRNA were uploaded to the IPA. Core analyses for each comparison group were performed. Highly restrained stringent filters were used for querying only those molecules and relationships very relevant to humans. Pathway tools were used to generate and test additional hypothesis. The “build” tool was used to determine which canonical or well‐established pathways were associated with the molecules in the study data. Experimentally validated mRNAs, regulated by the differentially expressed miRNA in the data were determined. The canonical pathways, associated with the differentially expressed miRNA experimentally validated to regulate mRNA expression in TargetScan, miRecords, Ingenuity Expert Findings, were investigated. IPA tools were used to customize the networks and pathways that permitted generation of additional hypotheses from the networks identified by these tools.33.13.1.1p = 0.0003),  signaling, IL8 signaling, regulation of epithelial mesenchymal transition by Growth Factors Pathway and leukocyte extravasation signaling. Additionally, pathways exclusively upregulated in Type 1 flares versus controls are NK cell signaling, integrin linked kinase signaling, NRF2‐mediated oxidative stress response, Th17 activation pathway, IL15 production and the coronavirus pathogenesis pathway.Pathways exclusively downregulated in this comparison group were inhibition of matrix metalloproteinase, phosphatase and tensin homolog (PTEN) signaling, erythropoietin signaling pathway.3.3.2β, IL1A, IL18, TNFSF12, CSF2, TGFβ1, IL4, C5, IL17α, MIF, and TREM1; regulators predicted to be inhibited in these samples were COL18A1, FBN1, TP53, and IL10. In addition, TNF, IL1β, IL1A were also predicted to be activated in HAE Type 1 baseline versus control samples implying a baseline activation of these regulators in HAE Type 1 even during quiescence.Figure 3.3.3Table Known Relationship” column indicates the percentage of relationship between upstream regulators and biological functions that have been previously reported in the literature. For the first network, 36% of the relationships between upstream regulators and the pathogenesis of acute HAE flares have been reported previously but the remaining relationships have not. Upstream regulators without the relationship lines connecting to a downstream function(s) imply novel associations. This is applicable for multiple pathways that are directed from the upstream regulators to the dataset molecules and the downstream functions. For example, there may be a novel relationship between IL1β (the target for canakinumab/colchicine) and differentiation of myeloid leukocytes, interaction of mononuclear leucocytes, lymphocyte migration and movement of endothelial cells  that were highly predicted to target 181 mRNAs, from which two miRNAs (miR‐200b‐3p and miR‐184) with expression fold‐change of 3.9 and 3.2, respectively, had been experimentally verified to target 6 mRNAs: MARCkS, AKT2, PLCG1, NFATC2, ELMO2, and PTEN . The enriched pathways associated with the corresponding mRNA relevant to the understanding of HAE, include phagosome formation, phospholipase C signaling, synaptogenesis signaling pathway (MARCKS), acute phase response, interleukin signaling (AKT2), CCR5 signaling (PLCG1), B‐cell activating factor signaling, B‐cell receptor signaling (NFATC2), CXCR4 signaling, phagosome formation (ELMO2), B‐cell receptor signaling, Fc‐gamma receptor‐mediated phagocytosis in macrophages/monocytes and ICOS‐ICOSL signaling in T‐helper cells. Signaling pathways such as IL‐7, IL‐9, IL‐15, signaling in lymphocytes, tight‐junction signaling, TLR signaling, and cellular immune response were specifically selected to filter these miRNAs and the targeted mRNA.miR‐1‐3p and miR‐184, with expression fold‐changes of 6.7 and 4.4 respectively, were two differentially expressed miRNAs between the blood samples collected during HAE Type I quiescence and flares. Together these miRNAs target 53 experimentally validated mRNAs that are enriched in several canonical pathways including IL‐15 production, IL‐17 signaling, VEGF‐signaling, IGF‐1 signaling, IL‐8 signaling, G‐protein coupled receptor signaling, LPS/IL‐1 mediated inhibition of RXR function, IL‐4 signaling, and Th17 activation pathway  and cell movement of mononuclear leucocytes but not between other downstream biological functions. However, there are links between VEGFA and cellular homeostasis and differentiation of myeloid leukocytes. Thus, there may be novel pathways mediated by IL1‐β via VEGFA not reported previously to be involved in the HAE pathogenesis. Drug targets for such upstream regulators could be effective as novel molecules for treating or preventing acute angioedema episodes in HAE Type 1. Thus, the regulator effects analysis as described in Figure Through the regulator effects analysis, this study determined the hypothetical role of IL1‐Analysis of the differentially expressed miRNAs between baseline  and control samples identifies AKT2, directly regulated by miRNA‐184, as one of the key mRNAs that might be involved in the pathogenesis of HAE ; Data curation (Lead); Formal analysis (Lead); Methodology ; Project administration ; Software (Lead); Validation ; Visualization (Lead); Writing – original draft ; Writing – review & editing . Jonathan A. Bernstein: Conceptualization ; Funding acquisition (Lead); Investigation (Lead); Methodology ; Project administration ; Resources (Lead); Supervision (Lead); Validation ; Writing – original draft ; Writing – review & editing .Umesh Singh: Nothing to disclose. Jonathan A. Bernstein: PI, consultant, speaker: Takeda/Shire, CSL Behring, Pharming, Biocyrst; PI, consultant: Biomarin, Kalvista, Ionis; Consultant:; Pharvaris, Astria.Supplementary Material S1Click here for additional data file.Supplementary Material S2Click here for additional data file.


Some of the most threatening human diseases are due to a blockage of the mitochondrial electron transport chain (ETC). In a variety of plants, fungi, and prokaryotes, there is a naturally evolved mechanism for such threats to viability, namely a bypassing of the blocked portion of the ETC by alternative enzymes of the respiratory chain. One such enzyme is the alternative oxidase (AOX). When AOX is expressed, it enables its host to survive life-threatening conditions or, as in parasites, to evade host defenses. In vertebrates, this mechanism has been lost during evolution. However, we and others have shown that transfer of AOX into the genome of the fruit fly and mouse results in a catalytically engaged AOX. This implies that not only is the AOX a promising target for combating human or agricultural pathogens but also a novel approach to elucidate disease mechanisms or, in several cases, potentially a therapeutic cure for human diseases. In this review, we highlight the varying functions of AOX in their natural hosts and upon xenotopic expression, and discuss the resulting need to develop species-specific AOX inhibitors. Cryptosporidium parvum [Blastocystis hominis [Novosphingobium aromaticivorans [Vibrio genus [Thiobacillus denitrificans [Blumeria graminis [Magnaporthe grisea [Septoria tritici [Ustilago maydis [Sclerotinia sclerotiorum [Botrytis cinerea [Arenicola marina [Sipunculus nudus [Genkendia demissa [Crassostrea gigas [Ciona intestinalis [Ciona intestinalis is now the closest known relative to humans still expressing a functional AOX enzyme. Remarkably, AOXs in fungi and Ciona appear to facilitate similar stress responses as has been described for plants, namely the conversion of cellular energy, redox homeostasis, and the control of mitochondrial ROS content [Ciona in particular have been utilized as a means to correct ETC impairments in humans, and we suggest that their use in vivo may even represent a potential novel therapeutic treatment for mitochondrial diseases [Enzymes act as catalysts for chemical reactions and thereby enable living organisms to adopt to their environment and convert energy from nutrients ready for all vital processes such as growth, maintenance, or reproduction . A classm parvum , Blastoc hominis  and the  hominis . The fulcivorans , membersio genus ,18,19 angraminis , Magnapoe grisea ,21, Sept tritici , Ustilago maydis ,23, Sclerotiorum ,25 and B cinerea ,26 to naa marina  and Sipuus nudus , the bivstinalis ,30. Sinc content ,30,31 , where AOX plays a crucial role in adapting the metabolism to facilitate its life-cycle either in the tsetse fly (genus Glossina) or within the human bloodstream. Originally, it was thought that the bloodstream form of Trypanosoma brucei relied on AOX for ATP production by OXPHOS [vice versa occurs rapidly and is likely regulated by substrate availability, eventually also leading to an altered mitochondrial proteome [pmf), a prerequisite for mitochondrial ATP formation [1F0-ATPase hydrolyses ATP to pump protons across the inner mitochondrial membrane [Trypanosoma brucei indeed relies on AOX activity for ATP production, albeit via glycolysis rather than OXPHOS [Numerous human pathogens express AOX, the activity of which facilitates a number of vital functions in the pathogen adaptation processes, which in turn can enhance their pathogenicity. One example is in y OXPHOS ,38. Thisy OXPHOS ,40. Thisy OXPHOS  while a y OXPHOS ,40. The proteome . Whilst ormation , suggestn OXPHOS ,38.Cryptosporidium parvum [C. parvum causes severe diarrheal disease (cryptosporidiosis), contributing significantly to mortality in children and immunocompromised individuals [C. parvum has a single-host life cycle in the intestine during which obligate asexual and sexual processes occur [in vitro [C. parvum possesses a degenerate mitochondrion and an incomplete F1-ATPase . It is Blastocystis hominis, a parasite generally considered to be an obligate anaerobe [B. hominis possess only mitochondria-like organelles with an incomplete ETC and a partial Krebs cycle alongside other metabolic pathways but does contain a functional AOX [A seemingly different situation is observed in anaerobe ,49. Intronal AOX . Accordional AOX  and sincAcanthamoeba [Acanthamoeba co-expresses two fully functional and competing quinol-oxidizing pathways, the phosphorylating canonical ETC and the cyanide-resistant AOX pathway [AOX is also expressed in thamoeba  which cathamoeba ,52. Acan pathway ,54. Both pathway . Moreove pathway ,57. Alth pathway , to buff pathway  or to ge pathway .Other emerging human pathogens that broadly express AOX include the microsporidia, a group of obligate intracellular parasitic eukaryotes . The ideCandida albicans (candidiasis) [Cryptococcus neoformans (cryptococcosis) [Aspergillus fumigatus (aspergillosis) [Candida albicans is of particular interest since its expression appears to be a reaction to different forms of increased stress resistance. In other words, the protein is expressed to protect against mitochondrial ROS generation during copper starvation [Candida auris [Candida, first isolated from a patient's auricle in Japan in 2009 [C. auris infections have been reported in India, South Africa, Kuwait, Venezuela, Brazil, the United States, Colombia, Pakistan, Spain, Germany, Israel, Norway, Oman, and the United Kingdom [C. auris appears not only resistant to fluconazole [AOX is also expressed in opportunistic human fungal pathogens, i.e. pathogens that do not represent a health threat unless an individual is immunocompromised or susceptible through other conditions, such as idiasis) ,69, Crypoccosis)  and Aspeillosis) . The bioarvation , to decrarvation , or to parvation . A worldda auris , a multi in 2009 . This mu in 2009 . Since i Kingdom . The latconazole , a drug conazole .Candida spp. enabling it to swiftly adapt to varying enviromental conditions and host niches [C. auris. Since AOX enables a metabolic stress resistance of C. auris, we have previously suggested that AOX inhibitors may become a safe and effective treatment approach and thereby could become a ‘game changer' in the clinic [Paracoccidioides brasiliensis is one of the causative agents of paracoccidioidomycosis, and treatment of P. brasiliensis [C. neoformans [aox1 gene, which is induced at 37 °C presumably after sensing the human body temperature, led to a decrease in C. neoformans virulence in a murine model. Finally, silencing of the A. fumigatus AOX has led to a decreased viability in the presence of ROS and a higher rate of killing by macrophages [Whilst metabolic flexibility is considered a key characteristic of t niches , little e clinic . In suppiliensis  with theoformans  aox1 genrophages .As an increasing number of mechanisms of action of antibiotics and antifungal drugs against human pathogens are bypassed, the presence of multidrug-resistant organisms thus poses a serious threat to human welfare. AOX is emerging as a suitable pharmacological target. To date many compounds have been reported as inhibitors of the AOX, with the earliest and most used historically being the alkyl-gallate and hydroxamic acid derivatives from the 1980s . UnfortuFusarium graminearum in wheat [Magnaporthe oryzae in rice [Ustilago maydis in maize [Sclerotinia sclerotiorum in potatoes [Moniliophthora perniciosa in cocoa [Mycosphaerella fijiensis in banana [Venturia spp. in apple [The rising presence of fungal infections is also recognized as a global threat to food safety in agriculture, which endangers both ecosystem and human health . Fungal in wheat , Magnapo in rice , Ustilagin maize , and Sclpotatoes  and soybpotatoes . Such inin cocoa ,92, Mycon banana , Venturiin apple , and varin apple . Disturbin apple ,25,96,97M. perniciosa, AOX is necessary for spore germination and host infection [M. grisea virulence in barley leaves appears to use different mechanisms [bc1 complex) of the ETC by strobilurins and other so-called quinone-outside inhibitor (QoI) fungicides [bc1 complex, AOX can restore mitochondrial functions  and the development of synthetic analogs with enhanced field performance [M. grisea that AOX allows fungal survival long enough for the emergence of point-mutations in the cytochrome bc1 complex, such as the change of glycine 143 to alanine (G143A), thereby rendering azoxystrobin completely ineffective [Unfortunately, only very few studies have addressed the biochemical differences between the various AOX enzymes and the different roles that fungal AOXs may play in plant pathogenesis. Hitherto, it has been demonstrated that in the cocoa pathogen nfection  while M.chanisms . A more ngicides . Due to unctions  and sustin wheat , and Gae tritici , the whee grisea , Sclerotrotiorum , M. pernrniciosa ,98, Ventaequalis ,103 and um majus . QoI funformance . Azoxystformance . An alarffective ,107. AOXffective ,108. MecAcremonium chrysogenum [Aspergillus nidulans [Aspergillus flavus but also by other Aspergillus species [https://t1p.de/8t67). Health and economic losses, however, may not only occur through direct contact with human beings but also by contamination and killing of poultry and livestock. In addition, many mycotoxins are highly stable meaning that they may remain present even after the toxin-producing fungus has been eliminated during the food processing [A further aspect which deserves attention is the possible role of AOX in the production of harmful mycotoxins by food colonizing fungi which can occur in the field or during post-harvest storage. In addition to AOX allowing fungal growth as discussed above, which by itself can increase mycotoxin incidence, this enzyme may contribute to the metabolic pathways associated with mycotoxin production. There is a known positive correlation between AOX expression in ysogenum  and Aspenidulans ,111 and  species . Since mocessing .Because AOX is present in both the fungal pathogen and the infected plant, putative AOX inhibitors that can be approved as fungicides must have exceptional specificity to inhibit the pathogen without harming the host, and this specificity must be accompanied by high efficacy and (food) safety. A key challenge in understanding the structure-function relationship of AOX has been the identification of its substrate- and inhibitor-binding site and its mechanisms of catalytic action. A detailed knowledge of the nature of this binding site is thus important, since it will reveal if there is a common architecture for the binding of its substrate or an inhibitor, thereby providing unprecendented insights into differential mechanisms of binding. Only such knowledge will allow the rational design of phytopathogenic and anti-parasitic drugs that target AOX in a species-specific manner.d-glucopyranoside [AOXs are self-folding di-iron carboxylate proteins harboring four highly conserved ExxH motifs, a characteristic of the common iron binding sequences found in non-heme iron binding proteins ,117. Iniranoside  increaseranoside . Notablyranoside . Althougranoside ,125 haveOne of the most important structural features for the protein with respect to both its species-specific functionality and inhibition is the hydrophobic substrate cavity. This cavity is located between the α1 and α4 helices and leads directly to the di-iron core from the proposed membrane binding region  providinpara position from the hydrophobic chain. In addition to ascofuranone-like compounds, the gallates are also considered to be effective inhibitors in high nanomolar region  deficient flies [In addition to the role of AOX in enabling human and fungal pathogens to seek opportunistic ways of causing diseases, there are also perspectives for the use of AOX to challenge disease paradigms of human pathologies. One such emerging opportunity is within the area of AOX gene therapy, in attempts to overcome mitochondrial dysfunctions . Howevernt flies ,144, andnt flies ,145. Mornt flies  , rigidity, tremor, and the inability to maintain an upright position . A mitochondrial involvement in the pathology has been postulated, since pharmacological inhibition of OXPHOS complexes such as the respiratory complex I inhibitor and neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) [Parkinson's disease (PD) is a neurodegenerative disease with an adult-onset that is characterized by the degeneration of dopaminergic neurons in the e (MPTP)  and the e (MPTP) ,150. Fure (MPTP)  supportiDrosophila melanogaster, in which the catalytic subunit of the mitochondrial DNA polymerase was down-regulated by RNAi technology [To ultimately identify the position of mitochondrial OXPHOS dysfunction in the hierarchy of PD, the combination of animal models and genetic tools became inevitable. One such approach used the fly model, chnology . Expectechnology . AOX resDrosophila disease model, the dj-1b mutant [Dictyostelium discoideum. The results showed that DJ1 controls extramitochondrial functions such as phagocytosis and, to a lesser extent, pinocytosis under physiologic conditions [dj-1b fly mutant [Further evidence for a positive effect of AOX on the course of PD arose in another b mutant . DJ1 is b mutant . The exab mutant , althougb mutant . Anotherb mutant , rather nditions . Loss ofy mutant , which ic oxidase) [postmortem observations in brains of AD patients [Alzheimer's disease (AD) is the most common form of dementia based on a chronic neurodegenerative process that worsens over time ,158. A coxidase) ,176 has oxidase) ,178 whicpatients ,180. Thepatients  and an ipatients ,183.bc1 complex inhibitor antimycin A. Furthermore, AOX partially alleviated a neurodegenerative phenotype, i.e. a decreased lifespan in fly models expressing human Aβ peptides specifically in neurons. This improvement in survival was associated with a decrease in marker expression indicative of oxidative stress pointing towards disruption of electron flow through the ETC and thus induced ROS production as causative factor for the development of AD.Despite the progress in understanding underlying disease mechanisms, no treatment has thus far has been developed which could stop or reverse the loss of cognitive functions. Based on current knowledge, however, mitochondria appear as a valuable target to shift fate ,185. ThiSepsis is defined as ‘life-threatening organ dysfunction caused by a dysregulated host response to infection' . Sepsis-2S) [The complex network of dysregulated signaling cascades observed during sepsis affects all levels of cellular metabolism and hence organs which rely heavily on OXPHOS, such as the heart, are particularly sensitive . Recentl2S) , all thr2S) . Althoug2S) ,204, nit2S) ,205,206.2S) .c partially restored complex IV activity and improved cardiac function [Cardiac contractile dysfunction is another important component towards fatality during sepsis. While systolic dysfunction paradoxically predicts a better outcome, diastolic dysfunction is a common and major predictor of mortality in sepsis . Again, function ,211. Intfunction ,213. A pOvercoming mitochondrial respiratory impairment has been identified as the key outcome of clinical and biochemical organ failure. Published data indicates that AOX expression provides protection against septic shock, based on mitochondrial protection or acceleration of the recovery process through stimulation of mitochondrial activity, e.g. in a mouse model of LPS-induced endotoxemia . MechaniChronic obstructive pulmonary disease (COPD) describes a number of conditions of chronic inflammatory lung disease which obstruct the airflow . Most prOne reason for the observed cigarette smoke toxicity may be its content of detrimental oxidants  which caBypassing a blocked ETC by AOX increases the viability of organisms. This mechanism helps plants, fungi and other organisms adapt to their ever-changing environment, but is also used by pathogens to evade the host's immune response . To prot