Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus induced disease-2019 (COVID-19), is a type of common cold virus responsible for a global pandemic which requires immediate measures for its containment. India has the world’s largest population aged between 10 and 40 years. At the same time, India has a large number of individuals with diabetes, hypertension and kidney diseases, who are at a high risk of developing COVID-19. A vaccine against the SARS-CoV-2, may offer immediate protection from the causative agent of COVID-19, however, the protective memory may be short-lived. Even if vaccination is broadly successful in the world, India has a large and diverse population with over one-third being below the poverty line. Therefore, the success of a vaccine, even when one becomes available, is uncertain, making it necessary to focus on alternate approaches of tackling the disease. In this review, we discuss the differences in COVID-19 death/infection ratio between urban and rural India; and the probable role of the immune system, co-morbidities and associated nutritional status in dictating the death rate of COVID-19 patients in rural and urban India. Also, we focus on strategies for developing masks, vaccines, diagnostics and the role of drugs targeting host-virus protein-protein interactions in enhancing host immunity. We also discuss India’s strengths including the resources of medicinal plants, good food habits and the role of information technology in combating COVID-19. We focus on the Government of India’s measures and strategies for creating awareness in the containment of COVID-19 infection across the country. India has also been largely affected by instances of COVID-19. SARS-CoV-2 viral protein interacts with various host proteins to mediate viral entry and replication in the human host . COVAXIN has currently gone into Phase III clinical trial after successful completion of Phase I and II clinical trials started by Bharat Biotech from July, 2020 onwards6. Recently, the Drug Controller General of India (DCGI) has granted emergency approval to COVAXIN in India7. The Ministry of Ayush under the Govt. of India has emphasized the importance of exploiting medicinal herbs in the context of COVID-19. Indian indigenous medicinal plants with immune regulating properties have often served to boost immunity and render protection against viral infections (Coronavirus disease 2019 (COVID-19) outbreak, caused by the novel coronavirus (SARS-CoV-2) has emerged as a global epidemic and posed serious worldwide public health concerns owing to the contagious nature of the virus and high death rate. Transmission through droplets facilitated its rapid spread and caused panic across the globe. There were 80,776,890 confirmed cases worldwide till December 30, 2020man host . Targetiman host . Convalehe world . The phafections . BesidesComputational bioinformatics and AI have been exploited for better management of COVID-19. Machine learning techniques (MLTs) have been employed for taxonomic and hierarchical classification of SARS-CoV-2 strains . ComputaIn a nutshell, this review highlights the current scenario of COVID-19 across India, with special emphasis on death/infection ratio in urban and rural India and disease association with co-morbidities. This review also deals with strains of SARS-CoV-2 circulating in India and the immuno-modulatory action of viral proteins. It discusses the various diagnostic kits, masks and disinfection techniques in use for diagnosing and combating COVID-19. This review further focuses on various approaches that may be followed to tackle the problem of SARS-CoV-2 infection  have shown high COVID-19 death rates. The total number of confirmed cases and death rates in Indian cities with important international airports has been tabulated in 9. Although, reduced to some extent by the Swachh Bharat Mission, a large proportion of rural households avail open defecation and public toilet facilities. Also, many rural households travel long distances to carry drinking water from community source. Social distancing becomes a difficult proposition in such situations , chronic obstructive pulmonary disease (COPD), obesity, chronic kidney disease (CKD), cerebro-vascular accident (CVA), malignancy, and inflammatory conditions have been noted to worsen health status in COVID-19 positive patients . MedicatCoronaviridae. Viruses belonging to this family have a single-stranded, (+) sense RNA genome of ∼30 kb  protein with host cell angiotensin-converting enzyme 2 (ACE2) aided by TMPRSS2 mediates viral entry. SARS-CoV-2 viral proteins  . In this assay, nasal swab is used as the specimen for detection12. Gargle lavage sample collected from COVID-19 patients was identified as an easy, alternative showing comparable efficiency as nasopharyngeal and oropharyngeal swab samples  put an extra burden on specialized COVID-19 diagnosis . Clinica10 . Accurat10 . India h10 . Diagnos10 . Initial10 . The limgy (NIV) . SOPs fogy (NIV) . To enhagy (NIV) . The progy (NIV) . Recentlgy (NIV) . However samples . Monitor13. Apart from RT-PCR based testing, other approaches have also been demonstrated which involve two-step detection methods involving more affordable thermal cyclers  and fluorescence spectrometers14.Standard diagnosis for infection requires real-time thermal cyclers which are used to perform RT-PCR, a robust and reliable detection technology . TechnolThe SARS-CoV-2 infection and the COVID-19 pandemic have posed an unprecedented challenge to the medical fraternity. The treatment is restricted to the best supportive care and experimental medications. Targeting the viral entry, interaction of the virus with its host and the downstream signaling pathways using novel or repurposed drugs, is one of the strategies for the management of COVID-19. Several agents (enlisted in Mycobacterium W (Mw)), on COVID-19 patients at PGIMER, Chandigarh in partnership with the Council of Scientific and Industrial Research (CSIR) to reduce the mortality of critically ill COVID-19 patients and have obtained promising results15.Indian Pharmaceuticals Cadila has tested the immunomodulator drug named Sepsivac  ratio at the typical dosages used is limiting their possible clinical use.Another approach for developing drugs targeting host immunity has been to express SARS-CoV-2 proteins in human cell lines and identify their human protein interacting partners. Of 332 interactions, 66 human proteins were found as druggable candidates that could be targeted by 29 FDA approved drugs, 12 compounds in clinical trials and 28 compounds in preclinical stage . FurtherNatural killer cells play a role in the clearance of SARS-CoV. NK cell based products are in various stages of trial as anti-COVID-19 agents. The US-based Company Celularity has developed placenta derived NK cells CYNK-001 . Recombi20. However, only three vaccines are available with the data published in peer reviewed journals till 5th January, 2021 namely, mRNA1273, BNT162, and chAdOX1nCOV19 . COVAX aims at fixed vaccination for 20% of population belonging to the LCMICs by 2021. The vaccine will be provided by the AstraZeneca22. A plasmid DNA based vaccine, ZyCoV-D has been developed by Ahmedabad-based pharma company Cadila Healthcare (Zydus Cadila). It has been claimed that this vaccine is stable for 3 months at a temperature of 30°C and longer at 2–8°C. This thermostability could be beneficial for nationwide vaccination program due to minimalistic cold storage requirements. Phase III human clinical trials are being initiated for this vaccine. Besides these indigenous vaccines, several non indigenous vaccines of foreign origin are presently in various stages of clinical trial in India. Covishield, the vaccine developed by Oxford University and AstraZeneca has entered phase III trials in collaboration with the Serum Institute, Pune, India. Serum Institute has applied to DCGI for emergency regulatory authorization for Covishield use in India and has submitted additional requisite vaccine datasheet in this regard. This vaccine has been approved for emergency use in United Kingdom and has become the first COVID-19 vaccine candidate to have obtained emergency approval in India (see text footnote 4)23. Covishield has the advantage of storage at 2–8°C24. Besides this, Dr. Reddy’s Laboratories Limited and Sputnik LLC (Russia) have been jointly conducting the clinical trial of Sputnik-V, the world’s first registered vaccine in India. This vaccine, ranking among world’s top 10 vaccine candidates is presently in Phase II Human Clinical Trial in India25. The Biological E’s novel Covid-19 vaccine is also in the Phase I/II Human Clinical Trial in India (see text footnote 5). Ecological studies have highlighted lower number of infections and reduced COVID-19 mortality in countries, where BCG vaccination is made mandatory . Serum Institute, Pune, India has conducted phase III trial of BCG vaccine VPM1002 to evaluate cross-protection to COVID-1926. BCG vaccine could serve as a booster of innate immunity against COVID-19 via metabolic and epigenetic changes in a process called trained immunity  has currently entered the Phase III Human clinical trial and has recently been given emergency approval in India by the DCGI (see text footnote 7)andatory . Randomiimmunity .27. Similarly, Moderna vaccine also has stringent storage requirement at −20°C28. Such stringent refrigeration needs may be difficult to achieve in developing countries and may render mass vaccination in India extremely challenging. Although India has cold storage facilities, they are limited and there are constraints especially in the case of handling large numbers of doses in a country as densely populated as India. Ramping up of cold chain and restructuring of cold storage facilities with synergistic aid from food storage and supply cold chain, may aid in overcoming vaccine storage issues to some extent29. Another important factor involved in mass vaccination is the economic burden. The COVAX facility  have emerged to financially support and enable equitable distribution of COVID-19 vaccines across the world30. The Government of India (GOI) has also taken initiatives to bear the entire cost of vaccination and ensure mass immunization at nominal price31. The Global Alliance for Vaccines and Immunizations (GAVI) has estimated an expenditure of $1.4 billion to $1.8 billion on part of India (the second most populous country after China) for the first phase vaccination, even after support from the COVAX facility32. Moderna vaccine, apart from its cold requirement, is highly priced (at $10–450 per dose), which might be difficult to cater to the Indian population. However, aid from COVAX alliance and Government may help Moderna reduce its cost for India. Covishield and COVAXIN are reasonably priced with respect to the Indian scenario. Covishield have been priced at $3 per dose for government and approximately, $10 for private entities. Because of their local origin and normal refrigeration temperatures, these two vaccines will be easy for handling and supply chain distribution in India33,34.Another important vaccine, BNT162 from Pfizer, which has already been rolled out in United Kingdom, United States and received emergency use approval in more than 10 countries, has extreme cold chain and storage requirement at −75°C to keep its potency intact35.COVID-19 mass vaccination drive in India shall soon be initiated with 30 crore people receiving the vaccines in the first phase. Healthcare workers, frontline workers and individuals aged above 50 will be vaccinated first according to the recommendations of the National Expert Group on Vaccine Administration for COVID-19 (NEGVAC). In this regard, the COVID Vaccine Intelligence Network (Co-WIN) system has been developed as a digital platform for registration and real time monitoring of vaccination to pre registered individuals in IndiaNeutralization of the virus by antibodies is an important strategy for containing SARS-CoV-2. In SARS-CoV, the RBD122 (amino acids 318 to 510) of the S protein is primarily being targeted by neutralizing antibodies . The RBD36. N95 respirators, surgical masks or cloth masks are recommended to prevent respiratory transmission. Cloth masks may possibly be cost-effective in preventing community transmission in densely populated Asian countries  including face piece respirators, gloves, shoe covers and face shields are necessary for the protection of health workers from infectionountries . Unlike ountries . To previn India .38,39  sensor, portable X-ray machine, fiberoptic bronchoscopes, video laryngoscopes, are required in monitoring and treatment of COVID-19 patients . The venlators43 . An altelators43 . Recentllators43 .Antiviral herbal therapy has made enormous progress in the past decade . Various44. Besides, the Ministry of Ayush has formulated a collection of four ayurvedic herbs namely, ashwagandha, guduchi, yasthimadhu, peepli; and a drug named Ayush 64 for combating COVID-19. The Ministry of Ayush along with the CSIR have initiated the process of validating the efficacy of these formulations against COVID-19 in the month of May, 2020 and the outcomes of these trials are expected to be available soon45,46,47.The Ministry of Ayush under the Govt. of India has recommended use of indigenous herbal plants and spices namely, tulsi, cinnamon, dry ginger, black pepper, turmeric, coriander, cumin and garlic for enhancing immunityThe worldwide outbreak of SARS-CoV-2 has resulted in a tremendous dearth of clinical equipment. In order to contain the pandemic effectively, large scale testing and diagnosis are required. This is evident from the successful containment of SARS-CoV-2 virus in countries that have been able to perform mass testing of possibly infected people and contact tracing. RT-PCR serves as the gold standard test for validating SARS-CoV-2 infection. Inadequate testing capability in most countries, along with the high dependency of the RT-PCR test on the swab technique, has spurred the need to search for alternative methods that allow COVID-19 diagnosis.The chest X-ray and thoracic computed tomography (CT) are examples of easily accessible medical imaging equipment, which assists clinicians in diagnosis. CT images may serve as a visual indicator of coronavirus infection for radiologists . While RDeep learning technology which lies central to current concept of Artificial Intelligence has been effective in automated detection of lung diseases with high diagnostic accuracy. However, there are challenges when developing AI-empowered deep learning technologies for COVID-19 screening . Most of48,49. The Delhi Government has also launched the Delhi Corona app to create public awareness regarding availability of hospital facilities for COVID-19 treatment and also for complaint redressal regarding refusal to admit COVID-19 patients by hospitals with available facilities50,51,52. Apart from these mobile applications, the Govt. of India has promoted strict social distancing to contain spread of COVID-19 amongst the Indian population  and CT scan severity score have been evaluated for highlighting disease progression and the risk for development of post recovery complications such as pulmonary fibrosis, ARDS, neurological disorder or even multi-organ failure . COVID-19 infection may exert detrimental long term effects on organs such as lungs, liver, kidney, brain. and heart . These m failure . IdentifAll the authors reviewed the literature, wrote the review, edited, and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Changes in total fruit intake (including fruit juice) were not significant. Results suggest a pediatric FVPP may have meaningful impacts on children’s dietary behaviors, particularly with regard to the intake of whole fruits.Public health recommendations suggest limiting child consumption of fruit juice in favor of whole fruit due to juice’s high sugar content, lack of fruit fiber, and potential for excess intake. However, replacing juice with whole fruit may be particularly challenging for low-income and minority children, who report the highest intake of 100% juice. To address access and affordability challenges among low-income children, researchers partnered with pediatricians in an urban food desert community, to introduce a fruit and vegetable prescription program (FVPP) that provided a $15 prescription for fresh produce to every child during each office visit. Participating vendors included a farmers’ market and local mobile market. This study assessed changes in daily consumption of total fruit and whole fruit among 108 pediatric patients following six months of exposure to the FVPP. Child-reported mean daily intake of whole fruit increased significantly from the baseline to the 6-month follow-up ( Although consumption of fruit has been associated with a reduced risk of obesity and chronic disease ,3,4,5,6,When considering intake of total fruit, which includes 100% fruit juice, it is important to note that fruit juice, which is often less expensive and more accessible, does not necessarily carry the same benefits of whole fruit . AlthougPediatricians care for individuals from infancy to early adulthood, and their influence on dietary behaviors during this period is likely to have long-term health, growth, and developmental impacts ,35,36,37Enduring challenges with child poverty  and inadThis was a non-controlled longitudinal intervention trial with a consecutive sample of caregiver–child dyads newly exposed to a pediatric fruit and vegetable prescription program. Self-reported data was collected from caregiver–child dyads at baseline and at the 6-month follow-up. Study recruitment and the prescription program are described in full in an earlier article sharing baseline food security and dietary patterns in a subsample exclusively of Flint children . All metThe pediatric fruit and vegetable prescriptions in the current study, similar to medical prescriptions, were written by pediatricians and given to patients. All patients received a $15 fruit and vegetable prescription at the conclusion of every clinic visit to be redeemed at either the Flint Farmers’ Market or a local mobile market. Vendors treated prescriptions as vouchers that could be redeemed only for fresh fruits and vegetables.The fruit and vegetable prescription program was introduced to all clinic patients (0–18 years of age) in August 2018. At that time, caregivers with children who were 7 to 18 years old and spoke English were invited to participate in the research study. Caregiver–child dyads consisted of one caregiver and one child from each family, who separately completed demographic questions followed by survey questions used to assess dietary patterns, participation in food assistance programs, and food security. At approximately the 6-month follow-up (6 months after baseline data collection), caregiver–child dyads completed the identical assessments via caregiver-selected telephone or in-person interview. All data were collected using a secure digital platform  after receiving caregiver consent and child assent.Child dietary patterns were measured with the 41-item Block Kids Food Screener, a tool that has been shown to have good relative validity for children and adolescents . The insCaregiver survey questions included information regarding participation in a variety of local, state, and national food assistance programs. In comparison to minimal participation in other food assistance programs, a high percentage of caregivers reported household participation in the Supplemental Nutrition Assistance Program (SNAP) (45.6%), which is the US government’s largest anti-hunger program , and/or To measure household food insecurity and hunger, caregivers completed the US Household Food Security Module: Six-Item Short Form, developed by the National Center for Health Statistics . A samplChildren 12–18 years of age completed the 9-item Self-Administered Food Security Survey Module for Youth . This agT-tests examined changes from baseline to 6-month follow-up in terms of mean daily intake of total fruit, whole fruit, 100% fruit juice, and vegetables . Independent samples T-tests were used to compare mean change in intake of whole fruit by child gender, child race, caregiver race, and child age, and one-way ANOVA was used to compare mean change by caregiver age and education groups. Caregiver-reported household food security and child-reported food security at 6-month follow-up were dichotomized into high/marginal or low/very low food security. Chi-square analysis was used to compare ¼ and ½ cup whole fruit change by food security category at 6-month follow-up. Independent samples T-tests compared mean change in whole fruit consumption by food assistance programs  as well as by caregiver-reported household food security and child-reported food security categories. All data were analyzed using SPSS statistical software .Paired samples n = 108) completing the Block Kids Food Screener at both time points. Most of the children  were African American (62.8%) and female (55.4%). Similarly, the majority of caregivers  were African American (60.5%) and female (94.7%).A total of 114 caregiver–child dyads completed baseline and 6-month assessments between August 2018 and July 2019, with 94.7% of the children (T-tests. Mean daily servings of whole fruit increased significantly (p = 0.029) from baseline  to 6-month follow-up . Of the 108 children who completed baseline and 6-month follow-up Block Kids Food Screeners, 47 (43.5%) reported an increase in mean daily consumption of whole fruit by at least ¼ cup, and 37 children (34.3%) reported an increase in mean daily consumption of whole fruit by at least ½ cup. The mean daily intake of total fruit increased from baseline  to 6-month follow-up , but the change was not significant (p = 0.548). The mean daily intake of 100% fruit juice decreased, but the change from baseline  to 6-month follow-up  was not significant (p = 0.388). There was no statistically significant change in children’s vegetable intake from baseline  to 6-month follow-up .Baseline and 6-month follow-up measures of mean daily servings of total fruit, whole fruit, and 100% fruit juice reported in cup equivalents were compared using paired samples To examine whether change in mean daily intake of whole fruit was associated with child or caregiver characteristics, we compared mean change in whole fruit consumption by key demographic variables. There was no significant difference in mean change of whole fruit intake by child gender, race, or age category . SimilarT-tests, we examined the difference in change in mean daily intake of whole fruit by participation in SNAP and free and reduced-price school meals. There was no significant difference in this change by participation in either SNAP or free and reduced-price school meals , which provides federal grants to states for supplemental foods, health care referrals, and nutrition education for low-income pregnant and postpartum women, and to infants and children up to 5 years of age who are at nutritional risk . Similarol meals .p = 0.644). Similarly, there was no significant difference in change in mean daily intake of whole fruit between child-reported food security categories at 6 months (p = 0.344).To further examine whether change in whole fruit intake was influenced by household or child food security, we examined the relationship between the food security category and the change from baseline to 6-month follow-up in daily intake of whole fruit. There was no significant difference in the mean daily intake of whole fruit between caregiver-reported household food security categories at 6 months (p = 0.102). When considering child-reported feelings of food security, 17 children (48.6%) who reported high/marginal food security increased daily whole fruit consumption by at least ¼ cup, compared to six children (33.3%) who reported low/very low food security (p = 0.289).As shown in n = 23) participated in SNAP, compared to 46.9% (n = 23) of those in the low/very food security group (p = 0.719). For households that reported high/marginal food security, 52.8% (n = 28) reported their children received free and reduced-price school meals, compared to 63.3% (n = 31) in the low/very food security group (p = 0.286). Data were similar when examining child-reported food security groups as there was no relationship between food security category and SNAP participation  or free and reduced-price school meals participation .To better understand the relationship between the food security category and participation in food assistance programs, a chi-square analysis assessed the difference in the proportion of high/marginal and low/very low food security groups by participation in SNAP and school lunch programs. For households that reported high/marginal food security, 43.4% (After only six months of exposure to a pediatric fruit and vegetable prescription program that provided a $15 prescription to every child at each office visit, children in our study reported a significant increase in mean daily consumption of whole fruits, without an analogous increase in consumption of fruit juice. These preliminary results suggest an important positive association between exposure to a pediatric fruit and vegetable prescription program and an increased consumption of whole fruit among children. The lack of association between exposure to the prescription program and intake of fruit juice among our sample of children is also noteworthy. In spite of recommendations to limit juice consumption , fruit jThe change in the intake of whole fruit was consistent across all subgroups in our sample, indicating program effectiveness regardless of child age, gender, race, or socioeconomic status. With childhood consistently identified as a critical period for the establishment of lifelong dietary patterns ,36,37, iConsistent evidence suggests that total fruit intake is significantly lower among economically disadvantaged children and adolescents ,24,28,66Research focused on low-income adults who participate in fruit and vegetable prescription programs has reported no significant changes in produce consumption or purchasing patterns . The curFinally, it has been well documented that, in comparison to higher-income neighborhoods, low-income neighborhoods have lower-quality food and fewer healthy food options ,71. SimiSeveral limitations to the current study should be noted. Our sample was small and specific to one low-income, urban community. As a result, findings may not be generalizable. Although there may have been selection bias, as responses from households that voluntarily elected to complete the survey could have differed from responses of households that did not, the study population’s characteristics strongly matched those of the source population, which largely consists of low-income, minority children receiving public health insurance. The accuracy of the Block Kids Food Screener may be limited by recall bias, but a trained research assistant was consistently available to children when completing this instrument in an effort to minimize this particular study limitation. Furthermore, this was the first study to use a dietary assessment tool, previously validated for use with children, to assess changes in dietary intake following exposure to a pediatric fruit and vegetable prescription program.Pediatricians and primary care physicians are in the unique and favorable position to address the poor access and affordability of fresh, whole fruits among children. The current study suggests that after only 6 months of exposure to a pediatric fruit and vegetable prescription program, mean daily consumption of whole fruits improves significantly among children. Findings are especially important to pediatricians and primary care physicians who recognize the deleterious consequences of food insecurity and poor access among young patients ,40,41,42


Myocardial infarction with non-obstructive coronary arteries (MINOCA) is a heterogeneous entity with relevant long-term major cardiovascular events. Several trials have demonstrated that dual antiplatelet therapy (DAPT), β-blocker, renin-angiotensin-aldosterone system (RAAS) inhibitor and statin therapy improve the prognosis in patients with obstructive myocardial infarction (ob-MI). However, evidence on the best medical therapy for secondary prevention in MINOCA patients is lacking.To investigate the effects of secondary prevention treatments at discharge on mid-term outcomes in MINOCA.Patients with acute myocardial infarction (MI) undergoing early coronary angiography between 2016 and 2018 were extracted from a clinical database. The diagnosis of MINOCA was made according to 2016 ESC MINOCA Position Paper criteria. Second-level diagnostic work-up including cardiac magnetic resonance was performed to exclude non-ischemic troponin elevation cause. The relationship between treatments and outcomes was evaluated by using Kaplan-Meier survival analysis and Cox regression models. All confirmed MINOCA were followed in our outpatient clinics. The primary end-points were all-cause mortality, re-hospitalization for MI and a composite outcome including all-cause mortality, hospitalization for MI and ischemic stroke (MACE).Out of 1,141 AMI who underwent coronary angiography, 134 were initially diagnosed as MINOCA. Patients with MINOCA were less likely to receive secondary prevention treatments than patients with obstructive coronary artery disease (CAD) MI . Based on the diagnostic work-up completed during the first month after discharge, a final sample of 88 patients had confirmed MINOCA. During an average follow-up of 19.35 ± 10.65 months, all-cause mortality occurred in 11 (12.5%) patients, recurrence of MI in 4 (4.5%), and MACE in 15 (17.0%) patients. Patients treated with RAAS inhibitors and statins had a significantly longer survival. On the contrary, no increase in survival was found in patients treated with β-blockers or DAPT. Cox multivariable analysis, including all secondary prevention drugs, showed that only RAAS inhibitors were associated with reduced all cause-mortality and MACE.This prospective study suggests that RAAS inhibitor therapy provides mid-term beneficial effects on outcomes in MINOCA patients; in contrast, dual antiplatelet, β-blocker and statin therapy had no effects on mortality and MACE. These results should be considered preliminary and warrant confirmation from larger studies. The 2018 ESC guidelines of the Fourth Universal definition of myocardial infarction (MI) differentiated myocardial infarction from myocardial injury and defined acute myocardial infarction with non-obstructive coronary arteries (MINOCA). According to the pathophysiological mechanisms, MINOCA were located between type I and type II MI: type 1 MI caused by atherosclerotic plaque disruption, and type 2 MI due to non athero-thrombosis plaque  .The diagnosis of MINOCA includes the criteria for acute MI, no evidence of angiographic coronary obstruction (coronary stenosis <50%) and of a clinically apparent alternative diagnosis for the acute presentation . In thisSimilarly to heart failure, MINOCA should be considered as a “working diagnosis” that requires further evaluation to investigate its underlying mechanisms, with important diagnostic and prognostic implications on secondary prevention therapy. Consequently, the term MINOCA designates a heterogeneous subgroup of patients with myocardial infarction, with the afore-mentioned characteristics, rather than a specific pathophysiological mechanism .Nowadays, the prevalence of MINOCA is estimated at 5–8%, with variations depending on the proportion of patients with MI who performed coronary angiography and on the high-sensitivity cardiac troponin (hs-cTn) assays used .Literature prognosis data showed that MINOCA is not a benign condition, with an overall estimated mortality around 3.5–4.5% . This unMoreover, most drugs used for secondary prevention in patients with myocardial infarction and obstructive coronary arteries are aimed at atherosclerotic disease prevention, that is a minor problem in MINOCA. Therefore, doubts have been raised about the use of standard therapy.In three observational studies, ACEI/angiotensin Receptor Blockers (ARB) therapy has demonstrated a beneficial effect on outcome in patients with MINOCA . HoweverOut of 1,523 consecutive patients with acute myocardial infarction admitted between January 2016 and December 2018 to Bologna University Hospital , 1,141 underwent coronary angiography (CAG) within the first 48 h of the index hospitalization. According to the extent of stenosis, patients were classified into three groups: a) obstructive CAD (ob-CAD), defined as a stenosis ≥50% of the lumen diameter in at least one coronary artery, b) non-obstructive CAD (non ob-CAD), defined as a stenosis 1–49% of the coronary artery diameter, and (c) normal coronary arteries (NCA), defined as no angiographic visible stenosis or atheromasia. The MINOCA diagnosis was performed according to 2016 ESC MINOCA Position Paper criteria .We excluded patients who did not perform coronary angiography or performed it after 48 h of hospital admission and patients with previous coronary revascularization , cardiovascular risk factors, family history of cardiovascular disease, and first admission diagnosis. We also collected information on major epicardial coronary arteries obstruction, based on visual assessment during acute CAG performed by an expert interventional cardiologist. Discharge medications were also recorded . Antiplatelet therapy was prescribed according to the current guideline recommendations .-lead ECG was performed and laboratory testing was carried out in all patients, including hemoglobin and creatinine. The glomerular filtration rate was calculated using CKD-EPI formula.A standard 12All patients underwent 2D-echocardiogram and the left ventricular ejection fraction (LVEF) was calculated, according to European recommendations .During hospitalization or within 1-month of discharge, patients underwent contrast cardiac magnetic resonance (CMR) and/or pulmonary and vascular computed tomography in order to exclude non-ischemic troponin elevation causes .MoreoverClinicalTrials.gov Identifier: NCT03883711). All patients were treated in accordance with the Declaration of Helsinki; all of them were informed about their participation in the registry and provided informed consent for the anonymous publication of scientific data.Our data are collected as part of an approved protocol regarding the observational study “Acute Myocardial Infarction; Prognostic and Therapeutic Evaluation” . Myocardial infarction was diagnosed according to current ESC guidelines; (1) stroke was defined as an ischemic cerebral infarction caused by embolic or thrombotic occlusion of a major intracranial artery.The main purpose of this study was to compare the effects of secondary prevention treatments  prescribed at discharge on mid-term prognosis.Patients were followed from discharge through outpatient clinical visits or telephone interviews every 6 months until the end of follow-up. During the follow-up visit, each patient underwent ECG, echocardiogram and was clinically evaluated by an experienced cardiologist, who recorded drug compliance and the clinical end-points.Patient characteristics were summarized using mean ± standard deviation for continuous variables and absolute and percentage frequencies for categorical variables. Patient subgroups were compared using χ² test or Fisher’s exact test for categorical variables and t-test or Mann-Whitney test for continuous variables, as appropriate.Patients’ survival at 36 months was obtained using Kaplan-Meier estimates and compared between subgroups using log-rank test. Univariate and multivariable Cox proportional-hazard models were used to analyze the association of pharmacological treatments with death, re-infarction, stroke and MACE (either of the three major cardiovascular events).Out of 143 patients with non-obstructive CAD or normal coronary arteries, 9 were excluded because a non-ischemic troponin elevation cause was found  during hospitalization. The diagnosis of MINOCA was made at discharge in 134 patients. Forty-one patients were further excluded because the diagnosis was not confirmed by second-level instrumental examination performed after discharge or for incomplete diagnostic work-up and 5 patients were not traced at follow-up. The final sample used to analyze the effects of secondary prevention treatments on outcomes consisted of 88 patients (We first compared the characteristics of patients with MINOCA and those with obstructive CAD (ob-CAD). Patients with MINOCA were younger, more frequently female, and had less frequently hypertension, dyslipidemia and type-2 diabetes than patients with ob-CAD .Of the 134 MINOCA patients  and only 42.1% with dual antiplatelet therapy (DAPT), compared with 94.6% and 81.8% ob-CAD patients. Similarly, the percentage of MINOCA patients treated with β-blockers, RAAS inhibitor and statins was lower compared with ob-CAD  (During an average follow up of 19.35 ± 10.6 months), all-cause death occurred in 11 (12.5%) patients, re-MI in 4 (4.5%) patients, and MACE in 15 (17%). No ischemic stroke was observed. Interestingly, all-cause death and recurrent MI were more frequent among patients with NCA than among patients with ob-CAD , although this difference was not statistically significant.Concerning medications, RAAS inhibitors and statins were the only treatments significantly associated with longer survival in a Kaplan-Meier analysis  The rate of MINOCA depends largely on the proportion of patients with MI who performed CAG and on hs-cTn assays used.In our study the prevalence of MINOCA is around 12%; a possible reason for this discrepancy is that 75% of our patients with acute MI underwent CAG. Indeed, not all patients presenting without ST-segment elevation acute coronary syndromes (ACS) undergo coronary angiography, which may lead to a selection bias . In factMINOCA long-term prognosis is not event-free, with an approximate 9.2% rate of MACE per year . InteresMany clinical studies suggested that MINOCA is an undertreated population;  however Consequently, discharging MINOCA patients is challenging for physicians due to the lack of guidelines regarding optimal treatment. Thus, in the absence of a clear alternative, clinicians tend to prescribe atherosclerotic prevention medication. This treatment choice is based on clinical trials that enrolled mostly patients with coronaries obstruction and cannot be extended to MINOCA patients, which represent a different population. Moreover, most drugs used for secondary prevention in patients with MI and obstructive CAD are aimed at counteracting atherosclerotic progression, that constitutes a minor problem in MINOCA in which atherosclerotic plaque disruption is only one of the several related underlying etiologies.Thus, the crucial issue is whether the prognosis of these patients is not event-free because they are under-treated or because the prescribed drugs are not targeted for this category of patients. Probably, both previous statements are true; in fact, our study confirmed that RAAS inhibitors were the only drug significantly associated with lower all-cause mortality and MACE; in contrast, β-blockers and anti-platelet agents had apparently no effects on outcomes.The long- term cardiovascular protection conferred by angiotensin-converting enzyme inhibitor-mediated was strongly supported by the results of the Heart Outcomes Prevention Evaluation (HOPE) trial, indicating a significant relative risk reduction in the combined endpoint of cardiovascular death, MI and stroke or cardiac arrest . ESC guiThe beneficial effects of RAAS inhibitors were already demonstrated in patients with non-obstructive coronary artery disease, however these studies had some limitations such as the definition of MINOCA and inclusion of patients with non-ischemic troponin elevation cause. Our study confirms that RAAS inhibitors are beneficial in selected patients who meet the 2016 ESC MINOCA Position Paper criteria .The RAAS inhibitors block the ACE-mediated formation of angiotensin (Ag) II (ACE-I) and the linkage of Ag II to receptor type 1 (AT1) (ARBs), in the circulation and peripheral tissues, producing blood pressure lowering, sympathetic inhibition, anti-atherosclerotic, and anti-thrombotic effects(10). In fact, RAAS inhibitors reduce macrophage accumulation, increase bradykinin bioavailability, improving non-endothelial and endothelial dependent coronary microvascular function . AdditioThe “pleiotropic” effects of RAAS inhibitors on the cardiovascular system and their various possible pathways support their benefit for MINOCA patients, a heterogeneous population with different etiology.As regards statins, survival analysis indicates that they are associated with mortality reduction. The plausible mechanism explaining this effect is the slowdown of the progress of atherosclerotic process and plaque stabilization;  this is Although findings from the literature indicate a significant risk reduction in patients with obstructive CAD treated with β-blockers and anti-platelets therapy, no benefits were proved in MINOCA patients. Probably, the many etiopathogenic mechanisms in the absence of athero-thrombotic phenomena associated with complicated plaque and coronary stents support the lack of benefit in antiplatelet prescription. The PROSPECT study demonstrated that ACS occurs from atheroma with definite histopathologic characteristics that are not necessarily dependent on the degree of angiographic stenosis . Indeed,First, although the prevalence of our population is consistent with that observed in the literature, the number of patients with non-obstructive CAD is relatively small; thus the study is not adequately powered to detect the effect of secondary prevention drugs. Thus, our results should be considered preliminary and warrant confirmation in larger prospective samples. Second, no information was available on the staging and duration of comorbid diseases , and on medication use before hospitalization, which prevents a more detailed investigation on the impact of each comorbidity on outcomes.Third, angiographic data were retrieved from clinical records and were not adjudicated by a core laboratory, having some degree of between-person variability.Fourth, no intracoronary investigation other than coronary angiography was performed. Fractional flow reserve or intravascular imaging techniques, like intravascular ultrasound or optical coherence tomography, would likely increase atherosclerotic plaque detection, not visible on CAG . NowadayOur data support that treatment with RAAS inhibitor provides mid-term beneficial effects on outcomes in patients with MINOCA; in contrast, we did not find an effect of dual antiplatelet, β-blocker and statin therapy. Our results should be interpreted with caution because the study is under-powered to detect drug effects in a population with a small event rate.Multicenter prospective adequately powered studies targeting this specific population and the potential benefit of guideline-recommended therapies are warranted.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Acute Myocardial Infarction; Prognostic and Therapeutic Evaluation' . The patients/participants provided their written informed consent to participate in this study.PP, LB, and CP contributed conception and design of the study. GS, IM, ED'A, ST, AS, AR, LB, FA, and FD organized the database. LB and PR performed the statistical analysis. PP wrote the first draft of the manuscript. PP, CP, and NT wrote sections of the manuscript. AM and NG revised the article. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.001) and increasing body weight  in weaned beef calves. Slaughter yield grades were higher for calves that had been fed Se-enriched alfalfa hay during the preconditioning period (PTreatment = 0.008). No significant differences were observed in fecal parasite load, which remained low. The nasal microbiome and microbiota diversity within calves and across calves expanded from weaning (week 0) to the feedlot period (week 12), which was promoted by feeding Se-biofortified alfalfa hay. Especially concerning was the expansion of nasal Mycoplasmataceae in the feedlot, which reached over 50% of the total microbiota in some calves. In conclusion, we identified dietary Se-biofortified alfalfa hay as a potential promoter of nasal microbiome genome and microbiota diversity, which may explain in part high-Se benefits for prevention of bovine respiratory disease complex in beef calves.We previously reported that feeding Se-biofortified alfalfa hay to weaned beef calves in a preconditioning program decreases morbidity and mortality during the feedlot period. To understand the mode of action by which supranutritional Se supplementation supports calf health, we examined the effect of agronomic Se-biofortification on nasal microbiome and fecal parasites. Recently weaned Angus-cross beef calves (n = 30) were randomly assigned to two groups and fed an alfalfa hay-based diet for 9 weeks in a preconditioning program. Alfalfa hay was harvested from fields fertilized with sodium selenate at a rate of 0 or 90 g Se/ha. Calculated Se intake from dietary sources was 1.09 and 27.45 mg Se/calf per day for calves consuming alfalfa hay with Se concentrations of 0.06 and 3.47 mg Se/kg dry matter, respectively. Feeding Se-biofortified alfalfa hay for 9 weeks was effective at increasing whole-blood Se concentrations (556 ± 11 vs 140 ± 11 ng/mL;  Selenium (Se) is an essential trace mineral important for immune function and overall health of cattle. Optimal immune function is critical for calves undergoing the stresses of weaning, relocation to feedlots, and commingling with animals of different origins. The National Agriculture Statistics Service reports that 630,000 calves were born in Oregon in 2017. The majority of Oregon grown calves enter a feedlot. Even with good vaccination programs, calves often experience negative health issues in the feedlot, including mortality. Bovine respiratory disease (BRD) complex is a major cause of morbidity , as both groups had all but 4 animals with choice grade (PTreatment = 0.53). However, yield grade  were improved in animals that were fed Se-enriched alfalfa hay during the preconditioning period (PTreatment = 0.008), as more calves in the group fed Se-enriched alfalfa hay had a 1 or 2 (86%) compared with calves in the control group (36%). Slaughter data were not available for 4 calves in the control group and 1 calf in the high-Se treatment group because ear tags were lost at slaughter.Feeding Se-fertilized alfalfa hay was effective at increasing BW in weaned beef calves  and trichostrongyle-type egg counts (PTreatment = 0.81). No significant time, nor treatment × time interactions were observed. At the beginning of the feedlot period, calves were dewormed. As a result, fecal counts were zero or ≤ 25 eggs/g feces in week 12.We evaluated fecal parasite load at weeks 0, 5, and 9 (end) of a preconditioning period as well as in the feedlot (week 12). Overall, fecal parasite counts were low; especially counts for We collected nasal microbiome genome at weeks 0 and 9 (end of the Se-supplementation preconditioning period) as well as in the feedlot (week 12). Sequencing of the bacterial 16S rRNA gene in the samples yielded 10,308,260 quality sequences . Rarefaction analysis was performed at a depth of 31,744 sequences . ExpansiThe microbial diversity increased within High-Se calves during the Se-treatment period, as indicated by Chao1 (+171%) and observed ASVs (+151%). Control calves caught up with High-Se calves in microbial diversity in the feedlot ; Table 2unweighted = +0.30, P = 0.001) of the unweighted UniFrac distances. Moreover, the nasal microbiome differed across time, as shown by distinct clustering for weeks 0, 9, and 12 within Control and High Se calves . Treatment groups did not differ in nasal microbiome at week 0  and week 12 .Nasal microbiome differed between High-Se and Control calves at the end of the Se-treatment preconditioning period (week 9), as shown by the distinct clustering of High-Se and Control calves within the principal coordinates  and ANOSe calves . There wThe microbial diversity of less abundant bacteria taxa increased between High-Se calves during the Se-treatment period, as indicated by the unweighted UniFrac distances. Control calves caught up with High-Se calves in microbial diversity in the feedlot ; Table 3The nasal microbiome changed during the preconditioning period in all calves, with greater changes in less abundant bacteria taxa observed in High-Se calves compared with Control calves . Both thTenericutes, which contains genera of the Mycoplasmataceae family, increased dramatically after calves entered the feedlot. Some calves had over 50% Tenericutes. The phylum most negatively impacted by the increase in Tenericutes was Bacteroidetes.We detected 23 bacterial phyla in nasal swabs across time and treatment. The profiles of the 19 most abundant bacterial phyla for individual calves across time are shown in Acidobacteria, Chlamydiae, OP11, TM7, and WPS-2. Control calves caught up with High-Se calves in phyla diversity after they entered the feedlot; however, Chlamydiae, TM7, and WPS-2 remained present only in High-Se calves.The number of nasal microbiota phyla within High-Se calves increased during the Se-treatment period . Five baFirmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Tenericutes, Euryarchaeota, and Verrucomicrobia  for higher proportions of Verrucomicrobia after feeding Se-biofortified hay in the preconditioning period.For the statistical analysis, we partitioned bacterial phyla into those that were detected in at least 14 of 15 calves per group across treatment and time  and thosmicrobia . The proAcidobacteria, Chlamydiae, OP11, TM7, and WPS-2, were only present in High-Se calves in week 9. In addition, Gemmatimonadetes and Other Bacteria were present in more High-Se than Control calves.The major effects of feeding Se-biofortified hay was on the minor bacterial phyla . FeedingBacteroidetes genera; 12 Firmicutes genera, 11 Proteobacteria genera, 9 Actinobacteria, 4 Verrucomicrobia genera, 2 Thermi genera, and one each for Chlamydiae, Fibrobacteres, Tenericutes, TM7 and WPS-2) were present only in High-Se calves in week 9. In contrast, 10 bacterial genera were present only in Control calves in week 9. Control calves caught up with High-Se calves in genera diversity after they entered the feedlot  remained present only in High-Se calves.We further queried on the genera level and detected a total of 322 bacterial genera in nasal swabs. The number of nasal microbiota genera within High-Se calves increased during the Se-treatment period . A total feedlot ; howeverBacteroidetes genera; 7 Firmicutes genera, 3 Proteobacteria genera, 2 Actinomycetales genera, and one Tenericutes genus. The Tenericutes genus Ureaplasma of the Mycoplasmataceae family was especially intriguing . In the feedlot, all calves had Ureaplasma and the proportions had dramatically increased: Ureaplasma was the sixth most abundant bacterial genus in Control calves and the third most abundant genus in High-Se calves with no treatment differences observed.Eighteen bacterial genera were present only in High-Se calves in week 9, and in both treatment groups in the feedlot: 5 triguing . At weanMycoplasmataceae family  increased dramatically after calves entered the feedlot (Tenericutes (results not shown). In the feedlot, Mycoplasma was the most abundant bacterial genus in both treatment groups with some calves having over 50% of their nasal microbiota being Mycoplasma  and R = -0.02 (P = 0.71) at weeks 0 and 9, respectively. There was a significant effect of time on nasal microbial metabolism, as indicated by PCoA plots of Bray Curtis Dissimilarities .The principal coordinates of Bray Curtis Dissimilarities for predicted metabolic pathways did not differ between Control and High-Se calves at weeks 0, 9, and 12 . The ANOlarities  and pairFertilizing alfalfa with sodium selenite at 90 g Se/ha increased the Se content of alfalfa hay over 50 fold. Feeding this Se-biofortified alfalfa hay to calves during the preconditioning period increased WB-Se concentrations almost 300%, improved growth and carcass weights, increased overall scores for slaughter yield grade, and enriched the nasal microbial diversity. There was no effect on parasite load because fecal egg counts remained low throughout.We have previously shown a linear relationship between Se fertilizer application rate and forage Se concentration , 33, 34.Blood Se concentrations were increasing throughout the 9-week preconditioning period, and remained high relative to control calves after three weeks in the feedlot. These results are consistent with our previous studies. Consumption of Se-biofortified alfalfa hay in this study resulted in WB-Se concentrations after 9 weeks of 556 ± 11 ng/mL, which was consistent with our previous range of 278 ± 7 ng/mL (7-weeks preconditioning period)  when fedP = 0.07), similar to what we previously reported [Consumption of Se-fertilized alfalfa hay resulted in increased BW in weaned beef calves and tended to increase hot carcass weight  to an alfalfa field as $158/ha; thus a calculated cost of $8/head for the Se enriched alfalfa hay consumed during the preconditioning period. Market prices for slaughter calves are currently approximately $3.54/kg hot carcass weight. Applying these market prices to the gain measured in our former study, feeding calves high Se enriched alfalfa hay resulted in an increase of $166/head over that of the control group of calves. In the current study, at the end of the feedlot period, calves in the high Se group had a 9 kg higher slaughter weight compared with control calves resulting in an increase of $32/head over that of control calves. Thus, it is economically justified to recommend feeding Se-biofortified alfalfa hay during the preconditioning period to beef calves.In this study, third cutting alfalfa hay was enriched with Se by mixing inorganic sodium-selenate with water and spraying it onto the soil and foliage of an alfalfa field at an application rate of 90 g Se/ha. In our previous study , 9, usinH. contortus gastrointestinal parasites [Although fecal parasite counts were assessed at baseline and weeks 5 and 9 of the preconditioning period, and again 3 weeks after entering the feedlot, no significant treatment effects were observed. This was likely because fecal parasite counts were very low throughout the preconditioning period, and essentially zero 3 weeks after entering the feedlot because all calves were dewormed at entry to the feedlot. We had previously shown in sheep that supranutritional Se-yeast supplementation may enhance resistance to naturally occurring arasites . Youngerarasites . In thisThe microbiome refers to the collection of genomes from all the microorganisms in the nasopharyngeal environment . In the Beta diversity measured using the phylogeny-based unweighted and weighted UniFrac distances, showed that calves receiving the High-Se-treatment clustered separately from Control calves, although clustering was not significant for weighted UniFrac distances. Beta diversity measures differences between nasopharyngeal samples/calves . UnweighSeveral studies have examined how some management practices decrease diversity within the nasopharyngeal bacterial communities and reported that weaning, transportation methods, on-arrival processing at the feedlot, time in the feedlot  and antiAnother important factor that decreases nasal microbiome diversity is respiratory health status. Alpha-diversity indices revealed a lower bacterial diversity in the nasopharynx of steers with bronchopneumonia compared to healthy pen-mates , 47. BacThe question arises how increasing the diversity of the nasopharyngeal bacterial community improves health status. Microbial biodiversity correlates to the efficiency of nutrient utilization by a bacterial community; a greater microbial diversity means greater diversity in microbial metabolism pathways, which allows for more efficient nutrient utilization. This, in turn, decreases the chances of bacterial respiratory pathogens adhering to and colonizing in the nasopharynx (reviewed in ). This bThere is limited information how dietary Se may increase the diversity of the nasopharyngeal bacterial community. We have reported over the last decade that Se-supplementation to Se-replete livestock can prevent infectious diseases , 48, 49.Firmicutes (35%), Proteobacteria (22%), Bacteroidetes (19%), and Actinobacteria (13%), and Tenericutes (2%). These were similar for Control calves and calves slated for High-Se treatment. We did not have baseline microbiota data in our former study [Proteobacteria (44%). The predominant bacterial phyla in the current study were similar to those reported by others for calves at entry to the feedlot [Tenericutes ranging from 47.4% [Tenericutes, as Tenericutes was the only phylum with relative abundance >1% altered by Se-biofortification in our current study and in our previous study [The microbiota refers to specific microorganisms found within the nasopharynx . At basermicutes %, Proteo feedlot , althougom 47.4%  to 22.5%om 47.4%  in differmicutes %, ProteoMollicutes of the phylum Tenericutes was the only bacterial class significantly altered by Se-biofortification. Mollicutes are unique in that they lack a cell wall and include the smallest bacterial species with the shortest genome sequences; as a result, Mollicutes species adhere to and sometimes enter nasal epithelial cells and rely heavily on nutrients from the host . Mollicllenging .Mollicutes genera, time and treatment affected primarily the 3 genera of the family Mycoplasmataceae: Ureaplasma, Mycoplasma, and Other Mycoplasmataceae, all of which increased dramatically after calves entered the feedlot. In the feedlot, the predominant genera in the present study were unclassified (24%), Mycoplasma (16%), Moraxella 6%, and Ureaplasma (6%). We did not see high numbers of Acinobacter as reported by others [Mycoplasmataceae has been reported previously after calves entered the feedlot [Ureaplasma after calves entered the feedlot and the effect of Se-biofortification on the presence of Ureaplasma during the preconditioning period.Of the 7 known y others . A simil feedlot , which c feedlot . Unique Ureaplasma and Mycoplasma are very similar in structure and function, and both are linked to a variety of inflammatory diseases in cattle; their differences are in their colony formation structure, the fact that only Ureaplasma can secrete urease, and in the number of species they contain [Mycoplasma species, there are only 11 identified bovine Ureaplasma species, the best known species being Ureaplasma parvum, which is prevalent in many herds and linked to a high incidence of abortions  [Ureaplasma parvum has been identified in the nose and the lung of cattle and its increased presence has been linked with Mycoplasma to BRD [Mycoplasma species, Ureaplasma species are usually benign; however, their ability to secrete toxins and induce a pro-inflammatory response in the host can cause extensive epithelial tissue damage [Ureaplasma or Mycoplasma species could decrease feed efficiency because of their nutrient dependency on the host. The question arises why Ureaplasma was present in 2/3 of High-Se calves, but none of the Control calves at the end of the preconditioning period. One hypothesis is that Ureaplasma species are able to evade detection by the immune system of the host by hiding in epithelial cells [ contain , 58. In asmosis) , 59, 60.asmosis) . Ureaplaa to BRD , 62, 63.e damage , 65. Moral cells , which gMycoplasma was the most abundant bacterial genus in both Control and High-Se treatment calves in the feedlot . In contrast to Ureaplasma, some Control and High-Se treatment calves had high proportions of Mycoplasma at baseline and 9 weeks. Others have reported a large increase in Mycoplasma after arrival at the feedlot [Mycoplasma contains over 120 species and their differentiation is challenging [Mycoplasma species differ in pathogenicity, polymerase chain reaction (PCR) techniques are the standard method for Mycoplasma diagnosis [Mycoplasma using a ASVs approach with DADA2, because assignment to specific variants are based on small differences in ASVs [Mycoplasma species [Mycoplasma by PCR techniques so we cannot further characterize the increase in Mycoplasma species.In the present study,  feedlot , which t feedlot . The genllenging , 66, 67.iagnosis . In the  in ASVs  and only species . We alsoMycoplasma [Mycoplasma species are opportunistic pathogens present in symptomatic and asymptomatic cattle with Mycoplasma bovis being the most pathogenic [Mycoplasma species and suggested the nasopharynx as the best target for management strategies against BRD [Mycoplasma adherence.Bovine respiratory disease is one of the most common causes of health and economic losses in the feedlot and closely linked to an animal’s commensal bacteria populations in the respiratory tract including coplasma , 69. Sevthogenic , 55, 69.inst BRD , as it cMycoplasmataceae.One interesting feature in the present study was the effect of feeding Se-biofortified hay on the number of minor phyla and genera present at the end of the Se-supplementation period, which persisted in the feedlot. After feeding Se-biofortified hay for 9 weeks, the number of phyla within High-Se calves increased compared with control calves from 13 to 19 phyla, whereas Control calves had no change in number of phyla compared with weaning (13 phyla). The increase in phyla numbers in High-Se calves was the result of increased numbers of the minor bacterial phyla . Five bacterial phyla were present only in High-Se calves at 9 weeks. Although Control calves caught up with High-Se calves in phyla diversity after they entered the feedlot (increased from 13 to 16 phyla), three of the minor bacterial phyla were still only present in the High-Se calves. Similarly, the number of microbiota genera within High-Se calves increased during the High-Se treatment period. A total of 56 bacterial genera (>90% in the major phyla) were present only after High-Se treatment, whereas only 10 genera were present solely in Control calves at 9 weeks. Again, Control calves caught up with High-Se calves in genera diversity after they entered the feedlot. None-the-less, 31 bacterial genera (>85% in the major phyla) remained present only in High-Se calves in the feedlot. Coincidently, WB-Se concentrations remained higher in High-Se treated calves after 3 weeks in the feedlot. We propose that the increased diversity of nasal microbiota present after 9 weeks of consuming Se-fortified alfalfa, and still present 3 weeks after entering the feedlot, may provide a bacterial community component for potentially regulating bacterial overgrowth and respiratory disease caused by The effect of feeding weaned beef calves Se-biofortified alfalfa hay was smaller on nasal microbial metabolism compared with its effect on the nasal microbiome and microbiota. There was a small increase in the number of predicted metabolic pathways in response to feeding Se-biofortified hay, but Control calves caught up with High-Se calves in the number of predicted metabolic pathways in the feedlot. Again, the primary effect of feeding Se-biofortified alfalfa hay was in the less abundant predicted metabolic pathways.Bray Curtis dissimilarities measures dissimilarities between samples with no difference being zero and maximum differences being one; this measure only considers differences in abundances and does not account for phylogenetic tree differences . BecauseThese results suggest that the nasopharyngeal microbiota in weaned beef calves can be modified by feeding Se-biofortified alfalfa hay in a preconditioning period prior to entering the feedlot. The nasopharyngeal microbiota is important for overall respiratory health, especially when stressors such as weaning, transportation, and feed adaptation impair the normal respiratory defenses (reviewed in ). BecausIn summary, Se fertilization of alfalfa fields in a region with Se-deficient soils increases Se content of alfalfa hay. We have now shown in two independent trials that feeding recently weaned beef calves Se-biofortified alfalfa hay during the preconditioning period is an effective management strategy to build Se-body reserves, optimize growth prior to entering the feedlot, and improve feedlot performance as evidenced by higher slaughter yield grades. The novel finding of the present study is that feeding recently weaned beef calves Se-biofortified alfalfa hay during the preconditioning period promotes nasal microbiome and microbiota diversity, which in turn may provide health benefits by ameliorating a risk factor for BRD.S1 TableMean and median relative percentages of the most abundant bacterial groups are shown, annotated to the level of phylum, family, and genus, based on sequencing of the 16S rRNA.(XLSX)Click here for additional data file.S2 TableMean and median relative percentages of the most abundant functional metabolism pathways based on sequencing of the 16S rRNA are shown. Bacterial taxa in bold significantly differ between treatment groups.(XLSX)Click here for additional data file.


Meiotic recombination commences with hundreds of programmed DNA breaks, however the degree to which they are accurately repaired remains poorly understood. We report that meiotic break repair is 8-fold more mutagenic for single-base substitutions than was previously understood, leading to de novo mutation in 1 in 4 sperm and 1 in 12 eggs. Its impact on indels and structural variants is even higher, with 100-1300-fold increases in rates per break. We uncover novel mutational signatures and footprints relative to break sites, which implicate unexpected biochemical processes and error-prone DNA repair mechanisms including translesion synthesis and end-joining in meiotic break repair. We provide evidence that these mechanisms drive mutagenesis in human germlines and lead to disruption of hundreds of genes genome-wide. Meiotic recombination is essential for creation of gametes in most sexually reproducing species . It shufRecombination and mutation are traditionally thought of as distinct mechanisms generating genetic variation. Nevertheless, work in yeast has shown that meiotic cells accumulate more mutations than mitotic cells , 14 in aIn humans, indirect population-based approaches have shown an excess of rare C>G, A>G and C>T single nucleotide polymorphisms (SNPs) in recombination hotspots , with C>Despite these developments, the provenance of mutations associated with meiotic breaks remains poorly understood across all size scales, especially in higher eukaryotes. The burden of single-base substitutions due to the vast majority (~90%) of DSBs, i.e., those not repaired with a crossover, is unknown. Indels (defined as insertions or deletions less than 50bp long) in unique DNA remain unexplored. The nature, provenance, and impact of SVs due to this process also remain uncharacterised in general.To address these questions, we have harnessed a range of population-scale resources to construct detailed base-pair resolution maps of mutation relative to human recombination hotspots. These data include de novo and extremely rare genetic variation, comprising 341 million SNPs, 64 million short indels, and 0.5 million SVs. These high-resolution maps enable us to characterize sequence properties of mutations and compare their footprints with the localization of distinct molecular processes taking place within hotspots . They reUnlike crossovers, other repair outcomes such as non-crossovers are either difficult or impossible to detect in gametes or pedigrees , 30, 31.-13, For each hotspot, we counted de novo single-base substitutions identified in 2,976 Icelandic trios -13, ). HoweveThe sex-specific DNM rates due to the recombination machinery  we inferred are 0.234  DNMs per paternal and 0.080  DNMs per maternal meiosis. These are 8-fold higher than the mutation burden per meiosis due to crossovers alone in both males and females  for males and 0.011  for females in these hotspots). They comprise ~0.5% of the total burden of DNMs genome-wide and imply that, on average, ~1 in 4 sperm and ~1 in 12 eggs has a DNM specifically due to the meiotic recombination machinery.The proportion of DNMs due to crossover  are comparable to the proportion of DSBs that are resolved as crossover. These data support the parsimonious view that single-base substitutions in hotspots are driven by DSBs, and are not strongly influenced by the particular repair outcome. Since the number of crossovers per meiosis is well understood , we can -4 in males and 2x10-4 in females on average  and their reverse complements are enriched with distinctive footprints , S2A-E. Further analysis reveals distinct footprints within these flanks. Several mutation types  have “off-centre” peaks 700-800 bp from the centre , S2A,B,EIn summary, our analyses have revealed unexpected complexity in mutagenesis, implicating three distinct factors, namely SPO11-mediated break machinery, ssDNA hypermutation, and DNA re-synthesis outside the D-loop, underlying the overall increase in single-base substitutions in hotspots. We investigate the mechanisms underlying these factors below.To understand the role of meiotic breaks in generating length polymorphisms, we extended the approach above to indels and SVs. For indels, we used whole-genome data from gnomAD, comprising 64 million indels after filtering for variant quality. For SVs, we included the following datasets: (i) gnomAD-SV  (ii) deCFirst, we consider indels, which are insertions and deletions less than 50 bp long. Insertions are ~399-fold  and deletions are 115-fold  higher per break than would be expected in these regions in the absence of the break, i.e., from genomic insults collectively in the germline and the zygote . The fooSVs are variants impacting 50+ bp of DNA and comprise mainly deletions and insertions , S3A-B. Strikingly, the bias towards insertions is reversed on the X chromosome . Whilst We characterise indel polymorphisms further below, stratifying them by the DNA context of their breakpoints (unique DNA or any of the known repetitive DNA families) to account for underlying differences in mutation propensity.-20, p=9x10-41, for insertions and deletions, respectively, -8, p=2x10-11, respectively) over and above the expectation from local mutation rates . These have a different composition relative to those in unique DNA, both inside and outside hotspots. Insertion and deletion rates are similar outside hotspots, consistent with evolutionary stability of TR sequences on average . HoweverWe conclude that bias towards insertions and higher mutation rates near telomeres are a consistent feature of mutations arising from meiotic breaks in the autosomes.Exons are over-represented near hotspots for the PRDM9 alleles mapped in humans , S7A-B, The number of gnomAD predicted loss of function (pLOF) mutations in hotspots is 75%  higher for indels and 67%  higher for single-base substitutions than expected from the proportion of DNA sequence in hotspots . To asseFor a mutation, we can estimate the probability that it arose from meiotic break repair based on its distance from a hotspot centre and the over-representation of that variant type in hotspots -D. Here,TMLHE (X-linked Autism), CDKL5 , FANCD2 (Fanconi anaemia), FLT4 , FTCD (Glutamate formiminotransferase deficiency), and DOCK8 (DOCK8 immunodeficiency syndrome)  . These gyndrome) . Amongstbination , 53, 54.-4 . The im-4 ).We identified 81 genes that have hotspot-overlapping exons with statistically significant increases in pathogenic mutations after Bonferroni-correction for multiple testing . These gCollectively, these data establish that meiotic breaks are a previously under-recognised cause of human disease.Many exogenous and endogenous factors that impact DNA have characteristic ‘mutational signatures’. These signatures have proved powerful in understanding the molecular processes driving many cancers . For exaWe therefore assessed the trinucleotide mutational signature of the central and off-centre peaks of single-base substitutions identified above  DNA polymerases, e.g., Rev1, Polη, PolϚ, are strong candidates for effecting this repair . TLS canIn addition to the off-centre peaks, C>G and A>G mutations exhibit long-range (>10kb) strand-asymmetric mutations . This si-16). The canonical model for generating insertions is “polymerase slippage” .For autosomal indels that have both breakpoints in unique DNA, we examined the inserted and deleted sequences for microhomology. The vast majority of indels in hotspots showed significant microhomology at the breakpoint, which is not expected by chance  , weA model that is consistent with these data allows for deletions to arise through TMEJ between resected sites flanking DSBs and insertions via TMEJ between sites where some DNA re-synthesis has already taken place . Polθ, wThe canonical model for SVs associated with the recombination machinery is non-allelic homologous recombination (NAHR), which posits that SVs are generated through ectopic pairing between large DNA segments with high sequence similarity . Althoug-22 for deletions, p=10-32 for insertions, Detailed sequence and breakpoint analysis of several complex SVs in autosomal hotspots revealed features observed in short indels above, specifically template-switching and signatures of TMEJ . This su-7) (-4) and similar to expectations from NHEJ. Microhomology in short deletions in X chromosome hotspots is also lower than autosomal hotspots and similar to deletions outside hotspots  . While t-7) . Consist-7) . The medhotspots .-15) and are over-represented on the X chromosome . Sub-telomeric hotspots have an intense burden of programmed breaks in male meiosis and exhibit distinct kinetics of repair . These 354 genes are involved in a range of developmental disorders and cancers and, to the best of our knowledge, only 3 of them have previously been linked with mutations generated as a consequence of meiotic break repair.Sexual reproduction requires homologous chromosomes to pair up in order to re-segregate into haploid gametes. Adaptation of the tools of DNA repair to achieve this challenging task lies at the heart of meiosis and multiple aspects of this process are conserved from yeast to human , 85. TheThe over-representation of programmed breaks near exons in humans and at transcription-start sites in many species is therefore surprising and suggests that there is an evolutionary benefit to positioning breaks near genes. It is possible that the chromatin environment in these regions promotes repair, thereby increasing the likelihood of successful chromosome pairing . If thisThe evolutionary cost of incorrectly repaired breaks is predicted to be particularly acute for the sex chromosomes, where lower effective population sizes and reduced crossing over imply that efficiency of natural selection will be lower. Concentration of breaks towards telomeres in males and lower gene density on the sex chromosomes may, in part, reflect an evolutionary response to the mutation burden of DSBs. Extensive DNA resection accompanying breaks, while incurring a clear mutational cost, likely contributes to correct chromosome pairing and safeguards against more catastrophic genome instability. The mechanisms underlying meiotic recombination thus perform a delicate evolutionary balancing act between the benefit of sexual reproduction and the burden of genetic disease.We used ChIP-seq data for single-stranded DNA bound to DMC1, which was measured in testes of a human male homozygous for the A-allele and one heterozygous for the C and L4 alleles , 39. HotWe used published data of de novo mutations (DNMs) and crossovers identified in 2,976 Icelandic trios .-3 as extremely rare SNPs are recent enough for the impact of selection and meiotic drive to be small and have proven to be a powerful source for research in human mutation  with the number of times a C base was observed at that position in the reference genome. The same approach was extended to mutations within each trinucleotide context. Some sites may have experienced more than one independent mutation in the genealogical history of the individuals in the gnomAD sample set . Since eWe used SV calls from the Icelandic population (deCODE-SV)  and the B be the event of a break in it in a meiosis and M be the event that it incurs a mutation at a specific position in that meiosis. We inferred the per-DSB fold excess in mutations as follows. Consider a hotspot and let P(M|B′) be bgr. Since P(B) ≪ 1, Let the background rate n hotspots and m meiosis in the sample, i.e., We wish to calculate the mutation rate per break averaged across P(ijB)) is not known. However, we have estimated the average probability of a break in an A-allele hotspot from a present-day Icelandic sample (0.8907%), as described in  and include microsatellites, minisatellites and other tandem repeats with a period size between 1 bp and 2 kb. In cases where the RepeatMasker and simpleRepeat track both annotated the same sequence, we used the Tandem Repeat annotation.Tandem Repeat (TR) annotations were downloaded using the simpleRepeat track from the UCSC Genome Browser, which is based on Tandem Repeat Finder (TRF) . Tandem Only indels with both breakpoints in the same context were used in context-specific analyses. Indels for which neither breakpoint overlapped a RepeatMaster or Tandem Repeat sequence were deemed to be in “unique DNA”.For specific analyses measuring indel homology and microhomology in unique DNA , we perfn, the true insertion point can be any one of n+1 possible sites: immediately upstream or downstream of the duplicated sequence or anywhere in between. The first of these positions is reported in gnomAD, as mentioned above. We also use this representation, without loss of generality. Imperfect duplications can also have multiple representations. In such cases, for consistency, we chose the representation that maximized homology with the right-flanking sequence, which was almost always the one reported in gnomAD (98%). We excluded insertions where the findings  were different for these different representations to avoid biasing the results. This was also frequently the case in complex SVs where the inserted sequence showed homology with more than one locus.For perfect side-by-side duplications, it is not possible to determine the true breakpoint. For a duplication of size We modelled the provenance of insertions using Markov Chain Monte Carlo (MCMC). Detailed methodology for inferring the provenance of insertions is provided in .Gene and exon annotations were downloaded from Gencode (v42). We restricted to protein coding genes and used Ensembl canonical transcripts to define gene and exon boundaries.f in the number of SV and indel breakpoints at a given distance d from the hotspot centre. We infer that the probability that a variant, which has its hotspot-proximal breakpoint at this distance, arose due to recombination is (f-1)/f. We calculated this value for each base pair distance d from hotspot centres on average (f decreases monotonically with distance from the hotspot centre (this assumption is well supported by the data, see d. The sum of squares of the deviation between the data and regression function was minimised, which is equivalent to maximum likelihood estimation under an assumption that errors are normally distributed. The resultant quadratic programme was solved in R using the quadprog package.Consider the fold-enrichment  average -D. We asWe downloaded the ClinVar data on January 19, 2023. We restricted to variants that had the ClinSigSimple field set to 1, flagging pathogenic and likely pathogenic variants.Data S1Supplementary materialTable S2Table S3


With the incredibly high incidence of Type 2 Diabetes in the current population of emergency department patients, it is critical for clinicians to understand the possible complications of the treatment of this disease. Medication like canagliflozin are more common to encounter on patient’s home medication lists and clinicians should be aware of how these medications, alone or combined with dietary modifications, can result in significant pathology and even mortality if not appropriately treated.We report a case of a patient with type II diabetes mellitus who presented with euglycemic diabetic ketoacidosis in the setting of concurrent use of canagliflozin, a sodium-glucose transporter-2 (SGLT-2) inhibitor, and strict adherence to a low-carbohydrate ketogenic diet for weight control.Euglycemic ketoacidosis has previously been observed in both diabetic and non-diabetic patients following strict ketogenic diets, as well as in diabetic patients being treated with SGLT-2 inhibitors.As more patients choose ketogenic diets for weight control and diabetes management, clinicians should be aware of this potentially life-threatening complication in patients concurrently taking SGLT-2 inhibitors. Very low-carbohydrate, or ketogenic, diets originated in the realm of fad weight-loss diets, but the practice has moved into the armamentarium of medicine in controlling weight, epilepsy, and diabetes. Sodium-glucose transporter-2 (SGLT-2) inhibitors have also become more heavily used in the control of diabetes, leading to an increased incidence of concomitant use of ketogenic diets and SGLT-2 inhibitors to control weight in diabetic patients.A 31-year-old female with a history of type II diabetes mellitus (T2DM) presented to a primary care office with dizziness and shortness of breath worsening over one week. She also noted slurring of her speech, nausea, pain radiating down the posterior aspect of both legs, and constipation. She had no history of previous episodes of diabetic ketoacidosis (DKA) or hyperosmolar hyperglycemic state (HHS). The patient had been attempting to control her T2DM by following a very low-carbohydrate, ketogenic diet for about two weeks, during which she had restricted her carbohydrate intake to 10–15 grams per day. Fluid intake included 3–4 liters of water per day and pickle juice. Over this time, she had a 12.2 kilogram weight loss. She had previously used long-acting insulin (insulin detemir) to control her glucose but had not needed to use insulin to maintain normoglycemia in weeks and was not using insulin at presentation. She stopped using insulin at the time of starting the ketogenic diet.At the time of presentation, her T2DM management regime consisted solely of canagliflozin and dietary modifications. The patient initially presented to her primary care physician, where she was found to have a blood glucose of 133 milligrams per deciliter (mg/dL) on fingerstick and positive urine ketones. She was sent to the emergency department (ED) for further evaluation. Initial vital signs in the ED were blood pressure 126/78 millimeters of mercury, pulse 137 beats per minute, temperature 97.7º Fahrenheit (36.5º Celsius), respirations 24 breaths per minute, and oxygen saturation 100% on room air. On physical exam, the patient appeared acutely ill. Neurological exam on presentation was notable for Glasgow Coma Scale of 15, drunken affect, gait instability, and mildly slurred speech without aphasia.Initial electrocardiogram showed sinus tachycardia with normal intervals. Chest radiograph was normal. Laboratory evaluation showed a pH on a venous blood gas of 7.056 with a bicarbonate of 8.0 milliequivalents per liter (mEq/L), blood glucose of 139 mg/dL, blood ketones of 80 mg/dL, lactate of 1.4 millimole per liter (mmol/L), and an anion gap of 29. The remainder of lab results are displayed in the The patient was transferred for admission to ICU level of care for severe metabolic acidosis, ketosis, and tachycardia. The patient had an uneventful course over the next few days with normalization of her ketones and acidosis while being treated with an insulin infusion with concomitant glucose-containing fluids to maintain euglycemia. She was discharged at baseline health and was lost to follow-up.This case demonstrates the occurrence of euglycemic ketoacidosis in a patient with T2DM concurrently following a low-carbohydrate, ketogenic diet and using an SGLT-2 inhibitor. This is an uncommon cause of altered mental status, and it is important for emergency physicians to be aware of this potential complication of diabetes management.What do we already know about this clinical entity?Euglycemic diabetic ketoacidosis (EDKA) and its relation to sodium-glucose transporter-2 (SLGT-2) inhibitors has been previously acknowledged and cases reported.What makes this presentation of disease reportable?Our case represents a novel combination of extreme EDKA in a patient on an SLGT-2 inhibitor in combination with a very-low carbohydrate (or ketogenic) diet.What is the major learning point?Ketogenic diets and SGLT-2 inhibitor use, both singly and in combination, can lead to severe, life-threatening EDKA.How might this improve emergency medicine practice?Clinicians should consider EDKA in acidotic patients for whom new dietary trends can lead to significant medication interactions and morbidity.Canagliflozin is an oral sodium-glucose cotransporter-2 (SGLT-2) inhibitor approved by the United States Food and Drug Administration (FDA) for treatment of T2DM and has been shown to improve glycemic control, weight loss, and hemoglobin A1c levels. SGLT-2 inhibitors decrease serum glucose concentrations by preventing glucose reabsorption in the proximal renal tubule of the kidney, thereby promoting glucosuria. SGLT-2 inhibitors also stimulate glucagon secretion from the alpha cells of the pancreas, minimizing the potential for hypoglycemic events. Although SGLT-2 inhibitors have shown efficacy in diabetes management, serious adverse events have been associated with their use. In 2017 the FDA initiated a boxed warning regarding the increased risk of leg and foot amputations in patients taking canagliflozin. Large surveillance studies have also noted increased risk of diabetic ketoacidosis with SGLT-2 inhibitors.4Euglycemic diabetic ketoacidosis (EDKA) is a previously rare clinical condition now showing increasing prevalence with the use of SGLT-2 inhibitors. The pathogenesis of EDKA involves an increased glucagon to insulin ratio, in which ketogenesis is stimulated without hyperglycemia. Increased glucagon levels contribute to gluconeogenesis, glycogenolysis and fatty acid metabolism to mobilize energy substrates.Low-carbohydrate, ketogenic diets significantly restrict the dietary supply of carbohydrates, promoting a ketogenic state of fatty acid metabolism. Although ketogenic diets induce a state of ketosis, they are not commonly associated with diabetic ketoacidosis.Since both low-carbohydrate, ketogenic diets and SGLT-2 inhibitors increase glucagon secretion while limiting serum glucose levels, a synergistic effect increasing the risk for EDKA is plausible. Cases of severe ketoacidosis associated with a low-carbohydrate diet and use of ipragliflozin and dapagliflozin have previously been reported.The patient’s initial altered mental status and speech changes may be attributable to her relative depletion of intracellular and intravascular glucose stores. Similar changes in mental status in patients with alcoholic ketoacidosis (AKA) have been previously attributed to intracellular hypoglycemia. As discussed above, this patient also experienced relative hypoglycemia due to her low relative insulin levels, creating a similar state as that of AKA.Euglycemic diabetic ketoacidosis is a recognized complication of SGLT-2 inhibitor use for weight and glucose control in patients with diabetes. However, there have been few if any previous published cases that were complicated by a concomitant ketogenic diet. Our goal is not to defame the practice of using ketogenic diets in an effort to control body weight and blood glucose, but to caution clinicians that ketogenic diets and SGLT-2 inhibitor use, both singly and in combination, can lead to severe, life-threatening EDKA.