Acute coronary syndrome (ACS) is a complex syndrome of clinical symptoms. In order to accurately diagnose the type of disease in ACS patients, this study is aimed at exploring the differentially expressed genes (DEGs) and biological pathways between acute myocardial infarction (AMI) and unstable angina (UA). The GSE29111 and GSE60993 datasets containing microarray data from AMI and UA patients were downloaded from the Gene Expression Omnibus (GEO) database. DEG analysis of these 2 datasets is performed using the “limma” package in R software. DEGs were also analyzed using protein-protein interaction (PPI), Molecular Complex Detection (MCODE) algorithm, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Correlation analysis and “cytoHubba” were used to analyze the hub genes. A total of 286 DEGs were obtained from GSE29111 and GSE60993, including 132 upregulated genes and 154 downregulated genes. Subsequent comprehensive analysis identified 20 key genes that may be related to the occurrence and development of AMI and UA and were involved in the inflammatory response, interaction of neuroactive ligand-receptor, calcium signaling pathway, inflammatory mediator regulation of TRP channels, viral protein interaction with cytokine and cytokine receptor, human cytomegalovirus infection, and cytokine-cytokine receptor interaction pathway. The integrated bioinformatical analysis could improve our understanding of DEGs between AMI and UA. The results of this study might provide a new perspective and reference for the early diagnosis and treatment of ACS. Acute coronary syndrome (ACS) is a complex syndrome of clinical symptoms, which is characterized by acute myocardial ischemia with severe coronary stenosis or occlusion caused by coronary plaque rupture and subsequent thrombosis, mainly including acute myocardial infarction (AMI) and unstable angina (UA) , 2. AMI Based on clinical epidemiology, the mechanism of the relationship between UA and AMI was studied, and great progress has been made –9. StudiWith the development of high-throughput gene chip and transcriptome sequencing methods, more and more gene chip technologies are used in the research of cancer and cardiovascular diseases, which enriches the biological markers for early diagnosis, classification, and prognosis of diseases and also provides references for the targeted therapy of diseases –15. Micrhttps://www.ncbi.nlm.nih.gov/gds/) developed and maintained by the National Center for Biotechnology Information (NCBI) not only collects and shares publicly available transcriptome data but also facilitates biomedical research [The GEO database (research . In the P < 0.05 (FC: fold change).First, the gene expression dataset is translated into common gene symbols by using annotation information from the platform. The “limma” package of R software was utilized to screen the original dataset for expression normalization and differential expression analysis . In addihttp://string-db.org, version: 11.0) is more than double to 5090 compared with the previous version. It collects, scores, and integrates all public sources of protein-protein interaction information and supplements this information through computational predictions, including direct and indirect correlations between proteins [https://cytoscape.org/) to establish a protein-protein interaction (PPI) network model [K‐core = 2, and Max.depth = 100). Then, the Maximal Clique Centrality (MCC) algorithm in “cytoHubba” was applied to screen the hub genes with high connectivity in PPI networks [The interaction between proteins is crucial for understanding the metabolic and molecular mechanisms of diseases. The number of organisms covered by the STRING database , “clusterProfiler” [GO provides a framework for describing the function of gene products in organisms, which can identify the biological characteristics of high-throughput genome and transcriptome data, including biological process (BP), cellular component (CC), and molecular function (MF) . KEGG carofiler” , and “GOrofiler”  in R sofhttp://www.ehbio.com/ImageGP/). In addition, in order to better understand the key DEGs, the Pearson correlation coefficient was used for correlation analysis, and the “Corrplot” package in R software was used for visualization [According to the MCC algorithm, 10 genes with the most stable and highest score in the network were selected as the key genes. The differences in hub gene expression between AMI and UA were shown using the boxplot tool in ImageGP , each of which contained AMI and UA samples, including 53 AMI samples and 25 UA samples . DEG anaThe obtained DEGs were imported into the STRING database, and the data with a confidence score greater than 0.7 were selected. Cytoscape was used for network visualization analysis of these interacting DEGs. The PPI network of GSE29111 includes 22 nodes and 34 edges . Using MThe PPI network of GSE60993 includes 20 nodes and 19 edges . The MCOGO and KEGG pathway enrichment analysis was performed on PPI data of GSE29111 differentially expressed genes using DAVID and “GOplot” package in R software. According to FDR < 0.05, 4 GO entries with significant enrichment were selected Figures , Table 4P adjust < 0.01, a total of 52 significantly enriched GO entries were screened  classification, the absolute values of 0-0.30, 0.30-0.50, 0.50-0.70, and 0.70-1.00 indicated “poor,” “medium,” “good,” and “strong” correlations. In addition, “r = 0” means completely irrelevant and “r = 1.00” means completely relevant. The key DEGs screened by GSE29111, BDKRB1, BDKRB2, CCL25, HRH1, KISS1, and NPBWR1 were highly expressed in UA patients and lowly expressed in AMI patients. GRPR, TACR3, HTR2B, and ORM1 were lowly expressed in UA patients and highly expressed in AMI patients . IFI44, RSAD2, and CLEC4D showed a poor positive correlation (r = 0.25).The key DEGs screened by GSE60993, CLEC4D, FFAR2, GNG10, IFI44, IFIT3, MCEMP1, PLSCR1, and RSAD2 were highly expressed in UA patients and lowly expressed in AMI patients . As showMany factors affect the occurrence and development of ACS, such as gender, fatigue, and frailty –33. AccoThe integrated bioinformatics analysis method was performed to screen the potential differential genes related to AMI and UA. Through retrieval, 2 datasets (GSE29111 and GSE60993) from the GEO database were included. Although datasets as GSE29111 and GSE60993 were already used and published by some authors, the main comparison is the DEGs between AMI and healthy controls. The difference is that this study used these two datasets to explore the DEGs between AMI and UA. A total of 242 DEGs were obtained from GSE29111, including 128 upregulated genes and 114 downregulated genes. There were 44 DEGs obtained from GSE60993, including 4 upregulated genes and 40 downregulated genes. According to the MCC algorithm, the 10 most stable and highest score genes in the PPI network were selected as core genes. The hub genes involved in GSE29111 included BDKRB1, BDKRB2, CCL25, HRH1, KISS1, NPBWR1, GRPR, TACR3, HTR2B, and ORM1. The core genes involved in GSE60993 include IFI44, FFAR2, GNG10, CXCR2, MCEMP1, FPR2, CLEC4D, IFIT3, RSAD2, and PLSCR1. These identified core genes act as a whole and may play an important role in the early diagnosis of ACS.α), while interleukin-1 beta (IL-1β) increased after infarction [GO enrichment analysis showed that the DEGs screened from GSE29111 were mainly enriched in the inflammatory response, G-protein-coupled receptor signaling pathway, integral component of plasma membrane, and plasma membrane. The GO entries of the DEGs screened from the GSE60993 were mainly related to neutrophil activation, immune response, inflammatory response, particles, plasma membrane, cytokine receptor activity, and cytokine binding. Over the past decades, the critical role of immune inflammatory processes in the occurrence, development, and deterioration of many cardiovascular diseases has received increasing attention, such as atherosclerosis, myocardial infarction, heart failure, myocarditis, and vasculitis , 37. At farction . The finThe enrichment analysis of the KEGG pathway showed that the main enriched pathways of DEGs screened from the GSE29111 dataset were related to the interaction of neuroactive ligand-receptor (hsa04080), calcium signaling pathway (hsa04020), and inflammatory mediator regulation of TRP channels hsa04750). Previous studies have shown that the nervous system regulation of cardiac activity has nothing to do with the physiological state of the heart . However50. Previ2+-mediated myocardial cell death in patients with myocardial infarction, which has important therapeutic significance and potential clinical effects [κB (NF-κB), signal transduction and transcription activation factor 3 (STAT3), and hypoxia-inducible factor-1 (NIF-1) are all related to the increase of ROS and intracellular Ca2+ concentration, and these characteristics make TRP channels effectively regulate inflammation [Calcium, as a common intracellular second messenger, is involved in regulating fertilization, gene transcription, secretion, and myocardial cells . Ruiz-Me effects . Consistammation . AlthougIn the current study, the core genes enriched in the neuroactive ligand-receptor interaction with the smallest FDR value were discussed and analyzed in detail. HRH1, NPBWR1, BDKRB2, HTR2B, TACR3, BDKRB1, and GRPR are 7 core genes enriched in the neuroactive ligand-receptor interaction. Histamine receptor H1 (HRH1) belongs to the G-protein-coupled receptor family. Its gene products are expressed in many tissues such as smooth muscle and neurons , 54. BhuAs a neurotransmitter, 5-hydroxytryptamine (5-HT) not only plays a signal transduction role in immune cells but also regulates mood, behavior, and cardiovascular and gastrointestinal functions , 66. A sα and IL-6 [The KEGG pathway enriched by the DEGs screened from the GSE60993 dataset is related to viral protein interaction with cytokine and cytokine receptor (hsa04061), human cytomegalovirus infection (hsa05163), and cytokine-cytokine receptor interaction (hsa04060). Cytokines are extracellular molecules that transmit intercellular signals, mainly involved in cell differentiation and inflammation through binding to specific receptors on the cell surface . Inflammand IL-6 . Boekholand IL-6 . TherefoIn this study, the hub genes enriched in these 3 pathways are C-X-C chemokine receptor type 2 (CXCR2), the receptor of interleukin-8 (IL-8). Neutrophils play an important role in innate immunity . CXC cheHowever, some limitations of our study should be considered. For instance, the differential genes analyzed by the two chips did not overlap, which may be caused by various reasons such as the source of the included samples, the annotation analysis platform, and the time of blood sampling. In addition, this study is based on the analysis of chip data; further experiments are needed to verify.In summary, the integrated bioinformatical analysis could improve our understanding of DEGs between AMI and UA. The integrated bioinformatical analysis could improve our understanding of DEGs between AMI and UA.


Zika virus (ZIKV), a re-emerging virus, causes congenital brain abnormalities and Guillain–Barré syndrome. It is mainly transmitted by Aedes mosquitoes, but infections are also linked to sexual transmissions. Infectious ZIKV has been isolated, and viral RNA has been detected in semen over a year after the onset of initial symptoms, but the mode of long-term persistence is not yet understood. ZIKV can proliferate in human Sertoli cells (HSerC) for several weeks in vitro, suggesting that it might be a reservoir for persistent ZIKV infection. This study determined proteomic changes in HSerC during ZIKV infections by TMT-mass spectrometry analysis. Levels of 4416 unique Sertoli cell proteins were significantly altered at 3, 5, and 7 days after ZIKV infection. The significantly altered proteins include enzymes, transcription regulators, transporters, kinases, peptidases, transmembrane receptors, cytokines, ion channels, and growth factors. Many of these proteins are involved in pathways associated with antiviral response, antigen presentation, and immune cell activation. Several immune response pathway proteins were significantly activated during infection, e.g., interferon signaling, T cell receptor signaling, IL-8 signaling, and Th1 signaling. The altered protein levels were linked to predicted activation of immune response in HSerC, which was predicted to suppress ZIKV infection. ZIKV infection also affected the levels of critical regulators of gluconeogenesis and glycolysis pathways such as phosphoglycerate mutase, phosphoglycerate kinase, and enolase. Interestingly, many significantly altered proteins were associated with cardiac hypertrophy, which may induce heart failure in infected patients. In summary, our research contributes to a better understanding of ZIKV replication dynamics and infection in Sertoli cells. Zika virus (ZIKV) belongs to the family Flaviviridae, and is a single-stranded positive-sense RNA virus. Other members of the family include Japanese encephalitis virus (JEV), Yellow fever virus (YFV), West Nile virus (WNV) and Dengue virus (DENV). ZIKV was first discovered in Rhesus monkeys in Uganda’s Zika rainforest in 1947 [However, the virus remained undiagnosed for a long time due to the disease’s non-specific flu-like symptoms and a lack of diagnostic screening ,3. In 20Aedes aegypti and A. albopictus) [In endemic regions, ZIKV is mainly transmitted by mosquito bites (opictus) . Howeveropictus) ,15,16. Aopictus)  and 69 dopictus) , respectopictus) . ZIKV waopictus) ,21,22,23opictus) ,25,26. Topictus) , ZIKV exopictus) , and hidSertoli, Leydig, and germ cells are the most prevalent cells found in human testes . SertoliWe previously used an aptamer-based assay and demonstrated that ZIKV infection caused alterations in the levels of proteins involved in spermatogenesis in HSerC . In orde2 in poly-l-lysine- coated culture plates in Sertoli Cell Medium . Following the company’s recommendation, Sertoli cells were trypsinized to detach them from the culture surface and passaged every 2–3 days for maintenance. All experiments were conducted using the cells that had reached passage 7.Primary Sertoli cells (HSerC) isolated from human testis were purchased from ScienCell Research Laboratories, CA, USA . The cells were cultured at 37 °C in 5% COThe Zika virus strain used in this study (ZIKV/Homo sapiens/PRI/PRVABC59/2015) was donated by Dr. David Safronetz, Chief of Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada. For future use, the ZIKV strain was expanded in Vero cells  and kept at −80 °C in 10% FBS . The stock virus was titered in Vero cells by plaque assay. 2 cell culture flasks treated with poly-l-lysine. At 70% confluency, cells were infected with ZIKV at a multiplicity of infection (MOI) of 3 plaque-forming units (PFU) per cell. Culture plates were rocked every 10–15 min for 2 h in a 37 °C incubator  to allow the virus to attach to cell surfaces, and cells were overlaid with Sertoli cell media containing 2.5% FBS. At 3, 5, and 7 days after infection, ZIKV-infected and mock, non-infected cells were collected. All experiments were done in three biological replicates. HSerC were grown in 75 cmg for 8 min pelleted the cells, which were then washed three times with sterile ice-cold PBS. The pelleted cells were then lysed in 4% SDS in 100 mM HEPES buffer pH 8.5 by sonication. Centrifugation at 14,000× g for 10 min at 4 °C was used to remove insoluble cellular components. Bradford Protein Assay was used to determine the protein concentrations in the supernatants .The Sertoli cells infected with ZIKV and time-matched mock-treated cells were scraped from the culture plates after 3, 5 and 7 days of infection. Centrifugation at 600× The protein concentrations in the cell lysates were measured using the Bradford Protein Assay, and 20 µg of protein were resolved in 10% SDS-PAGE gels and transferred to 0.2 µm nitrocellulose membranes. Anti-PSMA2 , anti-ZIKV NS1 , anti-ZIKV NS3 , anti-ZIKV Env , anti-STAT1 , and anti-Beta-Actin  antibodies were used to detect protein targets. Anti-rabbit  or anti-mouse  secondary antibody was used to identify the primary antibody conjugates to the targeted proteins. After overlaying with ECL reagents, protein bands were photographed with an Amersham Imager 680 . To quantify band intensities, Image J version. 1.53e  was used, and Graphpad Prism version 6.0.  was used to visualize them graphically.2 scale for easier differential analysis. To determine the impact of ZIKV infection on the cellular proteome, a total of 18 protein samples were collected from ZIKV- and mock-infected Sertoli cells at 3, 5 and 7 days post-infection (dpi). Proteins were digested into peptides by the SP3 (single-pot solid-phase-enhanced sample preparation) methods as described elsewhere ,39. In s2 values (Delta log) of an infected and time-matched mock sample. Then, the delta Log2 values were converted to fold-change for each of the proteins. The p-value was determined by the Students t-test (2 tails) and Z-score analyses based on the protein expression difference of all three replicates. p-value < 0.05 and Z-score values of ≥.96 σ and ≤−1.96 σ were considered significant as described before [p-value < 0.05 and fold change above 1.5 or below −1.5. The IPA core analysis predicted the top affected canonical pathways, bio-functions, interconnecting networks and upstream molecules based on these protein level changes. The Western blot image band intensities were quantified using Image J version1.53e software , and statistical analyses were performed by one-way or two-way ANOVA  in GraphPad Prism version 6.0. MORPHEUS , a free internet software program, was used to generate the heatmaps.Initially, alterations in the levels of any individual protein expression were determined by the differences between Logd before . The lisAfter infecting HSerC with ZIKV at MOI = 3, viral protein expression levels, which could result from increased expression, lower protein turnover, or a combination of both, and cytopathic effects were monitored at 3, 5 and 7 dpi, ZIKV infection did not induce any cytopathic effects A. Howevep value < 0.05) affected at 3, 5 and 7 dpi, respectively  revealed that certain protein levels were affected at specific time periods while other proteins were significantly affected at all times , 5 dpi , and 7 dpi , which includes cytokines, enzymes, G-protein coupled receptors, growth factors, kinases, microRNAs, peptidases, phosphatase, transcription regulators, transmembrane receptor and transporters  software to understand predicted impacts of ZIKV infection on cellular signaling pathways, bio-functions, and protein–protein networks. A total of 15, 31 and 13 Sertoli cell canonical pathways were predicted to be significantly affected at 3, 5 and 7 dpi, respectively, after ZIKV infection . GP6 sigsporters . The topsporters C. The fusporters . IPA also built protein–protein networks of the significantly affected proteins based on their direct or indirect interactions. There were 12, 9, and 10 protein–protein interaction networks  predicted by IPA analysis of the altered proteins at 3, 5 and 7 dpi, respectively. The predicted networks were associated with different cellular functions and diseases, including cell morphology, cellular assembly and organization, neurological disease, lipid metabolism, molecular transport, cardiovascular disease, connective tissue disorders, antimicrobial response, cell cycle, and endocrine system development . Cancer,To understand the impact of ZIKV infections across the three time points, we selected the list of 50 commonly affected proteins A,B and pThe immune response of antigen-presenting cells, immune response of phagocytes and leukocytes, activation of antiviral response, phagocytosis of cells and innate immune response also were predicted to be activated at 3, 5 and 7 days post-ZIKV infection A. SeveraWe identified several proteins associated with establishment of viral infection and replication that were significantly affected by ZIKV infection in HSerC A. Based d-glucose, d-hexose, monosaccharide, phospholipids and synthesis of carbohydrate and phospholipid. At 5 dpi ZIKV caused significant activation of glycolysis (Z-score = 2.44) and gluconeogenesis (Z-score = 2.0) pathways A. There ZIKV infection significantly affected 20 proteins D involveFive proteins  were selected for validation based on their expression fold-change and availability of commercial antibodies. Western blot was done A, and thThe mechanism(s) underlining the long-term persistence of ZIKV in male semen is not yet clearly understood. The sexual transmission of the virus raises the risk of initiating outbreaks in non-endemic regions, even in the absence of a mosquito vector. Furthermore, the pathologic consequences of viral persistence in the male genital tract are unclear. In our previous study, to investigate ZIKV infection-induced HSerC proteome alterations, we applied SOMAscan, a multi-plexed targeted technology that can identify over 1300 proteins from each sample . AlthougThe human testis maintains an immune-privileged environment, separated from the body cavity by the blood–testis barrier (BTB), made with Sertoli cells . Howeverd-fructose, monosaccharides, phosphatidic acid, phospholipids were also predicted to be significantly activated by the significantly altered proteins (Viruses use biomolecules from the host and induce anabolism to create the macromolecules needed for virion replication and assembly. As a result, it is no surprise that viral infection forces host cells to modify their metabolism in order to support effective virus replication . Many onproteins . Previouproteins A, as repSurprisingly, we found many proteins involved in cardiac disease that were significantly affected E–G durinUnderstanding the mechanisms of ZIKV persistence in the male genital tract is critical for treatment of the disease, antiviral/vaccine development and overall control of viral transmission. Multiple studies have suggested that Sertoli cells could be a potential reservoir for this viral persistence. Sertoli cells build the blood–testis barrier that maintains an isolated environment for optimal development of the germ cells and provides sufficient nutrients and signals for sequential differentiation into sperm by spermatogenesis . ZIKV in


Research on their roles in hypertension and kidney disease has greatly expanded recently. The developing kidney can be programmed by various adverse in utero conditions by so-called renal programming, giving rise to hypertension and kidney disease in adulthood. Accordingly, early gasotransmitter-based interventions may have therapeutic potential to revoke programming processes, subsequently preventing hypertension and kidney disease of developmental origins. In this review, we describe the current knowledge of NO, CO, and H2S implicated in pregnancy, including in physiological and pathophysiological processes, highlighting their key roles in hypertension and kidney disease. We summarize current evidence of gasotransmitter-based interventions for prevention of hypertension and kidney disease in animal models. Continued study is required to assess the interplay among the gasotransmitters NO, CO, and H2S and renal programming, as well as a greater focus on further clinical translation.Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H Although these gases are toxic in excess, they are endogenously generated and exert specific biological functions at the physiological level [2S, next to NO and CO, has emerged as a third gasotransmitter with key roles in the regulation of cardiovascular and other systems [Gasotransmitters, such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide  and hypertension are major non-communicable diseases, which are the leading causes of global deaths. According to the WHO, one in five women and one in four men have hypertension worldwide . An estiDuring kidney development, various early-life adverse environmental conditions can lead to hypertension and kidney disease in adulthood . The ideThe three gases and their roles in established kidney disease and hypertension have been extensively reviewed elsewhere ,15,16,17We searched the PubMed/MEDLINE databases for studies published in English using the following search terms: “gasotransmitter”, “kidney disease’, “developmental programming”, “DOHaD”, “nitric oxide”, “hydrogen sulfide”, “carbon monoxide”, “heme oxygenase”, “oxidative stress”, “nephron”, “nephrogenesis”, “mother”, “pregnancy”, “gestation”, “offspring”, “progeny”, “reprogramming”, and “hypertension”. We also used the reference lists of identified articles to find additional studies. The last search was made on 30 May 2021.2S, and signaling pathways able to maintain normal pregnancy and fetal development are depicted in A variety of adverse conditions during pregnancy can affect fetal development resulting in hypertension and kidney disease in adult offspring, including maternal malnutrition, maternal exposure to environmental chemicals/toxins, maternal illnesses, medication uses in pregnancy, etc. ,20,21,222+-activated K+ channel (BKCa) [NO plays a vital role in the regulation of feto-placental circulation, fetal development, and transfer of nutrients from mother to fetus in normal pregnancy . NO can l (BKCa) .G-Nitro arginine methyl ester  in pregnancy developed hypertension, proteinuria, and kidney disease [Maternal plasma arginine levels were reduced in pregnancies complicated by intrauterine growth retardation (IUGR) . Likewis disease ,40.Ca in rat vascular smooth muscle cells through a cGMP-independent mechanism [Like NO, CO is a diatomic low molecular weight gas with similar molecular size and structure . Howeverechanism .In pregnant women, low respiratory CO levels are associated with hypertension in pregnancy and preeclampsia . CO has 2S synthesis, including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) [2S [2S. In an alternative pathway, 3-mercaptopyruvate (3-MP), the substrate for 3MST to produce H2S, is provided by cysteine aminotransferase (CAT) and D-amino acid oxidase (DAO) [2S [2S can be produced via non-enzymatical pathway or by bacteria [e (3MST) . In the MST) [2S . CBS andse (DAO) . In the DAO) [2S . In addibacteria .Ca, uterine smooth muscle KATP channel is important for uterine quiescence [2S can mediate vasodilation via KATP channel to maintain feto-placental circulation [ATP levels increase in human uterine artery smooth muscle cells [2S level and placental CBS and CSE protein levels relate to preeclampsia [Uterine CBS and CSE levels increase during pregnancy and decrease during labor . Like BKiescence ,50. In hculation . During le cells . Howeverclampsia ,53,54.2S play crucial roles for normal pregnancy. Dysregulated gasotransmitter signaling has been linked to preeclampsia, IUGR, stillbirth, and preterm labor [Collectively, NO, CO, and Hrm labor ,25. Alth2S play key roles in the pathogenesis of hypertension. The first are observations on knockout mice lacking genes responsible for gasotransmitter synthesis. First, eNOS knockout mice displayed hypertension [2S-generating enzymes in hypertension has also been demonstrated using CSE, CBS, or 3MST knockout mice [Several lines of evidence indicate that NO, CO, and Hrtension . The impout mice ,56,57,58out mice .2S-generating enzymes and/or activity in hypertension has been established in various animal models, including the NO-deficient rats [The second line of evidences report dysregulated gasotransmitter signaling pathways in human and experimental models of hypertension. Prior research has addressed impaired L-Arginine–ADMA–NO pathway in the development of hypertension . Dysreguent rats , the Dahent rats , the spoent rats , and theent rats .Third, several therapeutic strategies targeting different gasotransmitters have demonstrated to be significant promising for beneficial effects against hypertension in various animal models ,66,67,68The gasotransmitter generating enzymes iNOS, eNOS, nNOS, HO-1, HO-2, CSE, CBS, and 3MST were detected in kidney cells comprising podocytes, glomerular endothelial cells, tubular cells, and mesangial cells, but not all of them are constitutively expressed in every cell type ,70. For In the kidney, NO performs important signaling functions including the modulation of renal sympathetic neural activity, control of renal hemodynamics, regulation of pressure-natriuresis, blunting of tubuloglomerular feedback, and inhibition of tubular sodium reabsorption . AccordiThe beneficial actions of CO in the kidney have also been recognized . Inhibit2S regulates basic physiologic mechanisms of the kidney such as sodium reabsorption, glomerular filtration, and renal homeostasis [Heostasis . In someeostasis , acute keostasis , cisplateostasis , obstruceostasis , and diaeostasis , it can 2S share the same sGC–cGMP pathway to elicit relaxation in kidney cells [2S inhibiting cGMP degradation [2S donor), which also rescues NO bioavailability [2S crosstalk in the control of blood pressure (BP).Although NO and Hey cells , they acradation . In ratslability . These d2S also employs post-translational modifications, namely, S-sulfhydration [2S physiologically S-sulfydrates proteins on the thiol group of cysteine residues , leading to the formation of the –SSH moiety. These observations lead to a hypothesis that there might be competition between S-nitrosylation and S-sulfhydration for the same cysteine residues in proteins, thus allowing the two gasotransmitters to regulate each other [One of the NO-based cellular signaling pathways is via protein S-nitrosylation, the covalent addition of NO moiety to the sulfur atom of cysteine residues . S-nitroydration . Endogench other .CO could also target sGC and regulate NO-mediated vasodilatation , which w2S can activate NRF2 via S-nitrosylation and S-sulfhydration of KEAP1, respectively [2S [2S are important determinants for kidney disease  . NRF2 isectively ,93. In aectively . As NFκB disease .2S signaling that may pose new opportunities for the therapeutic protection of hypertension and kidney disease.Although the beneficial actions of gasotransmitters against established kidney disease and hypertension have been established, their roles in mediating programmed responses behind developmental origins remain unclear. For this reason, this review will next outline the potential early-life interventions targeting NO, CO, and HSo far, little reliable information exists regarding the impact of gasotransmitters on the development of hypertension and kidney disease in humans. Animal models enable researchers to consider various adverse environmental conditions in developmental stages to determine underlying programming processes and long-term outcome in adult offspring.2S, and renal programming. The current review is solely restricted to early-life insults starting in pregnancy and lactation period with focusing on gasotransmitter-related renal programming. The developing kidney is extremely vulnerable to the effects of adverse environmental events, resulting in renal programming and eventually functional alterations and structural changes . As reviAs shown in Our previous study reported that ADMA (a reactive oxygen species (ROS) inducer and endogenous NOS inhibitor) impaired ureteric bud branching morphogenesis, consequently leading to decreases of nephron number . Additio2S [Most studies focused on gasotransmitter NO ,104,105,2S ,107,108 2S . Impaire2S ,97, stre2S , materna2S , high-fr2S , materna2S , prenata2S ,102, pre2S , prenata2S-synthesing enzyme expression, decreased renal H2S releasing activity, and low plasma H2S level have been reported in various models of programmed hypertension [However, there is only one report demonstrating HO-1 is involved in maternal diabetes-induced hypertension and kidney disease . Moreovertension ,107,108.Various early-life insults can cause similar renal phenotypes, implying the existence of common pathways behind renal programming that may contribute to hypertension and kidney disease of developmental origins. Although the pathogenetic mechanisms have not yet been fully disclosed, certain renal programming mechanisms have been documented, including but not limited to, oxidative stress, aberrant renin–angiotensin system (RAS), dysregulated nutrient sensing signals, epigenetic regulation, gut microbiota dysbiosis, and sex differences ,110,111.With a greater understanding of mechanisms behind renal programming, implementation of interventions for therapeutic prevention of hypertension and kidney disease in later life is feasible. An important message is that whereas therapeutic interventions can be delivered at any disease stage, reprogramming is barely restricted to key periods during early development. Here, we summarize the knowledge available today regarding gasotransmitters used as reprogramming strategies for developmental hypertension and kidney disease in various animal models ,120,121,Evidence from the studies reviewed indicates that rats are the most commonly used animal models. Rats become sexually mature at 6 weeks. In adulthood, one rat month is comparable to three human years . AccordiSeveral therapeutic interventions have been used to increase NO bioavailability, such as supplementation of NO substrate, NO donors, ADMA-lowering agents, and enhancement of the expression and/or activity of NOS . NeverthL-Arginine supplementation has been considered as a therapeutic approach to improve NO bioavailability in human diseases , whereasG-nitro–L-arginine methyl ester (L-NAME) exposure [Because L-Citrulline can be converted to L-Arginine and it can bypass hepatic metabolism, oral L-Citrulline supplementation has been used as an add-on therapy to increase L-Arginine concentrations, subsequently increasing NO production . Table 3exposure , maternaexposure , streptoexposure , and preexposure . In addiexposure .The use of NO donors is another way to increase NO. Two NO donors—pentaerythritol tetranitrate and molsidomine—have been reported to prevent the development of hypertension in SHRs and FFH rats, respectively ,114 Tab. HoweverAs opposed to NO, limited information is available about the CO-based interventions to study their roles on kidney disease and hypertension of developmental origins. Carbon monoxide releasing molecules (CORMs), a group of chemical compounds capable of controlled CO release directly in tissues or organs, have emerged as a therapeutic tool for human diseases . HoweverIn addition to CORMs, HO-1 or Nrf2 activators are potential CO-based modalities to activate the NRF2–HO-1–CO signaling pathway. Many natural compounds have shown to be effective activators of NRF2/HO-1 like resveratrol, curcumin, quercetin, anthocyanins, carnosic acid, epigallocatechin gallate, celastrol, isothiocyanates, garlic-derived organosulfur compounds, etc. . Aside fThere is general lack of studies investigating NRF2/HO-1 activators for the prevention of kidney disease of developmental origins. As shown in 2S-based modalities used for therapeutic protection of hypertension and kidney disease include H2S donors, precursors of H2S, and organosulfur compounds. Inorganic sulfide salts like sodium hydrosulfide (NaHS) are the most widely used H2S donors to evaluate the therapeutic potential of exogenous H2S [2S donors have not yet been tested in terms of their reprogramming effects on hypertension and kidney disease of developmental origins.So far, available Hnous H2S . NaHS hanous H2S , Dahl sanous H2S , and SHRnous H2S . In linenous H2S . Neverth2S include L-Cysteine, D-Cysteine, and NAC, a stable cysteine analog. 2S-producing enzymes in offspring kidneys [2S [Precursors of H kidneys . Organosneys [2S .2S concentrations, such as amlodipine, aspirin, carvediol, atorvastatin, digoxin, paracetamol, metformin, ramipril, testosterone, vitamin D, and 17β-estradiol [2S-releasing drugs. It would be interesting to see whether these H2S-releasing drugs would appear to be a practical approach to prevent hypertension and kidney disease from further clinical translation.Several clinically used medications have been shown to increase Hstradiol . AdditioCollectively, these findings indicate the potential impact of gasotransmitter-based interventions for therapeutic prevention of programmed kidney disease and hypertension. While these studies have also raised concern, the protective mechanisms behind some gasotransmitter-based interventions are not limited to only one gasotransmitter. For example, resveratrol has properties to lower ADMA and activate NRF2; however, to what extent its reprogramming effects on kidney disease and hypertension can be attributed to NO or CO deserves further clarification. Accordingly, a better understanding of each gasotransmitter-dependent and -independent mechanisms responsible for the reprogramming effects of various gasotransmitter-based interventions is therefore highly warranted.2S-based drugs have led to a significant progress in our understanding of established hypertension and kidney disease, attention must be paid to prevent (and not just to treat) these diseases; translation from animal models into clinical practice will be an additional challenge.Current evidences suggest a potential therapeutic role of gasotransmitter-based interventions for prevention of programmed hypertension and kidney disease. Although many NO-, CO-, and H2S, and most of them focused on hypertension of developmental origins. Nevertheless, there is little reliable information about the reprogramming effects of CO-based intervention. Meanwhile, we are aware that almost no studies have taken a holistic approach to simultaneous determinations of NO, CO, and H2S signaling pathway in one experiment. In view of the complex interplay between these three gasotransmitters, the reprogramming effect responding to each gasotransmitter-based intervention, either individually or in combination, are incomplete and difficult to predict. Furthermore, more attention should be paid to decide the optimal dosage and duration of gasotransmitter-based intervention using the appropriate animal models prior to clinical translation.Of note, much of the preclinical work investigated the reprogramming actions of NO and H


The COVID-19 pandemic has deeply influenced sanitization procedures, and high-level disinfection has been massively used to prevent SARS-CoV-2 spread, with potential negative impact on the environment and on the threat of antimicrobial resistance (AMR). Aiming to overcome these concerns, yet preserving the effectiveness of sanitization against enveloped viruses, we assessed the antiviral properties of the Probiotic Cleaning Hygiene System (PCHS), an eco-sustainable probiotic-based detergent previously proven to stably abate pathogen contamination and AMR. PCHS  was tested in comparison to common disinfectants (70% ethanol and 0.5% sodium hypochlorite), in suspension and carrier tests, according with the European UNI EN 14476:2019 and UNI EN 16777:2019 standards. Human alpha- and beta-coronaviruses hCoV-229E and SARS-CoV-2, human herpesvirus type 1, human and animal influenza viruses, and vaccinia virus were included in the study. The results showed that PCHS was able to inactivate 99.99% of all tested viruses within 1–2 h of contact, both in suspension and on surface. Notably, while control disinfectants became inactive within 2 h after application, the PCHS antiviral action persisted up to 24 h post-application, suggesting that its use may effectively allow a continuous prevention of virus spread via contaminated environment, without worsening environmental pollution and AMR concern.  The COVID-19 pandemic, caused by the new SARS-CoV-2 human coronavirus has deeply influenced the habits relative to hygiene and sanitization, shining light on the risk associated with environmental virus contamination, especially in the hospital environment. COVID-19 has in fact spread worldwide, causing at the moment over 244 million confirmed cases and 4.96 million deaths , suggestConsistently with these data, WHO has proposed preventive measures, and high-level virucidal chemical disinfectants have been mandatorily introduced by regulatory bodies for surface cleaning of indoor environments, including healthcare and non-healthcare settings, as interim recommendations to combat the COVID-19 health emergency ,15. Howereservoirs and transmission vectors to susceptible humans [Besides SARS-CoV-2, several enveloped viruses have been similarly shown to retain infectivity for long periods on hard surfaces, depending on virus type, surface characteristics, temperature, and humidity , includie humans ,31. Conse humans ,32,33, sAmong the viruses that might be transmitted by the contaminated environment, human coronaviruses , which can cause mild to severe respiratory infections , can per2O2), for sanitization [Based on the ability of many viruses to maintain infectivity on inanimate surfaces, the control of environmental viral contamination represents a key point to address. Current guidelines for COVID-19 management suggest using high level disinfectants, mostly 0.1–0.5% sodium hypochlorite (NaClO) and 1% hydrogen peroxide  [Aiming to obtain a long-term effective cleaning procedure, stably reducing viral contamination without impacting on environmental pollution and AMR, we tested the antiviral properties of an eco-sustainable probiotic-based sanitation system , that was previously shown to prevent pathogen recontamination by stably remodulating the hospital microbiome. Its action, based on the biological properties of selected probiotic s (−80%) ,46,47,48s (−80%) ,46, and s (−80%) , testinghttps://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32010R0066, accessed on 23 October 2021) detergent containing 107 CFU/mL spores of selected probiotics belonging to the Bacillus genus . PCHS was tested at 1:10, 1:50, and 1:100 dilution in sterile distilled water.The Probiotic Cleaning Hygiene System (PCHS) used in all the assays was previously described . BrieflyThe enveloped viruses and appropriate target cells used for virus inocula preparation, virus titration, and standard inactivation assays, are summarized in 2 in the appropriate culture medium supplemented with 10% foetal bovine serum (FBS), 2 mM L-Glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (cell culture complete medium) .MRC-5 cells were cultured in Eagle Minimal Essential Medium Eagle (EMEM), whereas the other cell lines were grown in Dulbecco Minimal Essential Medium (DMEM). All the cell lines were expanded at 37 °C + 5% CO2 in culture medium additioned with 2% FBS. Infected cell cultures were incubated for different times until appearance of cytopathic effect (CPE) involving >80% of cultured cells, that corresponded to: 2 days for MVA and HSV-1, 5 days for SARS-CoV-2, 3 days for influenza viruses, and 7 days for hCoV-229E. At the end of the incubation time, cells and culture supernatants were collected. Cells were lysed by 3 cycles of rapid freezing/thawing in liquid nitrogen and 37 °C, interspersed with 30 s pulse-vortex. Cell lysate was then added to culture supernatant and viral particles were recovered by centrifugation at 20,000× g for 45 min at 4 °C. The virus pellets were suspended in 1 mL of PBS + 1% bovine serum albumin (BSA), then frozen and maintained at −80 °C until use. The virus stock titre was determined by infecting the appropriate target cells seeded in 96-well plates, using the same culture conditions used for virus stock preparation, and evaluating the 50% tissue culture infectious dose (TCID50) per ml by the standard Spearman-Karber method, as previously described [Virus stocks were obtained by infecting specific 90% confluent target cells, subsequently incubated at the appropriate temperature  + 5% COescribed . Briefly8 TCID50/mL, as calculated by the Spearman-Karber method.All virus stocks contained around 105–107 TCID50, depending on virus type) were added to 90 µL of the appropriate dilution of PCHS , in the presence of 0.3% of BSA, to mimic the conditions that could be found on a hospital surface (“clean” conditions). Negative and positive controls were respectively represented by culture medium and 70% ethanol (EtOH). The suspension was incubated at room temperature for 1, 2, 4, 8, and 24 h; then it was collected, immediately diluted in 0.9 mL cold medium + 2% FBS , filtrated (0.45 µm) to remove the probiotic component, and then serially diluted (10-fold dilution) in cold medium + 2% FBS for titration of the residual virus amount by the Spearman-Karber method. Infected cells were then incubated at the appropriate temperature in the presence of 5% CO2 for the time needed to evidence virus CPE. Representative CPE pictures are shown in 50/mL. Each experimental condition was assessed in duplicate and the collected results represent the mean values of three independent assays. All the experiments with SARS-CoV-2 were performed in a BSL-III laboratory. Experimental controls and cytotoxicity evaluation of PCHS and standard disinfectants were performed in all cell types used in the assays, following the protocol indicated in the standard procedures.The antiviral activity of PCHS in suspension was assayed following the European standard procedure UNI EN 14476:2019, as indicated by the Technical Committee 216 (TC216) “Chemical disinfectants and antiseptics” of the European Committee for Standardization (CEN), which has been developing methods for testing the efficacy of disinfectants in Europe since 1989 ,52. Brie6 TCID50) were seeded on carrier surface with a micropipette, and the drop was spread on a surface area of about 1 cm diameter and left to dry at room temperature, then immediately covered with 100 µL of PCHS previously diluted 1:10, 1:50, and 1:100 in water. EtOH 70% and culture medium were used as positive and negative controls, respectively. After 1, 2, 4, 8, and 24h, 0.9 mL (10-fold dilution) of ice cold medium + 2% FCS were added to collect the residual virus. The collected medium was then filtered (0.45µm) to remove probiotic component and serially diluted (10-fold) in cold medium + 2% FCS. Each dilution was transferred to 96-well microplates (0.1 mL/well) containing a 90% confluent monolayer cells, which were then incubated at the appropriate conditions to evaluate the residual virus titre by the Spearman-Karber method. In the prevention assay, 100 µL of 1:10, 1:50, and 1:100 diluted PCHS were seeded on surface and left to dry at room temperature. EtOH 70% and culture medium were used as positive and negative controls, respectively. Immediately after drying, the surfaces were contaminated with 100 µL of virus inoculum (106 TCID50). After 1, 2, 4, 8, and 24h, samples were collected and titrated as described for the decontamination assay. The long-term ability of PCHS in preventing virus contamination was assayed by seeding 100 µL of 1:10, 1:50, and 1:100 diluted PCHS on surface and then contaminating the treated surface after 1, 2, 4, 8, and 24h with 100 µL of virus inoculum (106 TCID50). EtOH 70% and sodium hypochlorite (NaClO) 0.5% were used as positive controls; culture medium was used as a negative control. Virus inoculum was left in place for 2 h and then collected and titrated as described for the previous assays. Experimental controls and cytotoxicity evaluation of PCHS and standard disinfectants were performed on all cell types used in the assays, following that indicated in the standard procedures. Both EtOH and NaClO were cytotoxic at 10−1 dilution on all used cell lines, whereas PCHS did not show cell toxicity; direct comparison of virus titre was thus performed on the dilutions from 10−2 onwards.The antiviral ability of PCHS on hard non-porous surfaces was assessed following the European standard procedure UNI EN 16777:2019  and usinBacillus strains contained in the PCHS detergent , previously culturally isolated, were assessed by the API-ZYM system  for their ability to produce enzymes potentially useful to degrade virus components, following the manufacturer’s instructions.The https://www.r.project.org/, accessed on 10 May 2021) by Student’s t-test. A p-value ≤ 0.05 was considered significant.Statistical analyses were performed with Agilent GeneSpring GX v11.5 software  and R , showed that PCHS could efficiently inactivate all the tested viruses, regardless of the type of virus used , includiConsistently, PCHS exhibited a similar antiviral activity also against SARS-CoV-2 D, which Since in the suspension method the viruses come into contact with a large amount of the disinfectant, which may render them easier to inactivate, the antiviral activity of PCHS was also assessed in carrier tests, performed according to the UNI EN 16777:2019 standard European procedure. Based on the results obtained in suspension tests, only hCoV-229E and MVA viruses were used in the assays, to include a human coronavirus similar to SARS-CoV-2 and a highly resistant enveloped virus. Three different conditions were tested, to assess respectively: (1) the decontaminating ability of PCHS, (2) the PCHS ability to prevent virus contamination by PCHS, (3) the long-term stability of PCHS activity in preventing virus contamination.The results of decontamination-type assays showed that, similar to that observed in suspension tests, any dilution of PCHS could completely inactivate hCoV-229E (>4 Log decrease) within 1 h, whereas MVA was totally inactivated in 1 h by 1:10 and 1:50 dilutions and in 2 h by 1:100 diluted PCHS . The lowNext, the ability of PCHS-treated surfaces to prevent a subsequent virus contamination was assessed. To this purpose, PCHS was first applied on surface and left to dry, then the treated surface was contaminated with the virus inoculum, whose residual titre was evaluated after 1, 2, 4, 8, and 24 h of contact. The results, summarized in Lastly, the long-term ability of PCHS to prevent virus contamination was assessed on surfaces. PCHS  was applied on surfaces and left to dry, then virus inocula were added to treated surfaces after 1, 2, 4, 8, and 24 h. Viruses were left in place for 2 h and then collected to measure residual virus titre .Notably, PCHS-treated surfaces completely inactivated both viruses even 24 h after treatment, whereas EtOH- and NaClO-treated surfaces did not maintain their inactivating ability over time. EtOH lost its inactivating activity within 1 h and NaClO gradually lost its virucidal activity from 2 h on both MVA (−2.25 Logs at 2 h) and hCoV-229E (−3.4 Logs): at that time, in fact, the NaClO activity resulted below the threshold needed to define a compound as virucidal (−4 Logs). At longer times the antiviral activity of both chemical disinfectants disappeared completely.Bacillus strains were analyzed for their enzymatic activity, in order to highlight any eventual production of enzymes capable of degrading viral components. The three Bacillus strains included in the PCHS detergent  were individually tested by the API-ZYM system, allowing to identify and quantify simultaneously 19 different enzymatic activities. The results, summarized in B. pumilus and B. megaterium), α-chimotrypsin (B. pumilus and B. megaterium), naphtol-phosphohydrolase, α- and β-galactosidase (absent respectively in B. megaterium and B. subtilis), α- and β-glucosidase, N-acetyl-β-glucosamidase, and α-mannosidase (B. pumilus only). The presence of enzymes capable of processing lipids, proteins, and sugars, support the hypothesis that such probiotics could chemically degrade the outer components of enveloped viruses, achieving virus inactivation.To elucidate the possible contribution of probiotics in virus inactivation, PCHS-derived The COVID-19 pandemic, caused by SARS-CoV-2, has profoundly influenced sanitization procedures and the massive use of disinfectants has been indicated to prevent virus spread. On the other hand, contamination of viral origin has been reportedly evidenced for several viruses beside SARS-CoV-2 , possiblIn contrast, we recently reported that a sanitation approach based on the microbiome-balance principle , could stably and efficiently counteract pathogens compared to chemical disinfectants, through competitive exclusion mechanisms , decreas10 decrease of virus titre obtained within 1 h as the threshold to establish the antiviral activity of the product against a specific virus.Based on these premises, and in consideration of the current emergency linked to COVID-19, here we aimed to assess the antiviral potential of PCHS against the enveloped viruses potentially contaminating the healthcare setting, including SARS-CoV-2. PCHS was tested following suspension and surface procedures, according with the European standard UNI EN 14476:2019 and UNI EN 16777:2019. Indeed, both procedures prescribe the use of non-enveloped viruses and only MVA among the enveloped ones (since it is considered the most resistant enveloped virus); however, since our study aimed to analyze the enveloped viruses potentially able to persist on surfaces and similar to SARS-CoV-2, we included, beside MVA, the human coronaviruses hCoV-229E and SARS-CoV-2, the human herpesvirus HSV-1 and three human/animal type A influenza viruses. Three different concentrations of PCHS were tested, including a lower one that is used for routine cleaning (1:100 dilution) and two higher concentrations (1:10 and 1:50 dilutions), comparing their activity with that of 70% EtOH and 0.5% NaClO, two common disinfectants indicated for decontamination during the pandemic. Based on standard procedures evaluating the virucidal properties of a product, we considered a ≥4-LogThe results showed that PCHS was active on all enveloped viruses at all dilutions in a time-dependent manner. In particular, the 1:100 dilution completely inactivated within 1 h all tested viruses except for MVA, which was inactivated in 2 h. A variable situation was observed with influenza viruses, confirming that previously reported for chemical disinfectants, showing different activity depending on virus type and host origin . In factThe carrier/surface test results, performed on hCoV-229E and MVA, evidenced a high inactivation activity both in decontamination and prevention conditions. Interestingly, while 70% EtOH and 0.5% NaClO became inactive within 1 and 2 h, respectively, PCHS-treated surfaces maintained their antiviral properties even 24 h after PCHS application, suggesting that a daily PCHS sanitation  may assure a continuous stably decontaminated environment.Bacillus strains spread on the treated surface may be responsible for the continuous action. Accordingly, the analysis of the enzymes produced by the three PCHS-derived Bacillus species  revealed several enzymatic activities potentially able to degrade the virus shell components, including lipids, protein, and sugar residues. Consistent with this, the Bacillus genus is reportedly known as one of the most important bacterial sources of enzymes with remarkable properties, such as high resistance to extreme temperatures, pH, organic solvents, and oxidizing agents. Bacillus enzymes have been long used in medicine, biofilm destruction, animal feed, agriculture, degradation of feathers, wool, and hair [Bacillus genus possess sporulation capacity that renders them resistant to harsh environmental conditions and usable in concentrated detergents, which makes them interesting for medical and industrial purposes. Lastly, PCHS is a low-cost affordable system, which is also important from a cost-saving perspective [The surfactants contained in detergent are known to disrupt and damage the envelope of viruses ,68, and and hair ,70, thusand hair , suggestspective , and renspective . To thisspective . For nonIn light of its effective long-lasting antiviral activity and characteristics of sustainability, PCHS sanitation may be considered as a novel and safe perspective for the control and prevention of the spread of various enveloped viruses including SARS-CoV-2, also in light of the lack of negative side-effects on the environment health and on AMR concerns.


To identify factors associated with severe coronavirus disease 2019 (COVID-19), defined by hospitalization status, in patients with primary mitochondrial diseases (PMDs), thereby enabling future risk stratification and informed management decisions.We undertook a cross-sectional, international, registry-based study. Data were extracted from the International Neuromuscular COVID-19 Database and collected between May 1, 2020, and May 31, 2021. The database included subjects with (1) PMD diagnosis (any age), clinically/histopathologically suspected and/or genetically confirmed; and (2) COVID-19 diagnosis classified as “confirmed”, “probable”, or “suspected” based on World Health Organization definitions. The primary outcome was hospitalization because of COVID-19. We collected demographic information, smoking status, coexisting comorbidities, outcomes after COVID-19 infection, and PMD genotype-phenotype. Baseline status was assessed using the modified Rankin scale (mRS) and the Newcastle Mitochondrial Disease Adult Scale (NMDAS).p = 0.003) and mRS (p = 0.001), presence of respiratory dysfunction (p < 0.001), neurologic involvement (p = 0.003), and more than 4 comorbidities (p = 0.002). In multivariable analysis, respiratory dysfunction was independently associated with COVID-19 hospitalization .Seventy-nine subjects with PMDs from 10 countries were included (mean age 41.5 ± 18 years): 25 (32%) were hospitalized, 48 (61%) recovered fully, 28 (35%) improved with sequelae, and 3 (4%) died. Statistically significant differences in hospitalization status were observed in baseline status, including the NMDAS score (Respiratory dysfunction is an independent risk factor for severe COVID-19 in PMDs while high disease burden and coexisting comorbidities contribute toward COVID-19–related hospitalization. These findings will enable risk stratification and informed management decisions for this vulnerable population. Inclusion criteria were (1) a PMD diagnosis (any age) in the opinion of the recruiting healthcare provider, clinically/histopathologically suspected and/or genetically confirmed; and (2) a COVID-19 diagnosis stratified as “confirmed,” “probable,” or “suspected” based on World Health Organization (WHO) definitions.5 Baseline status was assessed using the modified Rankin scale (mRS) and the Newcastle Mitochondrial Disease Adult Scale . Key comorbidities included respiratory dysfunction, mitochondrial diabetes ,7 hypertension or other cardiovascular diseases, obesity (body mass index ≥ 30), and neurologic involvement. Respiratory dysfunction was defined by the presence of at least one of the following: (1) obstructive lung disease (chronic obstructive pulmonary disease or asthma), (2) restrictive lung disease, (3) obstructive sleep apnea, (4) use of noninvasive ventilation, and (5) tracheostomy. Neurologic involvement was determined by the presence of at least one of the following: (1) dysphagia, (2) epilepsy, (3) learning disabilities, (4) polyneuropathy, (5) skeletal muscle weakness, and (6) stroke/stroke-like episodes. All comorbidities included under the “neurologic involvement” section were reported as disease-specific neurologic features in the mitochondrial disease module of the database. Only anonymized, nonidentifiable data were collected; thus, participant consent was not required according to the UK Health Research Authority. Statistical analyses were performed using SPSS v19.0 (SPSS Inc). p value < 0.05 was considered significant. The primary outcome of this study was hospitalization status for COVID-19. See the eMethods in the Supplement for further details concerning database design and data storage, protocol approvals, and participants.The NHS England Highly Specialized Services for Rare Mitochondrial Disorders in London and Newcastle, UK, designed a PMD-specific module within the “International Neuromuscular COVID-19 Database”Dr. Pizzamiglio had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.5 COVID-19 symptoms were present in 75 patients (95%). As previously reported in PMDs,8 the most common symptoms comprised fever , fatigue , persistent cough , headache , myalgia (39%), anosmia/hyposmia , and dysgeusia . Only 3 current or former smokers were identified in the cohort. Consequently, further statistical analysis in this subgroup was not possible.From May 1, 2020, to May 31, 2021, 79 patients  across 10 countries . In patients with respiratory dysfunction, there were 14 subjects with restrictive lung disease, 6 with obstructive lung disease, 6 using noninvasive ventilation, 3 with obstructive sleep apnea, and 2 with a tracheostomy (see eTable 2 in the Supplement).Neurologic involvement was the most common comorbidity and was present in 58 subjects (73%), followed by mitochondrial diabetes , hypertension or other cardiovascular diseases , respiratory dysfunction , and obesity . Many patients had multiple single-system and/or multisystem comorbidities. Of those with neurologic problems, 36 had skeletal muscle weakness, 21 dysphagia, 16 epilepsy, 12 learning disabilities, 8 polyneuropathy, and 8 stroke-like episodes . The presence of ≥4 comorbidities was associated with hospitalization , in line with previous data from general adult population.9 Analysis of individual comorbidities showed that the presence of respiratory dysfunction  and neurologic involvement  were associated with COVID-19–related hospitalization, and were both present in the 3 deceased subjects. No significant difference in hospitalization status was detected by age, sex, ethnicity, or PMD diagnosis.Univariate analysis  showed tp = 0.002). The results of the sensitivity multivariate are the same when only confirmed cases (n = 65) are included .Multivariable analysis adjusted for age group, respiratory dysfunction, neurologic involvement, and the mRS, discerned respiratory dysfunction was independently associated with COVID-19 hospitalization , worsening of isolated fatigue (n = 4), and development of new symptoms (n = 13). Limitations of this study include the cross-sectional design and voluntary nature of the registry; thus, a selection bias toward severe, rather than asymptomatic and mild, COVID-19 could exist. Furthermore, the limited sample size does not allow outcomes to be correlated with specific genotypes or comorbidities.To date, this study represents the largest cohort of people with PMDs and COVID-19 ever reported. We have identified a group of patients with PMD who are extremely vulnerable to SARS-CoV-2 because of respiratory dysfunction. Their respiratory dysfunction is an independent risk factor for developing severe COVID-19, and coexisting comorbidities and high disease burden contribute toward the risk of COVID-19–related hospitalization, a factor now recognized in other chronic neurologic disorders.In conclusion, the data presented here were collected during the initial stages of the COVID-19 pandemic and were not available to influence clinical decision making during the initial wave. However, we consider that in future waves of COVID-19, or similar pandemics, this information will be crucial to clinicians worldwide managing patients with PMDs and other conditions where there is mitochondrial dysfunction and significant respiratory muscle weakness.


Salt stress adversely affected the growth parameters. In salt-treated plants, proline content and oxidative stress indicators such as malondialdehyde (MDA) content and hydrogen peroxide (H2O2) content were increased with the increment of salt concentration but the relative water content decreased. Due to salt stress catalase (CAT), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glyoxalase I (Gly I) and glyoxalase II (Gly II) activity decreased. However, the activity of ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST) and peroxidase (POD) increased under salt stress. On the contrary, supplementation of Se, B and Se+B enhanced the activities of APX, MDHAR, DHAR, GR, CAT, GPX, GST, POD, Gly I and Gly II which consequently diminished the H2O2 content and MDA content under salt stress, and also improved the growth parameters. The results reflected that exogenous Se, B and Se+B enhanced the enzymatic activity of the antioxidant defense system as well as the glyoxalase systems under different levels of salt stress, ultimately alleviated the salt-induced oxidative stress, among them Se+B was more effective than a single treatment.The present investigation was executed with an aim to evaluate the role of exogenous selenium (Se) and boron (B) in mitigating different levels of salt stress by enhancing the reactive oxygen species (ROS) scavenging, antioxidant defense and glyoxalase systems in soybean. Plants were treated with 0, 150, 300 and 450 mM NaCl at 20 days after sowing (DAS). Foliar application of Se (50 µM Na Abiotic stress or environmental stress is not a sole entity. Under the umbrella of abiotic stress, it comprises all types of hostile environmental conditions that a plant may face in nature . Salt st2SO4) are the most available soluble salts. An increase in salinity level, in most of the cases, indicates mainly an increase in Na+ and Cl− concentration. Both Na+ and Cl− ions produce critical conditions for plant survival, but between them, Cl− is more dangerous [2•−), hydrogen peroxide (H2O2), hydroxyl radical (•OH) etc. is needed to be stopped to protect the plants from oxidative damage and to regulate the proper physiological and biochemical activities. Plants maintain the balance between formation and detoxification of ROS by an antioxidant defense system [S-transferase (GST), and peroxidase (POD) coordinately act to control the ROS and ROS-induced oxidative stress, along with non-enzymatic components [In the soil solution, sodium chloride (NaCl) and sodium sulfate Na2SO are the angerous . Therefoangerous . The ovee system . Multiplmponents .+ accumulation in plant parts, Na+ compartmentalization, upregulating Na+ and Cl− ions transporter genes, chelation and boosting of the antioxidant defense system. Selenium protects plants from oxidative stress by triggering the detoxification of ROS, which is overgenerated due to salt stress [− content in sugar beet under salt stress by upregulating the transportation of Cl− ions [2O2) content under oxidative stress. Moreover, the application of B increased the antioxidant defense and decreased oxidative stress in different crops [Many investigations have been carried out and attempts have been taken to mitigate the hazardous effect of salt stress on crops ,10,11. At stress ,10,12. Bt stress . Boron it stress . Supplemt stress . ExogenoCl− ions . Supplemnt crops ,17. Howent crops .Glycine max L.) is a widely cultivated legume around the world because of its versatile uses and economic importance. It is a prominent source of proteins and edible oil, it has valuable uses as food, feed and oilseed crop [Soybean  detoxification was hardly studied. The present investigation was conducted to study the injurious effect of the different levels of salt stress on soybean and also to study the roles of Se, B and Se+B in mitigating salt-induced oxidative stress, and enhancing the salt stress tolerance in soybean by upregulating the antioxidant defense system and MG detoxification system.Upon exposure to 150 mM NaCl stress, the reduction in plant height was not significant but at 300 and 450 mM NaCl, stress plant height was reduced by 35 and 55%, respectively, in comparison to untreated control . However, supplementation of Se and Se+B increased the plant height under all salt treatments. At 150 and 300 mM NaCl stress B alone did not increase the plant height significantly, compared to a respective only salt-treated plant without Se, B and Se+B spray. Under mild, moderate and severe salinity Se+B spray increased the plant height by 17, 30 and 39%, respectively, compared to corresponding salt-treatment alone  was declined by 43, 63 and 78% at 150, 300 and 450 mM NaCl stress, in comparison to untreated control. Rather than single supplementation, Se+B combinedly increased the shoot FW significantly under salt stress. Application of Se+B spray increased the shoot FW by 22, 41 and 66% at 150, 300 and 450 mM NaCl stress, respectively, in comparison to corresponding only NaCl-treated plants B.−1 was statistically similar, in comparison to untreated control. However, Se and B alone did not increase the shoot DW significantly under salt stress. Combinedly Se+B increased the shoot DW plant−1 significantly by 85 and 93% at 150 and 300 mM NaCl stress but at 450 mM NaCl stress the increment of shoot DW was not significant, respectively, compared to respective salt treatment alone (Shoot dry weight (DW) was decreased by 41% at 150 mM NaCl stress, compared to untreated control. Moreover, at 300 and 450 mM NaCl stress the reduction in shoot DW plantnt alone C.In 150 mM NaCl-stressed plants, reduction in leaf area was not significant, compared to untreated control. Moreover, at 300 mM and 450 mM NaCl stress decrement of leaf area was statistically similar. Single supplementation of Se and B showed statistically similar results to Se+B and increased the leaf area at 300 and 450 mM NaCl stress but not at 150 mM NaCl stress. However, due to Se+B spray at 300 and 450 mM NaCl stress, leaf area was increased by 39 and 37%, respectively, compared to salt-treated plant alone . On the contrary, foliar application of Se, B and Se+B increased the leaf RWC and showed a statistically similar increment of leaf RWC under different levels of NaCl stress, compared to only salt-treated plants. Under mild, moderate and severe salinity Se+B spray increased the leaf RWC by 18, 21 and 20%, respectively, compared to corresponding salt-treatment alone  accumulation was observed in 150, 300 and 450 mM NaCl-stressed plants; 88, 131 and 180%, respectively, in comparison to untreated control. On the other hand, foliar-applied Se, B and Se+B diminished the Pro content in saline condition only. Moreover, the single and combined application of Se and B showed a statistically similar result in decreasing Pro content under salt stress. Application of Se+B decreased the Pro content by 20, 19 and 12% at 150, 300 and 450 mM NaCl stress, respectively, compared to corresponding only NaCl-treated plants . Moreover, foliar application of Se, B and Se+B further increased the APX activity in plants under different levels of salinity, compared to only salt-treated plants. Supplementation of Se+B enhanced the activity of APX at 150 mM NaCl stress which was statistically similar to the result of Se and B alone. However, at 300 and 450 mM NaCl stress, Se+B amplified the APX activity more than the individual supplementation of Se and B, and increased the APX activity by 19 and 19%, respectively, in comparison to corresponding salt treatment alone A.In response to 150, 300 and 450 mM NaCl stress, MDHAR activity was declined by 27, 42 and 54%, respectively, in comparison to untreated control. On the contrary, Se, B and Se+B treatments increased the MDHAR activity under salt stress, compared to corresponding salt treatment alone. Among them Se+B showed more increment in MDHAR activity than single supplementation. However, at 150, 300 and 450 mM NaCl stress exogenous Se+B enhanced the MDHAR activity by 43, 45 and 36%, respectively, in comparison to respective only salt-treated plants B.The DHAR activity was decreased by 12, 22 and 36% at 150, 300 and 450 mM NaCl, respectively, in comparison to untreated control. However, DHAR activity was enhanced under salt stress because of Se, B and Se+B spray, compared to the respective only salt treatment . A statistically similar enhancement was observed in the DHAR activity at 150 mM NaCl stress due to the application of Se, B and Se+B spray, compared to 150 mM NaCl stress . The combined application of Se+B increased the DHAR activity by 21 and 16%, respectively, at 300 and 450 mM NaCl, in comparison to the corresponding salt treatment alone which was followed by individual application of Se and B C.When exposed to 150, 300 and 450 mM NaCl, GR activity was increased by 45, 126 and 228%, respectively, compared to untreated control. However, exogenous supplementation of Se, B and Se+B further increased the GR activity at 150, 300 and 450 mM NaCl, compared to salt treatment alone. In combined Se+B increased the GR activity more than spraying with of Se or B alone, and due to exogenous Se+B the GR activity was accelerated by 54, 50 and 18% at 150, 300 and 450 mM NaCl, respectively, in comparison to respective only salt-treated plants D.All salt treatments caused a substantial reduction in the CAT activity, in comparison to untreated control. Under mild, moderate and severe salinity CAT activity was decreased by 33, 43 and 56%, respectively, compared to untreated control. On the contrary, foliar application of Se, B and Se+B enhanced the CAT activity under salt stress. Among the foliar supplementations, Se+B showed a higher increment in CAT activity than Se or B alone. Moreover, application of Se+B increased the CAT activity by 38, 25 and 27% under mild, moderate and severe salinity, respectively, in comparison to respective salt treatment alone A.In NaCl-treated plants, GPX activity was enhanced by 33, 108 and 125%, respectively, in comparison to untreated control. Moreover, foliar supplementation of Se, B and Se+B further stimulated the GPX activity under different levels of salinity. Among the treatments, under salt stress Se+B increased the GPX activity more than Se or B alone. Comparing with corresponding salt-treated plants , GPX activity was increased by 66, 20 and 29%, respectively, in Se+B-treated plants under mild, moderate and severe salinity B.Between untreated control plants and 150 mM salt-treated plants, no significant difference in GST activity was recorded. Moreover, at 300 and 450 mM salt stress GST activity was increased by 41 and 111%, in comparison to untreated control. However, at 150, 300 and 450 mM salt stress, application of Se+B further accelerated the GST activity by 21, 19 and 21%, respectively, compared to the respective only salt treatment. Although Se+B showed higher GST activity than an individual spray, but at 150 and 300 mM NaCl stress, B spray showed a similar increase in GST activity as Se+B spray, compared to only salt treatment C.When plants were exposed to 150, 300 and 450 mM salt stress, POD activity was increased by 58, 95 and 124%, respectively, in comparison to untreated control. Application of Se+B increased the POD activity by 15, 14 and 7% at 150, 300 and 450 mM salt stress, respectively, compared to respective salt treatment alone. Moreover, no significant increment in POD activity was observed at 150 and 450 mM NaCl stress because of single supplementation of B. Single supplementation of Se caused a statistically similar increment of POD activity as Se+B at 150 and 450 mM NaCl stress, compared to corresponding salt treatment alone D.The activity of Gly I and Gly II showed reverse relation with the salt concentration, Gly I and Gly II activity decreased with the gradual increase in the salt concentration.Under mild, moderate and severe salinity Gly I activity was declined by 26, 46 and 56%, respectively, in comparison to untreated control. On the contrary, foliar application of Se and B alone significantly increased the Gly I activity under mild and moderate salinity but not at severe salinity. Moreover, combined application of Se+B enhanced the Gly I activity by 31, 29 and 18% under mild, moderate and severe salinity, respectively, in comparison to corresponding salt treatment alone, followed by an individual spray of Se and B A.In response to mild, moderate and severe salinity the activity of Gly II was declined by 24, 46 and 59% in 150, 300 and 450 mM NaCl-treated plants, respectively, in comparison to untreated control . At 150 and 300 mM NaCl stress foliar application of Se+B increased the Gly II activity by 24 and 42%, respectively, compared to respective salt-treatment alone. Moreover, at 450 mM salt stress B alone and combined supplementation of Se+B showed a similar result, in the case of increasing the Gly II activity, respectively, in comparison to corresponding only salt-treated plant B.Salt stress has a detrimental effect on the growth, development and physiological activities of soybean ,21. In cIn response to salt stress shoot growth stunts . Akram e+ and Cl− inside the cell also restricts the water uptake. Therefore, the plant faces physiological drought (osmotic stress) under salt stress [− ion in xylem sap, thus improved the water uptake [Higher NaCl concentration in the soil solution of the root zone decreases the water potential and also hinders the water uptake through the root . Moreovet stress . Many ret stress ,29,30. Dt stress ,29,30. Ur uptake .P5CS) and proline dehydrogenase (PDH) genes and the activities of P5CS and PDH were involved in modulating Pro accumulation in Brassica napus [P5CS2 and PDH in fragrant rice [Higher accumulation of osmolytes occurs to protect the plant cells from NaCl-induced dehydration . Salt stca napus . Seleniuant rice . This ev2O2 is a ROS, overproduction of H2O2 results in higher lipid peroxidation which is indicated by higher MDA content [2O2 and MDA content in different soybean genotypes [2O2 content, as well as MDA content, increased with the increase in the NaCl concentrations. Supplementation of Se protected plants from salt-induced oxidative stress by triggering the detoxification of ROS, which were generated due to salt stress [2O2 content and as well as MDA content under salt stress [− transportation and protected plants from salt-induced oxidative stress [2O2 and MDA content) under salt stress due to Se, B and Se+B spray.Salt-induced osmotic stress, ionic stress, nutrient imbalance, disrupted ion homeostasis and excessive generation of ROS, altogether threaten the antioxidant defense system of plants. However, salt-induced oxidative stress deteriorates the cell membrane, cellular organelles, cellular components  and nucleic acids (DNA and RNA), and may also cause programmed cell death . As H2O2enotypes ,24,40. It stress . Moreovet stress . Boron pt stress . Supplemt stress . Foliar e stress . HoweverIn the antioxidant defense system four key enzymes viz. APX, MDHAR, DHAR and GR of the ascorbate-glutathione (AsA-GSH) pathway are crucial for enhancing stress tolerance as well as in minimizing the stress-induced oxidative damages, by detoxification of the ROS . Moreove2O and O2 by direct dismutation of H2O2. Thus, CAT plays a major role in ROS detoxification under abiotic stress [2O2 content under salt stress due to Se, B and Se+B spray indicated that Se and B alone and combinedly enhanced the antioxidant defense against salt-induced oxidative stress by increasing the ROS scavenging. The GPX enzymes are pivotal for cell protection and detoxification under oxidative stress. Selenium enhances the activity of GPX enzymes as it is a cofactor of GPX enzymes [2O2 by utilizing GSH [Catalase produces Hc stress . In prevc stress ,42. Simic stress ,29,43,44c stress . However enzymes ,46. Alonzing GSH . Upon exzing GSH ,34,47. Hzing GSH . Applicazing GSH . In the zing GSH . The actzing GSH .S-D-lactoyl-glutathione by utilizing GSH. In last step, S-D-lactoyl-glutathione is converted into D-lactate by Gly II [Methylglyoxal is a by-product of the glyoxalase system which is a highly reactive and cytotoxic compound. The enzymes of the glyoxalase system, viz. Gly I and Gly II transform MG into nontoxic compounds. In the glyoxalase system, MG detoxification occurs in two steps. First, Gly I converts MG into y Gly II . The acty Gly II ,42. Lowey Gly II . In the 2O2 and MDA content) increased. On the contrary, Se and B alone and combined improved the growth parameters, leaf RWC and decreased the ROS accumulation which was clear by the reduced H2O2 and MDA content (lipid peroxidation), and also decreased the Pro content when subjected to salt stress. All NaCl treatments decreased the MDHAR, DHAR and CAT activity and enhanced the APX, GR, GPX, GST and POD activity. However, exogenous Se, B and Se+B spray enhanced the enzymatic activities  as a part of the antioxidant defense system under salt stress. Along with other enzymatic activities, the activity of Gly I and Gly II were also increased due to single and combined application of Se and B in salt-treated plants. However, it is clear from the results of the present study that foliar-applied Se, B and Se+B mitigated the salt stress by modulating the antioxidant defense system, ROS metabolism and glyoxalase system  seeds were sown in plastic pots (14 L). Organic manure, urea, triple super phosphate and muriate of potash were applied as basal dose without B. At 20 days after sowing (DAS) along with control (0 mM NaCl), mild, moderate and severe salinity was imposed on plants by treating with 150, 300 and 450 mM NaCl, respectively. Single and combined supplementation of Se (50 µM Na2SeO4) and B (1 mM H3BO3) was accomplished at 16, 20, 24 and 28 DAS under control and saline conditions. The experiment was laid out in completely randomized design (CRD) with three replications.Healthy, matured, well-dried and uniform soybean  buffer (pH 7.0) and potassium iodide (KI). After that, H2O2 content was determined spectrophotometrically by measuring the optical absorption of supernatant at 390 nm by using an extinction coefficient of 0.28 μM−1 cm−1 which was expressed as nmol g−1 FW.According to Yang et al. , leaf sa−1 cm−1 and expressed as nmol g−1 FW.By following the method of Heath and Packer  lipid peProtein content was estimated by following the method of Bradford  in which2O2, ethylenediaminetetraacetic acid (EDTA) and extract for enzyme were used during the process where 2.8 mM–1 cm–1 was the extinction coefficient and it was expressed as μmol min−1 mg−1 protein.The APX (EC: 1.11.1.11) activity was estimated spectrophotometrically at 290 nm according to the method of Nakano and Asada, . The sol–1 cm–1 was the extinction coefficient [−1 mg−1 protein.The MDHAR (EC: 1.6.5.4) activity was determined from the reaction mixture consisting of Tris-HCl buffer (pH 7.5), AsA, ascorbate oxidase (AO), nicotinamide adenine dinucleotide phosphate (NADPH) along with enzyme and 6.2 mMfficient . The act–1 cm–1 was used during the enzyme calculation and DHAR activity was expressed as nmol min−1 mg−1 protein.The DHAR (EC: 1.8.5.1) activity was measured spectrophotometrically by following the method of Nahar et al.  at 265 n–1 cm–1 as the extinction coefficient [−1 mg−1 protein.The GR (EC: 1.6.4.2) activity was determined from the reaction of the reaction mixture which consists of K-P buffer (pH 7.0), oxidized glutathione (GSSG), EDTA, NADPH and enzyme solution at 340 nm using 6.2 mMfficient . The GR 2O2 and enzyme solution and computed using 39.4 M–1 cm–1 as the extinction coefficient and expressed as μmol min−1 mg−1 protein.The CAT (EC: 1.11.1.6) activity was estimated according to Hasanuzzaman et al.  at 240 n–1 cm–1 as the extinction coefficient [−1 mg−1 protein.The activity of GST (EC: 2.5.1.18) was measured from the reaction mixture of GSH, 1-chloro-2, 4-dinitrobenzene (CDNB), Tris-HCl buffer (pH 6.5) and enzyme extract, using 9.6 mMfficient . The GST3), NADPH, GR, H2O2 and enzyme at 340 nm. Extinction coefficient 6.62 mM–1 cm–1 was used during the calculation of enzyme activity [−1 mg−1 protein.The GPX (EC: 1.11.1.9) activity was read from the absorbance of the reaction between K-P buffer (pH 7.0), GSH, EDTA, sodium azide (NaNactivity . The GPX2O2. The activity was computed by using an extinction coefficient of 6.58 mM−1 cm−1 and it was expressed as μmol min−1 mg−1 protein.The POD activity (EC: 1.11.1.7) was determined according to Hemeda and Klein , at 470 –1 cm–1 as the extinction coefficient [−1 mg−1 protein.The Gly I (EC: 4.4.1.5) activity was read from the reaction of the mixture which consists of K-P buffer (pH 7.0), GSH, magnesium sulfate and MG at 240 nm by using 3.37 mMfficient . The GlyS-D-lactoylglutathione at 412 nm by using an extinction coefficient of 13.6 mM–1 cm–1 [−1 mg−1 protein.The Gly II (EC: 3.1.2.6) activity was measured from the reaction of enzyme, Tris-HCl buffer (pH 7.2), 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB) and M–1 cm–1 . The GlyAll data of three replications were statistically analyzed by using CoStat v.6. 400 . Data we2O2 content and MDA content. Moreover, growth parameters and leaf RWC were also improved due to supplementation of Se+B under salt stress. Though in the present study, Se and B alone showed similar results to the combined application, but it was evident that the combined application of Se+B was more effective than a single application to confer the different levels of salt stress. Therefore, further investigations should be performed to ascertain the doses of Se, B and Se+B under different levels of salinity to mitigate the salt stress, as a high concentration of Se and B is phytotoxic. The role of B in MG detoxification has rarely been studied, and the mechanism is not well known yet; thus, it needs advanced future investigations.The results of the present study clearly reveal that single and combined supplementation of Se and B mitigated the deleterious effect of salt stress and salt-induced oxidative stress in soybean, and also improved salt tolerance. However, exogenous Se, B and Se+B declined the salt-induced oxidative stress in soybean by upregulating the enzymatic activities of the antioxidant defense system and glyoxalase system, ultimately by diminishing the ROS accumulation which was indicated by the reduced H