Exome sequencing and functional analyses revealed CETN1 variants leads to impaired cell division and male fertility.

Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

Genomics empowering conservation action and improvement of celery in the face of climate change.

MAIN CONCLUSION: Integration of genomic approaches like whole genome sequencing, functional genomics, evolutionary genomics, and CRISPR/Cas9-based genome editing has accelerated the improvement of crop plants including leafy vegetables like celery in the face of climate change. The anthropogenic climate change is a real peril to the existence of life forms on our planet, including human and plant life. Climate change is predicted to be a significant threat to biodiversity and food security in the coming decades and is rapidly transforming global farming systems. To avoid the ghastly future in the face of climate change, the elucidation of shifts in the geographical range of plant species, species adaptation, and evolution is necessary for plant scientists to develop climate-resilient strategies. In the post-genomics era, the increasing availability of genomic resources and integration of multifaceted genomics elements is empowering biodiversity conservation action, restoration efforts, and identification of genomic regions adaptive to climate change. Genomics has accelerated the true characterization of crop wild relatives, genomic variations, and the development of climate-resilient varieties to ensure food security for 10 billion people by 2050. In this review, we have summarized the applications of multifaceted genomic tools, like conservation genomics, whole genome sequencing, functional genomics, genome editing, pangenomics, in the conservation and adaptation of plant species with a focus on celery, an aromatic and medicinal Apiaceae vegetable. We focus on how conservation scientists can utilize genomics and genomic data in conservation and improvement.

Heat stress induced piRNA alterations in pachytene spermatocytes and round spermatids.

BACKGROUND: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells. METHODS: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs. RESULTS: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts. CONCLUSIONS: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.

A comprehensive and conceptual overview of omics-based approaches for enhancing the resilience of vegetable crops against abiotic stresses.

MAIN CONCLUSION: Abiotic stresses adversely affect the productivity and production of vegetable crops. The increasing number of crop genomes that have been sequenced or re-sequenced provides a set of computationally anticipated abiotic stress-related responsive genes on which further research may be focused. Knowledge of omics approaches and other advanced molecular tools have all been employed to understand the complex biology of these abiotic stresses. A vegetable can be defined as any component of a plant that is eaten for food. These plant parts may be celery stems, spinach leaves, radish roots, potato tubers, garlic bulbs, immature cauliflower flowers, cucumber fruits, and pea seeds. Abiotic stresses, such as deficient or excessive water, high temperature, cold, salinity, oxidative, heavy metals, and osmotic stress, are responsible for the adverse activity in plants and, ultimately major concern for decreasing yield in many vegetable crops. At the morphological level, altered leaf, shoot and root growth, altered life cycle duration and fewer or smaller organs can be observed. Likewise different physiological and biochemical/molecular processes are also affected in response to these abiotic stresses. In order to adapt and survive in a variety of stressful situations, plants have evolved physiological, biochemical, and molecular response mechanisms. A comprehensive understanding of the vegetable's response to different abiotic stresses and the identification of tolerant genotypes are essential to strengthening each vegetable's breeding program. The advances in genomics and next-generation sequencing have enabled the sequencing of many plant genomes over the last twenty years. A combination of modern genomics (MAS, GWAS, genomic selection, transgenic breeding, and gene editing), transcriptomics, and proteomics along with next-generation sequencing provides an array of new powerful approaches to the study of vegetable crops. This review examines the overall impact of major abiotic stresses on vegetables, adaptive mechanisms and functional genomic, transcriptomic, and proteomic processes used by researchers to minimize these challenges. The current status of genomics technologies for developing adaptable vegetable cultivars that will perform better in future climates is also examined.

Genome-wide identification of DCL, AGO, and RDR gene families in wheat (Triticum aestivum L.) and their expression analysis in response to heat stress.

Key components of the RNA interference (RNAi) pathway include the Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) gene families. While these components have been studied in various plant species, their functional validation in wheat remains unexplored particularly under heat stress. In this study, a comprehensive genome-wide analysis to identify, and characterize DCL, AGO, and RDR genes in wheat and their expression patterns was carried out. Using phylogenetic analysis with orthologous genes from Arabidopsis and rice, we identified a total of 82 AGO, 31 DCL, and 31 RDR genes distributed across the 21 chromosomes of wheat. To understand the regulatory network, a network analysis of miRNAs that target RNA-silencing genes was performed. Our analysis revealed that 13 miRNAs target AGO genes, 8 miRNAs target DCL genes, and 10 miRNAs target RDR genes at different sites, respectively. Additionally, promoter analysis of the RNA-silencing genes was done and identified the presence of 132 cis-elements responsive to stress and phytohormones. To examine their expression patterns, we performed RNA-seq analysis in the flag leaf samples of wheat exposed to both normal and heat stress conditions. To understand the regulation of RNA silencing, we experimentally analysed the transcriptional changes in response to gradient heat stress treatments. Our results showed constitutive expression of the AGO1, AGO9, and DCL2 gene families, indicating their importance in the overall biological processes of wheat. Notably, RDR1, known to be involved in small interfering RNA (siRNA) biogenesis, exhibited higher expression levels in wheat leaf tissues. These findings suggest that these genes may play a role in responses to stress in wheat, highlighting their significance in adapting to environmental challenges. Overall, our study provides additional knowledge to understand the mechanisms underlying heat stress responses and emphasizes the essential roles of these gene families in wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01362-0.

Pediatric Scrub Typhus Manifesting with Multisystem Inflammatory Syndrome: A New Cause for Confusion or Concern-A Case Series.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has discovered a new disease called multisystem inflammatory syndrome in children (MIS-C). In developing nations, pediatricians must be mindful of the similarities between MIS-C and other tropical fevers such as scrub typhus. Not only should such patients be kept on high alert to rule out tropical diseases and receive appropriate treatment, such as steroids or immunomodulatory medications, but this is also concerning because, if rickettsial or bacterial infection is not detected through cultures and serology, steroid, or immunomodulatory treatment alone can be fatal. How to cite this article: Bhardwaj P, Sood M, Singh R. Pediatric Scrub Typhus Manifesting with Multisystem Inflammatory Syndrome: A New Cause for Confusion or Concern-A Case Series. Indian J Crit Care Med 2022;26(6):723-727.

Cell signaling in sperm midpiece ensures quiescence and survival in cauda epididymis.

Sperm in most mammalian species including rat, mice and human are kept completely quiescent (motionless) and viable for up to a few weeks in the cauda epididymis before ejaculation. Vigorous motility is initiated almost instantly upon sperm release from cauda during ejaculation. The molecular mechanisms that suppress sperm motility but increase cell survival during storage in cauda epididymis are not known. Intracellular signaling via phosphorylation cascades is quick events that may regulate motility and survival of transcriptionally inactive sperm. Pathscan intracellular signaling array provided the preliminary picture of cell signaling in quiescent and motile rat sperm, indicating upregulation of cell-survival pathways in quiescent sperm, which were downregulated during motility activation. Interactome of signaling proteins involved in motility activation was constructed by Search Tool for the Retrieval of Interacting Genes (STRING) software, which identified mitogen activated protein kinase-p38 (MAPK-p38), AKT, mTOR and their downstream target p70S6K as the key kinases regulating sperm function. Further validation was achieved by western blotting and pathway activators/inhibitors. Immunofluorescence localized the kinase proteins in the sperm mid-piece region (mitochondria), a known extra-nuclear target for these signaling pathways. Activators of these kinases inhibited sperm motility but increased viability, and vice versa was true for inhibitors, in most of the cases. Activators and inhibitors also affected sperm mitochondrial membrane potential, ATP content and reactive oxygen species (ROS) levels. Data suggest that sperm motility and survival are inversely complementary and critically regulated by intracellular cell signaling. Aberrant cell signaling in caudal sperm may affect cell survival (sperm concentration) and motility of ejaculated sperm.

Mitochondrial localization, import, and mitochondrial function of the androgen receptor.

Nuclear localization of androgen receptor (AR) directs transcriptional regulation of a host of genes, referred to as genomic signaling. Additionally, nonnuclear or nongenomic activities of the AR have long been described, but understanding of these activities remains elusive. Here, we report that AR is imported into and localizes to mitochondria and has a novel role in regulating multiple mitochondrial processes. Employing complementary experimental approaches of AR knockdown in AR-expressing cells and ectopic AR expression in AR-deficient cells, we demonstrate an inverse relationship between AR expression and mitochondrial DNA (mtDNA) content and transcription factor A, mitochondrial (TFAM), a regulator of mtDNA content. We show that AR localizes to mitochondria in prostate tissues and cell lines and is imported into mitochondria in vitro We also found that AR contains a 36-amino-acid-long mitochondrial localization sequence (MLS) capable of targeting a passenger protein (GFP) to the mitochondria and that deletion of the MLS abolishes the import of AR into the mitochondria. Ectopic AR expression reduced the expression of oxidative phosphorylation (OXPHOS) subunits. Interestingly, AR also controlled translation of mtDNA-encoded genes by regulating expression of multiple nuclear DNA-encoded mitochondrial ribosomal proteins. Consistent with these observations, OXPHOS supercomplexes were destabilized, and OXPHOS enzymatic activities were reduced in AR-expressing cells and restored upon AR knockdown. Moreover, mitochondrial impairment induced AR expression and increased its translocation into mitochondria. We conclude that AR localizes to mitochondria, where it controls multiple mitochondrial functions and mitonuclear communication. Our studies also suggest that mitochondria are novel players in nongenomic activities of AR.

Characterization of genetic diversity and population structure in wheat using array based SNP markers.

Genetic diversity is crucial for successful adaptation and sustained improvement in crops. India is bestowed with diverse agro-climatic conditions which makes it rich in wheat germplasm adapted to various niches. Germplasm repository consists of local landraces, trait specific genetic stocks including introgressions from wild relatives, exotic collections, released varieties, and improved germplasm. Characterization of genetic diversity is done using morpho-physiological characters as well as by analyzing variations at DNA level. However, there are not many reports on array based high throughput SNP markers having characteristics of genome wide coverage employed in Indian spring wheat germplasm. Amongst wheat SNP arrays, 35K Axiom Wheat Breeder's Array has the highest SNP polymorphism efficiency suitable for genetic mapping and genetic diversity characterization. Therefore, genotyping was done using 35K in 483 wheat genotypes resulting in 14,650 quality filtered SNPs, that were distributed across the B (~ 50%), A (~ 39%), and D (~ 10%) genomes. The total genetic distance coverage was 4477.85 cM with 3.27 SNP/cM and 0.49 cM/SNP as average marker density and average inter-marker distance, respectively. The PIC ranged from 0.09 to 0.38 with an average of 0.29 across genomes. Population structure and Principal Coordinate Analysis resulted in two subpopulations (SP1 and SP2). The analysis of molecular variance revealed the genetic variation of 2% among and 98% within subpopulations indicating high gene flow between SP1 and SP2. The subpopulation SP2 showed high level of genetic diversity based on genetic diversity indices viz. Shannon's information index (I) = 0.648, expected heterozygosity (He) = 0.456 and unbiased expected heterozygosity (uHe) = 0.456. To the best of our knowledge, this study is the first to include the largest set of Indian wheat genotypes studied exclusively for genetic diversity. These findings may serve as a potential source for the identification of uncharacterized QTL/gene using genome wide association studies and marker assisted selection in wheat breeding programs.

Small RNAs: an ideal choice as sperm quality biomarkers.

Spermatozoa were classically known as vehicles for the delivery of the paternal genome to the oocyte. However, in 1962, spermatozoa were discovered to carry significant amounts of RNA in them, which raised questions about the significance of these molecules in such a highly specialized cell. Scientific research in the last six decades has investigated the biological significance of sperm RNAs by various means. Irrespective of what sperm RNAs do, their presence in spermatozoa has attracted attention for their exploitation as biomarkers of fertility. Research in this direction started in the year 2000 and is still underway. A major hurdle in this research is the definition of the standard human sperm RNAome. Only a few normozoospermic samples have been analyzed to define the normal sperm RNAome. In this article, we provide a perspective on the suitability of sperm RNAs as biomarkers of fertility and the importance of defining the normal sperm RNAome before we can succeed in identifying RNA-based biomarkers of sperm quality and fertility. The identification of sperm RNA biomarkers of fertility can be exploited for quality screening of donor sperm samples, explain infertility in idiopathic cases, and RNA therapeutics for the treatment of male infertility.

Needle Stick Injury Among Healthcare Workers in a Tertiary Care Setting in Dehradun, Sub-Himalayan Region: A Four-Year Record-Based Study.

INTRODUCTION: Needlestick injuries (NSIs) represent a significant occupational health risk in healthcare settings. These injuries, caused by contaminated sharps such as needles, vials, and scalpel blades, can lead to percutaneous exposure to infectious materials. Despite the severity of NSIs, they often go unreported, highlighting a critical gap in occupational safety protocols. AIMS: This study aimed to investigate the occurrence of NSIs among healthcare workers (HCWs) by sex, profession, and working areas. It also sought to explore the underlying reasons for these injuries and the factors contributing to their underreporting. METHODOLOGY: Adhering to the RECORD guidelines (Reporting of studies Conducted using Observational Routinely Collected Data), this record-based study involved a retrospective analysis of reported NSIs. Data were collected from voluntary reports by HCWs who experienced NSIs or exposure to potentially infectious materials such as blood and body fluids. Statistical analysis was conducted using IBM SPSS Statistics for Windows, Version 16 (Released 2007; IBM Corp., Armonk, New York) and Microsoft Excel 2010 (Microsoft Corporation, Redmond, Washington). RESULTS: Data from 142 participants indicated a higher proportion of females experiencing NSIs compared to males, with rates of 57.7% pre-COVID and 60.6% during COVID. There were notable shifts in NSI rates across professions, with increases observed among staff nurses and ward attendants/helpers. Analysis of injury circumstances revealed a decrease in sampling procedure-related injuries but an increase during intravenous procedures and biomedical waste segregation. Injuries occurring on the right-hand index finger decreased from 52.1% pre-COVID to 31% during COVID, while those on the left-hand index finger increased from 19.7% pre-COVID to 39.4% during COVID. Statistically significant associations were found between the injury site and the place of occurrence (p=0.021). Healthcare professionals commonly cleansed the site with disinfectants and used personal protective equipment (PPE) kits, with increased PPE usage noted during the COVID-19 pandemic. These findings emphasize the evolving dynamics of NSIs among HCWs and underscore the importance of tailored preventive measures during pandemics.

An Observational Study of Fungal Infections in COVID-19: Highlighting the Role of Mucormycosis in Tertiary Healthcare Settings.

Background Fungal infections, especially mucormycosis, have remarkably surged during the coronavirus disease 2019 (COVID-19) era, especially during the second wave peak of the pandemic raising the concern of the clinicians for the admitted patients. Steroid therapy, diabetes, and other immunocompromised states are more commonly associated with COVID-19-associated mucormycosis (CAM). Aim and objective The aim of this study is to ascertain the prevalence of fungal infections amidst the second wave of the COVID-19 pandemic and discern the associated risk factors. Materials and methods During the second peak of COVID-19, samples were received in the microbiology laboratory from all clinically suspected mucormycosis patients. These samples underwent processing for potassium hydroxide (KOH) wet mount, fungal culture on Sabouraud's dextrose agar (SDA) medium, and COVID-19 reverse transcription-polymerase chain reaction (RT-PCR) testing. All relevant clinical and associated risk factors were tabulated and analyzed. Results Among the 107 suspected cases of mucormycosis, 39 (36.4%) were confirmed positive for COVID-19 via RT-PCR, while 68 (63.6%) tested negative. Males exhibited a predominant infection rate, with the rhinocerebral system being the most commonly affected site. Significantly higher mortality rates were observed in COVID-19-associated mucormycosis (CAM) patients (33.4%) compared to those without COVID-19 (5.9%), with a notable p-value of 0.0005. CAM patients also demonstrated a higher frequency of ICU admissions (77%) compared to non-COVID-19-associated mucormycosis patients (21.4%), a statistically significant finding (p-value of 0.007). Additionally, immunocompromised states, diabetes, and the administration of oxygen therapy were identified as significant risk factors in CAM (p < 0.05). Notably, mucormycosis accounted for the majority of fungal isolates (48.27%) among COVID-19 patients. Conclusion Mucormycosis infection is more commonly seen in COVID-19-infected patients as compared to non-COVID-19 patients, especially with comorbidities such as diabetes mellitus, steroid usage, and other immunocompromised states.

Comparative performance of biofire pneumonia panel and standard culture-based methods for diagnosing pneumonia in critically ill patients: Impact on antibiotic stewardship.

INTRODUCTION: Lower respiratory tract infections (LRTIs) are a common cause of morbidity and mortality worldwide. Accurate identification of the pathogens causing LRTIs is crucial for ensuring of diagnostic and antibiotic stewardship. The Biofire Pneumonia Panel (BFPP) is a molecular diagnostic test that allows rapid detection of various bacterial and viral pathogens. In this study, we compared the performance of BFPP with standard culture methods for the detection of pathogens. MATERIALS AND METHODS: Respiratory samples from 70 patient with suspected LRTIs were tested using both BFPP and standard culture methods. The distribution of isolated bacterial pathogens was analyzed, and the sensitivity and specificity of BFPP were calculated. Additionally, the performance of BFPP in detecting antimicrobial resistance genes was evaluated. The results were compared with those obtained from VITEK-2 antimicrobial susceptibility testing and culture-based methods. RESULTS: Among the suspected LRTI cases, BFPP identified a single pathogen in 32.8% of cases and multiple pathogens in 40% of cases. The standard culture method detected a single pathogen in 47.1% of cases. BFPP showed a sensitivity of 93.9% and a specificity of 45.9% for the total sample. The performance of BFPP in detecting antimicrobial resistance genes varied for different pathogens with overall sensitivity of 40.1% and specificity of 95.9%. CONCLUSION: The Biofire Pneumonia Panel (BFPP) demonstrated high sensitivity for several bacterial pathogens, indicating its potential as a rapid diagnostic tool. However, its performance varied for different microorganisms, and it had limitations in detecting certain pathogens and antimicrobial resistance genes for which still required more further studies to explore different resistance gene mechanism that can be incorporated in this panel in future. The BFPP can complement standard culture methods as a rapid tool in the diagnosis of LRTIs.

Monitoring deforestation, forest health, and environmental criticality in a protected area periphery using Geospatial Techniques.

Protected areas in South Asia face significant challenges due to human disturbance and deforestation. The ongoing debate surrounds the recent surge in illegal encroachment of forest buffer zones in the Musali divisional secretariat division (DSD), which has led to a significant loss of forest cover over the past three decades. In this context, detecting changes in forest cover, assessing forest health, and evaluating environmental quality are crucial for sustainable forest management. As such, our efforts focused on assessing forest cover dynamics, forest health, and environmental conditions in the DSD from 1988 to 2022. We employed standardized image processing techniques, utilizing Landsat-5 (TM) and Landsat-8 (OLI) images. However, the forest area in the DSD has shown minimal changes, and environmental conditions and forest health have illustrated considerable spatial-temporal variations over the 34 years. The results indicated that 8.5 km2 (1.9%) of forest cover in the DSD has been converted to other land use classes. Overall, the Normalized Difference Vegetation Index (NDVI) has declined over time, while Land Surface Temperature (LST) exhibits an increasing trend. The regression results demonstrated a robust inverse relationship between LST and NDVI. The declining vegetation conditions and the increasing LST contribute to an increase in environmental criticality. The derived maps and indices will be beneficial for forest authorities in identifying highly sensitive locations. Additionally, they could enable land use planners to develop sustainable land management strategies.

Optimizing forage harvest and the nutritive value of Italian ryegrass-based mixed forage cropping under northwestern Himalayan conditions.

The scarcity of high-quality forage has a significant influence on the productivity and profitability of livestock. Addressing this concern, an investigation was undertaken to assess the effects of distinct Italian ryegrass genotypes, namely, Punjab ryegrass-1, Kashmir collection, and Makhan grass, in conjunction with varying seeding ratios of Italian ryegrass to Egyptian clover. The seeding ratios considered were 100:0 (Italian ryegrass to Egyptian clover), 75:25, 50:50, and 25:75. All possible combinations of Italian ryegrass and Egyptian clover with seeding ratios were set up in a randomized complete block design and replicated thrice. Co-cultivating Italian ryegrass and Egyptian clover at a 75:25 seeding ratio yields the best yield benefit, as determined by the land equivalent ratio. It is noteworthy that in this configuration, real yield loss is higher for Egyptian clover and for Italian ryegrass when the seeding ratio is 25:75. The higher competitiveness of Italian ryegrass in comparison to Egyptian clover is highlighted by the competitive ratio. Notably, the nutritive parameter, crude protein yield, was significantly higher in the Makhan grass-based 50:50 and 75:25 seeding ratio. Results of the study ascertained the compatibility of grass-legume co-cultivation with significantly higher quantity and quality forage harvested under mixed cropping systems whereas Makhan grass as the superior and dominant genotype in comparison to Kashmir collection. The outcomes of this study revealed that the 100:0 seeding ratio, coupled with the Makhan grass genotype, exhibited superior performance in terms of cumulative forage harvest, dry matter accumulation, net returns, and benefit-cost ratio.

Computational insights into allosteric inhibition of focal adhesion kinase: A combined pharmacophore modeling and molecular dynamics approach.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that modulates integrin and growth factor signaling pathways and is implicated in cancer cell migration, proliferation, and survival. Over the past decade various, FAK kinase, FERM, and FAT domain inhibitors have been reported and a few kinase domain inhibitors are under clinical consideration. However, few of them were identified as multikinase inhibitors. In kinase drug design selectivity is always a point of concern, to improve selectivity allosteric inhibitor development is the best choice. The current research utilized a pharmacophore modeling (PM) approach to identify novel allosteric inhibitors of FAK. The all-available allosteric inhibitor bound 3D structures with PDB ids 4EBV, 4EBW, and 4I4F were utilized for the pharmacophore modeling. The validated PM models were utilized to map a database of 770,550 compounds prepared from ZINC, EXIMED, SPECS, ASINEX, and InterBioScreen, aiming to identify potential allosteric inhibitors. The obtained compounds from screening step were forwarded to molecular docking (MD) for the prediction of binding orientation inside the allosteric site and the results were evaluated with the known FAK allosteric inhibitor (REF). Finally, 14 FAK-inhibitor complexes were selected from the docking study and were studied under molecular dynamics simulations (MDS) for 500 ns. The complexes were ranked according to binding free energy (BFE) and those demonstrated higher affinity for allosteric site of FAK than REF inhibitors were selected. The selected complexes were further analyzed for intermolecular interactions and finally, three potential allosteric inhibitor candidates for the inhibition of FAK protein were identified. We believe that identified scaffolds may help in drug development against FAK as an anticancer agent.

Testicular piRNA Analysis Identified Dysregulated piRNAs in Non-obstructive Azoospermia.

Male infertility has remained idiopathic in a remarkable proportion of all cases. Gonadal expression of PIWI-interacting RNAs (piRNAs) has been shown to be vital to normal spermatogenesis, as they are expressed in almost all types of testicular germ cells. These molecules and their related Piwi proteins strictly regulate transposable elements' activity and gene expression. We aimed to identify dysregulated piRNAs in idiopathic non-obstructive azoospermic (NOA) testis by global expression analysis. Testis tissue samples from 18 azoospermic patients (ten NOA and eight OA) were studied by small RNA sequencing. To validate high-throughput sequencing data, quantitative real-time polymerase chain reactions for two differentially altered piRNAs were performed. Bioinformatics analyses were undertaken to identify pathways affected by piRNA dysregulation. In the NOA group, 1328 piRNAs were identified to be differentially expressed, of which 1322 were downregulated and 6 were upregulated. Bioinformatics analysis corroborated the involvement of dysregulated piRNA in spermatogenesis. We also identified 64 clusters of differentially expressed piRNAs, of which 42 clusters had a minimum of ten absolute piRNA hits. Our study suggests that piRNAs show significant dysregulation in infertility. Their target genes play a role in their self-biogenesis, probably by regulating their own production through a feedback mechanism. The downregulated piRNAs may find value as biomarkers for the presence of spermatozoa in the testis of azoospermic individuals, while the upregulated piRNAs are great candidates for further investigation of their precise functions in spermatogenesis.

Modelled impact of virtual fractional flow reserve in patients undergoing coronary angiography (VIRTU-4).

BACKGROUND: The practical application of 'virtual' (computed) fractional flow reserve (vFFR) based on invasive coronary angiogram (ICA) images is unknown. The objective of this cohort study was to investigate the potential of vFFR to guide the management of unselected patients undergoing ICA. The hypothesis was that it changes management in >10% of cases. METHODS: vFFR was computed using the Sheffield VIRTUheart system, at five hospitals in the North of England, on 'all-comers' undergoing ICA for non-ST-elevation myocardial infarction acute coronary syndrome (ACS) and chronic coronary syndrome (CCS). The cardiologists' management plan (optimal medical therapy, percutaneous coronary intervention (PCI), coronary artery bypass surgery or 'more information required') and confidence level were recorded after ICA, and again after vFFR disclosure. RESULTS: 517 patients were screened; 320 were recruited: 208 with ACS and 112 with CCS. The median vFFR was 0.82 (0.70-0.91). vFFR disclosure did not change the mean number of significantly stenosed vessels per patient (1.16 (±0.96) visually and 1.18 (±0.92) with vFFR (p=0.79)). A change in intended management following vFFR disclosure occurred in 22% of all patients; in the ACS cohort, there was a 62% increase in the number planned for medical management, and in the CCS cohort, there was a 31% increase in the number planned for PCI. In all patients, vFFR disclosure increased physician confidence from 8 of 10 (7.33-9) to 9 of 10 (8-10) (p<0.001). CONCLUSION: The addition of vFFR to ICA changed intended management strategy in 22% of patients, provided a detailed and specific 'all-in-one' anatomical and physiological assessment of coronary artery disease, and was accompanied by augmentation of the operator's confidence in the treatment strategy.

Effects of Varicocele Repair on Sperm DNA Fragmentation and Seminal Malondialdehyde Levels in Infertile Men with Clinical Varicocele: A Systematic Review and Meta-Analysis.

PURPOSE: Varicoceles can be a source of elevated seminal oxidative stress (OS) and sperm DNA fragmentation (SDF). However, it remains unclear whether varicocele repair (VR) could reduce these parameters. This systematic review and meta-analysis (SRMA) aims to investigate the impact of VR on SDF and seminal malondialdehyde (MDA). MATERIALS AND METHODS: A literature search was performed in Scopus, PubMed, Ovid, Embase, and Cochrane databases. This SRMA included randomized controlled trials and observational studies reporting the pre- and postoperative levels of SDF and seminal OS in infertile men with clinical varicocele that underwent VR. Subgroup analyses included techniques of VR and SDF testing. The effect size was expressed as standardized mean difference (SMD). RESULTS: Out of 1,632 abstracts assessed for eligibility, 29 studies with 1,491 infertile men were included. The analysis showed a significant reduction in SDF after VR, compared to preoperative values (SMD -1.125, 95% confidence interval [CI] -1.410, -0.840; p<0.0001) with high inter-study heterogeneity (I²=90.965%). Reduction in SDF was evident with microsurgical technique and non-microsurgical inguinal approaches (SMD -1.014, 95% CI -1.263, -0.765; p<0.0001, and SMD -1.495, 95% CI -2.116, -0.873; p<0.0001), respectively. Reduction in SDF was significant irrespective of testing was done by sperm chromatin dispersion (SMD -2.197, 95% CI -3.187, -1.207; p<0.0001), sperm chromatin structure assay (SMD -0.857, 95% CI -1.156, -0.559; p<0.0001) or TUNEL (SMD -1.599, 95% CI -2.478, -0.719; p<0.0001). A significant decrease in seminal MDA levels was observed following VR (SMD -2.450, 95% CI -3.903 to -0.997, p=0.001) with high inter-study heterogeneity (I²=93.7%). CONCLUSIONS: Using pre- and post-intervention data, this SRMA indicates a significant reduction in SDF and seminal MDA levels in infertile men with clinical varicocele treated with VR. These findings may have important implications for the future management of this selected group of infertile patients.

Does Varicocele Repair Improve Conventional Semen Parameters? A Meta-Analytic Study of Before-After Data.

PURPOSE: The purpose of this meta-analysis is to study the impact of varicocele repair in the largest cohort of infertile males with clinical varicocele by including all available studies, with no language restrictions, comparing intra-person conventional semen parameters before and after the repair of varicoceles. MATERIALS AND METHODS: The meta-analysis was performed according to PRISMA-P and MOOSE guidelines. A systematic search was performed in Scopus, PubMed, Cochrane, and Embase databases. Eligible studies were selected according to the PICOS model (Population: infertile male patients with clinical varicocele; Intervention: varicocele repair; Comparison: intra-person before-after varicocele repair; Outcome: conventional semen parameters; Study type: randomized controlled trials [RCTs], observational and case-control studies). RESULTS: Out of 1,632 screened abstracts, 351 articles (23 RCTs, 292 observational, and 36 case-control studies) were included in the quantitative analysis. The before-and-after analysis showed significant improvements in all semen parameters after varicocele repair (except sperm vitality); semen volume: standardized mean difference (SMD) 0.203, 95% CI: 0.129-0.278; p<0.001; I²=83.62%, Egger's p=0.3329; sperm concentration: SMD 1.590, 95% CI: 1.474-1.706; p<0.001; I²=97.86%, Egger's p<0.0001; total sperm count: SMD 1.824, 95% CI: 1.526-2.121; p<0.001; I²=97.88%, Egger's p=0.0063; total motile sperm count: SMD 1.643, 95% CI: 1.318-1.968; p<0.001; I²=98.65%, Egger's p=0.0003; progressive sperm motility: SMD 1.845, 95% CI: 1.537%-2.153%; p<0.001; I²=98.97%, Egger's p<0.0001; total sperm motility: SMD 1.613, 95% CI 1.467%-1.759%; p<0.001; l2=97.98%, Egger's p<0.001; sperm morphology: SMD 1.066, 95% CI 0.992%-1.211%; p<0.001; I²=97.87%, Egger's p=0.1864. CONCLUSIONS: The current meta-analysis is the largest to date using paired analysis on varicocele patients. In the current meta-analysis, almost all conventional semen parameters improved significantly following varicocele repair in infertile patients with clinical varicocele.