Microglia: a promising therapeutic target in spinal cord injury.

Microglia are present throughout the central nervous system and are vital in neural repair, nutrition, phagocytosis, immunological regulation, and maintaining neuronal function. In a healthy spinal cord, microglia are accountable for immune surveillance, however, when a spinal cord injury occurs, the microenvironment drastically changes, leading to glial scars and failed axonal regeneration. In this context, microglia vary their gene and protein expression during activation, and proliferation in reaction to the injury, influencing injury responses both favorably and unfavorably. A dynamic and multifaceted injury response is mediated by microglia, which interact directly with neurons, astrocytes, oligodendrocytes, and neural stem/progenitor cells. Despite a clear understanding of their essential nature and origin, the mechanisms of action and new functions of microglia in spinal cord injury require extensive research. This review summarizes current studies on microglial genesis, physiological function, and pathological state, highlights their crucial roles in spinal cord injury, and proposes microglia as a therapeutic target.

Vaccine-based immunotherapy and related preclinical models for glioma.

Glioma, the most common primary malignant tumor in the central nervous system (CNS), lacks effective treatments, and >60% of cases are glioblastoma (GBM), the most aggressive form. Despite advances in immunotherapy, GBM remains highly resistant. Approaches that target tumor antigens expedite the development of immunotherapies, including personalized tumor-specific vaccines, patient-specific target selection, dendritic cell (DC) vaccines, and chimeric antigen receptor (CAR) and T cell receptor (TCR) T cells. Recent studies show promising results in treating GBM and lower-grade glioma (LGG), fostering hope for future immunotherapy. This review discusses tumor vaccines against glioma, preclinical models in immunological research, and the role of CD4+ T cells in vaccine-induced antitumor immunity. We also summarize clinical approaches, challenges, and future research for creating more effective vaccines.

Whole transcriptome analysis to identify non-coding RNA regulators and hub genes in sperm of non-obstructive azoospermia by microarray, single-cell RNA sequencing, weighted gene co-expression network analysis, and mRNA-miRNA-lncRNA interaction analysis.

BACKGROUND: The issue of male fertility is becoming increasingly common due to genetic differences inherited over generations. Gene expression and evaluation of non-coding RNA (ncRNA), crucial for sperm development, are significant factors. This gene expression can affect sperm motility and, consequently, fertility. Understanding the intricate protein interactions that play essential roles in sperm differentiation and development is vital. This knowledge could lead to more effective treatments and interventions for male infertility. MATERIALS AND METHODS: Our research aim to identify new and key genes and ncRNA involved in non-obstructive azoospermia (NOA), improving genetic diagnosis and offering more accurate estimates for successful sperm extraction based on an individual's genotype. RESULTS: We analyzed the transcript of three NOA patients who tested negative for genetic sperm issues, employing comprehensive genome-wide analysis of approximately 50,000 transcript sequences using microarray technology. This compared gene expression profiles between NOA sperm and normal sperm. We found significant gene expression differences: 150 genes were up-regulated, and 78 genes were down-regulated, along with 24 ncRNAs up-regulated and 13 ncRNAs down-regulated compared to normal conditions. By cross-referencing our results with a single-cell genomics database, we identified overexpressed biological process terms in differentially expressed genes, such as "protein localization to endosomes" and "xenobiotic transport." Overrepresented molecular function terms in up-regulated genes included "voltage-gated calcium channel activity," "growth hormone-releasing hormone receptor activity," and "sialic acid transmembrane transporter activity." Analysis revealed nine hub genes associated with NOA sperm: RPL34, CYB5B, GOL6A6, LSM1, ARL4A, DHX57, STARD9, HSP90B1, and VPS36. CONCLUSIONS: These genes and their interacting proteins may play a role in the pathophysiology of germ cell abnormalities and infertility.

Unraveling the Significance of Nanog in the Generation of Embryonic Stem-like Cells from Spermatogonia Stem Cells: A Combined In Silico Analysis and In Vitro Experimental Approach.

Embryonic stem-like cells (ES-like cells) are promising for medical research and clinical applications. Traditional methods involve "Yamanaka" transcription (OSKM) to derive these cells from somatic cells in vitro. Recently, a novel approach has emerged, obtaining ES-like cells from spermatogonia stem cells (SSCs) in a time-related process without adding artificial additives to cell cultures, like transcription factors or small molecules such as pten or p53 inhibitors. This study aims to investigate the role of the Nanog in the conversion of SSCs to pluripotent stem cells through both in silico analysis and in vitro experiments. We used bioinformatic methods and microarray data to find significant genes connected to this derivation path, to construct PPI networks, using enrichment analysis, and to construct miRNA-lncRNA networks, as well as in vitro experiments, immunostaining, and Fluidigm qPCR analysis to connect the dots of Nanog significance. We concluded that Nanog is one of the most crucial differentially expressed genes during SSC conversion, collaborating with critical regulators such as Sox2, Dazl, Pou5f1, Dnmt3, and Cdh1. This intricate protein network positions Nanog as a pivotal factor in pathway enrichment for generating ES-like cells, including Wnt signaling, focal adhesion, and PI3K-Akt-mTOR signaling. Nanog expression is presumed to play a vital role in deriving ES-like cells from SSCs in vitro. Finding its pivotal role in this path illuminates future research and clinical applications.

Exploring the Molecular Tapestry: Organ-Specific Peptide and Protein Ultrafiltrates and Their Role in Therapeutics.

This study aims to characterize the proteome composition of organ-derived protein extracts from rabbits. Protein isolation was performed using soft homogenization and size exclusion via ultrafiltration. The proteome analysis of the ultrafiltrates was conducted using gel electrophoresis, and the mass spectrometry data were subjected to gene ontology analysis. Proteomic profiling revealed comprehensive protein profiles associated with RNA regulation, fatty acid binding, inflammatory response, oxidative stress, and metabolism. Additionally, our results demonstrate the presence of abundant small proteins, as observed in the mass spectrometry datasets. Small proteins and peptides are crucial in transcription modulation and various biological processes. The protein networks identified in the ultrafiltrates have the potential to enhance and complement biological therapeutic interventions. Data are available via ProteomeXchange with identifier PXD050039.

LRRK2 Gly2019Ser Mutation Promotes ER Stress via Interacting with THBS1/TGF-ß1 in Parkinson's Disease.

The gene mutations of LRRK2, which encodes leucine-rich repeat kinase 2 (LRRK2), are associated with one of the most prevalent monogenic forms of Parkinson's disease (PD). However, the potential effectors of the Gly2019Ser (G2019S) mutation remain unknown. In this study, the authors investigate the effects of LRRK2 G2019S on endoplasmic reticulum (ER) stress in induced pluripotent stem cell (iPSC)-induced dopamine neurons and explore potential therapeutic targets in mice model. These findings demonstrate that LRRK2 G2019S significantly promotes ER stress in neurons and mice. Interestingly, inhibiting LRRK2 activity can ameliorate ER stress induced by the mutation. Moreover, LRRK2 mutation can induce ER stress by directly interacting with thrombospondin-1/transforming growth factor beta1 (THBS1/TGF-ß1). Inhibition of LRRK2 kinase activity can effectively suppress ER stress and the expression of THBS1/TGF-ß1. Knocking down THBS1 can rescue ER stress by interacting with TGF-ß1 and behavior burden caused by the LRRK2 mutation, while suppression of TGF-ß1 has a similar effect. Overall, it is demonstrated that the LRRK2 mutation promotes ER stress by directly interacting with THBS1/TGF-ß1, leading to neural death in PD. These findings provide valuable insights into the pathogenesis of PD, highlighting potential diagnostic markers and therapeutic targets.

Sox2 Localization During Spermatogenesis and Its Association with other Spermatogenesis Markers Using Protein-Protein Network Analysis.

Background: Sox2 (SRY box2) is an essential transcription factor that plays a vital role in spermatogenesis and regulates the genes in this process. Sox2 is important for pluripotency, self-renewal, and even spermatogonial stem cell differentiation. This gene is found in pluripotent and specialized cells, and it is involved in their biological activities. Methods: Protein-protein interaction (PPI) network analysis was performed during spermatogenesis using NCBI, STRING, and Cytoscape databases. Then, after isolating spermatogonial stem cells from 6 C57BL/6 mice, mouse embryonic stem cells and ES-like cells were prepared. In the following, Sox2 expression was examined in differentiated and undifferentiated spermatogonia by immunohistochemistry (IMH), immunocytochemistry (ICC), and Fluidigm PCR (polymerase chain reaction). Finally, the results were compared using the Kruskal-Wallis and Dunn tests at the significance level of p<0.05. Results: The results of this experiment showed that contrary to expectations, Sox2 has cytoplasmic expression in undifferentiated cells and nuclear expression in differentiated cells in in vitro conditions. In addition, the expression of Sox2 increased during differentiation. Fluidigm PCR showed a significantly higher expression of Sox2 (p<0.05) in differentiated compared to undifferentiated spermatogonia. Sox2 has an interaction with other genes during spermatogenesis such as Oct4, Nanog, Klf4, Stra8, Smad1, Tcf3, and Osm. Conclusion: Sox2, which is known as a pluripotency marker, has a vital role in spermatogenesis and could be a differential marker. Sox2 has strong connections with other genes such as Oct4, Nanog, Klf4, Tcf3, Osm, Stra8, Lim2, Smad1, Gdnf, and Kit.

The Single-Dose Application of Interleukin-4 Ameliorates Secondary Brain Damage in the Early Phase after Moderate Experimental Traumatic Brain Injury in Mice.

Activation of the interleukin-4 (IL-4) pathway ameliorates secondary injury mechanisms after experimental traumatic brain injury (TBI); therefore, we assessed the effect of a therapeutic IL-4 administration on secondary brain damage after experimental TBI. We subjected 100 C57/Bl6 wildtype mice to controlled cortical impact (CCI) and administered IL-4 or a placebo control subcutaneously 15 min thereafter. Contusion volume (Nissl staining), neurological function (hole board, video open field, and CatWalkXT®), and the immune response (immunofluorescent staining) were analyzed up to 28 days post injury (dpi). Contusion volumes were significantly reduced after IL-4 treatment up to 14 dpi (e.g., 6.47 ± 0.41 mm3 vs. 3.80 ± 0.85 mm3, p = 0.011 3 dpi). Macrophage invasion and microglial response were significantly attenuated in the IL-4 group in the acute phase after CCI (e.g., 1.79 ± 0.15 Iba-1+/CD86+ cells/sROI vs. 1.06 ± 0.21 Iba-1/CD86+ cells/sROI, p = 0.030 in the penumbra 3 dpi), whereas we observed an increased neuroinflammation thereafter (e.g., mean GFAP intensity of 3296.04 ± 354.21 U vs. 6408.65 ± 999.54 U, p = 0.026 in the ipsilateral hippocampus 7 dpi). In terms of functional outcome, several gait parameters were improved in the acute phase following IL-4 treatment (e.g., a difference in max intensity of -7.58 ± 2.00 U vs. -2.71 ± 2.44 U, p = 0.041 3 dpi). In conclusion, the early single-dose administration of IL-4 significantly reduces secondary brain damage in the acute phase after experimental TBI in mice, which seems to be mediated by attenuation of macrophage and microglial invasion.

Altered G-Protein Transduction Protein Gene Expression in the Testis of Infertile Patients with Nonobstructive Azoospermia.

Recent studies have shown that several members of the G-protein-coupled receptors (GPCR) superfamily play crucial roles in the maintenance of ion-water homeostasis of the sperm and Sertoli cells, development of the germ cells, formation of the blood barrier, and maturation of sperm. The GPCR, guanyl-nucleotide exchange factor, membrane traffic protein, and small GTPase genes were analyzed by microarray and bioinformatics (3513 sperm and Sertoli cell genes). In the microarray analyses of three human cases with different nonobstructive azoospermia sperm, the expression of GOLGA8IP, OR2AT4, PHKA1, A2M, OR56A1, SEMA3G, LRRC17, APP, ARHGAP33, RABGEF1, NPY2R, GHRHR, LTB4R2, GRIK5, OR6K6, NAPG, OR6C65, VPS35, FPR3, and ARL4A was upregulated, while expression of MARS, SIRPG, OGFR, GPR150, LRRK1, and NGEF was downregulated. There was an increase in GBP3, GBP3, TNF, TGFB3, and CLTC expression in the Sertoli cells of three human cases with NOA, whereas expression of PAQR4, RRAGD, RAC2, SERPINB8, IRPB1, MRGPRF, RASA2, SIRPG, RGS2, RAP2A, RAB2B, ARL17, SERINC4, XIAP, DENND4C, ANKRA2, CSTA, STX18, and SNAP23 were downregulated. A combined analysis of Enrich Shiny Gene Ontology (GO), STRING, and Cytoscape was used to predict proteins' molecular interactions and then to recognize master pathways. Functional enrichment analysis showed that the biological process (BP), regulation of protein metabolic process, regulation of small GTPase-mediated signal transduction were significantly expressed in up-/downregulated differentially expressed genes (DEGs) in sperm. In molecular function (MF) experiments of DEGs that were up-/downregulated, it was found that GPCR activity, guanyl ribonucleotide binding, GTPase activity and nucleoside-triphosphatase activity were overexpressed. An analysis of GO enrichment findings of Sertoli cells showed BP and MF to be common DEGs. When these gene mutations have been validated, they can be used to create new GPCR antagonists or agonists that are receptor-selective.

Distinct neuroanatomical and neuropsychological features of Down syndrome compared to related neurodevelopmental disorders: a systematic review.

Objectives: We critically review research findings on the unique changes in brain structure and cognitive function characteristic of Down syndrome (DS) and summarize the similarities and differences with other neurodevelopmental disorders such as Williams syndrome, 22q11.2 deletion syndrome, and fragile X syndrome. Methods: We conducted a meta-analysis and systematic literature review of 84 studies identified by searching PubMed, Google Scholar, and Web of Science from 1977 to October 2022. This review focuses on the following issues: (1) specific neuroanatomic and histopathological features of DS as revealed by autopsy and modern neuroimaging modalities, (2) language and memory deficits in DS, (3) the relationships between these neuroanatomical and neuropsychological features, and (4) neuroanatomic and neuropsychological differences between DS and related neurodevelopmental syndromes. Results: Numerous post-mortem and morphometric neuroimaging investigations of individuals with DS have reported complex changes in regional brain volumes, most notably in the hippocampal formation, temporal lobe, frontal lobe, parietal lobe, and cerebellum. Moreover, neuropsychological assessments have revealed deficits in language development, emotional regulation, and memory that reflect these structural changes and are more severe than expected from general cognitive dysfunction. Individuals with DS also show relative preservation of multiple cognitive, linguistic, and social domains compared to normally developed controls and individuals with other neurodevelopmental disorders. However, all these neurodevelopment disorders exhibit substantial heterogeneity among individuals. Conclusion: People with Down syndrome demonstrate unique neurodevelopmental abnormalities but cannot be regarded as a homogenous group. A comprehensive evaluation of individual intellectual skills is essential for all individuals with neurodevelopment disorders to develop personalized care programs.

Correlating MRI-based brain volumetry and cognitive assessment in people with Down syndrome.

INTRODUCTION: Down syndrome (DS) is the most common genetic cause of intellectual disability. Children and adults with DS show deficits in language performance and explicit memory. Here, we used magnetic resonance imaging (MRI) on children and adults with DS to characterize changes in the volume of specific brain structures involved in memory and language and their relationship to features of cognitive-behavioral phenotypes. METHODS: Thirteen children and adults with the DS phenotype and 12 age- and gender-matched healthy controls (age range 4-25) underwent an assessment by MRI and a psychological evaluation for language and cognitive abilities. RESULTS: The cognitive profile of people with DS showed deficits in different cognition and language domains correlating with reduced volumes of specific regional and subregional brain structures, confirming previous related studies. Interestingly, in our study, people with DS also showed more significant parahippocampal gyrus volumes, in agreement with the results found in earlier reports. CONCLUSIONS: The memory functions and language skills affected in studied individuals with DS correlate significantly with the reduced volume of specific brain regions, allowing us to understand DS's cognitive-behavioral phenotype. Our results provide an essential basis for early intervention and the design of rehabilitation management protocols.

Effects of a neurokinin-1 receptor antagonist in the acute phase after thoracic spinal cord injury in a rat model.

Objective: Disruption of the blood-spinal cord barrier (BSCB) with subsequent edema formation and further neuroinflammation contributes to aggravation of spinal cord injury (SCI). We aimed to observe the effect of antagonizing the binding of the neuropeptide Substance-P (SP) to its neurokinin-1 (NK1) receptor in a rodent SCI model. Methods: Female Wistar rats were subjected to a T9 laminectomy with or without (Sham) a T9 clip-contusion/compression SCI, followed by the implantation of an osmotic pump for the continuous, seven-day-long infusion of a NK1 receptor antagonist (NRA) or saline (vehicle) into the intrathecal space. The animals were assessed via MRI, and behavioral tests were performed during the experiment. 7 days after SCI, wet & dry weight and immunohistological analyses were conducted. Results: Substance-P inhibition via NRA showed limited effects on reducing edema. However, the invasion of T-lymphocytes and the number of apoptotic cells were significantly reduced with the NRA treatment. Moreover, a trend of reduced fibrinogen leakage, endothelial and microglial activation, CS-GAG deposition, and astrogliosis was found. Nevertheless, only insignificant general locomotion recovery could be observed in the BBB open field score and the Gridwalk test. In contrast, the CatWalk gait analysis showed an early onset of recovery in several parameters. Conclusion: Intrathecal administration of NRA might reinforce the integrity of the BSCB in the acute phase after SCI, potentially attenuating aspects of neurogenic inflammation, reducing edema formation, and improving functional recovery.

Modulation of Cellular Senescence in HEK293 and HepG2 Cells by Ultrafiltrates UPla and ULu Is Partly Mediated by Modulation of Mitochondrial Homeostasis under Oxidative Stress.

Protein probes, including ultrafiltrates from the placenta (UPla) and lung (ULu) of postnatal rabbits, were investigated in premature senescent HEK293 and HepG2 cells to explore whether they could modulate cellular senescence. Tris-Tricine-PAGE, gene ontology (GO), and LC-MS/MS analysis were applied to describe the characteristics of the ultrafiltrates. HEK293 and HepG2 cells (both under 25 passages) exposed to a sub-toxic concentration of hydrogen peroxide (H2O2, 300 µM) became senescent; UPla (10 µg/mL), ULu (10 µg/mL), as well as positive controls lipoic acid (10 µg/mL) and transferrin (10 µg/mL) were added along with H2O2 to the cells. Cell morphology; cellular proliferation; senescence-associated beta-galactosidase (SA-ß-X-gal) activity; expression of senescence biomarkers including p16 INK4A (p16), p21 Waf1/Cip1 (p21), HMGB1, MMP-3, TNF-α, IL-6, lamin B1, and phospho-histone H2A.X (γ-H2AX); senescence-related gene expression; reactive oxygen species (ROS) levels; and mitochondrial fission were examined. Tris-Tricine-PAGE revealed prominent detectable bands between 10 and 100 kDa. LC-MS/MS identified 150-180 proteins and peptides in the protein probes, and GO analysis demonstrated a distinct enrichment of proteins associated with "extracellular space" and "proteasome core complex". UPla and ULu modulated senescent cell morphology, improved cell proliferation, and decreased beta-galactosidase activity, intracellular and mitochondrial ROS production, and mitochondrial fission caused by H2O2. The results from this study demonstrated that UPla and Ulu, as well as lipoic acid and transferrin, could protect HEK293 and HepG2 cells from H2O2-induced oxidative damage via protecting mitochondrial homeostasis and thus have the potential to be explored in anti-aging therapies.

A review of the potential of induced pluripotent stem cell-derived exosome as a novel treatment for male infertility.

Exosomes are a subset of Extracellular vesicles (EVs) released by most cells in the body and can play a significant role in the intercellular connection. Researchers today claim that exosomes secreted by induced pluripotent stem cells (iPSCs) alone can play the same role as direct cell transplantation and, unlike iPSCs, do not lead to tumorigenesis. As a result, iPSC-derived exosomes (iPSC-Exos) have many applications in cell-free treatments and therapeutic effects on various diseases. Male infertility due to a defect or deficiency of spermatogonia to maintain spermatogenesis is one of the diseases that iPSC-Exos seems to be a new way to cure. However, the studies on the effect of iPSC-Exos on male infertility are very limited. In this review, we intend to provide a broader perspective on understanding the mechanisms of iPSC-Exos on spermatogenesis by collecting and reviewing some of the research conducted in this field.

A Fundamental Research in In Vitro Spermatogonial Stem Cell Culturing: What Are Clump Cells?

Spermatogonial stem cells (SSCs) are a small group of testicular cells located in the basement membrane of seminiferous tubules and can balance self-renewal and differentiation during spermatogenesis. Our in vitro culture experiments of mouse SSCs indicated heterogeneity of cultured cells. Highly compact colonies were observed next to SSC colonies, which we call clump cells. We used immunocytochemical staining to identify SSCs and somatic cells with VASA and Vimentin antibodies. Subsequently, we compared mRNA expression levels of VASA, DAZL, PLZF, GFRA1, Lin28, Kit, Myc and Vimentin genes using Fluidigm real-time RT-polymerase chain reaction in clump cells, SSCs, and testicular stromal cells. To better understand the functions of selected genes, we created a protein-protein interaction network and performed an enrichment analysis using different databases. Based on the data collected, we state that clump cells do not express the molecular markers of SSCs, so we cannot consider them as SSCs; however, we claim that these cells are altered SSCs. The molecular mechanism of this conversion is still obscure. Therefore, this study can support the analysis of germ cell development both in vitro and in vivo. In addition, it can be effective in finding new and more efficient treatments for male infertility.

Assessment of hindlimb motor recovery after severe thoracic spinal cord injury in rats: classification of CatWalk XT® gait analysis parameters.

Assessment of locomotion recovery in preclinical studies of experimental spinal cord injury remains challenging. We studied the CatWalk XT® gait analysis for evaluating hindlimb functional recovery in a widely used and clinically relevant thoracic contusion/compression spinal cord injury model in rats. Rats were randomly assigned to either a T9 spinal cord injury or sham laminectomy. Locomotion recovery was assessed using the Basso, Beattie, and Bresnahan open field rating scale and the CatWalk XT® gait analysis. To determine the potential bias from weight changes, corrected hindlimb (H) values (divided by the unaffected forelimb (F) values) were calculated. Six weeks after injury, cyst formation, astrogliosis, and the deposition of chondroitin sulfate glycosaminoglycans were assessed by immunohistochemistry staining. Compared with the baseline, a significant spontaneous recovery could be observed in the CatWalk XT® parameters max intensity, mean intensity, max intensity at%, and max contact mean intensity from 4 weeks after injury onwards. Of note, corrected values (H/F) of CatWalk XT® parameters showed a significantly less vulnerability to the weight changes than absolute values, specifically in static parameters. The corrected CatWalk XT® parameters were positively correlated with the Basso, Beattie, and Bresnahan rating scale scores, cyst formation, the immunointensity of astrogliosis and chondroitin sulfate glycosaminoglycan deposition. The CatWalk XT® gait analysis and especially its static parameters, therefore, seem to be highly useful in assessing spontaneous recovery of hindlimb function after severe thoracic spinal cord injury. Because many CatWalk XT® parameters of the hindlimbs seem to be affected by body weight changes, using their corrected values might be a valuable option to improve this dependency.

Tendon-Specific Activation of Tenogenic Transcription Factors Enables Keeping Tenocytes' Identity In Vitro.

We generated a novel tetracycline-inducible transgenic mouse line with the tendon-specific expression of a series of tendon-critical transcription factors. Primary tenocytes derived from this mouse line consistently expressed green fluorescent protein reporter transcription factors in response to doxycycline. The tenocytes maintained their tendon cell properties for a longer time after the transient induction in the absence of growth factors and mechanical stress. Four key transcription factors for tendon development and the green fluorescent protein reporter were linked with different viral 2A self-cleaving peptides. They were expressed under the control of the tet-responsive element. In combination with the expression of BFP, which reports on the tendon-specific collagen I, and mScarlet, which reports on the tendon-specific transcription factor Scleraxis (Scx), we observed the more extended maintenance of the tendon cell identity of in vitro cultured tendon cells and Achilles tendon explants. This means that the Scleraxis bHLH transcription factor (Scx), mohawk homeobox (Mkx), early growth response 1 (Egr1) and early growth response 2 (Egr2) contributed to the maintenance of tenocytes' identity in vitro, providing a new model for studying extracellular matrix alterations and identifying alternative biomaterials in vitro.

VASA protein and gene expression analysis of human non-obstructive azoospermia and normal by immunohistochemistry, immunocytochemistry, and bioinformatics analysis.

VASA, also known as DDX4, is a member of the DEAD-box proteins and an RNA binding protein with an ATP-dependent RNA helicase. The VASA gene expression, which is required for human germ cell development, may lead to infertility. Immunocytochemistry and immunohistochemistry were used to examine the expression of VASA protein in the human testis sections of azoospermic patients, in-vitro and in-silico models. Some studies of fertile humans showed VASA expression in the basal and adluminal compartments of seminiferous tubules. Our Immunocytochemistry and immunohistochemistry in infertile humans showed expression of VASA in the luminal compartments of the seminiferous tubule. The immunohistochemical analysis of three human cases with different levels of non-obstructive azoospermia revealed a higher expression of VASA-positive cells. For this purpose, Enrichr and Shiny Gene Ontology databases were used for pathway enrichment analysis and gene ontology. STRING and Cytoscape online evaluation were applied to predict proteins' functional and molecular interactions and performed to recognize the master genes, respectively. According to the obtained results, the main molecular functions of the up-regulated and downregulated genes include the meiotic cell cycle, RNA binding, and differentiation. STRING and Cytoscape analyses presented seven genes, i.e., DDX5, TNP2, DDX3Y, TDRD6, SOHL2, DDX31, and SYCP3, as the hub genes involved in infertility with VASA co-function and protein-protein interaction. Our findings suggest that VASA and its interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

Whole Exome Sequencing and In Silico Analysis of Human Sertoli in Patients with Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA) is a serious cause of male infertility. The Sertoli cell responds to androgens and takes on roles supporting spermatogenesis, which may cause infertility. This work aims to enhance the genetic diagnosis of NOA via the discovery of new and hub genes implicated in human NOA and to better assess the odds of successful sperm extraction according to the individual's genotype. Whole exome sequencing (WES) was done on three NOA patients to find key genes involved in NOA. We evaluated genome-wide transcripts (about 50,000 transcripts) by microarray between the Sertoli of non-obstructive azoospermia and normal cells. The microarray analysis of three human cases with different non-obstructive azoospermia revealed that 32 genes were upregulated, and the expressions of 113 genes were downregulated versus the normal case. For this purpose, Enrich Shiny GO, STRING, and Cytoscape online evaluations were applied to predict the functional and molecular interactions of proteins and then recognize the master pathways. The functional enrichment analysis demonstrated that the biological process (BP) terms "inositol lipid-mediated signaling", "positive regulation of transcription by RNA polymerase II", and "positive regulation of DNA-templated transcription" significantly changed in upregulated differentially expressed genes (DEGs). The BP investigation of downregulated DEGs highlighted "mitotic cytokinesis", "regulation of protein-containing complex assembly", "cytoskeleton-dependent cytokinesis", and the "peptide metabolic process". Overrepresented molecular function (MF) terms in upregulated DEGs included "ubiquitin-specific protease binding", "protease binding", "phosphatidylinositol trisphosphate phosphatase activity", and "clathrin light chain binding". Interestingly, the MF analysis of the downregulated DEGs revealed overexpression in "ATPase inhibitor activity", "glutathione transferase activity", and "ATPase regulator activity". Our findings suggest that these genes and their interacting hub proteins could help determine the pathophysiologies of germ cell abnormalities and infertility.