Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actin.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

The planar cell polarity (PCP) protein Fat 1 in Sertoli cell function.

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin-related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid create an important intercellular bridge, whose adhesive function is in turn supported by Fjx1, a non-receptor Ser/Thr protein kinase. This concept is derived from earlier studies of Drosophila, which has been confirmed in this and earlier reports as well. Herein, we use the approach of knockdown of Fat1 by RNAi using primary cultures of Sertoli cells that mimicked the blood-testis barrier (BTB) in vivo, and a series of coherent experiments including functional assays to monitor Sertoli cell tight junction (TJ)-permeability barrier and a functional in vitro TJ integrity assay to assess the role of Fat1 in the testis. It was shown that planar cell polarity (PCP) protein Fat1 affected Sertoli cell function through its modulation of actin and microtubule cytoskeletal function, altering their polymerization activity through the Fat1/Fjx1 complex. Furthermore, Fat1 is intimately associated with ß-catenin and α-N-catenin, as well as with Prickle 1 of the Vangl1/Prickle 1 complex, another PCP Core Protein to support intercellular interactions to confer PCP. In summary, these findings support the notion that the Fat:Dchs and the Vangl2:Fzd PCP intercellular bridges are tightly associated with basal ES/TJ structural proteins to stabilize PCP function at the Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interface to sustain spermatogenesis.

UV-filter benzophenones suppress human, pig, rat, and mouse 11ß-hydroxysteroid dehydrogenase 1: Structure-activity relationship and in silico docking analysis.

Benzophenone chemicals (BPs) have been developed to prevent the adverse effects of UV radiation and they are widely contaminated. 11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) catalyze the conversion of inactive glucocorticoid to active glucocorticoid, playing critical role in many physiological function. However, the direct effect of BPs on human, pig, rat, and mouse 11ß-HSD1 remains unclear. In this study, we screened the inhibitory strength of 12 BPs on 4 species, and performed the structure-activity relationship (SAR) and in silico docking analysis. The inhibitory potency of BPs was: for human 11ß-HSD1, BP6 (IC50 = 18.76 µM) > BP8 (40.84 µM) > BP (88.89 µM) > other BPs; for pig 11ß-HSD1, BP8 (45.57 µM) > BP6 (59.44 µM) > BP2 (65.12 µM) > BP (135.56 µM) > other BPs; for rat 11ß-HSD1, BP7 (67.17 µM) > BP (68.83 µM) > BP8 (133.04 µM) > other BPs; and for mouse 11ß-HSD1, BP8 (41.41 µM) > BP (50.61 µM) > other BPs. These BP chemicals were mixed/competitive inhibitors of these 11ß-HSD1 enzymes. The 2,2'-dihydroxy substitutions in two benzene rings play a key role in enhancing the effectiveness of inhibiting 11ß-HSD1, possibly via increasing hydrogen bond interactions. Docking analysis shows that these BPs bind to NADPH/glucocorticoid binding sites and forms hydrogen bonds with catalytic residues Ser and/or Tyr. In conclusion, this study demonstrates that BP chemicals can inhibit 11ß-HSD1 from 4 species, and there are subtle species-dependent difference in the inhibitory strength and structural variations of BPs.

Demethoxylation of curcumin enhances its inhibition on human and rat 17ß-hydroxysteroid dehydrogenase 3: QSAR structure-activity relationship and in silico docking analysis.

Curcuminoids have many pharmacological effects. They or their metabolites may have side effects by suppressing 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3). Herein, we investigated the inhibition of curcuminoids and their metabolites on human and rat 17ß-HSD3 and analyzed their structure-activity relationship (SAR) and performed in silico docking. Curcuminoids and their metabolites ranked in terms of IC50 values against human 17ß-HSD3 were bisdemethoxycurcumin (0.61 µM) > curcumin (8.63 µM) > demethoxycurcumin (9.59 µM) > tetrahydrocurcumin (22.04 µM) > cyclocurcumin (29.14 µM), and those against rat 17ß-HSD3 were bisdemethoxycurcumin (3.94 µM) > demethoxycurcumin (4.98 µM) > curcumin (9.62 µM) > tetrahydrocurcumin (45.82 µM) > cyclocurcumin (143.5 µM). The aforementioned chemicals were mixed inhibitors for both enzymes. Molecular docking analysis revealed that they bind to the domain between the androstenedione and NADPH active sites of 17ß-HSD3. Bivariate correlation analysis showed a positive correlation between LogP and pKa of curcumin derivatives with their IC50 values. Additionally, a 3D-QSAR analysis revealed that a pharmacophore model consisting of three hydrogen bond acceptor regions and one hydrogen bond donor region provided a better fit for bisdemethoxycurcumin compared to curcumin. In conclusion, curcuminoids and their metabolites possess the ability to inhibit androgen biosynthesis by directly targeting human and rat 17ß-HSD3. The inhibitory strength of these compounds is influenced by their lipophilicity and ionization characteristics.

P2X7-Mediated Antigen-Independent Activation of CD8+ T Cells Promotes Salt-Sensitive Hypertension.

BACKGROUND: CD8+ T cells (CD8Ts) have been implicated in hypertension. However, the specific mechanisms are not fully understood. In this study, we explore the contribution of the P2X7 (purinergic receptor P2X7) receptor to CD8T activation and subsequent promotion of sodium retention in the kidney. METHODS: We used mouse models of hypertension. Wild type were used as genetic controls, OT1 and Rag2/OT1 mice were utilized to determine antigen dependency, and P2X7-knockout mice were studied to define the role of P2X7 in activating CD8Ts and promoting hypertension. Blood pressure was monitored continuously and kidneys were obtained at different experimental end points. Freshly isolated CD8Ts from mice for activation assays and ATP stimulation. CD8T activation-induced promotion of sodium retention was explored in cocultures of CD8Ts and mouse DCTs. RESULTS: We found that OT1 and Rag2/OT1 mice, which are nonresponsive to common antigens, still developed hypertension and CD8T-activation in response to deoxycorticosterone acetate/salt treatment, similar to wild-type mice. Further studies identified the P2X7 receptor on CD8Ts as a possible mediator of this antigen-independent activation of CD8Ts in hypertension. Knockout of the P2X7 receptor prevented calcium influx and cytokine production in CD8Ts. This finding was associated with reduced CD8T-DCT stimulation, reversal of excessive salt retention in DCTs, and attenuated development of salt-sensitive hypertension. CONCLUSIONS: Our findings suggest a novel mechanism by which CD8Ts are activated in hypertension to exacerbate salt retention and infer that the P2X7 receptor on CD8Ts may represent a new therapeutic target to attenuate T-cell-mediated immunopathology in hypertension.

BHLHE40 drives protective polyfunctional CD4 T cell differentiation in the female reproductive tract against Chlamydia.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.

Bisphenol a alternatives suppress human and rat aromatase activity: QSAR structure-activity relationship and in silico docking analysis.

The use of alternative substances to replace bisphenol A (BPA) has been encouraged. The objective of this study was to evaluate the effects of BPA and 9 BPA alternatives on human and rat aromatase (CYP19A1) in human and rat placental microsomes. The results revealed that bisphenol A, AP, B, C, E, F, FL, S, and Z, and 4,4'-thiodiphenol (TDP) inhibited human CYP19A1 and bisphenol A, AP, B, C, FL, Z, and TDP inhibited rat CYP19A1. The IC50 values of human CYP19A1 ranged from 3.3 to 172.63 µM and those of rat CYP19A1 ranged from 2.20 to over 100 µM. BPA alternatives were mixed/competitive inhibitors and inhibited estradiol production in BeWo placental cells. Molecular docking analysis showed that BPA alternatives bind to the domain between heme and steroid and form a hydrogen bond with catalytic residue Met374. Pharmacophore analysis showed that there were one hydrogen bond donor, one hydrophobic region, and one ring aromatic hydrophobic region. Bivariate correlation analysis showed that molecular weight, alkyl atom weight, and LogP of BPA alternatives were inversely correlated with their IC50 values. In conclusion, BPA alternatives can inhibit human and rat CYP19A1 and the lipophilicity and the substituted alkyl size determines their inhibitory strength.

The Chaperone Protein Cct5 is Essential for Hematopoietic Stem Cell Maintenance.

Proper proteostasis is indispensable for the long-term maintenance of hematopoietic stem and progenitor cells (HSPCs). The TRiC/CCT (chaperonin-containing TCP-1) complex, a key constituent of cellular machinery facilitating accurate protein folding, has remained enigmatic in its specific function within HSPCs. Here we show that conditional knockout (KO) of Cct5 significantly impairs the maintenance of murine HSPCs. Primary and secondary transplantation experiments unequivocally demonstrate the incapacity of Cct5 KO HSPCs to reconstitute both myeloid and lymphoid lineage cells in recipient mice, highlighting the pivotal role of the TRiC/CCT complex in governing these cellular lineages. Furthermore, leveraging an integrated approach that merges a Protein-Protein Interaction (PPI) database with RNA sequencing (RNA-seq) data of HSPCs, our analysis reveals intricate interactions between Cct5 and vital transcription factors crucial for HSC homeostasis. Notably, Cct5 engages with MYC, PIAS1, TP53, ESR1, HOXA1, and JUN, intricately regulating the transcriptional landscape governing autophagy, myeloid differentiation, and cytoskeleton organization within HSPCs. Our study unveils the profound significance of TRiC/CCT complex-mediated proteostasis in orchestrating the maintenance and functionality of HSPCs.

BHLHE40 drives protective polyfunctional CD4 T cell differentiation in the female reproductive tract against Chlamydia.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.

High-Power-Efficiency Readout Circuit Employing Average Capacitance-to-Voltage Converter for Micro-Electro-Mechanical System Capacitive Accelerometers.

A capacitance-to-voltage converter (CVC) is proposed in this paper and applied to a readout circuit for a micro-electro-mechanical system (MEMS) accelerometer to improve the power efficiency. In a traditional readout circuit, the front-end CVC has to operate at a high sampling frequency to resist thermal noise deterioration due to the large parasitic capacitance introduced by the mechanical sensing element. Thus, the back-end analog-to-digital converter (ADC) also has to operate at a high sampling frequency to avoid noise aliasing when sampling the output signal of the CVC, which leads to high power consumption. The average CVC technique is proposed in this paper to reduce the sampling frequency requirement of the back-end ADC and thus reduce the power consumption. Both the traditional readout circuit and the proposed readout circuit are simulated with a commercial 0.18 µm BCD process. The simulation results show that noise aliasing occurs, and the noise power spectral density (PSD) of the traditional readout circuit increases by 12 dB when the sampling frequency of back-end ADC is reduced by 24 dB. However, in the proposed readout circuit, a noise aliasing effect does not occur. Moreover, the proposed readout circuit reduces the power consumption by 53% without thermal noise deterioration. In addition, the proposed CVC circuits are fabricated in an 0.18 µm BCD process, and the test results show that the presented readout circuit based on the average CVC technique can obtain better performance than the traditional CVC-based readout circuit.

Azole fungicides inhibit human and rat gonadal 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Azole fungicides are widely used in the agricultural industry to control fungal infections in crops. However, recent studies have shown that some azole fungicides inhibit the activity of 3ß-hydroxysteroid dehydrogenases (3ß-HSDs) in the gonads. Out of the 16 azole fungicides tested, 8 were found to inhibit human KGN cell 3ß-HSD2 with IC50 values of less than 100 µM. The strongest inhibitor was difenoconazole, with an IC50 value of 1.88 µM. In contrast, only 3 of the azole fungicides inhibited rat testicular 3ß-HSD1, which was less sensitive to inhibition. Azole fungicides potently inhibited progesterone secretion by KGN cells under basal and forskolin stimulated conditions at ≥ 5 µM. The inhibitory strength of azole fungicides was determined by their lipophilicity (LogP), molecular weight, pKa, and binding energy. A pharmacophore analysis revealed that the hydrogen bond acceptor-lipid group was a critical feature required for inhibition. Overall, these findings suggest that the use of azole fungicides have unintended consequences on reproductive health due to their inhibition of gonadal 3ß-HSDs. Key words: Azole fungicides; steroid hormones; 3ß-hydroxysteroid dehydrogenase; docking analysis; lipophilicity.

Dual-edged effects and mechanisms of hydroxylamine in partial denitrification-anaerobic ammonium oxidation system.

The combination of partial denitrification (PD) and anaerobic ammonium oxidation (anammox) is a novel and promising nitrogen removal process. Regulating the synergistic reaction between denitrifiers and anammox bacteria (AnAOB) is the key to achieving stable and efficient PD-anammox performance. In this study, 10 mg/L of hydroxylamine (NH2OH) was considered to efficiently promote the bacterial activity, microbial energy flow, and the synergy of functional microflora. As a result, the nitrogen removal rate (NRR) significantly increased from 0.05 to 0.30 g N/L/d in parallel with an increase in the nitrogen loading rate (NLR) from 0.10 to 0.40 g N/L/d. However, the dual-edged effect of NH2OH was also confirmed. The long-term presence of NH2OH caused overgrowth of complete-denitrifying bacteria and decreased the NRR to 0.11 g N/L/d. Additionally, NH2OH enhanced nitrous oxide (N2O) emissions via chemical pathways as well as enhanced denitrification Fortunately, the inhibition caused by NH2OH was reversible by stopping the dosing, the reactor restored to stable operation with an NRR of 0.27 g N/L/d. Analysis of metabolic intensity and pathways revealed the effecting process and mechanism of NH2OH on the PD-anammox system. This study verified the dual-edged effects and mechanisms of NH2OH, therefore proving a theoretical basis and technical reference for the application of PD-anammox.

Regulation of Sertoli cell function by planar cell polarity (PCP) protein Fjx1.

Four-jointed box kinase 1 (Fjx1) is a planar cell polarity (PCP) protein and a member of the Fat (FAT atypical cadherin 1)/Dchs (Dachsous cadherin-related protein)/Fjx1 PCP complex. Fjx1 is also a non-receptor Ser/Thr protein kinase capable of phosphorylating Fat1 at is extracellular cadherin domains when it is being transported across the Golgi system. As such, Fjx1 is a Golgi-based regulator of Fat1 function by determining its extracellular deposition. Herein, Fjx1 was found to localize across the Sertoli cell cytoplasm, partially co-localized with the microtubules (MTs) across the seminiferous epithelium. It was most notable at the apical ES (ectoplasmic specialization) and basal ES, displaying distinctive stage-specific expression. The apical ES and basal ES are the corresponding testis-specific cell adhesion ultrastructures at the Sertoli-elongated spermatid interface and the Sertoli cell-cell interface, respectively, consistent with the role of Fjx1 as a Golgi-associated Ser/Thr kinase that modulates the Fat (and/or Dchs) integral membrane proteins. Its knockdown (KD) by RNAi using specific Fjx1 siRNA duplexes versus non-targeting negative control siRNA duplexes was found to perturb the Sertoli cell tight junction function, as well as perturbing the function and organization of MT and actin. While Fjx1 KD did not affect the steady-state levels of almost two dozens of BTB-associated Sertoli cell proteins, including structural and regulatory proteins, its KD was found to down-regulate Fat1 (but not Fat2, 3, and 4) and to up-regulate Dchs1 (but not Dchs2) expression. Based on results of biochemical analysis, Fjx1 KD was found to be capable of abolishing phosphorylation of its putative substrate Fat1 at its Ser/Thr sites, but not at its Tyr site, illustrating an intimate functional relationship of Fjx1 and Fat1 in Sertoli cells.

Cadmium-induced Sertoli Cell Injury Through p38-MAPK and Related Signaling Proteins-A Study by RNA Sequencing.

Environmental toxicants, such as cadmium, found in foods, water, and consumer products are known to induce male reproductive dysfunction. However, the underlying molecular mechanism(s) by which cadmium-induced Sertoli cell injury as manifested by a disruption of the blood-testis barrier (BTB) remains unknown. Interestingly, one of the primary targets of cadmium toxicity in the testis is the cytoskeletons of the Sertoli cells, which, in turn, impedes cell junctions in the seminiferous epithelium. In order to expand these earlier observations and to provide a roadmap for future studies, we embarked a study using RNA sequencing to identify the pertinent genes involved in cadmium-induced Sertoli cell injury. Using bioinformatics analyses, multiple gene sets that regulated actin and microtubule (MT) cytoskeletons were identified along with components of the mitogen-activated protein kinase (MAPK) signaling protein and several signaling pathways. More important, we have also discovered that while the gene expression of p38-MAPK (also JNK or c-Jun) was considerably up- or downregulated during cadmium-induced Sertoli cell injury, the activated (phosphorylated) form was upregulated. Importantly, doramapimod (also known as BIRB 796), a specific p38-MARK inhibitor, that was shown to selectively block cadmium-induced p-p38 MAPK activation via phosphorylation in Sertoli cells, was indeed capable of blocking cadmium-induced Sertoli cell injury including disruption of the Sertoli cell-permeability barrier function, disruptive distribution of BTB-associated proteins, and disruptive organization of the actin and MT cytoskeletons. These data provide a helpful source of information for investigators to probe the role of signaling proteins and/or their signaling cascades, besides MAPKs, that likely utilized by cadmium to induce reproductive dysfunction.

Curcuminoids inhibit human and rat placental 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, curcuma longa L has been applied to treat pain and tumour-related symptoms for over thousands of years. Curcuminoids, polyphenolic compounds, are the main pharmacological component from the rhizome of Curcuma longa L. Pharmacological investigations have found that curcuminoids have many pharmacological activities of anti-inflammatory, anti-tumour, and anti-metastasis. AIM OF THE STUDY: 3ß-Hydroxysteroid dehydrogenase (3ß-HSD1) catalyses the production of steroid precursors for androgens and estrogens, which play an essential role in cancer metastasis. We explored the potency and mode of action of curcuminoids and their metabolites of inhibiting 3ß-HSD1 activity and compared the species difference between human and rat. MATERIALS AND METHODS: In this study, we investigated the direct inhibition of 6 curcuminoids on human placental 3ß-HSD1 activity and compared the species-dependent difference in human 3ß-HSD1 and rat placental homolog 3ß-HSD4. RESULTS: The inhibitory potency of curcuminoids on human 3ß-HSD1 was demethoxycurcumin (IC50, 0.18 µM) > bisdemethoxycurcumin (0.21 µM)>curcumin (2.41 µM)> dihydrocurcumin (4.13 µM)>tetrahydrocurcumin (15.78 µM)>octahydrocurcumin (ineffective at 100 µM). The inhibitory potency of curcuminoids on rat 3ß-HSD4 was bisdemethoxycurcumin (3.34 µM)>dihydrocurcumin (5.12 µM)>tetrahydrocurcumin (41.82 µM)>demethoxycurcumin (88.10 µM)>curcumin (137.06 µM)> octahydrocurcumin (ineffective at 100 µM). Human choriocarcinoma JAr cells with curcuminoid treatment showed that these chemicals had similar potency to inhibit progesterone secretion under basal and 8bromo-cAMP stimulated conditions. Docking analysis showed that all chemicals bind pregnenolone-binding site with mixed/competitive mode for 3ß-HSD. CONCLUSION: Some curcuminoids are potent human placental 3ß-HSD1 inhibitors, possibly being potential drugs to treat prostate cancer and breast cancer.

Physiological Roles of ß-amyloid in Regulating Synaptic Function: Implications for AD Pathophysiology.

The physiological functions of endogenous amyloid-ß (Aß), which plays important role in the pathology of Alzheimer's disease (AD), have not been paid enough attention. Here, we review the multiple physiological effects of Aß, particularly in regulating synaptic transmission, and the possible mechanisms, in order to decipher the real characters of Aß under both physiological and pathological conditions. Some worthy studies have shown that the deprivation of endogenous Aß gives rise to synaptic dysfunction and cognitive deficiency, while the moderate elevation of this peptide enhances long term potentiation and leads to neuronal hyperexcitability. In this review, we provide a new view for understanding the role of Aß in AD pathophysiology from the perspective of physiological meaning.

Gene expression analysis in endometriosis: Immunopathology insights, transcription factors and therapeutic targets.

Background: Endometriosis is recognized as an estrogen-dependent inflammation disorder, estimated to affect 8%-15% of women of childbearing age. Currently, the etiology and pathogenesis of endometriosis are not completely clear. Underlying mechanism for endometriosis is still under debate and needs further exploration. The involvement of transcription factors and immune mediations may be involved in the pathophysiological process of endometriosis, but the specific mechanism remains to be explored. This study aims to investigate the underlying molecular mechanisms in endometriosis. Methods: The gene expression profile of endometriosis was obtained from the gene expression omnibus (GEO) database. Gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA) were applied to the endometriosis GSE7305 datasets. Cibersort and MCP-counter were used to explore the immune response gene sets, immune response pathway, and immune environment. Differentially expressed genes (DEGs) were identified and screened. Common biological pathways were being investigated using the kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Transcription factors were from The Human Transcription Factors. The least absolute shrinkage and selection operator (Lasso) model identified four differential expressions of transcription factors (AEBP1, HOXB6, KLF2, and RORB). Their diagnostic value was calculated by receiver operating characteristic (ROC) curve analysis and validated in the validation cohort (GSE11691, GSE23339). By constructing the interaction network of crucial transcription factors, weighted gene coexpression network analysis (WGCNA) was used to search for key module genes. Metascape was used for enrichment analysis of essential module genes and obtained HOXB6, KLF2. The HOXB6 and KLF2 were further verified as the only two intersection genes according to Support Vector Machine Recursive Feature Elimination (SVM-RFE) and random forest models. We constructed ceRNA (lncRNA-miRNA-mRNA) networks with four potential transcription factors. Finally, we performed molecular docking for goserelin and dienogest with four transcription factors (AEBP1, HOXB6, KLF2, and RORB) to screen potential drug targets. Results: Immune and metabolic pathways were enriched in GSVA and GSEA. In single sample gene set enrichment analysis (ssGSEA), most immune infiltrating cells, immune response gene sets, and immune response pathways are differentially expressed between endometriosis and non-endometriosis. Twenty-seven transcription factors were screened from differentially expressed genes. Most of the twenty-seven transcription factors were correlated with immune infiltrating cells, immune response gene sets and immune response pathways. Furthermore, Adipocyte enhancer binding protein 1 (AEBP1), Homeobox B6 (HOXB6), Kruppel Like Factor 2 (KLF2) and RAR Related Orphan Receptor B (RORB) were selected out from twenty-seven transcription factors. ROC analysis showed that the four genes had a high diagnostic value for endometriosis. In addition, KLF2 and HOXB6 were found to play particularly important roles in multiple modules (String, WGCNA, SVM-RFE, random forest) on the gene interaction network. Using the ceRNA network, we found that NEAT1 may regulate the expressions of AEBP1, HOXB6 and RORB, while X Inactive Specific Transcript (XIST) may control the expressions of HOXB6, RORB and KLF2. Finally, we found that goserelin and dienogest may be potential drugs to regulate AEBP1, HOXB6, KLF2 and RORB through molecular docking. Conclusions: AEBP1, HOXB6, KLF2, and RORB may be potential biomarkers for endometriosis. Two of them, KLF2 and HOXB6, are critical molecules in the gene interaction network of endometriosis. Discovered by molecular docking, AEBP1, HOXB6, KLF2, and RORB are targets for goserelin and dienogest.

Defects of microtubule cytoskeletal organization in NOA human testes.

The importance of actin and microtubule (MT) cytoskeletons in testis function in rodents is known to some extent, but its role in the etiology of azoospermia in humans remains unexplored. Here, we examined if MT cytoskeleton was defective in NOA (non-obstructive azoospermia) testes versus normal human testes based on histopathological, immunofluorescence (IF), and scRNA-Seq transcriptome profiling. Testis biopsy samples from n = 6 normal men versus n = 3 Sertoli cell only (SCO) and n = 3 MA (meiotic arrest) of NOA patients were used for histopathological analysis. IF analysis was also used to examine MT organization across the seminiferous epithelium, investigating the likely involvement of microtubule-associated proteins (MAPs). scRNA-Seq transcriptome profiling datasets from testes of 3 SCO patients versus 3 normal men in public domain in Gene Expression Omnibus (GEO) Sample (GSM) with identifiers were analyzed to examine relevant genes that regulate MT dynamics. NOA testes of MA and SCO patients displayed notable defects in MT organization across the epithelium with extensive truncation, mis-alignments and appeared as collapsed structures near the base of the tubules. These changes are in contrast to MTs in testes of normal men. scRNA-Seq analyses revealed considerable loss of spermatogenesis capacity in SCO testes of NOA patients versus normal men. An array of genes that support MT dynamics displayed considerable changes in expression and in spatial distribution. In summary, defects in MT cytoskeleton were noted in testes of NOA (SCO) patients, possibly mediated by defective spatial expression and/or distribution of MAPs. These changes, in turn, may impede spermatogenesis in SCO testes of NOA patients.

IFNγ and Antibody Synergize To Enhance Protective Immunity against Chlamydia Dissemination and Female Reproductive Tract Reinfections.

CD4 T cell-dependent IFNγ production and antibody are the two best known effectors for protective immunity against Chlamydia female reproductive tract (FRT) infection. Nevertheless, mice lacking either IFNγ or B cells can clear the vast majority of Chlamydia from the FRT, while suffering from varying degrees of disseminated infection. In this study, we investigated whether IFNγ and B cells play complementary roles in host defense against Chlamydia and evaluated their relative contributions in systemic and mucosal tissues. Using mice deficient in both IFNγ and B cells (IFNγ-/- x µMT), we showed that mice lacking both effectors were highly susceptible to lethal systemic bacterial dissemination following Chlamydia muridarum intravaginal infection. Passive transfer of immune convalescent serum, but not recombinant IFNγ, reduced bacterial burden in both systemic and mucosal tissues in IFNγ-/- x µMT mice. Notably, over the course of primary infection, we observed a reduction of bacterial shedding of more than 2 orders of magnitude in IFNγ-/- x µMT mice following both C. muridarum and C. trachomatis FRT infections. In contrast, no protective immunity against C. muridarum reinfection was detected in the absence of IFNγ and B cells. Together, our results suggest that IFNγ and B cells synergize to combat systemic Chlamydia dissemination, while additional IFNγ and B cell-independent mechanisms exist for host resistance to Chlamydia in the lower FRT.

The IFNγ-PDL1 Pathway Enhances CD8T-DCT Interaction to Promote Hypertension.

BACKGROUND: Renal T cells contribute importantly to hypertension, but the underlying mechanism is incompletely understood. We reported that CD8Ts directly stimulate distal convoluted tubule cells (DCTs) to increase NCC (sodium chloride co-transporter) expression and salt reabsorption. However, the mechanistic basis of this pathogenic pathway that promotes hypertension remains to be elucidated. METHODS: We used mouse models of DOCA+salt (DOCA) treatment and adoptive transfer of CD8+ T cells (CD8T) from hypertensive animals to normotensive animals in in vivo studies. Co-culture of mouse DCTs and CD8Ts was used as in vitro model to test the effect of CD8T activation in promoting NCC-mediated sodium retention and to identify critical molecular players contributing to the CD8T-DCT interaction. Interferon (IFNγ)-KO mice and mice receiving renal tubule-specific knockdown of PDL1 were used to verify in vitro findings. Blood pressure was continuously monitored via radio-biotelemetry, and kidney samples were saved at experimental end points for analysis. RESULTS: We identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFN-γ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFN-γ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in 2 different mouse models. CONCLUSIONS: Our results identified the role of activated CD8Ts in contributing to increased sodium retention in DCTS through the IFNγ-PDL1 pathway. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.