Perfluoropentane-based oxygen-loaded nanodroplets reduce microglial activation through metabolic reprogramming.

JOURNAL/nrgr/04.03/01300535-202504000-00032/figure1/v/2024-07-06T104127Z/r/image-tiff Microglia, the primary immune cells within the brain, have gained recognition as a promising therapeutic target for managing neurodegenerative diseases within the central nervous system, including Parkinson's disease. Nanoscale perfluorocarbon droplets have been reported to not only possess a high oxygen-carrying capacity, but also exhibit remarkable anti-inflammatory properties. However, the role of perfluoropentane in microglia-mediated central inflammatory reactions remains poorly understood. In this study, we developed perfluoropentane-based oxygen-loaded nanodroplets (PFP-OLNDs) and found that pretreatment with these droplets suppressed the lipopolysaccharide-induced activation of M1-type microglia in vitro and in vivo, and suppressed microglial activation in a mouse model of Parkinson's disease. Microglial suppression led to a reduction in the inflammatory response, oxidative stress, and cell migration capacity in vitro. Consequently, the neurotoxic effects were mitigated, which alleviated neuronal degeneration. Additionally, ultrahigh-performance liquid chromatography-tandem mass spectrometry showed that the anti-inflammatory effects of PFP-OLNDs mainly resulted from the modulation of microglial metabolic reprogramming. We further showed that PFP-OLNDs regulated microglial metabolic reprogramming through the AKT-mTOR-HIF-1α pathway. Collectively, our findings suggest that the novel PFP-OLNDs constructed in this study alleviate microglia-mediated central inflammatory reactions through metabolic reprogramming.

Human and rat gonadal 3ß-hydroxysteroid dehydrogenases are suppressed by dithiocarbamate fungicides via interacting with cysteine residues.

Dithiocarbamates have been widely used in various industrial applications, such as insecticides (ferbam) or drug (disulfiram). This study explored the inhibitory effects of dithiocarbamates on human and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD) and investigated the structure-activity relationship and mechanistic insights. The inhibitory activity of six dithiocarbamates and thiourea on the conversion of pregnenolone to progesterone was evaluated using human KGN cell and rat testicular microsomes, with subsequent progesterone measurement using HPLC-MS/MS. The study found that among the tested compounds disulfiram, ferbam, and thiram exhibited significant inhibitory activity against human 3ß-HSD2 and rat 3ß-HSD1, with ferbam demonstrating the highest potency. The mode of action for these compounds was characterized, showing mixed inhibition for human 3ß-HSD2 and mixed/noncompetitive inhibition for rat 3ß-HSD1. Additionally, it was observed that dithiothreitol dose-dependently reversed the inhibitory effects of dithiocarbamates on both human and rat gonadal 3ß-HSD enzymes. The study also delved into the penetration of these dithiocarbamates through the human KGN cell membrane and their impact on progesterone production, highlighting their potency in inhibiting human 3ß-HSD2. Furthermore, bivariate correlation analysis revealed a positive correlation of LogP (lipophilicity) with IC50 values for both enzymes. Docking analysis indicated that dithiocarbamates bind to NAD+ and steroid-binding sites, with some interactions with cysteine residues. In conclusion, this study provides valuable insights into the structure-activity relationship and mechanistic aspects of dithiocarbamates as inhibitors of human and rat gonadal 3ß-HSDs, suggesting that these compounds likely exert their inhibitory effects through binding to cysteine residues.

Structures of African swine fever virus topoisomerase complex and their implications.

African swine fever virus (ASFV) is the causal agent of African swine fever (ASF), which is contagious and highly lethal to domestic pigs and wild boars. The genome of ASFV encodes many proteins important for ASFV life cycle. The functional importance of topoisomerase AsfvTopII has been confirmed by in vivo and in vitro assays, but the structure of AsfvTopII is poorly studied. Here, we report four AsfvTopII complex structures. The ATPase domain structures reveal the detailed basis for ATP binding and hydrolysis, which is shared by AsfvTopII and eukaryotic TopIIs. The DNA-bound structures show that AsfvTopII follows conserved mechanism in G-DNA binding and cleavage. Besides G-DNA, a T-DNA fragment is also captured in one AsfvTopII structure. Mutagenesis and in vitro assays confirm that Pro852 and the T-DNA-binding residue Tyr744 are important for the function of AsfvTopII. Our study not only advances the understanding on the biological function of AsfvTopII, but also provides a solid basis for the development of AsfvTopII-specific inhibitors.

Motor proteins, spermatogenesis and testis function.

The role of motor proteins in supporting intracellular transports of vesicles and organelles in mammalian cells has been known for decades. On the other hand, the function of motor proteins that support spermatogenesis is also well established since the deletion of motor protein genes leads to subfertility and/or infertility. Furthermore, mutations and genetic variations of motor protein genes affect fertility in men, but also a wide range of developmental defects in humans including multiple organs besides the testis. In this review, we seek to provide a summary of microtubule and actin-dependent motor proteins based on earlier and recent findings in the field. Since these two cytoskeletons are polarized structures, different motor proteins are being used to transport cargoes to different ends of these cytoskeletons. However, their involvement in germ cell transport across the blood-testis barrier (BTB) and the epithelium of the seminiferous tubules remains relatively unknown. It is based on recent findings in the field, we have provided a hypothetical model by which motor proteins are being used to support germ cell transport across the BTB and the seminiferous epithelium during the epithelial cycle of spermatogenesis. In our discussion, we have highlighted the areas of research that deserve attention to bridge the gap of research in relating the function of motor proteins to spermatogenesis.

Knockout of C1q/tumor necrosis factor-related protein-9 aggravates cardiac fibrosis in diabetic mice by regulating YAP-mediated autophagy.

Introduction: Diabetic cardiomyopathy (DCM) is predominantly distinguished by impairment in ventricular function and myocardial fibrosis. Previous studies revealed the cardioprotective properties of C1q/tumor necrosis factor-related protein 9 (CTRP9). However, whether CTRP9 affects diabetic myocardial fibrosis and its underlying mechanisms remains unclear. Methods: We developed a type 1 diabetes (T1DM) model in CTRP9-KO mice via streptozotocin (STZ) induction to examine cardiac function, histopathology, fibrosis extent, Yes-associated protein (YAP) expression, and the expression of markers for autophagy such LC3-II and p62. Additionally, we analyzed the direct impact of CTRP9 on high glucose (HG)-induced transdifferentiation, autophagic activity, and YAP protein levels in cardiac fibroblasts. Results: In diabetic mice, CTRP9 expression was decreased in the heart. The absence of CTRP9 aggravated cardiac dysfunction and fibrosis in mice with diabetes, alongside increased YAP expression and impaired autophagy. In vitro, HG induced the activation of myocardial fibroblasts, which demonstrated elevated cell proliferation, collagen production, and α-smooth muscle actin (α-SMA) expression. CTRP9 countered these adverse effects by restoring autophagy and reducing YAP protein levels in cardiac fibroblasts. Notably, the protective effects of CTRP9 were negated by the inhibition of autophagy with chloroquine (CQ) or by YAP overexpression through plasmid intervention. Notably, the protective effect of CTRP9 was negated by inhibition of autophagy caused by chloroquine (CQ) or plasmid intervention with YAP overexpression. Discussion: Our findings suggest that CTRP9 can enhance cardiac function and mitigate cardiac remodeling in DCM through the regulation of YAP-mediated autophagy. CTRP9 holds promise as a potential candidate for pharmacotherapy in managing diabetic cardiac fibrosis.

Structural and functional insights into the helicase protein E5 of Mpox virus.

Mpox virus (MPXV) can cause mpox in humans. Due to its quick and wide spread in the past two years, mpox has turned into a significant public health concern. Helicase E5 is a multi-domain protein; its primer synthesis and DNA unwinding activity are required for genome uncoating and DNA replication of MPXV. However, the in vitro DNA unwinding activity has never been demonstrated. Here, we report the structural and biochemical studies of MPXV E5, showing that the full-length protein adopts an auto-inhibited conformation. Truncation of the N-terminus can recover the in vitro unwinding activity of E5 towards the forked DNA. Further structural analysis reveals that MPXV E5 shares a conserved mechanism in DNA unwinding and primer synthesis with the homologous proteins. These findings not only advance our understanding on the function of MPXV E5, but also provide a solid basis for the development of anti-poxvirus drugs.

Perfluorooctane sulfonate-induced Sertoli cell injury through c-Jun N-terminal kinase: a study by RNA-Seq.

Per- and polyfluoroalkyl substances (PFASs) are a family of "forever chemicals" including perfluorooctane sulfonate (PFOS). These toxic chemicals do not break down in the environment or in our bodies. In the human body, PFOS and perfluoroctanoic acid (PFOA) have a half-life (T1/2) of about 4-5 yr so low daily consumption of these chemicals can accumulate in the human body to a harmful level over a long period. Although the use of PFOS in consumer products was banned in the United States in 2022/2023, this forever chemical remains detectable in our tap water and food products. Every American tested has a high level of PFAS in their blood (https://cleanwater.org/pfas-forever-chemicals). In this report, we used a Sertoli cell blood-testis barrier (BTB) model with primary Sertoli cells cultured in vitro with an established functional tight junction (TJ)-permeability barrier that mimicked the BTB in vivo. Treatment of Sertoli cells with PFOS was found to perturb the TJ-barrier, which was the result of cytoskeletal disruption across the cell cytoplasm, disrupting actin and microtubule polymerization. These changes thus affected the proper localization of BTB-associated proteins at the BTB. Using RNA-Seq transcriptome profiling, bioinformatics analysis, and pertinent biochemical and cell biology techniques, it was discovered that PFOS -induced Sertoli cell toxicity through the c-Jun N-terminal kinase (JNK; also known as stress-activated protein kinase, SAPK) and its phosphorylated/active form p-JNK signaling pathway. More importantly, KB-R7943 mesylate (KB), a JNK/p-JNK activator, was capable of blocking PFOS-induced Sertoli cell injury, supporting the notion that PFOS-induced cell injury can possibly be therapeutically managed.NEW & NOTEWORTHY PFOS induces Sertoli cell injury, including disruption of the 1) blood-testis barrier function and 2) cytoskeletal organization, which, in turn, impedes male reproductive function. These changes are mediated by JNK/p-JNK signaling pathway. However, the use of KB-R7943, a JNK/p-JNK activator was capable of blocking PFOS-induced Sertoli cell injury, supporting the possibility of therapeutically managing PFOS-induced reproductive dysfunction.

Microglia-mediated pericytes migration and fibroblast transition via S1P/S1P3/YAP signaling pathway after spinal cord injury.

Platelet-derived growth factor receptor ß positive (PDGFRß+) pericytes detach from the microvascular wall and migrate into the injury center following spinal cord injury (SCI), which has been widely regarded as the main source of fibrotic scar, but the mechanism of migration and fibroblast transition remains elusive. Here we show the associated spatiotemporal distribution between microglia and pericytes at three and seven days post-injury (dpi). The increased expression of Sphingosine kinase-1 (SPHK1) in microglia significantly raised the concentration of Sphingosine-1-phosphate (S1P) in the spinal cord, which promotes migration and fibroblast transition of pericyte. In vitro experiments, we found the elevated Sphingosine 1-phosphate receptor 3 (S1P3), the S1P/S1PR3 axis inhibited the phosphorylation of YAP and promoted its nuclear translocation, which contributed to the formation of alpha-smooth muscle actin (α-SMA) and collagen type I (COL1) protein, This process can be blocked by an S1P3 specific inhibitor TY52156 in vitro. The S1P/S1P3/YAP pathway might be a potential target for treatment in SCI.

Triclosan inhibits testosterone biosynthesis in adult rats via inducing m6A methylation-mediated autophagy.

Triclosan is a potent antibacterial compound widely used in everyday products. Whether triclosan affects Leydig cell function in adult male rats remains unknown. In this study, 0, 50, 100, or 200 mg/kg/day triclosan was gavaged to Sprague-Dawley male rats from 56 to 63 days postpartum. Triclosan significantly reduced serum testosterone levels at ≥ 50 mg/kg/day via downregulating the expression of Leydig cell gene Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3 and regulatory transcription factor Nr3c2 at 100-200 mg/kg. Further analysis showed that triclosan markedly increased autophagy as shown by increasing LC3II and BECN1 and decreasing SQSTM1. The mRNA m6A modification analysis revealed that triclosan significantly downregulated Fto expression at 200 mg/kg while upregulating Ythdf1 expression at 100 and 200 mg/kg, leading to methylation of Becn1 mRNA as shown by MeRIP assay. Triclosan significantly inhibited testosterone output in rat R2C Leydig cells at ≥ 5 µM via downregulating Fto and upregulating Ythdf1. SiRNA Ythdf1 knockdown can reverse triclosan-mediated mitophagy in R2C cells, thereby reversing the reduction of testosterone output. In summary, triclosan caused Becn1 m6A methylation by downregulating Fto and upregulating Ythdf1, which accelerated Becn1 translation, thus leading to the occurrence of autophagy and the decrease of testosterone biosynthesis.

Metabolically active neutrophils represent a permissive niche for Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb)-infected neutrophils are often found in the airways of patients with active tuberculosis (TB), and excessive recruitment of neutrophils to the lung is linked to increased bacterial burden and aggravated pathology in TB. The basis for the permissiveness of neutrophils for Mtb and the ability to be pathogenic in TB has been elusive. Here, we identified metabolic and functional features of neutrophils that contribute to their permissiveness in Mtb infection. Using single-cell metabolic and transcriptional analyses, we found that neutrophils in the Mtb-infected lung displayed elevated mitochondrial metabolism, which was largely attributed to the induction of activated neutrophils with enhanced metabolic activities. The activated neutrophil subpopulation was also identified in the lung granulomas from Mtb-infected non-human primates. Functionally, activated neutrophils harbored more viable bacteria and displayed enhanced lipid uptake and accumulation. Surprisingly, we found that interferon-γ promoted the activation of lung neutrophils during Mtb infection. Lastly, perturbation of lipid uptake pathways selectively compromised Mtb survival in activated neutrophils. These findings suggest that neutrophil heterogeneity and metabolic diversity are key to their permissiveness for Mtb and that metabolic pathways in neutrophils represent potential host-directed therapeutics in TB.

Structure-activity relationship and mechanistic study of organotins as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases.

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3ß-HSD2 with IC50 values of 114.79, 106.98, and 5.40 µM, respectively. For pig 3ß-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 µM, respectively. Similarly, for rat 3ß-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 µM, respectively. They were mixed inhibitors of pig and rat 3ß-HSD, while triphenyltin was identified as a competitive inhibitor of human 3ß-HSD2. The mechanism underlying the inhibition of organotins on 3ß-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3ß-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites.

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 Protein Fat1 in Sertoli Cell Function.

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin-related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interfaces create an important intercellular bridge whose adhesive function is in turn supported by Fjx1, a nonreceptor 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 the 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.