Deficiency of primate-specific SSX1 induced asthenoteratozoospermia in infertile men and cynomolgus monkey and tree shrew models.

Primate-specific genes (PSGs) tend to be expressed in the brain and testis. This phenomenon is consistent with brain evolution in primates but is seemingly contradictory to the similarity of spermatogenesis among mammals. Here, using whole-exome sequencing, we identified deleterious variants of X-linked SSX1 in six unrelated men with asthenoteratozoospermia. SSX1 is a PSG expressed predominantly in the testis, and the SSX family evolutionarily expanded independently in rodents and primates. As the mouse model could not be used for studying SSX1, we used a non-human primate model and tree shrews, which are phylogenetically similar to primates, to knock down (KD) Ssx1 expression in the testes. Consistent with the phenotype observed in humans, both Ssx1-KD models exhibited a reduced sperm motility and abnormal sperm morphology. Further, RNA sequencing indicated that Ssx1 deficiency influenced multiple biological processes during spermatogenesis. Collectively, our experimental observations in humans and cynomolgus monkey and tree shrew models highlight the crucial role of SSX1 in spermatogenesis. Notably, three of the five couples who underwent intra-cytoplasmic sperm injection treatment achieved a successful pregnancy. This study provides important guidance for genetic counseling and clinical diagnosis and, significantly, describes the approaches for elucidating the functions of testis-enriched PSGs in spermatogenesis.

Bi-allelic variants in DNHD1 cause flagellar axoneme defects and asthenoteratozoospermia in humans and mice.

Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1‒/‒) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals.

Transmissible gastroenteritis virus induces inflammatory responses via RIG-I/NF-κB/HIF-1α/glycolysis axis in intestinal organoids and in vivo.

Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.

Improving the RNA velocity approach with single-cell RNA lifecycle (nascent, mature and degrading RNAs) sequencing technologies.

We presented an experimental method called FLOUR-seq, which combines BD Rhapsody and nanopore sequencing to detect the RNA lifecycle (including nascent, mature, and degrading RNAs) in cells. Additionally, we updated our HIT-scISOseq V2 to discover a more accurate RNA lifecycle using 10x Chromium and Pacbio sequencing. Most importantly, to explore how single-cell full-length RNA sequencing technologies could help improve the RNA velocity approach, we introduced a new algorithm called 'Region Velocity' to more accurately configure cellular RNA velocity. We applied this algorithm to study spermiogenesis and compared the performance of FLOUR-seq with Pacbio-based HIT-scISOseq V2. Our findings demonstrated that 'Region Velocity' is more suitable for analyzing single-cell full-length RNA data than traditional RNA velocity approaches. These novel methods could be useful for researchers looking to discover full-length RNAs in single cells and comprehensively monitor RNA lifecycle in cells.

C9orf131 and C10orf120 are not essential for male fertility in humans or mice.

Male infertility affects approximately 7% of childbearing couples and is a major health issue. Although nearly 50% idiopathic infertile men are assumed to have a genetic basis, the underlying causes remain largely unknown in most infertility cases. Here, we report two rare homozygous variants in two previously uncharacterized genes, C9orf131 and C10orf120, identified in two unrelated men with asthenozoospermia. Both genes were predominantly expressed in the testes. Furthermore, C9orf131 and C10orf120 knockout mice were successfully generated using the CRISPR-Cas9 technology. However, both C9orf131-/- and C10orf120-/- adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type mice. No overt differences were found between wild-type, C9orf131-/-, and C10orf120-/- mice regarding testicular/epididymal tissue morphology, sperm count, sperm motility, or sperm morphology. Moreover, TUNEL assays indicated that the number of apoptotic germ cells in testes was not significantly different between the three groups. In summary, these findings suggest that C9orf131 and C10orf120 are redundant genes in male infertility.

Ceramide analog C2-cer induces a loss in insulin sensitivity in muscle cells through the salvage/recycling pathway.

Ceramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes.

Comparative transcriptome analysis reveals transcriptional regulation of anthocyanin biosynthesis in purple radish (Raphanus sativus L.).

Radish exhibits significant variation in color, particularly in sprouts, leaves, petals, fleshy roots, and other tissues, displaying a range of hues such as green, white, red, purple, and black. Although extensive research has been conducted on the color variation of radish, the underlying mechanism behind the variation in radish flower color remains unclear. To date, there is a lack of comprehensive research investigating the variation mechanism of radish sprouts, leaves, fleshy roots, and flower organs. This study aims to address this gap by utilizing transcriptome sequencing to acquire transcriptome data for white and purple radish flowers. Additionally, the published transcriptome data of sprouts, leaves, and fleshy roots were incorporated to conduct a systematic analysis of the regulatory mechanisms underlying anthocyanin biosynthesis in these four radish tissues. The comparative transcriptome analysis revealed differential expression of the anthocyanin biosynthetic pathway genes DFR, UGT78D2, TT12 and CPC in the four radish tissues. Additionally, the WGCNA results identified RsDFR.9c and RsUGT78D2.2c as hub genes responsible for regulating anthocyanin biosynthesis. By integrating the findings from the comparative transcriptome analysis, WGCNA, and anthocyanin biosynthetic pathway-related gene expression patterns, it is hypothesized that genes RsDFR.9c and RsUGT78D2.2c may serve as pivotal regulators of anthocyanins in the four radish tissues. Furthermore, the tissue-specific expression of the four copies of RsPAP1 is deemed crucial in governing anthocyanin synthesis and accumulation. Our results provide new insights into the molecular mechanism of anthocyanin biosynthesis and accumulation in different tissues of radish.

Synthesis of Polar-functionalized Isotactic Polypropylenes Using Commercial Heterogeneous Ziegler-Natta Catalyst.

The efficient synthesis of polar-functionalized polypropylenes with high molecular weight and high stereoregularity represents a challenging task. This challenge becomes even more daunting when pursuing an industrially preferred heterogeneous process. This study demonstrated the realization of these goals through the use of commercial heterogeneous Ziegler-Natta catalysts in the copolymerization of propylene with ionic cluster polar monomers. The results revealed high copolymerization activity (∼1.1 × 107 g mol-1 h-1), moderate polar monomer incorporation ratios (∼4.9 mol %), high copolymer molecular weight (Mw > 105 g mol-1), high stereoregularity ([mmmm] ∼ 96%), and high melting temperature range (150-162 °C). The utilization of ionic cluster polar monomers improved the thermal stability as well as stereoselectivity of the catalyst. Moreover, the Ziegler-Natta catalyst can homopolymerize ionic cluster polar monomers with high activities (>104 g mol-1 h-1). The resulting polar-functionalized isotactic polypropylenes (iPP) exhibited superior tensile strength, impact strength, creep resistance, transparency, and crystallinity compared with nonpolar iPP. This enhancement was attributable to the dual roles of the ionic cluster polar monomer unit, serving as both a transparent nucleating agent and a dynamic cross-linking functionality. Furthermore, the polar-functionalized iPP exhibited improved compatibility with polar materials, offering benefits for applications in composites, recycling of mixed plastic wastes, 3D printing, and other fields. This study offered a comprehensive solution for the future industrial production of polar-functionalized iPP via copolymerization, bridging the gap between an efficient and practical copolymerization process from a synthetic chemistry perspective and enhanced material properties from an application perspective.

Bi-allelic mutations of DNAH10 cause primary male infertility with asthenoteratozoospermia in humans and mice.

Multiple morphological abnormalities of the sperm flagella (MMAF)-induced asthenoteratozoospermia is a common cause of male infertility. Previous studies have identified several MMAF-associated genes, highlighting the condition's genetic heterogeneity. To further define the genetic causes underlying MMAF, we performed whole-exome sequencing in a cohort of 643 Chinese MMAF-affected men. Bi-allelic DNAH10 variants were identified in five individuals with MMAF from four unrelated families. These variants were either rare or absent in public population genome databases and were predicted to be deleterious by multiple bioinformatics tools. Morphological and ultrastructural analyses of the spermatozoa obtained from men harboring bi-allelic DNAH10 variants revealed striking flagellar defects with the absence of inner dynein arms (IDAs). DNAH10 encodes an axonemal IDA heavy chain component that is predominantly expressed in the testes. Immunostaining analysis indicated that DNAH10 localized to the entire sperm flagellum of control spermatozoa. In contrast, spermatozoa from the men harboring bi-allelic DNAH10 variants exhibited an absence or markedly reduced staining intensity of DNAH10 and other IDA components, including DNAH2 and DNAH6. Furthermore, the phenotypes were recapitulated in mouse models lacking Dnah10 or expressing a disease-associated variant, confirming the involvement of DNAH10 in human MMAF. Altogether, our findings in humans and mice demonstrate that DNAH10 is essential for sperm flagellar assembly and that deleterious bi-allelic DNAH10 variants can cause male infertility with MMAF. These findings will provide guidance for genetic counseling and insights into the diagnosis of MMAF-associated asthenoteratozoospermia.

Molecular evolution analysis of MYB5 in Brassicaceae with specific focus on seed coat color of Brassica napus.

BACKGROUND: MYB transcription factors are splay a vital role in plant biology, with previous research highlighting the significant impact of the R2R3-MYB-like transcription factor MYB5 on seed mucilage biosynthesis, trichome branching, and seed coat development. However, there is a dearth of studies investigating its role in the regulation of proanthocyanidin (PA) biosynthesis. RESULTS: In this study, a total of 51 MYB5 homologous genes were identified across 31 species belonging to the Brassicaceae family, with particular emphasis on Brassica napus for subsequent investigation. Through phylogenetic analysis, these genes were categorized into four distinct subclasses. Protein sequence similarity and identity analysis demonstrated a high degree of conservation of MYB5 among species within the Brassicaceae family. Additionally, the examination of selection pressure revealed that MYB5 predominantly underwent purifying selection during its evolutionary history, as indicated by the Ka/Ks values of all MYB5 homologous gene pairs being less than one. Notably, we observed a higher rate of non-synonymous mutations in orthologous genes compared to paralogous genes, and the Ka/Ks value displayed a stronger correlation with Ka. In B. napus, an examination of expression patterns in five tissues revealed that MYB5 exhibited particularly high expression in the black seed coat. The findings from the WGCNA demonstrated a robust correlation between MYB5 and BAN(ANR) associated with PA biosynthesis in the black seed coat, providing further evidence of their close association and co-expression. Furthermore, the results obtained from of the analysis of protein interaction networks offer supplementary support for the proposition that MYB5 possesses the capability to interact with transcriptional regulatory proteins, specifically TT8 and TT2, alongside catalytic enzymes implicated in the synthesis of PAs, thereby making a contribution to the biosynthesis of PAs. These findings imply a plausible and significant correlation between the nuique expression pattern of MYB5 and the pigmentation of rapeseed coats. Nevertheless, additional research endeavors are imperative to authenticate and substantiate these findings. CONCLUSIONS: This study offers valuable insights into the genetic evolution of Brassicaceae plants, thereby serving as a significant reference for the genetic enhancement of Brassicaceae seed coat color.

Is postmastectomy radiotherapy necessary for breast cancer patients with clinically node-positive downstaging to ypN0 after neoadjuvant chemotherapy?

PURPOSE: The significance of postmastectomy radiotherapy (PMRT) in breast cancer patients who initially have clinically node-positive (cN +) status but achieve downstaging to ypN0 following neoadjuvant chemotherapy (NAC) remains uncertain. This study aims to assess the impact of PMRT in this patient subset. METHODS: Patients were enrolled from West China Hospital, Sichuan University from 2008 to 2019. Overall survival (OS), Locoregional recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), and breast cancer-specific survival (BCSS) were estimated using the Kaplan-Meier method and assessed with the log-rank test. The impact of PMRT was further analyzed by the Cox proportional hazards model. Propensity score matching (PSM) was performed to reduce the selection bias. RESULTS: Of the 333 eligible patients, 189 (56.8%) received PMRT, and 144 (43.2%) did not. At a median follow-up period of 71 months, the five-year LRFS, DMFS, BCSS, and OS rates were 99.1%, 93.4%, 96.4%, and 94.3% for the entire cohort, respectively. Additionally, the 5-year LRFS, DMFS, BCSS, and OS rates were 98.9%, 93.8%, 96.7%, and 94.5% with PMRT and 99.2%, 91.3%, 94.9%, and 92.0% without PMRT, respectively (all p-values not statistically significant). After multivariate analysis, PMRT was not a significant risk factor for any of the endpoints. When further stratified by stage, PMRT did not show any survival benefit for patients with stage II-III diseases. CONCLUSION: In the context of comprehensive treatments, PMRT might be exempted in ypN0 breast cancer patients. Further large-scale, randomized controlled studies are required to investigate the significance of PMRT in this patient subset.

Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/ß-catenin signaling via interaction with P27.

Avian leukemia virus subgroup J (ALV-J) causes various diseases associated with tumor formation and decreased fertility and induced immunosuppressive disease, resulting in significant economic losses in the poultry industry globally. Virus usually exploits the host cellular machinery for their replication. Although there are increasing evidences for the cellular proteins involving viral replication, the interaction between ALV-J and host proteins leading to the pivotal steps of viral life cycle are still unclear. Here, we reported that ribonucleoside-diphosphate reductase subunit M2 (RRM2) plays a critical role during ALV-J infection by interacting with capsid protein P27 and activating Wnt/ß-catenin signaling. We found that the expression of RRM2 is effectively increased during ALV-J infection, and that RRM2 facilitates ALV-J replication by interacting with viral capsid protein P27. Furthermore, ALV-J P27 activated Wnt/ß-catenin signaling by promoting ß-catenin entry into the nucleus, and RRM2 activated Wnt/ß-catenin signaling by enhancing its phosphorylation at Ser18 during ALV-J infection. These data suggest that the upregulation of RRM2 expression by ALV-J infection favors viral replication in host cells via activating Wnt/ß-catenin signaling. IMPORTANCE Our results revealed a novel mechanism by which RRM2 facilitates ALV-J growth. That is, the upregulation of RRM2 expression by ALV-J infection favors viral replication by interacting with capsid protein P27 and activating Wnt/ß-catenin pathway in host cells. Furthermore, the phosphorylation of serine at position 18 of RRM2 was verified to be the important factor regulating the activation of Wnt/ß-catenin signaling. This study provides insights for further studies of the molecular mechanism of ALV-J infection.

Landscape of New Nuclease-Containing Antiphage Systems in Escherichia coli and the Counterdefense Roles of Bacteriophage T4 Genome Modifications.

Many phages, such as T4, protect their genomes against the nucleases of bacterial restriction-modification (R-M) and CRISPR-Cas systems through covalent modification of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, raising the question of the role of phage genome modifications in countering these systems. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli and demonstrated the roles of T4 genome modifications in countering these systems. Our analysis identified at least 17 nuclease-containing defense systems in E. coli, with type III Druantia being the most abundant system, followed by Zorya, Septu, Gabija, AVAST type 4, and qatABCD. Of these, 8 nuclease-containing systems were found to be active against phage T4 infection. During T4 replication in E. coli, 5-hydroxymethyl dCTP is incorporated into the newly synthesized DNA instead of dCTP. The 5-hydroxymethylcytosines (hmCs) are further modified by glycosylation to form glucosyl-5-hydroxymethylcytosine (ghmC). Our data showed that the ghmC modification of the T4 genome abolished the defense activities of Gabija, Shedu, Restriction-like, type III Druantia, and qatABCD systems. The anti-phage T4 activities of the last two systems can also be counteracted by hmC modification. Interestingly, the Restriction-like system specifically restricts phage T4 containing an hmC-modified genome. The ghmC modification cannot abolish the anti-phage T4 activities of Septu, SspBCDE, and mzaABCDE, although it reduces their efficiency. Our study reveals the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of T4 genomic modification in countering these defense systems. IMPORTANCE Cleavage of foreign DNA is a well-known mechanism used by bacteria to protect themselves from phage infections. Two well-known bacterial defense systems, R-M and CRISPR-Cas, both contain nucleases that cleave the phage genomes through specific mechanisms. However, phages have evolved different strategies to modify their genomes to prevent cleavage. Recent studies have revealed many novel nuclease-containing antiphage systems from various bacteria and archaea. However, no studies have systematically investigated the nuclease-containing antiphage systems of a specific bacterial species. In addition, the role of phage genome modifications in countering these systems remains unknown. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli using all 2,289 genomes available in NCBI. Our studies reveal the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of genomic modification of phage T4 in countering these defense systems.

Deleterious variants in X-linked RHOXF1 cause male infertility with oligo- and azoospermia.

Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.

A hemizygous loss-of-function variant in BCORL1 is associated with male infertility and oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a common type of male infertility; however, its genetic causes remain largely unknown. Some of the genetic determinants of OAT are gene defects affecting spermatogenesis. BCORL1 (BCL6 corepressor like 1) is a transcriptional corepressor that exhibits the OAT phenotype in a knockout mouse model. A hemizygous missense variant of BCORL1 (c.2615T > G:p.Val872Gly) was reported in an infertile male patient with non-obstructive azoospermia (NOA). Nevertheless, the correlation between BCORL1 variants and OAT in humans remains unknown. In this study, we used whole-exome sequencing to identify a novel hemizygous nonsense variant of BCORL1 (c.1564G > T:p.Glu522*) in a male patient with OAT from a Han Chinese family. Functional analysis showed that the variant produced a truncated protein with altered cellular localization and a dysfunctional interaction with SKP1 (S-phase kinase-associated protein 1). Further population screening identified four BCORL1 missense variants in subjects with both OAT (1 of 325, 0.31%) and NOA (4 of 355, 1.13%), but no pathogenic BCORL1 variants among 362 fertile subjects. In conclusion, our findings indicate that BCORL1 is a potential candidate gene in the pathogenesis of OAT and NOA, expanded its disease spectrum and suggested that BCORL1 may play a role in spermatogenesis by interacting with SKP1.

Systemic neutrophils are triggered by respiratory Bacillus Calmette- Guérin and mediate pulmonary mycobacterial clearance in synergy with the triggering receptor expressed on myeloid cells 1.

Tuberculosis remains a threat to public health. The only approved vaccine, Bacillus Calmette-Guérin (BCG), is administered intradermally and provides limited protection, and its effect on innate immunity via the respiratory route has not been fully elucidated. A mouse model with genetically depleted TREM1 and seven-color flow cytometry staining were used to characterize the comprehensive immune response induced by respiratory BCG, through evaluating organ bacterial loads, lung histopathology, and lung immunohistochemistry. During respiratory BCG infection, the murine lungs displayed effective bacterial clearance. Notably, marked differences in neutrophils were observed between thymus and bone marrow cells, characterized by a significant increase in the expression of the triggering receptor expressed on myeloid cells 1 (TREM1). Subsequently, upon depletion of TREM1, a reduction in pulmonary neutrophils was observed, which further exacerbated bacterial loads and resulted in worsened pathology following respiratory BCG infection. In summary, up-regulated expression of TREM1 in rapidly increasing circulating neutrophil by pulmonary BCG is required for an efficient host response to BCG infection, and suggests the important role of TREM1 in neutrophil-related pulmonary bacteria clearance and pathology.

Egr-1 is a key regulator of the blood-brain barrier damage induced by meningitic Escherichia coli.

Bacterial meningitis remains a leading cause of infection-related mortality worldwide. Although Escherichia coli (E. coli) is the most common etiology of neonatal meningitis, the underlying mechanisms governing bacterial blood-brain barrier (BBB) disruption during infection remain elusive. We observed that infection of human brain microvascular endothelial cells with meningitic E. coli triggers the activation of early growth response 1 (Egr-1), a host transcriptional activator. Through integrated chromatin immunoprecipitation sequencing and transcriptome analysis, we identified Egr-1 as a crucial regulator for maintaining BBB integrity. Mechanistically, Egr-1 induced cytoskeletal changes and downregulated tight junction protein expression by directly targeting VEGFA, PDGFB, and ANGPTL4, resulting in increased BBB permeability. Meanwhile, Egr-1 also served as a master regulator in the initiation of neuroinflammatory response during meningitic E. coli infection. Our findings support an Egr-1-dependent mechanism of BBB disruption by meningitic E. coli, highlighting a promising therapeutic target for bacterial meningitis.

TGFß1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli.

BACKGROUND: Meningitic Escherichia coli (E. coli) is the major etiological agent of bacterial meningitis, a life-threatening infectious disease with severe neurological sequelae and high mortality. The major cause of central nervous system (CNS) damage and sequelae is the bacterial-induced inflammatory storm, where the immune response of the blood-brain barrier (BBB) is crucial. METHODS: Western blot, real-time PCR, enzyme-linked immunosorbent assay, immunofluorescence, and dual-luciferase reporter assay were used to investigate the suppressor role of transforming growth factor beta 1 (TGFß1) in the immune response of brain microvascular endothelial cells elicited by meningitic E. coli. RESULT: In this work, we showed that exogenous TGFß1 and induced noncanonical Hedgehog (HH) signaling suppressed the endothelial immune response to meningitic E. coli infection via upregulation of intracellular miR-155. Consequently, the increased miR-155 suppressed ERK1/2 activation by negatively regulating KRAS, thereby decreasing IL-6, MIP-2, and E-selectin expression. In addition, the exogenous HH signaling agonist SAG demonstrated promising protection against meningitic E. coli-induced neuroinflammation. CONCLUSION: Our work revealed the effect of TGFß1 antagonism on E. coli-induced BBB immune response and suggested that activation of HH signaling may be a potential protective strategy for future bacterial meningitis therapy. Video Abstract.

IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity.

Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function1-4. However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer-an aggressive malignancy that is refractory to standard treatments and current immunotherapies5-8-induces endoplasmic reticulum stress and activates the IRE1α-XBP1 arm of the unfolded protein response9,10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, which triggered IRE1α-XBP1 activation that suppressed mitochondrial activity and IFNγ production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N-linked protein glycosylation, abrogating IRE1α-XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1α-XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts.

Biallelic CFAP61 variants cause male infertility in humans and mice with severe oligoasthenoteratozoospermia.

BACKGROUND: The genetic causes for most male infertility due to severe oligoasthenoteratozoospermia (OAT) remain unclear. OBJECTIVE: To identify the genetic cause of male infertility characterised by OAT. METHODS: Variant screening was performed by whole-exome sequencing from 325 infertile patients with OAT and 392 fertile individuals. In silico and in vitro analyses were performed to evaluate the impacts of candidate disease-causing variants. A knockout mouse model was generated to confirm the candidate disease-causing gene, and intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of clinical treatment. RESULTS: We identified biallelic CFAP61 variants (NM_015585.4: c.1654C>T (p.R552C) and c.2911G>A (p.D971N), c.144-2A>G and c.1666G>A (p.G556R)) in two (0.62%) of the 325 OAT-affected men. In silico bioinformatics analysis predicted that all four variants were deleterious, and in vitro functional analysis confirmed the deleterious effects of the mutants. Notably, H&E staining and electron microscopy analyses of the spermatozoa revealed multiple morphological abnormalities of sperm flagella, the absence of central pair microtubules and mitochondrial sheath malformation in sperm flagella from man with CFAP61 variants. Further immunofluorescence assays revealed markedly reduced CFAP61 staining in the sperm flagella. In addition, Cfap61-deficient mice showed the OAT phenotype, suggesting that loss of function of CFAP61 was the cause of OAT. Two individuals accepted ICSI therapy using their own ejaculated sperm, and one of them succeeded in fathering a healthy baby. CONCLUSIONS: Our findings indicate that CFAP61 is essential for spermatogenesis and that biallelic CFAP61 variants lead to male infertility in humans and mice with OAT.