Staphylococcus aureus foldase PrsA contributes to the folding and secretion of protein A.

BACKGROUND: Staphylococcus aureus secretes a variety of proteins including virulence factors that cause diseases. PrsA, encoded by many Gram-positive bacteria, is a membrane-anchored lipoprotein that functions as a foldase to assist in post-translocational folding and helps maintain the stability of secreted proteins. Our earlier proteomic studies found that PrsA is required for the secretion of protein A, an immunoglobulin-binding protein that contributes to host immune evasion. This study aims to investigate how PrsA influences protein A secretion. RESULTS: We found that in comparison with the parental strain HG001, the prsA-deletion mutant HG001ΔprsA secreted less protein A. Deleting prsA also decreased the stability of exported protein A. Pulldown assays indicated that PrsA interacts with protein A in vivo. The domains in PrsA that interact with protein A are mapped to both the N- and C-terminal regions (NC domains). Additionally, the NC domains are essential for promoting PrsA dimerization. Furthermore, an immunoglobulin-binding assay revealed that, compared to the parental strain HG001, fewer immunoglobulins bound to the surface of the mutant strain HG001ΔprsA. CONCLUSIONS: This study demonstrates that PrsA is critical for the folding and secretion of protein A. The information derived from this study provides a better understanding of virulent protein export pathways that are crucial to the pathogenicity of S. aureus.

Immunoglobulin-like transcript 2 as an impaired anti-tumor cytotoxicity marker of natural killer cells in patients with hepatocellular carcinoma.

Introduction: Natural killer (NK) cells play a pivotal role in immune surveillance in the liver. We aimed to identify potential targets for NK cell-mediated immune intervention by revealing the functional molecules on NK cells in HCC patients. Methods: To evaluate the impact of aging on NK cell phenotypes, we examined NK cells from healthy volunteers (HVs) of various ages. Because ILT2 expression on CD56dim NK cells increased with increasing age, we enrolled age-matched HCC patients and HVs. We determined the NK cell phenotypes in blood mononuclear cells (PBMCs) and intrahepatic lymphocytes (IHLs) from cancerous and non-cancerous tissues. We evaluated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells in vitro. Results: ILT2-positive CD56dim NK cells in PBMCs were increased in HCC patients compared with HVs. In HCC patients, ILT2-positive CD56dim NK cells were increased in cancerous IHLs compared with non-cancerous IHLs and PBMCs. We examined the impact of macrophage migration inhibitory factor (MIF) on ILT2 expression in co-cultures of HCC cells and NK cells. The enhanced expression of ILT2 on CD56dim NK cells from HCC patients was inhibited by masking antibodies against MIF and CXCR4. ILT2-positive CD56dim NK cells exhibited lower capacities for cytotoxicity and ADCC than ILT2-negative cells, which were partially restored by ILT2 blockade. Conclusions: In HCC patients, ILT2 is a signature molecule for cancerous CD56dim NK cells with impaired cytolytic capacity. The MIF-CXCR4 interaction is associated with ILT2 induction on CD56dim NK cells and ILT2 serves as a target for functional NK cell restoration.

Immunoglobulins and serum proteins impair anti-tumor NK cell effector functions in malignant ascites.

Introduction: Malignant ascites indicates ovarian cancer progression and predicts poor clinical outcome. Various ascites components induce an immunosuppressive crosstalk between tumor and immune cells, which is poorly understood. In our previous study, imbalanced electrolytes, particularly high sodium content in malignant ascites, have been identified as a main immunosuppressive mechanism that impaired NK and T-cell activity. Methods: In the present study, we explored the role of high concentrations of ascites proteins and immunoglobulins on antitumoral NK effector functions. To this end, a coculture system consisting of healthy donor NK cells and ovarian cancer cells was used. The anti-EGFR antibody Cetuximab was added to induce antibody-dependent cellular cytotoxicity (ADCC). NK activity was assessed in the presence of different patient ascites samples and immunoglobulins that were isolated from ascites. Results: Overall high protein concentration in ascites impaired NK cell degranulation, conjugation to tumor cells, and intracellular calcium signaling. Immunoglobulins isolated from ascites samples competitively interfered with NK ADCC and inhibited the conjugation to target cells. Furthermore, downregulation of regulatory surface markers CD16 and DNAM-1 on NK cells was prevented by ascites-derived immunoglobulins during NK cell activation. Conclusion: Our data show that high protein concentrations in biological fluids are able to suppress antitumoral activity of NK cells independent from the mechanism mediated by imbalanced electrolytes. The competitive interference between immunoglobulins of ascites and specific therapeutic antibodies could diminish the efficacy of antibody-based therapies and should be considered in antibody-based immunotherapies.

Expression of IZUMO1 and JUNO in the gonads of domestic cats (Felis catus).

Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets.

Profiling of microglia nodules in multiple sclerosis reveals propensity for lesion formation.

Microglia nodules (HLA-DR+ cell clusters) are associated with brain pathology. In this post-mortem study, we investigated whether they represent the first stage of multiple sclerosis (MS) lesion formation. We show that microglia nodules are associated with more severe MS pathology. Compared to microglia nodules in stroke, those in MS show enhanced expression of genes previously found upregulated in MS lesions. Furthermore, genes associated with lipid metabolism, presence of T and B cells, production of immunoglobulins and cytokines, activation of the complement cascade, and metabolic stress are upregulated in microglia nodules in MS. Compared to stroke, they more frequently phagocytose oxidized phospholipids and possess a more tubular mitochondrial network. Strikingly, in MS, some microglia nodules encapsulate partially demyelinated axons. Taken together, we propose that activation of microglia nodules in MS by cytokines and immunoglobulins, together with phagocytosis of oxidized phospholipids, may lead to a microglia phenotype prone to MS lesion formation.

Human serum-derived α-synuclein auto-antibodies mediate NMDA receptor-dependent degeneration of CNS neurons.

BACKGROUND: Presence of autoantibodies against α-synuclein (α-syn AAb) in serum of the general population has been widely reported. That such peripheral factors may be involved in central nervous system pathophysiology was demonstrated by detection of immunoglobulins (IgGs) in cerebrospinal fluid and brain of Parkinson's disease (PD) patients. Thus, blood-borne IgGs may reach the brain parenchyma through an impaired blood-brain barrier (BBB). FINDINGS: The present study aims to evaluate the patho-physiological impact of α-syn AAbs on primary brain cells, i.e., on spontaneously active neurons and on astrocytes. Exposure of neuron-astrocyte co-cultures to human serum containing α-syn AAbs mediated a dose-dependent reduction of spontaneous neuronal activity, and subsequent neurodegeneration. Removal specifically of α-syn AAbs from the serum prevented neurotoxicity, while purified, commercial antibodies against α-syn mimicked the neurodegenerative effect. Mechanistically, we found a strong calcium flux into neurons preceding α-syn AAbs-induced cell death, specifically through NMDA receptors. NMDA receptor antagonists prevented neurodegeneration upon treatment with α-syn (auto)antibodies. α-syn (auto)antibodies did not affect astrocyte survival. However, in presence of α-syn, astrocytes reacted to α-syn antibodies by secretion of the chemokine RANTES. CONCLUSION: These findings provide a novel basis to explain how a combination of BBB impairment and infiltration of IgGs targeting synuclein may contribute to neurodegeneration in PD and argue for caution with α-syn immunization therapies for treatment of PD.

The B7:CD28 family and friends: Unraveling coinhibitory interactions.

Immune responses must be tightly regulated to ensure both optimal protective immunity and tolerance. Costimulatory pathways within the B7:CD28 family provide essential signals for optimal T cell activation and clonal expansion. They provide crucial inhibitory signals that maintain immune homeostasis, control resolution of inflammation, regulate host defense, and promote tolerance to prevent autoimmunity. Tumors and chronic pathogens can exploit these pathways to evade eradication by the immune system. Advances in understanding B7:CD28 pathways have ushered in a new era of immunotherapy with effective drugs to treat cancer, autoimmune diseases, infectious diseases, and transplant rejection. Here, we discuss current understanding of the mechanisms underlying the coinhibitory functions of CTLA-4, PD-1, PD-L1:B7-1 and PD-L2:RGMb interactions and less studied B7 family members, including HHLA2, VISTA, BTNL2, and BTN3A1, as well as their overlapping and unique roles in regulating immune responses, and the therapeutic potential of these insights.

HHLA2 is more significantly associated with poor prognosis in TSCC than PD-L1.

BACKGROUND: The incidence and mortality of tongue squamous cell carcinoma have shown an alarming increase in recent years. This study aimed to investigate the potential of HHLA2 as an immune checkpoint in comparison to PD-L1. METHODS: We obtained RNA-seq data from TCGA to study HHLA2 and PD-L1 expression across various tissues. Using the CIBERSORT package, we estimated cell type abundances within mixed populations based on gene expression profiles. Immunohistochemistry was performed to analyze HHLA2 and PD-L1 expression in Tongue squamous cell carcinoma. Prognostic evaluation was carried out with Kaplan-Meier curves and the log-rank test. To explore factors affecting HHLA2, univariate and multivariate Cox regression analyses were conducted with the COX regression model. Additionally, we used single-cell RNA sequencing data from the GEO database for gene set enrichment analysis with genes strongly correlated with HHLA2. RESULTS: Our analysis of RNA-seq data unveiled a significant upregulation of HHLA2 and PD-L1 expression in primary tumors when compared with normal tissue. HHLA2 exhibited a positive expression rate of 36.9%, while PD-L1 had a positive expression rate of 24.6%. HHLA2 emerged as a noteworthy independent risk factor impacting the overall survival of Tongue squamous cell carcinoma patients. The analysis of scRNA-seq data shed light on the involvement of HHLA2 in key pathways related to cell cycle regulation and interferon alpha/beta signaling. CONCLUSIONS: This study suggests that in the context of Tongue squamous cell carcinoma, HHLA2 may represent a more promising target for immunotherapy when compared with PD-L1.

The Immune Suppressor IGSF1 as a Potential Target for Cancer Immunotherapy.

The development of first-generation immune-checkpoint inhibitors targeting PD-1/PD-L1 and CTLA-4 ushered in a new era in anticancer therapy. Although immune-checkpoint blockade therapies have shown clinical success, a substantial number of patients yet fail to benefit. Many studies are under way to discover next-generation immunotherapeutic targets. Immunoglobulin superfamily member 1 (IGSF1) is a membrane glycoprotein proposed to regulate thyroid function. Despite containing 12 immunoglobin domains, a possible role for IGSF1, in immune response, remains unknown. Here, our studies revealed that IGSF1 is predominantly expressed in tumors but not normal tissues, and increased expression is observed in PD-L1low non-small cell lung cancer (NSCLC) cells as compared with PD-L1high cells. Subsequently, we developed and characterized an IGSF1-specific human monoclonal antibody, WM-A1, that effectively promoted antitumor immunity and overcame the limitations of first-generation immune-checkpoint inhibitors, likely via a distinct mechanism of action. We further demonstrated high WM-A1 efficacy in humanized peripheral blood mononuclear cells (PBMC), and syngeneic mouse models, finding additive efficacy in combination with an anti-PD-1 (a well-characterized checkpoint inhibitor). These findings support IGSF1 as an immune target that might complement existing cancer immunotherapeutics.

Sperm induction of somatic cell-cell fusion as a novel functional test.

The fusion of mammalian gametes requires the interaction between IZUMO1 on the sperm and JUNO on the oocyte. We have recently shown that ectopic expression of mouse IZUMO1 induces cell-cell fusion and that sperm can fuse to fibroblasts expressing JUNO. Here, we found that the incubation of mouse sperm with hamster fibroblasts or human epithelial cells in culture induces the fusion between these somatic cells and the formation of syncytia, a pattern previously observed with some animal viruses. This sperm-induced cell-cell fusion requires a species-matching JUNO on both fusing cells, can be blocked by an antibody against IZUMO1, and does not rely on the synthesis of new proteins. The fusion is dependent on the sperm's fusogenic capacity, making this a reliable, fast, and simple method for predicting sperm function during the diagnosis of male infertility.

Pentapeptide PYRAE triggers ER stress-mediated apoptosis of breast cancer cells in mice by targeting RHBDF1-BiP interaction.

Reinforced cellular responses to endoplasmic reticulum (ER) stress are caused by a variety of pathological conditions including cancers. Human rhomboid family-1 protein (RHBDF1), a multiple transmembrane protein located mainly on the ER, has been shown to promote cancer development, while the binding immunoglobulin protein (BiP) is a key regulator of cellular unfolded protein response (UPR) for the maintenance of ER protein homeostasis. In this study, we investigated the role of RHBDF1 in maintaining ER protein homeostasis in breast cancer cells. We showed that deleting or silencing RHBDF1 in breast cancer cell lines MCF-7 and MDA-MB-231 caused marked aggregation of unfolded proteins in proximity to the ER. We demonstrated that RHBDF1 directly interacted with BiP, and this interaction had a stabilizing effect on the BiP protein. Based on the primary structural motifs of RHBDF1 involved in BiP binding, we found a pentapeptide (PE5) targeted BiP and inhibited BiP ATPase activity. SPR assay revealed a binding affinity of PE5 toward BiP (Kd = 57.7 µM). PE5 (50, 100, 200 µM) dose-dependently promoted ER protein aggregation and ER stress-mediated cell apoptosis in MCF-7 and MDA-MB-231 cells. In mouse 4T1 breast cancer xenograft model, injection of PE5 (10 mg/kg, s.c., every 2 days for 2 weeks) significantly inhibited the tumor growth with markedly increased ER stress and apoptosis-related proteins in tumor tissues. Our results suggest that the ability of RHBDF1 to maintain BiP protein stability is critical to ER protein homeostasis in breast cancer cells, and that the pentapeptide PE5 may serve as a scaffold for the development of a new class of anti-BiP inhibitors.

Histological study of Bactrian camel cryptorchidism and expression of immunoglobulin λ light chain in the testicular and epididymis of cryptorchid Bactrian camel.

Lambda light chains (λ-LCs) are frequently responsible for triggering the activation of inflammatory factors in autoimmune disorders, and an increase in their levels will cause various pathological changes in serum. The aim of this study was to determine the histological differences between the epididymis and testis of normal and cryptorchid Bactrian camels and the differences in λ-LC expression in the epididymis and testis of normal and cryptorchid Bactrian camels. Haematoxylin and eosin (H&E) staining was used to examine the pathological changes in cryptorchidism. The gene and protein levels of λ-LC were determined using RT-qPCR and western blot. The distribution of λ-LCs was assessed by immunohistochemistry and immunofluorescence. Compared with that in normal Bactrian camels, the diameter of the epididymal lumen and the thickness of the epithelium were decreased in the epididymis of cryptorchidic animals. Additionally, no sperm was detected in the cavity of the cryptorchidic epididymis. Meanwhile, the expression of λ-LC was significantly increased in the cryptorchidic epididymis at both the mRNA and protein levels (p < .05). The highest protein expression of λ-LC was found in epididymal epithelial halo cells and testicular Sertoli cells. These findings suggested that the structural changes observed in the epididymal epithelium of cryptorchidic camels affect its secretory and absorptive functions. Additionally, the high level of λ-LC expression recorded in halo cells suggested that these cells play an important role in epithelial immunity in cryptorchidic Bactrian camels. Furthermore, the high λ-LC expression levels detected in normal testicular Sertoli cells indicated that λ-LCs may be involved in spermatogenesis. The results of this study provide clues for an in-depth study of immunological sterility in cryptorchidic Bactrian camels.

Effects of microalgae, with or without xylanase supplementation, on serum immunoglobulins, cecal short-chain fatty acids, microbial diversity, and metabolic pathways of broiler chickens.

Modern broilers are highly susceptible to environmental and pathogenic threats, leading to gut disorders and poor nutrient utilization if not managed properly. Nutritional programming using several feedstuffs and coproducts to manage gut health has been studied. This study used microalgae as a functional compound and xylanase enzyme in broilers' diets as a strategy to manage gut health. A total of 162 one-day-old unsexed Cobb 500 broiler chicks were randomly assigned to 1 of the 3 dietary treatments: a) corn-soybean meal-based control diet (CON), b) 3% microalgae (MAG), and c) MAG with xylanase enzyme (MAG+XYN). The chicks were reared for 35 days (d) on a floor pen system maintaining standard environment conditions to evaluate the effects of microalgae, with or without xylanase supplementation, on serum immunoglobulins, cecal short-chain fatty acids (SCFA) production, cecal microbial diversity, and metabolic pathways. No significant differences were found for serum immunoglobulin and cecal SCFA among the treatment groups (P > 0.05). Relative microbial abundance at the genus level showed that MAG and MAG+XYN groups had a diverse microbial community on d 3 and d 35. However, no bacterial genus had a significant difference (P > 0.05) in their relative abundance on d 3, but 16 genera showed significant differences (P < 0.05) in their relative abundance among the dietary treatments on d 35. Most of these bacteria were SCFA-producing bacteria. Moreover, MAG and MAG+XYN-fed broilers had better responses than CON groups for metabolic pathways (D-mannose degradation, pectin degradation I and II, ß-1-4-mannan degradation, tetrahydrofolate biosynthesis, glutathione biosynthesis, glutathione-peroxide redox reactions, lactate fermentation to propionate, acetate, and hydrogen, etc.) both on d 3 and d 35. The results suggest that using microalgae, with or without xylanase, had no statistical impact on serum immunoglobulins and cecal SCFA production in broilers. However, an improvement in the cecal microbial diversity and metabolic pathways, which are essential indicators of gut health and nutrient utilization, was observed. Most of the improved metabolic pathways were related to fiber utilization and oxidative stress reduction.

Regulation of CD47 expression on CD14+ monocytes by interferon-α in PBC patients.

Background: Primary biliary cholangitis (PBC) is a chronic intrahepatic cholestatic autoimmune liver disease characterized by inflammatory injury of small and medium-sized bile ducts in the liver. The pathogenesis of PBC has yet to be entirely understood. CD47/signal-regulatory protein alpha (SIRPα) is closely related to developing autoimmune diseases by promoting inflammatory response. However, the effect of CD47/SIRPα on inflammatory response in PBC patients is still unclear. Objective: We investigated the expression of CD47/SIRPα and the effect of inflammatory cytokines on the CD47 expression, analyzed potential autoantibodies against CD47 and the effect of anti-CD47 antibody on the inflammatory response in PBC, provided laboratory basis for the study of the pathogenesis and targets for non-invasive diagnosis and treatment on PBC. Methods: The expression levels of CD47 and SIRPα on peripheral blood mononuclear cells (PBMC) were measured in 14 patients with PBC (the PBC group) and 13 healthy subjects (the Control group) by flow cytometry (FCM). The PBMC derived from healthy subjects were stimulated with healthy subjects' serum, PBC patients' serum, IFN-α or TNF-α, and the CD47 expression level on CD14+ monocytes was detected by FCM. The level of serum anti-CD47 antibody or IFN-α in PBC patients and healthy subjects was analyzed by ELISA. FCM was used to examine the TNF-α expression level in CD14+ monocytes of healthy subjects stimulated with isotype control antibody, anti-CD47 antibody, LPS or LPS combined with CD47 antibody. Results: The CD47 expression level on the CD14+ monocytes in PBC patients was statistically higher than that in the Control group (P<0.01). Compared with the Control group (PBMC+healthy serum), the CD47 expression on CD14+ monocyte stimulated with the PBC patients' serum (PBMC+PBC patients' serum) was increased (P<0.001); the CD47 expression on CD14+ monocyte stimulated with IFN-α (PBMC + IFN-α) increased gradually with the increased concentration of IFN-α (P<0.05). However, there was no similar trend on CD14+ monocyte stimulated with the TNF-α (PBMC+TNF-α) (P>0.05). The levels of serum anti-CD47 antibody and IFN-α in the PBC patients were higher than those in healthy subjects (P<0.05). The TNF-α expression level in CD14+ monocyte stimulated with the LPS (PBMC+LPS) or anti-CD47 antibody+LPS group (PBMC+LPS+anti-CD47 antibody) was significantly increased than that in the Control group (PBMC+isotype control antibody) (P<0.01 and P<0.001, respectively). The TNF-α expression level in CD14+ monocyte stimulated with the anti-CD47 antibody + LPS was higher than that with the LPS (P< 0.05). Conclusion: The CD47 may be related to the pathogenesis of PBC by inflammatory response. The CD47/SIRPα signal were imbalanced in PBC patients. The presence of serum anti-CD47 antibodies in PBC patients provides a laboratory basis for clinical diagnosis and treatment.

[Research progress on the role and clinical application of Siglecs in tumor immunity].

Lectins are proteins responsible for recognizing the signals of sugar molecules in the body. Sialic acid-binding immunoglobulin-like lectins (Siglecs) regulate the innate and adaptive immune responses in the tumor microenvironment by recognizing the glycan structure containing sialic acid and mediating downstream signals through immune receptor tyrosine inhibitory motifs. In recent years, a variety of tumor treatment strategies targeting the sialic acid-Siglecs axis have been introduced, including sialoglycoprotein-mediated drug delivery and antibody mediated inhibition of Siglecs from recognizing tumor surface ligands. In the future, by combining with glycoprotein nanotherapy, antibody therapy and gene therapy, Siglecs can be used to accurately locate tumor targets and release the anti-tumor immunity, so as to achieve the purpose of effective cure of tumors.

ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs.

In addition to the canonical ISGF3 and non-canonical STAT2/IRF9 complexes, evidence is emerging of the role of their unphosphorylated counterparts in IFN-dependent and -independent ISG transcription. To better understand the relation between ISGF3 and U-ISGF3 and STAT2/IRF9 and U-STAT2/IRF9 in IFN-I-stimulated transcriptional responses, we performed RNA-Seq and ChIP-Seq, in combination with phosphorylation inhibition and antiviral experiments. First, we identified a group of ISRE-containing ISGs that were commonly regulated in IFNα-treated WT and STAT1-KO cells. Thus, in 2fTGH and Huh7.5 WT cells, early and long-term IFNα-inducible transcription and antiviral activity relied on the DNA recruitment of the ISGF3 components STAT1, STAT2 and IRF9 in a phosphorylation- and time-dependent manner. Likewise, in ST2-U3C and Huh-STAT1KO cells lacking STAT1, delayed IFN responses correlated with DNA binding of phosphorylated STAT2/IRF9 but not U-STAT2/IRF9. In addition, comparative experiments in U3C (STAT1-KO) cells overexpressing all the ISGF3 components (ST1-ST2-IRF9-U3C) revealed U-ISGF3 (and possibly U-STAT2/IRF9) chromatin interactions to correlate with phosphorylation-independent ISG transcription and antiviral activity. Together, our data point to the dominant role of the canonical ISGF3 and non-canonical STAT2/IRF9, without a shift to U-ISGF3 or U-STAT2/IRF9, in the regulation of early and prolonged ISG expression and viral protection. At the same time, they suggest the threshold-dependent role of U-ISFG3, and potentially U-STAT2/IRF9, in the regulation of constitutive and possibly long-term IFNα-dependent responses.

Molecular dynamics of JUNO-IZUMO1 complexation suggests biologically relevant mechanisms in fertilization.

JUNO-IZUMO1 binding is the first known physical link created between the sperm and egg membranes in fertilization, however, how this initiates sperm-egg fusion remains elusive. As advanced structural insights will help to combat the infertility crisis, or advance fertility control, we employed all-atom Molecular Dynamics (MD) to derive dynamic structural insights that are difficult to obtain experimentally. We found that the hydrated JUNO-IZUMO1 interface is composed of a large set of short-lived non-covalent interactions. The contact interface is destabilized by strategically located point mutations, as well as by Zn2+ ions, which shift IZUMO1 into the non-binding "boomerang" conformation. We hypothesize that the latter might explain how the transient zinc spark, as released after sperm entry into the oocyte, might contribute to block polyspermy. To address a second mystery, we performed another set of simulations, as it was previously suggested that JUNO in solution is unable to bind to folate despite it belonging to the folate receptor family. MD now suggests that JUNO complexation with IZUMO1 opens up the binding pocket thereby enabling folate insertion. Our MD simulations thus provide crucial new hypotheses how the dynamics of the JUNO-IZUMO1 complex upon solvation might regulate fertility.

A1BG-AS1 promotes adriamycin resistance of breast cancer by recruiting IGF2BP2 to upregulate ABCB1 in an m6A-dependent manner.

Adriamycin (ADR) resistance is an obstacle for chemotherapy of breast cancer (BC). ATP binding cassette subfamily B member 1 (ABCB1) expression is indicated to be closely related to the drug resistance of cancer cells. The current work intended to explore the molecular mechanisms to regulate ABCB1 in BC cells with ADR resistance. We found that long noncoding RNA (lncRNA) A1BG antisense RNA 1 (A1BG-AS1) is upregulated in ADR resistant BC cell lines (MCF-7/ADR, MDA-MB-231/ADR). A1BG-AS1 knockdown enhanced the ADR sensitivity by suppressing the viability, proliferation potential and migration ability, and facilitating cell apoptosis in BC. Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is known to be an m6A reader to modulate the stability of mRNA transcripts in an m6A-dependent manner, which was a shared RNA binding protein (RBP) for A1BG-AS1 and ABCB1. The interaction of IGF2BP2 with A1BG-AS1 or ABCB1 was explored and verified using RNA pulldown and RNA immunoprecipitation (RIP) assays. ABCB1 mRNA and protein expression was positively regulated by A1BG-AS1 and IGF2BP2 in BC cells. ABCB1 mRNA expression was stabilized by A1BG-AS1 via recruiting IGF2BP2 in an m6A-dependent manner. Moreover, rescue assays demonstrated that A1BG-AS1 enhanced BC ADR resistance by positively modulating ABCB1. Xenograft mouse models were used to explore whether A1BG-AS1 affected the ADR resistance in BC in vivo. The findings indicated that A1BG-AS1 silencing inhibited tumor growth and alleviated ADR resistance in vivo. In conclusion, A1BG-AS1 enhances the ADR resistance of BC by recruiting IGF2BP2 to upregulate ABCB1 in an m6A-dependent manner.

Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

Perspectives of targeting LILRB1 in innate and adaptive immune checkpoint therapy of cancer.

Immune checkpoint blockade is a compelling approach in tumor immunotherapy. Blocking inhibitory pathways in T cells has demonstrated clinical efficacy in different types of cancer and may hold potential to also stimulate innate immune responses. A novel emerging potential target for immune checkpoint therapy is leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1). LILRB1 belongs to the superfamily of leukocyte immunoglobulin-like receptors and exerts inhibitory functions. The receptor is expressed by a variety of immune cells including macrophages as well as certain cytotoxic lymphocytes and contributes to the regulation of different immune responses by interaction with classical as well as non-classical human leukocyte antigen (HLA) class I molecules. LILRB1 has gained increasing attention as it has been demonstrated to function as a phagocytosis checkpoint on macrophages by recognizing HLA class I, which represents a 'Don't Eat Me!' signal that impairs phagocytic uptake of cancer cells, similar to CD47. The specific blockade of the HLA class I:LILRB1 axis may provide an option to promote phagocytosis by macrophages and also to enhance cytotoxic functions of T cells and natural killer (NK) cells. Currently, LILRB1 specific antibodies are in different stages of pre-clinical and clinical development. In this review, we introduce LILRB1 and highlight the features that make this immune checkpoint a promising target for cancer immunotherapy.