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Objective: Micronutrient status, specifically vitamin D and iron, represent modifiable factors for optimizing military readiness. The primary purpose of this investigation was to determine associations between micronutrient deficiency (i.e., iron status and 25-hydroxy-vitamin D [25(OH)D]) and operationally relevant outcomes (i.e., skeletal health, musculoskeletal injury) at baseline and post-10 weeks of arduous military training. Methods: A total of 227 (177 men, 50 women) Marine Officer Candidates School (OCS) candidates who completed OCS training with complete data sets were included in this analysis. Vitamin D and iron status indicators were collected at two timepoints, pre (baseline) and post OCS. Musculoskeletal outcomes at the mid- and proximal tibial diaphysis were assessed via peripheral quantitative computed tomography. Results: Micronutrient status declined following OCS training in men and women and was associated with musculoskeletal outcomes including greater bone strength (strength strain index) at the mid-diaphysis site in those with optimal status (M = 38.26 mm3, SE = 15.59) versus those without (M = −8.03 mm3, SE = 17.27). In women (p =.037), endosteal circumference was greater in the deficient group (M = 53.26 mm, SE = 1.19) compared with the optimal group (M = 49.47 mm, SE = 1.31) at the proximal diaphysis. In men, greater baseline hepcidin concentrations were associated with an increased likelihood of suffering musculoskeletal injury during training. Conclusions: Vitamin D and iron status declined over the course of training, suggesting impaired micronutrient status. Differences in musculoskeletal outcomes by micronutrient group suggests optimal vitamin D and ferritin concentrations may exert beneficial effects on bone fatigability and fracture reduction during military training. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the effect of interleukin-6 (IL-6) inhibitor (tocilizumab) on bacterial infection-associated bone resorption around implants during osseointegration in rabbits. Materials and Methods: At total of 24 male, 9-monthold New Zealand white rabbits were included, and their two mandibular anterior teeth were extracted. Three months after extraction, 24 one-piece Dentium implants (Ø 2.5 mm, intraosseous length of 12 mm) were inserted in the anterior mandible, and the rabbits were divided into four groups (n = 6 per group). Different treatment methods were used in each group: blank control group (BC); only silk ligation (negative control [NC]); silk ligation and injection with minocycline hydrochloride ointment (positive control [PC]); and silk ligation and injection with tocilizumab at 8 mg/kg via the auricle vein (experimental [EP]). Eight weeks later, the animals were sacrificed, and samples were collected and then analyzed using microcomputed tomography (microCT) scanning, immunohistochemical analysis, and histologic analysis. Results: From the microCT measurement, the ratio of the bone volume to the total volume (BV/TV) in the EP group was 67.00% ± 2.72%, which was higher than that in the other three groups (58.85% ± 2.43% in the BC group, 55.72% ± 2.48% in the PC group, and 36.52% ± 3.02% in the NC group). From immunohistochemical analysis, the expression of IL-6 was found to be higher in the NC group than in the BC, PC, and EP groups, but there was no statistical difference between these three groups. Furthermore, the RANKL (receptor activator of nuclear factor-κB ligand) expression was the lowest in the EP group, followed by the BC group, the PC group, and the NC group, which had the highest expression; there was no difference between the NC and PC groups. Upon histologic analysis, significant new bone was found on the implant surfaces in the EP group, sparse and less new bone could be seen in the BC and PC groups, and the most serious bone resorption occurred in the NC group. Conclusions: Tocilizumab, an inhibitor of IL-6, has a certain effect in preventing bone loss around implants caused by bacterial infection during the osseointegration period. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.

Glioblastoma (GBM) is the most common malignant primary brain tumor and remains incurable. Previous work has shown that systemic administration of Decitabine (DAC) induces sufficient expression of cancer-testis antigens (CTA) in GBM for targeting by adoptive T-cell therapy in vivo. However, the mechanisms by which DAC enhances immunogenicity in GBM remain to be elucidated. Using New York esophageal squamous cell carcinoma 1 (NY-ESO-1) as a representative inducible CTA, we demonstrate in patient tissue, immortalized glioma cells, and primary patient-derived gliomaspheres that basal CTA expression is restricted by promoter hypermethylation in gliomas. DAC treatment of glioma cells specifically inhibits DNA methylation silencing to render NY-ESO-1 and other CTA into inducible tumor antigens at single-cell resolution. Functionally, NY-ESO-1 T-cell receptor-engineered effector cell targeting of DAC-induced antigen in primary glioma cells promotes specific and polyfunctional T-cell cytokine profiles. In addition to induction of CTA, DAC concomitantly reactivates tumor-intrinsic human endogenous retroviruses, interferon response signatures, and MHC-I. Overall, we demonstrate that DAC induces targetable tumor antigen and enhances T-cell functionality against GBM, ultimately contributing to the improvement of targeted immune therapies in glioma.SignificanceThis study dissects the tumor-intrinsic epigenetic and transcriptional mechanisms underlying enhanced T-cell functionality targeting decitabine-induced cancer-testis antigens in glioma. Our findings demonstrate concomitant induction of tumor antigens, reactivation of human endogenous retroviruses, and stimulation of interferon signaling as a mechanistic rationale to epigenetically prime human gliomas to immunotherapeutic targeting.

BACKGROUND: Fibrosis-4 (FIB4) is a recommended noninvasive test to assess hepatic fibrosis among patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we used FIB4 trajectory over time (ie, slope of FIB4) as a surrogate marker of liver fibrosis progression and examined if FIB4 slope is associated with clinical and genetic factors among individuals with clinically defined MASLD within the Million Veteran Program Cohort. METHODS: In this retrospective cohort study, FIB4 slopes were estimated through linear regression for participants with clinically defined MASLD and FIB4

Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7β1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies.

We report a multi-ancestry genome-wide association study on liver cirrhosis and its associated endophenotypes, alanine aminotransferase (ALT) and γ-glutamyl transferase. Using data from 12 cohorts, including 18,265 cases with cirrhosis, 1,782,047 controls, up to 1 million individuals with liver function tests and a validation cohort of 21,689 cases and 617,729 controls, we identify and validate 14 risk associations for cirrhosis. Many variants are located near genes involved in hepatic lipid metabolism. One of these, PNPLA3 p.Ile148Met, interacts with alcohol intake, obesity and diabetes on the risk of cirrhosis and hepatocellular carcinoma (HCC). We develop a polygenic risk score that associates with the progression from cirrhosis to HCC. By focusing on prioritized genes from common variant analyses, we find that rare coding variants in GPAM associate with lower ALT, supporting GPAM as a potential target for therapeutic inhibition. In conclusion, this study provides insights into the genetic underpinnings of cirrhosis.

UNLABELLED: The bacterial chemotaxis system is a well-understood signaling pathway that promotes bacterial success. Chemotaxis systems comprise chemoreceptors and the CheA kinase, linked by CheW or CheV scaffold proteins. Scaffold proteins provide connections between chemoreceptors and CheA and also between chemoreceptors to create macromolecular arrays. Chemotaxis is required for host colonization by many microbes, including the stomach pathogen Helicobacter pylori. This bacterium builds chemoreceptor-CheA contacts with two distinct scaffold proteins, CheW and CheV1. H. pylori cheW or cheV1 deletion mutants both lose chemoreceptor array formation, but show differing semisolid agar chemotaxis assay behaviors: ∆cheW mutants exhibit total migration failure, whereas ∆cheV1::cat mutants display a 50% reduction. On investigating these varied responses, we found that both mutants initially struggle with migration. However, over time, ∆cheV1::cat mutants develop a stable, enhanced migration capability, termed migration-able (Mig+). Whole-genome sequencing analysis of four distinct ∆cheV1::cat Mig+ strains identified single-nucleotide polymorphisms (SNPs) in hpg27_252 (hp0273) that were predicted to truncate the encoded protein. Computational analysis of the hpg27_252-encoded protein revealed it encoded a hypothetical protein that was a remote homolog of the PilO Type IV filament membrane alignment complex protein. Although H. pylori lacks Type IV filaments, our analysis showed it retains an operon of genes for homologs of PilO, PilN, and PilM. Deleting hpg27_252 in the ∆cheV1::cat or wild type strain resulted in enhanced migration in semisolid agar. Our study thus reveals that while cheV1 mutants initially have significant migration defects, they can recover the migration ability through genetic suppressors, highlighting a complex regulatory mechanism in bacterial migration. IMPORTANCE: Chemotactic motility, present in over half of bacteria, depends on chemotaxis signaling systems comprising receptors, kinases, and scaffold proteins. In Helicobacter pylori, a stomach pathogen, chemotaxis is crucial for colonization, with CheV1 and CheW as key scaffold proteins. While both scaffolds are essential for building chemoreceptor complexes, their roles vary in other assays. Our research reexamines cheV1 mutants behavior in semisolid agar, a standard chemotaxis test. Initially, cheV1 mutants exhibited defects similar to those of cheW mutants, but they evolved genetic suppressors that enhanced migration. These suppressors involve mutations in a previously uncharacterized gene, unknown in motility behavior. Our findings highlight the significant chemotaxis defects in cheV1 mutants and identify new elements influencing bacterial motility.

Stromal fibroblasts are a major stem cell niche component essential for organ formation and cancer development. Fibroblast heterogeneity, as revealed by recent advances in single-cell techniques, has raised important questions about the origin, differentiation, and function of fibroblast subtypes. In this study, we show in mammary stromal fibroblasts that loss of the receptor tyrosine kinase (RTK) negative feedback regulators encoded by Spry1, Spry2, and Spry4 causes upregulation of signaling in multiple RTK pathways and increased extracellular matrix remodeling, resulting in accelerated epithelial branching. Single-cell transcriptomic analysis demonstrated that increased production of FGF10 due to Sprouty (Spry) loss results from expansion of a functionally distinct subgroup of fibroblasts with the most potent branching-promoting ability. Compared to their three independent lineage precursors, fibroblasts in this subgroup are activated, as they are located immediately adjacent to the epithelium that is actively undergoing branching and invasion. Spry genes are downregulated, and activated fibroblasts are expanded, in all three of the major human breast cancer subtypes. Together, our data highlight the regulation of a functional subtype of mammary fibroblasts by Spry genes and their essential role in epithelial morphogenesis and cancer development.

IntroductionAlthough dementia-related proteinopathy has a strong negative impact on public health, and is highly heritable, understanding of the related genetic architecture is incomplete.MethodsWe applied multidimensional generalized partial credit modeling (GPCM) to test genetic associations with dementia-related proteinopathies. Data were analyzed to identify candidate single nucleotide variants for the following proteinopathies: Aβ, tau, α-synuclein, and TDP-43.ResultsFinal included data comprised 966 participants with neuropathologic and WGS data. Three continuous latent outcomes were constructed, corresponding to TDP-43-, Aβ/Tau-, and α-synuclein-related neuropathology endophenotype scores. This approach helped validate known genotype/phenotype associations: for example, TMEM106B and GRN were risk alleles for TDP-43 pathology; and GBA for α-synuclein/Lewy bodies. Novel suggestive proteinopathy-linked alleles were also discovered, including several (SDHAF1, TMEM68, and ARHGEF28) with colocalization analyses and/or high degrees of biologic credibility.DiscussionA novel methodology using GPCM enabled insights into gene candidates for driving misfolded proteinopathies.HighlightsLatent factor scores for proteinopathies were estimated using a generalized partial credit model. The three latent continuous scores corresponded well with proteinopathy severity. Novel genes associated with proteinopathies were identified. Several genes had high degrees of biologic credibility for dementia risk factors.

BackgroundRecently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress.MethodsWe performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling.ResultsWe first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction.ConclusionsCollectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

Throughout history, coronaviruses have posed challenges to both public health and the global economy; nevertheless, methods to combat them remain rudimentary, primarily due to the absence of experiments to understand the function of various viral components. Among these, membrane (M) proteins are one of the most elusive because of their small size and challenges with expression. Here, we report the development of an expression system to produce tens to hundreds of milligrams of M protein per liter of Escherichia coli culture. These large yields render many previously inaccessible structural and biophysical experiments feasible. Using cryo-electron microscopy and atomic force microscopy, we image and characterize individual membrane-incorporated M protein dimers and discover membrane thinning in the vicinity, which we validated with molecular dynamics simulations. Our results suggest that the resulting line tension, along with predicted induction of local membrane curvature, could ultimately drive viral assembly and budding.

Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

Membrane protein biogenesis poses acute challenges to protein homeostasis, and how they are selectively escorted to the target membrane is not well understood. Here we address this question in the guided-entry-of-tail-anchored protein (GET) pathway, in which tail-anchored membrane proteins (TAs) are relayed through an Hsp70-Sgt2-Get3 chaperone triad for targeting to the endoplasmic reticulum. We show that the Hsp70 ATPase cycle and TA substrate drive dimeric Sgt2 from a wide-open conformation to a closed state, in which TAs are protected by both substrate binding domains of Sgt2. Get3 is privileged to receive TA from closed Sgt2, whereas off-pathway chaperones remove TAs from open Sgt2. Sgt2 closing is less favorable with suboptimal GET substrates, which are rejected during or after the Hsp70-to-Sgt2 handover. Our results demonstrate how fine-tuned conformational dynamics in Sgt2 enable hydrophobic TAs to be effectively funneled onto their dedicated targeting factor while also providing a mechanism for substrate selection.

Toxoplasma gondii divides by endodyogeny, in which two daughter buds are formed within the cytoplasm of the maternal cell using the inner membrane complex (IMC) as a scaffold. During endodyogeny, components of the IMC are synthesized and added sequentially to the nascent daughter buds in a tightly regulated manner. We previously showed that the early recruiting proteins IMC32 and IMC43 form an essential daughter bud assembly complex which lays the foundation of the daughter cell scaffold in T. gondii. In this study, we identify the essential, early recruiting IMC protein BCC0 as a third member of this complex by using IMC32 as bait in both proximity labeling and yeast two-hybrid screens. We demonstrate that BCC0's localization to daughter buds depends on the presence of both IMC32 and IMC43. Deletion analyses and functional complementation studies reveal that residues 701-877 of BCC0 are essential for both its localization and function and that residues 1-899 are sufficient for function despite minor mislocalization. Pairwise yeast two-hybrid assays additionally demonstrate that BCC0's essential domain binds to the coiled-coil region of IMC32 and that BCC0 and IMC43 do not directly interact. This data supports a model for complex assembly in which an IMC32-BCC0 subcomplex initially recruits to nascent buds via palmitoylation of IMC32 and is locked into the scaffold once bud elongation begins by IMC32 binding to IMC43. Together, this study dissects the organization and function of a complex of three early recruiting daughter proteins which are essential for the proper assembly of the IMC during endodyogeny.

BackgroundTrichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized.ResultsTo address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose.ConclusionThis study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, using quantitative live-cell microscopy in genome-edited Jurkat cells, we investigate the impact of Nef on clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment is associated with an increase in the recruitment and lifetime of the CME coat protein AP-2 and the late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2 and transferrin, suggesting that Nef recruitment to CME sites promotes site maturation to ensure high efficiency in host protein downregulation. Implications of these observations for HIV-1 infection are discussed.

Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, transmembrane PCP proteins are trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in the HCC1569 breast cancer cell line, and in undifferentiated, but not differentiated, HC11 cells that serve as a model for mammary lactogenic differentiation. The loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate recruitment of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a, Ccnd1 and Csn2, larger acini and organoids, and precocious differentiation; phenotypes are rescued by overexpression of Vangl2, but not Vangl2ΔNLS. Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.

Cancer immunotherapy has emerged as a promising strategy for the treatment of cancer, with the tumor microenvironment (TME) playing a pivotal role in modulating the immune response. CD47, a cell surface protein, has been identified as a crucial regulator of the TME and a potential therapeutic target for cancer therapy. However, the precise functions and implications of CD47 in the TME during immunotherapy for cancer patients remain incompletely understood. This comprehensive review aims to provide an overview of CD47's multifaced role in TME regulation and immune evasion, elucidating its impact on various types of immunotherapy outcomes, including checkpoint inhibitors and CAR T-cell therapy. Notably, CD47-targeted therapies offer a promising avenue for improving cancer treatment outcomes, especially when combined with other immunotherapeutic approaches. The review also discusses current and potential CD47-targeted therapies being explored for cancer treatment and delves into the associated challenges and opportunities inherent in targeting CD47. Despite the demonstrated effectiveness of CD47-targeted therapies, there are potential problems, including unintended effects on healthy cells, hematological toxicities, and the development if resistance. Consequently, further research efforts are warranted to fully understand the underlying mechanisms of resistance and to optimize CD47-targeted therapies through innovative combination approaches, ultimately improving cancer treatment outcomes. Overall, this comprehensive review highlights the significance of CD47 as a promising target for cancer immunotherapy and provides valuable insight into the challenges and opportunities in developing effective CD47-targeted therapies for cancer treatment.

The multifaceted chemo-immune resistance is the principal barrier to achieving cure in cancer patients. Identifying a target that is critically involved in chemo-immune-resistance represents an attractive strategy to improve cancer treatment. iRhom1 plays a role in cancer cell proliferation and its expression is negatively correlated with immune cell infiltration. Here we show that iRhom1 decreases chemotherapy sensitivity by regulating the MAPK14-HSP27 axis. In addition, iRhom1 inhibits the cytotoxic T-cell response by reducing the stability of ERAP1 protein and the ERAP1-mediated antigen processing and presentation. To facilitate the therapeutic translation of these findings, we develop a biodegradable nanocarrier that is effective in codelivery of iRhom pre-siRNA (pre-siiRhom) and chemotherapeutic drugs. This nanocarrier is effective in tumor targeting and penetration through both enhanced permeability and retention effect and CD44-mediated transcytosis in tumor endothelial cells as well as tumor cells. Inhibition of iRhom1 further facilitates tumor targeting and uptake through inhibition of CD44 cleavage. Co-delivery of pre-siiRhom and a chemotherapy agent leads to enhanced antitumor efficacy and activated tumor immune microenvironment in multiple cancer models in female mice. Targeting iRhom1 together with chemotherapy could represent a strategy to overcome chemo-immune resistance in cancer treatment.

Comprehensive Summary: TAG‐assisted peptide synthesis technology enables optimal conservation of Fmoc amino acid raw materials and chemical solvents while eliminating the need for intricate chromatographic purification processes. This work presents a 4,4'‐diphenylphosphonoxy diphenylcarbinol tag‐mediated liquid‐phase synthesis approach for preparing side‐to‐tail cyclopeptides macolacin which has strong activity against gram‐negative bacteria, and its 15 analogues containing four N‐methylation modified cyclopeptides, as well as an investigation of their structure‐activity relationship (SAR). The synthesis of macolacin analogues primarily focuses on the modifications of the N‐methylation group of Ile‐7 and the tail fatty acyl chain of macolacin. The incorporation of N‐methylation for Ile‐7, along with the dihalogenated or monohalogenated benzoic acids for tail modification, exhibited remarkable antibacterial efficacy and minimal hepatocellular toxicity in vitro. The present study identified an N‐methylation‐modified antimicrobial cyclopeptide Ma14 that exhibits rapid bactericidal efficacy against A. baumanii, etc., while showing reduced hepatocellular toxicity. Molecular docking simulations were conducted to investigate the binding of cyclopeptides to the outer membrane protein BamA of A. baumannii. The findings demonstrated the stable binding interactions of the cyclopeptides with the BamA protein and then presented a novel approach to explain the bacteriostatic mechanism of macolacin‐based cyclopeptide antibiotics. [ABSTRACT FROM AUTHOR]

Viroporins possess important potential as antiviral targets due to their critical roles during virus life cycles, spanning from virus entry to egress. Although the antiviral amantadine targets the M2 viroporin of influenza A virus, successful progression of other viroporin inhibitors into clinical use remains challenging. These challenges relate in varying proportions to a lack of reliable full‐length 3D‐structures, difficulties in functionally characterising individual viroporins, and absence of verifiable direct binding between inhibitor and viroporin. This review offers perspectives to help overcome these challenges. We provide a comprehensive overview of the viroporin family, including their structural and functional features, highlighting the moldability of their energy landscapes and actions. To advance the field, we suggest a list of best practices to aspire towards unambiguous viroporin identification and characterisation, along with considerations of potential pitfalls. Finally, we present current and future scenarios of, and prospects for, viroporin targeting drugs. [ABSTRACT FROM AUTHOR]

[Display omitted] Hypothesis: We hypothesize that simultaneous incorporation of ion channel peptides (in this case, potassium channel as a model) and hydrophobic magnetite Fe 3 O 4 nanoparticles (hFe 3 O 4 NPs) within lipidic hexagonal mesophases, and aligning them using an external magnetic field can significantly enhance ion transport through lipid membranes. Experiments: In this study, we successfully characterized the incorporation of gramicidin membrane ion channels and hFe 3 O 4 NPs in the lipidic hexagonal structure using SAXS and cryo-TEM methods. Additionally, we thoroughly investigated the conductive characteristics of freestanding films of lipidic hexagonal mesophases, both with and without gramicidin potassium channels, utilizing a range of electrochemical techniques, including impedance spectroscopy, normal pulse voltammetry, and chronoamperometry. Findings: Our research reveals a state-of-the-art breakthrough in enhancing ion transport in lyotropic liquid crystals as matrices for integral proteins and peptides. We demonstrate the remarkable efficacy of membranes composed of hexagonal lipid mesophases embedded with K+ transporting peptides. This enhancement is achieved through doping with hFe 3 O 4 NPs and exposure to a magnetic field. We investigate the intricate interplay between the conductive properties of the lipidic hexagonal structure, hFe 3 O 4 NPs, gramicidin incorporation, and the influence of Ca2+ on K+ channels. Furthermore, our study unveils a new direction in ion channel studies and biomimetic membrane investigations, presenting a versatile model for biomimetic membranes with unprecedented ion transport capabilities under an appropriately oriented magnetic field. These findings hold promise for advancing membrane technology and various biotechnological and biomedical applications of membrane proteins. [ABSTRACT FROM AUTHOR]

Film-electrochemical electron paramagnetic resonance spectroscopy (FE-EPR) enables simultaneous electrochemical and spectroscopic characterisation of paramagnetic electron-transfer centres, including in soluble proteins. We now report a modified set-up FE-EPR with tuneable macroporous working electrodes and demonstrate the feasibility to investigate electron transfer in membrane proteins in their native membrane environment. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the similarities and differences in milk fat globule membrane (MFGM) proteins from porcine colostrum (PC) and human colostrum (HC) based on a label‐free proteomics approach. In total, 3901 and 4027 MFGM proteins were identified in PC and HC, respectively, including 3452 common proteins. Similar biological functions and pathways were found in two groups, whereas the quantities and types of MFGM proteins involved were different. This study compared the differences between MFGM proteins in PC and HC, effectively expanding the MFGM proteome and its bioinformatic database and providing new insights for the exploration of porcine milk. [ABSTRACT FROM AUTHOR]

One of the main hallmarks of Parkinson's disease (PD) pathology is the spread of the aggregate‐prone protein α‐synuclein (α‐syn), which can be detected in the plasma and cerebrospinal fluid of patients as well as in the extracellular environment of neuronal cells. The secreted α‐syn can exhibit “prion‐like” behavior and transmission to naïve cells can promote conformational changes and pathology. The precise role of plasma membrane proteins in the pathologic process of α‐syn is yet to be fully resolved. The TMEM16 family of lipid scramblases and ion channels has been recently associated with cancer and infectious diseases but is less known for its role in aging‐related diseases. To elucidate the role of TMEM16F in α‐syn spread, we transduced neurons derived from TMEM16F knockout mice with a reporter system that enables the distinction between donor and recipient neurons of pathologic α‐synA53T. We found that the spread of α‐synA53T was reduced in neurons derived from TMEM16F‐knockout mice. These findings were recapitulated in vivo in a mouse model of PD, where attenuated α‐synA53T spread was observed when TMEM16F was ablated. Moreover, we identified a single nucleotide polymorphism in TMEM16F of Ashkenazi Jewish PD patients resulting in a missense Ala703Ser mutation with enhanced lipid scramblase activity. This mutation is associated with altered regulation of α‐synA53T extracellular secretion in cellular models of PD. Our study highlights TMEM16F as a novel regulator of α‐syn spread and as a potential therapeutic target in synucleinopathies. [ABSTRACT FROM AUTHOR]

Valbenazine is a highly potent and selective inhibitor of synaptic vesicular monoamine transporter 2. The current therapeutic doses of valbenazine for tardive dyskinesia (TD) are 40, 60, or 80 mg capsules, given orally, once daily (QD). While 40 and 80 mg were investigated in phase 3 KINECT® 3 trial and initially approved, the approval of valbenazine 60 mg was based on the analysis utilizing the Model‐informed drug development (MIDD) approach, facilitated through the US Food and Drug Administration's MIDD Pilot Program. This study aimed to demonstrate the efficacy of 60 mg QD dose through model simulations using an established exposure‐response (E‐R) relationship between valbenazine active metabolite [+]‐α‐dihydrotetrabenazine exposure and the change from baseline in Abnormal Involuntary Movement Scale total score (AIMS‐CFB). A longitudinal E–R model was constructed based on the 40 and 80 mg data from the KINECT 3 trial. The final Emax model adequately predicted dose‐dependent improvement in the primary endpoint and was used to interpolate AIMS‐CFB for 60 mg at week 6. The efficacy of the unstudied 60 mg dose regimen is expected to be within the range of doses studied clinically with predicted mean AIMS‐CFB (95% confidence interval) of −2.69 (−3.30, −2.13) between observed mean AIMS‐CFB for 40 mg of −1.92 and 80 mg of −3.39. Results from this analysis provided the key evidence to establish efficacy of 60 mg QD without the need for an additional clinical trial. The availability of valbenazine 60 mg dose fills an existing medical need for patients with TD who could benefit from this third effective dose. [ABSTRACT FROM AUTHOR]

Gliomagenesis induces profound changes in the composition of the extracellular matrix (ECM) of the brain. In this study, we identified a cellular population responsible for the increased deposition of collagen I and fibronectin in glioblastoma. Elevated levels of the fibrillar proteins collagen I and fibronectin were associated with the expression of fibroblast activation protein (FAP), which is predominantly found in pericyte‐like cells in glioblastoma. FAP+ pericyte‐like cells were present in regions rich in collagen I and fibronectin in biopsy material and produced substantially more collagen I and fibronectin in vitro compared to other cell types found in the GBM microenvironment. Using mass spectrometry, we demonstrated that 3D matrices produced by FAP+ pericyte‐like cells are rich in collagen I and fibronectin and contain several basement membrane proteins. This expression pattern differed markedly from glioma cells. Finally, we have shown that ECM produced by FAP+ pericyte‐like cells enhances the migration of glioma cells including glioma stem‐like cells, promotes their adhesion, and activates focal adhesion kinase (FAK) signaling. Taken together, our findings establish FAP+ pericyte‐like cells as crucial producers of a complex ECM rich in collagen I and fibronectin, facilitating the dissemination of glioma cells through FAK activation. [ABSTRACT FROM AUTHOR]

Summary: Granado beans, widely cultivated in Chile, offer plant‐based proteins with functional benefits for healthier food formulations. These legumes have a 28% protein content (dry basis) with peptides called lectins that have the potential of reducing type‐II diabetes. Our lab has developed a membrane‐based method to produce protein isolates from oilseeds. This method was adapted for granado beans. The process involves alkaline extraction at pH 10, followed by ultrafiltration, diafiltration, acid precipitation at pH 4 and freeze‐drying. The resulting precipitated protein isolate contains 81% protein, comparable to other legumes. However, the acid‐soluble protein isolate (SPI) has a protein content of only 53% due to co‐recovery of high molecular weight carbohydrates. The total phenolic compound contents of the protein products were low resulting in both products having an attractive light colour with no astringent taste. The characterisation of the SPI by High‐Performance Liquid Chromatography found that it contained about 40% starch and other carbohydrates. To increase the protein content of SPI to ~90%, further purification steps to remove the carbohydrates are necessary. [ABSTRACT FROM AUTHOR]

Background: There is insufficient replicated data to establish a relationship between the polymorphisms of SLC6A2 and CYP2D6 and the treatment responses of atomoxetine (ATX) in ADHD. We focused on evaluating the effect of top-line single nucleotide polymorphisms (SNPs) in SLC6A2 and CYP2D6 on the ATX treatment response in attention deficit and hyperactivity disorder (ADHD). Methods: Of 160 patient records, 34 patients who met the inclusion criteria were evaluated to determine the relationship between genotypes of ten SNPs (six of SLC6A2 and four of CYP2D6) and ATX treatment response. Additionally, the connection between SNPs of CYP2D6 and the severity of side effects associated with ATX was analyzed in 37 patients, including the 34 study patients, and three patients discontinued because of ATX-dependent side effects. Results: All six polymorphisms we studied in SLC6A2 were associated with the treatment response of ATX. Clinical improvement in oppositional defiant disorder symptoms of patients with ADHD was only observed in carriers of the homozygous "C" allele of rs3785143 (podd = 0.026). We detected an association between higher CGI-side-effect severity scores and the "TT" genotype of rs1065852 polymorphism in CYP2D6 (p = 0.043). Conclusions: The findings of this study suggest that genotypes of polymorphisms within the SLC6A2 and CYP2D6 may play an influential role in treatment response or the severity of side effects associated with ATX in ADHD patients. [ABSTRACT FROM AUTHOR]

Summary: The retromer complex is a conserved sorting machinery that maintains cellular protein homeostasis by transporting vesicles containing cargo proteins to defined destinations. It is known to sort proteins at the vacuole membranes for retrograde trafficking, preventing their degradation in the vacuole. However, the detailed mechanism of retromer recruitment to the vacuole membrane has not yet been elucidated.Here, we show that the vacuolar SNARE complex MoPep12‐MoVti1‐MoVam7‐MoYkt6 regulates retromer‐mediated vesicle trafficking by recruiting the retromer to the vacuole membrane, which promotes host invasion in Magnaporthe oryzae.Such recruitment is also essential for the retrieval of the autophagy regulator MoAtg8 and enables appressorium‐mediated host penetration. Furthermore, the vacuolar SNARE subunits are involved in suppressing the host defense response by regulating the deployment of retromer‐MoSnc1‐mediated effector secretion.Altogether, our results provide insights into the mechanism of vacuolar SNAREs‐dependent retromer recruitment which is necessary for pathogenicity‐related membrane trafficking events in the rice blast fungus. [ABSTRACT FROM AUTHOR]

Intellectual disability (ID) is a kind of nervous developmental disorder and affects more than 1% of people worldwide. SLC45A1 as a transmembrane protein is implicated in the regulation of glucose homoeostasis. Through trio‐based exome sequencing, the missense mutations of SLC45A1 c.103G>A (p.V35M) and c.1211T>G (p.F404C) were identified in the proband with syndromic ID. The distribution, expression and activity of SLC45A1 wild‐type (WT) and variants were assayed in transfected COS7 cells. In SLC45A1 variants, the hydrogen bonds surrounding the 35th and 404th amino acid were changed, location on the cytomembrane was failed, their activity to transport glucose was also significantly decreased to contrast with SLC45A1‐WT. No difference was observed at the mRNA and protein level. In conclusion, the compound heterozygous variants of SLC45A1 might be the genetic etiology for syndromic ID. These novel mutations probably attenuated its activity to transport glucose by the alteration of tertiary structure and failure of intracellular location. [ABSTRACT FROM AUTHOR]

Background: Triple‐negative breast cancer, a subtype of breast cancer, is characterized by a poor prognosis. Recent studies have shown that miRNA30b acts as an oncogene and is vital for the proliferation of malignancies across various systems. This study aimed to elucidate the impact of miRNA30b on the proliferation, migration, and invasion capabilities of breast cancer cells in vitro. Methods: Triple‐negative breast cancer cell lines MDA‐MB‐231 were transiently transfected with miRNA30b inhibitor, mimic, or the negative control by Lipofectamine 2000. Successful transfection was confirmed by quantitative real‐time polymerase chain reaction (qRT‐PCR). Functional assays, including CCK8, clone formation, scratch, and transwell assays, were conducted to evaluate the proliferation, invasion, and migration ability of MDA‐MB‐231 cells in each group. The target protein of miRNA30b was determined using an online prediction data website, and the dual‐luciferase assay confirmed whether there was a binding site between miRNA30b and ADAM12. The effect was further verified by Western blot analysis. Results: MDA‐MB‐231 cells were transfected with miRNA30b inhibitor, mimic, and negative control. miRNA30b expression was downregulated in the cells. Relative to the negative control group, miRNA30b expression significantly increased in the mimic group and decreased in the miRNA30b inhibitor group, with the differences being statistically significant. The miRNA30b mimic group exhibited a significant increase in miRNA30b expression and a corresponding promotion of cell proliferation, colony formation, and migration. Conversely, the miRNA30b inhibitor group displayed significantly reduced miRNA30b expression and suppressed cell proliferation, colony formation, and migration abilities compared to the negative control cells. Bioinformatics software predicted ADAM12 as a potential target of miRNA30b. Dual‐luciferase assays confirmed the presence of a binding site between miRNA30b and ADAM12. Western blot analysis revealed that overexpression of miRNA30b downregulated ADAM12 expression in MDA‐MB‐231 cells. Conclusions: miRNA30b could promote proliferation, migration, and invasion of TNBC cell lines MDA‐MB‐231. The effect of miRNA30b on triple‐negative breast cancer would be achieved partly at least through inhibiting the expression of ADAM12. [ABSTRACT FROM AUTHOR]

New hematopoietic cell models have recently emerged through immortalization of CD34 cells to study and understand various molecular mechanisms of erythropoiesis. Here, we characterize the JK-1 CML-derived cell line, previously shown to spontaneously differentiate without cytokines. Using an epigenetic differentiation inhibitor that keeps JK-1 in an early differentiation phase, we characterized 2 progenitor stages: BFU-E JK-1 and CFU-E JK-1 with CD34+/CD36− and CD34−/CD36 + phenotypes respectively. Then, using the PFI-1 inducer known to synchronously control the terminal differentiation of JK-1 cells, 5 precursor stages were obtained (ProE, Baso1-2, PolyC, OrthoC) and characterized via cell morphology, CD49a and Band3 markers. Enlarged phenotyping was carried out for the earlier phase, and expression kinetics of membrane proteins such as RhAG, RhD/CE, CD47, DARC and CD44 were performed on each stage of the terminal phase. Furthermore, since JK-1 offers the unique property of covering a broad spectrum of differentiation stages, we explored deeper the GATA2/GATA1 and the non-erythroid/erythroid spectrin 'switching'. The possibility of obtaining large quantities of JK-1 cells at each stage of differentiation, as shown in this study, as well as the potential to genetically modify these cells, via CrisprCas9, makes their use of considerable interest for studying pathologies occurring during erythropoiesis. [ABSTRACT FROM AUTHOR]

Outer membrane proteins (OMPs) play a key role in facilitating the survival of Helicobacter pylori within the gastric tissue by mediating adherence. Among these proteins, Outer inflammatory protein A (OipA) is a critical factor in H. pylori colonization of the host gastric epithelial cell surface. While the role of OipA in H. pylori attachment and its association with clinical outcomes have been established, the structural mechanisms underlying OipA's action in adherence to gastric epithelial cells remain limited. Our study employed experimental and computational approaches to investigate the interaction partners of OipA on the gastric epithelial cell surface. Initially, we conducted a proteomic analysis using a pull-down assay with recombinant OipA and gastric epithelial cell membrane proteins to identify the OipA interactome. This analysis revealed 704 unique proteins that interacted with OipA. We subsequently analyzed 16 of these OipA partners using molecular modeling tools. Among these 16 partners, we highlight three human proteins, namely Hepatocyte growth factor (HGF), Mesenchymal epithelial transition factor receptor (Met), and Adhesion G Protein-Coupled Receptor B1 (AGRB1) that could play a role in H. pylori adherence to the gastric epithelial cell surface with OipA. Collectively, these findings reveal novel host interactions mediated by OipA, suggesting their potential as therapeutic targets for combating H. pylori infection. [ABSTRACT FROM AUTHOR]

To enable transmission of information in the brain, synaptic vesicles fuse to presynaptic membranes, liberating their content and exposing transiently a myriad of vesicular transmembrane proteins. However, versatile methods for quantifying the synaptic translocation of endogenous proteins during neuronal activity remain unavailable, as the fast dynamics of synaptic vesicle cycling difficult specific isolation trafficking proteins during such a transient surface exposure. Here, we developed a novel approach using synaptic cleft proximity labeling to capture and quantify activity-driven trafficking of endogenous synaptic proteins at the synapse. We show that accelerating cleft biotinylation times to match the fast dynamics of vesicle exocytosis allows capturing endogenous proteins transiently exposed at the synaptic surface during neural activity, enabling for the first time the study of the translocation of nearly every endogenous synaptic protein. As proof-of-concept, we further applied this technology to obtain direct evidence of the surface translocation of noncanonical trafficking proteins, such as ATG9A and NPTX1, which had been proposed to traffic during activity but for which direct proof had not yet been shown. The technological advancement presented here will facilitate future studies dissecting the molecular identity of proteins exocytosed at the synapse during activity, helping to define the molecular machinery that sustains neurotransmission in the mammalian brain. The fast dynamics of synaptic vesicle release present a challenge for the quantification of synaptic protein translocation during neuronal activity. This study presents a method for quantifying the translocation of endogenous neuronal proteins to the synaptic surface and visualizes the translocation of proteins of interest when neurons are firing. [ABSTRACT FROM AUTHOR]

Zinc transporters (ZnTs) act as H+/Zn2+ antiporters, crucial for zinc homeostasis. Brain‐specific ZnT3 expressed in synaptic vesicles transports Zn2+ from the cytosol into vesicles and is essential for neurotransmission, with ZnT3 dysfunction associated with neurological disorders. Ubiquitously expressed ZnT4 localized to lysosomes facilitates the Zn2+ efflux from the cytosol to lysosomes, mitigating the cell injury risk. Despite their importance, the structures and Zn2+ transport mechanisms remain unclear. We characterized the three‐dimensional structures of human ZnT3 (inward‐facing) and ZnT4 (outward‐facing) using cryo‐electron microscopy. By combining these structures, we assessed the conformational changes that could occur within the transmembrane domain during Zn2+ transport. Our results provide a structural basis for a more comprehensive understanding of the H+/Zn2+ exchange mechanisms exhibited by ZnTs. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Material and Methods Results Conclusions Hereditary spherocytosis (HS) is a congenital haemolytic disorder, resulting from plasma membrane protein deficiency of red blood cells (RBCs). Typical pathological signs are anemia, jaundice, and splenomegaly; in newborns, jaundice is the main symptom.This study focused on the state of art about the HS diagnosis, from traditional to innovative methods, including diagnostic algorithms that can be applied for pediatric and adult patients, for different laboratory diagnostic levels.The first erythrocyte parameters used for HS diagnosis were the mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), and red blood cell distribution width (RDW); nowadays new parameters are used in blood cell counter. Advia analyzers (Siemens Medical Solutions) supply the hyper‐dense cell percentage (% Hyper), which reflects the red blood cells hyperchromia. Sysmex instruments (i.e. XT‐4000i, XE‐5000, XN‐Series) provide the MicroR, that is the percentage of erythrocytes smaller than 60 fL, Hypo‐He, which is the percentage of erythrocytes with a content of hemoglobin less than 17 pg and % Hyper‐He, which represents the percentage of RBC with cellular hemoglobin content higher than 49 pg. CELL‐DYN Sapphire (Abbott Diagnostics) introduced the HPR parameter (% HPR), which represents the erythrocytes with hemoglobin > 410 g/L. Beckman Coulter instruments supply the mean sphered corpuscular volume (MSCV), which is the average volume of all erythrocytes, including mature erythrocytes and reticulocytes. Other reference tests for screening and diagnosis of HS are the acidified glycerol lysis test (AGLT), the eosin‐5‐maleimide (EMA) binding test and genetic testing by next‐generation sequencing.The diagnostic workup of hereditary spherocytosis could be improved thanks to all the available tests, including new molecular tools. However, it requires synergy between clinicians and laboratory staff, evaluating clinical manifestations, all available data related to the disease and the prognosis to fill the diagnostic gaps in the near future. [ABSTRACT FROM AUTHOR]

ABSTRACT Nitrate (NO3−) deficiency decreases root water uptake and root hydraulic conductance. This adaptive response is correlated with reduced abundance and activity of plasma membrane intrinsic protein (PIP) aquaporins. We therefore screened changes in the root architecture of a complete set of Arabidopsis pip loss‐of‐function mutants grown under NO3− deficiency to systematically approach the impact of PIPs under these conditions. NO3− deprivation led to attenuated responses of specific pip single mutants compared to the strongly altered LR parameters of wild‐type plants. In particular, pip1;1 exhibited a lower relative reduction in LR length and LR density, revealing that PIP1;1 represses LR development when NO3− is scarce. Indeed, PIP1;1 compromises root and shoot NO3− accumulation during early developmental stages. A fluorescent VENUS‐PIP1;1 fusion revealed that PIP1;1 is specifically repressed in the pericycle, endodermis and at the flanks of emerging LRs upon NO3− deficiency. Thus, LR plasticity and NO3− uptake are affected by an interactive mechanism involving aquaporins (PIP1;1) and nitrate accumulation during seedling development under NO3−‐deficient conditions. [ABSTRACT FROM AUTHOR]

Background: Malignant gliomas exhibit rapid tumor progression and resistance to treatment, leading to high lethality. One of the causes is the reduced progression of apoptosis in glioma cells. Layilin is a type 1 transmembrane protein with a C-type lectin motif in its extracellular domain. We previously reported that layilin is mainly localized to mitochondria or their close proximity and that layilin is essential for maintaining of the fragmented type of mitochondria. This study investigates the effects of layilin on mitochondria-mediated apoptosis, focusing on B cell lymphoma (BCL)-2 family proteins in a glioma cell line of A172 cells. Results: We compared the levels of pro-apoptotic BCL-2 family proteins of BAD, BAK, BAX, and BIM and anti-apoptotic BCL-2 family proteins of BCL-2 and BCL-XL between layilin- knockdown (KD) cells and control cells using western blot. The protein levels of BAD were significantly smaller in layilin-KD cells than in control cells, while those of BCL-2 were significantly larger. We then compared the mitochondrial membrane potential (ΔΨm) under p-trifluoromethoxyphenyl hydrazone (FCCP)-treated conditions using MT-1 staining. In layilin-KD cells, ΔΨm was significantly larger and FCCP-induced ΔΨm reduction was significantly lower than in control cells. Furthermore, we examined the levels of cell membrane-bound Annexin V and DNA-bound propidium idodide (PI) in layilin-KD cells with/without staurosporine (STS) treatment. Layilin-KD significantly decreased levels of cell membrane-bound Annexin V with/without STS treatment. On the other hand, PI levels were not changed by layilin-KD. We also investigated the amounts of the active caspase (CASP)-3, CASP-6, CASP-7, and poly (ADP-ribose) polymerase-1 (PARP1, cleaved form), as well as DNA fragmentation in layilin-KD cells under apoptotic conditions induced by STS, using western blot and the DNA ladder method, respectively. Under STS-treated conditions, the amounts of active CASP-3, CASP-7, and poly (ADP-ribose) PARP1 were significantly smaller in layilin-KD cells than in control cells. Accordingly, DNA fragmentation was significantly suppressed in layilin-KD cells compared to control cells under STS-treated conditions. Conclusion: This study demonstrates that layilin contributes to ΔΨm reduction to promote apoptosis by up-regulating BAD and down-regulating BCL-2 in glioma cells. Our data elucidates a new function of layilin: regulation of mitochondria-mediated apoptosis. [ABSTRACT FROM AUTHOR]

Presenilin-associated protein (PSAP) is a mitochondrial proapoptotic protein as established in cell biology studies. It remains unknown whether it involves in neurodegenerative diseases. Here, we explored PASP expression in adult and aged human brains and its alteration relative to Alzheimer-disease (AD)-type neuropathology. In pathology-free brains, light PASP immunoreactivity (IR) occurred among largely principal neurons in the cerebrum and subcortical structures. In the brains with AD pathology, enhanced PSAP IR occurred in neuronal and neuritic profiles with a tangle-like appearance, with PSAP and pTau protein levels elevated in neocortical lysates relative to control. Neuronal/neuritic profiles with enhanced PSAP IR partially colocalized with pTau, but invariably with Amylo-Glo labelled tangles. The neuronal somata with enhanced PASP IR also showed diminished IR for casein kinase 1 delta (Ck1δ), a marker of granulovacuolar degeneration; and diminished IR for sortilin, which is normally expressed in membrane and intracellular protein sorting/trafficking organelles. In old 3xTg-AD mice with β-amyloid and pTau pathologies developed in the brain, PSAP IR in the cerebral sections exhibited no difference relative to wildtype mice. These findings indicate that PSAP upregulation is involved in the course of tangle formation especially in the human brain during aging and in AD pathogenesis. [ABSTRACT FROM AUTHOR]

The anti-Shigella vaccine is one of the WHO's top priorities. Every year the disease kills more than 200,000 people worldwide and poses a serious threat to children under 5 years of age and the elderly. Increasing antibiotic resistance and limitations in diagnostics emphasize the need to develop an effective vaccine. Recent research and clinical trials report multiple approaches used in Shigella-vaccine development. However, despite the efforts of researchers, pharmaceutical companies and health care organizations, there is no licensed vaccine against shigellosis available to the community. Here, we expressed, broadly characterized and demonstrated the protective properties of outer membrane protein C as an effective molecule serving as a universal antigen for Shigella vaccine. Most of the current approaches to the development of Shigella vaccine are based on the polysaccharide antigens, which are serotype specific and have always been challenging in terms of their high specificity, targeting the most exposed surface antigens identified for certain Shigella serotypes. Here, we confirm immunogenic and protective properties of the recombinant OmpC protein, which protects mice against a lethal dose of a virulent strain 2 weeks after active immunization. [ABSTRACT FROM AUTHOR]

Phosphatase of regenerating liver (PRL) is an oncogenic protein that promotes tumor progression by directly binding to cyclin M (CNNM) membrane proteins and inhibiting their Mg2+ efflux activity. In this study, we have developed a high-throughput screening system to detect the interactions between PRL and CNNM proteins based on homogenous time-resolved fluorescence resonance energy transfer (HTR-FRET, HTRF). We optimized the tag sequences attached to the recombinant proteins of the CNNM4 CBS domains and PRL3 lacking the carboxyl terminal CAAX motif, and successfully detected the interaction by observing the FRET signal in the mixture of the tagged proteins and fluorophore-conjugated antibodies. Moreover, we performed compound library screening using this system and discovered several compounds that could efficiently inhibit the PRL-CNNM interaction. Characterization of one candidate compound revealed that it was relatively stable compared with thienopyridone, a known inhibitor of the PRL-CNNM interaction. The candidate compound can also inhibit PRL function in cells: suppression of CNNM-dependent Mg2+ efflux, and has sufficient in vitro drug metabolism and pharmacokinetic properties. Overall, these results demonstrate the effectiveness of this screening system for identifying novel inhibitors of the PRL-CNNM interaction, which could contribute to the development of novel anti-cancer drugs. [ABSTRACT FROM AUTHOR]

Influenza virus pandemics are caused by viruses from animal reservoirs that adapt to efficiently infect and replicate in human hosts. Here, we investigate whether Interferon-Induced Transmembrane Protein 3 (IFITM3), a host antiviral factor with known human deficiencies, plays a role in interspecies virus infection and adaptation. We find that IFITM3-deficient mice and human cells can be infected with low doses of avian influenza viruses that fail to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious virus dose threshold. Remarkably, influenza viruses passaged through Ifitm3−/− mice exhibit enhanced host adaptation, a result that is distinct from viruses passaged in mice deficient for interferon signaling, which exhibit attenuation. Our data demonstrate that IFITM3 deficiency uniquely facilitates potentially zoonotic influenza virus infections and subsequent adaptation, implicating IFITM3 deficiencies in the human population as a vulnerability for emergence of new pandemic viruses. This study highlights that IFITM3 deficiency lowers the minimum infectious dose of influenza virus, enhances adaptation of influenza viruses to a new host species, and broadly increases infection of human cells by avian and swine influenza viruses. [ABSTRACT FROM AUTHOR]

Phytopathogens often secrete effectors to enhance their infection of plants. In the case of Sclerotinia sclerotiorum, a necrotrophic phytopathogen, a secreted protein named SsPEIE1 (Sclerotinia sclerotiorum Plant Early Immunosuppressive Effector 1) plays a crucial role in its virulence. During the early stages of infection, SsPEIE1 is significantly up-regulated. Additionally, transgenic plants expressing SsPEIE1 exhibit increased susceptibility to different phytopathogens. Further investigations revealed that SsPEIE1 interacts with a plasma membrane protein known as hypersensitive induced reaction (HIR) that dampens immune responses. SsPEIE1 is required for S. sclerotiorum virulence on wild-type Arabidopsis but not on Arabidopsis hir4 mutants. Moreover, Arabidopsis hir2 and hir4 mutants exhibit suppressed pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) bursts and salicylic acid (SA)-associated immune gene induction, all of which are phenocopied by the SsPEIE1 transgenic plants. We find that the oligomerization of AtHIR4 is essential for its role in mediating immunity, and that SsPEIE1 inhibits its oligomerization through competitively binding to AtHIR4. Remarkably, both Arabidopsis and rapeseed plants overexpress AtHIR4 display significantly increased resistance to S. sclerotiorum. In summary, these results demonstrate that SsPEIE1 inhibits AtHIR4 oligomerization-mediated immune responses by interacting with the key immune factor AtHIR4, thereby promoting S. sclerotiorum infection. SsPEIE1 is an essential virulence factor of Sclerotinia sclerotiorum. SsPEIE1 interacts with the key immune factor AtHIR4 and inhibits AtHIR4 oligomerization-mediated immune responses in Arabidopsis, thereby promoting S. sclerotiorum infection. [ABSTRACT FROM AUTHOR]

Peroxisomes are organelles that are central to lipid metabolism and chemical detoxification. Despite advances in our understanding of peroxisome biogenesis, the mechanisms maintaining peroxisomal membrane proteins remain to be fully elucidated. We show here that mammalian FAF2/UBXD8, a membrane-associated cofactor of p97/VCP, maintains peroxisomal homeostasis by modulating the turnover of peroxisomal membrane proteins such as PMP70. In FAF2-deficient cells, PMP70 accumulation recruits the autophagy adaptor OPTN (Optineurin) to peroxisomes and promotes their autophagic clearance (pexophagy). Pexophagy is also induced by p97/VCP inhibition. FAF2 functions together with p97/VCP to negatively regulate pexophagy rather than as a factor for peroxisome biogenesis. Our results strongly suggest that p97/VCPFAF2-mediated extraction of ubiquitylated peroxisomal membrane proteins (e.g., PMP70) prevents peroxisomes from inducing nonessential autophagy under steady state conditions. These findings provide insight into molecular mechanisms underlying the regulation of peroxisomal integrity by p97/VCP and its associated cofactors. The mechanisms maintaining peroxisomal membrane proteins remain to be fully elucidated. Here, the authors report that p97/VCP and FAF2/UBXD8 modulate the turnover of peroxisomal membrane proteins to prevent autophagic degradation of peroxisomes. [ABSTRACT FROM AUTHOR]

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition. At the Golgi, the Dsc ubiquitin ligase complex targets proteins with shorter transmembrane domains for proteasomal or lysosomal degradation. This quality control at the sorting center of cells restricts the uncontrolled spreading of orphaned proteins. [ABSTRACT FROM AUTHOR]