Membrane Proteins Research Articles

Evaluation of the anti-arthritic properties of Litsea glutinosa (L.) C.B. Rob Bark: An integrated analysis utilizing in-silico, in-vitro, and in-vivo approaches, along with identification of major compounds

The current study explored the anti-arthritic properties of Litsea glutinosa bark extracts. The dried bark of L. glutinosa was defatted with n-hexane and extracted by methanol using a soxhlet apparatus. The bioactive compounds present in the extract were analysed by GC-MS and LC-MS. The methanol extract of L. glutinosa (MELG) was evaluated at different concentrations (25–400 mg/ml) for its in-vitro anti-arthritic efficacy using membrane stabilization and protein denaturation techniques. In in-vivo evaluation paw diameter, arthritic score, arthritic index, body weight, organ weight, and haematological and biochemical parameters were measured to assess anti-arthritic potential. Subsequently, pro-inflammatory and inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), IL-1β, cyclooxygenase-2 (COX-2), IL-13, and IL-10 were determined. The Compounds identified by GC–MS and LC-MS were subjected to an in-silico molecular docking analysis utilizing AutoDock 4.2 and BIOVIA-Discovery Studio Visualizer against COX-2, IL-1β, IL-6, and TNF-α. The in vitro anti-arthritic effect of MELG exhibited a dose-dependent reduction in membrane stabilization (67.9 % at 400 µg/ml) and protein denaturation assay (66.6 % at 400 µg/ml). MELG treated rats improved all arthritic parameters. Significant restoration of pro-inflammatory cytokines was observed in rats administered with MELG (200 & 400 mg/kg). In the MELG (400 mg/kg) treated group, there was a significant reduction in IL-10 (P˂0.001) and IL-13 (P˂0.05) levels and a substantial rise in IL-6 (P˂0.01), TNF-α (P̂˂0.001), and IL-1β (P˂0.01) levels. The presence of phytoconstituents was analyzed by GC–MS (Fourteen compounds) and LC-MS (Ten compounds). Boldine identified by LC-MS analysis, exhibited good docking scores against COX-2 (-8.7), IL-1β (-6.3 kcal/mol), IL-6 (-6.2 kcal/mol), and TNF-α (-6.7 kcal/mol).The presence of bioactive compound, boldine in MELG might be responsible for its anti-arthritic activity.

Ultrasonication outperforms electroporation for extracellular vesicle cargo depletion

Extracellular vesicles (EVs), submicron-sized membranous structures released by cells, serve as vehicles of tissue-specific proteins and nucleic acids, facilitating intercellular communication and playing roles in pathophysiological processes. Leveraging their unique characteristics, EVs have emerged as promising drug delivery nanocarriers. Electroporation (EP) and ultrasonication (US) are among the prevalent techniques used for loading exogenous drugs into EVs owing to their simplicity and efficiency. However, the effectiveness of the two methods in depleting initial EV cargo has been overlooked. But this information is indispensable, as the bioactive residuals of EVs, notably derived from tumor or stem donor cells, may impact downstream therapeutic effects. Bridging this knowledge gap, therefore, can guide the selection of optimal drugs and loading methods tailored to therapeutic objectives. Here, we used high-throughput sequencing to investigate the protein and small RNA cargo of EVs treated with EP and US, respectively. We found that US exhibits higher efficacy in depleting EV cargo compared to EP, while US may also deplete essential endogenous molecules for combination therapy. Neither method demonstrated significant selectivity in cargo depletion, but they might preferentially retain few specific molecules. Additionally, membrane proteins are more prone to loss during US and EP treatments than cytoplasmic proteins.

The role of microRNAs regulation of endoplasmic reticulum stress in ischemia-reperfusion injury: A review.

The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells, responsible for a range of biological functions such as the secretion, modification and folding of proteins, maintaining Ca2+ homeostasis and the synthesis of steroids/lipids, secreted proteins and membrane proteins. When cells are affected by internal or external factors, including abnormal energy metabolism, disrupted Ca2+ balance, altered glycosylation, drug toxicity, and so on, the unfolded or misfolded proteins accumulate in the ER, leading to the unfolded protein response (UPR) and ER stress. The abnormal ER stress has been reported to be involved in various pathological processes. MicroRNAs (miRNAs) are non-coding RNAs with the length of approximately 19-25 nucleotides. They control the expression of multiple genes through posttranscriptional gene silencing in eukaryotes or some viruses. Increasing evidence indicates that miRNAs are involved in various cellular functions and biological processes, such as cell proliferation and differentiation, growth and development, and metabolic homeostasis. Hence, miRNAs participate in multiple pathological processes. Recently, many studies have shown that miRNAs play an important role by regulating ER stress in ischemia-reperfusion (I/R) injury, but the relevant mechanisms are not fully understood. In this review, we reviewed the current understanding of ER stress, as well as the biogenesis and function of miRNAs, and focused on the role of miRNAs regulation of ER stress in I/R injury, with the aim of providing new targets for the treatment of I/R injury.

Comprehensive analysis of the oligopeptide transporter gene family in maize: Genome-wide identification, structural characterization, and stress-responsive expression

Oligopeptide transporter (OPT) proteins are integral proteins in cell membranes that perform a crucial role in cellular metal homeostasis, mineral uptake, transport of small peptides and abiotic stress tolerance. Hence, in-depth evolutionary, structural, molecular and expression analysis could guide in exploring the mechanism of how these transporters contribute to nutrient balance and abiotic stress tolerance in maize. Following a series of bioinformatic analyses, eight OPTs in maize were identified. These transporters showed significant variations in molecular weight, and have one to five introns, 11 to 15 motifs, 11 to 18 transmembrane domains, multiple beta-strands, multiple alpha and transmembrane helices. ZmOPT1, 5, and 6 are plasma membranes and the rest are endoplasmic reticulum localized. All ZmOPTs are distributed in six different chromosomes having significant phytohormone-responsive cis-elements might be due to their great roles in hormonal regulation in maize. The significant transport activity of these transporters plays a great role in biological processes, cellular components and molecular functions in maize during gene ontology analysis. Co-expression of five genes among eight OPTs with 56 genes might be due to their great role in regulating other genes in multiple signaling pathways or vice-versa. Significant tissue-specific and drought, salinity, nitrogen starvation, and heat stress induced in silico expression following real time PCR validation of ZmOPT1, 7, and 8 genes might guide to elucidate the potential roles of these transporters in specific tissues and abiotic stress tolerance. Hence, the findings of the research might guide plant biologists to develop new varieties of maize having higher efficiency of peptide and metal ion transport, balance, and abiotic stress tolerance. Findings of these structural and expression analyzes might guide the plant biologist for single transgenesis, or developing and transforming programming-based synthetic genetic circuits in maize for advancing research in peptide transport, metal hemostasis, and increasing abiotic stress tolerance in maize.

Membrane tension-dependent conformational change of Isoleucine 106 of loop B diminishes water permeability in FaPIP2;1.

Aquaporins (AQPs) are membrane proteins specialized in facilitating water transport across membranes. Mechanical stress is one of the various stimuli that regulate AQPs. Briefly, there are several studies that report a decrease in permeability upon an increase in membrane tension. However, the molecular details of this mechanosensitive (MS) response are still a matter of debate. Our work attempts to close that gap in knowledge by providing evidence of a conformational change that occurs inside the pore of the strawberry aquaporin FaPIP2;1. Via osmotic shock experiments and molecular dynamics (MD) simulations, we found that a residue of loop B, I106, is key to the blocking of the permeation pathway and such a change is almost exclusively found under membrane tensile stress. In detail, osmotic shock experiments exhibited a nonlinear increment in water fluxes for increasing osmolarities, evidencing a decrease in the FaPIP2;1 permeability. MD simulations under membrane tension showed the same trend, with a significant increase in states with a low water permeability. The latter was correlated with a conformational change in I106 that generates a permeation barrier of around 18 kJ mol-1, effectively closing the pore. This work constitutes the first report of a PIP type aquaporin reacting to tensile stress in the membrane. Our findings could pave the way to test whether this conformational change is also responsible for mechanical gating in the other MS aquaporins, both those already reported and those still waiting to be found.

Experimental and computational biophysics to identify vasodilator drugs targeted at TRPV2 using agonists based on the probenecid scaffold

TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in Pichia pastoris using a novel medium-throughput Ca2+ influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target.

Mesomycoplasma (Mycoplasma) ovipneumoniae dihydrolipoamide dehydrogenase is an immunogenic plasminogen binding protein and a putative adhesin

The interaction of Mesomycoplasma (Mycoplasma) ovipneumoniae (M. ovipneumoniae) with host cells is a pivotal step in the infection process, underlining the necessity to develop vaccines and therapeutic approaches targeting the pathogen's key invasion mechanisms. The bacterium's capacity for adherence, invasion, and subsequent evasion of the host immune response underpins its pathogenicity, rendering adherence genes feasible vaccine targets. This study focuses on pyruvate dehydrogenase complex component E3 (PdhD), a membrane-anchored surface protein implicated in these pathogenic processes. Bioinformatics analysis reveals the conservation of PdhD sequence within M. ovipneumoniae. Membrane protein extraction, immunoblotting and ELISA assay have confirmed the presence of PdhD on the M. ovipneumoniae surface and cytoplasm, suggesting its multifunctionality. Our research employed antibody inhibition assays to characterize the bacterial adhesion suppression by anti-PdhD antibodies, complemented by bactericidal complement assays, supporting its candidacy as a putative vaccine target. The ELISA binding assay substantiated that PdhD binded to plasminogen (Plg) in a dose-dependent manner. Notably, PdhD is also involved in biofilm formation. The inhibitory effect of anti-PdhD sera on biofilm formation is congruent with novel therapeutic strategies targeting related mycoplasmas. This study reports the characterization of the first virulence-associated protein PdhD of M. ovipneumoniae and suggests its potential as a vaccine target to combat M. ovipneumoniae infection.

Dihydrolipoamide acetyltransferase is a key factor mediating adhesion and invasion of host cells by Mycoplasma synoviae.

Mycoplasma synoviae is a significant avian pathogen responsible for chronic respiratory diseases, arthritis, and infectious synovitis in chickens and turkeys. These infections result in substantial economic losses to the global poultry industry. Dihydrolipoamide acetyltransferase (E2) is a multifunctional protein that plays an indispensable role in energy metabolism and redox balance and is also a key virulence factor of various pathogens. In this study, we used the avian pathogen M. synoviae as a model to identify the role of the E2 protein in the colonization and invasion of host cells. First, we prepared the polyclonal antibody of recombinant E2 (rE2) protein and found that the rE2 antibody had a strong complement-activating ability. E2 was found to be distributed in the cytoplasm and cell membrane of M. synoviae by immunoelectron microscopy. E2 localized on the cell membrane is a key factor in the adhesion of M. synoviae and has good immunogenicity. Enzyme-linked immunosorbent assay showed that the binding of rE2 to membrane proteins of chicken embryo fibroblasts (DF-1) was dose-dependent, and antiserum effectively inhibited this binding ability. Furthermore, E2 interacted with various components of the host extracellular matrix (ECM) and promoted the conversion of plasminogen to plasmin through terephthalic acid (tPA). In addition, E2 can enhance the ability of M. synoviae to invade DF-1 cells, which was significantly reduced after treatment with anti-E2 serum. These results indicate that E2 is an adhesion- and invasion-related protein and may be involved in the pathogenesis of M. synoviae, which provides new ideas for studying the pathogenesis of M. synoviae and preparing subunit vaccines.

Deciphering Cronobacter sakazakii pathogenicity: Exploring virulence factors and host interactions": A short review

Opportunistic human pathogen called Cronobacter spp. can cause severe illnesses in immunocompromised individuals and newborns. These species are mostly responsible for disorders like meningitis, necrotizing enterocolitis, and bacteremia because they can survive in very dry foods like PIF and have also been identified in various environmental niches, including plants. These species' contamination is ascribed to biofilms, which are biotic or abiotic surface communities. Because they build biofilms on a variety of surfaces, identifying and eliminating them is extremely difficult. For the purpose of creating efficient treatment interventions and preventative measures, it is essential to comprehend the processes of virulence and the relationship between this pathogen and its host. A variety of virulence factors, such as adhesion factors, invasion proteins, biofilm formation components, and secreted toxins, contribute to Cronobacter spp. pathogenicity. Genes involved in capsule formation—such as those involved in exopolysaccharide synthesis—are essential to its pathogenicity because they are critical in desiccation resistance and resistance to host immunological defenses. Furthermore, genes that encode flagella, fimbriae, and outer membrane proteins are linked to host cell adherence and invasion. This review also explores the emerging link between Cronobacter spp. and plants, highlighting the bacterium's ability to survive and persist in the environment because Plants may act as reservoirs, contributing to the contamination of raw agricultural products, which can subsequently enter the food supply chain. Developing focused preventative measures and treatment approaches requires knowledge of the various biological as well as genetic factors that contribute to Cronobacter spp. virulence and how it affects the host. This review clarifies the effects of Cronobacter spp. on the host and investigates the genes linked to virulence in Cronobacter.

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the timescale of ps-μs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of μs-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and ms-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the ms timescale.

MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7cells.

V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75mM and 15°C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7cells, with its optimal concentration determined to be 7.5μg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p<0.05) in NO levels. Additionally, the relative mRNA levels of IL-1β, IL-6, and TNF-α in RAW264.7cells were measured by qRT-PCR, showing a remarkable elevation (p<0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.

SLMAP3 is essential for neurulation through mechanisms involving cytoskeletal elements, ABP, and PCP.

SLMAP3 is a tail-anchored membrane protein that targets subcellular organelles and is believed to regulate Hippo signaling. The global loss of SLMAP3 causes late embryonic lethality in mice, with some embryos exhibiting neural tube defects such as craniorachischisis. We show here that SLMAP3 -/- embryos display reduced length and increased width of neural plates, signifying arrested convergent extension. The expression of planar cell polarity (PCP) components Dvl2/3 and the activity of the downstream targets ROCK2, cofilin, and JNK1/2 were dysregulated in SLMAP3 -/- E12.5 brains. Furthermore, the cytoskeletal proteins (γ-tubulin, actin, and nestin) and apical components (PKCζ and ZO-1) were mislocalized in neural tubes of SLMAP3 -/- embryos, with a subsequent decrease in colocalization of PCP proteins (Fzd6 and pDvl2). However, no changes in PCP or cytoskeleton proteins were found in cultured neuroepithelial cells depleted of SLMAP3, suggesting an essential requirement for SLMAP3 for these processes in vivo for neurulation. The loss of SLMAP3 had no impact on Hippo signaling in SLMAP3 -/- embryos, brains, and neural tubes. Proteomic analysis revealed SLMAP3 in an interactome with cytoskeletal components, including nestin, tropomyosin 4, intermediate filaments, plectin, the PCP protein SCRIB, and STRIPAK members in embryonic brains. These results reveal a crucial role of SLMAP3 in neural tube development by regulating the cytoskeleton organization and PCP pathway.

Whole genome-based characterization of extended-spectrum β-lactamase-producing Enterobacter cloacae from orthopedic patients and environment of a tertiary referral hospital in Tanzania

ObjectivesWe investigated the genomic epidemiology of extended-spectrum β-lactamase-producing Enterobacter cloacae (ESBL-Ec) isolates from patients and hospital environment to better understand their distribution to help devising effective strategies for infection prevention and control. MethodsWe screened ESBL-Ec at Bugando Medical Center (BMC) in Mwanza, Tanzania. Rectal swabs from orthopedic patients on admission and swabs from the neighboring inanimate environment were collected. Following microbial culture, DNA was extracted from pure ESBL-Ec, and whole-genome sequencing was done. Sequence typing (ST), plasmid replicons, drug resistance, and virulence genes were deciphered using the Rapid Microbial Analysis Pipeline (rMAP). ResultsWe obtained 209 ESBL isolates, of which 15 (7.2%) were ESBL-Ec [8 (53.30%) from patients and 7 (46.7%) from the environment]. Seven isolates were novel and eight were diverse each with a unique ST. All isolates harbored two to five β-lactamase genes, with the predominance of blaCTX-M-15(15/15), blaOXA-1(14/15), blaTEM(14/15) and blaACT(12/15). The most common non β-lactam drug resistance genes were aac(3)-IIa (14/15), aac(6’)-Ib-cr (14/15), fosA (14/15), and qnrB1 (12/15), aph(3’’)-Ib (10/15) and aph(6)-Id (10/15). Eleven different types of plasmid replicons were identified in 14/15 of the isolates, harboring one to five plasmids, with the most common plasmids being IncFII (11/15) and IncFIB (10/15). All isolates harbored the outer membrane protein (ompA), and curli protein (csg) was in 14/15 isolates. ConclusionAdmitted orthopedic patients and the hospital environment act as a reservoir of ESBL-Ec with diverse STs and endowed with drug resistance and arsenals of virulence genes, calling for their routine screening on admission for mitigation of potential subsequent infections.

A defined tubby domain β-barrel surface region of TULP3 mediates ciliary trafficking of diverse cargoes.

The primary cilium is a paradigmatic subcellular compartment at the nexus of numerous cellular and morphogenetic pathways. The tubby family protein TULP3 acts as an adapter of the intraflagellar transport complex-A in transporting integral membrane and membrane-associated lipidated proteins into cilia. However, the mechanisms by which TULP3 coordinates ciliary transport of diverse cargoes is not well understood. Here, we provide molecular insights into TULP3-mediated ciliary cargo recognition. We screened for critical TULP3 residues by proximity biotinylation-mass spectrometry, structural analysis, and testing TULP3 variants in human patients with hepatorenal fibrocystic disease and spina bifida. The TULP3 residues we identified (a) were located on one side of the β-barrel of the tubby domain away from the phosphoinositide binding site, (b) mediated ciliary trafficking of lipidated and transmembrane cargoes, and (c) determined proximity with these cargoes in vivo without affecting ciliary localization, phosphoinositide binding or hydrodynamic properties of TULP3. Overall, these findings implicate a specific region of one of the surfaces of the TULP3 β-barrel in ciliary trafficking of diverse cargoes. This region overlooks the β strands 8-12 of the β-barrel and is away from the membrane anchoring phosphoinositide binding site. Targeting the TULP3-cargo interactions could provide therapeutics in ciliary trafficking diseases.

Optimized simple culture protocol for inducing mature myotubes from MYOD1-overexpressed human iPS cells.

The forced expression system of MYOD1, a master gene for myogenic differentiation, can efficiently and rapidly reproduce muscle differentiation of human induced pluripotent stem cells (hiPSCs). Despite these advantages of the MYOD1 overexpression system, developed myotubes are relatively immature and do not recapitulate several aspects of striated muscle fibers. Here, we developed a simple optimized protocol using an alternative culture medium for maximizing the advantages of the MYOD1 overexpression system, and successfully improved the formation of multinucleated mature myotubes within 10 days. In this study, we generated hiPSCs derived from healthy donors and an individual with congenial muscular dystrophy caused by LMNA mutation (laminopathy), and compared disease-associated phenotypes in differentiated myotubes generated by the conventional method and by our new optimized culture method. Using our optimized method, abnormal myonuclear shape was pronounced in the patient-derived iPSCs. In addition, abnormal accumulation of the nuclear membrane protein emerin was observed in LMNA-mutant hiPSCs. Our new culture method is expected to be widely applicable as a MYOD1 overexpression model of hiPSC-derived skeletal muscle cells for the analysis of a variety of muscle diseases.

Broadly inhibitory antibodies to severe malaria virulence proteins.

Malaria pathology is driven by the accumulation of Plasmodium falciparum-infected erythrocytes in microvessels1. This process is mediated by the polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins of the parasite. A subset of PfEMP1 variants that bind to human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis2. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here we describe two broadly reactive and inhibitory human monoclonal antibodies to CIDRα1. The antibodies isolated from two different individuals exhibited similar and consistent EPCR-binding inhibition of diverse CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins, as well as parasite sequestration in bioengineered 3D human brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with three different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies are likely to represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

Adhesive interactions within microbial consortia can be differentiated at the single-cell level through expansion microscopy

Investigating microbe-microbe interactions at the single-cell level is critical to unraveling the ecology and dynamics of microbial communities. In many situations, microbes assemble themselves into densely packed multispecies biofilms. The density and complexity pose acute difficulties for visualizing individual cells and analyzing their interactions. Here, we address this problem through an unconventional application of expansion microscopy, which allows for the "decrowding" of individual bacterial cells within a multispecies community. Expansion microscopy generally has been carried out under isotropic expansion conditions and used as a resolution-enhancing method. In our variation of expansion microscopy, we carry out expansion under heterotropic conditions; that is, we expand the space between bacterial cells but not the space within individual cells. The separation of individual bacterial cells from each other reflects the competition between the expansion force pulling them apart and the adhesion force holding them together. We employed heterotropic expansion microscopy to study the relative strength of adhesion in model biofilm communities. These included mono- and dual-species Streptococcus biofilms and a three-species synthetic community (Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sanguinis) under conditions that facilitated interspecies coaggregation. Using adhesion mutants, we investigated the interplay between F. nucleatum outer membrane protein RadD and different Streptococcus species. We also examined the Schaalia-TM7 epibiont association. Quantitative proximity analysis was used to evaluate the separation of individual microbial members. Our study demonstrates that heterotropic expansion microscopy can "decrowd" dense biofilm communities, improve visualization of individual bacterial members, and enable analysis of microbe-microbe adhesive interactions at the single-cell level.

Ankyrin-B is required for the establishment and maintenance of lens cytoarchitecture, mechanics, and clarity.

The transparent ocular lens is essential for vision by focusing light onto the retina. Despite recognizing the importance of its unique cellular architecture and mechanical properties, the molecular mechanisms governing these attributes remain elusive. This study aims to elucidate the role of ankyrin-B (AnkB), a membrane scaffolding protein, in lens cytoarchitecture, growth, and function using a conditional knockout (cKO) mouse model. AnkB cKO mouse has no defects in lens morphogenesis but exhibited changes that supported a global role for AnkB in maintenance of lens clarity, size, cytoarchitecture, membrane organization, and stiffness. Notably, absence of AnkB led to nuclear cataract formation, evident from P16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, cell adhesion and channel proteins, loss and degradation of several membrane proteins (e.g., NrCAM. N-cadherin and aquaporin-0) along with a disorganized plasma membrane and impaired membrane interdigitations. Furthermore, absence of AnkB led to decreased lens stiffness. Collectively, these results illustrate the essential role for AnkB in lens architecture, growth and function through its involvement in membrane skeletal and protein organization and stability.

EP152R-mediated endoplasmic reticulum stress contributes to African swine fever virus infection via the PERK-eIF2α pathway.

African swine fever virus (ASFV) is a large, icosahedral, double-stranded DNA virus in the Asfarviridae family and the causative agent of African swine fever (ASF). ASFV causes a hemorrhagic fever with high mortality rates in domestic and wild pigs. ASFV contains an open reading frame named EP152R, previous research has shown that EP152R is an essential gene for virus rescue in swine macrophages. However, the detailed functions of ASFV EP152R remain elusive. Herein, we demonstrate that EP152R, a membrane protein located in the endoplasmic reticulum (ER), induces ER stress and swelling, triggering the PERK/eIF2α pathway, and broadly inhibiting host protein synthesis invitro. Additionally, EP152R strongly promotes immune evasion, reduces cell proliferation, and alters cellular metabolism. These results suggest that ASFV EP152R plays a critical role in the intracellular environment, facilitating viral replication. Furthermore, virus-level experiments have shown that the knockdown of EP152R or PERK inhibitors efficiently affects viral replication by decreasing viral gene expression. In summary, these findings reveal a series of novel functions of ASFV EP152R and have important implications for understanding host-pathogen interactions.

Conditionally Activatable Chimeras for Tumor-Specific Membrane Protein Degradation.

The recent advancements on membrane protein degraders (MPDs) have broadened the applicability of proteolysis-targeting chimeras (PROTACs) beyond intracellular proteins to include the previously "undruggable" cell-surface targets. However, the potential toxicity of MPDs caused by undesired off-target degradation poses a significant challenge to clinical deployment, mirroring concerns associated with PROTACs. Here, we introduce a conditionally activatable membrane protein degrader (Pro-MPD), which leverages the specificity and high affinity of biparatopic nanobodies combined with a tumor microenvironment-activated cell-penetrating peptide (Pro-CPP) to achieve on-target activated internalization and degradation of PD-L1 within tumor sites. This modularly designed Pro-MPD demonstrated a high target degradation efficiency and T cell reactivation, as well as sustained inhibition of tumor growth in xenograft models, highlighting its potential as a safer and highly efficient MPD for in vivo applications. Our work provides a general strategy for the development of conditionally activatable MPDs, which offers a new avenue for reducing the undesired systemic toxicity of MPDs due to the off-tumor degradation.