Structural basis for coupling of the WASH subunit FAM21 with the endosomal SNX27-Retromer complex.

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains.

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4.

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2.

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously in vitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.

Engineering Platelet Membrane-Coated Bimetallic MOFs as Biodegradable Nanozymes for Efficient Antibacterial Therapy.

Nanocatalytic-based wound therapeutics present a promising strategy for generating reactive oxygen species (ROS) to antipathogen to promote wound healing. However, the full clinical potential of these nanocatalysts is limited by their low reactivity, limited targeting ability, and poor biodegradability in the wound microenvironment. Herein, a bio-organic nanozyme is developed by encapsulating a FeZn-based bimetallic organic framework (MOF) (MIL-88B-Fe/Zn) in platelet membranes (PM@MIL-88B-Fe/Zn) for antimicrobial activity during wound healing. The introduction of Zn in MIL-88B-Fe/Zn modulates the electronic structure of Fe thus accelerating the catalytic kinetics of its peroxidase-like activity to catalytically generate powerful ROS. The platelet membrane coating of MOF innovatively enhanced the interaction between nanoparticles and the biological environment, further developing bacterial-targeted therapy with excellent antibacterial activity against both gram-positive and gram-negative bacteria. Furthermore, this nanozyme markedly suppressed the levels of inflammatory cytokines and promoted angiogenesis in vivo to effectively treat skin surface wounds and accelerate wound healing. PM@MIL-88B-Fe/Zn exhibited superior biodegradability, favourable metabolism and non-toxic accumulation, eliminating concerns regarding side effects from long-term exposure. The high catalytic reactivity, excellent targeting features, and biodegradability of these nanoenzymes developed in this study provide useful insights into the design and synthesis of nanocatalysts/nanozymes for practical biomedical applications.

Isolation, whole genome sequencing and application of a broad-spectrum Salmonella phage.

Salmonella is considered as one of the most common zoonotic /foodborne pathogens in the world. The application of bacteriophages as novel antibacterial agents in food substrates has become an emerging strategy. Bacteriophages have the potential to control Salmonella contamination.We have isolated and characterized a broad-spectrum Salmonella phage, SP154, which can lyse 9 serotypes, including S. Enteritidis, S. Typhimurium, S. Pullorum, S. Arizonae, S. Dublin, S. Cholerasuis, S. Chester, S. 1, 4, [5], 12: i: -, and S. Derby, accounting for 81.9% of 144 isolates. SP154 showed a short latent period (40 min) and a high burst size (with the first rapid burst size at 107 PFUs/cell and the second rapid burst size at approximately 40 PFUs/cell). Furthermore, SP154 activity has higher survival rates across various environmental conditions, including pH 4.0-12.0 and temperatures ranging from 4 to 50 °C for 60 min, making it suitable for diverse food processing and storage applications. Significant reductions in live Salmonella were observed in different foods matrices such as milk and chicken meat, with a decrease of up to 1.9 log10 CFU/mL in milk contamination and a 1 log10 CFU/mL reduction in chicken meat. Whole genome sequencing analysis revealed that SP154 belongs to the genus Ithacavirus, subfamily Humphriesvirinae, within the family Schitoviridae. Phylogenetic analysis based on the terminase large subunit supported this classification, although an alternate tree using the tail spike protein gene suggested affiliation with the genus Kuttervirus, underscoring the limitations of relying on a single gene for phylogenetic inference. Importantly, no virulence or antibiotic resistance genes were detected in SP154. Our research highlights the potential of using SP154 for biocontrol of Salmonella in the food industry.

vB_EcoM-P896 coliphage isolated from duck sewage can lyse both intestinal pathogenic Escherichia coli and extraintestinal pathogenic E. coli.

Pathogenic Escherichia coli strains cause diseases in both humans and animals. The limiting factors to prevent as well as control infections from pathogenic E. coli strains are their pathotypes, serotypes, and drug resistance. Herein, a bacteriophage (vB_EcoM-P896) has been isolated from duck sewage. Furthermore, aside from targeting intestinal pathogenic E. coli strains like enteropathogenic E. coli, Shiga toxin-producing E. coli, entero-invasive E. coli, and enteroaggregative E. coli, vB_EcoM-P896 can cause lysis in extraintestinal pathogenic E. coli strains such as avian pathogenic E. coli. Stability analysis revealed that vB_EcoM-P896 was stable under the following conditions: temperature, 4℃-50℃; pH, 3-11. The sequencing of the vB_EcoM-P896 genome was conducted utilizing an HiSeq system (Illumina, San Diego, CA) and subjected to de novo assembling with the aid of Spades 3.11.1. The characteristics of the DNA genome were as follows: size, 170,656 bp; GC content, 40.4%; the number of putative coding regions, 294. Transmission electron microscopy analysis of morphology and genome analysis revealed that the phage vB_EcoM-P896 belonged to the order Caudovirales and the family Myoviridae. The pan-genome analysis of vB_EcoM-P896 was divided into two levels. The first level involved the analysis of 91 strains of muscle tail phages, which were mainly divided into 5 groups. The second level involved the analysis of 24 strains of myophage with high homology. Of the 1480 gene clusters, 23 were shared core genes. Neighbor-joining phylogenetic trees were constructed using the Poisson model with MEGA6.0 based on the conserved sequences of phage proteins, the amino acid sequence of the terminase large subunit, and tail fibrin. Further analysis revealed that vB_EcoM-P896 was a typical T4-like potent phage with potential clinical applications.

Identification of Subunits for Novel Universal Vaccines against Three Predominant Serogroups and the Emerging O145 among Avian Pathogenic Escherichia coli by Pan-RV Pipeline.

Avian pathogenic Escherichia coli, a causative agent of avian colibacillosis, has been causing serious economic losses in the poultry industry. The increase in multidrug-resistant isolates and the complexity of the serotypes of this pathogen, especially the recently reported emergence of a newly predominant serogroup of O145, make the control of this disease difficult. To address this challenge, a high-throughput screening approach, called Pan-RV (Reverse vaccinology based on pangenome analysis), is proposed to search for universal protective antigens against the three traditional serogroups and the newly emerged O145. Using this approach, a total of 61 proteins regarded as probable antigens against the four important serogroups were screened from the core genome of 127 Avian pathogenic Escherichia coli (APEC) genomes, and six were verified by Western blots using antisera. Overall, our research will provide a foundation for the development of an APEC subunit vaccine against avian colibacillosis. Given the exponential growth of whole-genome sequencing (WGS) data, our Pan-RV pipeline will make screening of bacterial vaccine candidates inexpensive, rapid, and efficient. IMPORTANCE With the emergence of drug resistance and the newly predominant serogroup O145, the control of Avian pathogenic Escherichia coli is facing a serious challenge; an efficient immunological method is urgently needed. Here, for the first time, we propose a high-throughput screening approach to search for universal protective antigens against the three traditional serogroups and the newly emerged O145. Importantly, using this approach, a total of 61 proteins regarded as probable antigens against the four important serogroups were screened, and three were shown to be immunoreactive with all antisera (covering the four serogroups), thereby providing a foundation for the development of APEC subunit vaccines against avian colibacillosis. Further, our Pan-RV pipeline will provide immunological control strategies for pathogens with complex and variable genetic backgrounds such as Escherichia coli and will make screening of bacterial vaccine candidates more inexpensive, rapid, and efficient.

Accuracy of contrast-enhanced ultrasound in diagnosing extracranial carotid occlusion: A meta-analysis.

AIM: This study aimed to assess the accuracy of contrast-enhanced ultrasound (CEUS) in detecting extracranial carotid artery occlusion. MATERIALS AND METHODS: A systematic literature search was conducted in the Cochrane, PubMed, and EMBASE databases. Prospective or retrospective studies that reported sensitivity and specificity of CEUS for the diagnosis of carotid artery occlusion were selected. Eight studies (354 arteries) were included in the meta-analysis. A bivariate random-effect model was used to estimate overall sensitivity and specificity. The results were also summarized by developing a summary receiver operating characteristic (SROC) curve. RESULTS: The overall sensitivity, specificity, positive, and negative likelihood ratios were 0.99 (95% CI: 0.83-1.00), 0.97 (95% CI: 0.90-0.99), 30.0 (95% CI: 9.8-91.4), and 0.01 (95% CI: 0.00-0.21), respectively; the odds ratio for diagnosis was 4,796 (95% CI: 119-192,584). CONCLUSION: The diagnostic test accuracy suggests that CEUS is a reliable tool for diagnosis of extracranial carotid artery occlusion.

Nanoreactor based on Cu nanoparticles confined in B, N co-doped porous carbon nanotubes for glutathione biosensing.

A cost-effective approach has been developed to synthesize Cu nanoparticles encapsulated into B and N double-doped carbon nanotubes (Cu@BCNNTs) by one-step pyrolysis. According to the specific binding of Cu-Cl and Cu-glutathione (GSH), we employed Cu@BCNNTs to build an electrochemical sensing platform to detect GSH. The unique space-confined structure can prevent Cu nanoparticles from agglomeration. In addition, B and N co-doped porous hollow tubes can improve the electrochemical conductivity, expand the number of active sites, enhance surface adsorption, and shorten the transport path. These favorable characteristics of Cu@BCNNTs make them have excellent electrocatalytic properties. These results display that the prepared sensor can detect GSH from 0.5 to 120 µM with a detection limit of 0.024 µM. The obtained sensors can be successfully applied in the human serum with recovery of GSH ranging from 100.2 to 103.9%. This work provides a new vision to synthesize nanoparticles confined in a hollow tube for the applications in biosensing and medical diagnostics.

Bone marrow-derived mesenchymal stem cells microvesicles stabilize atherosclerotic plaques by inhibiting NLRP3-mediated macrophage pyroptosis.

Rupture of atherosclerotic plaques constitutes the major cause of thrombosis and acute ischemic coronary syndrome. Bone marrow-derived mesenchymal stem cells microvesicles (BMSCs-MVs) are reported to promote angiogenesis. This study investigated the role of BMSCs-MVs in stabilizing atherosclerotic plaques. BMSCs-MVs in mice were isolated and identified. The mouse model of atherosclerosis was established, and mice were injected with BMSCs-MVs via the tail vein. The macrophage model with high glucose and oxidative damage was established and then incubated with BMSCs-MVs. Nod-like receptor protein 3 (NLRP3) expression, pyroptosis-related proteins, and inflammatory factors were detected. Actinomycin D was used to inhibit the secretion of BMSCs-MVs to verify the source of microRNA-223 (miR-223). The binding relationship between miR-223 and NLRP3 was predicted and verified. BMSCs-MVs with knockdown of miR-223 were cocultured with bone marrow-derived macrophages with knockdown of NLRP3, and then levels of miR-223, NLRP3, pyroptosis-related proteins, and inflammatory factors were detected. BMSCs-MVs could reduce the vulnerability index of atherosclerotic plaques and intima-media thickness in mice, and inhibit pyroptosis and inflammation. BMSCs-MVs inhibited pyroptosis and inflammatory factors in macrophages. BMSCs-MVs carried miR-223 to inhibit NLRP3 expression and reduce macrophage pyroptosis, thereby stabilizing the atherosclerotic plaques.