Crystal structure of the outer membrane protein OmpU from Vibrio cholerae at 2.2 Å resolution

Vibrio cholerae causes a severe disease that kills thousands of people annually. The outer membrane protein OmpU is the most abundant outer membrane protein in V. cholerae, and has been identified as an important virulence factor that is involved in host-cell interaction and recognition, as well as being critical for the survival of the pathogenic V. cholerae in the host body and in harsh environments. The mechanism of these processes is not well understood owing to a lack of the structure of V. cholerae OmpU. Here, the crystal structure of the V. cholerae OmpU trimer is reported to a resolution of 2.2 Å. The protomer forms a 16-β-stranded barrel with a noncanonical N-terminal coil located in the lumen of the barrel that consists of residues Gly32–Ser42 and is observed to participate in forming the second gate in the pore. By mapping the published functional data onto the OmpU structure, the OmpU structure reinforces the notion that the long extracellular loop L4 with a β-hairpin-like motif may be critical for host-cell binding and invasion, while L3, L4 and L8 are crucially implicated in phage recognition by V. cholerae

Comparative efficacy of different exercise methods to improve cardiopulmonary function in stroke patients: a network meta-analysis of randomized controlled trials

BackgroundAlthough some studies have shown that exercise has a good effect on improving the cardiopulmonary function of stroke patients, it still needs to be determined which exercise method does this more effectively. We, therefore, aimed to evaluate the effectiveness of different exercise methods in improving cardiovascular function in stroke patients through a network meta-analysis (NMA), providing a basis to select the best treatment plan for stroke patients.MethodsWe systematically searched CNKI, WanFang, VIP, CBM, PubMed, Embase, Web of Science, and The Cochrane Library databases from establishment to 30 April 2023. Randomized controlled trials (RCTS) on exercise improving cardiopulmonary function in stroke patients were included, and we screened the included articles and extracted the relevant data. RevMan (version 5.4) and Stata (version 17.0) were used for data analysis.ResultsWe included 35 RCTs and a total of 2,008 subjects. Intervention measures included high-intensity interval training (HIIT), aerobic training (AT), resistance training (RT), combined aerobic and resistance exercise (CE), and conventional therapy (CT). In the network meta-analysis, the surface under the cumulative ranking area (SUCRA) ranking result indicated that HIIT improved peak oxygen uptake (VO2peak) and 6 mins walking distance (6MWD) optimally, with rankings of HIIT (100.0%) > CE (70.5%) > AT (50.2%) > RT (27.7%) > CT (1.6%), and HIIT (90.9%) > RT (60.6%) > AT (48.9%) > RT (48.1%) > CT (1.5%), respectively. The SUCRA ranking result showed that CE improved systolic blood pressure (SBP) and diastolic blood pressure (DBP) optimally, with rankings of CE (82.1%) > HIIT (49.8%) > AT (35.3%) > CT (32.8%), and CE (86.7%) > AT (45.0%) > HIIT (39.5%) > CT (28.8%), respectively.ConclusionWe showed that exercise can effectively improve the cardiopulmonary function of stroke patients. HIIT was the most effective in improving VO2peak and 6MWD in stroke patients. CE was the most effective in improving SBP and DBP in stroke patients. However, due to the limitations of existing clinical studies and evidence, larger sample size, multi-center, and high-quality RCTs are needed to verify the above conclusions in the future.Systematic review registrationhttps://www.crd.york.ac.uk/prospero/, identifier [CRD42023436773]

The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3 associated phagocytosis

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP −/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting

Buchnera breaks the specialization of the cotton-specialized aphid (Aphis gossypii) by providing nutrition through zucchini

The cotton aphid, Aphis gossypii Glover, is a species of polyphagous aphid with many biotypes, and its host transfer has always been the focus of research on the control of cotton aphid. An important factor affecting aphid specialization is the nutritional association with microbial symbionts that provide the host with nutrients lacking in the diet. We analyzed the microbial composition and biodiversity of reared on zucchini for 10 generations (T1–T10) and cotton as a control (CK), by high-throughput Illumina sequencing of 16S ribosomal RNA genes. The findings showed that the change in plant hosts decreased the richness and variety of microbial species. Regardless of whether the plant host is altered or not, Proteobacteria and Firmicutes are the predominate phyla in cotton-specialized aphid. Additionally, cotton-specialized aphids that live in zucchini had considerably lower relative abundances of non-dominant phyla (Bacteroidetes) than cotton hosts. At the genus level the dominant communities were Buchnera, Acinetobacter, and Arsenophonus. The relative abundance of Buchnera was significantly higher in aphids reared on zucchini than those on cotton, whereas the opposite was observed for Acinetobacter, as well as for some non-dominant communities (Stenotrophomonas, Pseudomons, Flavobacterium, Novosphingobium). Collectively, this study clarifies the dynamic changes of symbiotic bacteria in cotton-specialized aphids reared on zucchini for multiple generations. Among them, Buchnera is crucial for the cotton-specialized aphid to get nutrients during the transfer of the host and has a favorable impact on the colonization of cotton-specialized aphid populations on zucchini hosts. It not only enriches our understanding of the relationship between the bacterial microbiota of aphids and their adaptability to new hosts, zucchini, but also expands the current body of research on the mechanisms underlying the host shifting ability of cotton-specialized aphids

Benzyl isothiocyanate induces apoptosis and inhibits tumor growth in canine mammary carcinoma via down-regulation of the cyclin B1/Cdk1 pathway

Background: Canine mammary carcinoma is common in female dogs, and its poor prognosis remains a serious clinical challenge, especially in developing countries. Benzyl isothiocyanate (BITC) has attracted great interest because of its inhibitory effect against tumor activity. However, its effect and the underlying mechanisms of action in canine mammary cancer are not well-understood. Here, we show that BITC suppresses mammary tumor growth, both in vivo and in vitro, and reveal some of the potential mechanisms involved. Methods: The effect of BITC on canine mammary cancer was evaluated on CIPp and CMT-7364, canine mammary carcinoma lines. The cell lines were treated with BITC and then subjected to wound healing and invasion assays. Cell cycles and apoptosis were measured using flow cytometry; TUNEL assay; immunohistochemistry (IHC) for caspase 3, caspase 9, and cyclin D1; hematoxylin and eosin (H&E) staining; and/or quantitative polymerase chain reaction (qPCR). Results: BITC showed a strong suppressive effect in both CIPp and CMT-7364 cells by inhibiting cell growth in vitro; these effects were both dose- and time-dependent. BITC also inhibited migration and invasion of CIPp and CMT-7364 cells. BITC induced G2 arrest and apoptosis, decreasing tumor growth in nude mice by downregulation of cyclin B1 and Cdk1 expression. Conclusion: BITC suppressed both invasion and migration of CIPp and CMT-7364 cells and induced apoptosis. BITC inhibited canine mammary tumor growth by suppressing cyclinB1 and Cdk1 expression in nude mice

Control of infection by LC3-associated phagocytosis, CASM, and detection of raised vacuolar pH by the V-ATPase-ATG16L1 axis

The delivery of pathogens to lysosomes for degradation provides an important defense against infection. Degradation is enhanced when LC3 is conjugated to endosomes and phagosomes containing pathogens to facilitate fusion with lysosomes. In phagocytic cells, TLR signaling and Rubicon activate LC3-associated phagocytosis (LAP) where stabilization of the NADPH oxidase leads to sustained ROS production and raised vacuolar pH. Raised pH triggers the assembly of the vacuolar ATPase on the vacuole membrane where it binds ATG16L1 to recruit the core LC3 conjugation complex (ATG16L1:ATG5-12). This V-ATPase-ATG16L1 axis is also activated in nonphagocytic cells to conjugate LC3 to endosomes containing extracellular microbes. Pathogens provide additional signals for recruitment of LC3 when they raise vacuolar pH with pore-forming toxins and proteins, phospholipases, or specialized secretion systems. Many microbes secrete virulence factors to inhibit ROS production and/or the V-ATPase-ATG16L1 axis to slow LC3 recruitment and avoid degradation in lysosomes

Non‐canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces

The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal influenza A virus infection at epithelial surfaces

Phagocytosis and autophagy represent two evolutionarily ancient pathways that provide an important defense against infection by delivering pathogens to lysosomes for degradation. Phagocytosis and autophagy are linked by non-canonical autophagy pathways that conjugate LC3 to endo-lysosome compartments to facilitate phagosome maturation and lysosome fusion. A role for non-canonical autophagy in host defence is implied from cellular studies in vitro, but critically, these studies have rarely been extended to infection of model organisms with intact epithelial barriers and complex immune systems. To address this, we developed a mouse model with specific loss of non-canonical autophagy by removing the WD and linker domain of ATG16L1 required for recruitment of LC3 to endo-lysosome compartments. The mice retain the coiled-coiled domain of ATG16L1 required for conventional autophagy and maintain tissue and immunological homeostasis. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity murine-adapted influenza A virus leading to extensive viral replication throughout the lungs, cytokine dysregulation, fulminant pneumonia and lung inflammation leading to high mortality associated with virulent strains. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung required non-canonical autophagy within epithelial barriers but was independent of phagocytes and other leukocytes. This establishes non-canonical autophagy pathways in epithelial cells as a novel innate defence mechanism that can restrict IAV infection at mucosal surfaces

Role played by the WD domain of ATG16L1 in defense against Salmonella infection in vivo

Non-canonical autophagy, or LC3-associated phagocytosis (LAP), conjugates autophagy protein LC3 to endo-lysosome compartments to facilitate delivery of extracellular materials to lysosomes. The WD domain of autophagy protein ATG16L1 is required for conjugation of LC3 to endo-lysosomes during LAP, but is not required for LC3 conjugation during autophagy. Mice lacking the WD domain of ATG16L1 (WD mice) are therefore defective in LAP, but can activate autophagy to maintain tissue homeostasis. This study has used WD mice to determine the role played by the WD domain of ATG16L1 during S. Typhimurium infection "in vivo". S. Typhimurium showed increased replication and virulence in δWD mice characterized by high mortality rate, severe weight loss, and enhanced dissemination of bacteria and lymphocytes to liver and spleen. Crosses with LysMcre and villincre mice showed that expression of the WD domain of ATG16L1 in intestinal epithelial cells, rather than myeloid cells, protected against S. Typhimurium. SopF is an S. Typhimurium SPI-1 T3SS effector thought to increase virulence by blocking the binding of the WD domain of ATG16L1 to the endo-lysosome v-ATPase to prevent recruitment of LC3 to Salmonella containing vacuoles. A SopF knockout S. Typhimurium strain (JH3009δSOPF), generated by lambda recombination, showed reduced virulence in control mice, but both wild type S. Typhimurium and JH3009δSOPF showed increased virulence in mice lacking the WD domain. This suggested that the WD domain restricted S. Typhimurium replication by additional pathways that are independent of the WD-v-ATPase axis blocked by Sop-F. Fluorescent probes and biosensors showed that cells and tissues of WD mice had raised intracellular cholesterol that accumulated at sites of replication. Replication was reduced by cholesterol-lowering drugs. Taken together the results suggest that WD domain of ATG16L1 restricts S. Typhimurium replication in intestinal epithelial cells partly by reducing intracellular cellular cholesterol levels

Mapping Rice Fields in Urban Shanghai, Southeast China, Using Sentinel-1A and Landsat 8 Datasets

Sentinel-1A and Landsat 8 images have been combined in this study to map rice fields in urban Shanghai, southeast China, during the 2015 growing season. Rice grown in paddies in this area is characterized by wide inter-field variability in addition to being fragmented by other landuses. Improving rice classification accuracy requires the use of multi-source and multi-temporal high resolution data for operational purposes. In this regard, we first exploited the temporal backscatter of rice fields and background land-cover types at the vertical transmitted and vertical received (VV) and vertical transmitted and horizontal received (VH) polarizations of Sentinel-1A. We observed that the temporal backscatter of rice increased sharply at the early stages of growth, as opposed to the relatively uniform temporal backscatter of the other land-cover classes. However, the increase in rice backscatter is more sustained at the VH polarization, and two-class separability measures further indicated the superiority of VH over VV in discriminating rice fields. We have therefore combined the temporal VH images of Sentinel-1A with the normalized difference vegetation index (NDVI) and the modified normalized difference water index (MNDWI) derived from a single-date cloud-free Landsat 8 image. The integration of these optical indices with temporal backscatter eliminated all commission errors in the Rice class and increased overall accuracy by 5.3%, demonstrating the complimentary role of optical indices to microwave data in mapping rice fields in subtropical and urban landscapes such as Shanghai