Physiological temperature drives TRPM4 ligand recognition and gating.

Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity1,2. However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca2+-activated ion channel3-7. By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a 'warm' conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca2+-binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator)8 and ATP (an inhibitor)9, bind to different locations of TRPM4 at physiological temperatures than at lower temperatures10,11, and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes.

Identification of Novel Bisamide-Decorated Benzotriazole Derivatives as Anti-Phytopathogenic Virus Agents: Bioactivity Evaluation and Computational Simulation.

As a notorious phytopathogenic virus, the tobacco mosaic virus (TMV) severely reduced the quality of crops worldwide and caused critical constraints on agricultural production. The development of novel virucides is a persuasive strategy to address this predicament. Herein, a series of novel bisamide-decorated benzotriazole derivatives were elaborately prepared and screened. Biological tests implied that the optimized compound 7d possessed the most brilliant antiviral inactive profile (EC50 = 157.6 µg/mL) and apparently surpassed that of commercial ribavirin (EC50 = 442.1 µg/mL) 2.8-fold. The preliminary antiviral mechanism was elaborately investigated via transmission electron microscopy, microscale thermophoresis (MST) determination, RT-qPCR, and Western blot analysis. The results showed that compound 7d blocked the assembly of TMV by binding with coat protein (Kd = 0.7 µM) and suppressed TMV coat protein gene expression and biosynthesis process. Computational simulations indicated that 7d displayed strong H-bonds and pi interactions with TMV coat protein, affording a lower binding energy (ΔGbind = -17.8 kcal/mol) compared with Ribavirin (ΔGbind = -10.7 kcal/mol). Overall, current results present a valuable perception of bisamide decorated benzotriazole derivatives with appreciably virustatic competence and should be profoundly developed as virucidal candidates in agrochemical.

Identification of natural Rutaecarpine as a potent tobacco mosaic virus (TMV) helicase candidate for managing intractable plant viral diseases.

BACKGROUND: Naturally occurring alkaloids are particularly suitable for use as pesticide precursors and further modifications due to their cost-effectiveness, unique mechanism of action, tolerable degradation, and environmental friendliness. The famous tobacco mosaic virus (TMV) is a persistent plant pathogenic virus that can parasitize many plants and severely reduce crop production. To treat TMV disease, TMV helicase acts as a crucial target by hydrolyzing adenosine triphosphate (ATP) to provide energy for double-stranded RNA unwinding. RESULTS: To seek novel framework alkaloid leads targeting TMV helicase, this work successfully established an efficient screening platform for TMV helicase inhibitors based on natural alkaloids. In vivo activity screening, enzyme activity detection, and binding assays showed that Rutaecarpine from Evodia rutaecarpa (Juss.) Benth exhibited excellent TMV helicase inhibitory properties [dissociation constant (Kd ) = 1.1 µm, half maximal inhibitory concentration (IC50 ) = 227.24 µm] and excellent anti-TMV ability. Molecular docking and dynamic simulations depicted that Rutaecarpine could stably bind in active pockets of helicase with low binding energy (ΔGbind = -17.8 kcal/mol) driven by hydrogen bonding and hydrophobic interactions. CONCLUSION: Given Rutaecarpine's laudable bioactivity and structural modifiability, it can serve as a privileged building block for further pesticide discovery.

Insights into a class of natural eugenol and its optimized derivatives as potential tobacco mosaic virus helicase inhibitors by structure-based virtual screening.

Plant diseases caused by malignant and refractory phytopathogenic viruses have considerably restricted crop yields and quality. To date, drug design targeting functional proteins or enzymes of viruses is an efficient and viable strategy to guide the development of new pesticides. Herein, a series of novel eugenol derivatives targeting the tobacco mosaic virus (TMV) helicase have been designed using structure-based virtual screening (SBVS). Structure-activity relationship indicated that 2 t displayed the most powerful bonding capability (Kd = 0.2 µM) along with brilliant TMV helicase ATPase inhibitory potency (IC50 = 141.9 µM) and applausive anti-TMV capability (EC50 = 315.7 µg/mL), ostentatiously outperforming that of commercial Acyclovir (Kd = 23.0 µM, IC50 = 183.7 µM) and Ribavirin (EC50 = 624.3 µg/mL). Molecular dynamics simulations and docking suggested ligand 2 t was stable and bound in the active pocket of the TMV helicase by multiple interactions. Given these superior properties, eugenol-based derivatives could be considered as the novel potential plant viral helicase inhibitors. Furthermore, this effective and feasible SBVS strategy established a valuable screening platform for helicase-targeted drug development.

Therapeutic action of Kushen recipe extractive and its inhibitory effect on eotaxin in mouse models with contact dermatitis.

BACKGROUND: Treatment of skin allergic diseases remains a challenging research topic. OBJECTIVE: To investigate the effect of Kushen recipe extractive (KS) gel on contact dermatitis (CD) of mouse. METHODS: Allergic contact dermatitis (ACD) model of mouse was established. Immunohistochemical method (ICH) and flow cytometry method (FCM) were used to detect CD4+ and CD8+ T lymphocytes and explore the regulation effect of KS on the immune status of the organism. The expression status of eotaxin tissue was evaluated by real-time polymerase chain reaction (RT-PCR), ICH, and western blotting method. The survival rates of HaCaT cell and Fibroblasts affected by KS were detected by methyl thiazolyl tetrazolium (MTT) method. The inhibitory effect of KS on eotaxin produced by HaCaT cell and FBs induced by TNF-α and interleukin (IL)-4 were evaluated using RT-PCR and enzyme-linked immunosorbent assay methods. The inhibitory effect of KS on nuclear factor-κB (NF-κB) and Signal transducers and activators of transcription 6 (STAT6) activation induced by TNF-α and IL-4 was detected by electrophoretic mobility shift assay and western blotting methods. RESULTS: We confirmed that KS shows favorable therapeutic effect on CD, which can obviously inhibit eotaxin expression and Eosinophils recruitment in allergic skin of mouse, as well as regulate the immune status of the organism. Furthermore, KS and its main effective components can inhibit TNF-α and IL-4 induced upregulation of eotaxin via the two signal transduction pathways, NF-κB and STAT6. CONCLUSIONS: The great importance of traditional Chinese recipe KS is evidenced by its therapeutic effect and mechanism in ACD of mouse.

Obesity, but not high-fat diet, is associated with bone loss that is reversed via CD4+CD25+Foxp3+ Tregs-mediated gut microbiome of non-obese mice.

Osteoporosis is characterized by decreased bone mass, microarchitectural deterioration, and increased bone fragility. High-fat diet (HFD)-induced obesity also results in bone loss, which is associated with an imbalanced gut microbiome. However, whether HFD-induced obesity or HFD itself promotes osteoclastogenesis and consequent bone loss remains unclear. In this study, we developed HFD-induced obesity (HIO) and non-obesity (NO) mouse models to evaluate the effect of HFD on bone loss. NO mice were defined as body weight within 5% of higher or lower than that of chow diet fed mice after 10 weeks HFD feeding. NO was protected from HIO-induced bone loss by the RANKL /OPG system, with associated increases in the tibia tenacity, cortical bone mean density, bone volume of cancellous bone, and trabecular number. This led to increased bone strength and improved bone microstructure via the microbiome-short-chain fatty acids (SCFAs) regulation. Additionally, endogenous gut-SCFAs produced by the NO mice activated free fatty acid receptor 2 and inhibited histone deacetylases, resulting in the promotion of Treg cell proliferation in the HFD-fed NO mice; thereby, inhibiting osteoclastogenesis, which can be transplanted by fecal microbiome. Furthermore, T cells from NO mice retain differentiation of osteoclast precursors of RAW 264.7 macrophages ex vivo. Our data reveal that HFD is not a deleterious diet; however, the induction of obesity serves as a key trigger of bone loss that can be blocked by a NO mouse-specific gut microbiome.

Novel spiro[chromanone-2,4'-piperidine]-4-one derivatives as potential inhibitors of fatty acid synthesis in pathogens: Design, synthesis, and biological evaluation.

Bacterial survival depends on membrane lipid homeostasis that enables to regulate lipid composition to adapt and optimize their growth in diverse environments. Therefore, the development of inhibitors that interfere with the bacterial fatty acid synthesis process is considered to be a promising tactic. In this study, 58 novel spirochromanone derivatives were prepared and their structure-activity relationship (SAR) was investigated. The bioassay results showed that all most of the compounds showed excellent biological activities, exampled by compounds B14, C1, B15, and B13, which had outstanding inhibitory activities toward various pathogenic bacteria with EC50 values of 0.78 µg/mL ∼3.48 µg/mL. Preliminary antibacterial behavior was studied by a series of biochemical assays including, but not limited to, fluorescence imaging patterns, GC-MS analysis, TEM images, and fluorescence titration experiments. Notably, compound B14 decreased the lipid content of the cell membrane, and increased cell membrane permeability, thereby destroying the integrity of the bacterial cell membrane. Further qRT-PCR results indicated that compound B14 interfered with the mRNA expression levels of fatty acid synthesis process-related genes including ACC, ACP, and Fab family genes. Herein, we highlight the promising bactericidal skeleton based on the spiro[chromanone-2,4'-piperidine]-4-one as a potential inhibitor of fatty acid synthesis.

Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.

Regulation of eotaxin expression in skin allergic diseases.

Introduction: As a key chemotactic factor during Eos recruitment on the allergic inflammation site, eotaxin is regarded as one of the important therapeutic targets. Aim: To address the expression and regulation mechanism of eotaxin, which constitutes an important procedure in skin allergic disease and a target for drug therapy. Material and methods: An allergic contact dermatitis (ACD) model of mouse was established. Immunohistochemical method (ICH) and flow cytometry method (FCM) were used to determine the amounts of CD4+ and CD8+ T cells and their ratios. The eotaxin mRNA and protein were evaluated by real-time PCR, ICH and western-blotting method. Nuclear factor-κB (NF-κB) nuclear translocation and STAT6 phosphorylation were studied by EMSA and western-blotting methods. Results: We confirmed that both CD4+ and CD8+ T cells in mouse blood and tissue increased during the allergic process, FBs was the main source for eotaxin under the allergic condition. Both TNF-α and IL-4 showed synergic effects on the up-regulation of eotaxin mRNA and protein in KC and FBs. Eotaxin can be expressed via NF-κB and STAT6 transcription after KC and FBs were stimulated by TNF-α and IL-4. Conclusions: The obvious up-regulation of eotaxin expression in skin tissue of the mouse ACD model was confirmed, the exact expression site and dynamic process was determined both in vivo and in vitro. The eotaxin expression ability of FBs outperformed that of KC, and eotaxin expression can be regulated by TNF-α and IL-4 via NF-κB and STAT6. The overall findings may pave the way for discovering targets for new drugs and new therapeutic drugs for treating allergic diseases.

Effect of Drought Stress on Degradation and Remodeling of Membrane Lipids in Nostoc flagelliforme.

Nostoc flagelliforme is a kind of terrestrial edible cyanobacteria with important ecological and economic value which has developed special mechanisms to adapt to drought conditions. However, the specific mechanism of lipidome changes in drought tolerance of N. flagelliforme has not been well understood. In this study, the ultra-high-performance liquid chromatography and mass spectrometry were employed to analyze the lipidome changes of N. flagelliforme under dehydration. A total of 853 lipid molecules were identified, of which 171 were significantly different from that of the control group. The digalactosyldiacylglycerol/monogalactosyldiacylglycerol (DGDG/MGDG) ratio was increased. The amount of wax ester (WE) was sharply decreased during drought stress, while Co (Q10) was accumulated. The levels of odd chain fatty acids (OCFAs) were increased under dehydration, positively responding to drought stress according to the energy metabolism state. In conclusion, the lipidomic data corroborated that oxidation, degradation, and biosynthesis of membrane lipids took place during lipid metabolism, which can respond to drought stress through the transformation of energy and substances. Besides, we constructed a lipid metabolic model demonstrating the regulatory mechanism of drought stress in N. flagelliforme. The present study provides insight into the defense strategies of cyanobacteria in lipid metabolic pathways.

Structural characterization and anti-photoaging activity of a polysaccharide from Sargassum fusiforme.

In this study, a purified algal polysaccharide (P1) was isolated from Sargassum fusiforme and its structural characteristics and anti-photoaging activity were studied. Results showed that P1 had a molecular weight of 289 kDa and was mainly composed of mannuronic acid, guluronic acid and fucose with molar ratio of 7.67:2.35:1.00. The backbone of P1 was →4)-ß-ManA-(1→4)-α-GulA-(1→4)-ß-ManA-(1→4)-ß-ManA-(1→4)-α-GulA-(1→4)-ß-ManA-(1→3,4)-ß-ManA-(1→ with a terminal group of α-Fucp-(1→ linked to O-3 position of →3,4)-ß-ManA-(1→. In addition, P1 could inhibit the expressions of MMPs (MMP-1, MMP-3 and MMP-9) in the UVB-irradiated HaCaT cells, indicating that P1 could reduce collagen loss caused by UVB irradiation. It also reduced the contents of ROS and inflammatory factors (TNF-α, IL-6 and IL-1ß), indicating that P1 could reduce the oxidative stress and inflammation response. Thus, Sargassum fusiforme polysaccharide P1 could be used as a potential functional food to relieve skin photoaging.

Proteome profiling reveals changes in energy metabolism, transport and antioxidation during drought stress in Nostoc flagelliforme.

BACKGROUND: Drought is an important abiotic stress that constrains the growth of many species. Despite extensive study in model organisms, the underlying mechanisms of drought tolerance in Nostoc flagelliforme remain elusive. RESULTS: We characterized the drought adaptation of N. flagelliforme by a combination of proteomics and qRT-PCR. A total of 351 differentially expressed proteins involved in drought stress adaptation were identified. It was found that the expression of several nutrient influx transporters was increased, including molybdate ABC transporter substrate binding protein (modA), sulfate ABC transporter substrate-binding protein (sbp) and nitrate ABC transporter (ntrB), while that of efflux transporters for toxic substances was also increased, including arsenic transporting ATPase (ArsA), potassium transporter (TrkA) and iron ABC transporter substrate-binding protein (VacB). Additionally, photosynthetic components were reduced while sugars built up during drought stress. Non-enzymatic antioxidants, orange carotenoid protein (OCP) homologs, cytochrome P450 (CYP450), proline (Pro) and ascorbic acid (AsA) were all altered during drought stress and may play important roles in scavenging reactive oxygen species (ROS). CONCLUSION: In this study, N. flagelliforme may regulates its adaptation to drought stress through the changes of protein expression in photosynthesis, energy metabolism, transport, protein synthesis and degradation and antioxidation. HIGHLIGHTS: • A total of 351 DEPs involved in adaptation to drought stress were identified. • Changes in the expression of six OCP homologs were found in response to drought stress. • Differential expression of transporters played an important role in drought stress adaptation. • Most PSII proteins were downregulated, while PSI proteins were unchanged in response to drought stress. • Sugar metabolism was upregulated in response to drought stress.

Carbon Metabolism and the ROS Scavenging System Participate in Nostoc flagelliforme's Adaptive Response to Dehydration Conditions through Protein Acetylation.

Acetylation represents an extensively occurring protein post-translational modification (PTM) that plays a key role in many cellular physiological and biochemical processes. However, studies on PTMs such as acetylation of lysine (LysAc) in cyanobacteria are still rare. In this study, a quantitative LysAc approach (acetylome) on the strains of Nostoc flagelliforme subjected to different dehydration treatments was conducted. We observed that starch contents were significantly accumulated due to dehydration treatments, and we identified 2474 acetylpeptides and 1060 acetylproteins based on acetylome analysis. Furthermore, an integrative analysis was performed on acetylome and nontargeted metabolism, and the results showed that many KEGG terms were overlapped for both omics analyses, including starch and sucrose metabolism, transporter activity, and carbon metabolism. In addition, time series clustering was analyzed, and some proteins related to carbon metabolism and the ROS scavenging system were significantly enriched in the list of differentially abundant acetylproteins (DAAPs). These protein expression levels were further tested by qPCR. A working model was finally proposed to show the biological roles of protein acetylation from carbon metabolism and the ROS scavenging system in response to dehydration in N. flagelliforme. We highlighted that LysAc was essential for the regulation of key metabolic enzymes in the dehydration stress response.

Lactobacillus alleviated obesity induced by high-fat diet in mice.

Obesity is closely related to dyslipidaemia, diabetes and other metabolic syndromes. Long-term consumption of a high-fat diet (HFD) is an important risk factor that can lead to obesity. In the current research, three Lactobacillus strains, namely, Loigolactobacillus coryniformis subsp. torquens T3 (T3), Lacticasebacillus paracasei subsp. paracasei M5 (M5), and Lacticaseibacillus paracasei subsp. paracasei X12 (X12), were tested to determine their inhibitory effects on HFD-induced obesity. The results showed that M5, T3, and X12 significantly decreased the body weight gain, Lee's index and adipose index. T3 showed significant effects on reducing serum TG levels to 0.92 mmol/ml and increasing HDL-C levels to 2.18 mmol/ml. The M5 treatment significantly reduced the serum TG level and leptin content to 1.11 mmol/ml and 3.7 ng/ml, respectively, and it increased the HDL-C level and adiponectin content to 2.35 mmol/ml and 7 ng/ml, respectively. M5 and T3 dramatically ameliorated hepatic steatosis in HFD-treated mice by reducing the liver index, lipid droplet number in the liver and TC levels in the liver. Gene expression of PPAR-γ and TNF-α was notably downregulated and FAS was upregulated by T3 and M5 treatment. Additionally, administration of M5 and T3 modified the diversity of the gut microbiota with increased OTU number, ACE index, and Chao1, and decreased the Shannon index and the Bacteroidetes /Firmicutes ratio. Overall, our results indicate that Lactobacillus may be used to prevent obesity and gut dysbiosis. PRACTICAL APPLICATION: Lactobacillus from traditional Chinese foods showed strong anti-obesity effects on high-fat diet-fed mice through the regulation of adipocytokines. Additionally, administration of certain Lactobacilli modified the diversity of the gut microbiota. The results indicate that Lactobacillus may be promising functional materials in healthy foods.

Comprehensive Phosphoproteomic Analysis of Nostoc flagelliforme in Response to Dehydration Provides Insights into Plant ROS Signaling Transduction.

Terrestrial cyanobacteria, originated from aquatic cyanobacteria, exhibit a unique mechanism for drought adaptation during long-term evolution. To elucidate this diverse adaptive mechanism exhibited by terrestrial cyanobacteria from the post-translation modification aspect, we performed a global phosphoproteome analysis on the abundance of phosphoproteins in response to dehydration using Nostoc flagelliforme, a kind of terrestrial cyanobacteria having strong ecological adaptability to xeric environments. A total of 329 phosphopeptides from 271 phosphoproteins with 1168 phosphorylation sites were identified. Among these, 76 differentially expressed phosphorylated proteins (DEPPs) were identified for each dehydration treatment (30, 75, and 100% water loss), compared to control. The identified DEPPs were functionally categorized to be mainly involved in a two-component signaling pathway, photosynthesis, energy and carbohydrate metabolism, and an antioxidant system. We concluded that protein phosphorylation modifications related to the reactive oxygen species (ROS) signaling pathway might play an important role in coordinating enzyme activity involved in the antioxidant system in N. flagelliforme to adapt to dehydration stress. This study provides deep insights into the extensive modification of phosphorylation in terrestrial cyanobacteria using a phosphoproteomic approach, which may help to better understand the role of protein phosphorylation in key cellular mechanisms in terrestrial cyanobacteria in response to dehydration.

Oxidative stress and endoplasmic reticulum stress contribute to L. paracasei subsp. paracasei M5L exopolysaccharide-induced apoptosis in HT-29 cells.

Colorectal cancer is the third most malignant cancer occurring around the world. Effective prevention and treatment have been increasingly the focus of global attention. Long-term diet of fermented dairy inhibits proliferation of colon cancer cell, which is considered that not only live lactic acid bacteria but also the secreted exopolysaccharides exert the function. In this scenario, this study aimed to investigate the mechanism of growth inhibition on HT-29 cells induced in vitro by exopolysaccharides isolated from Lactobacillus paracasei subsp. paracasei M5L (M5-EPSs). HT-29 cells which were treated by a set of concentrations of M5-EPSs have been investigated of cell viability, characteristic changes, cell cycle distribution, and redox system. The results demonstrated that M5-EPSs treatments induced HT-29 cell apoptosis and resulted in upregulation of ROS levels and downregulation of antioxidant enzyme activities, leading to an imbalance in the oxidation system in HT-29 cells. In response to M5-EPSs, endogenous ER stress (ERS) markers, including GRP78, ATF4, and CHOP, were transcriptionally altered so that activating the ERS in HT-29 cells. After NAC treatment, the oxidative stress was inhibited, and the expression of GRP78 and CHOP was significantly decreased, indicating that oxidative stress can significantly affect the ERS pathway. Furthermore, it suggested that the occurrence of apoptosis was associated with Bcl-2 gene family. In conclusion, this study demonstrated that M5-EPSs can induce HT-29 cells apoptosis by destroying the redox system through activation of the ERS signaling pathway.

Aligning the Symmetry of the Type III Secretion System Needle Complex.

Advances in cryo-EM single-particle analysis have resulted in the routine determination of molecular structures to resolutions rivalling X-ray crystallography. Determining a reconstruction to high resolution requires a homogeneous particle data set; heterogeneity in conformation, occupancy, or even symmetry-mismatched components within a protein complex can present a challenge in data processing and affect the achievable resolution. The bacterial type III secretion system, or injectisome, is a macromolecular nanomachine used by some Gram-negative bacteria to inject effector proteins into a eukaryotic host to aid bacterial survival. The core dual-membrane-spanning needle complex has been the focus of structural study for the last two decades; however, the varied and mismatched internal symmetries of the highly oligomeric constituent components have presented numerous challenges for cryo-EM single-particle data processing. Here we give an overview of the history of cryo-EM studies of the prototypical Salmonella SPI-1 needle complex and discuss the workflow we recently employed in the successful determination of the entire complex.

Towards capture of dynamic assembly and action of the T3SS at near atomic resolution.

The T3SS is a syringe-shaped nanomachine essential for the progression of many Gram-negative bacterial infections including plague, typhoid fever, and dysentery. It spans both bacterial membranes and that of the host allowing delivery of proteins that modulate cell function to aid bacterial survival. Its structure has been the focus of scrutiny for 20 years; however, limitations in purification and structure determination techniques have restricted understanding to atomic structures of individual components and subcomplexes or lower resolution information of the more complete assembly. The recent cryo-EM resolution revolution has facilitated dramatic advances in our structural understanding of the T3SS with complimentary techniques of single particle cryo-EM and cryo-ET revealing structures of isolated complexes to near-atomic resolutions or the architecture of the entire T3SS in its native cellular environment. Here we present an overview of these advances and discuss how these structures further understanding of the dynamic process of injectisome assembly.

T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly.

The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. Bacterial proteins destined for self-assembly and host-cell targeting are translocated by the injectisome in a process known as type III secretion (T3S). The core structure is the ~4 MDa needle complex (NC), built on a foundation of three highly oligomerized ring-forming proteins that create a hollow scaffold spanning the bacterial inner membrane (IM) (24-mer ring-forming proteins PrgH and PrgK in the Salmonella enterica serovar Typhimurium Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS)) and outer membrane (OM) (15-mer InvG, a member of the broadly conserved secretin pore family). An internalized helical needle projects from the NC and bacterium, ultimately forming a continuous passage to the host, for delivery of virulence effectors. Here, we have captured snapshots of the entire prototypical SPI-1 NC in four distinct needle assembly states, including near-atomic resolution, and local reconstructions in the absence and presence of the needle. These structures reveal the precise localization and molecular interactions of the internalized SpaPQR 'export apparatus' complex, which is intimately encapsulated and stabilized within the IM rings in the manner of a nanodisc, and to which the PrgJ rod directly binds and functions as an initiator and anchor of needle polymerization. We also describe the molecular details of the extensive and continuous coupling interface between the OM secretin and IM rings, which is remarkably facilitated by a localized 16-mer stoichiometry in the periplasmic-most coupling domain of the otherwise 15-mer InvG oligomer.

Indirubin attenuates mouse psoriasis-like skin lesion in a CD274-dependent manner: an achievement of RNA sequencing.

It was previously reported that the expression of CD274 was down-regulated in psoriatic epidermis, leading to immune disorders of psoriasis. However, the regulatory mechanisms of CD274 were rarely elucidated. We aimed to explore the regulatory mechanisms of CD274. Skin samples were collected from 18 patients with psoriasis vulgaris and 9 healthy participants for RNA sequencing. Candidate genes were chosen based on degree and k-core difference of genes in the co-expression network. The relations between candidate genes and CD274 were validated by flow cytometry and real-time PCR in primary human epidermal keratinocytes. The therapeutic effect of indirubin was assessed in an imiquimod-treated mouse model. Interferon-γ (IFN-γ), cyclin-dependent kinase (CDK) 1, Toll-like receptor 3 (TLR3), TLR4 and interleukin (IL)-17A were considered as candidate genes. In primary human epidermal keratinocytes, the level of CD274 was obviously increased under the stimulation of IFN-γ and CDK1 inhibitor (indirubin), independent of TLR4, TLR3 or IL-17A. Indirubin alleviated the severity of psoriatic mice in a CD274-dependent manner. Co-expression network analysis served as an effective method for the exploration of molecular mechanisms. We demonstrated for the first time that CD274 was the regulator of indirubin-mediated effect on mouse psoriasis-like skin lesion based on co-expression network analysis, contributing to the alleviation of mouse psoriasis-like skin lesion.