Salmonella spvC gene suppresses macrophage/neutrophil antibacterial defense mediated by gasdermin D.

OBJECTIVE: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a representative model organism for investigating host-pathogen interactions. It was reported that S. Typhimurium spvC gene alleviated intestinal inflammation to aggravate systemic infection, while the precise mechanisms remain unclear. In this study, the influence of spvC on the antibacterial defense of macrophage/neutrophil mediated by gasdermin D (GSDMD) was investigated. METHODS: Mouse macrophage-like cell lines J774A.1 and RAW264.7, neutrophil-like cells derived from HL-60 cells (human promyletic leukemia cell lines) were infected with S. Typhimurium wild type, spvC deletion and complemented strains. Cell death was evaluated by LDH release and Annexin V-FITC/PI staining. Macrophage pyroptosis and neutrophil NETosis were detected by western blotting, live cell imaging and ELISA. Flow cytometry was used to assess the impact of spvC on macrophage-neutrophil cooperation in macrophage (dTHP-1)-neutrophil (dHL-60) co-culture model pretreated with GSDMD inhibitor disulfiram. Wild-type and Gsdmd-/- C57BL/6J mice were utilized for in vivo assay. The degree of phagocytes infiltration and inflammation were analyzed by immunofluorescence and transmission electron microscopy. RESULTS: Here we find that spvC inhibits pyroptosis in macrophages via Caspase-1/Caspase-11 dependent canonical and non-canonical pathways, and restrains neutrophil extracellular traps extrusion in GSDMD-dependent manner. Moreover, spvC could ameliorate macrophages/neutrophils infiltration and cooperation in the inflammatory response mediated by GSDMD to combat Salmonella infection. CONCLUSIONS: Our findings highlight the antibacterial activity of GSDMD in phagocytes and reveal a novel pathogenic mechanism employed by spvC to counteract this host defense, which may shed new light on designing effective therapeutics to control S. Typhimurium infection.

Impact of cell wall polysaccharide modifications on the performance of Pichia pastoris: novel mutants with enhanced fitness and functionality for bioproduction applications.

BACKGROUND: Pichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities. RESULTS: Two novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in ß-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage. CONCLUSIONS: Two novel P. pastoris chassis hosts with impaired ß-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.

Effects and mechanisms of Salmonella plasmid virulence gene spv on host-regulated cell death.

Salmonella is responsible for the majority of food poisoning outbreaks around the world. Pathogenic Salmonella mostly carries a virulence plasmid that contains the Salmonella plasmid virulence gene (spv), a highly conserved sequence encoding effector proteins that can manipulate host cells. Intestinal epithelial cells are crucial components of the innate immune system, acting as the first barrier of defense against infection. When the barrier is breached, Salmonella encounters the underlying macrophages in lamina propria, triggering inflammation and engaging in combat with immune cells recruited by inflammatory factors. Host regulated cell death (RCD) provides a variety of means to fight against or favour Salmonella infection. However, Salmonella releases effector proteins to regulate RCD, evading host immune killing and neutralizing host antimicrobial effects. This review provides an overview of pathogen-host interactions in terms of (1) pathogenicity of Salmonella spv on intestinal epithelial cells and macrophages, (2) mechanisms of host RCD to limit or promote pathogenic Salmonella expansion, and (3) effects and mechanisms of Salmonella spv gene on host RCD.

Inactivation of Carotenogenic-Biosynthesizing Genes Altered Lipids Composition and Intensity in Cronobacter sakazakii.

Cronobacter sakazakii, an opportunistic milk-borne pathogen responsible for severe neonatal meningitis and bacteremia, can synthesize yellow pigment (various carotenoids) benefiting for bacterial survival, while little literature was available about the influence of various carotenoids on bacterial resistance to a series of stresses and the characteristics of cell membrane, obstructing the development of novel bactericidal strategies overcoming the strong tolerance of C. sakazakii. Thus in this study, for the first time, five carotenogenic genes of C. sakazakii BAA-894 were inactivated, respectively, to construct a series of mutants producing various carotenoids and their effects on the cell membrane properties, and resistances to food- and host-related stresses, were investigated systematically. Furthermore, to explore its possible mode of action, comparative lipidomics analysis was performed to reveal the change of lipids that were mainly located at cell membranes. The results showed that five mutants (ΔcrtB, ΔcrtI, ΔcrtY, ΔcrtZ, and ΔcrtX) displayed negligible change in growth rate but higher permeability of the outer membrane and lower fluidity of cell membrane compared to the wild type. Besides, these mutants exhibited poorer ability of biofilm formation and lower resistances to acid, oxidative, osmotic, and desiccation stresses, indicating that different carotenoid composition significantly affected environmental tolerance of C. sakazakii. To discover the possible causes, lipidomics analysis of C. sakazakii was conducted and more than 500 lipid species belonging to 27 classes had been identified at first. Compared to that of BAA-894, the composition and relative intensity of lipid species in five mutants varied significantly, especially the monounsaturated and biunsaturated phosphatidylethanolamine. The evidence presented in this study demonstrated that the varied composition of carotenoids in C. sakazakii significantly altered the lipid profile and intensity, which maybe a crucial means to influencing the characteristics of cell membranes and resistance to environmental stresses.

Comparative lipidomics of Pichia pastoris using constitutive promoter reveals lipid diversity and variability at different growth phases.

Pichia pastoris is an expression platform widely used for foreign protein expression, while it is unknown how the global lipid profiles changed during the cultivation process, which is crucial for fermentation optimization and chassis design. Therefore, this study aimed to reveal the diverse lipid profiles of P. pastoris controlled by constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase gene and to unravel their change in the lag, logarithmic, stationary, and death phases, using ultra-performance liquid chromatography/nano-electrospray ionization-tandem mass spectrometry. Two hundred forty lipid species across 11 lipid classes were detected, including various glycerolipids, glycerophospholipids, and sphingolipids. Pichia cells displayed high diversity and variability of lipids in lipid profile, relative intensity, phosphatidylinositol/phosphatidylserine ratio, fatty acid chain length, and unsaturation degree. Notably, increase of unsaturated triacylglycerol level was accompanied by rise of malondialdehyde level under oxidative stress. The increased ceramide with long fatty acid chain could be a key feature at death phase. This work deepened our understanding of the physiology of P. pastoris during cultivation and provided valuable information for further improvement of the P. pastoris expression system.

Growth medium- and strain-dependent bactericidal efficacy of blue light against Shiga toxin-producing Escherichia coli on food-grade stainless steel and plastic.

Shiga toxin-producing Escherichia coli (STEC) are a major group of human pathogens and may persist on both abiotic and biotic surfaces. In this report, two blue-light prototypes were used to evaluate the antimicrobial efficacy against STEC on food processing surfaces (stainless steel and polyoxymethylene plastic). Investigation using a light-bulb prototype (Prototype 1 at 405 nm, 26 mW/cm2) showed significant antimicrobial effects in nutrient deficient condition but not in nutrient rich condition, demonstrating that the presence of organic matters from rich nutrient medium was thought to be light-absorptive and reduce the bactericidal efficacy of blue light, as evident from the lack of bacterial reduction when suspended in cooked meat broth. An advanced (surface-mounted-diode) light panel, Prototype 2 with high light intensity (405 nm; 50 mW/cm2) was able to inactivate a cocktail of seven STEC strains (from seven major serotypes O26, O45, O103, O111, O121, O145 and O157) on type 304 stainless steel (1.66 log10 CFU) and polyoxymethylene plastic (4.25 log10 CFU) at light dosages of 720 and 45 J/cm2, respectively when cells were illuminated in a nutrient-deficient medium (M9 broth). Post-treatment, no STEC cells were recoverable from plastic, both when tested on plates (agar or petrifilms) and by polymerase chain reaction (PCR). In contrast, surviving colonies were identified on samples taken from stainless steel, albeit only four strains could be detected by PCR analysis - those belonging to serotypes O26, O45, O103 and O157 - which indicated that the susceptibility of STEC to blue light varied across the tested strains.

Cell Models in Autophagy Research.

Autophagy is highly conserved in organisms ranging from yeast to humans. C. elegans, D. melanogaster, zebrafish, and mice have been extensively used to study autophagy, though each of them has shortcomings. Suitable cell models are very important, and there is considerable potential for them to help advance autophagy research. Cell models have advantages in speed, stability, economy, etc. Moreover, experimental conditions are more easily controlled in cell models than in animal models. More than 40 ATG genes have been found in budding yeast and other fungi since 1992. As a model organism, yeast has a unique place in autophagy research and has become the most widely used cell model. It is almost equal to E. coli in terms of rapid proliferation, ease of culture, and handling. Yeast is also a good host for eukaryotic gene expression and can be used for screens that help clarify the function of unknown genes. However, as a lower unicellular organism, it is unable to show tissue-specific regulation of autophagy. Cells from higher organisms, such as humans or other animals, are indispensable. Deeper and more extensive study of autophagy using cell models such as nervous tissue-derived cell models, epithelial tissue-derived cell models, muscle tissue-derived cell models, blood cell, and immune cell models has made significant progress.

Genetic Factors Affect the Survival and Behaviors of Selected Bacteria during Antimicrobial Blue Light Treatment.

Antimicrobial resistance is a global, mounting and dynamic issue that poses an immediate threat to human, animal, and environmental health. Among the alternative antimicrobial treatments proposed to reduce the external use of antibiotics is electromagnetic radiation, such as blue light. The prevailing mechanistic model is that blue light can be absorbed by endogenous porphyrins within the bacterial cell, inducing the production of reactive oxygen species, which subsequently inflict oxidative damages upon different cellular components. Nevertheless, it is unclear whether other mechanisms are involved, particularly those that can affect the efficacy of antimicrobial blue light treatments. In this review, we summarize evidence of inherent factors that may confer protection to a selected group of bacteria against blue light-induced oxidative damages or modulate the physiological characteristics of the treated bacteria, such as virulence and motility. These include descriptions of three major photoreceptors in bacteria, chemoreceptors, SOS-dependent DNA repair and non-SOS protective mechanisms. Future directions are also provided to assist with research efforts to increase the efficacy of antimicrobial blue light and to minimize the development of blue light-tolerant phenotypes.

Interconnections among major forms of regulated cell death.

As a basic biological phenomenon of cells, regulated cell death (RCD) has irreplaceable influence on the occurrence and development of many processes of life and diseases. RCD plays an important role in the stability of the homeostasis, the development of multiple systems and the evolution of organisms. Thus comprehensively understanding of RCD is undoubtedly helpful in the innovation of disease treatment. Recently, research on the underlying mechanisms of the major forms of RCD, such as apoptosis, autophagy, necroptosis, pyroptosis, paraptosis and neutrophils NETosis has made significant breakthroughs. In addition, the interconnections among them have attracted increasing attention from global scholars in the field of life sciences. Here, recent advances in RCD research field are discussed.

Identification and characterization of an actin filament-associated Anaplasma phagocytophilum protein.

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA) is an obligate intracellular Gram-negative bacterium. During intracellular replication, A. phagocytophilum interacts with many host cell components including actin cytoskeleton. However the bacterial factors contributing to the interaction between A. phagocytophilum and actin filaments remain unknown. In this study we identified a novel type IV secretion system substrate of A. phagocytophilum by employing TEM-1 ß-lactamase based protein translocation assay, and found it is an actin filament-associated protein. Here, we name this protein as an actin filament-associated Anaplasma phagocytophilumprotein (AFAP). Further analysis showed that the middle region of AFAP harboring four tandem repeats is involved in its interaction with actin filaments. The identification and characterization of an actin filament-associated A. phagocytophilum protein in this study may help understand the interaction between A. phagocytophilum and actin cytoskeleton of its host cells, facilitating the elucidation of HGA pathogenesis.

Salmonella effector SpvB interferes with intracellular iron homeostasis via regulation of transcription factor NRF2.

Iron is a necessary nutrient for humans and nearly all bacterial species. During Salmonella infection, macrophages limit the availability of iron to intracellular pathogens in one of the central components of nutritional immunity. However, Salmonella also have mechanisms to interfere with the antimicrobial effect of host iron withdrawal and meet their own nutrient requirements by scavenging iron from the host. Here, we provide what is, to our knowledge, the first report that SpvB, a pSLT-encoded cytotoxic protein whose function is associated with the intracellular stage of salmonellosis, perturbs macrophage iron metabolism, thereby facilitating Salmonella survival and intracellular replication. In investigating the underlying mechanism, we observed that the Salmonella effector SpvB down-regulated nuclear factor erythroid-derived 2-related factor 2 (NRF2), and its C-terminal domain was necessary and sufficient for NRF2 degradation via the proteasome pathway. Decreased NRF2 expression in the nucleus resulted in a decrease in its transcriptional target ferroportin, encoding the sole macrophage iron exporter, thus ultimately decreasing iron efflux and increasing the intracellular iron content. Additionally, SpvB contributes to the pathogenesis of Salmonella including severe serum hypoferremia, increased splenic and hepatic bacterial burden, and inflammatory injury in vivo. Together, our observations uncovered a novel contribution of SpvB to Salmonella pathology via interference with host intracellular iron metabolism.-Yang, S., Deng, Q., Sun, L., Dong, K., Li, Y., Wu, S., Huang, R. Salmonella effector SpvB interferes with intracellular iron homeostasis via regulation of transcription factor NRF2.

Salmonella spv locus affects type I interferon response and the chemotaxis of neutrophils via suppressing autophagy.

Salmonella is a facultative intracellular pathogen that can cause significant morbidity and mortality in humans and animals. Salmonella plasmid virulence (spv) gene sequence is a highly conserved 6.8 kb region which exists in the plasmid of most pathogenic Salmonella. Autophagy is a degradation process of unnecessary and dysfunctional cytoplasm components to maintain cellular homeostasis, which could affect host inflammatory responses, such as type I interferon response. Type I interferon response can promote the antibacterial activity of macrophage as well as the secretion of cytokines and neutrophil chemokines. We previously reported that spv locus could suppress autophagy and the aggregation of neutrophils in zebrafish larvae. To explore the influence of spv locus on Salmonella escaping from the innate immune responses and the underlying mechanism, the models of Salmonella enterica serovar Typhimurium infected macrophages in vitro and zebrafish larvae in vivo were used in this study. The interactions among spv locus, autophagy, type I interferon response and the chemotaxis of neutrophils were investigated. Western blot was used to detect the expression levels of autophagy related proteins and RT-qPCR was used to measure the mRNA levels of type I interferon response and the neutrophil chemokines. The chemotaxis of neutrophils were observed by Laser Scanning confocal microscopy. Autophagy agonist Torin 1 was also involved to interfere the autophagy influx. Results showed that spv locus could restrain type I interferon response and the chemotaxis of neutrophils via suppressing autophagy, which provided substantial foundation to study the mechanism of Salmonella escaping the innate immunity.

Identification of novel immunoreactive proteins and delineation of a specific epitope of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA), an increasingly recognized febrile tick-borne illness, is caused by a gram-negative obligate intracellular bacterium Anaplasma phagocytophilum. Because of nonspecific clinical manifestations, diagnosis of HGA highly depends on laboratory tests. Identification of immunoreactive proteins is prerequisite for development of specific and sensitive immunoassays for HGA. In this study, we identified novel immunoreactive proteins of A. phagocytophilum. Previous studies indicated that secreted proteins of A. phagocytophilum and other bacteria can be immunoreactive antigens. Here we in silico screened A. phagocytophilum genome for encoding proteins which bear features of type IV secretion system substrates. Among seventy seven predicted proteins, fourteen proteins were determined for antigenicity and nine proteins were showed to be immunoreactive antigens. In addition, an APH1384 peptide harboring a B cell epitope predicted by bioinformatics was found specifically reacting with anti-A. phagocytophilum sera. Hereby, we identified novel immunoreactive proteins and delineated a specific epitope of A. phagocytophilum, which might be employed for HGA diagnosis.

Synstable Fusion: A Network-Based Algorithm for Estimating Driver Genes in Fusion Structures.

Gene fusion structure is a class of common somatic mutational events in cancer genomes, which are often formed by chromosomal mutations. Identifying the driver gene(s) in a fusion structure is important for many downstream analyses and it contributes to clinical practices. Existing computational approaches have prioritized the importance of oncogenes by incorporating prior knowledge from gene networks. However, different methods sometimes suffer different weaknesses when handling gene fusion data due to multiple issues such as fusion gene representation, network integration, and the effectiveness of the evaluation algorithms. In this paper, Synstable Fusion (SYN), an algorithm for computationally evaluating the fusion genes, is proposed. This algorithm uses network-based strategy by incorporating gene networks as prior information, but estimates the driver genes according to the destructiveness hypothesis. This hypothesis balances the two popular evaluation strategies in the existing studies, thereby providing more comprehensive results. A machine learning framework is introduced to integrate multiple networks and further solve the conflicting results from different networks. In addition, a synchronous stability model is established to reduce the computational complexity of the evaluation algorithm. To evaluate the proposed algorithm, we conduct a series of experiments on both artificial and real datasets. The results demonstrate that the proposed algorithm performs well on different configurations and is robust when altering the internal parameter settings.

InvS Coordinates Expression of PrgH and FimZ and Is Required for Invasion of Epithelial Cells by Salmonella enterica serovar Typhimurium.

Deep sequencing has revolutionized our understanding of the bacterial RNA world and has facilitated the identification of 280 small RNAs (sRNAs) in Salmonella Despite the suspicions that sRNAs may play important roles in Salmonella pathogenesis, the functions of most sRNAs remain unknown. To advance our understanding of RNA biology in Salmonella virulence, we searched for sRNAs required for bacterial invasion into nonphagocytic cells. After screening 75 sRNAs, we discovered that the ablation of InvS caused a significant decrease of Salmonella invasion into epithelial cells. A proteomic analysis showed that InvS modulated the levels of several type III secreted Salmonella proteins. The level of PrgH, a type III secretion apparatus protein, was significantly lower in the absence of InvS, consistent with the known roles of PrgH in effector secretion and bacterial invasion. We discovered that InvS modulates fimZ expression and hence flagellar gene expression and motility. We propose that InvS coordinates the increase of PrgH and decrease in FimZ that promote efficient Salmonella invasion into nonphagocytic cells.IMPORTANCE Salmonellosis continues to be the most common foodborne infection reported by the CDC in the United States. Central to Salmonella pathogenesis is the ability to invade nonphagocytic cells and to replicate inside host cells. Invasion genes are known to be regulated by protein transcriptional networks, but little is known about the role played by small RNAs (sRNAs) in this process. We have identified a novel sRNA, InvS, that is involved in Salmonella invasion. Our result will likely provide an opportunity to better understand the fundamental question of how Salmonella regulates invasion gene expression and may inform strategies for therapeutic intervention.

spv locus aggravates Salmonella infection of zebrafish adult by inducing Th1/Th2 shift to Th2 polarization.

Salmonella enterica serovar typhimurium (S. typhimurium) are facultative intracellular enteric pathogens causing disease with a broad range of hosts. It was known that Th1-type cytokines such as IFN-γ, IL-12, and TNF-α etc. could induce protective immunity against intracellular pathogens, while Th2-type cytokines such as IL-4, IL-10, and IL-13 etc. are proved to help pathogens survive inside hosts and cause severe infection. One of the critical virulence factor attributes to the pathogenesis of S. typhimurium is Salmonella plasmid virulence genes (spv). Until now, the interaction between spv locus and the predictable generation of Th1 or Th2 immune responses to Salmonella has not been identified. In this study, zebrafish adults were employed to explore the effect of spv locus on Salmonella pathogenesis as well as host adaptive immune responses especially shift of Th1/Th2 balance. The pathological changes of intestines and livers in zebrafish were observed by hematoxylin-eosin (HE) staining and electron microscopy. Levels of the transcription factors of Th1 (Tbx21) and Th2 (GATA3) were measured by real-time quantitative PCR (RT-qPCR). Expression of cytokines were determined by using RT-qPCR and ELISA, respectively. Results showed that spv operon aggravates damage of zebrafish. Furthermore, it demonstrated that spv locus could inhibit the transcription of tbx21 gene and suppress the expression of cytokines IFN-γ, IL-12 and TNF-α. On the contrary, the transcription of gata3 gene could be promoted and the expression of cytokines IL-4, IL-10 and IL-13 were enhanced by spv locus. Taken together, our data revealed that spv locus could aggravate Salmonella infection of zebrafish adult by inducing an imbalance of Th1/Th2 immune response and resulting in a detrimental Th2 bias of host.

Meta-analysis of genome-wide association data identifies novel susceptibility loci for obesity.

Obesity is a major public health problem with strong genetic determination. Multiple genetic variants have been implicated for obesity by conducting genome-wide association (GWA) studies, primarily focused on body mass index (BMI). Fat body mass (FBM) is phenotypically more homogeneous than BMI and is more appropriate for obesity research; however, relatively few studies have been conducted on FBM. Aiming to identify variants associated with obesity, we carried out meta-analyses of seven GWA studies for BMI-related traits including FBM, and followed these analyses by de novo replication. The discovery cohorts consisted of 21 969 individuals from diverse ethnic populations and a total of over 4 million genotyped or imputed SNPs. The de novo replication cohorts consisted of 6663 subjects from two independent samples. To complement individual SNP-based association analyses, we also carried out gene-based GWA analyses in which all variations within a gene were considered jointly. Individual SNP-based association analyses identified a novel locus 1q21 [rs2230061, CTSS (Cathepsin S)] that was associated with FBM after the adjustment of lean body mass (LBM) (P = 3.57 × 10(-8)) at the genome-wide significance level. Gene-based association analyses identified a novel gene NLK (nemo-like kinase) in 17q11 that was significantly associated with FBM adjusted by LBM. In addition, we confirmed three previously reported obesity susceptibility loci: 16q12 [rs62033400, P = 1.97 × 10(-14), FTO (fat mass and obesity associated)], 18q22 [rs6567160, P = 8.09 × 10(-19), MC4R (melanocortin 4 receptor)] and 2p25 [rs939583, P = 1.07 × 10(-7), TMEM18 (transmembrane protein 18)]. We also found that rs6567160 may exert pleiotropic effects to both FBM and LBM. Our results provide additional insights into the molecular genetic basis of obesity and may provide future targets for effective prevention and therapeutic intervention.

Multistage genome-wide association meta-analyses identified two new loci for bone mineral density.

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10(-8)) level: 14q24.2 (rs227425, P-value 3.98 × 10(-13), SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10(-9), CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n = 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis.

Volatile Fatty Acids Production from Codigestion of Food Waste and Sewage Sludge Based on ß-Cyclodextrins and Alkaline Treatments.

Volatile fatty acids (VFAs) are preferred valuable resources, which can be produced from anaerobic digestion process. This study presents a novel technology using ß-cyclodextrins (ß-CD) pretreatment integrated alkaline method to enhance VFAs production from codigestion of food waste and sewage sludge. Experiment results showed that optimized ratio of food waste to sewage sludge was 3 : 2 because it provided adequate organic substance and seed microorganisms. Based on this optimized ratio, the integrated treatment of alkaline pH 10 and ß-CD addition (0.2 g/g TS) performed the best enhancement on VFAs production, and the maximum VFAs production was 8631.7 mg/L which was 6.13, 1.38, and 1.57 times higher than that of control, initial pH 10, and 0.2 g ß-CD/g TS treatment, respectively. Furthermore, the hydrolysis rate of protein and polysaccharides was greatly improved in integration treatment, which was 1.18-3.45 times higher than that of other tests. Though the VFAs production and hydrolysis of polymeric organics were highly enhanced, the primary bacterial communities with different treatments did not show substantial differences.

Salmonella plasmid virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish infection model.

Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S. typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis in vivo. We found that spvB gene could decrease the LD50 of S. typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S. typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response.