Anti-thermal quenching of luminescence in Y2W3O12:Yb3+/RE3+ (RE = Er/Ho/Tm) and its temperature sensing application.

Herein, a series of Y2W3O12:10%Yb3+/x%RE3+ (RE = Er/Ho/Tm) phosphors is prepared via a solid-state reaction. The upconversion and downshift luminescence properties of the phosphors were investigated under an excitation of 980 nm. The bright blue light emission from Tm3+ ion and the green and red light emissions from Ho3+(Er3+) ions were observed. The near-infrared light intensity of NIR-I (Tm3+, ∼850 nm), NIR-II (Er3+: ∼1550 nm; Tm3+: ∼1783 nm) and NIR-III (Ho3+: ∼2050 nm) were analyzed. In particular, the dramatic thermal enhancement phenomenon in visible and NIR regions was exhibited by the Y2W3O12:10%Yb3+/x%RE3+ (RE = Er/Ho/Tm) phosphors. Among them, the green light intensity of Er3+ ions increased 26.77 times, from 303 to 573 K. The NIR-II emission band (∼1783 nm) intensity of Tm3+ ions at 533 K increased 168.7 times compared to that at 313 K. The possible thermal enhancement mechanism is illustrated by the negative thermal expansion (NTE) and Frenkel defect of the Y2W3O12 host. Finally, the optical temperature sensing performances of Y2W3O12:10%Yb3+/x%RE3+ (RE = Er/Ho/Tm) samples are investigated according to the luminescence intensity dependence relationship on temperature. The maximum value of SR reached 4.24% K-1 at 353 K for Y2W3O12:10%Yb3+/0.6%Ho3+ phosphor. The results indicate that the Y2W3O12:10%Yb3+/x%RE3+ (RE = Er/Ho/Tm) phosphors possess anti-thermal quenching properties and are suitable for developing optical temperature sensors.

ArcA positively regulates the expression of virulence genes and contributes to virulence of porcine Shiga toxin-producing enterotoxigenic Escherichia coli.

IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in humans and animals, leading to death and huge economic loss worldwide. Thus, elucidation of ETEC's pathogenic mechanisms will provide powerful data for the discovery of drugs serving as prevention or therapeutics against ETEC-caused diarrheal diseases. Here, we report that ArcA plays an essential role in the pathogenicity and virulence regulation in ETEC by positively regulating the expression of several key virulence factors including F18 fimbriae, heat-labile and heat-stable toxins, Shiga toxin 2e, and hemolysin, under microaerobic conditions and in vivo. Moreover, we found that positive regulation of several virulence genes by ArcA requires a global repressor H-NS (histone-like nucleoid structuring), implying that ArcA may exert positive effects by antagonizing H-NS. Collectively, our data established a key role for ArcA in the pathogenicity of porcine ETEC and ETEC strains isolated from human infections. Moreover, our work reveals another layer of regulation in relation to oxygen control of virulence factors in ETEC.

NhaA facilitates the maintenance of bacterial envelope integrity and the evasion of complement attack contributing to extraintestinal pathogenic Escherichia coli virulence.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for severe bloodstream infections in humans and animals. However, the mechanisms underlying ExPEC's serum resistance remain incompletely understood. Through the transposon-directed insertion-site sequencing approach, our previous study identified nhaA, the gene encoding a Na+/H+ antiporter, as a crucial factor for infection in vivo. In this study, we investigated the role of NhaA in ExPEC virulence utilizing both in vitro models and systemic infection models involving avian and mammalian animals. Genetic mutagenesis analysis revealed that nhaA deletion resulted in filamentous bacterial morphology and rendered the bacteria more susceptible to sodium dodecyl sulfate, suggesting the role of nhaA in maintaining cell envelope integrity. The nhaA mutant also displayed heightened sensitivity to complement-mediated killing compared to the wild-type strain, attributed to augmented deposition of complement components (C3b and C9). Remarkably, NhaA played a more crucial role in virulence compared to several well-known factors, including Iss, Prc, NlpI, and OmpA. Our findings revealed that NhaA significantly enhanced virulence across diverse human ExPEC prototype strains within B2 phylogroups, suggesting widespread involvement in virulence. Given its pivotal role, NhaA could serve as a potential drug target for tackling ExPEC infections.

Genomic Island-Encoded Histidine Kinase and Response Regulator Coordinate Mannose Utilization with Virulence in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429-5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429-5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429-5434) involved in mannose utilization. The TCS directly upregulates EDL5429-5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.

Maximum expected survival rate model for public access defibrillator placement.

AIM: Mathematical optimization of automated external defibrillator (AED) placement has demonstrated potential to improve survival of out-of-hospital cardiac arrest (OHCA). Existing models mostly aim to improve accessibility based on coverage radius and do not account for detailed impact of delayed defibrillation on survival. We aimed to predict OHCA survival based on time to defibrillation and developed an AED placement model to directly maximize the expected survival rate. METHODS: We stratified OHCAs occurring in Singapore (2010-2017) based on time to defibrillation and developed a regression model to predict the Utstein survival rate. We then developed a novel AED placement model, the maximum expected survival rate (MESR) model. We compared the performance of MESR with a maximum coverage model developed for Canada that was shown to be generalizable to other settings (Denmark). The survival gain of MESR was assessed through 10-fold cross-validation for placement of 20 to 1000 new AEDs in Singapore. Statistical analysis was performed using χ2 and McNemar's tests. RESULTS: During the study period, 15,345 OHCAs occurred. The power-law approximation with R2 of 91.33% performed best among investigated models. It predicted a survival of 54.9% with defibrillation within the first two minutes after collapse that was reduced by more than 60% without defibrillation within the first 4 minutes. MESR outperformed the maximum coverage model with P-value < 0.05 (<0.0001 in 22 of 30 experiments). CONCLUSION: We developed a novel AED placement model based on the impact of time to defibrillation on OHCA outcomes. Mathematical optimization can improve OHCA survival.

Characterization of Shiga toxin-producing Escherichia coli isolated from Cattle and Sheep in Xinjiang province, China, using whole-genome sequencing.

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen capable of causing severe gastrointestinal diseases in humans. Cattle and sheep are the natural reservoir hosts of STEC strains. Previously, we isolated 56 STEC strains from anal and carcass swab samples of cattle and sheep in farms and slaughterhouses. In this study, we performed whole-genome sequencing of these isolates and determined their serotypes, virulence profiles, sequence types (STs) and genetic relationships. Our results showed that the 56 isolates belong to 20 different STs, 29 O:H serotypes and 8 stx subtype combinations. The highly prevalent serotypes for bovine and ovine isolates were O8:H25 and O87:H16, respectively. Five serotypes of cattle or sheep isolates are novel. The majority (63%) of cattle isolates contain stx1 + stx2, subtyped into stx1a, stx2a and stx2c. In contrast, most of the sheep isolates contain stx1 only, primarily subtyped into stx1a and stx1c. None of the isolates tested eae-positive, but virulence factors such as ehxA and espP were present with variable prevalence rates. The prevalence of saa (19.6%) and espP (12.5%) in cattle isolates is much higher than that in sheep isolates, whereas that of subA (34%), katP (14.3%) and ireA (28.6%) in sheep isolates is considerably higher than that in cattle isolates. Core-genome SNP analysis revealed that the majority of isolates could be clustered based on their serotypes or STs, whereas some clustering is associated with more than one ST or serotype. Five sheep isolates (4 belonging to ST675 and serotype O76:H19 and 1 belonging to ST25 and serotype O128:H2) share STs, serotypes and stx profiles with two hemolytic uremic syndrome-associated enterohemorrhagic E. coli (HUSEC) isolates; a cattle isolate belonging to the same ST as HUSEC isolate HUSEC001 contains all the nine virulence genes tested. These data suggest a potential of the six isolates for causing severe human infections. Collectively, we described the characteristics of cattle and sheep STEC isolates from Xinjiang, China, which may be utilized in comparative studies of other geographic regions and sources of isolation, and for surveillance as well.

A previously uncharacterized two-component signaling system in uropathogenic Escherichia coli coordinates protection against host-derived oxidative stress with activation of hemolysin-mediated host cell pyroptosis.

Uropathogenic Escherichia coli (UPEC) deploy an array of virulence factors to successfully establish urinary tract infections. Hemolysin is a pore-forming toxin, and its expression correlates with the severity of UPEC infection. Two-component signaling systems (TCSs) are a major mechanism by which bacteria sense environmental cues and respond by initiating adaptive responses. Here, we began this study by characterizing a novel TCS (C3564/C3565, herein renamed orhK/orhR for oxidative resistance and hemolysis kinase/regulator) that is encoded on a UPEC pathogenicity island, using bioinformatic and biochemical approaches. A prevalence analysis indicates that orhK/orhR is highly associated with the UPEC pathotype, and it rarely occurs in other E. coli pathotypes tested. We then demonstrated that OrhK/OrhR directly activates the expression of a putative methionine sulfoxide reductase system (C3566/C3567) and hemolysin (HlyA) in response to host-derived hydrogen peroxide (H2O2) exposure. OrhK/OrhR increases UPEC resistance to H2O2 in vitro and survival in macrophages in cell culture via C3566/C3567. Additionally, OrhK/OrhR mediates hemolysin-induced renal epithelial cell and macrophage death via a pyroptosis pathway. Reducing intracellular H2O2 production by a chemical inhibitor impaired OrhK/OrhR-mediated activation of c3566-c3567 and hlyA. We also uncovered that UPEC links the two key virulence traits by cotranscribing the c3566-c3567 and hlyCABD operons. Taken together, our data suggest a paradigm in which a signal transduction system coordinates both bacterial pathogen defensive and offensive traits in the presence of host-derived signals; and this exquisite mechanism likely contributes to hemolysin-induced severe pathological outcomes.

Bis-molybdopterin guanine dinucleotide modulates hemolysin expression under anaerobiosis and contributes to fitness in vivo in uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs). Successful urinary tract colonization requires appropriate expression of virulence factors in response to host environmental cues, such as limited oxygen and iron availability. Hemolysin is a pore-forming toxin, and its expression correlates with the severity of UPEC infection. Previously, we showed that hemolysin expression is enhanced under anaerobic conditions; however, the genetic basis and regulatory mechanisms involved remain undefined. Here, a transposon-based forward screen identified bis-molybdopterin guanine dinucleotide cofactor (bis-MGD) biosynthesis as an important factor for a full transcription of hemolysin under anaerobiosis but not under aerobiosis. bis-MGD positively influences hemolysin transcription via c3566-c3568, an operon immediately upstream of and cotranscribed with hlyCABD. Furthermore, suppressor mutation analysis identified the nitrogen regulator NtrC as a direct repressor of c3566-c3568-hlyCABD expression, and intact bis-MGD biosynthesis downregulated ntrC expression, thus at least partially explaining the positive role of bis-MGD in modulating hemolysin expression. Finally, bis-MGD is involved in hemolysin-mediated uroepithelial cell death and contributes to the competitive fitness of UPEC in a murine model of UTI. Collectively, our data establish that bis-MGD biosynthesis plays a crucial role in UPEC fitness in vivo, thus providing a potential target for combatting UTIs.

Multi-Perspective Analysis of Building Orientation Effects on Microstructure, Mechanical and Surface Properties of SLM Ti6Al4V with Specific Geometry.

Building orientation is important in selective laser melting (SLM) processes. Current studies only focus on the horizontal and vertical building orientations without considering different modes of horizontal orientations. In fact, for horizontal orientation, different surfaces of the sample that contact the substrate will affect the heat transfer mode and efficiency, and in turn affect the microstructure and material properties. In this paper, the effect of two modes of horizontal building orientations on microstructure, mechanical and surface properties of SLM Ti6Al4V was studied. Current research about building orientation is deficient because the geometry of samples or test surfaces are not strictly defined, which seriously influences the results due to their different heat transfer efficiency and mode. Therefore, the geometry of the samples and test surfaces were clearly defined, and its necessity was proved in this study. To achieve the research goal, three test samples were prepared: sample SLM-PB-S with the building orientation parallel to the substrate and the shorter side L1 contacts it, sample SLM-PB-L with the building orientation parallel to the substrate and the longer side L2 contacts it and sample SLM-VB with the building orientation vertical to the substrate. Subsequently, the microstructure, grain information, densification, residual stress, micro-hardness, tensile properties and surface topography of different samples were analyzed and compared. In the results, SLM-PB-S exhibited denser microstructure and better mechanical properties than SLM-PB-L, including smaller grain size, stronger texture, higher density, micro-hardness, tensile strength, plasticity and better surface quality. It originates from a higher cooling rate and shorter scanning time between layers during SLM-PB-S fabrication, leading to finer grains, lower porosity and better interlayer metallurgical bonding, thus resulting in better material properties. This study can provide a reference to select the proper building orientation in SLM.

Proteomics Investigation of the Time Course Responses of RAW264.7 Macrophages to Infections With the Wild-Type and Twin-Arginine Translocation Mutant Strains of Brucella melitensis.

Brucella, a notorious intracellular pathogen, causes chronic infections in many mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane; protein substrates translocated by Brucella include ABC transporters, oxidoreductases, and cell envelope biosynthesis proteins. Previously, we showed that a Tat mutant of Brucella melitensis M28 exhibits reduced survival within murine macrophages. In this study, we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo. We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants. A total of 6085 macrophage proteins were identified with high confidence, and 79, 50, and 99 proteins were differentially produced upon infection with the Tat mutant at 4, 24, and 48 hpi, respectively, relative to the wild-type infection. Gene ontology and KEGG enrichment analysis indicated that immune response-related proteins were enriched among the upregulated proteins. Compared to the wild-type M28 infection, the most upregulated proteins upon Tat-mutant infection included the cytosolic nucleic acid signaling pathway-related proteins IFIH1, DHX58, IFI202, IFI204, and ISG15 and the NF-κB signaling pathway-related proteins PTGS2, CD40, and TRAF1, suggesting that the host increases the production of these proteins in response to Tat mutant infection. Upregulation of some proteins was further verified by a parallel reaction monitoring (PRM) assay. ELISA and qRT-PCR assays indicated that Tat mutant infection significantly induced proinflammatory cytokine (TNF-α and IL-6) and nitric oxide (NO) production. Finally, we showed that the Tat mutant displays higher sensitivity to nitrosative stress than the wild type and that treatment with the NO synthase inhibitor L-NMMA significantly increases the intracellular survival of the Tat mutant, indicating that NO production contributes to restricting Tat mutant survival within macrophages. Collectively, this work improves our understanding of host immune responses to Tat mutants and provides insights into the mechanisms underlying the attenuated virulence of Tat mutants.

Whole genome sequencing analysis of avian pathogenic Escherichia coli from China.

Avian pathogenic Escherichia coli (APEC) can cause localized or systemic infection in poultry herds, i.e., colibacillosis, which is an economically devastating bacterial disease of the poultry industry worldwide. Additionally, some APEC may have zoonotic potential. In this study, we sequenced 125 APEC isolates from chickens and ducks with obvious clinical symptoms in poultry farms in China and performed genomic epidemiological analysis along with 16 APEC reference genomes downloaded from NCBI. The phylogenetic analysis indicated a great diversity of APEC isolates, and a total of 35 different O types, 22 H types, and 29 ST types were identified. Several virulence-associated genes (VAGs), such as ompT (96.45 %), iss (97.87 %), and hlyF (90.78 %), as well as four complete siderophore gene clusters, including the Sit transport system (86.52 %), aerobactin (89.36 %), salmochelin (79.43 %), and yersiniabactin (54.61 %), were detected in APEC isolates with high prevalence, which could serve as virulence markers of APEC. Several virulence-associated gene clusters, including the two T6SS systems and the K1 capsule biosynthesis gene clusters, were significantly associated with APEC of phylogroups B2, D, and F but very rarely encoded by the APEC from phylogroups C and E. In addition, several virulence-associated genes, which have been reported in other E. coli pathotypes but have not been reported in APEC, were identified in this study. Our findings in this study have implications for a better understanding of APEC evolution and pathogenesis and may lead to the development of new diagnostic tools for APEC.

An Extracytoplasmic Function Sigma/Anti-Sigma Factor System Regulates Hypochlorous Acid Resistance and Impacts Expression of the Type IV Secretion System in Brucella melitensis.

The intracellular bacterial pathogen Brucella causes persistent infections in various mammalian species. To survive and replicate within macrophages, these bacteria must be able to withstand oxidative stresses and express the type IV secretion system (T4SS) to evade host immune responses. The extracytoplasmic function (ECF) sigma factor system is a major signal transduction mechanism in bacteria that senses environmental cues and responds by regulating gene expression. In this study, we defined an ECF σ bcrS and its cognate anti-σ factor abcS in Brucella melitensis M28 by conserved domain analysis and a protein interaction assay. BcrS directly activates an adjacent operon, bcrXQP, that encodes a methionine-rich peptide and a putative methionine sulfoxide reductase system, whereas AbcS is a negative regulator of bcrS and bcrXQP. The bcrS-abcS and bcrXQP operons can be induced by hypochlorous acid and contribute to hypochlorous acid resistance in vitro. Next, RNA sequencing analysis and genome-wide recognition sequence search identified the regulons of BcrS and AbcS. Interestingly, we found that BcrS positively influences T4SS expression in an AbcS-dependent manner and that AbcS also affects T4SS expression independently of BcrS. Last, we demonstrate that abcS is required for the maintenance of persistent infection, while bcrS is dispensable in a mouse infection model. Collectively, we conclude that BcrS and AbcS influence expression of multiple genes responsible for Brucella virulence traits. IMPORTANCEBrucella is a notorious intracellular pathogen that induces chronic infections in animals and humans. To survive and replicate within macrophages, these bacteria require a capacity to withstand oxidative stresses and to express the type IV secretion system (T4SS) to combat host immune responses. In this study, we characterized an extracytoplasmic function sigma/anti-sigma factor system that regulates resistance to reactive chlorine species and T4SS expression, thereby establishing a potential link between two crucial virulence traits of Brucella. Furthermore, the anti-sigma factor AbcS contributes to Brucella persistent infection of mice. Thus, this work provides novel insights into Brucella virulence regulation as well as a potential drug target for fighting Brucella infections.

Optimisation of Planning Parameters for Machining Blade Electrode Micro-Fillet with Scallop Height Modelling.

Aero-engine blades are manufactured by electroforming process with electrodes. The blade electrode is usually machined with five-axis micromilling to get required profile roughness. Tool path planning parameters, such as cutting step and tool tilt angle, have a significant effect on the profile roughness of the micro-fillet of blade electrode. In this paper, the scallop height model of blade electrode micro-fillet processed by ball-end milling cutter was proposed. Effects of cutting step and tool tilt angle the machined micro-fillet profile roughness were predicted with the proposed scallop height model. The cutting step and tool tilt angle were then optimised to ensure the contour precision of the micro-fillet shape requirement. Finally, the tool path planning was generated and the machining strategy was validated through milling experiments. It was also found that the profile roughness was deteriorated due to size effect when the cutting step decreased to a certain value.

Transcriptomic and Metabolomic Profiling Reveals That KguR Broadly Impacts the Physiology of Uropathogenic Escherichia coli Under in vivo Relevant Conditions.

Urinary tract infections are primarily caused by uropathogenic Escherichia coli (UPEC). In contrast to the intestinal E. coli strains that reside in nutrient-rich gut environment, UPEC encounter distinct niches, for instance human urine, which is an oxygen- and nutrient-limited environment. Alpha-ketoglutarate (KG) is an abundant metabolite in renal proximal tubule cells; and previously we showed that two-component signaling system (TCS) KguS/KguR contributes to UPEC colonization of murine urinary tract by promoting the utilization of KG as a carbon source under anaerobic conditions. However, knowledge about the KguR regulon and its impact on UPEC fitness is lacking. In this work, we analyzed transcriptomic and metabolomic changes caused by kguR deletion under anaerobiosis when KG is present. Our results indicated that 620 genes were differentially expressed in the ΔkguR mutant, as compared to the wild type; of these genes, 513 genes were downregulated and 107 genes were upregulated. Genes with substantial changes in expression involve KG utilization, acid resistance, iron uptake, amino acid metabolism, capsule biosynthesis, sulfur metabolism, among others. In line with the transcriptomics data, several amino acids (glutamate, lysine, etc.) and uridine 5'-diphosphogalactose (involved in capsule biosynthesis) were significantly less abundant in the ΔkguR mutant. We then confirmed that the ΔkguR mutant, indeed, was more sensitive to acid stress than the wild type, presumably due to downregulation of genes belonging to the glutamate-dependent acid resistance system. Furthermore, using gene expression and electrophoretic mobility shift assays (EMSAs), we demonstrate that KguR autoregulates its own expression by binding to the kguSR promoter region. Lastly, we performed a genome-wide search of KguR binding sites, and this search yielded an output of at least 22 potential binding sites. Taken together, our data establish that in the presence of KG, KguR broadly impacts the physiology of UPEC under anaerobiosis. These findings greatly further our understanding of KguS/KguR system as well as UPEC pathobiology.

The Twin-Arginine Translocation System Is Important for Stress Resistance and Virulence of Brucella melitensis.

Brucella, the causative agent of brucellosis, is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane and has been implicated in virulence in many bacterial pathogens. However, the roles of the Tat system and related substrates in Brucella remain unclear. We report here that disruption of Tat increases the sensitivity of Brucella melitensis M28 to the membrane stressor sodium dodecyl sulfate (SDS), indicating cell envelope defects, as well as to EDTA. In addition, mutating Tat renders M28 bacteria more sensitive to oxidative stress caused by H2O2 Further, loss of Tat significantly attenuates B. melitensis infection in murine macrophages ex vivo Using a mouse model for persistent infection, we demonstrate that Tat is required for full virulence of B. melitensis M28. Genome-wide in silico prediction combined with an in vivo amidase reporter assay indicates that at least 23 proteins are authentic Tat substrates, and they are functionally categorized into solute-binding proteins, oxidoreductases, cell envelope biosynthesis enzymes, and others. A comprehensive deletion study revealed that 6 substrates contribute significantly to Brucella virulence, including an l,d-transpeptidase, an ABC transporter solute-binding protein, and a methionine sulfoxide reductase. Collectively, our work establishes that the Tat pathway plays a critical role in Brucella virulence.

Leveraging Machine Learning Techniques and Engineering of Multi-Nature Features for National Daily Regional Ambulance Demand Prediction.

The accurate prediction of ambulance demand provides great value to emergency service providers and people living within a city. It supports the rational and dynamic allocation of ambulances and hospital staffing, and ensures patients have timely access to such resources. However, this task has been challenging due to complex multi-nature dependencies and nonlinear dynamics within ambulance demand, such as spatial characteristics involving the region of the city at which the demand is estimated, short and long-term historical demands, as well as the demographics of a region. Machine learning techniques are thus useful to quantify these characteristics of ambulance demand. However, there is generally a lack of studies that use machine learning tools for a comprehensive modeling of the important demand dependencies to predict ambulance demands. In this paper, an original and novel approach that leverages machine learning tools and extraction of features based on the multi-nature insights of ambulance demands is proposed. We experimentally evaluate the performance of next-day demand prediction across several state-of-the-art machine learning techniques and ambulance demand prediction methods, using real-world ambulatory and demographical datasets obtained from Singapore. We also provide an analysis of this ambulatory dataset and demonstrate the accuracy in modeling dependencies of different natures using various machine learning techniques.

Murine SIGNR1 (CD209b) Contributes to the Clearance of Uropathogenic Escherichia coli During Urinary Tract Infections.

Uropathogenic Escherichia coli (UPEC), a Gram-negative bacterial pathogen, is a major causative agent of urinary tract infections (UTIs). However, the molecular mechanisms of how UPEC causes infections have not been determined. Recent studies indicated that certain enteric Gram-negative bacteria interact with and hijack innate immune receptors DC-SIGN (CD209a) and SIGNR1 (CD209b), often expressed by antigen-presenting cells (APCs), such as macrophages, leading to dissemination and infection. It was not known whether UPEC could utilize DC-SIGN receptors to promote its infection and dissemination similarly to the enteric pathogens. The results of this study reveal that UPEC interacts with CD209-expressing macrophages and transfectants. This interaction is inhibited by anti-CD209 antibody, indicating that CD209s are receptors for UPEC. Additionally, in contrast to the results of previous studies, mice lacking SIGNR1 are more susceptible to infection of this uropathogen, leading to prolonged bacterial persistence. Overall, the results of our study indicate that the innate immune receptor CD209s participate in the clearance of UPEC during UTIs.

A novel small RNA Bmsr1 enhances virulence in Brucella melitensis M28.

Brucellosis, caused by Brucella spp., is one of the most serious zoonotic bacterial diseases. Small RNAs (sRNAs) are recognized as a key player in bacterial post-transcription regulation, since they participate in many biological processes with high efficiency and may govern the intracellular biochemistry and virulence of some pathogenic bacteria. Here, a novel small regulatory RNA, Bmsr1 (Brucella melitensis M28 small RNA 1), was identified in a virulent Brucella melitensis M28 strain based on bioinformatic analysis, reverse transcription PCR (RT-PCR), and Northern blot. The Bmsr1 expression level was highly induced after infection of macrophage cells RAW264.7 at 48 h, suggesting a role for Bmsr1 during in vitro infection. Indeed, bmsr1 deletion mutant of M28 attenuated its intracellular survival in RAW264.7 at 24 h and 48 h post-infection. In a mouse model of chronic infection, bmsr1 deletion strain displayed decreased colonization in the spleen while Bmsr1-overexpressed strain showed higher colonization levels than wild type pathogen. Isobaric tags for relative and absolute quantification (iTRAQ) revealed that 314 proteins were differentially expressed in M28Δbmsr1 compared with wild type. Functional annotation analysis demonstrated that most of those proteins are involved in biological processes and those proteins in the ribosome and nitrogen metabolism pathways were enriched. iTRAQ results combined with target prediction identified several potential target genes related to virulence, including virB2, virB9, virB10, virB11, and vjbR and many metabolism genes. Taken together, this study revealed the contribution of a novel sRNA Bmsr1 to virulence of B. melitensis M28, probably by influencing genes involved in T4SS, virulence regulator VjbR and other metabolism genes.

Transcriptional Control of Dual Transporters Involved in α-Ketoglutarate Utilization Reveals Their Distinct Roles in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) are the primary causative agents of urinary tract infections. Some UPEC isolates are able to infect renal proximal tubule cells, and can potentially cause pyelonephritis. We have previously shown that to fulfill their physiological roles renal proximal tubule cells accumulate high concentrations of α-ketoglutarate (KG) and that gene cluster c5032-c5039 contribute to anaerobic utilization of KG by UPEC str. CFT073, thereby promoting its in vivo fitness. Given the importance of utilizing KG for UPEC, this study is designed to investigate the roles of two transporters KgtP and C5038 in KG utilization, their transcriptional regulation, and their contributions to UPEC fitness in vivo. Our phylogenetic analyses support that kgtP is a widely conserved locus in commensal and pathogenic E. coli, while UPEC-associated c5038 was acquired through horizontal gene transfer. Global anaerobic transcriptional regulators Fumarate and nitrate reduction (FNR) and ArcA induced c5038 expression in anaerobiosis, and C5038 played a major role in anaerobic growth on KG. KgtP was required for aerobic growth on KG, and its expression was repressed by FNR and ArcA under anaerobic conditions. Analyses of FNR and ArcA binding sites and results of EMS assays suggest that FNR and ArcA likely inhibit kgtP expression through binding to the -35 region of kgtP promoter and occluding the occupancy of RNA polymerases. Gene c5038 can be specifically induced by KG, whereas the expression of kgtP does not respond to KG, yet can be stimulated during growth on glycerol. In addition, c5038 and kgtP expression were further shown to be controlled by different alternative sigma factors RpoN and RpoS, respectively. Furthermore, dual-strain competition assays in a murine model showed that c5038 mutant but not kgtP mutant was outcompeted by the wild-type strain during the colonization of murine bladders and kidneys, highlighting the importance of C5038 under in vivo conditions. Therefore, different transcriptional regulation led to distinct roles played by C5038 and KgtP in KG utilization and fitness in vivo. This study thus potentially expanded our understanding of UPEC pathobiology.

FNR regulates expression of important virulence factors contributing to pathogenicity of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTIs), which are some of the world's most common bacterial infections of humans. Here, we examined the role of FNR (fumarate and nitrate reduction), a well-known global regulator, in the pathogenesis of UPEC infections. We constructed an fnr deletion mutant of UPEC CFT073 and compared it to the wild type for changes in virulence, adherence, invasion, and expression of key virulence factors. Compared to the wild type, the fnr mutant was highly attenuated in the mouse model of human UTI and showed severe defects in adherence to and invasion of bladder and kidney epithelial cells. Our results showed that FNR regulates motility and multiple virulence factors, including expression of type I and P fimbriae, modulation of hemolysin expression, and expression of a novel pathogenicity island involved in α-ketoglutarate metabolism under anaerobic conditions. Our results demonstrate that FNR is a key global regulator of UPEC virulence and controls expression of important virulence factors that contribute to UPEC pathogenicity.