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Background: Metabolic reprogramming caused by SHMT1/2, as key enzymes in one-carbon metabolism, has been considered to play a critical role in occurrence and progression of hepatocellular carcinoma (HCC). However, the associations between single nucleotide polymorphisms (SNPs) in SHMT1/2 genes and susceptibility of HCC remains unknown. The purpose of this study was to investigate the relation between four candidate SNPs in SHMT1/2 and risk of HCC in Chinese population. Materials and methods: A case-control study including 514 HCC patients and 234 controls was performed to determine associations between SNPs of SHMT1/2 and HCC risk,isolated via mass spectrometry. Results: Our data revealed that SHMT2 rs2229717 was significantly associated with HCC risk when compared to healthy controls under multiple genetic models (Allele T: odds ratio (OR) = 0.73, P=0.023; Dominant Model: OR = 0.72, P=0.044). When our cohort was stratified by sex, male subjects with the T allele and TT genotype for rs2229717 exhibited a decreased risk for HCC risk (OR=0.67, 95%CI=0.50-0.91; OR=0.43, 95%CI=0.18-0.99). In addition, after age stratification was executed, the results showed that both the T allele and the GT genotype for rs2229717 were less common in HCC patients under 50 years old. Conclusion: These data indicate that rs2229717is a predisposing factor for HCC and may affect the occurrence of HCC in various age or sex subgroups, suggesting that SHMT2 rs2229717may be as a useful predictive or diagnostic tool for HCC in some populations.

The motility of macrophages in response to microenvironment stimuli is a hallmark of innate immunity, where macrophages play pro-inflammatory or pro-reparatory roles depending on their activation status during wound healing. Cell size and shape have been informative in defining macrophage subtypes, but their link to motility properties is unknown, despite M1 and M2 macrophages exhibiting distinct migratory behaviors,in vitro, in 3D andin vivo. We apply both morphology and motility-based image processing approaches to analyze live cell images consisting of macrophage phenotypes. Macrophage subtypes are differentiated from primary murine bone marrow derived macrophages using a potent lipopolysaccharide (LPS) or cytokine interleukin-4 (IL-4). We show that morphology is tightly linked to motility, which leads to our hypothesis that motility analysis could be used alone or in conjunction with morphological features for improved prediction of macrophage subtypes. We train a support vector machine (SVM) classifier to predict macrophage subtypes based on morphology alone, motility alone, and both morphology and motility combined. We show that motility has comparable predictive capabilities as morphology. However, using both measures can enhance predictive capabilities. While motility and morphological features can be individually ambiguous identifiers, together they provide significantly improved prediction accuracies (>79%) using only phase contrast time-lapse microscopy and a small unique cell count for training (∼250). Thus, the approach combining cell motility and cell morphology information can accurately assess functionally diverse macrophage phenotypes quickly and efficiently. Our approach offers a cost efficient and high through-put method for screening biochemicals targeting macrophage polarization with small datasets.SignificancePrevious work has shown that macrophage phenotypes can be distinguished by their morphological characteristics. We extend this work to show that distinct motility patterns are linked to macrophage morphology. Thus, motility patterns can be used to differentiate phenotypes. This can enable high-throughput classification of cell phenotypes without regard for the high-resolution images needed to back out morphological characteristics. Furthermore, combining motility-based features with morphological information improves prediction of macrophage subtypes by a machine learning based classification model.

The risk of chronic hepatitis B (CHB) infection is often affected by polyunsaturated fatty acids (PUFAs) metabolism which is strongly influenced by single nucleotide polymorphisms (SNPs) within the PUFA metabolic pathway. Given this, we designed this study to determine the relationship between specific polymorphisms within fatty acid desaturase 2 (FADS2), a key enzyme in PUFA metabolism, and CHB infection. We completed this evaluation using a case-control study comprising 230 CHB patients and 234 unrelated healthy controls in which the genetic relationships between three previously identified SNPs, isolated via mass spectrometry, and CHB infection. Our data revealed that none of these three SNPs (rs174568, rs174601, and rs2727270) were significantly associated with susceptibility to CHB infection when compared to healthy controls. However, when we stratified our cohort by sex, male subjects with the TC genotype for FADS2 exhibited a decreased risk for CHB infection (OR = 0.62, 95%CI = 0.39-0.96; OR = 0.64, 95%CI = 0.41-1.00; OR = 0.57, 95%CI = 0.36-0.90). Furthermore, age stratification revealed that both the T allele and the TC genotypes for each of the three target SNPs were less common in Chinese CHB cases in people younger than 50 years old. Correlation analysis also revealed that there was no statistically significant relationship between these three SNPs and HBV-DNA replication or hepatitis B surface antigen (HBsAg) levels. Thus, our data suggests that rs174568, rs174601, and rs2727270 may affect the CHB outcomes in various age or sex subgroups, suggesting that they may be useful predictive or diagnostic biomarkers of CHB infection in some populations.

BACKGROUND The risk of chronic hepatitis B (CHB) infection is influenced by aberrant DNA methylation and altered nucleotide synthesis and repair, possibly caused by polymorphic variants in one-carbon metabolism genes. In the present study, we investigated the relationship between polymorphisms belonging to the one-carbon metabolic pathway and CHB infection. METHODS A case-control study using 230 CHB patients and 234 unrelated healthy controls was carried out to assess the genetic association of 24 single nucleotide polymorphisins (SNPs) determined by mass spectrometry. RESULTS Three SNPs, comprising rs10717122 and rs2229717 in serine hydroxymethyltransferase1/2 (SHMT2) and rs585800 in betaine-homocysteine S-methyltransferase (BHMT), were associated with the risk of CHB. Patients with DEL allele, DEL.DEL and DEL.T genotypes of rs10717122 had a 1.40-, 2.00- and 1.83-fold increased risk for CHB, respectively. Cases inheriting TA genotype of rs585800 had a 2.19-fold risk for CHB infection. The T allele of rs2229717 was less represented in the CHB cases (odds ratio = 0.66, 95% confidence interval = 0.48-0.92). The T allele of rs2229717 was less in patients with a low hepatitis B virus-DNA level compared to the control group (odds ratio = 0.49, 95% confidence interval = 0.25-0.97) and TT genotype of rs2229717 had a significant correlation with hepatitis B surface antigen level (p = 0.0195). Further gene-gene interaction analysis showed that subjects carrying the rs10717122 DEL.DEL/DEL.T and rs585800 TT/TA genotypes had a 2.74-fold increased risk of CHB. CONCLUSIONS The results of the present study suggest that rs10717122, rs585800 and rs2229717 and gene-gene interactions of rs10717122 and rs585800 affect the outcome of CHB infection, at the same time as indicating their usefulness as a predictive and diagnostic biomarker of CHB infection.

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

Directed cardiomyogenesis from human induced pluripotent stem cells (hiPSCs) has been greatly improved in the last decade but directed differentiation to pacemaking cardiomyocytes (CMs) remains incompletely understood. In this study, we demonstrated that inhibition of NODAL signaling by a specific NODAL inhibitor (SB431542) in the cardiac mesoderm differentiation stage downregulated PITX2c, a transcription factor that is known to inhibit the formation of the sinoatrial node in the left atrium during cardiac development. The resulting hiPSC-CMs were smaller in cell size, expressed higher pro-pacemaking transcription factors, TBX3 and TBX18, and exhibited pacemaking-like electrophysiological characteristics compared to control hiPSC-CMs differentiated from established Wnt-based protocol. The pacemaker-like subtype increased up to 2.4-fold in hiPSC-CMs differentiated with the addition of SB431542 relative to the control. Hence, Nodal inhibition in the cardiac mesoderm stage promoted pacemaker-like CM differentiation from hiPSCs. Improving the yield of human pacemaker-like CMs is a critical first step in the development of functional human cell-based biopacemakers.

Hepatocellular carcinoma (HCC), a type of malignant liver tumor, has a grim prognosis. As a functional protein, synaptopodin-2 (SYNPO2) has been associated with malignancy; however, the expression profile and function of SYNPO2 in HCC remains unknown. Herein, we revealed that SYNPO2 was transcriptionally downregulated in HCC tissues from both The Cancer Genome Atlas cohort and our cohort, and was also decreased at the translational level as determined by western blotting and immunohistochemical staining. Furthermore, reduced SYNPO2 expression correlated significantly with short overall survival and recurrence free survival of HCC patients. Restoring SYNPO2 expression inhibited the proliferation and aggressiveness of hepatocarcinoma cells. Mechanistically, increasing the ratio of cytoplasmic SYNPO2 to nuclear SYNPO2 was positively associated with recurrence rate in HCC patients; calcineurin (CaN) activity positively correlated with cytoplasmic SYNPO2 levels in HCC tissues; and nuclear-cytoplasmic translocation of SYNPO2 was induced by CaN to facilitate metastasis of HCC through assembly of peripheral actin bundles. In short, our findings uncover a novel role of SYNPO2 in HCC metastasis via the CaN/SYNPO2/F-actin axis, and indicate that SYNPO2 may serve as a possible prognostic marker and novel therapeutic target.

The present study describes a method of using a nonlinear energy sink (NES) to realize vibration reduction of the flywheel system, and integrates the NES with the flywheel support structure consisting of a simply supported plate and the flywheels to achieve vibration control. In the micro-vibration environment of the laser communication satellite, the device is proposed on the vibration attenuation of the momentum wheel system. In the system, the external excitation consists of a superposition of several discrete sinusoidal harmonics generated by the radial micro-vibration of the flywheel. A finite-element model is developed and then verified by the equivalent model results. The governing equations of the plate are discretized via the Galerkin method. The complexification-averaging (CX-A) method is employed to obtain approximate solutions. The amplitude-frequency responses of the system are obtained. The vibration suppression of the system is analyzed, and the approximate solutions are verified by numerical results. To systematically analyze the dynamic behaviors of the designed nonlinear vibration absorber, different parameters are selected for NES, and saddle-node bifurcations and Hopf bifurcations are obtained by selecting some specific parameters. The RMS ratio of the transmitted force to the excitation force is used to calculate the transmissibility of the system equipped with the NES. A parametric study is conducted for further optimization. Results illustrate that NES has a good vibration reduction effect on the micro-vibration of the system.

© 2017 Wiley Periodicals, Inc. Stationary symmetrical fish keratocyte cells break symmetry and become motile spontaneously but slowly. We found that applying electric field (EF) accelerates the polarization by an order of magnitude. While spontaneously polarized cells move persistently for hours, the EF-induced polarity is lost in a majority of cells when the EF is switched off. However, if the EF is applied for a long time and then switched off, the majority of cell move stably. Myosin inhibition abolishes spontaneous polarization, but does not slow down EF-induced polarization, and after the EF is turned off, motility does not stop; however, the cell movements are erratic. Our results suggest that the EF rapidly polarizes the cells, but that resulting polarization becomes stable slowly, and that the EF bypasses the requirement for myosin action in motility initiation.

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, since a flagellin-deficient mutant of B. melitensis was found to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunologic standoff between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.

The refueling process of a high-pressure vessel fixed on the rocket-propelled spacecraft was analyzed by the gas kinetics theories and the thermodynamics of variable mass system. A mathematical model was built up to simulate and analyze the refueling process so as to get rules of the pressure and temperature changes and lay the foundation for the design of the fast refueling control algorithm. During the simulation, the thermodynamic model based on natural convection was adopted to make the simulation close to the actual process. The studies shows that the best way to decrease the temperature of the gas within cylinder during refueling and keep the whole time prolong slightly is increase the heat transfer coefficient of cylinder and decrease the volume of stationary tank within measure.

Systemic infection with Neisseria meningitidis leads to a devastating, septicaemic illness with a high mortality, particularly in children. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B, the commonest isolate in developed countries. Here, we describe the identification of vaccine candidates that protect mice against lethal challenge with this bacterium. A total 11 meningococcal proteins that are necessary for establishing systemic infection were expressed as recombinant antigens and assessed for their ability to protect animals against live bacterial challenge; the lactate permease (LctP) and ExbB, which is required for iron acquisition, elicited protective immunity. Both LctP and ExbB are expressed by a wide range of pathogenic isolates of N. meningitidis. Targeting bacterial factors required for pathogenesis may lead to new vaccines to protect individuals from this important human pathogen.

Recent studies have demonstrated distinctive motility and responses to extracellular cues of cells in isolation, cells collectively in groups, and cell fragments. Here we provide a protocol for generating cell sheets, isolated cells, and cell fragments of keratocytes from zebrafish scales. The protocol starts with a comprehensive fish preparation, followed by critical steps for scale processing and subsequent cell sheet generation, single cell isolation, and cell fragment induction, which can be accomplished in just 3 days including a 36-48 h incubation time. Compared to other approaches that usually produce single cells only or together with either fragments or cell groups, this facile and reliable methodology allows generation of all three motile forms simultaneously. With the powerful genetics in zebrafish our model system offers a useful tool for comparison of the mechanisms by which cell sheets, single cells, and cell fragments respond to extracellular stimuli.

Survival and replication inside host cells by Brucella spp. requires a type IV secretion system (T4SS), encoded by the virB locus. However, the identity of the molecules secreted by the T4SS has remained elusive. We hypothesized that proteins translocated by the T4SS would be co-regulated with the virB operon. The LuxR family regulator VjbR, known to regulate virB, bound a fragment of the virB promoter containing an 18 bp palindromic motif (virB promoter box), showing that VjbR regulated the virB operon directly. To identify virB co-regulated genes, we searched the Brucella suis 1330 and B. abortus 2308 genomes for genes with an upstream virB promoter box. One hundred and forty-four promoters in the two genomes contained the virB promoter box, including those of fliC encoding flagellin and cgs encoding cyclic beta-glucan synthetase. Thirteen of these proteins were tested for VirB-dependent translocation into macrophages using a beta-lactamase reporter assay. This analysis resulted in the identification of the proteins encoded by BAB1_1652 (VceA) and BR1038/BAB1_1058 (VceC) as novel protein substrates of the Brucella T4SS. VceC could also be translocated by the Legionella pneumophila Dot/Icm T4SS into host cells. Our results suggest that VjbR co-ordinates expression of the T4SS and at least two of its secreted substrates.

Bacterial flagellins are potent inducers of innate immunity. Three signaling pathways have been implicated in the sensing of flagellins; these involve toll-like receptor 5 (TLR5) and the cytosolic proteins Birc1e/Naip5 and Ipaf. Although the structural basis of TLR5-flagellin interaction is known, little is known about how flagellin enters the host cell cytosol to induce signaling via Birc1e/Naip5 and Ipaf. Here we demonstrate for the first time the translocation of bacterial flagellin into the cytosol of host macrophages by the vacuolar pathogen, Salmonella enterica serotype Typhimurium. Translocation of flagellin into the host cell cytosol was directly demonstrated using beta-lactamase reporter constructs. Flagellin translocation required the Salmonella Pathogenicity Island 1 Type III secretion system (SPI-1 T3SS) but not the flagellar T3SS.

Salmonella enterica serotype Typhi is a strictly human adapted pathogen that does not cause disease in nonprimate vertebrate hosts, while Salmonella enterica serotype Typhimurium is a broad-host-range pathogen. Serotype Typhi lacks some of the proteins (effectors) exported by the invasion-associated type III secretion system that are required by serotype Typhimurium for eliciting fluid secretion and inflammation in bovine ligated ileal loops. We investigated whether the remaining serotype Typhi effectors implicated in enteropathogenicity (SipA, SopB, and SopD) are functionally exchangeable with their serotype Typhimurium homologues. Serotype Typhi elicited fluid accumulation in bovine ligated ileal loops at levels similar to those elicited by a noninvasive serotype Typhimurium strain (the sipA sopABDE2 mutant) or by sterile culture medium. However, introduction of the cloned serotype Typhi sipA , sopB , and sopD genes complemented the ability of a serotype Typhimurium sipA sopABDE2 mutant to elicit fluid secretion in bovine ligated ileal loops. Introduction of the cloned serotype Typhi sipA , sopB , and sopD genes increased the invasiveness of a serotype Typhimurium sipA sopABDE2 mutant for human colon carcinoma epithelial (HT-29 and T84) cells and bovine kidney (MDBK) cells. Translational fusions between the mature TEM-1 β-lactamase reporter and SipA or SopD demonstrated that serotype Typhi translocates these effectors into host cells. We conclude that the inability of serotype Typhi to cause fluid accumulation in bovine ligated ileal loops is not caused by a functional alteration of its SipA, SopB, and SopD effector proteins with respect to their serotype Typhimurium homologues.

The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11 , and two putative genes, orf12 ( virB12 ) and orf13 , encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 ( virB12 ) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B. abortus grown to stationary phase. Mice infected with B. abortus 2308 produced an antibody response to the protein encoded by virB12 , showing that this gene is expressed during infection. Expression of virB12 was not required for survival in J774 macrophages. VirB12 was also dispensable for the persistence of B. abortus , B. melitensis , and B. suis in mice up to 4 weeks after infection, since deletion mutants lacking virB12 were recovered from splenic tissue at wild-type levels. These results show that VirB12 is not essential for the persistence of the human-pathogenic Brucella spp. in the mouse and macrophage models of infection.

Neisseria meningitidis is an important cause of septicaemia and meningitis. To cause disease, the bacterium must acquire essential nutrients for replication in the systemic circulation, while avoiding exclusion by host innate immunity. Here we show that the utilization of carbon sources by N. meningitidis determines its ability to withstand complement-mediated lysis, through the intimate relationship between metabolism and virulence in the bacterium. The gene encoding the lactate permease, lctP, was identified and disrupted. The lctP mutant had a reduced growth rate in cerebrospinal fluid compared with the wild type, and was attenuated during bloodstream infection through loss of resistance against complement-mediated killing. The link between lactate and complement was demonstrated by the restoration of virulence of the lctP mutant in complement (C3−/−)-deficient animals. The underlying mechanism for attenuation is mediated through the sialic acid biosynthesis pathway, which is directly connected to central carbon metabolism. The findings highlight the intimate relationship between bacterial physiology and resistance to innate immune killing in the meningococcus.

TheBrucella abortus virBlocus is required for establishing chronic infection in the mouse. Using in vitro and in vivo models, we investigated whethervirBis involved in evasion of the bactericidal activity of NADPH oxidase and the inducible nitric oxide synthase (iNOS) in macrophages. Elimination of NADPH oxidase or iNOS activity in macrophages in vitro increased recovery of wild-typeB. abortusbut not recovery of avirBmutant. In mice lacking either NADPH oxidase or iNOS, however,B. abortusinfected and persisted to the same extent as it did in congenic C57BL/6 mice up until 60 days postinfection, suggesting that these host defense mechanisms are not critical for limiting bacterial growth in the mouse. AvirBmutant did not exhibit increased survival in either of the knockout mouse strains, indicating that this locus does not contribute to evasion of nitrosative or oxidative killing mechanisms in vivo.

Numerous pathogenic bacteria contain luxS , which is required for autoinducer-2 production. Here, we demonstrate that Neisseria meningitidis contains a functional copy of luxS that is necessary for full meningococcal virulence; strains with a luxS deletion are defective for bacteremia, a prerequisite of meningococcal pathogenesis.

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, since a flagellin-deficient mutant of B. melitensis was found to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunologic standoff between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.

A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3' end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H(2)O(2), decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.

Many types of cells migrate directionally in direct current (DC) electric fields (EFs), a phenomenon termed galvanotaxis or electrotaxis. The directional sensing mechanisms responsible for this response to EFs, however, remain unknown. Exposing cells to an EF causes changes in plasma membrane potentials ( V m ). Exploiting the ability of Dictyostelium cells to tolerate drastic V m changes, we investigated the role of V m in electrotaxis and, in parallel, in chemotaxis. We used three independent factors to control V m : extracellular pH, extracellular [K + ], and electroporation. Changes in V m were monitored with microelectrode recording techniques. Depolarized V m was observed under acidic (pH 5.0) and alkaline (pH 9.0) conditions as well as under higher extracellular [K + ] conditions. Electroporation permeabilized the cell membrane and significantly reduced the V m , which gradually recovered over 40 min. We then recorded the electrotactic behaviors of Dictyostelium cells with a defined V m using these three techniques. The directionality (directedness of electrotaxis) was quantified and compared to that of chemotaxis (chemotactic index). We found that a reduced V m significantly impaired electrotaxis without significantly affecting random motility or chemotaxis. We conclude that extracellular pH, [K + ], and electroporation all significantly affected electrotaxis, which appeared to be mediated by the changes in V m . The initial directional sensing mechanisms for electrotaxis therefore differ from those of chemotaxis and may be mediated by changes in resting V m .

Damage to the respiratory epithelium is one of the most critical steps to many life-threatening diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. The mechanisms underlying repair of the damaged epithelium have not yet been fully elucidated. Here we provide experimental evidence suggesting a novel mechanism for wound repair: endogenous electric currents. It is known that the airway epithelium maintains a voltage difference referred to as the transepithelial potential. Using a noninvasive vibrating probe, we demonstrate that wounds in the epithelium of trachea from rhesus monkeys generate significant outward electric currents. A small slit wound produced an outward current (1.59 μA/cm2), which could be enhanced (nearly doubled) by the ion transport stimulator aminophylline. In addition, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) with CFTR(Inh)-172 significantly reduced wound currents (0.17 μA/cm2), implicating an important role of ion transporters in wound induced electric potentials. Time-lapse video microscopy showed that applied electric fields (EFs) induced robust directional migration of primary tracheobronchial epithelial cells from rhesus monkeys, towards the cathode, with a threshold of

Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.

This unit provides protocols for growth of Brucella abortus on solid or in liquid media and for long-term storage of laboratory stocks. Two issues affecting the culture and storage of isolates of this slow-growing bacterium are emphasized: contamination of cultures and outgrowth of attenuated variants lacking a complete lipopolysaccharide. Laboratories planning to work with B. abortus should be aware that Biosafety Level 3 facilities are required. Furthermore, this organism is classified in the U.S. as a Select Agent, which restricts its use to laboratories registered with the U.S. government's Select Agent programs.

We have identified genes required for competence of Neisseria meningitidis , a naturally transformable human pathogen. Although not comprehensive, our analysis identified competence-defective mutants with transposon insertions in genes not previously implicated in this process in Neisseria .

The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2 , are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2 . Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.

Meningococcal disease is a life-threatening infection caused by Neisseria meningitidis . Currently, there are no vaccines to prevent infection with serogroup B N. meningitidis strains, the leading cause of meningococcal meningitis in Europe and North America. Here we describe the construction and characterization of two attenuated serogroup B N. meningitidis strains, YH102 (MC58Δ sia Δ rfaF ) and YH103 (MC58Δ sia Δ metH ). Both strains are markedly attenuated in their capacity to cause bacteremia in rodent models and have a reduced ability to survive in a human whole-blood assay. Immunization of adult mice with these strains leads to the development of bactericidal antibodies and confers sterilizing protection against challenge with homologous live bacteria. Furthermore, we show that the strains confer protection against infection by other serogroups. Use of the attenuated strains in animals with gene knockouts or after depletion of immunological effectors could be used to define the basis of protection, and human volunteer studies could be undertaken to examine the immune response following exposure to this important human pathogen.

The pathogenic bacterium Neisseria meningitidis is an important cause of septicemia and meningitis, especially in childhood1. The establishment and maintenance of bacteremic infection is a pre-requisite for all the pathological sequelae of meningococcal infection. To further understand the genetic basis of this essential step in pathogenesis, we analyzed a library of 2,850 insertional mutants of N. meningitidis for their capacity to cause systemic infection in an infant rat model. The library was constructed by in vitro modification of Neisseria genomic DNA with the purified components of Tn10 transposition2. We identified 73 genes in the N. meningitidis genome that are essential for bacteremic disease. Eight insertions were in genes encoding known pathogenicity factors. Involvement of the remaining 65 genes in meningocoocal pathogenesis has not been demonstrated previously, and the identification of these genes provides insights into the pathogenic mechanisms that underlie meningococcal infection. Our results provide a genome-wide analysis of the attributes of N. meningitidis required for disseminated infection, and may lead to new interventions to prevent and treat meningococcal infection.