A novel sORF gene mutant strain of Yersinia pestis vaccine EV76 offers enhanced safety and improved protection against plague.

We recently identified two virulence-associated small open reading frames (sORF) of Yersinia pestis, named yp1 and yp2, and null mutants of each individual genes were highly attenuated in virulence. Plague vaccine strain EV76 is known for strong reactogenicity, making it not suitable for use in humans. To improve the immune safety of EV76, three mutant strains of EV76, Δyp1, Δyp2, and Δyp1&yp2 were constructed and their virulence attenuation, immunogenicity, and protective efficacy in mice were evaluated. All mutant strains were attenuated by the subcutaneous (s.c.) route and exhibited more rapid clearance in tissues than the parental strain EV76. Under iron overload conditions, only the mice infected with EV76Δyp1 survived, accompanied by less draining lymph nodes damage than those infected by EV76. Analysis of cytokines secreted by splenocytes of immunized mice found that EV76Δyp2 induced higher secretion of multiple cytokines including TNF-α, IL-2, and IL-12p70 than EV76. On day 42, EV76Δyp2 or EV76Δyp1&yp2 immunized mice exhibited similar protective efficacy as EV76 when exposed to Y. pestis 201, both via s.c. or intranasal (i.n.) routes of administration. Moreover, when exposed to 200-400 LD50 Y. pestis strain 201Δcaf1 (non-encapsulated Y. pestis), EV76Δyp2 or EV76Δyp1&yp2 are able to afford about 50% protection to i.n. challenges, significantly better than the protection afforded by EV76. On 120 day, mice immunized with EV76Δyp2 or EV76Δyp1&yp2 cleared the i.n. challenge of Y. pestis 201-lux as quickly as those immunized with EV76, demonstrating 90-100% protection. Our results demonstrated that deletion of the yp2 gene is an effective strategy to attenuate virulence of Y. pestis EV76 while improving immunogenicity. Furthermore, EV76Δyp2 is a promising candidate for conferring protection against the pneumonic and bubonic forms of plague.

Amyloid and Tau as cerebrospinal fluid biomarkers in anti-N-Methyl-D-aspartate receptor encephalitis.

INTRODUCTION: Neuroinfection is associated with the deposition of amyloid-beta (Aß) peptides, and subsequent decrease in cerebrospinal fluid (CSF) amyloid levels. However, whether autoimmune encephalitis involves extracellular deposition of Aß peptides in the brain is unreported. METHODS: We examined CSF amyloid and tau values in adults with anti-N-methyl-D-aspartate receptor encephalitis (NMDAR-E). Forty-two patients with NMDAR-E, 35 patients with viral and bacterial neuroinfections, and 16 controls were included. We measured CSF Aß1-42 (cAß1-42), Aß1-40 (cAß1-40), t-Tau (ct-Tau), and p-Tau181 (cp-Tau181) levels and assessed their efficacies regarding differential diagnosis and predicting prognosis. RESULTS: NMDAR-E patients had lower cAß1-42 levels; however, they were higher than those of patients with bacterial meningitis. ct-Tau levels in NMDAR-E patients were lower than those in patients with neuroinfections. No changes were observed in controls. cAß1-42 and ct-Tau were combined as an excellent marker to distinguish NMDAR-E from neuroinfections. cAß1-42 levels in NMDAR-E patients were positively correlated with Montreal Cognitive Assessment scores. We observed an inverse relationship between cAß1-42 levels and modified Rankin Scale scores. Patients with poor outcomes exhibited low cAß1-42 levels and high levels of several blood parameters. cAß1-42 was the highest quality biomarker for assessing NMDAR-E prognosis. Correlations were found between cAß1-42 and some inflammatory indicators. CONCLUSION: cAß1-42 was decreased in NMDAR-E patients. cAß1-42 levels indicated NMDAR-E severity and acted as a biomarker for its prognosis. Combining cAß1-42 and ct-Tau levels could serve as a novel differential diagnostic marker for NMDAR-E.

Secretome and Comparative Proteomics of Yersinia pestis Identify Two Novel E3 Ubiquitin Ligases That Contribute to Plague Virulence.

Plague is a zoonotic disease that primarily infects rodents via fleabite. Transmission from flea to host niches requires rapid adaption of Yersinia pestis to the outer environments to establish infection. Here, quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking the two typical niches, i.e., the mammalian host (Mh) and the flea vector (Fv), were performed to understand the adaption strategies of this deadly pathogen. A secretome of Y. pestis containing 308 proteins has been identified using TMT-labeling mass spectrometry analysis. Although some proteins are known to be secreted, such as the type III secretion substrates, PsaA and F1 antigen, most of them were found to be secretory proteins for the first time. Comparative proteomic analysis showed that membrane proteins, chaperonins and stress response proteins are significantly upregulated under the Mh condition, among which the previously uncharacterized proteins YP_3416∼YP_3418 are remarkable because they cannot only be secreted but also translocated into HeLa cells by Y. pestis. We further demonstrated that the purified YP_3416 and YP_3418 exhibited E3 ubiquitin ligase activity in in vitro ubiquitination assay and yp_3416∼3418 deletion mutant of Y. pestis showed significant virulence attenuation in mice. Taken together, our results represent the first Y. pestis secretome, which will promote the better understanding of Y. pestis pathogenesis, as well as the development of new strategies for treatment and prevention of plague.

Methylene blue usage for determining accessory artery ligation in donor kidneys.

Objectives. We present a technique for determining whether to ligate or preserve accessory arteries in donor kidneys before implantation. Methods. Forty-three living-related donor kidneys in patients from January 2014 to February 2018 at our institution were included, all of which had dual arteries without the same stem. Among them, 19 cases of accessory arterial blood supply were evaluated using methylene blue (MB) perfusion, and accessory arteries supplying less than 10% of the total MB perfusion volume were ligated. The other 24 cases were assessed using a conventional method in which arteries with diameters less than 2 mm were ligated. The back-table surgical time, Doppler ultrasonography index, renal function and complications were compared between the 2 groups. Results. All patients underwent successful kidney transplantation. The back-table surgical time in the MB group was longer than that in the conventional group (42.70 ± 4.70 min vs 34.64 ± 5.30 min, P < .05). The serum creatinine level in the MB group was significantly lower than that in the conventional group 1 month after the operation (103.15 ± 19.26 µmol/L vs 119.17 ± 28.32 µmol/L, P < .05). No differences in the Doppler ultrasonography index or postoperative complications were noted. Conclusions. MB perfusion provides an easy and effective method to make decisions regarding arterial ligation and helps preserve renal function without increasing the number of complications after transplantation.

Plasmids of novel incompatibility group IncpRBL16 from Pseudomonas species.

OBJECTIVES: To dissect genomic features of IncpRBL16 plasmids from Pseudomonas. METHODS: An extensive genomic comparison was applied to all 17 available sequenced IncpRBL16 plasmids, including 8 sequenced in this study and another 2 sequenced in two of our previous studies. RESULTS: Conserved IncpRBL16 backbone markers repAIncpRBL16 together with its iterons, parB2-parA, che, pil and ter were present in all 17 plasmids. At least 18 regions or sites across IncpRBL16 genomes exhibited major modular differences, including insertion of accessory modules, deletion of backbone regions surrounding insertion sites and substitution of multiple-gene backbone regions. Ten plasmids carried a sole IncpRBL16 replicon, while exogenous acquisition of an auxiliary replicon (located in an accessory module) besides the primary IncpRBL16 replicon was observed in each of the remaining seven plasmids. The 17 IncpRBL16 plasmids carried at least 71 different accessory modules, notably including Tn1403-related regions, Tn7-family transposons, Tn6571-family transposons, integrative and conjugative elements, and integrative and mobilizable elements. There were a total of 40 known resistance genes, which were involved in resistance to 15 categories of antibiotics and heavy metals, notably including blaIMP-9, blaIMP-45, blaVIM-2, blaDIM-2, blaOXA-246, blaPER-1, aphA and armA. CONCLUSIONS: Different IncpRBL16 plasmids contain different profiles of accessory modules and thus diverse collections of resistance genes. To the best of our knowledge, this is the first report of fully sequenced blaOXA-246-carrying (p12939-PER) and blaPER-1-carrying (p12939-PER and pA681-IMP) IncpRBL16 plasmids and also that of 14 novel (first identified in this study) and additionally 31 newly named (first designated in this study, but with previously determined sequences) mobile elements.

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid and quantitative detection of Coxiella burnetii phase I strains.

BACKGROUND: Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes a zoonotic disease commonly called Q fever globally. In this study, an up-converting phosphor technology-based lateral flow (UPT-LF) assay was established for the rapid and specific detection of phase I strains of C. burnetii. RESULTS: Specific monoclonal antibodies (10B5 and 10G7) against C. burnetii phase I strains were prepared and selected for use in the UPT-LF assay by the double-antibody-sandwich method. The detection sensitivity of the Coxiella-UPT-LF was 5 × 104 GE/ml for a purified C. burnetii phase I strain and 10 ng/ml for LPS of C. burnetii Nine Mile phase I (NMI). Good linearity was observed for C. burnetii phase I and NMI LPS quantification (R2 ≥ 0.989). The UPT-LF assay also exhibited a high specificity to C. burnetii, without false-positive results even at 108 GE/ml of non-specific bacteria, and good inclusivity for detecting different phase I strains of C. burnetii. Moreover, the performance of the Coxiella-UPT-LF assay was further confirmed using experimentally and naturally infected samples. CONCLUSIONS: Our results indicate that Coxiella-UPT-LF is a sensitive and reliable method for rapid screening of C. burnetii, suitable for on-site detection in the field.

Pretreatment with Retro-2 protects cells from death caused by ricin toxin by retaining the capacity of protein synthesis.

The current study explores the detoxification effect of Retro-2 on ricin toxin (RT) cytotoxicity, as well as the mechanisms underlying such effects, to provide a basis for follow-up clinical applications of Retro-2. The mouse-derived mononuclear/macrophage cell line, RAW264.7, was used to evaluate the detoxification effect of Retro-2 on RT by detecting cell viability, capacity for protein synthesis and the expression of cytokines, as well as endoplasmic reticulum stress (ERS)-related mRNA. The results indicated that many cells died when challenged with concentrations of RT ≥50ng/mL. The protein synthesis capacity of cells decreased when challenged with 200ng/mL RT for 2hours. Furthermore, the synthesis and release of many cytokines decreased, while the expression of cytokines or ERS-related mRNA increased when challenged with 200ng/mL of RT for 12 or more hours. However, cell viability, capacity for protein synthesis and release levels of many cytokines were higher, while the expression levels of cytokine, or ERS-related mRNA, were lower in cells pretreated with 20µm Retro-2 and challenged with RT, compared with those that had not been pretreated with Retro-2. In conclusion, Retro-2 retained the capacity for protein synthesis inhibited by RT, alleviated ERS induced by RT and increased the viability of cells challenged with RT. Retro-2 shows the potential for clinical applications.

Inhibition of microRNA-132 attenuates inflammatory response and detrusor fibrosis in rats with interstitial cystitis via the JAK-STAT signaling pathway.

Interstitial cystitis (IC) is a heterogeneous syndrome with unknown etiology, and microRNAs (miRs) were found to be involved in IC. In our study, we aim to explore the role of miR-132 in the inflammatory response and detrusor fibrosis in IC through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway in rat models. A rat model of IC was established and treated with the miR-132 mimic, miR-132 inhibitor, and/or JAK-STAT signaling pathway inhibitor AG490. Enzyme-linked immunosorbent assay was applied to measure the expression of interleukin (IL)-6, IL-10, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule-1 (ICAM-1). The urodynamic test was performed to assess urodynamic parameters, and reverse transcription quantitative polymerase chain reaction and Western blot analysis for the expression of miR-132, STAT4, suppressors of cytokine signaling 3 (SOCS3), JAK2, vascular endothelial growth factor (VEGF), IFN-γ, and TNF-α. IC rats treated with miR-132 inhibitor and AG490 had decreased collagen fiber, inflammatory cell infiltration, and mast cells, lower expression of IL-6, IL-10, IFN-γ, TNF-α, ICAM-1, collagens I and III, and alleviated urodynamic parameters and decreased expression of STAT4, VEGF, JAK2, IFN-γ, TNF-α, and increased expression of SOCS3. Taken together, our data indicate that downregulation of miR-132 alleviates inflammatory response and detrusor fibrosis in IC via the inhibition of the JAK-STAT signaling pathway.

Reversible Gene Expression Control in Yersinia pestis by Using an Optimized CRISPR Interference System.

Many genes in the bacterial pathogen Yersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Streptococcus pyogenes Cas9-derived dCas9 for gene knockdown in Y. pestis Multiple genes harbored on either the chromosome or plasmids of Y. pestis were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of hmsH (responsible for biofilm formation) or cspB (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance in in vitro phenotypic assays. Furthermore, silencing of the virulence-associated genes yscB or ail using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for yscB and ail null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in Y. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening of Y. pestis genes with unknown functions.IMPORTANCEYersiniapestis is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, many Y. pestis genes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported in Y. pestis Here, we systematically optimized the CRISPRi approach for use in Y. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected by in vitro assays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach in Y. pestis and highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.

An Interaction between the Inner Rod Protein YscI and the Needle Protein YscF Is Required to Assemble the Needle Structure of the Yersinia Type Three Secretion System.

The type III secretion system is a highly conserved virulence mechanism that is widely distributed in Gram-negative bacteria. It has a syringe-like structure composed of a multi-ring basal body that spans the bacterial envelope and a projecting needle that delivers virulence effectors into host cells. Here, we showed that the Yersinia inner rod protein YscI directly interacts with the needle protein YscF inside the bacterial cells and that this interaction depends on amino acid residues 83-102 in the carboxyl terminus of YscI. Alanine substitution of Trp-85 or Ser-86 abrogated the binding of YscI to YscF as well as needle assembly and the secretion of effectors (Yops) and the needle tip protein LcrV. However, yscI null mutants that were trans-complemented with YscI mutants that bind YscF still assembled the needle and secreted Yops, demonstrating that a direct interaction between YscF and YscI is critical for these processes. Consistently, YscI mutants that did not bind YscF resulted in greatly decreased HeLa cell cytotoxicity. Together, these results show that YscI participates in needle assembly by directly interacting with YscF.

Protein Acetylation Mediated by YfiQ and CobB Is Involved in the Virulence and Stress Response of Yersinia pestis.

Recent studies revealed that acetylation is a widely used protein modification in prokaryotic organisms. The major protein acetylation acetyltransferase YfiQ and the sirtuin-like deacetylase CobB have been found to be involved in basic physiological processes, such as primary metabolism, chemotaxis, and stress responses, in Escherichia coli and Salmonella However, little is known about protein acetylation modifications in Yersinia pestis, a lethal pathogen responsible for millions of human deaths in three worldwide pandemics. Here we found that Yp_0659 and Yp_1760 of Y. pestis encode the major protein acetylation acetyltransferase YfiQ and the sirtuin-like deacetylase CobB, respectively, which can acetylate and deacetylate PhoP enzymatically in vitro Protein acetylation impairment in cobB and yfiQ mutants greatly decreased bacterial tolerance to cold, hot, high-salt, and acidic environments. Our comparative transcriptomic data revealed that the strongly decreased tolerance to stress stimuli was probably related to downregulation of the genes encoding the heat shock proteins (HtpG, HslV, HslR, and IbpA), cold shock proteins (CspC and CspA1), and acid resistance proteins (HdeB and AdiA). We found that the reversible acetylation mediated by CobB and YfiQ conferred attenuation of virulence, probably partially due to the decreased expression of the psaABCDEF operon, which encodes Psa fimbriae that play a key role in virulence of Y. pestis This is the first report, to our knowledge, on the roles of protein acetylation modification in stress responses, biofilm formation, and virulence of Y. pestis.

Prevalence and associated knowledge of hepatitis B infection among healthcare workers in Freetown, Sierra Leone.

BACKGROUND: Hepatitis B virus (HBV) is considered highly prevalent in West Africa. However, major gaps in surveillance exist in Sierra Leone. Although healthcare workers (HCWs) are at high risk for HBV infection, little is known about the prevalence and knowledge of hepatitis B among HCWs in Sierra Leone. METHODS: A cross-sectional study of all HCWs at the No. 34 Military Hospital located in Freetown, Sierra Leone, was conducted from March 20 to April 10, 2017. Whole blood was collected and screened for HBV markers using a one-step rapid immunochromatographic test with positive samples tested for HBV DNA. Additionally, questionnaires assessing self-reported knowledge of HBV infections were administered to all participants. Data were processed and analyzed using SPSS (version 17.0) software. RESULTS: A total of 211 HCWs were included in this study with a median age of 39.0 years (range: 18-59). Of the participating HCWs, 172 (81.5%) participants were susceptible (all markers negative), 21(10.0%) were current HBV (HBsAg positive) and nine (4.3%) were considered immune because of past infection (HBsAg negative and anti-HBc positive; anti-HBs positive). Additionally, nine (4.3%) participants displayed immunity to the virus as a result of prior hepatitis B vaccination (only anti-HBs positive). Of the 21 HCWs with positive HBsAg, 13 (61.9%) had detectable HBV DNA. There was a significantly lower risk for current HBV infection among HCWs older than 39 years (OR 0.337, p = 0.046). In addition, only 14 (6.6%), 73 (34.6%) and 82 (38.9%) participants in this survey had adequate knowledge about the clinical outcome, routes of transmission, and correct preventive measures of HBV infection, respectively. CONCLUSIONS: HCWs in Sierra Leone lacked adequate knowledge of the hepatitis B virus. Additionally, the low coverage rate of hepatitis B vaccination among HCWs fails to meet WHO recommendations, leaving many of the sampled HCWs susceptible to infection. This study reaffirms the need for more intensive training for HCWs in addition to strengthening vaccination programmes to protect HCWs against HBV in Sierra Leone.

Yersinia pestis YopK Inhibits Bacterial Adhesion to Host Cells by Binding to the Extracellular Matrix Adaptor Protein Matrilin-2.

Pathogenic yersiniae harbor a type III secretion system (T3SS) that injects Yersinia outer protein (Yop) into host cells. YopK has been shown to control Yop translocation and prevent inflammasome recognition of the T3SS by the innate immune system. Here, we demonstrate that YopK inhibits bacterial adherence to host cells by binding to the extracellular matrix adaptor protein matrilin-2 (MATN2). YopK binds to MATN2, and deleting amino acids 91 to 124 disrupts binding of YopK to MATN2. A yopK null mutant exhibits a hyperadhesive phenotype, which could be responsible for the established Yop hypertranslocation phenotype of yopK mutants. Expression of YopK, but not YopKΔ91-124, in a yopK mutant restored the wild-type phenotypes of adhesion and Yop translocation, suggesting that binding to MATN2 might be essential for YopK to inhibit bacterial adhesion and negatively regulate Yop translocation. A green fluorescent protein (GFP)-YopK fusion specifically binds to the endogenous MATN2 on the surface of HeLa cells, whereas GFP-YopKΔ91-124 cannot. Addition of purified YopK protein during infection decreased adhesion of Y. pestis to HeLa cells, while YopKΔ91-124 protein showed no effect. Taking these results together, we propose a model that the T3SS-secreted YopK hinders bacterial adhesion to HeLa cells by binding to MATN2, which is ubiquitously exposed on eukaryotic cells.

Epidemic Clones, Oceanic Gene Pools, and Eco-LD in the Free Living Marine Pathogen Vibrio parahaemolyticus.

We investigated global patterns of variation in 157 whole-genome sequences of Vibrio parahaemolyticus, a free-living and seafood associated marine bacterium. Pandemic clones, responsible for recent outbreaks of gastroenteritis in humans, have spread globally. However, there are oceanic gene pools, one located in the oceans surrounding Asia and another in the Mexican Gulf. Frequent recombination means that most isolates have acquired the genetic profile of their current location. We investigated the genetic structure in the Asian gene pool by calculating the effective population size in two different ways. Under standard neutral models, the two estimates should give similar answers but we found a 27-fold difference. We propose that this discrepancy is caused by the subdivision of the species into a hundred or more ecotypes which are maintained stably in the population. To investigate the genetic factors involved, we used 51 unrelated isolates to conduct a genome-wide scan for epistatically interacting loci. We found a single example of strong epistasis between distant genome regions. A majority of strains had a type VI secretion system associated with bacterial killing. The remaining strains had genes associated with biofilm formation and regulated by cyclic dimeric GMP signaling. All strains had one or other of the two systems and none of isolate had complete complements of both systems, although several strains had remnants. Further "top down" analysis of patterns of linkage disequilibrium within frequently recombining species will allow a detailed understanding of how selection acts to structure the pattern of variation within natural bacterial populations.

Yersinia protein kinase A phosphorylates vasodilator-stimulated phosphoprotein to modify the host cytoskeleton.

Pathogenic Yersinia species evolved a type III secretion system that injects a set of effectors into the host cell cytosol to promote infection. One of these effectors, Yersinia protein kinase A (YpkA), is a multidomain effector that harbours a Ser/Thr kinase domain and a guanine dissociation inhibitor (GDI) domain. The intercellular targets of the kinase and GDI domains of YpkA were identified to be Gαq and the small GTPases RhoA and Rac1, respectively, which synergistically induce cytotoxic effects on infected cells. In this study, we demonstrate that vasodilator-stimulated phosphoprotein (VASP), which is critical for regulation of actin assembly, cell adhesion and motility, is a direct substrate of YpkA kinase activity. Ectopic co-expression of YpkA and VASP in HEK293T cells leads to the phosphorylation of VASP at S157, and YpkA kinase activity is essential for VASP phosphorylation at this site. Moreover, YpkA directly phosphorylates VASP in in vitro kinase assay. YpkA-mediated VASP phosphorylation significantly inhibits actin polymerization and promotes the disruption of actin cytoskeleton, which inhibits the phagocytosis. Taken together, our study found a novel molecular mechanism used by YpkA to disrupt cytoskeleton dynamics, thereby promoting the anti-phagocytosis ability of pathogenic Yersiniae.

IL-17A produced by neutrophils protects against pneumonic plague through orchestrating IFN-γ-activated macrophage programming.

Innate immune cells, including neutrophils and macrophages, are critically involved in host antimicrobial defense responses. Intrinsic regulatory mechanisms controlling neutrophil and macrophage activities are poorly defined. In this study, we found that IL-17A, a natural signal factor, could provide protection against early pneumonic plague inflammation by coordinating the functions of neutrophils and programming of macrophages. The IL-17A level is promptly increased during the initial infection. Importantly, abrogation of IL-17A or IL-17AR significantly aggravated the infection, but mIL-17A treatment could significantly alleviate inflammatory injury, revealing that IL-17A is a critical requirement for early protection of infection. We also demonstrated that IL-17A was predominantly produced by CD11b(+)Ly6G(+) neutrophils. Although IL-17A could not significantly affect the antimicrobial responses of neutrophils, it could target the proinflammatory macrophage (M1) programming and potentiate the M1's defense against pneumonic plague. Mechanistically, IFN-γ treatment or IFN-γ-activated M1 macrophage transfer could significantly mitigate the aggravated infection of IL-17A(-/-) mice. Finally, we showed that IL-17A and IFN-γ could synergistically promote macrophage anti-infection immunity. Thus, our findings identify a previously unrecognized function of IL-17A as an intrinsic regulator in coordinating neutrophil and macrophage antimicrobial activity to provide protection against acute pneumonic plague.

Genome and Evolution of Yersinia pestis.

This chapter summarizes researches on genome and evolution features of Yersinia pestis, the young pathogen that evolved from Y. pseudotuberculosis at least 5000 years ago. Y. pestis is a highly clonal bacterial species with closed pan-genome. Comparative genomic analysis revealed that genome of Y. pestis experienced highly frequent rearrangement and genome decay events during the evolution. The genealogy of Y. pestis includes five major branches, and four of them seemed raised from a "big bang" node that is associated with the Black Death. Although whole genome-wide variation of Y. pestis reflected a neutral evolutionary process, the branch length in the genealogical tree revealed over dispersion, which was supposedly caused by varied historical molecular clock that is associated with demographical effect by alternate cycles of enzootic disease and epizootic disease in sylvatic plague foci. In recent years, palaeomicrobiology researches on victims of the Black Death, and Justinian's plague verified that two historical pandemics were indeed caused by Y. pestis, but the etiological lineages might be extinct today.

Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis.

The genetic diversity of Yersinia pestis, the etiologic agent of plague, is extremely limited because of its recent origin coupled with a slow clock rate. Here we identified 2,326 SNPs from 133 genomes of Y. pestis strains that were isolated in China and elsewhere. These SNPs define the genealogy of Y. pestis since its most recent common ancestor. All but 28 of these SNPs represented mutations that happened only once within the genealogy, and they were distributed essentially at random among individual genes. Only seven genes contained a significant excess of nonsynonymous SNP, suggesting that the fixation of SNPs mainly arises via neutral processes, such as genetic drift, rather than Darwinian selection. However, the rate of fixation varies dramatically over the genealogy: the number of SNPs accumulated by different lineages was highly variable and the genealogy contains multiple polytomies, one of which resulted in four branches near the time of the Black Death. We suggest that demographic changes can affect the speed of evolution in epidemic pathogens even in the absence of natural selection, and hypothesize that neutral SNPs are fixed rapidly during intermittent epidemics and outbreaks.

Ureteroscopy and cystoscopy training: comparison between transparent and non-transparent simulators.

BACKGROUND: Simulators have been widely used to train operational skills in urology, how to improve its effectiveness deserves further investigation. In this paper, we evaluated training using a novel transparent anatomic simulator, an opaque model or no simulator training, with regard to post-training ureteroscopy and cystoscopy proficiency. METHODS: Anatomically correct transparent and non-transparent endourological simulators were fabricated. Ten experienced urologists provided a preliminary evaluation of the models as teaching tools. 36 first-year medical students underwent identical theoretical training and a 50-point examination of theoretical knowledge. The students were randomly assigned to receive training with the transparent simulator (Group 1), the non-transparent simulator (Group 2) or detailed verbal instruction only (Group 3). 12 days after the training session, the trainees' skills at ureteral stent insertion and removal were evaluated using the Uro-Scopic Trainer and rated on an Objective Structured Assessment of Technical Skills (OSATS) scale. RESULTS: The new simulators were successfully fabricated in accordance with the design parameters. Of the ten urologists invited to evaluate the devices, 100% rated the devices as anatomically accurate, 90% thought both models were easy to use and 80% thought they were good ureteroscopy and cystoscopy training tools. The scores on the theoretical knowledge test were comparable among the training groups, and all students were able to perform ureteral stent insertion and removal. The mean OSATS scores of groups 1, 2 and 3 were 21.83 ± 3.64, 18.50 ± 4.03 and 15.58 ± 2.23 points, respectively, (p = 0.001). CONCLUSIONS: Simulator training allowed students to achieve higher ureteroscopic and cystoscopic proficiency, and transparent simulators were more effective than non-transparent simulators.

Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4.

An outbreak caused by Shiga-toxin­producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance.