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PDF file - 24K, Supplementary Figure 1. Complement system inhibition by CVF.

PDF file - 135K, Supplementary Figure 3. Effectiveness of NK cell depletion.

Although the role of the complement system in cancer development has been studied, its involvement in the development of an antitumoral immune response remains poorly understood. Using cobra venom factor (CVF) to inhibit the complement cascade via C3 molecule exhaustion in immunocompetent mice bearing B16gp33 melanoma tumors, we show that transient inhibition of the complement system allowed for the development of a more robust gp33-specific antitumoral CD8+ T-cell response. This immune response proved to be natural killer (NK) dependent, suggesting an interaction of complement proteins with this cellular subset leading to T lymphocyte activation and enhanced cytotoxic T-cell activity against tumor cells. This study demonstrates for the first time the implication of the complement system in the development of NK-mediated cytotoxic T-cell–dependent antitumoral immune responses. The complement pathway could therefore be a potent therapeutic target to improve NK-dependent antitumoral immune responses in patients with cancer. Cancer Immunol Res; 2(3); 200–6. ©2013 AACR.

PDF file - 26K, Supplemental Figure 2. MDSC proportions following CVF treatment.

PDF file - 86K

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infectingBorrelia burgdorferispirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derivedB. burgdorferisensu stricto (Bbss) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification ofBbssisolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectiousBbssisolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ∼800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm acrossBbssisolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Genome-scale analyses have revealed many transcription factor binding sites within, rather than upstream of genes, raising questions as to the function of these binding sites. Here, we use complementary approaches to map the regulon of theEscherichia colitranscription factor PhoB, a response regulator that controls transcription of genes involved in phosphate homeostasis. Strikingly, the majority of PhoB binding sites are located within genes, but these intragenic sites are not associated with detectable transcription regulation and are not evolutionarily conserved. Many intragenic PhoB sites are located in regions bound by H-NS, likely due to shared sequence preferences of PhoB and H-NS. However, these PhoB binding sites are not associated with transcription regulation even in the absence of H-NS. We propose that for many transcription factors, including PhoB, binding sites not associated with promoter sequences are transcriptionally inert, and hence are tolerated as genomic “noise”.IMPORTANCERecent studies have revealed large numbers of transcription factor binding sites within the genes of bacteria. The function, if any, of the vast majority of these binding sites has not been investigated. Here, we map the binding of the transcription factor PhoB across theEscherichia coligenome, revealing that the majority of PhoB binding sites are within genes. We show that PhoB binding sites within genes are not associated with regulation of the overlapping genes. Indeed, our data suggest that bacteria tolerate the presence of large numbers of non-regulatory, intragenic binding sites for transcription factors, and that these binding sites are not under selective pressure.

Nontuberculous mycobacteria (NTM) are environmental bacteria commonly found in soil and water in almost every part of the world. While usually non-pathogenic, they can cause acute respiratory and cutaneous infections under certain circumstances or in patients with underlying medical conditions. Contrary to members of the Mycobacterium tuberculosis complex, documented human-to-human transmissions of NTM have been rarely reported and most cases result from direct environmental exposure. Here we describe the identification of a new NTM species isolated from a hand laceration of a New York State patient after a fall. This new NTM forms rough, orange pigmented colonies and is naturally resistant to doxycycline and tobramycin. Whole genome analysis reveal no close relatives present in public databases, and our findings are in accordance with the recognition of a new taxonomic species of NTM. We propose the name Mycobacterium salfingeri sp. nov. for this new NTM representative. The type strain is 20-157661T (DSM = 113368T, BCCM = ITM 501207T).

Two types of autoimmune hepatitis (AIH) are recognized; AIH-1 is characterized by the presence of anti-nuclear and/or anti-smooth muscle autoantibodies, while AIH-2 is associated with the presence of anti-Liver kidney microsome and/or anti-Liver Cytosol antibodies. The autoantigens targeted by AIH-2 autoantibodies are the cytochrome P450 2D6 and Formiminotransferase-cyclodeaminase for anti-LKM1 and anti-LC1 respectively. Both autoantigens are expressed in hepatocytes at higher levels than in any other cell type. Therefore, compared to AIH-1, the autoantigens targeted in AIH-2 are predominantly tissue-specific. Distinct clinical features are specific to AIH-2 compared to AIH-1, including diagnosis in younger patients (mean age 6.6 years), onset as fulminant hepatitis in very young patients (3 years of age or less), higher frequency in children than in adults and is frequently associated with extrahepatic T cell-mediated autoimmune diseases. AIH-2 is also often diagnosed in patients with primary immunodeficiency. AIH-2 is associated with specific HLA class II susceptibility alleles; DQB1*0201 is considered the main determinant of susceptibility while DRB1*07/DRB1*03 is associated with the type of autoantibody present. HLA DQB1*0201 is in strong linkage disequilibrium with both HLA DRB1*03 and DRB1*07. Interestingly, as in humans, MHC and non-MHC genes strongly influence the development of the disease in an animal model of AIH-2. Altogether, these findings suggest that AIH-2 incidence is likely dependent on specific genetic susceptibility factors combined with distinct environmental triggers.

Immune checkpoint inhibitors (ICI) are being increasingly used to successfully treat several types of cancer. However, due to their mode of action, these treatments are associated with several immune-related adverse events (irAEs), including immune-mediated autoimmune-like hepatitis in 5 to 10% of cases. The specific immune mechanism responsible for the development of immune-mediated liver injury caused by immune checkpoint inhibitors (ILICI) is currently unknown. This review summarizes the current knowledge on hepatic irAEs during cancer immunotherapy. It also addresses the clinical management of ILICI and how it is becoming an increasingly important clinical issue. Clinical, histological, and laboratory features of autoimmune hepatitis (AIH) and ILICI are compared, and their shared and distinctive traits are discussed in an effort to better understand the development of hepatic irAEs. Finally, based on the current knowledge of liver immunology and AIH pathogenesis, we propose a series of events that could trigger the observed liver injury in ICI-treated patients. This model could be useful in the design of future studies aiming to identify the specific immune mechanism(s) at play in ILICI and improve immune checkpoint inhibitor cancer immunotherapy.

9 páginas, 4 figuras, 1 tabla., Background: Molecular diagnostics are considered the most promising route to achieving rapid, universal drug susceptibility testing for Mycobacterium tuberculosiscomplex (MTBC). We aimed to generate a WHO endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: A candidate gene approach was used to identify mutations as associated with resistance, or consistent with susceptibility, for 13 WHO endorsed anti-tuberculosis drugs. 38,215 MTBC isolates with paired whole-genome sequencing and phenotypic drug susceptibility testing data were amassed from 45 countries. For each mutation, a contingency table of binary phenotypes and presence or absence of the mutation computed positive predictive value, and Fisher's exact tests generated odds ratios and Benjamini-Hochberg corrected p-values. Mutations were graded as Associated with Resistance if present in at least 5 isolates, if the odds ratio was >1 with a statistically significant corrected p-value, and if the lower bound of the 95% confidence interval on the positive predictive value for phenotypic resistance was >25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: 15,667 associations were computed for 13,211 unique mutations linked to one or more drugs. 1,149/15,667 (7·3%) mutations were classified as associated with phenotypic resistance and 107/15,667 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was >80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were classified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: This first WHO endorsed catalogue of molecular targets for MTBC drug susceptibility testing provides a global standard for resistance interpretation. Its existence should encourage the implementation of molecular diagnostics by National Tuberculosis Programmes. Funding: UNITAID, Wellcome, MRC, BMGF., Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

During 2016-2017, three rabid terrestrial animals were discovered in the raccoon rabies virus-free zone of Long Island, New York, USA. Whole-genome sequencing and phylogenetic analyses revealed the likely origins of the viruses, enabling the rabies outbreak response (often costly and time-consuming) to be done less expensively and more efficiently.

In 2017, the New York State Department of Health investigated a large Klebsiella pneumoniae outbreak in a health care facility. A retrospective analysis was conducted to compare the use of multiple molecular typing methods for characterizing the outbreak. Forty-four isolates were characterized using the rapid real-time PCR OpGen Acuitas® AMR Gene Panel. Additionally, short-read whole genome sequencing (WGS) analysis was used to identify antimicrobial resistance (AMR) genes and assess isolate relatedness. Long-read Oxford Nanopore MinION WGS was used to characterize the plasmid content of a subset of isolates. All methods showed overall concordance, identifying four clusters, with a few discrepancies in the clustering of individual isolates. Though short- and long-read WGS results provided a more nuanced understanding of the molecular epidemiology of this outbreak, this study highlights the utility of the Acuitas® PCR-based approach, which can more easily be performed by health care facilities, for rapid clustering of patient isolates.

Liver ischemia/reperfusion injury (IRI) is an important cause of liver damage especially early after liver transplantation, following liver resection, and in other clinical situations. Using rat experimental models, we identified oxaloacetate (OAA) as a key metabolite able to protect hepatocytes from hypoxia and IRI. In vitro screening of metabolic intermediates beneficial for hepatocyte survival under hypoxia was performed by measures of cell death and injury. In vivo, the effect of OAA was evaluated using the left portal vein ligation (LPVL) model of liver ischemia and a model of warm IRI. Liver injury was evaluated in vivo by serum transaminase levels, liver histology, and liver weight (edema). Levels and activity of caspase 3 were also measured. In vitro, the addition of OAA to hepatocytes kept in a hypoxic environment significantly improved cell viability (P < 0.01), decreased cell injury (P < 0.01), and improved energy metabolism (P < 0.01). Administration of OAA significantly reduced the extent of liver injury in the LPVL model with lower levels of alanine aminotransferase (ALT; P < 0.01), aspartate aminotransferase (AST; P < 0.01), and reduced liver necrosis (P < 0.05). When tested in a warm IRI model, OAA significantly decreased ALT (P < 0.001) and AST levels (P < 0.001), prevented liver edema (P < 0.001), significantly decreased caspase 3 expression (P < 0.01), as well as histological signs of cellular vesiculation and vacuolation (P < 0.05). This was associated with higher adenosine triphosphate (P < 0.05) and energy charge levels (P < 0.01). In conclusion, OAA can significantly improve survival of ischemic hepatocytes. The hepatoprotective effect of OAA was associated with increased levels of liver bioenergetics both in vitro and in vivo. These results suggest that it is possible to support mitochondrial activity despite the presence of ischemia and that OAA can effectively reduce ischemia-induced injury in the liver.

Background Transmission of pathogens by vector mosquitoes is intrinsically linked with mosquito’s reproductive strategy because anautogenous mosquitoes require vertebrate blood to develop a batch of eggs. Each cycle of egg maturation is tightly linked with the intake of a fresh blood meal for most species. Mosquitoes that acquire pathogens during the first blood feeding can transmit the pathogens to susceptible hosts during subsequent blood feeding and also vertically to the next generation via infected eggs. Large-scale gene-expression changes occur following each blood meal in various tissues, including ovaries. Here we analyzed mosquito ovary transcriptome following a blood meal at three different time points to investigate blood-meal induced changes in gene expression in mosquito ovaries. Results We collected ovaries from Aedes aegypti that received a sugar meal or a blood meal on days 3, 10 and 20 post blood meal for transcriptome analysis. Over 4000 genes responded differentially following ingestion of a blood meal on day 3, and 660 and 780 genes on days 10 and 20, respectively. Proteins encoded by differentially expressed genes (DEGs) on day 3 include odorant binding proteins (OBPs), defense-specific proteins, and cytochrome P450 detoxification enzymes. In addition, we identified 580 long non-coding RNAs that are differentially expressed at three time points. Gene ontology analysis indicated that genes involved in peptidase activity, oxidoreductase activity, extracellular space, and hydrolase activity, among others were enriched on day 3. Although most of the DEGs returned to the nonsignificant level compared to the sugar-fed mosquito ovaries following oviposition on days 10 and 20, there remained differences in the gene expression pattern in sugar-fed and blood-fed mosquitoes. Conclusions Enrichment of OBPs following blood meal ingestion suggests that these genes may have other functions besides being part of the olfactory system. The enrichment of immune-specific genes and cytochrome P450 genes indicates that ovaries become well prepared to protect their germ line from any pathogens that may accompany the blood meal or from environmental contamination during oviposition, and to deal with the detrimental effects of toxic metabolites.

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multi-drug resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of rpoB mutations which do not confer high levels of RIF resistance as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF minimum inhibitory concentrations (MICs) compared to fully susceptible strains, however remain phenotypically susceptible by mycobacteria growth indicator tube (MGIT) testing and have been associated with poor patient outcomes. Here we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 21/2-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 μg/mL in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT, however were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial-gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.

Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays., Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis. To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis. This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulcerans.

Next-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnostic and outbreak investigation in public health laboratories. However, costs of operation and the need for specialized staff remain major hurdles for laboratories with limited resources for implementing these technologies. This project aimed to assess the feasibility of using Oxford Nanopore MinION whole-genome sequencing data of Mycobacterium tuberculosis isolates for species identification, in silico spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases. The results were compared prospectively in real time to those obtained with our current clinically validated Illumina MiSeq sequencing assay for M. tuberculosis and phenotypic drug susceptibility testing results when available. Our assessment of 431 sequenced samples over a 32-week period demonstrates that, when using the proper quality controls and thresholds, the MinION can achieve levels of genotyping analysis and phenotypic resistance predictions comparable to those of the Illumina MiSeq at a very competitive cost per sample. Our results indicate that nanopore sequencing can be a suitable alternative to, or complement, currently used sequencing platforms in a clinical setting and has the potential to be widely adopted in public health laboratories in the near future.

Carbapenemase-producing Enterobacteriaceae are a major threat to global public health. Klebsiella pneumoniae carbapenemase (KPC) is the most commonly identified carbapenemase in the United States and is frequently found on mobile genetic elements including plasmids, which can be horizontally transmitted between bacteria of the same or different species. Here we describe the results of an epidemiological investigation of KPC-producing bacteria at two healthcare facilities. Using a combination of short-read and long-read whole-genome sequencing, we identified an identical 44 kilobase plasmid carrying the blaKPC–2 gene in four bacterial isolates belonging to three different species (Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli). The isolates in this investigation were collected from patients who were epidemiologically linked in a region in which KPC was uncommon, suggesting that the antibiotic resistance plasmid was transmitted between these bacterial species. This investigation highlights the importance of long-read sequencing in investigating the relatedness of bacterial plasmids, and in elucidating potential plasmid-mediated outbreaks caused by antibiotic resistant bacteria.

Legionnaires’ disease, a severe lung infection caused by the bacterium Legionella pneumophila, occurs as single cases or in outbreaks that are actively tracked by public health departments. To determine the point source of an outbreak, clinical isolates need to be compared to environmental samples to find matching isolates. One confounding factor is the genome plasticity of L. pneumophila, making an exact sequence comparison by whole-genome sequencing (WGS) challenging. Here, we present a WGS analysis pipeline, LegioCluster, that is designed to circumvent this problem by automatically selecting the best matching reference genome prior to mapping and variant calling. This approach reduces the number of false-positive variant calls, maximizes the fraction of all genomes that are being compared, and naturally clusters the isolates according to their reference strain. Isolates that are too distant from any genome in the database are added to the list of candidate references, thereby creating a new cluster. Short insertions or deletions are considered in addition to single-nucleotide polymorphisms for increased discriminatory power. This manuscript describes the use of this automated and “locked down” bioinformatic pipeline deployed at the New York State Department of Health’s Wadsworth Center for investigating relatedness between clinical and environmental isolates. A similar pipeline has not been widely available for use to support these critically important public health investigations.

Whole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterize Mycobacterium tuberculosis and other M. tuberculosis complex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, paired-end WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptible-predictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.

Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and comprehensive results at a cost that is advantageous compared with traditional diagnostic methods. Sequencing pathogens directly from a primary clinical specimen would help circumvent the need for culture and, in the process, substantially shorten the time to diagnosis and public health reporting. Unfortunately, this approach poses significant challenges because of the mixture of multiple sequences from a complex fecal biomass. The aim of this project was to develop a proof of concept protocol for the sequencing and genotyping of Shiga toxin-producing Escherichia coli (STEC) directly from stool specimens. We have developed an enrichment protocol that reliably achieves a substantially higher DNA yield belonging to E. coli, which provides adequate next-generation sequencing (NGS) data for downstream bioinformatics analysis. A custom bioinformatics pipeline was created to optimize and remove non-E. coli reads, assess the STEC versus commensal E. coli population in the samples, and build consensus sequences based on population allele frequency distributions. Side-by-side analysis of WGS from paired STEC isolates and matched primary stool specimens reveal that this method can reliably be implemented for many clinical specimens to directly genotype STEC and accurately identify clusters of disease outbreak when no STEC isolate is available for testing.

Mycobacteria encompass several slow-growing pathogens, including organisms that cause leprosy and tuberculosis. Mycobacteria use a component of their ESX (or Type VII) secretion system for a distinctive form of genetic exchange called distributive conjugal DNA transfer. Gray et al. investigated a quicker-growing model species, Mycobacterium smegmatis. They found that the secretory apparatus, ESX-4, is essential for DNA transfer into the recipient but is not required for donor cells to pass along their DNA. The ESX-1 secretory apparatus was required in the recipient for ESX-4 induction, but it did not appear to provide the physical channel for DNA. Rather, ESX-1 may secrete cell-surface “mating factor” receptors. More research will be needed to understand the details of this intriguing means of DNA exchange in mycobacteria. Science , this issue p. [347][1] [1]: /lookup/doi/10.1126/science.aag0828

Summary Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti-smooth muscle actin and/or anti-nuclear, anti-liver kidney microsomal type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)-DR3, -DR7 and -DR13. HLA-DR4 has the second strongest association with adult AIH, after HLA-DR3. We investigated the role of HLA-DR4 in the development of AIH by immunization of HLA-DR4 (DR4) transgenic non-obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti-LKM1/anti-LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (Tregs), which had decreased programmed death (PD)-1 expression. Splenic Tregs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8+ T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild-type (WT) NOD mice. Our results demonstrate that HLA-DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of Tregs and reduced PD-1 expression may result in spontaneous activation of key immune cell subsets, such as antigen-presenting cells and CD8+ T effectors, facilitating the induction of AIH and persistent liver damage.

A total of 30 Legionella pneumophila serogroup 1 isolates representing 10 separate legionellosis laboratory investigations (“outbreaks”) that occurred in New York State between 2004 and 2012 were selected for evaluation of whole-genome sequencing (WGS) approaches for molecular subtyping of this organism. Clinical and environmental isolates were available for each outbreak and were initially examined by pulsed-field gel electrophoresis (PFGE). Sequence-based typing alleles were extracted from WGS data yielding complete sequence types (ST) for isolates representing 8 out of the 10 outbreaks evaluated in this study. Isolates from separate outbreaks sharing the same ST also contained the fewest differences in core genome single nucleotide polymorphisms (SNPs) and the greatest proportion of identical allele sequences in a whole-genome multilocus sequence typing (wgMLST) scheme. Both core SNP and wgMLST analyses distinguished isolates from separate outbreaks, including those from two outbreaks sharing indistinguishable PFGE profiles. Isolates from a hospital-associated outbreak spanning multiple years shared indistinguishable PFGE profiles but displayed differences in their genome sequences, suggesting the presence of multiple environmental sources. Finally, the rtx gene demonstrated differences in the repeat region sequence among ST1 isolates from different outbreaks, suggesting that variation in this gene may be useful for targeted molecular subtyping approaches for L. pneumophila . This study demonstrates the utility of various genome sequence analysis approaches for L. pneumophila for environmental source attribution studies while furthering the understanding of Legionella ecology. IMPORTANCE We demonstrate that whole-genome sequencing helps to improve resolution of Legionella pneumophila isolated during laboratory investigations of legionellosis compared to traditional subtyping methods. These data can be important in confirming the environmental sources of legionellosis outbreaks. Moreover, we evaluated various methods to analyze genome sequence data to help resolve outbreak-related isolates.

We report a case of lymphadenitis caused by Mycobacterium orygis in an immunocompetent person in Stony Brook, New York, USA. Initial real-time PCR assay failed to provide a final subspecies identification within the M. tuberculosis complex, but whole-genome sequencing characterized the isolate as M. orygis.

We investigated the value of whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses in determining the relationships among and evolutionary rates of Legionella species with long-term persistence in three healthcare facilities. We examined retrospective clinical and environmental isolates of Legionella micdadei and Legionella pneumophila serogroup 1 isolates with identical PFGE DNA fingerprints sampled over the course of up to 18 years. WGS analyses demonstrated that heterogeneous populations of Legionella were present within each facility despite displaying the same PFGE profiles. Additionally, clustering of some clinical isolates with those from a separate but related institution exposed a source of infection not previously detected, underscoring the importance of considering phylogenetic relationships when assessing epidemiological links. The data supported an average substitution rate of 0.80 SNPs per genome per year for L. micdadei but a reliable estimate for L. pneumophila serogroup 1 could not be obtained due to complicating factors such as non-chronological links among isolates and inadequate sampling depths. While the substitution rate for L. micdadei is consistent with previous estimates for L. pneumophila, the lack of a temporal signal in our sequence data for L. pneuomphila serogroup 1 isolates suggests either insufficient change to provide an estimate or variable evolutionary rates, which could reflect the presence of both actively dividing and viable but non-culturable Legionella spp. in the built environment. This study highlights the increased discriminatory power of WGS SNP analysis as compared to PFGE, emphasizes the need for extended sampling, and provides insight into the evolution of Legionella from longitudinal investigations.

Hepatocellular carcinoma (HCC) is a metabolically heterogeneous cancer and the use of glucose by HCC cells could impact their tumorigenicity. Dt81Hepa1-6 cells display enhanced tumorigenicity compared to parental Hepa1-6 cells. This increased tumorigenicity could be explained by a metabolic adaptation to more restrictive microenvironments. When cultured at high glucose concentrations, Dt81Hepa1-6 displayed an increased ability to uptake glucose (P

HCC (Hepatocellular carcinoma) cells exhibit greater metabolic plasticity than normal hepatocytes since they must survive in a dynamic microenvironment where nutrients and oxygen are often scarce. Using a metabolomic approach combined with functional in vitro and in vivo assays, we aimed to identify an HCC metabolic signature associated with increased tumorigenicity and patient mortality. Metabolite profiling of HCC Dt81Hepa1-6 cells revealed that their increased tumorigenicity was associated with elevated levels of glycolytic metabolites. Tumorigenic Dt81Hepa1-6 also had an increased ability to uptake glucose leading to a higher glycolytic flux that stemmed from an increased expression of glucose transporter GLUT-1. Dt81Hepa1-6-derived tumors displayed increased mRNA expressions of glycolytic genes, Hypoxia-inducible factor-1alpha and of Cyclin D1. HCC tumors also displayed increased energy charge, reduced antioxidative metabolites and similar fatty acid biosynthesis compared to healthy liver. Increased tumoral expression of glycolytic and hypoxia signaling pathway mRNAs was associated with decreased survival in HCC patients. In conclusion, HCC cells can rapidly alter their metabolism according to their environment and switch to the use of glucose through aerobic glycolysis to sustain their tumorigenicity and proliferative ability. Therefore, cancer metabolic reprogramming could be essential for the tumorigenicity of HCC cells during cancer initiation and invasion.

Recent work has revealed that large numbers of promoters in bacteria are located inside genes. In contrast, almost all studies of transcription have focused on promoters upstream of genes. Bacterial promoters are recognized by Sigma factors that associate with initiating RNA polymerase. In Escherichia coli, one Sigma factor recognizes the majority of promoters, and six “alternative” Sigma factors recognize specific subsets of promoters. One of these alternative Sigma factors, FliA (σ28), recognizes promoters upstream of many flagellar genes. We previously showed that most E. coli FliA binding sites are located inside genes. However, it was unclear whether these intragenic binding sites represent active promoters. Here, we construct and assay transcriptional promoter-lacZ fusions for all 52 putative FliA promoters previously identified by ChIP-seq. These experiments, coupled with integrative analysis of published genome-scale transcriptional datasets, reveal that most intragenic FliA binding sites are active promoters that transcribe highly unstable RNAs. Additionally, we show that widespread intragenic FliA-dependent transcription is a conserved phenomenon, but that the specific promoters are not themselves conserved. We conclude that intragenic FliA-dependent promoters and the resulting RNAs are unlikely to have important regulatory functions. Nonetheless, one intragenic FliA promoter is broadly conserved, and constrains evolution of the overlapping protein-coding gene. Thus, our data indicate that intragenic regulatory elements can influence protein evolution in bacteria, and suggest that the impact of intragenic regulatory sequences on genome evolution should be considered more broadly.AUTHOR SUMMARYRecent genome-scale studies of bacterial transcription have revealed thousands of promoters inside genes. In a few cases, these promoters have been shown to transcribe functional RNAs. However, it is unclear whether most intragenic promoters have important biological function. Similarly, there are likely to be thousands of intragenic binding sites for transcription factors, but very few have been functionally characterized. Moreover, it is unclear what impact intragenic promoters and transcription factor binding sites have on evolution of the overlapping genes. In this study, we focus on FliA, a broadly conserved Sigma factor that is responsible for initiating transcription of many flagellar genes. We previously showed that FliA directs RNA polymerase to ~50 genomic sites in Escherichia coli. In our current study, we show that while most intragenic FliA promoters are actively transcribed, very few are conserved in other species. This suggests that most FliA promoters are not functional. Nonetheless, one intragenic FliA promoter is highly conserved, and we show that this promoter constrains evolution of the overlapping protein-coding gene. Given the enormous number of regulatory DNA sites within genes, we propose that the evolution of many genes is constrained by these elements.

Salmonella enterica serovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis of S . Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmental S . Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of the S . Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method for S . Enteritidis.

Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease characterized by interface hepatitis, the presence of circulating autoantibodies, and hyper-gammaglobulinemia. There are two types of AIH, type 1 (AIH-1) and type 2 (AIH-2), characterized by distinct autoimmune serology. Patients with AIH-1 are positive for anti–smooth muscle and/or antinuclear autoantibodies, whereas patients with AIH-2 have anti–liver kidney microsomal type 1 and/or anti–liver cytosol type 1 autoantibodies. Cytochrome P4502D6 is the antigenic target of anti–liver kidney microsomal type 1, and formiminotransferase cyclodeaminase is the antigenic target of anti–liver cytosol type 1. It is known that AIH, both types 1 and 2, is strongly linked to the human leukocyte antigen (HLA) alleles -DR3, -DR4, and -DR7. However, direct evidence of the association of HLA with AIH is lacking. We developed a novel mouse model of AIH using the HLA-DR3 transgenic mouse on the nonobese-diabetic background by immunization of HLA-DR3– and HLA-DR3+ nonobese-diabetic mice with a DNA plasmid, coding for human cytochrome P4502D6/formiminotransferase cyclodeaminase fusion protein. Immunization with cytochrome P4502D6/formiminotransferase cyclodeaminase leads to a sustained elevation of alanine aminotransferase, development of antinuclear autoantibodies and anti–liver kidney microsomal type 1/anti–liver cytosol type 1 autoantibodies, chronic immune cell infiltration, and parenchymal fibrosis on liver histology in HLA-DR3+ mice. Immunized mice also showed an enhanced T helper 1 immune response and paucity of the frequency of regulatory T cells in the liver. Moreover, HLA-DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion: Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. (Hepatology 2015;62:1536–1550)

During the summer of 2015, New York, New York, USA, had one of the largest and deadliest outbreaks of Legionnaires’ disease in the history of the United States. A total of 138 cases and 16 deaths were linked to a single cooling tower in the South Bronx. Analysis of environmental samples and clinical isolates showed that sporadic cases of legionellosis before, during, and after the outbreak could be traced to a slowly evolving, single-ancestor strain. Detection of an ostensibly virulent Legionella strain endemic to the Bronx community suggests potential risk for future cases of legionellosis in the area. The genetic homogeneity of the Legionella population in this area might complicate investigations and interpretations of future outbreaks of Legionnaires’ disease.

Objectives:Infections caused by Legionella are the leading cause of waterborne disease outbreaks in the United States. We investigated a large outbreak of Legionnaires’ disease in New York City in summer 2015 to characterize patients, risk factors for mortality, and environmental exposures.Methods:We defined cases as patients with pneumonia and laboratory evidence of Legionella infection from July 2 through August 3, 2015, and with a history of residing in or visiting 1 of several South Bronx neighborhoods of New York City. We describe the epidemiologic, environmental, and laboratory investigation that identified the source of the outbreak.Results:We identified 138 patients with outbreak-related Legionnaires’ disease, 16 of whom died. The median age of patients was 55. A total of 107 patients had a chronic health condition, including 43 with diabetes, 40 with alcoholism, and 24 with HIV infection. We tested 55 cooling towers for Legionella, and 2 had a strain indistinguishable by pulsed-field gel electrophoresis from 26 patient isolates. Whole-genome sequencing and epidemiologic evidence implicated 1 cooling tower as the source of the outbreak.Conclusions:A large outbreak of Legionnaires’ disease caused by a cooling tower occurred in a medically vulnerable community. The outbreak prompted enactment of a new city law on the operation and maintenance of cooling towers. Ongoing surveillance and evaluation of cooling tower process controls will determine if the new law reduces the incidence of Legionnaires’ disease in New York City.

Autoimmune hepatitis (AIH) is a multifactorial autoimmune disease of unknown pathogenesis, characterized by a loss of immunological tolerance against liver autoantigens resulting in the progressive destruction of the hepatic parenchyma. Current treatments are based on non-specific immunosuppressive drugs. Although tremendous progress has been made using specific biological agents in other inflammatory diseases, progress has been slow to come for autoimmune hepatitis patients. While current treatments are successful in the majority of patients, treatment discontinuation is difficult to achieve and relapses are frequent. Lifelong immunosuppression is not without risks, especially in the pediatric population; 4% of patient with type 1 AIH will eventually develop hepatocellular carcinoma with a 2.9% probability after 10 years of treatment. Therefore, future treatments should aim to restore tolerance to hepatic autoantigens and induce long-term remission. Promising new immunotherapies have been tested in experimental models of autoimmune hepatitis including T and B cell depletion and regulatory CD4+ T cells infusion. Clinical studies on limited numbers of patients have also shown encouraging results using B-cell depleting (Rituximab) and anti-TNF-alpha (Infliximab) antibodies. A better understanding of key molecular targets in AIH combined with effective site-specific immunotherapies could lead to long-term remission without blanket immunosuppression and with minimal deleterious side effects.

There are limited numbers of models to study hepatocellular carcinoma (HCC) in vivo in immunocompetent hosts. In an effort to develop a cell line with improved tumorigenicity, we derived a new cell line from Hepa1-6 cells through an in vivo passage in C57BL/6 mice. The resulting Dt81Hepa1-6 cell line showed enhanced tumorigenicity compared to Hepa1-6 with more frequent (28±12 vs. 0±0 lesions at 21 days) and more rapid tumor development (21 (100%) vs. 70 days (10%)) in C57BL/6 mice. The minimal Dt81Hepa1-6 cell number required to obtain visible tumors was 100,000 cells. The Dt81Hepa1-6 cell line showed high hepatotropism with subcutaneous injection leading to liver tumors without development of tumors in lungs or spleen. In vitro, Dt81Hepa1-6 cells showed increased anchorage-independent growth (34.7±6.8 vs. 12.3±3.3 colonies; P

International audience; Cancer therapy using oncolytic viruses has gained interest in the last decade. Vesicular stomatitis virus is an attractive candidate for this alternative treatment approach. The importance of the immune response against tumor antigens in virotherapy efficacy is now well recognized, however, its relative contribution versus the intrinsic oncolytic capacity of viruses has been difficult to evaluate. To start addressing this question, we compared glycoprotein and matrix mutants of vesicular stomatitis virus (VSV), showing different oncolytic potentials for B16/B16gp33 melanoma tumor cells in vitro, with the wild-type virus in their ability to induce tumor-specific CD8(+) T cell responses and control tumor progression in vivo. Despite the fact that wild-type and G mutants induced a stronger gp33-specific immune response compared to the MM51R mutant, all VSV strains showed a similar capacity to slow down tumor progression. The effectiveness of the matrix mutant treatment proved to be CD8(+) dependent and directed against tumor antigens other than gp33 since adoptive transfer of isolated CD8(+) T lymphocytes from treated B16gp33-bearing mice resulted in significant protection of naive mice against challenge with the parental tumor. Remarkably, the VSV matrix mutant induced the upregulation of major histocompatibility class-I antigen at the tumor cell surface thus favoring recognition by CD8(+) T cells. These results demonstrate that VSV mutants induce an antitumor immune response using several mechanisms. A better understanding of these mechanisms will prove useful for the rational design of viruses with improved therapeutic efficacy.

Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. Here, we identify an additional cellular function for the Nus factor complex in Escherichia coli: repression of the Nus factor-encoding gene, suhB. This repression occurs primarily by translation inhibition, followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Conservation of putative NusB/E binding sites upstream of Nus factor genes suggests that Nus factor autoregulation occurs in many bacterial species. Additionally, many putative NusB/E binding sites are also found upstream of other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory., The Nus factor complex regulates rRNA folding and prevents Rho-dependent transcription termination in bacteria. Here, Baniulyte et al. show that it also inhibits translation of one of the Nus factor-encoding genes, suhB, and probably regulates the expression of other genes in diverse bacterial species.

International audience; Elicitation of type I interferon (IFN-I) has been shown to both enhance and impair cell-mediated immune responses in acute and persistent viral infections, respectively. Here, we show that, in addition to its effect on T cells, IFN-I drives impairment of specific antibody responses through interaction with B cells in the acute phase of lymphocytic choriomeningitis virus (LCMV) infection. This impairment was limited to the T cell-dependent B cell response and was associated with disruption of B cell follicles, development of hypergammaglobulinemia (HGG), and expansion of the T follicular helper cell population. Antigen-specific antibody responses were restored by ablation of IFN-I signaling through antibody-mediated IFN-I receptor blockade and B cell-specific IFN-I receptor knockout. Importantly, IFN-I receptor deficiency in B cells also accelerated the development of LCMV neutralizing antibodies and alleviated HGG. These results provide a potential therapeutic target toward efficient treatment measures that limit immunopathology in persistent viral infections.

We report here the complete genome sequences of three Legionella pneumophila isolates that are associated with a Legionnaires' disease outbreak in New York in 2012. Two clinical isolates (D7630 and D7632) and one environmental isolate (D7631) were recovered from this outbreak. A single isolate-specific virulence gene was found in D7632. These isolates were included in a large study evaluating the genomic resolution of various bioinformatics approaches for L. pneumophila serogroup 1 isolates.

Here, we report the release of a draft genome assembly of a Gram-positive cocci Branchiibius sp. NY16-3462-2 with a high-GC content, sequenced from a mixed clinical sample containing Mycobacterium tuberculosis . This genome is the first publicly available sequence from a representative of the genus Branchiibius .

Collagen produced during the process of liver fibrosis can induce a hepatocellular protective response through ERK1 signalling. However, the influence of T cells and associated cytokine production on this protection is unknown. In addition, athymic mice are frequently used in hepatocellular carcinoma xenograft experiments but current methods limit our ability to study the impact of liver fibrosis in this setting due to high mortality. Therefore, a mouse model of liver fibrosis lacking T cells was developed using Foxn1 nu/nu mice and progressive oral administration of thioacetamide (TAA) [0.01-0.02%] in drinking water. Fibrosis developed over a period of 16 weeks (alpha-SMA positive area: 20.0 ± 2.2%, preCol1a1 mRNA expression: 11.7 ± 4.1 fold changes, hydroxyproline content: 1041.2 ± 77μg/g of liver) at levels comparable to that of BALB/c mice that received intraperitoneal TAA injections [200 μg/g of body weight (bw)] (alpha-SMA positive area: 20.9 ± 2.9%, preCol1a1 mRNA expression: 13.1 ± 2.3 fold changes, hydroxyproline content: 931.6 ± 14.8μg/g of liver). No mortality was observed. Athymic mice showed phosphorylation of ERK1/2 during fibrogenesis (control 0.03 ± 0.01 vs 16 weeks 0.22 ± 0.06AU; P

Similar to human immunodeficiency virus (HIV)-1, vaccine-induced elicitation of broadly neutralizing (bNt) antibodies (Abs) is gaining traction as a key goal toward the eradication of the hepatitis C virus (HCV) pandemic. Previously, the significance of the Ab response against HCV was underappreciated given the prevailing evidence advancing the role of the cellular immune response in clearance and overall control of the infection. However, recent findings have driven growing interest in the humoral arm of the immune response and in particular the role of bNt responses due to their ability to confer protective immunity upon passive transfer in animal models. Nevertheless, the origin and development of bNt Abs is poorly understood and their occurrence is rare as well as delayed with emergence only observed in the chronic phase of infection. In this review, we characterize the interplay between the host immune response and HCV as it progresses from the acute to chronic phase of infection. In addition, we place these events in the context of current hypotheses on the origin of bNt Abs against the HIV-1, whose humoral immune response is better characterized. Based on the increasing significance of the humoral immune response against HCV, characterization of these events may be critical in understanding the development of the bNt responses and, thus, provide strategies toward effective vaccine design.

Supermatrix and supertree analyses are frequently used to more accurately recover vertical evolutionary history but debate still exists over which method provides greater reliability. Traditional methods that resolve relationships among organisms from single genes are often unreliable because of the frequent lack of strong phylogenetic signal and the presence of systematic artifacts. Methods developed to reconstruct organismal history from multiple genes can be divided into supermatrix and supertree approaches. A supermatrix analysis consists of the concatenation of multiple genes into a single, possibly partitioned alignment, from which phylogenies are reconstructed using a variety of approaches. Supertrees build consensus trees from the topological information contained within individual gene trees. Both methods are now widely used and have been demonstrated to solve previously ambiguous or unresolved phylogenies with high statistical support. However, the amount of misleading signal needed to induce erroneous phylogenies for both strategies is still unknown. Using genome simulations, we test the accuracy of supertree and supermatrix approaches in recovering the true organismal phylogeny under increased amounts of horizontally transferred genes and changes in substitution rates. Our results show that overall, supermatrix approaches are preferable when a low amount of gene transfer is suspected to be present in the dataset, while supertrees have greater reliability in the presence of a moderate amount of misleading gene transfers. In the face of very high or very low substitution rates without horizontal gene transfers, supermatrix approaches outperform supertrees as individual gene trees remain unresolved and additional sequences contribute to a congruent phylogenetic signal.

Summary Speciation of sexually reproducing organisms requires reproductive barriers. Prokaryotes reproduce asexually but often exchange DNA by lateral gene transfer mechanisms and recombination [1], yet distinct lineages are still observed. Thus, barriers to gene flow such as geographic isolation, genetic incompatibility or a physiological inability to transfer DNA represent potential underlying mechanisms behind preferred exchange groups observed in prokaryotes [2–6]. In Bacteria, experimental evidence showed that sequence divergence impedes homologous recombination between bacterial species [7–11]. Here we study interspecies gene exchange in halophilic archaea that possess a parasexual mechanism of genetic exchange that is functional between species [12, 13]. In this process, cells fuse forming a diploid state containing the full genetic repertoire of both parental cells, which facilitates genetic exchange and recombination. Later, cells separate, occasionally resulting in hybrids of the parental strains [14]. We show high recombination frequencies between Haloferax volcanii and Haloferax mediterranei , two species that have an average nucleotide sequence identity of 86.6%. Whole genome sequencing of Haloferax interspecies hybrids revealed the exchange of chromosomal fragments ranging from 310Kb to 530Kb. These results show that recombination barriers may be more permissive in halophilic archaea than they are in bacteria.

Centromeres are essential cis-elements on chromosomes that are crucial for the stable transmission of genetic information during mitotic and meiotic cell divisions. Different species employ a variety of centromere configurations, from small genetically defined centromeres in budding yeast to holocentric centromeres that occupy entire chromosomes in Caenorhabditis, yet the incorporation of nucleosomes containing the essential centromere-specific histone H3 variant CENP-A is a common feature of centromeres in all eukaryotes. In vertebrates and fungi, CENP-A is specifically deposited at centromeres by a conserved chaperone, called HJURP or Scm3, respectively. Surprisingly, homologs of these proteins have not been identified in Drosophila, Caenorhabditis, or plants. How CENP-A is targeted to centromeres in these organisms is not known. The Drosophila centromeric protein CAL1, found only in the Diptera genus, is essential for CENP-A localization, is recruited to centromeres at a similar time as CENP-A, and interacts with CENP-A in both chromatin and pre-nucleosomal complexes, making it a strong candidate for a CENP-A chaperone in this lineage. Here, we discuss the conservation and evolution of this essential centromere factor and report the identification of a "Scm3-domain"-like region with similarity to the corresponding region of fungal Scm3 as well as a shared predicted alpha-helical structure. Given the lack of common ancestry between Scm3 and CAL1, we propose that an optimal CENP-A binding region was independently acquired by CAL1, which caused the loss of an ancestral Scm3 protein from the Diptera lineage.

BACKGROUND: Hepatitis B virus (HBV) infections are responsible for the development of chronic hepatitis in 400 million people worldwide. Currently, no consensus exists as to when treatment should be initiated for pediatric patients.OBJECTIVES: To evaluate the risks and predictive factors of success of lamivudine treatment in children with chronic, active HBV infection.METHODS: Forty-three children (22 male, median age 9.6 years) chronically infected with HBV and treated between 1998 and 2008 at CHU Ste-Justine (Montreal, Quebec) were included in the present chart review study. Inclusion criteria were detectable hepatitis B surface antigen and hepatitis B e antigen (HBeAg), minimum serum alanine aminotransferase (ALT) level of two times the upper limit of normal and detectable serum HBV DNA for at least three months. Patients received lamivudine for a minimum of six months (median 14 months). Genotyping was performed.RESULTS: Lamivudine treatment was effective in 35% of cases (15 of 43) and overall virological response (during or after treatment) was achieved in 51% of patients. Three patients harboured suspected lamivudine-resistant mutations and five progressed to HBeAg-chronic HBV. Predictive factors for success of treatment were: younger age at beginning of treatment (P=0.05), elevated ALT levels throughout treatment duration (P=0.003) and loss of HBeAg during treatment (P=0.016). Asian origin did not affect treatment success or spontaneous viral control during follow-up. HBV genotype did not influence treatment success.CONCLUSIONS: Lamivudine treatment in a carefully selected cohort of HBV patients demonstrated a good rate of success and low incidence of mutation. Younger age at the beginning of treatment and high ALT levels during treatment predicted a positive outcome.

Vesicular stomatitis virus (VSV) has been widely used to characterize cellular processes, viral resistance, and cytopathogenicity. Recently, VSV has also been used for oncolytic virotherapy due to its capacity to selectively lyse tumor cells. Mutants of the matrix (M) protein of VSV have generally been preferred to the wild-type virus for oncolysis because of their ability to induce type I interferon (IFN) despite causing weaker cytopathic effects. However, due to the large variability of tumor types, it is quite clear that various approaches and combinations of multiple oncolytic viruses will be needed to effectively treat most cancers. With this in mind, our work focused on characterizing the cytopathogenic profiles of four replicative envelope glycoprotein (G) VSV mutants. In contrast to the prototypic M mutant, VSV G mutants are as efficient as wild-type virus at inhibiting cellular transcription and host protein translation. Despite being highly cytopathic, the mutant G 6R triggers type I interferon secretion as efficiently as the M mutant. Importantly, most VSV G mutants are more effective at killing B16 and MC57 tumor cells in vitro than the M mutant or wild-type virus through apoptosis induction. Taken together, our results demonstrate that VSV G mutants retain the high cytopathogenicity of wild-type VSV, with G 6R inducing type I IFN secretion at levels similar to that of the M mutant. VSV G protein mutants could therefore prove to be highly valuable for the development of novel oncolytic virotherapy strategies that are both safe and efficient for the treatment of various types of cancer.

Ions near the high-end border of a mass defect distribution plot for native peptide fragment ions have potential as signature markers that are based on mass-to-charge ratio determination. The specificity of these marker ions, including phosphoryl ions, can be improved by removing interfering isobaric ions from the border region on the distribution plot. These interfering ions are rich in Asp and Glu content. The masses of amino acid residues and peptides are rescaled from the IUPAC scale (12C = 12 u as the mass reference) to the averagine scale (averagine mass = 111 u* as the mass reference with zero mass defect; u*: the mass unit on the averagine scale), using a scaling factor of 0.999493894. It is theoretically predicted that esterification of Asp and Glu side-chain carboxylates with n-butanol can achieve a sufficient retreat of the high-end border on a mass defect distribution plot based on the use of mass spectrometers with better-than-medium resolution. Theoretical calculations and laboratory experiments are performed to examine effects of various esterifications on the averagine-scale mass defect distribution of peptide fragment ions and on the specificity of two positive phosphoryl ions: the phosphotyrosine immonium ion and a cyclophosphoramidate ion.