A Simple Screen to Identify Promoters Conferring High Levels of Phenotypic Noise

Genetically identical populations of unicellular organisms often show marked variation in some phenotypic traits. To investigate the molecular causes and possible biological functions of this phenotypic noise, it would be useful to have a method to identify genes whose expression varies stochastically on a certain time scale. Here, we developed such a method and used it for identifying genes with high levels of phenotypic noise in Salmonella enterica ssp. I serovar Typhimurium (S. Typhimurium). We created a genomic plasmid library fused to a green fluorescent protein (GFP) reporter and subjected replicate populations harboring this library to fluctuating selection for GFP expression using fluorescent-activated cell sorting (FACS). After seven rounds of fluctuating selection, the populations were strongly enriched for promoters that showed a high amount of noise in gene expression. Our results indicate that the activity of some promoters of S. Typhimurium varies on such a short time scale that these promoters can absorb rapid fluctuations in the direction of selection, as imposed during our experiment. The genomic fragments that conferred the highest levels of phenotypic variation were promoters controlling the synthesis of flagella, which are associated with virulence and host–pathogen interactions. This confirms earlier reports that phenotypic noise may play a role in pathogenesis and indicates that these promoters have among the highest levels of noise in the S. Typhimurium genome. This approach can be applied to many other bacterial and eukaryotic systems as a simple method for identifying genes with noisy expression

The Mych Gene Is Required for Neural Crest Survival during Zebrafish Development

Background: Amomg Myc family genes, c-Myc is known to have a role in neural crest specification in Xenopus and in craniofacial development in the mouse. There is no information on the function of other Myc genes in neural crest development, or about any developmental role: of zebrafish Myc genes. Principal Findings: We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to sever defects in craniofacial development and in certain other tissues including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain. Significance: Mych is a novel factor required for neural crest cell survival in zebrafish

SeqGene: a comprehensive software solution for mining exome- and transcriptome- sequencing data

Abstract Background The popularity of massively parallel exome and transcriptome sequencing projects demands new data mining tools with a comprehensive set of features to support a wide range of analysis tasks. Results SeqGene, a new data mining tool, supports mutation detection and annotation, dbSNP and 1000 Genome data integration, RNA-Seq expression quantification, mutation and coverage visualization, allele specific expression (ASE), differentially expressed genes (DEGs) identification, copy number variation (CNV) analysis, and gene expression quantitative trait loci (eQTLs) detection. We also developed novel methods for testing the association between SNP and expression and identifying genotype-controlled DEGs. We showed that the results generated from SeqGene compares favourably to other existing methods in our case studies. Conclusion SeqGene is designed as a general-purpose software package. It supports both paired-end reads and single reads generated on most sequencing platforms; it runs on all major types of computers; it supports arbitrary genome assemblies for arbitrary organisms; and it scales well to support both large and small scale sequencing projects. The software homepage is http://seqgene.sourceforge.net.</p

Monocyte Derived Microvesicles Deliver a Cell Death Message via Encapsulated Caspase-1

Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1β and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death

Identification of CD4+ T Cell Epitopes in C. burnetii Antigens Targeted by Antibody Responses

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes acute Q fever and chronic infections in humans. A killed, whole cell vaccine is efficacious, but vaccination can result in severe local or systemic adverse reactions. Although T cell responses are considered pivotal for vaccine derived protective immunity, the epitope targets of CD4+ T cell responses in C. burnetii vaccination have not been elucidated. Since mapping CD4+ epitopes in a genome with over 2,000 ORFs is resource intensive, we focused on 7 antigens that were known to be targeted by antibody responses. 117 candidate peptides were selected from these antigens based on bioinformatics predictions of binding to the murine MHC class II molecule H-2 IAb. We screened these peptides for recognition by IFN-γ producing CD4+ T cell in phase I C. burnetii whole cell vaccine (PI-WCV) vaccinated C57BL/6 mice and identified 8 distinct epitopes from four different proteins. The identified epitope targets account for 8% of the total vaccination induced IFN-γ producing CD4+ T cells. Given that less than 0.4% of the antigens contained in C. burnetii were screened, this suggests that prioritizing antigens targeted by antibody responses is an efficient strategy to identify at least a subset of CD4+ targets in large pathogens. Finally, we examined the nature of linkage between CD4+ T cell and antibody responses in PI-WCV vaccinated mice. We found a surprisingly non-uniform pattern in the help provided by epitope specific CD4+ T cells for antibody production, which can be specific for the epitope source antigen as well as non-specific. This suggests that a complete map of CD4+ response targets in PI-WCV vaccinated mice will likely include antigens against which no antibody responses are made

A Novel Non-Lens βγ−Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications

In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens betagamma-crystallin (alpha-subunit) and trefoil factor (beta-subunit), named betagamma-CAT, was identified from frog Bombina maxima skin secretions. Its alpha-subunit and beta-subunit (containing three trefoil factor domains), with a non-covalently linked form of alphabeta(2), show significant sequence homology to ep37 proteins, a group of non-lens betagamma-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. betagamma-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. betagamma-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K(+) efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the alpha-subunit with Western blotting. Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of betagamma-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC(50) 10 nM) and apoptosis. betagamma-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting that betagamma-CAT endocytosis is important for its activities.These findings illustrate novel cellular functions of non-lens betagamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals

Cell Type-Specific Neuroprotective Activity of Untranslocated Prion Protein

Background: A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions. Principal Findings: Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells. Significance: These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function

Interferon-γ and Proliferation Responses to Salmonella enterica Serotype Typhi Proteins in Patients with S. Typhi Bacteremia in Dhaka, Bangladesh

Salmonella enterica serotype Typhi infection is a significant global public health problem and the cause of typhoid fever. Salmonella are intracellular pathogens, and cellular immune responses are required to control and clear Salmonella infections. Despite this, there are limited data on cellular immune responses during wild type S. Typhi infection in humans. Here we report the assessment of cellular immune responses in humans with S. Typhi bacteremia through a screening approach that permitted us to evaluate interferon-γ and proliferation responses to a number of S. Typhi antigens. We detected significant interferon-γ CD4 and CD8 responses, as well as proliferative responses, to a number of recombinantly purified S. Typhi proteins as well as membrane preparation in infected patients. Antigen-specific interferon-γ responses were present at the time of clinical presentation in patients and absent in healthy controls. These observations could assist in the development of interferon-γ-based diagnostic assays for typhoid fever

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field