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  • 1
    Language: English
    In: Applied and environmental microbiology, October 2015, Vol.81(19), pp.6800-11
    Description: Common salt (NaCl) is frequently used by the food industry to add flavor and to act as a humectant in order to reduce the water content of a food product. The improved health awareness of consumers is leading to a demand for food products with reduced salt content; thus, manufacturers require alternative water activity-reducing agents which elicit the same general effects as NaCl. Two examples include KCl and glycerol. These agents lower the water activity of a food matrix and also contribute to limit the growth of the microbiota, including foodborne pathogens. Little is currently known about how foodborne pathogens respond to these water activity-lowering agents. Here we examined the response of Salmonella enterica serovar Typhimurium 4/74 to NaCl, KCl, and glycerol at three time points, using a constant water activity level, compared with the response of a control inoculum. All conditions induced the upregulation of gluconate metabolic genes after 6 h of exposure. Bacteria exposed to NaCl and KCl demonstrated the upregulation of the osmoprotective transporter mechanisms encoded by the proP, proU, and osmU (STM1491 to STM1494) genes. Glycerol exposure elicited the downregulation of these osmoadaptive mechanisms but stimulated an increase in lipopolysaccharide and membrane protein-associated genes after 1 h. The most extensive changes in gene expression occurred following exposure to KCl. Because many of these genes were of unknown function, further characterization may identify KCl-specific adaptive processes that are not stimulated by NaCl. This study shows that the response of S. Typhimurium to different humectants does not simply reflect reduced water activity and likely involves systems that are linked to specific humectants.
    Keywords: Food Industry ; Hygroscopic Agents -- Pharmacology ; Salmonella Typhimurium -- Drug Effects
    ISSN: 00992240
    E-ISSN: 1098-5336
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 15 May 2012, Vol.109(20), pp.E1277-86
    Description: More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ(70) (including phoP, slyA, and invF) from which we identified the -10 and -35 motifs of σ(70)-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and 〈20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.
    Keywords: Gene Expression Regulation, Bacterial -- Genetics ; RNA, Small Untranslated -- Genetics ; Regulatory Sequences, Ribonucleic Acid -- Genetics ; Salmonella Typhimurium -- Genetics ; Transcription, Genetic -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: English
    In: Journal of bacteriology, 01 July 2017, Vol.199(13)
    Description: Deep sequencing has revolutionized our understanding of the bacterial RNA world and has facilitated the identification of 280 small RNAs (sRNAs) in Despite the suspicions that sRNAs may play important roles in pathogenesis, the functions of most sRNAs remain unknown. To advance our understanding of RNA biology in virulence, we searched for sRNAs required for bacterial invasion into nonphagocytic cells. After screening 75 sRNAs, we discovered that the ablation of InvS caused a significant decrease of invasion into epithelial cells. A proteomic analysis showed that InvS modulated the levels of several type III secreted proteins. The level of PrgH, a type III secretion apparatus protein, was significantly lower in the absence of InvS, consistent with the known roles of PrgH in effector secretion and bacterial invasion. We discovered that InvS modulates expression and hence flagellar gene expression and motility. We propose that InvS coordinates the increase of PrgH and decrease in FimZ that promote efficient invasion into nonphagocytic cells. Salmonellosis continues to be the most common foodborne infection reported by the CDC in the United States. Central to pathogenesis is the ability to invade nonphagocytic cells and to replicate inside host cells. Invasion genes are known to be regulated by protein transcriptional networks, but little is known about the role played by small RNAs (sRNAs) in this process. We have identified a novel sRNA, InvS, that is involved in invasion. Our result will likely provide an opportunity to better understand the fundamental question of how regulates invasion gene expression and may inform strategies for therapeutic intervention.
    Keywords: Salmonella ; Gene Regulation ; Host Cell Invasion ; Noncoding RNA ; Bacterial Proteins -- Metabolism ; Epithelial Cells -- Microbiology ; Salmonella Typhimurium -- Physiology
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 4
    Language: English
    In: Science (New York, N.Y.), 09 June 2017, Vol.356(6342)
    Description: Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies-single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)-to identify human blood CD123CD33CD45RA DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123 pre-DC subset and two CD45RACD123 lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.
    Keywords: Cell Lineage ; Dendritic Cells -- Cytology
    ISSN: 00368075
    E-ISSN: 1095-9203
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  • 5
    Language: English
    In: Cell, 11 January 2018, Vol.172(1-2), pp.162-175.e14
    Description: Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed “trained immunity,” causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, / mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases. Systemic inflammation induced by a Western diet is largely blunted by dietary changes, but myeloid cell-induced innate immune responses remain augmented and could potentially contribute to inflammatory disease.
    Keywords: Trained Immunity ; Innate Immune Memory ; Sterile Inflammation ; Granulocyte Macrophage Progenitors ; Western Diet Feeding ; Epigenetic Reprogramming ; Nlrp3 Inflammasome ; Atherosclerosis ; Non-Communicable Diseases ; ASC ; Biology
    ISSN: 0092-8674
    E-ISSN: 1097-4172
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  • 6
    Language: English
    In: Current Opinion in Biotechnology, 2011, Vol.22(2), pp.200-210
    Description: ► Overview of 50 . Typhimurium transcriptomic studies dating from 2003 to 2010. ► Wealth of transcriptomics but few datasets from . Typhimurium infection. ► Genes that are highly expressed during infection often play a direct role in virulence. ► Lack of published studies of Salmonella gene expression during food processing. ► Understanding global gene expression datasets continues to be a challenge. The first decade of transcriptomic studies of serovar Typhimurium focused upon gene expression , and during the infection of mammalian cells. The published regulons and stimulons show that the three Type Three Secretion Systems of Typhimurium respond to a diverse range of environmental conditions, and are controlled by a hierarchy of regulatory proteins. The integration of generated transcriptomic data with global gene expression of Typhimurium during infection is beginning to yield valuable information. The coordinated regulation of Salmonella gene expression is a key process for survival, adaptation and virulence capacities of the pathogen.
    Keywords: Engineering
    ISSN: 0958-1669
    E-ISSN: 1879-0429
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  • 7
    Language: English
    In: Applied and environmental microbiology, July 2013, Vol.79(14), pp.4376-84
    Description: Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate- and low-moisture foods.
    Keywords: Desiccation ; Stainless Steel ; Transcriptome ; Amino Acid Transport Systems, Neutral -- Genetics ; Bacterial Proteins -- Genetics ; Salmonella Typhimurium -- Physiology
    ISSN: 00992240
    E-ISSN: 1098-5336
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  • 8
    Language: English
    In: Scientific reports, 19 July 2018, Vol.8(1), pp.10950
    Description: Next-generation sequencing (NGS) is the method of choice when large numbers of sequences have to be obtained. While the technique is widely applied, varying error rates have been observed. We analysed millions of reads obtained after sequencing of one single sequence on an Illumina sequencer. According to our analysis, the index-PCR for sample preparation has no effect on the observed error rate, even though PCR is traditionally seen as one of the major contributors to enhanced error rates in NGS. In addition, we observed very persistent pre-phasing effects although the base calling software corrects for these. Removal of shortened sequences abolished these effects and allowed analysis of the actual mutations. The average error rate determined was 0.24 ± 0.06% per base and the percentage of mutated sequences was found to be 6.4 ± 1.24%. Constant regions at the 5'- and 3'-end, e.g., primer binding sites used in in vitro selection procedures seem to have no effect on mutation rates and re-sequencing of samples obtains very reproducible results. As phasing effects and other sequencing problems vary between equipment and individual setups, we recommend evaluation of error rates and types to all NGS-users to improve the quality and analysis of NGS data.
    Keywords: High-Throughput Nucleotide Sequencing -- Methods ; Polymerase Chain Reaction -- Methods
    E-ISSN: 2045-2322
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  • 9
    Language: English
    In: Current Opinion in Systems Biology, June 2017, Vol.3, pp.147-153
    Description: Cellular phenotyping, in particular immune cell phenotyping, has become an integral part of personalized and stratified medicine approaches in order to facilitate classification of patient cohorts according to proteomic, transcriptomic or genomic information, with the ultimate goal to increase treatment efficiency and outcome. However, choosing the optimal and most informative phenotyping approach to discover novel and predictive biomarkers for patient cohorts has become a major challenge and greatly hampers knowledge gain to successfully develop and tailor new and existing therapies to suitable patient collectives [1]. Recent technological innovations, such as single-cell proteomics (Mass Cytometry) and single-cell transcriptomics have become available which possess the power to measure thousands of features for thousands to millions of cells in parallel, thereby allowing the deep characterization of complex cellular networks in homeostasis as well as perturbations under disease conditions [2]. These multidimensional approaches dramatically accelerate the discovery of novel biomarkers for disease prediction and progression within personalized medicine approaches. These approaches now allow for the characterization of small amounts of patient material both on the protein and on the transcriptome level to allow for an unbiased, high-dimensional, and bioinformatically supported systems biology approach which enables discovery, design and implementation of novel biomarkers into the clinical routine in a rapid fashion. In this review, we will discuss the available technologies and recent applications and scientific advances enabled by these technologies highlighting our view of how to integrate these technologies into translational research to achieve a more reliable, more rapid and better informed approach to molecular phenotyping ultimately achieving the level of knowledge needed to implement personalized medicine approaches for a wider patient base.
    Keywords: Biomarker Discovery ; Single Cell Analysis ; Mass Cytometry ; Single Cell Transcriptomics ; High Dimensional Phenotyping
    ISSN: 2452-3100
    E-ISSN: 2452-3100
    Source: ScienceDirect Journals (Elsevier)
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  • 10
    Language: English
    In: Frontiers in microbiology, 2014, Vol.5, pp.373
    Description: Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24(CHX)) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent.
    Keywords: Snp Typing ; Salmonella ; Biocide Tolerance ; Chlorhexidine ; Proteomics ; Transcriptomics ; Whole Genome Sequencing
    ISSN: 1664-302X
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