Inadequate drinking water quality is among the major causes of preventable mortality, predominantly in young children. Identifying contaminated water sources remains a significant challenge, ...especially where resources are limited. The current methods for measuring Escherichia coli (E. coli), the WHO preferred indicator for measuring fecal contamination of water, involve overnight incubation and require specialized training. In 2016, UNICEF released a Target Product Profile (TPP) to incentivize product innovations to detect low levels of viable E. coli in water samples in the field in less than 6 h. Driven by this challenge, we developed a phage-based assay to detect and semi-quantify E. coli. We formulated a phage cocktail containing a total of 8 phages selected against an extensive bacterial strain library and recombined with the sensitive NanoLuc luciferase reporter. The assay was optimized to be processed in a microfluidic chip designed in-house and was tested against locally sourced sewage samples and on drinking water sources in Nairobi, Kenya. With this assay, combined with the microfluidic chip platform, we propose a complete automated solution to detect and semi-quantify E. coli at less than 10 MPN/100 mL in 5.5 h by minimally trained personnel.
•Non-genetic cellular heterogeneity drives persisters in bacteria, fungi, and cancer.•Gene expression variability is a fundamental feature enabling persister formation.•Persisters act as precursors ...for genetic adaptation under high-stress conditions.
A common feature of bacterial, fungal and cancer cell populations upon treatment is the presence of tolerant and persistent cells able to survive, and sometimes grow, even in the presence of usually inhibitory or lethal drug concentrations, driven by non-genetic differences among individual cells in a population. Here we review and compare data obtained on drug survival in bacteria, fungi and cancer cells to unravel common characteristics and cellular pathways, and to point their singularities. This comparative work also allows to cross-fertilize ideas across fields. We particularly focus on the role of gene expression variability in the emergence of cell-cell non-genetic heterogeneity because it represents a possible common basic molecular process at the origin of most persistence phenomena and could be monitored and tuned to help improve therapeutic interventions.
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We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of ...mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4(+) memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4(+) cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a ...single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis.
This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.
Phenotypic heterogeneity is a hallmark of aggressive cancer behaviour and a clinical challenge. Despite much characterisation of this heterogeneity at a multi-omics level in many cancers, we have a ...limited understanding of how this heterogeneity emerges spontaneously in an isogenic cell population. Some longitudinal observations of dynamics in epithelial-mesenchymal heterogeneity, a canonical example of phenotypic heterogeneity, have offered us opportunities to quantify the rates of phenotypic switching that may drive such heterogeneity. Here, we offer a mathematical modeling framework that explains the salient features of population dynamics noted in PMC42-LA cells: (a) predominance of EpCAM
subpopulation, (b) re-establishment of parental distributions from the EpCAM
and EpCAM
subpopulations, and (c) enhanced heterogeneity in clonal populations established from individual cells. Our framework proposes that fluctuations or noise in content duplication and partitioning of SNAIL-an EMT-inducing transcription factor-during cell division can explain spontaneous phenotypic switching and consequent dynamic heterogeneity in PMC42-LA cells observed experimentally at both single-cell and bulk level analysis. Together, we propose that asymmetric cell division can be a potential mechanism for phenotypic heterogeneity.
Reducing noise has always been one of the standard problems of the image analysis and processing community. Often though, at the same time as reducing the noise in a signal, it is important to ...preserve the edges. Edges are of critical importance to the visual appearance of images. So, it is desirable to preserve important features, such as edges, corners and other sharp structures, during the denoising process. This paper presents a review of some significant work in the area of image denoising. It provides a brief general classification of image denoising methods. The main aim of this survey is to provide evolution of research in the direction of edge-preserving image denoising. It characterizes some of the well known edge-preserving denoising methods, elaborating each of them, and discusses the advantages and drawbacks of each. Basic ideas and improvement of the denoising methods are also comprehensively summarized and analyzed in depth. Often, researchers face difficulty in selecting an appropriate denoising method that is specific to their purpose. We have classified and systemized these denoising methods. The key goal of this paper is to provide researchers with background on a progress of denoising methods so as to make it easier for researchers to choose the method best suited to their aims.
Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that ...antibiotic. In infections with Mycobacterium tuberculosis, such persisters underlie the need for long-term antibiotic therapy and contribute to treatment failure in tuberculosis cases. Here, we demonstrate the value of dual-reporter mycobacteriophages (Φ
DRMs) for characterizing M. tuberculosis persisters. The addition of isoniazid (INH) to exponentially growing M. tuberculosis cells consistently resulted in a 2- to 3-log decrease in CFU within 4 days, and the remaining ≤1% of cells, which survived despite being INH sensitive, were INH-tolerant persisters with a distinct transcriptional profile. We fused the promoters of several genes upregulated in persisters to the red fluorescent protein tdTomato gene in Φ
GFP10, a mycobacteriophage constitutively expressing green fluorescent protein (GFP), thus generating Φ
DRMs. A population enriched in INH persisters exhibited strong red fluorescence, by microscopy and flow cytometry, using a Φ
DRM with tdTomato controlled from the dnaK promoter. Interestingly, we demonstrated that, prior to INH exposure, a population primed for persistence existed in M. tuberculosis cells from both cultures and human sputa and that this population was highly enriched following INH exposure. We conclude that Φ
DRMs provide a new tool to identify and quantitate M. tuberculosis persister cells.
Tuberculosis (TB) is again the leading cause of death from a single infectious disease, having surpassed HIV. The recalcitrance of the TB pandemic is largely due to the ability of the pathogen Mycobacterium tuberculosis to enter a persistent state in which it is less susceptible to antibiotics and immune effectors, necessitating lengthy treatment. It has been difficult to study persister cells, as we have lacked tools to isolate these rare cells. In this article, we describe the development of dual-reporter mycobacteriophages that encode a green fluorescent marker of viability and in which the promoters of genes we have identified as induced in the persister state are fused to a gene encoding a red fluorescent protein. We show that these tools can identify heterogeneity in a cell population that correlates with propensity to survive antibiotic treatment and that the proportions of these subpopulations change in M. tuberculosis cells within human sputum during the course of treatment.
Many researchers have worked on scalable coding for unencrypted images, and there is more space for research in scalable coding for encrypted images. This paper exposits a new method of scalable ...coding for encrypted images, especially for lossy compression images, using the Modified Absolute Moment Block Truncation Code (MAMBTC) technique. The given input image is compressed using MAMBTC and then encrypted using a Pseudo-Random Number (PRNG) at the encryption phase. The PRNG is shared between the encoder and the decoder. At the decryption phase, the compressed pixel value is obtained by decryption using the PRNG and then reconstructed using MAMBTC, scaled by factor 2, and the Bilinear Interpolation Technique to acquire the original image. MAMBTC gives better image quality than Block Truncation Code (BTC), a higher PSNR of 36.32 dB, and a Compression ratio of 1.09, which makes the proposed system ready for signal processing applications.
Significance A mutant strain of Mycobacterium smegmatis mc ²155 provided the first plasmid transformation platform for any mycobacteria. It has since been used extensively as a surrogate host to ...study mycobacterial gene functions in Mycobacterium tuberculosis and Mycobacterium leprae . In this report, we discovered that the efficient plasmid transformation (ept) phenotype of mc ²155 is caused by a loss-of-function mutation in a gene, namely eptC , which encodes a protein that shares homology with the structural maintenance of chromosomes (SMC) family. While SMCs generally promote DNA segregation to daughter cells, we show that EptC selectively inhibits plasmid segregation and thereby establish a new plasmid restrictive role of this mycobacterial SMC homolog.
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