Real-Time PCR-Based Mismatch Amplification Mutation Assay for Specific Detection of CS6-Expressing Allelic Variants of Enterotoxigenic Escherichia coli and Its Application in Assessing Diarrheal Cases and Asymptomatic Controls

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies

Spectrally resolved ion imaging from laser produced plasmas using CR-39 detectors

Intense laser-produced plasmas generate bright, ultrashort bursts of accelerated ions. Reducing the required laser intensity and increasing the repetition rate of the laser to generate high energy ions is important, and mesoscopic particle targets are an attractive option to address this issue. Newer experimental strategies to measure ion energies and their angular distribution are needed in studies of such systems. In this paper, we outline a method to simultaneously measure these quantities using a single CR39 film. Although CR-39 detectors are known for ion imaging or spectroscopy, combining these specially for lower ion energies and applications to low-intensity laser experiments is not common. The paradigm chosen in our study is to consider the spatial distribution of nuclear tracks on a CR-39 sheet, while simultaneously separating them by their track diameter. Our method achieves an energy resolution of about 100 keV and a spatial resolution of tens of micrometers. In addition, ion species other than protons, i.e., carbon and oxygen, can also be imaged in an energy-resolved manner. © 2021 Author(s)

Laser structured micro-targets generate MeV electron temperature at 4 x10^16 W/cm^2

Relativistic temperature electrons higher than 0.5 MeV are generated typically with laser intensities of about 10^18 W/cm^2. Their generation with high repetition rate lasers that operate at non-relativistic intensities (~10^16W/cm^2) is cardinal for the realization of compact, ultra-short, bench-top electron sources. New strategies, capable of exploiting different aspects of laser-plasma interaction, are necessary for reducing the required intensity. We report here, a novel technique of dynamic target structuring of microdroplets, capable of generating 200 keV and 1 MeV electron temperatures at 1/100th of the intensity required by ponderomotive scaling(10^18 W/cm^2) to generate relativistic electron temperature. Combining the concepts of pre-plasma tailoring, optimized scale length and micro-optics, this method achieves two-plasmon decay boosted electron acceleration with "non-ideal" ultrashort (25 fs) pulses at 4 x10^16 W/cm^2 only. With shot repeatability at kHz, this precise in-situ targetry produces directed, imaging quality beam-like electron emission up to 6 MeV with milli-joule class lasers, that can be transformational for time-resolved, microscopic studies in all fields of science

Shaped liquid drops generate MeV temperature electron beams with millijoule class laser

MeV temperature electrons are typically generated at laser intensities of 1018 W cm−2. Their generation at non-relativistic intensities (~1016 W cm−2) with high repetition rate lasers is cardinal for the realization of compact, ultra-fast electron sources. Here we report a technique of dynamic target structuring of micro-droplets using a 1 kHz, 25 fs, millijoule class laser, that uses two collinear laser pulses; the first to create a concave surface in the liquid drop and the second, to dynamically-drive electrostatic plasma waves that accelerate electrons to MeV energies. The acceleration mechanism, identified as two plasmon decay instability, is shown to generate two beams of electrons with hot electron temperature components of 200 keV and 1 MeV, respectively, at an intensity of 4 × 1016 Wcm−2, only. The electron beams are demonstrated to be ideal for single shot high resolution (tens of μm) electron radiography

Shaped liquid drops generate MeV temperature electron beams with millijoule class laser

MeV temperature electrons are typically generated at laser intensities of 1018 W cm−2. Their generation at non-relativistic intensities (~1016 W cm−2) with high repetition rate lasers is cardinal for the realization of compact, ultra-fast electron sources. Here we report a technique of dynamic target structuring of micro-droplets using a 1 kHz, 25 fs, millijoule class laser, that uses two collinear laser pulses; the first to create a concave surface in the liquid drop and the second, to dynamically-drive electrostatic plasma waves that accelerate electrons to MeV energies. The acceleration mechanism, identified as two plasmon decay instability, is shown to generate two beams of electrons with hot electron temperature components of 200 keV and 1 MeV, respectively, at an intensity of 4 × 1016 Wcm−2, only. The electron beams are demonstrated to be ideal for single shot high resolution (tens of μm) electron radiography

MicroRNA-103a regulates sodium-dependent vitamin C transporter-1 expression in intestinal epithelial cells.

Intestinal absorption of ascorbic acid (AA) occurs via a Na+-dependent carrier-mediated process facilitated through the human sodium-dependent vitamin C transporters-1 &-2 (hSVCT1 and hSVCT2). Many studies have shown that hSVCT1 (product of the SLC23A1 gene) is expressed on the apical membrane of polarized enterocytes where it mediates AA absorption. hSVCT1 expression levels are therefore an important determinant of physiological vitamin C homeostasis. However, little is known about posttranscriptional mechanisms that regulate hSVCT1 expression in intestinal epithelia. In this study, we investigated regulation of hSVCT1 by microRNA (miRNA). A pmirGLO-SLC23A1-3'-UTR construct transfected into human intestinal cell lines (Caco-2 and NCM460 cells) showed markedly reduced luciferase activity. Bioinformatic analysis of the SLC23A1-3'-UTR predicted five miRNA binding sites (miR-103a, miR-107, miR-328, miR-384, and miR-499-5p) in the 3'-UTR. Expression of mature miR-103a was markedly higher compared to the other four putative miRNA regulators in both intestinal cell lines and mouse jejunal mucosa. Addition of a miR-103a mimic, but not a miR-103a mutant construct, markedly reduced the luminescence of the pmirGLO-SLC23A1-3'-UTR reporter. Reciprocally, addition of a miR-103a inhibitor significantly increased luciferase reporter activity. Addition of the miR-103a mimic led to a significant inhibition in AA uptake, associated with decreased hSVCT1 mRNA and protein expression in Caco-2 cells. In contrast, the miR-103a inhibitor increased AA uptake, associated with increased levels of hSVCT1 mRNA and protein. These findings provide the first evidence for posttranscriptional regulation of hSVCT1 by miRNA in intestinal epithelial cells