A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specifc enumeration of E. coli in contaminated drinking water is critical to mitigate signifcant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane fltration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifcally engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specifc to cellulose. These constructs were then inserted into the E. coli-specifc phage T7 which were used to infect E. coli trapped on a cellulose flter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose flter in proximity to the lysed cells. Following substrate addition, the location and quantifcation of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed

Cigarette Smoke Affects Keratinocytes SRB1 Expression and Localization via H2O2 Production and HNE Protein Adducts Formation

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H2O2) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H2O2, induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake

New final focus system for the SLAC linear collider

The final focus system of the SLC has been upgraded by replacing the final quadrupole magnets with higher gradient superconducting magnets positioned closer to the interaction point. The parameters of the new system have been chosen to be compatible with the SLD detector with a minimum of changes to other final focus components. Commissioning plans for the new system are also presented. 5 refs., 1 fig

Language development after cochlear implantation: an epigenetic model

Growing evidence supports the notion that dynamic gene expression, subject to epigenetic control, organizes multiple influences to enable a child to learn to listen and to talk. Here, we review neurobiological and genetic influences on spoken language development in the context of results of a longitudinal trial of cochlear implantation of young children with severe to profound sensorineural hearing loss in the Childhood Development after Cochlear Implantation study. We specifically examine the results of cochlear implantation in participants who were congenitally deaf (N = 116). Prior to intervention, these participants were subject to naturally imposed constraints in sensory (acoustic–phonologic) inputs during critical phases of development when spoken language skills are typically achieved rapidly. Their candidacy for a cochlear implant was prompted by delays (n = 20) or an essential absence of spoken language acquisition (n = 96). Observations thus present an opportunity to evaluate the impact of factors that influence the emergence of spoken language, particularly in the context of hearing restoration in sensitive periods for language acquisition. Outcomes demonstrate considerable variation in spoken language learning, although significant advantages exist for the congenitally deaf children implanted prior to 18 months of age. While age at implantation carries high predictive value in forecasting performance on measures of spoken language, several factors show significant association, particularly those related to parent–child interactions. Importantly, the significance of environmental variables in their predictive value for language development varies with age at implantation. These observations are considered in the context of an epigenetic model in which dynamic genomic expression can modulate aspects of auditory learning, offering insights into factors that can influence a child’s acquisition of spoken language after cochlear implantation. Increased understanding of these interactions could lead to targeted interventions that interact with the epigenome to influence language outcomes with intervention, particularly in periods in which development is subject to time-sensitive experience

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis

A Joint Fermi-GBM and Swift-BAT Analysis of Gravitational-wave Candidates from the Third Gravitational-wave Observing Run

We present Fermi Gamma-ray Burst Monitor (Fermi-GBM) and Swift Burst Alert Telescope (Swift-BAT) searches for gamma-ray/X-ray counterparts to gravitational-wave (GW) candidate events identified during the third observing run of the Advanced LIGO and Advanced Virgo detectors. Using Fermi-GBM onboard triggers and subthreshold gamma-ray burst (GRB) candidates found in the Fermi-GBM ground analyses, the Targeted Search and the Untargeted Search, we investigate whether there are any coincident GRBs associated with the GWs. We also search the Swift-BAT rate data around the GW times to determine whether a GRB counterpart is present. No counterparts are found. Using both the Fermi-GBM Targeted Search and the Swift- BAT search, we calculate flux upper limits and present joint upper limits on the gamma-ray luminosity of each GW. Given these limits, we constrain theoretical models for the emission of gamma rays from binary black hole mergers

A gravitational-wave standard siren measurement of the Hubble constant

The detection of GW170817 (ref. 1) heralds the age of gravitational-wave multi-messenger astronomy, with the observations of gravitational-wave and electromagnetic emission from the same transient source. On 17 August 2017 the network of Advanced Laser Interferometer Gravitational-wave Observatory (LIGO)2 and Virgo3 detectors observed GW170817, a strong signal from the merger of a binary neutron-star system. Less than two seconds after the merger, a γ-ray burst event, GRB 170817A, was detected consistent with the LIGO–Virgo sky localization region4–6). The sky region was subsequently observed by optical astronomy facilities7, resulting in the identification of an optical transient signal within about 10 arcseconds of the galaxy NGC 4993 (refs 8–13). GW170817 can be used as a standard siren14–18, combining the distance inferred purely from the gravitational-wave signal with the recession velocity arising from the electromagnetic data to determine the Hubble constant. This quantity, representing the local expansion rate of the Universe, sets the overall scale of the Universe and is of fundamental importance to cosmology. Our measurements do not require any form of cosmic ‘distance ladder’19; the gravitational-wave analysis directly estimates the luminosity distance out to cosmological scales. Here we report H0 = kilometres per second per megaparsec, which is consistent with existing measurements20,21, while being completely independent of them

Search for gravitational waves associated with gamma-ray bursts detected by Fermi and Swift during the LIGO–Virgo run O3b

We search for gravitational-wave signals associated with gamma-ray bursts (GRBs) detected by the Fermi and Swift satellites during the second half of the third observing run of Advanced LIGO and Advanced Virgo (2019 November 1 15:00 UTC–2020 March 27 17:00 UTC). We conduct two independent searches: a generic gravitational-wave transients search to analyze 86 GRBs and an analysis to target binary mergers with at least one neutron star as short GRB progenitors for 17 events. We find no significant evidence for gravitational-wave signals associated with any of these GRBs. A weighted binomial test of the combined results finds no evidence for subthreshold gravitational-wave signals associated with this GRB ensemble either. We use several source types and signal morphologies during the searches, resulting in lower bounds on the estimated distance to each GRB. Finally, we constrain the population of low-luminosity short GRBs using results from the first to the third observing runs of Advanced LIGO and Advanced Virgo. The resulting population is in accordance with the local binary neutron star merger rate

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specifc enumeration of E. coli in contaminated drinking water is critical to mitigate signifcant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane fltration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifcally engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specifc to cellulose. These constructs were then inserted into the E. coli-specifc phage T7 which were used to infect E. coli trapped on a cellulose flter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose flter in proximity to the lysed cells. Following substrate addition, the location and quantifcation of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed.This article is published as Hinkley, T.C., Singh,S., Garing, S., Le Ny,A.L.M., Nichols, K.P., Peters, J.E., Talbert, J.N., Nugen, S.R., A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water. Scientific Reports, 2018 (8); 14630. DOI: 10.1038/s41598-018-33097-4. </p