RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF

Old Drugs To Treat Resistant Bugs: Methicillin-Resistant Staphylococcus aureus Isolates with mecC Are Susceptible to a Combination of Penicillin and Clavulanic Acid.

β-Lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by the expression of an alternative penicillin-binding protein 2a (PBP2a) (encoded by mecA) with a low affinity for β-lactam antibiotics. Recently, a novel variant of mecA, known as mecC, was identified in MRSA isolates from both humans and animals. In this study, we demonstrate that mecC-encoded PBP2c does not mediate resistance to penicillin. Rather, broad-spectrum β-lactam resistance in MRSA strains carrying mecC (mecC-MRSA strains) is mediated by a combination of both PBP2c and the distinct β-lactamase encoded by the blaZ gene of strain LGA251 (blaZLGA251), which is part of mecC-encoding staphylococcal cassette chromosome mec (SCCmec) type XI. We further demonstrate that mecC-MRSA strains are susceptible to the combination of penicillin and the β-lactam inhibitor clavulanic acid in vitro and that the same combination is effective in vivo for the treatment of experimental mecC-MRSA infection in wax moth larvae. Thus, we demonstrate how the distinct biological differences between mecA- and mecC-encoded PBP2a and PBP2c have the potential to be exploited as a novel approach for the treatment of mecC-MRSA infections.This work was supported by a Medical Research Council (MRC) Partnership Grant (G1001787/1) held between the Department of Veterinary Medicine, University of Cambridge (M. A. H.), the School of Clinical Medicine, University of Cambridge (S. J. P.), the Moredun Research Institute (R. N. Z.) and the Wellcome Trust Sanger Institute (J. P. and S. J. P.).This is the author accepted manuscript. The final version is available from American Society for Microbiology via http://dx.doi.org/10.1128/AAC.01469-1

A modern multicentennial record of radiocarbon variability from an exactly dated bivalve chronology at the Tree Nob site (Alaska coastal current)

This is the final version. Available from Cambridge University Press via the DOI in this record. Quantifying the marine radiocarbon reservoir effect, offsets (ΔR), and ΔR variability over time is critical to improving dating estimates of marine samples while also providing a proxy of water mass dynamics. In the northeastern Pacific, where no high-resolution time series of ΔR has yet been established, we sampled radiocarbon (14C) from exactly dated growth increments in a multicentennial chronology of the long-lived bivalve, Pacific geoduck (Paneopea generosa) at the Tree Nob site, coastal British Columbia, Canada. Samples were taken at approximately decadal time intervals from 1725 CE to 1920 CE and indicate average ΔR values of 256 ± 22 years (1σ) consistent with existing discrete estimates. Temporal variability in ΔR is small relative to analogous Atlantic records except for an unusually old-water event, 1802–1812. The correlation between ΔR and sea surface temperature (SST) reconstructed from geoduck increment width is weakly significant (r2 = .29, p = .03), indicating warm water is generally old, when the 1802–1812 interval is excluded. This interval contains the oldest (–2.1σ) anomaly, and that is coincident with the coldest (–2.7σ) anomalies of the temperature reconstruction. An additional 32 14C values spanning 1952–1980 were detrended using a northeastern Pacific bomb pulse curve. Significant positive correlations were identified between the detrended 14C data and annual El Niño Southern Oscillation (ENSO) and summer SST such that cooler conditions are associated with older water. Thus, 14C is generally relatively stable with weak, potentially inconsistent associations to climate variables, but capable of infrequent excursions as illustrated by the unusually cold, old-water 1802–1812 interval.National Science FoundationNational Science Foundatio

Mechanisms of β-lactam resistance of Streptococcus uberis isolated from bovine mastitis cases.

A number of veterinary clinical pathology laboratories in New Zealand have been reporting emergence of increased minimum in inhibitory concentrations for β-lactams in the common clinical bovine mastitis pathogen Streptococcus uberis. The objective of this study was to determine the genetic basis of this increase in MIC for β-lactams amongst S. uberis. Illumina sequencing and determination of oxacillin MIC was performed on 265 clinical isolates. Published sequences of the five penicillin binding proteins pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x were used to identify, extract and align these sequences from the study isolates. Amino acid substitutions resulting from single nucleotide polymorphisms (SNP) within these genes were analysed for associations with elevated (≥ 0.5 mg/L) oxacillin MIC together with a genome wide association study. The population structure of the study isolates was approximated using a phylogenetic tree generated from an alignment of the core genome. A total of 53 % of isolates had MIC ≥ 0.5 mg/L for oxacillin. A total of 101 substitutions within the five pbp were identified, of which 11 were statistically associated with an MIC ≥ 0.5 mg/L. All 140 isolates which exhibited an increased β-lactam MIC had SNPs leading to pbp2x E381K and Q554E substitutions. The phylogenetic tree indicated that the genotype and phenotype associated with the increased MIC for oxacillin were present in several different lineages suggesting that acquisition of this increased β-lactam MIC had occurred in multiple geographically distinct regions. Reanalysis of the data from the intervention studies from which the isolates were originally drawn found a tendency for the pbp2x E381K substitution to be associated with lower cure rates. It is concluded that there is geographically and genetically widespread presence of pbp substitutions associated with reduced susceptibility to β-lactam antimicrobials. Additionally, presence of pbp substitutions tended to be associated with poorer cure rate outcomes following antimicrobial therapy for clinical mastitis

Design of low-cost ionic liquids for lignocellulosic biomass pretreatment

The cost of ionic liquids (ILs) is one of the main impediments to IL utilization in the cellulosic biorefinery, especially in the pretreatment step. In this study, a number of ionic liquids were synthesized with the goal of optimizing solvent cost and stability whilst demonstrating promising processing potential. To achieve this, inexpensive feedstocks such as sulfuric acid and simple amines were combined into a range of protic ionic liquids containing the hydrogen sulfate [HSO] anion. The performance of these ionic liquids was compared to a benchmark system containing the IL 1-ethyl-3-methylimidazolium acetate [CCim][OAc]. The highest saccharification yields were observed for the triethylammonium hydrogen sulfate IL, which was 75% as effective as the benchmark system. Techno-economic modeling revealed that this promising and yet to be optimized yield was achieved at a fraction of the processing cost. This study demonstrates that some ILs can compete with the cheapest pretreatment chemicals, such as ammonia, in terms of effectiveness and process cost, removing IL cost as a barrier to the economic viability of IL-based biorefineries

Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is an important global health problem. MRSA resistance to β-lactam antibiotics is mediated by the mecA or mecC genes, which encode an alternative penicillin-binding protein (PBP) 2a that has a low affinity to β-lactam antibiotics. Detection of mec genes or PBP2a is regarded as the gold standard for the diagnosis of MRSA. We identified four MRSA isolates that lacked mecA or mecC genes, but were still phenotypically resistant to pencillinase-resistant β-lactam antibiotics. METHODS: The four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays. RESULTS: We identified a number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 that were found in the resistant isolates but were absent in closely related susceptible isolates and which may be the basis of resistance. Of particular interest are three identical amino acid substitutions in PBPs 1, 2 and 3, occurring independently in isolates from at least two separate multilocus sequence types. Two different non-conservative substitutions were also present in the same amino acid of PBP1 in two isolates from two different sequence types. CONCLUSIONS: This work suggests that phenotypically resistant MRSA could be misdiagnosed using molecular methods alone and provides evidence of alternative mechanisms for β-lactam resistance in MRSA that may need to be considered by diagnostic laboratories

The dynamics of apparent horizons in Robinson-Trautman spacetimes

We present an alternative scheme of finding apparent horizons based on spectral methods applied to Robinson-Trautman spacetimes. We have considered distinct initial data such as representing the spheroids of matter and the head-on collision of two non-rotating black holes. The evolution of the apparent horizon is presented. We have obtained in some cases a mass gap between the final Bondi and apparent horizon masses, whose implications were briefly commented in the light of the thermodynamics of black holes.Comment: 9 pages, 7 figure

Global emergence of a hypervirulent carbapenem-resistant <i>Escherichia coli </i>ST410 clone

Carbapenem-resistant Escherichia coli (CREC) ST410 has recently emerged as a major global health problem. Here, we report a shift in CREC prevalence in Chinese hospitals between 2017 and 2021 with ST410 becoming the most commonly isolated sequence type. Genomic analysis identifies a hypervirulent CREC ST410 clone, B5/H24RxC, which caused two separate outbreaks in a children's hospital. It may have emerged from the previously characterised B4/H24RxC in 2006 and has been isolated in ten other countries from 2015 to 2021. Compared with B4/H24RxC, B5/H24RxC lacks the blaOXA-181-bearing X3 plasmid, but carries a F-type plasmid containing blaNDM-5. Most of B5/H24RxC also carry a high pathogenicity island and a novel O-antigen gene cluster. We find that B5/H24RxC grew faster in vitro and is more virulent in vivo. The identification of this newly emerged but already globally disseminated hypervirulent CREC clone, highlights the ongoing evolution of ST410 towards increased resistance and virulence. </p