Rapid in situ imaging and whole genome sequencing of biofilm in neonatal feeding tubes: a clinical proof of concept

The bacterial flora of nasogastric feeding tubes and faecal samples were analysed for a low-birth weight (725g) neonate EGA 25 weeks in intensive care. Samples were collected at age 6 and 8 weeks of life. Optical coherence tomography (OCT) was used to visualise bacterial biofilms inside the nasogastric feeding tubes. The biofilm was heterogeneously distributed along the tube lumen wall, and had a depth of up to 500µm. The bacterial biofilm and faecal samples included Enterococcus faecalis and Enterobacter hormaechei. Representative strains, recovered from both feeding tubes and faecal samples, were whole genome sequenced using Illumina, Mi-Seq, which revealed indistinguishable strains, each with less than 28 SNP differences, of E. faecalis and E. hormaechei. The E. faecalis strains were from two sequence types (ST191 and ST211) and encoded for a number of traits related to biofilm formation (BopD), adherence (Epb pili), virulence (cps loci, gelatinase, SprE) and antibiotic resistances (IsaA, tetM). The E. hormaechei were all ST106, and encoded for blaACT-15 β–lactamase and fosfomycin resistance (fosA). This proof of concept study demonstrates that bacterial flora within the neonatal feeding tubes may influence the bacterial colonisation of the intestinal tract and can be visualised nondestructively using OCT

Evidence of fatal skeletal injuries on Malapa Hominins 1 and 2

Malapa is one of the richest early hominin sites in Africa and the discovery site of the hominin species, Australopithecus sediba. The holotype and paratype (Malapa Hominin 1 and 2, or MH1 and MH2, respectively) skeletons are among the most complete in the early hominin record. Dating to approximately two million years BP, MH1 and MH2 are hypothesized to have fallen into a natural pit trap. All fractures evident on MH1 and MH2 skeletons were evaluated and separated based on wet and dry bone fracture morphology/characteristics. Most observed fractures are post-depositional, but those in the right upper limb of the adult hominin strongly indicate active resistance to an impact, while those in the juvenile hominin mandible are consistent with a blow to the face. The presence of skeletal trauma independently supports the falling hypothesis and supplies the first evidence for the manner of death of an australopith in the fossil record that is not attributed to predation or natural death

Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes

Optical Coherence Tomography in the UK Biobank Study – Rapid Automated Analysis of Retinal Thickness for Large Population-Based Studies

Purpose: To describe an approach to the use of optical coherence tomography (OCT) imaging in large, population-based studies, including methods for OCT image acquisition, storage, and the remote, rapid, automated analysis of retinal thickness. Methods: In UK Biobank, OCT images were acquired between 2009 and 2010 using a commercially available “spectral domain” OCT device (3D OCT-1000, Topcon). Images were obtained using a raster scan protocol, 6 mm x 6 mm in area, and consisting of 128 B-scans. OCT image sets were stored on UK Biobank servers in a central repository, adjacent to high performance computers. Rapid, automated analysis of retinal thickness was performed using custom image segmentation software developed by the Topcon Advanced Biomedical Imaging Laboratory (TABIL). This software employs dual-scale gradient information to allow for automated segmentation of nine intraretinal boundaries in a rapid fashion. Results: 67,321 participants (134,642 eyes) in UK Biobank underwent OCT imaging of both eyes as part of the ocular module. 134,611 images were successfully processed with 31 images failing segmentation analysis due to corrupted OCT files or withdrawal of subject consent for UKBB study participation. Average time taken to call up an image from the database and complete segmentation analysis was approximately 120 seconds per data set per login, and analysis of the entire dataset was completed in approximately 28 days. Conclusions: We report an approach to the rapid, automated measurement of retinal thickness from nearly 140,000 OCT image sets from the UK Biobank. In the near future, these measurements will be publically available for utilization by researchers around the world, and thus for correlation with the wealth of other data collected in UK Biobank. The automated analysis approaches we describe may be of utility for future large population-based epidemiological studies, clinical trials, and screening programs that employ OCT imaging

Mercury dynamics in a San Francisco estuary tidal wetland : assessing dynamics using in situ measurements

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Estuaries and Coasts 35 (2012): 1036-1048, doi:10.1007/s12237-012-9501-3.We used high-resolution in situ measurements of turbidity and fluorescent dissolved organic matter (FDOM) to quantitatively estimate the tidally driven exchange of mercury (Hg) between the waters of the San Francisco estuary and Browns Island, a tidal wetland. Turbidity and FDOM—representative of particle-associated and filter-passing Hg, respectively—together predicted 94 % of the observed variability in measured total mercury concentration in unfiltered water samples (UTHg) collected during a single tidal cycle in spring, fall, and winter, 2005–2006. Continuous in situ turbidity and FDOM data spanning at least a full spring-neap period were used to generate UTHg concentration time series using this relationship, and then combined with water discharge measurements to calculate Hg fluxes in each season. Wetlands are generally considered to be sinks for sediment and associated mercury. However, during the three periods of monitoring, Browns Island wetland did not appreciably accumulate Hg. Instead, gradual tidally driven export of UTHg from the wetland offset the large episodic on-island fluxes associated with high wind events. Exports were highest during large spring tides, when ebbing waters relatively enriched in FDOM, dissolved organic carbon (DOC), and filter-passing mercury drained from the marsh into the open waters of the estuary. On-island flux of UTHg, which was largely particle-associated, was highest during strong winds coincident with flood tides. Our results demonstrate that processes driving UTHg fluxes in tidal wetlands encompass both the dissolved and particulate phases and multiple timescales, necessitating longer term monitoring to adequately quantify fluxes.This work was supported by funding from the California Bay Delta Authority Ecosystem Restoration and Drinking Water Programs (grant ERP-00- G01) and matching funds from the United States Geological Survey Cooperative Research Program

T-Cell Epitope Prediction: Rescaling Can Mask Biological Variation between MHC Molecules

Theoretical methods for predicting CD8+ T-cell epitopes are an important tool in vaccine design and for enhancing our understanding of the cellular immune system. The most popular methods currently available produce binding affinity predictions across a range of MHC molecules. In comparing results between these MHC molecules, it is common practice to apply a normalization procedure known as rescaling, to correct for possible discrepancies between the allelic predictors. Using two of the most popular prediction software packages, NetCTL and NetMHC, we tested the hypothesis that rescaling removes genuine biological variation from the predicted affinities when comparing predictions across a number of MHC molecules. We found that removing the condition of rescaling improved the prediction software's performance both qualitatively, in terms of ranking epitopes, and quantitatively, in the accuracy of their binding affinity predictions. We suggest that there is biologically significant variation among class 1 MHC molecules and find that retention of this variation leads to significantly more accurate epitope prediction

Toll-like receptor gene polymorphisms are associated with susceptibility to graves' ophthalmopathy in Taiwan males

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs) are a family of pattern-recognition receptors, which plays a role in eliciting innate/adaptive immune responses and developing chronic inflammation. The polymorphisms of TLRs have been associated with the risk of various autoimmune diseases, including systemic lupus erythematosus (SLE), multiple sclerosis and rheumatorid arthritis. The aim of this study was to evaluate whether TLR genes could be used as genetic markers for the development of Graves' ophthalmopathy (GO).</p> <p>Methods</p> <p>6 TLR-4 and 2 TLR-9 gene polymorphisms in 471 GD patients (200 patients with GO and 271 patients without GO) from a Taiwan Chinese population were evaluated.</p> <p>Results</p> <p>No statistically significant difference was observed in the genotypic and allelic frequencies of TLR-4 and TLR-9 gene polymorphisms between the GD patients with and without GO. However, sex-stratified analyses showed that the association between TLR-9 gene polymorphism and GO phenotype was more pronounced in the male patients. The odds ratios (ORs) was 2.11 (95% confidence interval [CI] = 1.14-3.91) for rs187084 AàG polymorphism and 1.97 (95% CI = 1.07-3.62) for rs352140 AàG polymorphism among the male patients. Increasing one G allele of rs287084 and one A allele of rs352140 increased the risk of GO (<it>p </it>values for trend tests were 0.0195 and 0.0345, respectively). Further, in haplotype analyses, the male patients carrying the GA haplotype had a higher risk of GO (odds ratio [OR] = 2.02, 95% confidence interval [CI] = 1.09-3.73) than those not carrying the GA haplotype.</p> <p>Conclusion</p> <p>The present data suggest that TLR-9 gene polymorphisms were significantly associated with increased susceptibility of ophthalmopathy in male GD patients.</p

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

Expanding the Repertoire of Modified Vaccinia Ankara-Based Vaccine Vectors via Genetic Complementation Strategies

nkara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines. during infection, and that the processes governing the generation of antiviral antibody responses are more readily saturated by viral antigen than are those that elicit CD8+ T cell responses. deletion, enables the generation of novel replication-defective MVA mutants and expands the repertoire of genetic viral variants that can now be explored as improved vaccine vectors