3D media stabilizes membrane and prolongs lifespan of defolliculated Xenopus laevis oocytes

Xenopus laevis oocytes are commonly used in many fundamental biological studies. One of the major limitations of X. laevis oocytes is their short storage lifespan with most defolliculated oocytes physically deteriorating in 10 days or less. Herein, we identified a 3D Cultrex-based storage media that incorporates extracellular membrane-based hydrogels to maintain oocyte integrity. Under these treatments, the lifespan of the oocytes increased to more than 20 days compared to standard conditions. The treatment preserved the oocytes membrane integrity and did not interfere with mRNA- or cDNA-derived protein expression

High glucose enhances store-operated calcium entry by upregulating ORAI/STIM via calcineurin-NFAT signalling

© 2014, Springer-Verlag Berlin Heidelberg. Abstract: ORAI and stromal interaction molecule (STIM) are store-operated channel molecules that play essential roles in human physiology through a coupling mechanism of internal Ca 2+ store to Ca 2+ influx. However, the roles of ORAI and STIM in vascular endothelial cells under diabetic conditions remain unknown. Here, we investigated expression and signalling pathways of ORAI and STIM regulated by high glucose or hyperglycaemia using in vitro cell models, in vivo diabetic mice and tissues from patients. We found that ORAI1-3 and STIM1-2 were ubiquitously expressed in human vasculatures. Their expression was upregulated by chronic treatment with high glucose (HG, 25 mM d-glucose), which was accompanied by enhanced store-operated Ca 2+ influx in vascular endothelial cells. The increased expression was also observed in the aortae from genetically modified Akita diabetic mice (C57BL/6-Ins2 Akita /J) and streptozocin-induced diabetic mice, and aortae from diabetic patients. HG-induced upregulation of ORAI and STIM genes was prevented by the calcineurin inhibitor cyclosporin A and NFATc3 siRNA. Additionally, in vivo treatment with the nuclear factor of activated T cells (NFAT) inhibitor A-285222 prevented the gene upregulation in Akita mice. However, HG had no direct effects on ORAI1-3 currents and the channel activation process through cytosolic STIM1 movement in the cells co-expressing STIM1-EYFP/ORAIs. We concluded that upregulation of STIM/ORAI through Ca 2+ -calcineurin-NFAT pathway is a novel mechanism causing abnormal Ca 2+ homeostasis and endothelial dysfunction under hyperglycaemia. Key message: ORAI1-3 and STIM1-2 are ubiquitously expressed in vasculatures and upregulated by high glucose.Increased expression is confirmed in Akita (Ins2 Akita /J) and STZ diabetic mice and patients.Upregulation mechanism is mediated by Ca 2+ /calcineurin/NFATc3 signalling.High glucose has no direct effects on ORAI1-3 channel activity and channel activation process

Individualized Clinical Practice Guidelines for Pressure Injury Management: Development of an Integrated Multi-Modal Biomedical Information Resource

Background: Pressure ulcers (PU) and deep tissue injuries (DTI), collectively known as pressure injuries are serious complications causing staggering costs and human suffering with over 200 reported risk factors from many domains. Primary pressure injury prevention seeks to prevent the first incidence, while secondary PU/DTI prevention aims to decrease chronic recurrence. Clinical practice guidelines (CPG) combine evidence-based practice and expert opinion to aid clinicians in the goal of achieving best practices for primary and secondary prevention. The correction of all risk factors can be both overwhelming and impractical to implement in clinical practice. There is a need to develop practical clinical tools to prioritize the multiple recommendations of CPG, but there is limited guidance on how to prioritize based on individual cases. Bioinformatics platforms enable data management to support clinical decision support and user-interface development for complex clinical challenges such as pressure injury prevention care planning. Objective: The central hypothesis of the study is that the individual’s risk factor profile can provide the basis for adaptive, personalized care planning for PU prevention based on CPG prioritization. The study objective is to develop the Spinal Cord Injury Pressure Ulcer and Deep Tissue Injury (SCIPUD+) Resource to support personalized care planning for primary and secondary PU/DTI prevention. Methods: The study is employing a retrospective electronic health record (EHR) chart review of over 75 factors known to be relevant for pressure injury risk in individuals with a spinal cord injury (SCI) and routinely recorded in the EHR. We also perform tissue health assessments of a selected sub-group. A systems approach is being used to develop and validate the SCIPUD+ Resource incorporating the many risk factor domains associated with PU/DTI primary and secondary prevention, ranging from the individual’s environment to local tissue health. Our multiscale approach will leverage the strength of bioinformatics applied to an established national EHR system. A comprehensive model is being used to relate the primary outcome of interest (PU/DTI development) with over 75 PU/DTI risk factors using a retrospective chart review of 5000 individuals selected from the study cohort of more than 36,000 persons with SCI. A Spinal Cord Injury Pressure Ulcer and Deep Tissue Injury Ontology (SCIPUDO) is being developed to enable robust text-mining for data extraction from free-form notes. Results: The results from this study are pending. Conclusions: PU/DTI remains a highly significant source of morbidity for individuals with SCI. Personalized interactive care plans may decrease both initial PU formation and readmission rates for high-risk individuals. The project is using established EHR data to build a comprehensive, structured model of environmental, social and clinical pressure injury risk factors. The comprehensive SCIPUD+ health care tool will be used to relate the primary outcome of interest (pressure injury development) with covariates including environmental, social, clinical, personal and tissue health profiles as well as possible interactions among some of these covariates. The study will result in a validated tool for personalized implementation of CPG recommendations and has great potential to change the standard of care for PrI clinical practice by enabling clinicians to provide personalized application of CPG priorities tailored to the needs of each at-risk individual with SCI

Kepler-93b: A Terrestrial World Measured to within 120 km, and a Test Case for a New Spitzer Observing Mode

We present the characterization of the Kepler-93 exoplanetary system, based on three years of photometry gathered by the Kepler spacecraft. The duration and cadence of the Kepler observations, in tandem with the brightness of the star, enable unusually precise constraints on both the planet and its host. We conduct an asteroseismic analysis of the Kepler photometry and conclude that the star has an average density of 1.652+/-0.006 g/cm^3. Its mass of 0.911+/-0.033 M_Sun renders it one of the lowest-mass subjects of asteroseismic study. An analysis of the transit signature produced by the planet Kepler-93b, which appears with a period of 4.72673978+/-9.7x10^-7 days, returns a consistent but less precise measurement of the stellar density, 1.72+0.02-0.28 g/cm^3. The agreement of these two values lends credence to the planetary interpretation of the transit signal. The achromatic transit depth, as compared between Kepler and the Spitzer Space Telescope, supports the same conclusion. We observed seven transits of Kepler-93b with Spitzer, three of which we conducted in a new observing mode. The pointing strategy we employed to gather this subset of observations halved our uncertainty on the transit radius ratio R_p/R_star. We find, after folding together the stellar radius measurement of 0.919+/-0.011 R_Sun with the transit depth, a best-fit value for the planetary radius of 1.481+/-0.019 R_Earth. The uncertainty of 120 km on our measurement of the planet's size currently renders it one of the most precisely measured planetary radii outside of the Solar System. Together with the radius, the planetary mass of 3.8+/-1.5 M_Earth corresponds to a rocky density of 6.3+/-2.6 g/cm^3. After applying a prior on the plausible maximum densities of similarly-sized worlds between 1--1.5 R_Earth, we find that Kepler-93b possesses an average density within this group.Comment: 20 pages, 9 figures, accepted for publication in Ap

Analysis of the prostate cancer cell line LNCaP transcriptome using a sequencing-by-synthesis approach

BACKGROUND: High throughput sequencing-by-synthesis is an emerging technology that allows the rapid production of millions of bases of data. Although the sequence reads are short, they can readily be used for re-sequencing. By re-sequencing the mRNA products of a cell, one may rapidly discover polymorphisms and splice variants particular to that cell. RESULTS: We present the utility of massively parallel sequencing by synthesis for profiling the transcriptome of a human prostate cancer cell-line, LNCaP, that has been treated with the synthetic androgen, R1881. Through the generation of approximately 20 megabases (MB) of EST data, we detect transcription from over 10,000 gene loci, 25 previously undescribed alternative splicing events involving known exons, and over 1,500 high quality single nucleotide discrepancies with the reference human sequence. Further, we map nearly 10,000 ESTs to positions on the genome where no transcription is currently predicted to occur. We also characterize various obstacles with using sequencing by synthesis for transcriptome analysis and propose solutions to these problems. CONCLUSION: The use of high-throughput sequencing-by-synthesis methods for transcript profiling allows the specific and sensitive detection of many of a cell's transcripts, and also allows the discovery of high quality base discrepancies, and alternative splice variants. Thus, this technology may provide an effective means of understanding various disease states, discovering novel targets for disease treatment, and discovery of novel transcripts

JIB-04 has broad-spectrum antiviral activity and inhibits SARS-CoV-2 replication and coronavirus pathogenesis

Pathogenic coronaviruses are a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified small-molecule inhibitors that potently block the replication of severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with a 50% effective concentration of 695 nM, with a specificity index of greater than 1,000. JIB-04 showe

Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle

gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle

Identification of plasma lipid biomarkers for prostate cancer by lipidomics and bioinformatics

Background: Lipids have critical functions in cellular energy storage, structure and signaling. Many individual lipid molecules have been associated with the evolution of prostate cancer; however, none of them has been approved to be used as a biomarker. The aim of this study is to identify lipid molecules from hundreds plasma apparent lipid species as biomarkers for diagnosis of prostate cancer. Methodology/Principal Findings: Using lipidomics, lipid profiling of 390 individual apparent lipid species was performed on 141 plasma samples from 105 patients with prostate cancer and 36 male controls. High throughput data generated from lipidomics were analyzed using bioinformatic and statistical methods. From 390 apparent lipid species, 35 species were demonstrated to have potential in differentiation of prostate cancer. Within the 35 species, 12 were identified as individual plasma lipid biomarkers for diagnosis of prostate cancer with a sensitivity above 80%, specificity above 50% and accuracy above 80%. Using top 15 of 35 potential biomarkers together increased predictive power dramatically in diagnosis of prostate cancer with a sensitivity of 93.6%, specificity of 90.1% and accuracy of 97.3%. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) demonstrated that patient and control populations were visually separated by identified lipid biomarkers. RandomForest and 10-fold cross validation analyses demonstrated that the identified lipid biomarkers were able to predict unknown populations accurately, and this was not influenced by patient's age and race. Three out of 13 lipid classes, phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (ePE) and ether-linked phosphatidylcholine (ePC) could be considered as biomarkers in diagnosis of prostate cancer. Conclusions/Significance: Using lipidomics and bioinformatic and statistical methods, we have identified a few out of hundreds plasma apparent lipid molecular species as biomarkers for diagnosis of prostate cancer with a high sensitivity, specificity and accuracy