Maintenance of glucose-sensitive insulin secretion of cryopreserved human islets with University of Wisconsin solution and ascorbic acid-2 glucoside

Normal human islet cells are an ideal source for pancreas-targeted cell therapies, but the availability of human donor pancreata for islet isolation is severely limited. To effectively utilize such scarce donor organs for cell therapies, it is crucial to develop an excellent isolation, effective cryopreservation, and efficient gene transfer techniques for the transportation of isolated cells. In the present study, we investigate the effect of University of Wisconsin (UW) solution and ascorbic acid-2 glucoside (AA2G) on the cryopreservation of human islets. We also evaluate the gene transfer efficiency of a lentiviral vector expressing the E. coli LacZ gene, Lt-NLS/LacZ, in human islets. Human islets were isolated with a standard digestion method at the University of Alberta. Isolated islets were transported to Japan for 40 h and then subjected to cryopreservation experiments. The following preservation solutions were tested: UW solution with 100 mug/mL of AA2G, UW solution, 100% fetal bovine serum (FBS), and CMRL supplemented with 10% FBS. Following three months of cryopreservation, the islets were thawed and analyzed for viability, glucose-sensitive insulin secretion, proinsulin gene expression profile, and in vivo engraftment. The islets were also subjected to monolayer formation with 804G-cell-line-derived extracellular matrix (ECM), followed by Lt-NLS/LacZ transduction. The viability, morphology, glucose-sensitive insulin secretion, proinsulin gene expression, and monolayer formation efficiency of the thawed cryopreserved islets are significantly better maintained by the use of UW solution. When AA2G (100 mug/mL) is combined with UW, such parameters are further improved. The adequate engraftment of UW + AA2G-cryopreserved human islets is achieved in the liver of nude mice. Efficient Lt-NLS/LacZ transduction is identified in monolayered islets cryopreserved with UW solution with AA2G. The present work demonstrates that the combination of UW solution with AA2G (100 mug/mL) would be a useful cryopreservation means for human islets. Human islets monolayer-cultured with 804G-derived ECM are efficiently transduced with a lentiviral vector Lt-NLS/LacZ

Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets

Using a unique microarray platform for cytosine methylation profiling, the DNA methylation landscape of the human genome was monitored at more than 21,000 sites, including 79% of the annotated transcriptional start sites (TSS). Analysis of an oligodendroglioma derived cell line LN-18 revealed more than 4000 methylated TSS. The gene-centric analysis indicated a complex pattern of DNA methylation exists along each autosome, with a trend of increasing density approaching the telomeres. Remarkably, 2% of CpG islands (CGI) were densely methylated, and 17% had significant levels of 5 mC, whether or not they corresponded to a TSS. Substantial independent verification, obtained from 95 loci, suggested that this approach is capable of large scale detection of cytosine methylation with an accuracy approaching 90%. In addition, we detected large genomic domains that are also susceptible to DNA methylation reinforced inactivation, such as the HOX cluster on chromosome 7 (CH7). Extrapolation from the data suggests that more than 2000 genomic loci may be susceptible to methylation and associated inactivation, and most have yet to be identified. Finally, we report six new targets of epigenetic inactivation (IRX3, WNT10A, WNT6, RARalpha, BMP7 and ZGPAT). These targets displayed cell line and tumor specific differential methylation when compared with normal brain samples, suggesting they may have utility as biomarkers. Uniquely, hypermethylation of the CGI within an IRX3 exon was correlated with over-expression of IRX3 in tumor tissues and cell lines relative to normal brain samples

An accurate in vitro model of the E. coli envelope

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir-Blodgett and Langmuir-Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development

The effects of perceived and received support on objective performance outcome.

This is a postprint of an article published in European Journal of Sport Science, 2008, Vol. 8, Issue 6, pp. 359 – 368 © 2008 copyright Taylor & Francis. European Journal of Sport Science is available online at: http://www.tandfonline.com/loi/tejs20In this study, we examined the main and stress-buffering effects of perceived and received support upon objective performance outcome. The sample consisted of 123 male British high performance golfers, mean age 25.3 years (SD = 5.4). Participants completed measures of perceived support, stressors, stress, and received support before competitions. After the competitions, performance outcome (number of shots) was recorded. When both types of support were considered separately, there were significant main effects for perceived (ΔR2 = .08, b = -.81, p < .01) and received support (ΔR2 = .05, b = -.68, p < .01) on performance. There were also significant stress-buffering effects for perceived (ΔR2 = .03, b = -.48, p = .02) and received support (ΔR2 = .06, b = -.61, p < .01). When both types of support were considered simultaneously, the significant main effect (DR2 = .09, p < .01) was primarily attributable to perceived support (b = -.63, p = .02). The significant stress-buffering effect (DR2 = .06, p = .01) was primarily attributable to received support (b = -.56, p = .04). These results demonstrate the beneficial influence of social support on performance. The findings highlight the need to recognise the distinction between perceived and received support, both in terms of theory and the design of social support interventions with athletes

Job searching with a history of drugs and crime

This article explores the experiences and aspirations of offenders with histories of substance misuse in job searching. The analysis is based upon qualitative data from a localised study of 27 men and two women who were undertaking community-based court orders in Scotland. Their perspectives on job searching, job-readiness and aspirations for sustained employment including the role of self-employment are presented. The article concludes that with adequate support, greater tolerance and flexibility by employers and job searchers could contribute to reducing the vicious cycle of suspicion and dishonesty. Policy action needs to be sustained and possibly augmented to include enterprise training

Sars-Cov-2 Serostatus and Covid-19 Illness Characteristics By Variant Time Period in Non-Hospitalized Children and adolescents

OBJECTIVE: to describe COVID-19 illness characteristics, risk factors, and SARS-CoV-2 serostatus by variant time period in a large community-based pediatric sample. DESIGN: Data were collected prospectively over four timepoints between October 2020 and November 2022 from a population-based cohort ages 5 to 19 years old. SETTING: State of Texas, USA. PARTICIPANTS: Participants ages 5 to 19 years were recruited from large pediatric healthcare systems, Federally Qualified Healthcare Centers, urban and rural clinical practices, health insurance providers, and a social media campaign. EXPOSURE: SARS-CoV-2 infection. MAIN OUTCOME(S) AND MEASURE(S): SARS-CoV-2 antibody status was assessed by the Roche Elecsys RESULTS: Over half (57.2%) of the sample (N = 3911) was antibody positive. Symptomatic infection increased over time from 47.09% during the pre-Delta variant time period, to 76.95% during Delta, to 84.73% during Omicron, and to 94.79% during the Omicron BA.2. Those who were not vaccinated were more likely (OR 1.71, 95% CI 1.47, 2.00) to be infected versus those fully vaccinated. CONCLUSIONS: Results show an increase in symptomatic COVID-19 infection among non-hospitalized children with each progressive variant over the past two years. Findings here support the public health guidance that eligible children should remain up to date with COVID-19 vaccinations

Third WHO Global Consultation on regulatory requirements for xenotransplantation clinical trials, Changsha, Hunan, China December 12-14, 2018: "The 2018 Changsha Communiqué" The 10-Year Anniversary of The International Consultation on Xenotransplantation

After feedback from the working parties, the final session focused on drafting proposed revisions of the WHO documents, and resulted in the formulation of the draft “Third WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials, The 2018 Changsha Communiqué.” This draft was submitted to WHO in February 2019 for WHO and World Health Assembly consideration. If approved, the 2018 Changsha Communiqué will then be posted on the websites of WHO, IXA, and TTS, and published in Xenotransplantation. This report includes summaries of the various sessions, followed by the abstracts of invited speakers from the update sessions

Antibody Duration after infection From Sars-Cov-2 in the Texas Coronavirus antibody Response Survey

Understanding the duration of antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that causes COVID-19 is important to controlling the current pandemic. Participants from the Texas Coronavirus Antibody Response Survey (Texas CARES) with at least 1 nucleocapsid protein antibody test were selected for a longitudinal analysis of antibody duration. A linear mixed model was fit to data from participants (n = 4553) with 1 to 3 antibody tests over 11 months (1 October 2020 to 16 September 2021), and models fit showed that expected antibody response after COVID-19 infection robustly increases for 100 days postinfection, and predicts individuals may remain antibody positive from natural infection beyond 500 days depending on age, body mass index, smoking or vaping use, and disease severity (hospitalized or not; symptomatic or not)

Roles of residues in the interface of transient protein-protein complexes before complexation

Transient protein-protein interactions play crucial roles in all facets of cellular physiology. Here, using an analysis on known 3-D structures of transient protein-protein complexes, their corresponding uncomplexed forms and energy calculations we seek to understand the roles of protein-protein interfacial residues in the unbound forms. We show that there are conformationally near invariant and evolutionarily conserved interfacial residues which are rigid and they account for ∼65% of the core interface. Interestingly, some of these residues contribute significantly to the stabilization of the interface structure in the uncomplexed form. Such residues have strong energetic basis to perform dual roles of stabilizing the structure of the uncomplexed form as well as the complex once formed while they maintain their rigid nature throughout. This feature is evolutionarily well conserved at both the structural and sequence levels. We believe this analysis has general bearing in the prediction of interfaces and understanding molecular recognition