Creating Reporter Systems within Drosophila melanogaster

The objective of these projects was to generate two fluorescently based systems within Drosophila melanogaster for the advancement of molecular research. The first project was aimed to create fluorescent phosphomimetic constructs of m8, a regulator in eye formation. The second project was to create a fluorescent visual marker for heterochromatic gene silencing in Drosophila. Proper formation of the eye requires that only one cell from a group of equipotent cells assumes a neural fate (the R8 cell). The cells surrounding the R8 cell are prevented from assuming this outcome through a process known as lateral inhibition. Lateral inhibition uses the regulation of proteins from the Enhancer of split complex E(spl)C, to down regulate the neural protein, Atonal. The functionality of a specific E(spl) protein, M8, is regulated by phosphorylation by CK2 at a specific site within the phosphorylation domain of M8. It was the goal of this project to create fluorescently labeled phosphomimetic variants of M8 with aspartic acid and alanine substitutions at key phosphorylation sites (the CK2 phosphorylation site and all four phosphorylation sites within the M8 phosphorylation domain). These constructs were used to generate preliminary data in a Drosophila Expression System. The initial data indicated that M8S 159A showed statistical differences between the control and the treatment group, while M8, M8S159D, and M8SDDDD, showed no statistical difference between treatments and controls. The location of these constructs in Schneider 2 cells suggested that M8 and M8S159A proteins were at times located in the nucleus, while the other phosphomimetic variants were restricted to the cytoplasm. This project generated a solid foundation of data collection methods and gave some insight into the location of M8 that can give rise to future studies within this system.;The second project was aimed at creating a fluorescent marker for an epigenetic phenomenon known as Position Effect Variegation (PEV) within Drosophila. PEV is caused by the relocation of a gene close to, if not adjacent to heterochromatin. When in close proximity, the heterochromatin can spread to silence the gene in some cells, rendering the gene silent; while failing to spread in others, allowing for expression resulting in a variegated phenotype within Drosophila. Male flies with a miniwhite gene adjacent to Gal4/UAS driven Yellow Fluorescent Protein were mutated in hopes of causing a translocation or inversion which would bring the fluorescent marker in close proximity to heterochromatin to observe epigenetic silencing. Although no variegated flies were found, this tool would serve as an effective marker to visually observe, using different UAS drivers within Drosophila, where PEV is occurring spatially and at what developmental time points within the fly as well. Such a variegating mutant would provide a molecular tool for future research on how CK2 and other modifiers of PEV act within the cascade of epigenetic changes within Drosophila

Io’s Volcanic Activity from Time Domain Adaptive Optics Observations: 2013–2018

We present measurements of the near-infrared brightness of Io's hot spots derived from 2 to 5 μm imaging with adaptive optics on the Keck and Gemini N telescopes. The data were obtained on 271 nights between 2013 August and the end of 2018, and include nearly 1000 detections of over 75 unique hot spots. The 100 observations obtained between 2013 and 2015 have been previously published in de Kleer & de Pater the observations since the start of 2016 are presented here for the first time, and the analysis is updated to include the full five-year data set. These data provide insight into the global properties of Io's volcanism. Several new hot spots and bright eruptions have been detected, and the preference for bright eruptions to occur on Io's trailing hemisphere noted in the 2013–2015 data is strengthened by the larger data set and remains unexplained. The program overlapped in time with Sprint-A/EXCEED and Juno observations of the Jovian system, and correlations with transient phenomena seen in other components of the system have the potential to inform our understanding of the impact of Io's volcanism on Jupiter and its neutral/plasma environment

Io’s Volcanic Activity from Time Domain Adaptive Optics Observations: 2013–2018

We present measurements of the near-infrared brightness of Io's hot spots derived from 2 to 5 μm imaging with adaptive optics on the Keck and Gemini N telescopes. The data were obtained on 271 nights between 2013 August and the end of 2018, and include nearly 1000 detections of over 75 unique hot spots. The 100 observations obtained between 2013 and 2015 have been previously published in de Kleer & de Pater the observations since the start of 2016 are presented here for the first time, and the analysis is updated to include the full five-year data set. These data provide insight into the global properties of Io's volcanism. Several new hot spots and bright eruptions have been detected, and the preference for bright eruptions to occur on Io's trailing hemisphere noted in the 2013–2015 data is strengthened by the larger data set and remains unexplained. The program overlapped in time with Sprint-A/EXCEED and Juno observations of the Jovian system, and correlations with transient phenomena seen in other components of the system have the potential to inform our understanding of the impact of Io's volcanism on Jupiter and its neutral/plasma environment

Clinical outcomes and response to treatment of patients receiving topical treatments for pyoderma gangrenosum: a prospective cohort study

Background: pyoderma gangrenosum (PG) is an uncommon dermatosis with a limited evidence base for treatment. Objective: to estimate the effectiveness of topical therapies in the treatment of PG. Methods: prospective cohort study of UK secondary care patients with a clinical diagnosis of PG suitable for topical treatment (recruited July 2009 to June 2012). Participants received topical therapy following normal clinical practice (mainly Class I-III topical corticosteroids, tacrolimus 0.03% or 0.1%). Primary outcome: speed of healing at 6 weeks. Secondary outcomes: proportion healed by 6 months; time to healing; global assessment; inflammation; pain; quality-of-life; treatment failure and recurrence. Results: Sixty-six patients (22 to 85 years) were enrolled. Clobetasol propionate 0.05% was the most commonly prescribed therapy. Overall, 28/66 (43.8%) of ulcers healed by 6 months. Median time-to-healing was 145 days (95% CI: 96 days, ∞). Initial ulcer size was a significant predictor of time-to-healing (hazard ratio 0.94 (0.88;80 1.00); p = 0.043). Four patients (15%) had a recurrence. Limitations: No randomised comparator Conclusion: Topical therapy is potentially an effective first-line treatment for PG that avoids possible side effects associated with systemic therapy. It remains unclear whether more severe disease will respond adequately to topical therapy alone

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Human whole-exome genotype data for Alzheimer’s disease

The heterogeneity of the whole-exome sequencing (WES) data generation methods present a challenge to a joint analysis. Here we present a bioinformatics strategy for joint-calling 20,504 WES samples collected across nine studies and sequenced using ten capture kits in fourteen sequencing centers in the Alzheimer’s Disease Sequencing Project. The joint-genotype called variant-called format (VCF) file contains only positions within the union of capture kits. The VCF was then processed specifically to account for the batch effects arising from the use of different capture kits from different studies. We identified 8.2 million autosomal variants. 96.82% of the variants are high-quality, and are located in 28,579 Ensembl transcripts. 41% of the variants are intronic and 1.8% of the variants are with CADD &gt; 30, indicating they are of high predicted pathogenicity. Here we show our new strategy can generate high-quality data from processing these diversely generated WES samples. The improved ability to combine data sequenced in different batches benefits the whole genomics research community.</p