A Drosophila functional evaluation of candidates from human genome-wide association studies of type 2 diabetes and related metabolic traits identifies tissue-specific roles for dHHEX

BACKGROUND: Genome-wide association studies (GWAS) identify regions of the genome that are associated with particular traits, but do not typically identify specific causative genetic elements. For example, while a large number of single nucleotide polymorphisms associated with type 2 diabetes (T2D) and related traits have been identified by human GWAS, only a few genes have functional evidence to support or to rule out a role in cellular metabolism or dietary interactions. Here, we use a recently developed Drosophila model in which high-sucrose feeding induces phenotypes similar to T2D to assess orthologs of human GWAS-identified candidate genes for risk of T2D and related traits. RESULTS: Disrupting orthologs of certain T2D candidate genes (HHEX, THADA, PPARG, KCNJ11) led to sucrose-dependent toxicity. Tissue-specific knockdown of the HHEX ortholog dHHEX (CG7056) directed metabolic defects and enhanced lethality; for example, fat-body-specific loss of dHHEX led to increased hemolymph glucose and reduced insulin sensitivity. CONCLUSION: Candidate genes identified in human genetic studies of metabolic traits can be prioritized and functionally characterized using a simple Drosophila approach. To our knowledge, this is the first large-scale effort to study the functional interaction between GWAS-identified candidate genes and an environmental risk factor such as diet in a model organism system

A <em>Drosophila</em> Model of High Sugar Diet-Induced Cardiomyopathy

<div><p>Diets high in carbohydrates have long been linked to progressive heart dysfunction, yet the mechanisms by which chronic high sugar leads to heart failure remain poorly understood. Here we combine diet, genetics, and physiology to establish an adult <em>Drosophila melanogaster</em> model of chronic high sugar-induced heart disease. We demonstrate deterioration of heart function accompanied by fibrosis-like collagen accumulation, insulin signaling defects, and fat accumulation. The result was a shorter life span that was more severe in the presence of reduced insulin and P38 signaling. We provide evidence of a role for hexosamine flux, a metabolic pathway accessed by glucose. Increased hexosamine flux led to heart function defects and structural damage; conversely, cardiac-specific reduction of pathway activity prevented sugar-induced heart dysfunction. Our data establish <em>Drosophila</em> as a useful system for exploring specific aspects of diet-induced heart dysfunction and emphasize enzymes within the hexosamine biosynthetic pathway as candidate therapeutic targets.</p> </div

<i>Drosophila</i> model of diabetic cardiomyopathy.

<p>(A) Ventral view of the heart tube. 3D structures are shown. The heart was stained for F-Actin with phalloidin (magenta) and for nuclei with DAPI; Cypher-GFP (green) labeled Z-lines of myofibers within cardiomyocytes. Arrowheads indicate the non-myocardial longitudinal muscle fibers, asterisks the alary muscles that support the heart, and arrows the abdominal muscles. (B) Dorsal view of the heart tube. Myocardial cells wrap in a circular fashion around the central cavity. Arrows show the ostia through which hemolymph from the abdomen enters into the heart tube and circulates. (C) HSD significantly reduced life span. <i>w¹¹¹⁸</i> male flies were raised in 0.15 M or 1.0 M sucrose diet, food was changed every 2–3 days, and flies were counted every 10 days. HSD-fed flies displayed decreased median life span, 10 days shorter than flies fed an LSD. Mean ± SE are shown; n = 50 total flies in two separate experiments. The estimated median life span of HSD flies was also expressed as the percentage of LSD flies (*p = 7.61E-08 by log rank test). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003175#pgen.1003175.s001" target="_blank">Figure S1</a>. (D) Hemolymph glucose concentrations in 3-week-old, control and HSD-fed <i>w¹¹¹⁸</i> adult flies. n≥6. (E) Hemolymph trehalose concentrations in 3-week-old, control and high sucrose-fed <i>w¹¹¹⁸</i> adult flies. n≥6. (F) Bodies from <i>w¹¹¹⁸</i> adults fed LSD or HSD for 3 weeks were treated with insulin (1 µM) or vehicle and visualized using antibodies against <i>Drosophila</i> PO₄-Akt or Syntaxin. n = 10. Bands from Western blot experiments were quantified, and PO₄-Akt was normalized to Syntaxin as a loading control. (G) Total triglycerides (TAG) were assayed enzymatically in 3-week-old control and high sugar-fed <i>w¹¹¹⁸</i> adult flies, and normalized to weight. n≥12. Mean ± SE are shown. An unpaired, two-tailed t-test was used to derive p-values.</p

High sucrose shortened <i>Drosophila</i> life span and was associated with increased cardiac arrhythmia and heart deterioration.

<p>(A) Representative M-mode (5 seconds) from flies fed LSD and HSD. Three-week-old adults fed an HSD showed moderate cardiac arrhythmia; at six weeks arrhythmicity was increased. (B) Arrhythmia index obtained from <i>w¹¹¹⁸</i> flies fed LSD and HSD. Arrhythmias observed in M-mode can be quantified as arrhythmia index, which is the standard deviation of all heart periods in each record normalized to the median heart period for each fly. Mean ± SE are shown. At week three, a significant increase in arrhythmia index was observed in HSD fed flies (0.44) compared to low sucrose fed flies (0.16) (*P = 1.54E-17 by F-test). Arrhythmia index of six-week-old flies increased to 0.66 in HSD and 0.26 in low sucrose diet, respectively (*P = 6.01E-12 by F-test). Data are means ± SE. (C) Heart period of adult files fed low vs. high dietary sucrose. At three weeks of age, no difference was observed between HSD- and LSD-fed flies. Heart period was significantly increased at six weeks of age in both HSD- and LSD-fed flies (*P = 9.64E-06 and 1.44E-09, respectively, by t-test). Interestingly at six weeks of age, heart period of HSD-fed flies was shorter than that of low sucrose fed flies (*P = 0.015 by t-test). Data are means ± SE. (D) Fractional shortening of adult flies fed low <i>vs.</i> high dietary sucrose. At three weeks of age, no difference was observed between HSD- and LSD-fed flies; however, fractional shortening was significantly decreased in flies fed HSD- <i>vs.</i> LSD-fed (*P = 0.043 by t-test). Data are means ± SE. (E) Quantification of Pericardin level of adult heart by Western blot. Eight hearts from three-week-old LSD- and HSD-fed flies, respectively, were loaded. Pericardin level was detected by a monoclonal antibody against Pericardin, and normalized to Actin level. (F,G) Representative confocal images of three-week-old adult fly hearts expressing Cypher-GFP (posterior A2/anterior A3 segment) and stained with anti-Pericardin (magenta) antibody. Pericardin levels in hearts of flies fed an HSD were increased compared to those fed low sucrose. Note that Pericardin was detected in heart tissue but not abdominal muscles. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003175#pgen.1003175.s004" target="_blank">Figure S4</a>. (H) Fly hearts from <i>w¹¹¹⁸</i> adults fed LSD or HSD were treated with insulin (2.5 µM) or vehicle and visualized using antibodies against <i>Drosophila</i> PO₄-Akt, PO₄-4EBP or Actin, showing the response of the heart to exogenous insulin challenge. n = 3. Bands from Western blot experiments were quantified, and PO₄-Akt and PO₄-4EBP were normalized to Actin as a loading control. The ratio of HSD fed flies was then normalized to that of LSD fed flies. PO₄-Akt and PO₄-4EBP level were 74.3% and 13.9%, respectively, in HSD-fed flies compared to LSD-fed flies (P = 0.049 and 0.0003, respectively, by t-test). (I) Heart accumulated triglycerides (TAG) were assayed enzymatically in 15 hearts from 3-week-old LSD- and HSD-fed <i>w¹¹¹⁸</i> adult flies, and normalized to protein level. n = 2. (P = 0.028 by t-test). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003175#pgen.1003175.s004" target="_blank">Figure S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003175#pgen.1003175.s010" target="_blank">Videos S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003175#pgen.1003175.s011" target="_blank">S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003175#pgen.1003175.s012" target="_blank">S3</a>.</p

Efficacy and Safety of COVID-19 Convalescent Plasma in Hospitalized Patients: A Randomized Clinical Trial

There is clinical equipoise for COVID-19 convalescent plasma (CCP) use in patients hospitalized with COVID-19. To determine the safety and efficacy of CCP compared with placebo in hospitalized patients with COVID-19 receiving noninvasive supplemental oxygen. CONTAIN COVID-19, a randomized, double-blind, placebo-controlled trial of CCP in hospitalized adults with COVID-19, was conducted at 21 US hospitals from April 17, 2020, to March 15, 2021. The trial enrolled 941 participants who were hospitalized for 3 or less days or presented 7 or less days after symptom onset and required noninvasive oxygen supplementation. A unit of approximately 250 mL of CCP or equivalent volume of placebo (normal saline). The primary outcome was participant scores on the 11-point World Health Organization (WHO) Ordinal Scale for Clinical Improvement on day 14 after randomization; the secondary outcome was WHO scores determined on day 28. Subgroups were analyzed with respect to age, baseline WHO score, concomitant medications, symptom duration, CCP SARS-CoV-2 titer, baseline SARS-CoV-2 serostatus, and enrollment quarter. Outcomes were analyzed using a bayesian proportional cumulative odds model. Efficacy of CCP was defined as a cumulative adjusted odds ratio (cOR) less than 1 and a clinically meaningful effect as cOR less than 0.8. Of 941 participants randomized (473 to placebo and 468 to CCP), 556 were men (59.1%); median age was 63 years (IQR, 52-73); 373 (39.6%) were Hispanic and 132 (14.0%) were non-Hispanic Black. The cOR for the primary outcome adjusted for site, baseline risk, WHO score, age, sex, and symptom duration was 0.94 (95% credible interval [CrI], 0.75-1.18) with posterior probability (P[cOR<1] = 72%); the cOR for the secondary adjusted outcome was 0.92 (95% CrI, 0.74-1.16; P[cOR<1] = 76%). Exploratory subgroup analyses suggested heterogeneity of treatment effect: at day 28, cORs were 0.72 (95% CrI, 0.46-1.13; P[cOR<1] = 93%) for participants enrolled in April-June 2020 and 0.65 (95% CrI, 0.41 to 1.02; P[cOR<1] = 97%) for those not receiving remdesivir and not receiving corticosteroids at randomization. Median CCP SARS-CoV-2 neutralizing titer used in April to June 2020 was 1:175 (IQR, 76-379). Any adverse events (excluding transfusion reactions) were reported for 39 (8.2%) placebo recipients and 44 (9.4%) CCP recipients (P = .57). Transfusion reactions occurred in 2 (0.4) placebo recipients and 8 (1.7) CCP recipients (P = .06). In this trial, CCP did not meet the prespecified primary and secondary outcomes for CCP efficacy. However, high-titer CCP may have benefited participants early in the pandemic when remdesivir and corticosteroids were not in use. ClinicalTrials.gov Identifier: NCT04364737