DNA methylation, epialleles and gene regulation: Insights from the honey bee

The development of a multicellular organism is dependent upon interactions between the underlying genetic sequence, epigenomic processes and environmental cues. These interactions are complex, and understanding the exact role that each plays in directing transcription remains difficult. One of the more well studied epigenomic modifications, cytosine DNA methylation, plays a fundamental role in modulating gene activity. This modification has primarily been associated with gene repression, but recent evidence has highlighted that DNA methylation also correlates with gene activation, and that variation in methylation patterning occurs across species. In the honey bee DNA methylation predominately occurs intragenically, and is associated with active transcription. The discovery that DNA methylation is critical to caste determination in the bee and additional correlations between DNA methylation, behaviour and phenotype, has made the honey bee an interesting model organism from which we can gain insight into the role of DNA methylation. Whilst it has been shown extensively in plants and mammals that DNA methylation patterns are frequently dependent on genotype, few studies of the honey bee have interpreted differential DNA methylation patterns in the context of the underlying genetic sequence. Here, we show that differences in the underlying genetic sequence correlate with differential methylation of the gene coding for lysosomal α-mannosidase (LAM), demonstrating that several LAM epialleles exist in the honey bee population. These epialleles correlate with context-dependent transcriptional changes, with significant effects on LAM expression observed during larval development. To understand how such changes might affect larval growth and phenotype we used a knockdown approach, inhibiting LAM activity during early larval stages using the indolizidine alkaloid swainsonine. We show that such treatment causes sub-lethal effects during larval and pupal stages, and causes loco-like symptoms and death in newly emerged bees. Our findings suggest that LAM function in the honey bee is conserved, and we propose that the LAM epialleles might affect larval metabolism and growth. By influencing metabolism in the bee the LAM epialleles could generate substantial variation in complex traits, which would be beneficial to a colony whose stability is dependent upon high levels of phenotypic variation

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Developmental and loco-like effects of a swainsonine-induced inhibition of a-mannosidase in the honey bee, Apis mellifera

Background Deficiencies in lysosomal a-mannosidase (LAM) activity in animals, caused either by mutations or by consuming toxic alkaloids, lead to severe phenotypic and behavioural consequences. Yet, epialleles adversely affecting LAM expression exist in the honey bee population suggesting that they might be beneficial in certain contexts and cannot be eliminated by natural selection. Methods We have used a combination of enzymology, molecular biology and metabolomics to characterise the catalytic properties of honey bee LAM (AmLAM) and then used an indolizidine alkaloid swainsonine to inhibit its activity in vitro and in vivo. Results We show that AmLAM is inhibited in vitro by swainsonine albeit at slightly higher concentrations than in other animals. Dietary exposure of growing larvae to swainsonine leads to pronounced metabolic changes affecting not only saccharides, but also amino acids, polyols and polyamines. Interestingly, the abundance of two fatty acids implicated in epigenetic regulation is significantly reduced in treated individuals. Additionally, swainsonie causes loco-like symptoms, increased mortality and a subtle decrease in the rate of larval growth resulting in a subsequent developmental delay in pupal metamorphosis. Discussion We consider our findings in the context of cellular LAM function, larval development, environmental toxicity and colony-level impacts. The observed developmental heterochrony in swainsonine-treated larvae with lower LAM activity offer a plausible explanation for the existence of epialleles with impaired LAM expression. Individuals carrying such epialleles provide an additional level of epigenetic diversity that could be beneficial for the functioning of a colony whereby more flexibility in timing of adult emergence might be useful for task allocation

Differentially methylated obligatory epialleles modulate context-dependent <i>LAM</i> gene expression in the honeybee <i>Apis mellifera</i>

<p>Differential intragenic methylation in social insects has been hailed as a prime mover of environmentally driven organismal plasticity and even as evidence for genomic imprinting. However, very little experimental work has been done to test these ideas and to prove the validity of such claims. Here we analyze in detail differentially methylated obligatory epialleles of a conserved gene encoding lysosomal α-mannosidase (<i>AmLAM</i>) in the honeybee. We combined genotyping of progenies derived from colonies founded by single drone inseminated queens, ultra-deep allele-specific bisulfite DNA sequencing, and gene expression to reveal how sequence variants, DNA methylation, and transcription interrelate. We show that both methylated and non-methylated states of <i>AmLAM</i> follow Mendelian inheritance patterns and are strongly influenced by polymorphic changes in DNA. Increased methylation of a given allele correlates with higher levels of context-dependent <i>AmLAM</i> expression and appears to affect the transcription of an antisense long noncoding RNA. No evidence of allelic imbalance or imprinting involved in this process has been found. Our data suggest that by generating alternate methylation states that affect gene expression, sequence variants provide organisms with a high level of epigenetic flexibility that can be used to select appropriate responses in various contexts. This study represents the first effort to integrate DNA sequence variants, gene expression, and methylation in a social insect to advance our understanding of their relationships in the context of causality.</p

Variation in Racial Disparities in Liver Transplant Outcomes Across Transplant Centers in the United States.

Little is known about the role that transplant centers may play in perpetuating racial disparities after liver transplantation, which are unexplained by patient-level factors. We examined variation in between-center and within-center disparities among 34,114 Black and White liver transplant recipients in the United States from 2010 to 2017 using Scientific Registry of Transplant Recipient (SRTR) data. We used Cox proportional hazards models to calculate transplant center-specific Black-White hazard ratios and hierarchical survival analysis to examine potential effect modification of the race-survival association by transplant center characteristics, including transplant volume, proportion of Black patients, SRTR quality rating, and region. Models were sequentially adjusted for clinical, socioeconomic, and center characteristics. After adjustment, Black patients experienced 1.11 excess deaths after liver transplant per 100 person-years compared with White patients (95% confidence interval [CI], 0.65-1.56), corresponding to a 21% increased mortality risk (95% CI, 1.12-1.31). Although there was substantial variation in this disparity across transplant centers, there was no evidence of effect modification by transplant center volume, proportion of minority patients seen, quality rating, or region. We found significant racial disparities in survival after transplant, with substantial variation in this disparity across transplant centers that was not explained by selected center characteristics. This is the first study to directly evaluate the role transplant centers play in racial disparities in transplant outcomes. Further assessment of the qualitative factors that may drive disparities, such as selection processes and follow-up care, is needed to create effective center-level interventions to address health inequity

Molecular Aspects of a Robust Assay for Ferroxidase Function of Ceruloplasmin

Ceruloplasmin (Cp) is one of the most complex multicopper oxidase enzymes and plays an essential role in the metabolism of iron in mammals. Ferrous ion supplied by the ferroportin exporter is converted by Cp to ferric ion that is accepted by plasma metallo-chaperone transferrin. Study of the enzyme at the atomic and molecular level has been hampered by the lack of a suitable ferrous substrate. We have developed the classic chromophoric complex Fe^IIH_<i>x</i>(Tar)₂ (H₂Tar, 4-(2-thiazolylazo)­resorcinol; <i>x</i> = 0–2; overall charge omitted) as a robust substrate for evaluation of the ferroxidase function of Cp and related enzymes. The catalysis can be followed conveniently in real time by monitoring the solution absorbance at 720 nm, a fingerprint of Fe^IIH_<i>x</i>(Tar)₂. The complex is oxidized to its ferric form Fe^IIIH_<i>x</i>(Tar)₂ via the overall reaction sequence Fe^IIH_<i>x</i>(Tar)₂ → Fe^II-Cp → Fe^III-Cp → Fe^IIIH_<i>x</i>(Tar)₂: i.e., Fe­(II) is transferred formally from Fe^IIH_<i>x</i>(Tar)₂ to the substrate docking/oxidation (SDO) site(s) in Cp, followed by oxidation to product Fe­(III) that is trapped again by the ligand. Each Tar ligand in the above bis-complex coordinates the metal center in a meridional tridentate mode involving a pH-sensitive −OH group (p<i>K</i>_a > 12), and this imposes rapid Fe­(II) and Fe­(III) transfer kinetics to facilitate the catalytic process. The formation constants of both the ferrous and ferric complexes at pH 7.0 were determined (log β₂′ = 13.6 and 21.6, respectively), as well as an average dissociation constant of the SDO site(s) in Cp (log <i>K</i>_D′ = −7.2)