Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus.

PURPOSE: Systemic lupus erythematous (SLE) is a systemic autoimmune inflammatory disease with both genetic and epigenetic etiologies. Evidence suggests that deregulation of specific genes through epigenetic mechanisms may be a contributing factor to SLE pathology. There is increasing evidence that DNA methyltransferase activity may be involved. This study demonstrated modulation in expression of DNA methyltransferases (DNMTs) according to ethnicity in patients diagnosed with SLE. Furthermore, differential expression in one of the DNMTs was found in a subset of lupus patients on dehydroepiandrosterone (DHEA) therapy. METHODS: Real-time PCR analyses of DNMT1, DNMT3A and DNMT3B in peripheral blood mononuclear cells from a cohort of African American and European American lupus and non-lupus women were conducted. Also, global DNA methylation was assessed using the MethylFlash(TM) methylated quantification colorimetric assay. RESULTS: Significant increase in DNMT3A (p < 0.001) was shown in lupus patients when compared to age-matched healthy controls. This increase was associated with a higher SLEDI index. More striking was that expression levels for African American (AA) women were higher than European American women in the lupus populations. A subset of AA women on DHEA therapy showed a significant decrease (p < 0.05) in DNMT3A expression in comparison to lupus patients not on the therapy. DHEA is an androgenic steroid found in low levels in the serum of lupus patients. Supplementation of this hormone has been shown to be beneficial to some lupus patients. DHEA was not shown to effect DNMT1 or DNMT3B expression. Increased expression was also noted in DNMT3B (p < 0.05) in lupus patients compared to age-matched healthy controls. However, no significant difference was noted in DNMT1 (p = 0.2148) expression between lupus patients and healthy controls. Although increases were detected in de novo methyltransferases, a global decrease (p < 0.001) in 5-methycytosine was observed in lupus patients when compared to age-matched healthy controls. CONCLUSION: These findings suggest that epigenetic changes may play a critical role in the manifestations of the disease observed among ethnic groups, particularly African American women who often have a higher incidence of lupus. DHEA therapy effects on DNMT3A expression in AA women warrant further investigation in a larger population.

Decentralized clinical trials in the trial innovation network: Value, strategies, and lessons learned.

New technologies and disruptions related to Coronavirus disease-2019 have led to expansion of decentralized approaches to clinical trials. Remote tools and methods hold promise for increasing trial efficiency and reducing burdens and barriers by facilitating participation outside of traditional clinical settings and taking studies directly to participants. The Trial Innovation Network, established in 2016 by the National Center for Advancing Clinical and Translational Science to address critical roadblocks in clinical research and accelerate the translational research process, has consulted on over 400 research study proposals to date. Its recommendations for decentralized approaches have included eConsent, participant-informed study design, remote intervention, study task reminders, social media recruitment, and return of results for participants. Some clinical trial elements have worked well when decentralized, while others, including remote recruitment and patient monitoring, need further refinement and assessment to determine their value. Partially decentralized, or "hybrid" trials, offer a first step to optimizing remote methods. Decentralized processes demonstrate potential to improve urban-rural diversity, but their impact on inclusion of racially and ethnically marginalized populations requires further study. To optimize inclusive participation in decentralized clinical trials, efforts must be made to build trust among marginalized communities, and to ensure access to remote technology.

Leveraging the Expertise of the CTSA Program to Increase the Impact and Efficiency of Clinical Trials.

Importance: Multicenter clinical trials play a critical role in the translational processes that enable new treatments to reach all people and improve public health. However, conducting multicenter randomized clinical trials (mRCT) presents challenges. The Trial Innovation Network (TIN), established in 2016 to partner with the Clinical and Translational Science Award (CTSA) Consortium of academic medical institutions in the implementation of mRCTs, consists of 3 Trial Innovation Centers (TICs) and 1 Recruitment Innovation Center (RIC). This unique partnership has aimed to address critical roadblocks that impede the design and conduct of mRCTs, in expectation of accelerating the translation of novel interventions to clinical practice. The TIN's challenges and achievements are described in this article, along with examples of innovative resources and processes that may serve as useful models for other clinical trial networks providing operational and recruitment support. Observations: The TIN has successfully integrated more than 60 CTSA institution program hubs into a functional network for mRCT implementation and optimization. A unique support system for investigators has been created that includes the development and deployment of novel tools, operational and recruitment services, consultation models, and rapid communication pathways designed to reduce delays in trial start-up, enhance recruitment, improve engagement of diverse research participants and communities, and streamline processes that improve the quality, efficiency, and conduct of mRCTs. These resources and processes span the clinical trial spectrum and enable the TICs and RIC to serve as coordinating centers, data centers, and recruitment specialists to assist trials across the National Institutes of Health and other agencies. The TIN's impact has been demonstrated through its response to both historical operational challenges and emerging public health emergencies, including the national opioid public health crisis and the COVID-19 pandemic. Conclusions and Relevance: The TIN has worked to reduce barriers to implementing mRCTs and to improve mRCT processes and operations by providing needed clinical trial infrastructure and resources to CTSA investigators. These resources have been instrumental in more quickly and efficiently translating research discoveries into beneficial patient treatments.

Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study.

In a previous communication, kinetic ß-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic ß-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 ± 0.03, (D3)k(cat) = 1.12 ± 0.02 and (D3)ßk(cat) = 0.97 ± 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.

Digital Food Records in Community-Based Interventions: Mixed-Methods Pilot Study.

BACKGROUND: A pressing need exists to understand and optimize the use of dietary assessment tools that can be used in community-based participatory research (CBPR) interventions. A digital food record, which uses a mobile device to capture the dietary intake through text and photography inputs, is a particularly promising mobile assessment method. However, little is understood about the acceptability and feasibility of digital food records in CBPR and how to best tailor dietary assessment tools to the needs of a community. OBJECTIVE: The objective of our study was to evaluate the acceptability and feasibility of digital food records among church-based populations in resource-limited wards of Washington, DC, USA, using a mixed-methods approach. METHODS: This community-based pilot study was conducted as part of the Washington, DC Cardiovascular Health and Needs Assessment. Participants (n=17) received a mobile device (iPod Touch) to photodocument their dietary intake for a 3-day digital food record using a mobile app, FitNinja (Vibrent Health). The acceptability of the digital food record was explored through the thematic analysis of verbatim transcripts from a moderated focus group (n=8). In addition, the feasibility was evaluated by the percentage of participants complying with instructions (ie, capturing both before and after meal photos for at least 2 meals/day for 3 days). RESULTS: Qualitative themes identified were related to (1) the feasibility and acceptability of the mobile device and app, including issues in recording the dietary information and difficulty with photodocumentation; (2) suggestions for additional support and training experiences; and (3) comparisons with other mobile apps. Overall, the participants accepted the digital food record by demonstrating satisfaction with the tool and intent to continue the use (eg, participants recorded an average of 5.2, SD 7, consecutive days). Furthermore, of the 17 participants, 15 photodocumented at least 1 meal during the study period and 3 fully complied with the digital food record instructions. CONCLUSIONS: This study demonstrated digital food records as an acceptable tool in CBPR and identified contributors and barriers to the feasibility of digital food records for future research. Engaging community members in the implementation of novel assessment methods allows for the tailoring of technology to the needs of the community and optimizing community-based interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT01927783; https://www.clinicaltrials.gov/ct2/show/NCT01927783 (Archived by WebCite at http://www.webcitation.org/70WzaFWb6).

Adherence with physical activity monitoring wearable devices in a community-based population: observations from the Washington, D.C., Cardiovascular Health and Needs Assessment.

Wearable mobile health (mHealth) technologies offer approaches for targeting physical activity (PA) in resource-limited, community-based interventions. We sought to explore user characteristics of PA tracking, wearable technology among a community-based population within a health and needs assessment. In 2014-2015, we conducted the Washington, D.C., Cardiovascular Health and Needs Assessment in predominantly African-American churches among communities with higher obesity rates and lower household incomes. Participants received a mHealth PA monitor and wirelessly uploaded PA data weekly to church data collection hubs. Participants (n = 99) were 59 ± 12 years, 79% female, and 99% African-American, with a mean body mass index of 33 ± 7 kg/m2. Eighty-one percent of participants uploaded PA data to the hub and were termed "PA device users." Though PA device users were more likely to report lower household incomes, no differences existed between device users and non-users for device ownership or technology fluency. Findings suggest that mHealth systems with a wearable device and data collection hub may feasibly target PA in resource-limited communities.

Community Engagement to Optimize the Use of Web-Based and Wearable Technology in a Cardiovascular Health and Needs Assessment Study: A Mixed Methods Approach.

BACKGROUND: Resource-limited communities in Washington, D.C. have high rates of obesity-related cardiovascular disease in addition to inadequate physical activity (PA) facilities and limited Internet access. Engaging community members in the design and implementation of studies to address these health disparities is essential to the success of community-based PA interventions. OBJECTIVE: The objective of the study was to use qualitative and quantitative methods to evaluate the feasibility and acceptability of PA-monitoring wristbands and Web-based technology by predominantly African American, church-based populations in resource-limited Washington, D.C. neighborhoods. METHODS: To address cardiovascular health in at-risk populations in Washington, D.C., we joined community leaders to establish a community advisory board, the D.C. Cardiovascular Health and Obesity Collaborative (D.C. CHOC). As their first initiative, the Washington, D.C. Cardiovascular Health and Needs Assessment intends to evaluate cardiovascular health, social determinants of health, and PA-monitoring technologies. At the recommendation of D.C. CHOC members, we conducted a focus group and piloted the proposed PA-monitoring system with community members representing churches that would be targeted by the Cardiovascular Health and Needs Assessment. Participants (n=8) agreed to wear a PA-monitoring wristband for two weeks and to log cardiovascular health factors on a secure Internet account. Wristbands collected accelerometer-based data that participants uploaded to a wireless hub at their church. Participants agreed to return after two weeks to participate in a moderated focus group to share experiences using this technology. Feasibility was measured by Internet account usage, wristband utilization, and objective PA data. Acceptability was evaluated through thematic analysis of verbatim focus group transcripts. RESULTS: Study participants (5 males, 3 females) were African American and age 28-70 years. Participant wristbands recorded data on 10.1±1.6 days. Two participants logged cardiovascular health factors on the website. Focus group transcripts revealed that participants felt positively about incorporating the device into their church-based populations, given improvements were made to device training, hub accessibility, and device feedback. CONCLUSIONS: PA-monitoring wristbands for objectively measuring PA appear to be a feasible and acceptable technology in Washington, D.C., resource-limited communities. User preferences include immediate device feedback, hands-on device training, explicit instructions, improved central hub accessibility, and designation of a church member as a trained point-of-contact. When implementing technology-based interventions in resource-limited communities, engaging the targeted community may aid in early identification of issues, suggestions, and preferences. TRIAL REGISTRATION: ClinicalTrials.gov NCT01927783; https://clinicaltrials.gov/ct2/show/NCT01927783 (Archived by WebCite at http://www.webcitation.org/6f8wL117u).

A secondary isotope effect study of equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

beta-Secondary deuterium isotope effects have been measured for equine serum butyrylcholinesterase-catalyzed hydrolysis of acetyl-L(3)-thiocholine (L=H or (2)H). The dependencies of initial rates on isotopic substrate concentrations show close adherence to Michaelis-Menten kinetics, and yield the following isotope effects: (D3)k(cat)/K(m)=0.98+/-0.02 and (D3)k(cat)=1.10+/-0.02. The modestly inverse isotope effect on k(cat)/K(m) is consistent with partial rate limitation by a step that converts the sp(2)-hybridized ester carbonyl of the E+A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis. On the other hand, the markedly normal isotope effect on k(cat) indicates that the Michaelis complex that accumulates at substrate saturation of the active site during catalytic turnover is a tetrahedral intermediate, whose decomposition is the rate-limiting step. These results compliment a previous report [J.R. Tormos et al., J. Am. Chem. Soc. 127 (2005) 14538-14539] that showed that substrate-activated hydrolysis of acetylthiocholine (ATCh), catalyzed by recombinant human butyrylcholinesterase, is also rate limited by decomposition of an accumulating tetrahedral intermediate.

Revisiting the protein-coding gene catalog of Drosophila melanogaster using 12 fly genomes.

The availability of sequenced genomes from 12 Drosophila species has enabled the use of comparative genomics for the systematic discovery of functional elements conserved within this genus. We have developed quantitative metrics for the evolutionary signatures specific to protein-coding regions and applied them genome-wide, resulting in 1193 candidate new protein-coding exons in the D. melanogaster genome. We have reviewed these predictions by manual curation and validated a subset by directed cDNA screening and sequencing, revealing both new genes and new alternative splice forms of known genes. We also used these evolutionary signatures to evaluate existing gene annotations, resulting in the validation of 87% of genes lacking descriptive names and identifying 414 poorly conserved genes that are likely to be spurious predictions, noncoding, or species-specific genes. Furthermore, our methods suggest a variety of refinements to hundreds of existing gene models, such as modifications to translation start codons and exon splice boundaries. Finally, we performed directed genome-wide searches for unusual protein-coding structures, discovering 149 possible examples of stop codon readthrough, 125 new candidate ORFs of polycistronic mRNAs, and several candidate translational frameshifts. These results affect >10% of annotated fly genes and demonstrate the power of comparative genomics to enhance our understanding of genome organization, even in a model organism as intensively studied as Drosophila melanogaster.

The reactant state for substrate-activated turnover of acetylthiocholine by butyrylcholinesterase is a tetrahedral intermediate.

Secondary beta-deuterium kinetic isotope effects have been measured as a function of substrate concentration for recombinant human butyrylcholinesterase-catalyzed hydrolysis of acetyl-L3-thiocholine (L = 1H or 2H). The isotope effect on V/K is inverse, D3V/K = 0.93 +/- 0.03, which is consistent with conversion of the sp2 hybridized carbonyl carbon of the scissile ester bond of the E + A reactant state to a quasi-tetrahedral structure in the acylation transition state. In contrast, the isotope effect on Vmax under conditions of substrate activation is markedly normal, D3(betaVmax) = 1.29 +/- 0.06, an observation that is consistent with accumulation of a tetrahedral intermediate as the reactant state for catalytic turnover. Generally, tetrahedral intermediates for nonenzymatic ester hydrolyses are high-energy steady-state intermediates. Apparently, butyrylcholinesterase displays an unusual ability to stabilize such intermediates. Hence, the catalytic power of cholinesterases can largely be understood in terms of their ability to stabilize tetrahedral intermediates in the multistep reaction mechanism.