Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA.

Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

Pathways of thymidine hypermodification.

The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.

Bystander preference for naloxone products: a field experiment.

BACKGROUND: Bystander administration of naloxone is a critical strategy to mitigate opioid overdose mortality. To ensure bystanders' willingness to carry and administer naloxone in response to a suspected overdose, it is critical to select products for community distribution with the highest likelihood of being utilized. This study examines bystanders' preference for and willingness to administer three naloxone products approved by the FDA for bystander use and identify product features driving preference. METHODS: The population was a convenience sample of individuals who attended the Kentucky State Fair, August 18-28, 2022, in Louisville, Kentucky. Participants (n = 503) watched a standardized overdose education and naloxone training video, rated their willingness to administer each of three products (i.e., higher-dose nasal spray, lower-dose nasal spray, intramuscular injection), selected a product to take home, and rated factors affecting choice. RESULTS: After training, 44.4% chose the higher-dose nasal spray, 30.1% chose the intramuscular injection, and 25.5% chose the lower-dose nasal spray. Factors most influencing choice on a 10-point Likert scale were ease of use (9 [7-10]), naloxone dose (8 [5-10]), and product familiarity (5 [5-9]). CONCLUSIONS: Bystanders expressed high willingness to administer all studied formulations of naloxone products. Product choice preference varied as a function of product features. As the number and variety of available products continue to increase, continuous evaluation of formulation acceptability, in addition to including individuals with lived experience who are receiving and administering overdose reversal agents, is critical to support adoption and save lives.

Subepidermal calcified nodule presenting as a cutaneous horn: two cases and a review of the literature.

Subepidermal calcified nodules are an uncommon subtype of idiopathic calcinosis cutis. Morphologically, this entity typically present as a single, well-circumscribed, white-yellow nodule. Based on clinical appearance alone, subepidermal calcified nodules are frequently misdiagnosed and often requires histological confirmation. We describe two cases of subepidermal calcified nodules presenting atypically as cutaneous horns. Subepidermal calcified nodules presenting as a cutaneous horn has rarely been reported; on review, there are fewer than 10 such cases have been described within the past 30 years. The cases described here illustrate the clinical variety and should increase awareness of subepidermal calcified nodules presented.

Effects of low-frequency repetitive transcranial magnetic stimulation in adductor laryngeal dystonia: a safety, feasibility, and pilot study.

PURPOSE: The effects of neuromodulation are virtually unexplored in adductor laryngeal dystonia (AdLD), a disorder characterized by involuntary contraction of intrinsic laryngeal muscles. Recent findings indicated that intracortical inhibition is reduced in people with AdLD. Low-frequency repetitive transcranial magnetic stimulation (rTMS) induces prolonged intracortical inhibition, but the effects in AdLD are unexplored. This pilot and feasibility study aimed to examine the safety, feasibility, and effects of a single session 1 Hz rTMS over the laryngeal motor cortex (LMC) in people with AdLD and healthy individuals. METHODS: The stimulation location was individualized and determined through TMS-evoked responses in the thyroarytenoid muscles using fine-wire electrodes. 1200 pulses of 1 Hz rTMS were delivered to the left LMC in two groups: Control (n = 6) and AdLD (n = 7). Tolerance, adverse effects, intracortical inhibition, and voice recordings were collected immediately before and after rTMS. Voice quality was assessed with acoustic-based and auditory-perceptual measures. RESULTS: All participants tolerated the procedures, with no unexpected adverse events or worsening of symptoms. No significant effects on intracortical inhibition were observed. In the AdLD group, there was a large-effect size after rTMS in vocal perturbation measures and a small-effect size in decreased phonatory breaks. CONCLUSIONS: One rTMS session over the LMC is safe and feasible, and demonstrated trends of beneficial effects on voice quality and phonatory function in AdLD. These preliminary findings support further investigation to assess clinical benefits in a future randomized sham-controlled trial. CLINICALTRIALS.GOV: NCT02957942, registered on November 8, 2016.

Researcher perspectives on including people who use drugs in clinical research.

Background: People who use drugs are commonly excluded from clinical research despite being disproportionately impacted by numerous health conditions. Recent work indicates that community-recruited individuals report low support of research inclusion for persons reporting substance use.Objective: This study conducted a mixed-method assessment of researchers' attitudes on including persons reporting substance use in clinical research.Methods: Respondents (N = 168; 58% female; 58% psychologists) recruited via scientific society listservs and Twitter completed a survey containing two hypothetical research vignettes. Quantitative items evaluated respondents' endorsement of research participation by healthy adults, people who use drugs, and vulnerable populations. Qualitative items included open-ended questions asking reasons why people who use drugs should and should not participate in research.Results: Respondents reported significantly lower support for research participation by people who use drugs compared to healthy adults (p <.001). Open-ended responses concerning the inclusion of people who use drugs included themes relevant to the Belmont Principles (e.g., capacity to consent) and data quality (e.g., "bad data," poor compliance).Conclusion: Although lower support for research participation by people who use drugs was observed compared to healthy adults, the magnitude of this difference was smaller than reported for prior community-recruited respondents. These findings emphasize salient factors that may serve as both protections for and barriers to inclusion of people who use drugs in research. Initiatives including adoption of person-first language, addressing stereotyping of people who use drugs, and emphasizing the benefits of including these populations in clinical research should be explored to reduce bias while retaining needed protections.

Ambulatory monitoring of Lombard-related vocal characteristics in vocally healthy female speakers.

Speakers typically modify their voice in the presence of increased background noise levels, exhibiting the classic Lombard effect. Lombard-related characteristics during everyday activities were recorded from 17 vocally healthy women who wore an acoustic noise dosimeter and ambulatory voice monitor. The linear relationship between vocal sound pressure level and environmental noise level exhibited an average slope of 0.54 dB/dB and value of 72.8 dB SPL at 50 dBA when correlation coefficients were greater than 0.4. These results, coupled with analyses of spectral and cepstral vocal function measures, provide normative ambulatory Lombard characteristics for comparison with patients with voice-use related disorders.

Automatic speech and singing classification in ambulatory recordings for normal and disordered voices.

Ambulatory voice monitoring is a promising tool for investigating phonotraumatic vocal hyperfunction (PVH), associated with the development of vocal fold lesions. Since many patients with PVH are professional vocalists, a classifier was developed to better understand phonatory mechanisms during speech and singing. Twenty singers with PVH and 20 matched healthy controls were monitored with a neck-surface accelerometer-based ambulatory voice monitor. An expert-labeled ground truth data set was used to train a logistic regression on 15 subject-pairs with fundamental frequency and autocorrelation peak amplitude as input features. Overall classification accuracy of 94.2% was achieved on the held-out test set.

D-immunized blood donors who are female and who possess at least one HLA-DRB1*15 allele show a propensity for high serum RhIG production.

BACKGROUND: D- individuals with previous D-incompatible pregnancies and/or blood transfusions, as well as those who are actively immunized with small-volume D+ red blood cells (RBCs), are stimulated to produce RhIG. Many factors could influence the stimulation of immunoglobulin production in response to foreign antigen (such as antigen immunogenicity and genetic factors), and it is unknown whether genetic markers could potentially identify responder anti-D donors. STUDY DESIGN AND METHODS: Anti-D donors were assigned a responder profile based on their serum RhIG levels (n = 431). A subset of donors (n = 272) had DNA extracted for polymerase chain reaction genotyping assays for target genes in antigen presentation and pathogen recognition receptors (TLR2, TLR4, CD14, FcγRIIA, and the MHC Class II locus HLA-DRB1). Statistical tests for associations between anti-D donor responder profiles and genetic factors were performed. RESULTS: A large proportion of our donors (38.7%) were classified as nonresponder donors, despite receiving multiple D+ RBC immunizations, whereas female sex was significantly associated with an all-responder profile (p < 0.001). The presence of the DRB1*15 allele and absence of the DRB1*04 allele were more likely to be associated with a responder anti-D donor, although not significantly after Bonferroni correction. A combination of the DRB1*15 allele and female sex was significantly associated with an anti-D donor responder profile. CONCLUSION: This study has identified female sex and the HLA-DRB1*15 allele as potentially useful markers that could be used to screen donors before entry into D immunization programs.

Localized hypertrichosis of intermammary cleft in monozygotic twins.

Hypertrichosis is a condition in which hair is longer and denser than what is considered normal for an individual based on age, sex, ethnicity, and location on the body and can be classified based on its distribution (generalized vs localized), age of onset (congenital vs acquired), and type of hair (lanugo or vellus vs terminal). We describe a rare case of monozygotic female twins who presented for localized hypertrichosis of the intermammary cleft that developed during puberty. Endocrine examination was unremarkable. Discussion of various treatment modalities should be considered, because localized hypertrichosis may have a considerable psychosocial effect.

The Telescoping Phenomenon: Origins in Gender Bias and Implications for Contemporary Scientific Inquiry.

BACKGROUND: In an article published in International Journal of the Addictions in 1989, Nick Piazza and his coauthors described "telescoping," an accelerated progression through "landmark symptoms" of alcoholism, among a sample of recovering women. OBJECTIVES: The aim of this critical analysis is to apply a feminist philosophy of science to examine the origins of the framework of telescoping research and its implications for contemporary scientific inquiry. METHODS: A feminist philosophy of science framework is outlined and applied to key source publications of telescoping literature drawn from international and United States-based peer-reviewed journals published beginning in 1952. RESULTS: A feminist philosophy of science framework identifies gender bias in telescoping research in three ways. First, gender bias was present in the early conventions that laid the groundwork for telescoping research. Second, a "masculine" framework was present in the methodology guiding telescoping research. Third, gender bias was present in the interpretation of results as evidenced by biased comparative language. CONCLUSIONS: Telescoping research contributed to early evidence of critical sex and gender differences helping to usher in women's substance abuse research more broadly. However, it also utilized a "masculine" framework that perpetuated gender bias and limited generative, novel research that can arise from women-focused research and practice. A feminist philosophy of science identifies gender bias in telescoping research and provides an alternative, more productive approach for substance abuse researchers and clinicians.

Influence of Cocaine-Related Images and Alcohol Administration on Inhibitory Control in Cocaine Users.

BACKGROUND: Alcohol use and impulsivity, including decreased inhibitory control, predict poor treatment outcomes for individuals with cocaine use disorders. This study sought to determine the effects of alcohol administration on inhibitory control following cocaine-related and neutral cues on the Attentional Bias-Behavioral Activation (ABBA) task in cocaine users. We hypothesized that the proportion of inhibitory failures would increase following cocaine, compared to neutral, cues. We further hypothesized that there would be an interaction between alcohol administration and task version, such that alcohol would impair inhibitory control following cocaine, but not neutral cues. METHODS: Fifty current cocaine users completed this mixed-model, double-blind, placebo-controlled, crossover study over 2 experimental sessions. The ABBA task was completed following alcohol administration (0.0 and 0.65 g/kg). Subject-rated drug effect and physiological measures were collected prior to and after alcohol administration. RESULTS: Proportion of inhibitory failures was increased following cocaine-related cues compared to neutral cues independent of alcohol dose. Alcohol administration also produced prototypical subject-rated drug effects. CONCLUSIONS: A better understanding of the relationship between alcohol consumption and inhibitory control in cocaine users could direct the development of interventions to decrease the risk of relapse in individuals who drink and display impaired inhibitory control.

Distributive Processing by the Iron(II)/α-Ketoglutarate-Dependent Catalytic Domains of the TET Enzymes Is Consistent with Epigenetic Roles for Oxidized 5-Methylcytosine Bases.

The ten-eleven translocation (TET) proteins catalyze oxidation of 5-methylcytosine ((5m)C) residues in nucleic acids to 5-hydroxymethylcytosine ((5hm)C), 5-formylcytosine ((5f)C), and 5-carboxycytosine ((5ca)C). These nucleotide bases have been implicated as intermediates on the path to active demethylation, but recent reports have suggested that they might have specific regulatory roles in their own right. In this study, we present kinetic evidence showing that the catalytic domains (CDs) of TET2 and TET1 from mouse and their homologue from Naegleria gruberi, the full-length protein NgTET1, are distributive in both chemical and physical senses, as they carry out successive oxidations of a single (5m)C and multiple (5m)C residues along a polymethylated DNA substrate. We present data showing that the enzyme neither retains (5hm)C/(5f)C intermediates of preceding oxidations nor slides along a DNA substrate (without releasing it) to process an adjacent (5m)C residue. These findings contradict a recent report by Crawford et al. ( J. Am. Chem. Soc. 2016 , 138 , 730 ) claiming that oxidation of (5m)C by CD of mouse TET2 is chemically processive (iterative). We further elaborate that this distributive mechanism is maintained for TETs in two evolutionarily distant homologues and posit that this mode of function allows the introduction of (5m)C forms as epigenetic markers along the DNA.

Separate and Combined Effects of Naltrexone and Extended-Release Alprazolam on the Reinforcing, Subject-Rated, and Cardiovascular Effects of Methamphetamine.

Opioid antagonists (eg, naltrexone) and positive modulators of γ-aminobutyric acid type A receptors (eg, alprazolam) each modestly attenuate the abuse-related effects of stimulants. A previous study demonstrated that acute pretreatment with the combination of naltrexone and alprazolam attenuated a greater number of the subject-rated effects of D-amphetamine than the constituent drugs alone. This study tested the hypothesis that maintenance on the combination of naltrexone and alprazolam XR would attenuate the reinforcing and "positive" subject-rated effects of methamphetamine to a greater extent than the constituent drugs alone.Eight non-treatment-seeking, stimulant-using individuals completed a placebo-controlled, crossover, double-blind inpatient protocol. Participants were maintained on naltrexone (0 and 50 mg), alprazolam XR (0 and 1 mg), and the combination of naltrexone and alprazolam XR (50 mg and 1 mg, respectively) for 6 to 7 days. Under each maintenance condition, participants sampled intranasal doses of methamphetamine (0, 10, and 30 mg), and were then offered the opportunity to work for the sampled dose on a modified progressive-ratio procedure. Subject-rated drug effect questionnaires, psychomotor, and physiology assessments were collected.Intranasal methamphetamine functioned as a reinforcer and produced prototypical stimulant-like "positive" subject-rated and physiological effects. Maintenance on naltrexone significantly decreased the reinforcing, but not subject-rated drug effects of 10-mg methamphetamine. Alprazolam XR and the combination of naltrexone and alprazolam XR did not impact methamphetamine self-administration or subject-rated drug effects. The results support the continued evaluation of naltrexone for methamphetamine dependence, as well as the identification of other drugs that enhance its ability to reduce drug-taking behavior.

Cigarette Cue Attentional Bias in Cocaine-Smoking and Non-Cocaine-Using Cigarette Smokers.

INTRODUCTION: Cigarette smoking in cocaine users is nearly four times higher than the national prevalence and cocaine use increases cigarette smoking. The mechanisms underlying cigarette smoking in cocaine-using individuals need to be identified to promote cigarette and cocaine abstinence. Previous studies have examined the salience of cigarette and cocaine cues separately. The present aim was to determine whether cigarette attentional bias (AB) is higher in cigarettes smokers who smoke cocaine relative to individuals who only smoke cigarettes. METHODS: Twenty cigarette smokers who smoke cocaine and 20 non-cocaine-using cigarette smokers completed a visual probe task with eye-tracking technology. During this task, the magnitude of cigarette and cocaine AB was assessed through orienting bias, fixation time, and response time. RESULTS: Cocaine users displayed an orienting bias towards cigarette cues. Cocaine users also endorsed a more urgent desire to smoke to relieve negative affect associated with cigarette craving than non-cocaine users (g = 0.6). Neither group displayed a cigarette AB, as measured by fixation time. Cocaine users, but not non-cocaine users, displayed a cocaine AB as measured by orienting bias (g = 2.0) and fixation time (g = 1.2). There were no significant effects for response time data. CONCLUSIONS: Cocaine-smoking cigarettes smokers display an initial orienting bias toward cigarette cues, but not sustained cigarette AB. The incentive motivation underlying cigarette smoking also differs. Cocaine smokers report more urgent desire to smoke to relieve negative affect. Identifying differences in motivation to smoke cigarettes may provide new treatment targets for cigarette and cocaine use disorders. IMPLICATIONS: These results suggest that cocaine-smoking cigarette smokers display an initial orienting bias towards cigarette cues, but not sustained attention towards cigarette cues, relative to non-cocaine-using smokers. Smoked cocaine users also report a more urgent desire to smoke to relieve negative affect than non-cocaine users. Identifying differences in motivation to smoke cigarettes may provide new treatment targets for both cigarette and cocaine use disorders.

Alcohol Administration Increases Cocaine Craving But Not Cocaine Cue Attentional Bias.

BACKGROUND: Alcohol consumption is a known antecedent to cocaine relapse. Through associative conditioning, it is hypothesized that alcohol increases incentive motivation for cocaine and thus the salience of cocaine-related cues, which are important in maintaining drug-taking behavior. Cocaine-using individuals display a robust cocaine cue attentional bias as measured by fixation time during the visual probe task. The purpose of this study was to evaluate the influence of alcohol administration on cocaine cue attentional bias using eye-tracking technology to directly measure attentional allocation. METHODS: Twenty current cocaine users completed a double-blind, placebo-controlled, within-subjects study that tested the effect of 3 doses of alcohol (0.00, 0.325, and 0.65 g/kg) on cocaine cue attentional bias using the visual probe task with eye-tracking technology. The participant-rated and physiological effects of alcohol were also assessed. RESULTS: Participants displayed a robust cocaine cue attentional bias following both placebo and alcohol administration as measured by fixation time, but not response time. Alcohol administration did not influence cocaine cue attentional bias, but increased craving for cocaine in a dose-dependent manner. Alcohol produced prototypic psychomotor and participant-rated effects. CONCLUSIONS: Alcohol administration increases cocaine craving but not cocaine cue attentional bias. Alcohol-induced cocaine craving suggests that alcohol increases incentive motivation for cocaine but not the salience of cocaine-related cues.

Analysis of two candidate genes for Basan syndrome.

Basan syndrome is an extremely rare ectodermal dysplasia with autosomal dominant inheritance and variable expressivity. The etiology of Basan syndrome remains unknown. To identify the Basan syndrome gene, we sequenced keratin 14 (KRT14) and SMARCAD1 in a previously unreported kindred with the disease. Sequencing of the coding regions and splice junctions of KRT14 and SMARCAD1 was performed using PCR-amplified genomic DNA isolated from blood or saliva and standard PCR protocols. In vitro functional studies were performed for a variant identified in SMARCAD1. While direct sequencing of KRT14 failed to reveal any likely pathogenic sequence alterations or splice site variants, a heterozygous splicing variant (c.378+3A>T) that segregated with the disease was identified in the skin-specific isoform of SMARCAD1. In vitro studies failed to demonstrate a splicing defect in SMARCAD1. We screened two candidate genes for Basan syndrome in a 3-generation pedigree. The skin-specific isoform of SMARCAD1 remains a good candidate for this disease.

Wisconsin's Lifecourse Initiative for Healthy Families: application of the maternal and child health life course perspective through a regional funding initiative.

National experts are calling for more integrated approaches such as the life course perspective to reduce health disparities and achieve greater health equity. The translation and application of the life course perspective is therefore of great interest to public health planners, policy makers and funders to promote community-wide improvements in maternal and child health. However, few organizations have applied the life course perspective in designing strategic funding initiatives. For over three decades, Wisconsin has observed persistent racial disparities in birth outcomes. This complex public health issue led to the development of the Lifecourse Initiative for Health Families, a regional multi-million dollar funding initiative created and supported by the Wisconsin Partnership Program of the University of Wisconsin School of Medicine and Public Health (Created by the UW SMPH from an endowment following the conversion of Blue Cross Blue Shield United of Wisconsin, the Partnership Program makes investments in research, education, and public health and prevention initiatives that improve health and reduce health disparities in the state.). Over a 2-year period, the program funded four collaboratives to adopt a life course perspective and develop strategic plans for improving African American birth outcomes. The Twelve-point plan to close the black-white gap in birth outcomes provided the framework for the planning process. Despite the conceptual challenges, the life course perspective was embraced by the collaboratives, challenged community assumptions on the root causes of poor birth outcomes and provided a unifying funding construct for organizing and planning complementary individual-level interventions with social and physical environmental change strategies. These integrated and complimentary approaches provide a long-term opportunity to address the persistent racial birth outcome disparity in Wisconsin.

The Impact of Foreign Language Accent on Expert Listeners' Auditory-Perceptual Evaluations of Dysphonia.

INTRODUCTION: Auditory-perceptual evaluations of dysphonia, though essential for comprehensive voice evaluation, are subject to listener bias. Knowledge of an underlying voice disorder can influence auditory-perceptual ratings. Accented speech results in increased listener effort and delays in word identification. Yet, little is known about the impact of foreign language accents on auditory-perceptual ratings for dysphonic speakers. The purpose of this work was to determine the impact of a foreign language accent on experts' auditory-perceptual ratings of dysphonic speakers. METHODS: Twelve voice-specializing SLPs who spoke with a General American English (GAE) accent rated vocal percepts of 28 speakers with a foreign language accent and 28 with a GAE accent, all of whom had been diagnosed with a voice disorder. Speaker groups were matched based on sex, age, and mean smoothed cepstral peak prominence. Four linear mixed-effects models assessed the impact of a foreign language accent on expert auditory-perceptual ratings of the overall severity of dysphonia, roughness, breathiness, and strain. RESULTS: The twelve raters demonstrated good inter- and intra-rater reliability (ICC[3, k] = .89; mean ICC = .89). The linear mixed-effects models revealed no significant impact of foreign language accent on ratings of overall severity of dysphonia, roughness, breathiness, or strain. CONCLUSION: Despite the possibility of increased listener effort and bias, foreign language accent incongruence had no effect on expert listeners' auditory-perceptual evaluations for dysphonic speakers. Findings support the use of auditory-perceptual evaluations for voice disorders across sociolinguistically diverse populations. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:2272-2276, 2024.