Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Isolation and Characterization of Cell-Free DNA from Cerebral Organoids.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

CYP11B2 inhibitor dexfadrostat phosphate suppresses the aldosterone-to-renin ratio, an indicator of sodium retention, in healthy volunteers.

AIMS: High aldosterone is a key driver of hypertension and long-term negative sequelae. We evaluated the safety and efficacy of dexfadrostat phosphate (DP13), a novel aldosterone synthase (CYP11B2) inhibitor, in healthy participants. METHODS: This randomized, double-blind, placebo-controlled study was conducted in two parts. In part A, a single-ascending dose escalation, 16 participants received oral DP13 1-16 mg. Part B was a multiple-ascending dose, sequential group study in which 32 participants received oral DP13 4, 8 or 16 mg once daily for 8 days. Safety and tolerability were monitored throughout. An adrenocorticotropic hormone (ACTH) stimulation test at maximal blood drug concentrations defined the dose range for multiple dosing. RESULTS: DP13 was well tolerated at all doses, with no serious adverse events. In part B, all DP13 doses (4, 8 and 16 mg) over 8 days effectively suppressed aldosterone production, increased the urinary sodium/potassium ratio, decreased plasma sodium and increased plasma potassium and renin levels compared with placebo, resulting in potent suppression of the aldosterone-to-renin ratio (ARR). Endocrine counter-regulation resulted in the 4 mg dose no longer sustaining 24-h aldosterone suppression after 8 days of treatment, unlike the 8- and 16 mg doses. There was no evidence of drug-induced adrenal insufficiency (ACTH stress challenge). CONCLUSIONS: In patients with excess aldosterone and ensuing sodium retention driving hypertension, managing sodium balance is critical. A CYP11B2 inhibitor like DP13, whose effectiveness can be monitored by a reduction in ARR, may prove valuable in managing aldosterone-dependent hypertension and primary aldosteronism.

Insights into Repeated Renal Injury Using RNA-Seq with Two New RPTEC Cell Lines.

Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25-2.5 µM cisplatin (CisPt) or 12.5-100 µM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 µM CisPt from 3 to 96 h expressing 2000-5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity.

Omecamtiv mecarbil does not prolong QTc intervals at therapeutic concentrations.

AIMS: Omecamtiv mecarbil (OM) is a novel selective cardiac myosin activator under investigation for the treatment of heart failure. This study aimed to evaluate the effect of therapeutic concentrations of OM on electrocardiogram (ECG) parameters and exclude a clinically concerning effect on the rate-corrected QT (QTc) interval. METHODS: In part A, 70 healthy subjects received a 25 mg oral dose of OM, and pharmacokinetics were assessed. Only subjects with maximum observed plasma concentration ≤ 350 ng/mL (n = 60) were randomized into part B, where they received a single oral dose of placebo, 50 mg OM and 400 mg moxifloxacin in a 3-period, 3-treatment, 6-sequence crossover study with continuous ECG collection. RESULTS: After a 50-mg dose of OM, mean placebo-corrected change from baseline QTcF (∆∆QTcF; Fridericia correction) ranged from -6.7 ms at 1 hour postdose to -0.8 ms at 4 hours postdose. The highest upper bound of the 1-sided 95% confidence interval (CI) was 0.7 ms (4 h postdose). Moxifloxacin resulted in a clear increase in mean ∆∆QTcF, with a peak value of 13.1 ms (90% CI: 11.71-14.57) at 3 hours; lower bound of the 1-sided 95% CI was > 5 ms at all of the 3 prespecified time points. Based on a concentration-QTc analysis, an effect on ∆∆QTcF exceeding 10 ms can be excluded up to OM plasma concentrations of ~800 ng/mL. There were no serious or treatment-emergent adverse events leading to discontinuation from the study. CONCLUSION: OM does not have a clinically relevant effect on the studied ECG parameters.

Arabidopsis DNA Replication Initiates in Intergenic, AT-Rich Open Chromatin.

The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2'-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.

Oral semaglutide improves postprandial glucose and lipid metabolism, and delays gastric emptying, in subjects with type 2 diabetes.

AIM: To assess the effects of oral semaglutide on postprandial glucose and lipid metabolism, and gastric emptying, in subjects with type 2 diabetes (T2D). MATERIALS AND METHODS: In this randomized, double-blind, single-centre, crossover trial, subjects with T2D received once-daily oral semaglutide (escalated to 14 mg) followed by placebo, or vice versa, over two consecutive 12-week periods. Glucose and lipid metabolism, and gastric emptying (paracetamol absorption) were assessed before and after two types of standardized meals (standard and/or fat-rich) at the end of each treatment period. The primary endpoint was area under the glucose 0-5-h curve (AUC0-5h ) after the standard breakfast. RESULTS: Fifteen subjects were enrolled (mean age 58.2 years, HbA1c 6.9%, body weight 93.9 kg, diabetes duration 3.1 years; 13 [86.7%] males). Fasting concentrations of glucose were significantly lower, and C-peptide significantly greater, with oral semaglutide versus placebo. Postprandial glucose (AUC0-5h ) was significantly lower with oral semaglutide versus placebo (estimated treatment ratio, 0.71; 95% CI, 0.63, 0.81; p < .0001); glucose incremental AUC (iAUC0-5h/5h ) and glucagon AUC0-5h were also significantly reduced, with similar results after the fat-rich breakfast. Fasting concentrations of triglycerides, very low-density lipoprotein (VLDL) and apolipoprotein B48 (ApoB48) were significantly lower with oral semaglutide versus placebo. AUC0-8h for triglycerides, VLDL and ApoB48, and triglycerides iAUC0-8h/8h , were significantly reduced after oral semaglutide versus placebo. During the first postprandial hour, gastric emptying was delayed (a 31% decrease in paracetamol AUC0-1h ) with oral semaglutide versus placebo. One serious adverse event (acute myocardial infarction) occurred during oral semaglutide treatment. CONCLUSION: Oral semaglutide significantly improved fasting and postprandial glucose and lipid metabolism, and delayed gastric emptying.

A Highly Durable RNAi Therapeutic Inhibitor of PCSK9.

BACKGROUND: Inclisiran (ALN-PCSsc) is a long-acting RNA interference (RNAi) therapeutic agent that inhibits the synthesis of proprotein convertase subtilisin-kexin type 9 (PCSK9), a target for the lowering of low-density lipoprotein (LDL) cholesterol. METHODS: In this phase 1 trial, we randomly assigned healthy volunteers with an LDL cholesterol level of at least 100 mg per deciliter in a 3:1 ratio to receive a subcutaneous injection of inclisiran or placebo in either a single-ascending-dose phase (at a dose of 25, 100, 300, 500, or 800 mg) or a multiple-dose phase (125 mg weekly for four doses, 250 mg every other week for two doses, or 300 or 500 mg monthly for two doses, with or without concurrent statin therapy); each dose cohort included four to eight participants. Safety, the side-effect profile, and pharmacodynamic measures (PCSK9 level, LDL cholesterol level, and exploratory lipid variables) were evaluated. RESULTS: The most common adverse events were cough, musculoskeletal pain, nasopharyngitis, headache, back pain, and diarrhea. All the adverse events were mild or moderate in severity. There were no serious adverse events or discontinuations due to adverse events. There was one grade 3 elevation in the γ-glutamyltransferase level, which was considered by the investigator to be related to statin therapy. In the single-dose phase, inclisiran doses of 300 mg or more reduced the PCSK9 level (up to a least-squares mean reduction of 74.5% from baseline to day 84), and doses of 100 mg or more reduced the LDL cholesterol level (up to a least-squares mean reduction of 50.6% from baseline). Reductions in the levels of PCSK9 and LDL cholesterol were maintained at day 180 for doses of 300 mg or more. All multiple-dose regimens reduced the levels of PCSK9 (up to a least-squares mean reduction of 83.8% from baseline to day 84) and LDL cholesterol (up to a least-squares mean reduction of 59.7% from baseline to day 84). CONCLUSIONS: In this phase 1 trial, no serious adverse events were observed with inclisiran. Doses of 300 mg or more (in single or multiple doses) significantly reduced levels of PCSK9 and LDL cholesterol for at least 6 months. (Funded by Alnylam Pharmaceuticals and the Medicines Company; ClinicalTrials.gov number, NCT02314442 .).

Evidence-based Classification in Track Athletics for Athletes with a Vision Impairment: A Delphi Study.

SIGNIFICANCE: The Delphi analysis presented here highlights the need for a sport-specific evidence-based classification system for track athletics for athletes with a vision impairment (VI). This system may differ for different race distances. Further research is required to develop a useful test battery of vision tests for classification. The issue of intentional misrepresentation during classification needs particular attention. PURPOSE: At present, athletes with VI are placed into competition classes developed on the basis of legal definitions of VI. The International Paralympic Committee Athlete Classification Code states that all sports should have their own classification system designed to reflect the (visual) demands of that individual sport. This project gathered expert opinion on the specific requirements for an evidence-based sport-specific classification system for VI track athletics and to identify any particular issues within track athletics that require further research into their impact on sport performance. METHODS: A three-round Delphi review was conducted with a panel of 17 people with expertise in VI track athletics. RESULTS: The panel agreed that the current classification system in VI track athletics does not completely minimize the impact of impairment on competition outcome, highlighting the need for improvements. There was clear agreement that the existing measures of vision may fail to adequately reflect the type of vision loss that would impact running performance, with additional measures required. Intentional misrepresentation, where athletes "cheat" on classification tests, remains a serious concern. CONCLUSIONS: The panel has identified measures of vision and performance that will inform the development of an evidence-based classification system by better understanding the relationship between VI and performance in track athletics. Issues such as the use of guides and whether the current class system was equitable gave rise to differing opinions within the panel, with these varying across the different running distances.

Genome-Wide Analysis of the Arabidopsis Replication Timing Program.

Eukaryotes use a temporally regulated process, known as the replication timing program, to ensure that their genomes are fully and accurately duplicated during S phase. Replication timing programs are predictive of genomic features and activity and are considered to be functional readouts of chromatin organization. Although replication timing programs have been described for yeast and animal systems, much less is known about the temporal regulation of plant DNA replication or its relationship to genome sequence and chromatin structure. We used the thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to describe, at high-resolution, the genome-wide replication timing program for Arabidopsis (Arabidopsis thaliana) Col-0 suspension cells. We identified genomic regions that replicate predominantly during early, mid, and late S phase, and correlated these regions with genomic features and with data for chromatin state, accessibility, and long-distance interaction. Arabidopsis chromosome arms tend to replicate early while pericentromeric regions replicate late. Early and mid-replicating regions are gene-rich and predominantly euchromatic, while late regions are rich in transposable elements and primarily heterochromatic. However, the distribution of chromatin states across the different times is complex, with each replication time corresponding to a mixture of states. Early and mid-replicating sequences interact with each other and not with late sequences, but early regions are more accessible than mid regions. The replication timing program in Arabidopsis reflects a bipartite genomic organization with early/mid-replicating regions and late regions forming separate, noninteracting compartments. The temporal order of DNA replication within the early/mid compartment may be modulated largely by chromatin accessibility.

Semaglutide improves postprandial glucose and lipid metabolism, and delays first-hour gastric emptying in subjects with obesity.

AIM: To investigate the effects of semaglutide on fasting and postprandial glucose and lipid responses, and on gastric emptying. MATERIALS AND METHODS: This was a randomized, double-blind, placebo-controlled, 2-period, crossover trial. Subjects with obesity (N = 30) received once-weekly subcutaneous semaglutide, dose-escalated to 1.0 mg, or placebo. After each 12-week treatment period, glucose and lipid metabolism were assessed before and after standardized meals. Gastric emptying (paracetamol absorption test) and peptide YY (PYY) response were also assessed. RESULTS: Semaglutide treatment significantly lowered fasting concentrations of glucose and glucagon, and increased insulin vs placebo (estimated treatment ratio: 0.95 [95% confidence interval: 0.91, 0.98]; 0.86 [0.75, 0.98]; 1.45 [1.20, 1.75], respectively). Postprandial glucose metabolism significantly improved with semaglutide vs placebo (incremental area under the curve 0 to 5 hours [iAUC0-5h ]; estimated treatment difference: glucose -1.34 mmol h/L [-2.42, -0.27]; insulin -921 pmol h/L [-1461, -381]; C-peptide -1.42 nmol h/L [-2.33, -0.51]). Fasting and postprandial lipid metabolism improved with semaglutide vs placebo. First-hour gastric emptying after the meal was delayed with semaglutide vs placebo (AUC0-1h ; estimated treatment ratio: 0.73 [0.61, 0.87]); this may have contributed to the lower postprandial glucose increase in semaglutide-treated subjects. Overall gastric emptying (AUC0-5h ) was not statistically different between treatments. Fasting and postprandial PYY responses were significantly lower with semaglutide vs placebo (P = .0397 and P = .0097, respectively). CONCLUSION: Semaglutide improved fasting and postprandial glucose and lipid metabolism. Overall gastric emptying was similar to that with placebo; however, the observed first-hour delay with semaglutide may contribute to a slower entry of glucose into the circulation.

Functional Characteristics of Health Coalitions in Local Public Health Systems: Exploring the Function of County Health Councils in Tennessee.

CONTEXT: Partnerships are emerging as critically important vehicles for addressing health in local communities. Coalitions involving local health departments can be viewed as the embodiment of a local public health system. Although it is known that these networks are heavily involved in assessment and community planning activities, limited studies have evaluated whether health coalitions are functioning at an optimal capacity. OBJECTIVE: This study assesses the extent to which health coalitions met or exceeded expectations for building functional capacity within their respective networks. DESIGN: An evaluative framework was developed focusing on 8 functional characteristics of coalitions previously identified by Erwin and Mills. Twenty-nine indicators were identified that served as "proxy" measures of functional capacity within health coalitions. SETTING AND PARTICIPANTS: Ninety-three County Health Councils (CoHCs) in Tennessee. MAIN OUTCOME MEASURE(S): Diverse member representation; formal rules, roles, and procedures; open, frequent interpersonal communication; task-focused climate; council leadership; resources; active member participation; and external linkages were assessed to determine the level of functionality of CoHCs. Scores across all CoHCs were analyzed using descriptive statistics such as frequency distributions, measures of central tendency, and measures of variability. Data were analyzed using SAS 9.3. RESULTS: Of 68 CoHCs (73% response rate), the total mean score for the level of functional characteristics was 30.5 (median = 30.5; SD = 6.3; range, 18-44). Of the 8 functional characteristics, CoHCs met or exceeded all indicators associated with council leadership, tasked-focused climate, and external linkages. Lowest scores were for having a written communications plan, written priorities or goals, and opportunities for training. CONCLUSION: This study advances the research on health coalitions by establishing a process for quantifying the functionality of health coalitions. Future studies will be conducted to examine the association between health coalition functional capacity, local health departments' community health assessment and planning efforts, and changes in community health status.

Absence of clinically relevant cardiovascular interaction upon add-on of mirabegron or tamsulosin to an established tamsulosin or mirabegron treatment in healthy middle-aged to elderly men.

OBJECTIVE: Tamsulosin and mirabegron may be used concomitantly in patients with lower urinary tract symptoms. Since alpha1-adrenoceptor antagonists are associated with cardiovascular side effects, potential pharmacokinetic and cardiovascular interactions were evaluated. MATERIALS AND METHODS: This was an open-label, randomized, 2-arm, 2-sequence study in 48 healthy men (24/arm) aged 44 - 72 years. In arm 1, subjects received single-dose tamsulosin hydrochloride modified release capsules (0.4 mg) alone and with steady-state mirabegron oral controlled absorption system tablets (100 mg once daily) in random sequence. In arm 2, subjects received single-dose mirabegron alone and with steady-state tamsulosin. Samples for mirabegron and tamsulosin plasma concentrations were collected. Blood pressure (BP) and pulse rate (PR) were measured and orthostatic stress tests were performed. RESULTS: Mirabegron increased tamsulosin C(max) to 159% (90% confidence interval (CI) 143 - 177%), AUC(∞) to 161% (90% CI 149 - 173%), and t(1/2) to 116%. Tamsulosin reduced mirabegron C(max) to 85% (90% CI 71 - 103%) and AUC(∞) to 84% (90% CI 74 - 95%) without effect on t1/2. Mirabegron and tamsulosin co-treatment caused no statistically significant changes (p > 0.05) in PR or systolic BP versus mono-treatment up to 12 hours post-dose. Mean diastolic BP decreases of -2.1 (95% CI -4.1, -0.1) to -4.2 (-7.5, -0.9) mmHg in arm 1 and -3.0 (-5.7, -0.3) to -4.2 (-7.4, -1.0) mmHg in arm 2 were observed, statistically significant (p < 0.05) at several time points, not accompanied by orthostatic symptoms or increases in positive orthostatic stress tests. Adverse and orthostatic events were balanced across treatments. CONCLUSIONS: The observed pharmacokinetic interactions upon add-on of mirabegron or tamsulosin to existing tamsulosin or mirabegron therapy did not cause clinically relevant changes in cardiovascular safety or safety profiles.

A role for the stringent response in ciprofloxacin resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major cause of nosocomial infections and the leading cause of chronic lung infections in cystic fibrosis and chronic obstructive pulmonary disease patients. Antibiotic treatment remains challenging because P. aeruginosa is resistant to high concentrations of antibiotics and has a remarkable ability to acquire mutations conferring resistance to multiple groups of antimicrobial agents. Here we report that when P. aeruginosa is plated on ciprofloxacin (cipro) plates, the majority of cipro-resistant (ciproR) colonies observed at and after 48 h of incubation carry mutations in genes related to the Stringent Response (SR). Mutations in one of the major SR components, spoT, were present in approximately 40% of the ciproR isolates. Compared to the wild-type strain, most of these isolates had decreased growth rate, longer lag phase and altered intracellular ppGpp content. Also, 75% of all sequenced mutations were insertions and deletions, with short deletions being the most frequently occurring mutation type. We present evidence that most of the observed mutations are induced on the selective plates in a subpopulation of cells that are not instantly killed by cipro. Our results suggests that the SR may be an important contributor to antibiotic resistance acquisition in P. aeruginosa.

Genetic profiling of rat gliomas and cardiac schwannomas from life-time radiofrequency radiation exposure study using a targeted next-generation sequencing gene panel.

The cancer hazard associated with lifetime exposure to radiofrequency radiation (RFR) was examined in Sprague Dawley (SD) rats at the Ramazzini Institute (RI), Italy. There were increased incidences of gliomas and cardiac schwannomas. The translational relevance of these rare rat tumors for human disease is poorly understood. We examined the genetic alterations in RFR-derived rat tumors through molecular characterization of important cancer genes relevant for human gliomagenesis. A targeted next-generation sequencing (NGS) panel was designed for rats based on the top 23 orthologous human glioma-related genes. Single-nucleotide variants (SNVs) and small insertion and deletions (indels) were characterized in the rat gliomas and cardiac schwannomas. Translational relevance of these genetic alterations in rat tumors to human disease was determined through comparison with the Catalogue of Somatic Mutations in Cancer (COSMIC) database. These data suggest that rat gliomas resulting from life-time exposure to RFR histologically resemble low grade human gliomas but surprisingly no mutations were detected in rat gliomas that had homology to the human IDH1 p.R132 or IDH2 p.R172 suggesting that rat gliomas are primarily wild-type for IDH hotspot mutations implicated in human gliomas. The rat gliomas appear to share some genetic alterations with IDH1 wildtype human gliomas and rat cardiac schwannomas also harbor mutations in some of the queried cancer genes. These data demonstrate that targeted NGS panels based on tumor specific orthologous human cancer driver genes are an important tool to examine the translational relevance of rodent tumors resulting from chronic/life-time rodent bioassays.

Mitochondrial membrane potential regulates nuclear DNA methylation and gene expression through phospholipid remodeling.

Maintenance of the mitochondrial inner membrane potential (ΔΨM) is critical for many aspects of mitochondrial function, including mitochondrial protein import and ion homeostasis. While ΔΨM loss and its consequences are well studied, little is known about the effects of increased ΔΨM. In this study, we used cells deleted of ATPIF1, a natural inhibitor of the hydrolytic activity of the ATP synthase, as a genetic model of mitochondrial hyperpolarization. Our data show that chronic ΔΨM increase leads to nuclear DNA hypermethylation, regulating transcription of mitochondria, carbohydrate and lipid metabolism genes. Surprisingly, remodeling of phospholipids, but not metabolites or redox changes, mechanistically links the ΔΨM to the epigenome. These changes were also observed upon chemical exposures and reversed by decreasing the ΔΨM, highlighting them as hallmark adaptations to chronic mitochondrial hyperpolarization. Our results reveal the ΔΨM as the upstream signal conveying the mitochondrial status to the epigenome to regulate cellular biology, providing a new framework for how mitochondria can influence health outcomes in the absence of canonical dysfunction.

Safety, tolerability, and pharmacokinetics of single and multiple ascending Oral doses of DA-8010 in healthy subjects: First-in-human phase I study.

This study assessed the safety, tolerability, and pharmacokinetics of single and multiple oral doses of DA-8010, a muscarinic M3 receptor antagonist, in healthy subjects. This was a randomized, double-blind, placebo-controlled, ascending single (Part A: 1, 2.5, 5, 20, and 40 mg QD fasted and 10 mg QD fasted and fed) and multiple doses (Part B: 5, 10, and 20 mg QD from Days 1 to 7 fasted), sequential-group study. Safety data were analyzed descriptively, time to maximum plasma concentration (tmax ) nonparametrically, and pharmacokinetic parameters using power and mixed models and ANOVA. Of 109 subjects randomized (Part A = 69 and Part B = 40; each part consisted a female group), 31 (44.9%) in Part A and 29 (72.5%) in Part B experienced treatment-emergent adverse events (TEAEs) in a dose-related manner. Common drug-related TEAEs in Part A and B were dizziness (8.7% and 15.0%), headache (5.8% and 12.5%) and blurred vision (8.7% and 20%). One male (20 mg) and one female (10 mg) from Part B discontinued the study due to a confusional state, and nausea and vomiting. Irrespective of sex, DA-8010 was steadily absorbed following single and multiple doses in the fasted state with increased systemic exposure in a dose-proportional manner with maximum plasma concentration occurring at a median tmax between 4.0 and 6.0 h. A high-fat meal increased systemic exposure. DA-8010 was safe, well tolerated, and well absorbed at lower doses and moderately tolerated at higher doses without any notable effects of food and sex.

Cholesterol biosynthesis modulates differentiation in murine cranial neural crest cells.

Cranial neural crest cells (cNCC) are a multipotent embryonic cell population that give rise to a diverse set of cell types. These cells are particularly vulnerable to external metabolic stressors, as exemplified by the association between maternal hyperglycemia and congenital malformations. We were interested in studying the effect of various concentrations of glucose and pyruvate on cNCC metabolism, migration, and differentiation using an established murine neural crest cell model (O9-1). We unexpectedly observed a pattern of gene expression suggestive of cholesterol biosynthesis induction under glucose depletion conditions in O9-1 cells. We further showed that treatment with two different cholesterol synthesis inhibitors interfered with cell migration and differentiation, inhibiting chondrogenesis while enhancing smooth muscle cell differentiation. As congenital arhinia (absent external nose), a malformation caused by mutations in SMCHD1, appears to represent, in part, a defect in cNCC, we were also interested in investigating the effects of glucose and cholesterol availability on Smchd1 expression in O9-1 cells. Smchd1 expression was induced under high glucose conditions whereas cholesterol synthesis inhibitors decreased Smchd1 expression during chondrogenesis. These data highlight a novel role for cholesterol biosynthesis in cNCC physiology and demonstrate that human phenotypic variability in SMCHD1 mutation carriers may be related, in part, to SMCHD1's sensitivity to glucose or cholesterol dosage during development.

Safety and pharmacokinetics of GB1211, an oral galectin-3 inhibitor: a single- and multiple-dose first-in-human study in healthy participants.

PURPOSE: Galectin-3, a ß-galactoside-binding lectin, plays a key role in several cellular pathways involved in chronic inflammation, heart disease and cancer. GB1211 is an orally bioavailable galectin-3 inhibitor, developed to be systemically active. We report safety and pharmacokinetics (PK) of GB1211 in healthy participants. METHODS: This phase 1, double-blind, placebo-controlled, first-in-human study (NCT03809052) included a single ascending-dose phase (with a food-effect cohort) where participants across seven sequential cohorts were randomized 3:1 to receive oral GB1211 (5, 20, 50, 100, 200 or 400 mg) or placebo. In the multiple ascending-dose phase, participants received 50 or 100 mg GB1211 or placebo twice daily for 10 days. All doses were administered in the fasted state except in the food-effect cohort where doses were given 30 min after a high-fat meal. RESULTS: All 78 participants received at least one GB1211 dose (n = 58) or placebo (n = 20) and completed the study. No safety concerns were identified. Following single and multiple oral doses under fasted conditions, maximum GB1211 plasma concentrations were reached at 1.75-4 h (median) post-dose; mean half-life was 11-16 h. There was a ~ twofold GB1211 accumulation in plasma with multiple dosing, with steady-state reached within 3 days; 30% of the administered dose was excreted in urine as unchanged drug. Absorption in the fed state was delayed by 2 h but systemic exposure was unaffected. CONCLUSION: GB1211 was well tolerated, rapidly absorbed, and displayed favorable PK, indicating a potential to treat multiple disease types. These findings support further clinical development of GB1211. CLINICAL TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov (identifier: NCT03809052).

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.