A single N6-methyladenosine site regulates lncRNA HOTAIR function in breast cancer cells.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes.

BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment.

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction.

In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.

Cost analysis of one-time intravenous antibiotic doses in the emergency department.

BACKGROUND: Research shows that one-time doses of intravenous (IV) antibiotics do not improve resolution of infection. Providers, however, continue to use them - especially in the emergency department. Very few studies have aimed to quantify the cost of this practice. OBJECTIVES: The primary objective was to evaluate the difference in average total cost of emergency department (ED) stay between patients who received a one-time dose of intravenous antibiotics in the ED before discharging on oral antibiotics and patients who were just discharged on oral antibiotics. Secondary objectives were to evaluate the differences in durations of stay between the two groups, as well as the differences in adverse drug effects and need for healthcare contact after discharge. METHODS: Chart review was conducted to identify patients who received and did not receive a one-time dose of IV antibiotics in the ED between April 30, 2020, and April 30, 2022. A micro-costing approach was used to determine ED-associated costs per patient. Comparisons in primary and secondary outcomes were performed using statistical inferential tests. RESULTS: A total of 102 patients were analyzed in each group. Patients who received a one-time dose of intravenous antibiotics in the emergency department before being discharged on oral antibiotics had an average length of stay of 4.55 hours, as opposed to patients who did not receive a one-time dose of intravenous antibiotics before being discharged on oral antibiotics who had an average length of stay of 2.82 hours (absolute difference: 1.73 hours, p < 0.001). One-time dosing of intravenous antibiotics in the emergency department incurred an additional cost of approximately $556 per patient, totaling to over $56,000 in our study cohort. CONCLUSION: The use of one-time intravenous antibiotics in the emergency department did not confer any additional benefits to patients. Use of one-time doses resulted in significantly reduced throughput in the emergency department and significantly increased healthcare costs.

Cross-adaptation from heat stress to hypoxia: A systematic review and exploratory meta-analysis.

Cross-adaptation (CA) refers to the successful induction of physiological adaptation under one environmental stressor (e.g., heat), to enable subsequent benefit in another (e.g., hypoxia). This systematic review and exploratory meta-analysis investigated the effect of heat acclimation (HA) on physiological, perceptual and physical performance outcome measures during rest, and submaximal and maximal intensity exercise in hypoxia. Database searches in Scopus and MEDLINE were performed. Studies were included when they met the Population, Intervention, Comparison, and Outcome criteria, were of English-language, peer-reviewed, full-text original articles, using human participants. Risk of bias and study quality were assessed using the COnsensus based Standards for the selection of health status Measurement INstruments checklist. Nine studies were included, totalling 79 participants (100 % recreationally trained males). The most common method of HA included fixed-intensity exercise comprising 9 ± 3 sessions, 89 ± 24-min in duration and occurred within 39 ± 2 °C and 32 ± 13 % relative humidity. CA induced a moderate, beneficial effect on physiological measures at rest (oxygen saturation: g = 0.60) and during submaximal exercise (heart rate: g = -0.65, core temperature: g = -0.68 and skin temperature: g = -0.72). A small effect was found for ventilation (g = 0.24) and performance measures (peak power: g = 0.32 and time trial time: g = -0.43) during maximal intensity exercise. No effect was observed for perceptual outcome measures. CA may be appropriate for individuals, such as occupational or military workers, whose access to altitude exposure prior to undertaking submaximal activity in hypoxic conditions is restricted. Methodological variances exist within the current literature, and females and well-trained individuals have yet to be investigated. Future research should focus on these cohorts and explore the mechanistic underpinnings of CA.

PFKFB3 Inhibits Fructose Metabolism in Pulmonary Microvascular Endothelial Cells.

Pulmonary microvascular endothelial cells contribute to the integrity of the lung gas exchange interface, and they are highly glycolytic. Although glucose and fructose represent discrete substrates available for glycolysis, pulmonary microvascular endothelial cells prefer glucose over fructose, and the mechanisms involved in this selection are unknown. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is an important glycolytic enzyme that drives glycolytic flux against negative feedback and links glycolytic and fructolytic pathways. We hypothesized that PFKFB3 inhibits fructose metabolism in pulmonary microvascular endothelial cells. We found that PFKFB3 knockout cells survive better than wild-type cells in fructose-rich medium under hypoxia. Seahorse assays, lactate and glucose measurements, and stable isotope tracing showed that PFKFB3 inhibits fructose-hexokinase-mediated glycolysis and oxidative phosphorylation. Microarray analysis revealed that fructose upregulates PFKFB3, and PFKFB3 knockout cells increase fructose-specific GLUT5 (glucose transporter 5) expression. Using conditional endothelial-specific PFKFB3 knockout mice, we demonstrated that endothelial PFKFB3 knockout increases lung tissue lactate production after fructose gavage. Last, we showed that pneumonia increases fructose in BAL fluid in mechanically ventilated ICU patients. Thus, PFKFB3 knockout increases GLUT5 expression and the hexokinase-mediated fructose use in pulmonary microvascular endothelial cells that promotes their survival. Our findings indicate that PFKFB3 is a molecular switch that controls glucose versus fructose use in glycolysis and help better understand lung endothelial cell metabolism during respiratory failure.

Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline.

Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-m6A antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single-nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.

Beetroot juice ingestion does not improve neuromuscular performance and match-play demands in elite female hockey players: a randomized, double-blind, placebo-controlled study.

PURPOSE: Beetroot juice is a dietary supplement that contains high levels of inorganic nitrate (NO3-) and that its intake has proven effective at increasing blood nitric oxide (NO) concentrations improving endurance performance. However, the effect of this supplement in team sport performance, especially in female athletes, has been barely studied. This study aimed to compare the acute effects of beetroot juice supplementation on neuromuscular performance and match-play demands in elite female field hockey players. METHODS: Eleven elite female hockey players (22.8 ± 5.1 years) belonging to a bronze team medal in Eurohockey Club Champions Cup participated in this study. Participants were randomly divided into two groups undergoing a test battery with beetroot juice (70 mL, 6.4 mmol NO3-) or placebo (70 mL, 0.04 mmol NO3-) in two different days with one week between protocols. The neuromuscular test battery consisted of a countermovement jump, isometric handgrip strength (i.e., dominant hand), 20 m-sprint and repeated sprint ability test (RSA). Afterward, a simulated hockey match play (2 × 12.5 min) was performed and recorded by Global Positioning System (GPS). RESULTS: No statistically significant improvements were observed in any physical parameters analysed comparing beetroot juice compared to placebo ingestion, countermovement jump (p = 0.776, ES = 0.16), isometric handgrip strength (p = 0.829; ES = - 0.08), 20 m sprint test (p = 0.227; ES = - 0.23), mean repeated sprint ability (p = 0.955, ES = 0.03) and in any physical match demands measured by GPS (p = 0.243-1.000; ES = 0.02-0.47). CONCLUSION: Acute beetroot juice supplementation did not produce any statistically significant improvement in neuromuscular performance or match-play demands in elite female field hockey players. TRIAL REGISTRATION: The study was registered in ClinicalTrials.gov with the following ID: NCT05209139. The study was retrospectively registered by 26 January 2022.

Transcription control by the ENL YEATS domain in acute leukaemia.

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.

Effects of protein-carbohydrate vs. carbohydrate alone supplementation on immune inflammation markers in endurance athletes: a randomized controlled trial.

PURPOSE: The impact of ingesting carbohydrates alone or combined with proteins to support exercise immune adaptation in endurance athletes is scarcely investigated. The present study compares the effect of ingesting a combined protein-carbohydrate supplement vs. a carbohydrate-only supplement post-workout on immune inflammation markers following a 10 week periodized endurance training program in well-trained athletes. METHODS: Twenty-five men completed the study after being randomly assigned to one of the following intervention groups: combined protein-carbohydrate (PRO-CHO n = 12, 31 ± 9 years, [Formula: see text]O2peak 61.0 ± 5.6 ml.kg-1.min-1) or non-protein isoenergetic carbohydrate (CHO, n = 13, 33 ± 8 years, [Formula: see text]O2peak 60.6 ± 6.9 ml.kg-1.min-1). Treatment consisted of ingesting 24 g of assigned supplement, mixed with 250 ml of orange juice, once a day for 10 weeks immediately post-workout (or before breakfast on non-training days). Measurements were conducted pre- and post-intervention on total leukocytes, leukocyte subsets (i.e., neutrophils, eosinophils, basophils, monocytes, and lymphocytes), and platelets. The inflammatory status was assessed by the neutrophil-to-lymphocyte ratio (NLR), the platelet-to-lymphocyte ratio (PLR), and the systemic-immune inflammation index (SII). RESULTS: Post-intervention, significant increases were observed for CHO group only for the three inflammatory markers: NLR (p = 0.050, d = 0.58), PLR (p = 0.041, d = 0.60), and SII (p = 0.004, d = 0.81) but not for PRO-CHO (p > 0.05). CONCLUSION: Ingesting a post-workout protein-carbohydrate combined beverage promoted a more favourable immune status than carbohydrate-only ingestion by attenuating cellular inflammation over a 10 week training period in endurance male athletes. TRIAL REGISTRATION: The study was registered in ClinicalTrials.gov with the following ID: NCT02954367. The study was registered by 3 November 2016.

Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.

Changes in 25-hydroxyvitamin D levels post-vitamin D supplementation in people of Black and Asian ethnicities and its implications during COVID-19 pandemic: A systematic review.

BACKGROUND: People of Black and Asian ethnicities have a higher infection rate and mortality as a result of COVID-19. It has also been reported that vitamin D deficiency may play a role in this, possibly because of the multi-gene regulatory function of the vitamin D receptor. As a result, increased dietary intake and/or supplementation to attain adequate 25-hydroxyvitamin D (25(OH)D) levels could benefit people in these ethnicities. The present study aimed to review the literature examining the changes in 25(OH)D in different types of vitamin D supplementation from randomised controlled trials in this population. METHODS: This systematic review was conducted using the PRISMA guidelines. Electronic databases were systematically searched using keywords related to vitamin D supplementation in Black and Asian ethnicities. RESULTS: Eight studies were included in the review. All the included studies found that supplementation of vitamin D (D2 and D3 ), regardless of dosage, increased 25(OH)D levels compared to a placebo. All trials in which participants were vitamin D deficient at baseline showed increased 25(OH)D levels to a level considered adequate. Two studies that used food fortification yielded smaller 25(OH)D increases compared to similar studies that used oral supplementation (10.2 vs. 25.5 nmol L-1 , respectively). Furthermore, vitamin D2 supplementation yielded significantly lower 25(OH)D increases than vitamin D3 supplementation. CONCLUSIONS: Oral vitamin D supplementation may be more efficacious in increasing 25(OH)D levels than food fortification of Black and Asian ethnicities, with vitamin D3 supplementation possibly being more efficacious than vitamin D2 . It is recommended that people with darker skin supplement their diet with vitamin D3 through oral tablet modes where possible, with recent literature suggesting a daily intake of 7000-10,000 IU to be potentially protective from unfavourable COVID-19 outcomes. As a result of the paucity of studies, these findings should be treated as exploratory.

RNA matchmaking in chromatin regulation.

Beyond being the product of gene expression, RNA can also influence the regulation of chromatin. The majority of the human genome has the capacity to be transcribed and the majority of the non-protein-coding transcripts made by RNA Polymerase II are enriched in the nucleus. Many chromatin regulators can bind to these ncRNAs in the nucleus; in some cases, there are clear examples of direct RNA-mediated chromatin regulation mechanisms stemming from these interactions, while others have yet to be determined. Recent studies have highlighted examples of chromatin regulation via RNA matchmaking, a term we use broadly here to describe intermolecular base-pairing interactions between one RNA molecule and an RNA or DNA match. This review provides examples of RNA matchmaking that regulates chromatin processes and summarizes the technical approaches used to capture these events.

The dTAG system for immediate and target-specific protein degradation.

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

Effect of an acute blood donation on oxygen uptake kinetics in moderate and heavy domains over a period of 96 hours.

BACKGROUND: Studies determining the effects of blood donation (BD) on oxygen uptake kinetics are limited. This study aims to ascertain the effects of BD (~470 mL) over a period of 96 hours on oxygen uptake kinetics in moderate and heavy exercise domains. STUDY DESIGN AND METHODS: Twelve participants (nine males and three females; 31.1 ± 11.7 years, mass 79.9 ± 12.8 kg, height 175.5 ± 7.5 cm) completed four consecutive days (24-96 hours) of moderate and heavy V˙O2 on-kinetics trials pre BD and post BD. Visit one (0 hour), pre BD established hematological levels, V˙O2max and Gas Exchange Threshold (GET). Subsequent visits comprised two 6-minute moderate (∆ 50% rest-GET) and 1 heavy (∆ 20% GET-V˙O2max ) trial. Post BD 0 hour the participants donated blood post hematological testing only. RESULTS: Despite non-significances for V˙O2 amplitude, time constant-2 (tau2 ) for V˙O2 showed significant decreases at 24 and 48 hours, and tau3 showed significant increases at 72 and 96 hours pre to post BD (P < .05). Hemoglobin (Hb) values reduced (P < .05) pre (14.48 ± 0.16 g·dL-1 ) to post BD (13.47 ± 0.66 g·dL- 1). Hb significantly decreased at 24, 48, 72, and 96 hours compared to 0 hour post BD (P < .05). CONCLUSION: BD has no effect on V˙O2 amplitude, but time-based components show sensitivity to reduced circulating O2 ; with a decreased PO2 a slower O2 exchange across the blood myocyte barrier could result in altering O2 kinetics.

Case Report: Glaucoma-associated Peripapillary Retinoschisis with Corresponding Lamina Cribrosa Defect.

SIGNIFICANCE: Peripapillary retinoschisis is associated with primary and secondary glaucoma. It is important that clinicians are familiar with the presentation and management of peripapillary retinoschisis to understand its effects on the patient's glaucoma and to avoid unnecessary referral when the macula is not involved. PURPOSE: We present a case of peripapillary retinoschisis found incidentally on routine optical coherence tomographic (OCT) surveillance of primary open-angle glaucoma. CASE REPORT: A 70-year-old man presented for his annual diabetic eye examination. Surveillance with OCT revealed a splitting of the inner peripapillary retina corresponding to a previously noted notch in the right optic nerve. Further imaging of the right eye using enhanced depth imaging OCT revealed a defect in the lamina cribrosa that may have contributed to the formation and persistence of peripapillary retinoschisis. Retinal nerve fiber layer analysis showed a 5-year history of progressive temporal and inferotemporal thickening in the right eye. The patient was managed conservatively with instruction on regular Amsler grid testing. CONCLUSIONS: As seen in this case, peripapillary retinoschisis typically alters retinal nerve fiber layer thickness on OCT and can be mistakenly attributed to glaucomatous change. Glaucoma-associated peripapillary retinoschisis is usually not vision threatening and can be managed conservatively; in rare cases of progression to macular involvement, patients should be referred to a retina specialist.

Characterization of novel small RNAs (sRNAs) contributing to the desiccation response of Salmonella enterica serovar Typhimurium.

Noncoding RNA (ncRNA) modulation of gene expression has now been ubiquitously observed across all domains of life. An increasingly apparent role of ncRNAs is to coordinate changes in gene expressions in response to environmental stress. Salmonella enterica, a common food-born pathogen, is known for its striking ability to survive, adapt, and thrive in various unfavourable environments which makes it a particularly difficult pathogen to eliminate as well as an interesting model in which to study ncRNA contributions to cellular stress response. Mounting evidence now suggests that small RNAs (sRNAs) represent key regulators of Salmonella stress adaptation. Approximately 50-500 nucleotides in length, sRNAs regulate gene expression through complementary base pairing with molecular targets and have recently been suggested to outnumber protein-coding genes in bacteria. In this work, we employ small RNA transcriptome sequencing to characterize changes in the sRNA profiles of Salmonella in response to desiccation. In all, we identify 102 previously annotated sRNAs significantly differentially expressed during desiccation; and excitingly, 71 novel sRNAs likewise differentially expressed. Small transcript northern blotting and qRT-PCRs confirm the identities and expressions of several of our novel sRNAs, and computational analyses indicate the majority are highly conserved and structurally related to characterized sRNAs. Predicted sRNA targets include several proteins necessary for desiccation survival and this, in part, suggests a role for desiccation-regulated sRNAs in this stress response. Furthermore, we find individual knock-outs of two of the novel sRNAs identified herein, either sRNA1320429 or sRNA3981754, significantly impairs the ability of Salmonella to survive desiccation, confirming their involvements (and suggesting the potential involvements of other sRNAs we identify in this work) in the Salmonella response to desiccation.

Design and characterization of bivalent BET inhibitors.

Cellular signaling is often propagated by multivalent interactions. Multivalency creates avidity, allowing stable biophysical recognition. Multivalency is an attractive strategy for achieving potent binding to protein targets, as the affinity of bivalent ligands is often greater than the sum of monovalent affinities. The bromodomain and extraterminal domain (BET) family of transcriptional coactivators features tandem bromodomains through which BET proteins bind acetylated histones and transcription factors. All reported antagonists of the BET protein BRD4 bind in a monovalent fashion. Here we describe, to our knowledge for the first time, a bivalent BET bromodomain inhibitor-MT1-which has unprecedented potency. Biophysical and biochemical studies suggest MT1 is an intramolecular bivalent BRD4 binder that is more than 100-fold more potent, in cellular assays, than the corresponding monovalent antagonist, JQ1. MT1 significantly (P < 0.05) delayed leukemia progression in mice, as compared to JQ1. These data qualify a powerful chemical probe for BET bromodomains and a rationale for further development of multidomain inhibitors of epigenetic reader proteins.

Global profiling of hnRNP A2/B1-RNA binding on chromatin highlights LncRNA interactions.

Long noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

Levels of Physical Activity in Lithuanian Adolescents.

BACKGROUND AND OBJECTIVE: Population levels of physical activity are an international concern. The purpose of the present study was to describe and analyse physical activity levels in Lithuanian adolescents. MATERIALS AND METHODS: With this aim in mind, the Physician-based Assessment and Counselling for Exercise (PACE) questionnaire was administered to 5141 adolescents residing in Lithuania, 2502 boys (48.7%) and 2639 girls (51.3%), aged between 11 and 19 years. RESULTS: It was found that adolescents studied met the physical activity guideline, of 60 min of moderate-to-vigorous physical activity a day, on average 3.6 days/week (SD = 2.1). A total of 3426 adolescents (66.6%) were inactive as classified by the PACE questionnaire (at least 1 h of physical activity/day < 5 days/week). In the present sample there were more active (at least 1 h of physical activity/day ≥ 5 days/week) boys (n = 994, 39.7%) than girls (n = 721, 27.3%) (p < 0.001; OR 1.75, 95% CI 1.56 to 1.97), and, on average, boys were more likely to meet daily recommendations of physical activity than girls, 0.7 days more a week (p < 0.001; IRRs 1.21, 95% CI 1.17 to 1.25). According to age, younger adolescents (11⁻12 years) were significantly more active than older adolescents (13⁻19 years) and a curvilinear relationship between age and physical activity was observed with significant linear (unstandardized beta (B) = -0.807; standardized beta (ß) = -0.796; p < 0.001) and quadratic terms (unstandardized beta (B) = 0.024; standardized beta (ß) = 0.704; p < 0.001). CONCLUSIONS: It is necessary to increase the level of physical activity in Lithuanian adolescents and intervention programs should be carried out considering these results.