Dissecting the cis-regulatory syntax of transcription initiation with deep learning.

Despite extensive characterization of mammalian Pol II transcription, the DNA sequence determinants of transcription initiation at a third of human promoters and most enhancers remain poorly understood. Hence, we trained and interpreted a neural network called ProCapNet that accurately models base-resolution initiation profiles from PRO-cap experiments using local DNA sequence. ProCapNet learns sequence motifs with distinct effects on initiation rates and TSS positioning and uncovers context-specific cryptic initiator elements intertwined within other TF motifs. ProCapNet annotates predictive motifs in nearly all actively transcribed regulatory elements across multiple cell-lines, revealing a shared cis-regulatory logic across promoters and enhancers mediated by a highly epistatic sequence syntax of cooperative and competitive motif interactions. ProCapNet models of RAMPAGE profiles measuring steady-state RNA abundance at TSSs distill initiation signals on par with models trained directly on PRO-cap profiles. ProCapNet learns a largely cell-type-agnostic cis-regulatory code of initiation complementing sequence drivers of cell-type-specific chromatin state critical for accurate prediction of cell-type-specific transcription initiation.

Defining the value of faculty primary care clinical pharmacists.

BACKGROUND: Demonstrating the value of comprehensive medication management (CMM) within clinical faculty sites is a challenge when balancing patient volume with academic responsibilities. Utilizing an evidence-based implementation system for CMM, faculty primary care clinical pharmacists (PCCPs) standardized CMM within their practice sites. OBJECTIVE: The primary objective of this project was to define the value of faculty PCCPs. METHODS: An Ambulatory Care Summit was hosted to identify opportunities for consistency of CMM. Following the summit, the CMM implementation team (faculty PCCPs and project manager) utilized CMM implementation tools from the Comprehensive Medication Management in Primary Care Research Team. Additionally, a strategic plan was developed to enhance practice management, improve fidelity, and determine key performance indicators (KPIs). Five faculty-mentored student projects assessed value of faculty-delivered CMM in primary care clinics. Data included medication adherence metrics, clinic quality metrics, diabetes metrics, acute healthcare utilization rates, and a physician satisfaction survey. RESULTS: Among those receiving CMM, adherence improved 14% (P = 0.022), 119 clinic quality metrics were achieved, HbA1c ≤ 9% improved 45% (p < 0.001), average HbA1c decreased by 1.73% (p < 0.001), and medication preventable acute care utilization within the referral reason decreased. Over 90% of physicians surveyed agreed the faculty PCCP is a valuable team member, improved patients' health, and improved effectiveness/efficiency. Four student posters were presented at national conferences and 18 student pharmacists were engaged in various aspects of the project. CONCLUSION: Incorporating CMM at faculty primary care clinics provides value. To demonstrate this value, faculty must align KPIs with institution-specific payer contracts.

Rewriting regulatory DNA to dissect and reprogram gene expression.

Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory effects and programmability of such DNA sequences remain difficult to map or predict because we have lacked scalable methods to precisely edit regulatory DNA and quantify the effects in an endogenous genomic context. Here we present an approach to measure the quantitative effects of hundreds of designed DNA sequence variants on gene expression, by combining pooled CRISPR prime editing with RNA fluorescence in situ hybridization and cell sorting (Variant-FlowFISH). We apply this method to mutagenize and rewrite regulatory DNA sequences in an enhancer and the promoter of PPIF in two immune cell lines. Of 672 variant-cell type pairs, we identify 497 that affect PPIF expression. These variants appear to act through a variety of mechanisms including disruption or optimization of existing transcription factor binding sites, as well as creation of de novo sites. Disrupting a single endogenous transcription factor binding site often led to large changes in expression (up to -40% in the enhancer, and -50% in the promoter). The same variant often had different effects across cell types and states, demonstrating a highly tunable regulatory landscape. We use these data to benchmark performance of sequence-based predictive models of gene regulation, and find that certain types of variants are not accurately predicted by existing models. Finally, we computationally design 185 small sequence variants (≤10 bp) and optimize them for specific effects on expression in silico. 84% of these rationally designed edits showed the intended direction of effect, and some had dramatic effects on expression (-100% to +202%). Variant-FlowFISH thus provides a powerful tool to map the effects of variants and transcription factor binding sites on gene expression, test and improve computational models of gene regulation, and reprogram regulatory DNA.

Practical strategies to enhance resident engagement in clinical quality improvement.

BACKGROUND: Engaging residents in meaningful quality improvement (QI) is difficult. Challenges include competing demands, didactics which lack connection to meaningful work, suboptimal experiential learning, unclear accountability, absence of timely and relevant data, and lack of faculty coaches and role models. Various strategies to address these challenges for engagement have been described, but not as a unified approach. This paper describes a bundle of practical strategies to address common challenges to resident engagement in QI, illustrated through the experience of one residency education program. METHODS: 62 categorical residents in the University of Missouri Internal Medicine residency participated in a longitudinal QI curriculum integrated into residency clinic assignments with dedicated QI work sessions and brief just-in-time didactics with mentorship from faculty coaches. Residents completed at least two PDSA (Plan-Do-Study-Act) cycles for their projects. The experience included clear expectations and tools for accountability. Project criteria included importance to patients, residents, and the institution. Residents had access to data related to their own practice. A pre-post survey asked residents to self-assess their level of interest and engagement in QI on a 5-point Likert scale, with 1 = least desired and 5 = most desired result. Data were analyzed by paired t-test. RESULTS: All 62 residents participated in the program as members of ten QI teams. 40/62 residents completed both pre- and post-surveys. Items related to self-assessment of QI in clinical work all changed in the desired direction: likelihood of participation (3.7 to 4.1, p = 0.03), frequency of QI use (3.3 to 3.9, p = 0.001), and opinion about using QI in clinical work (3.9 to 4.0, p = 0.21). Resident assessment of QI priority in clinical work did not change. CONCLUSIONS: We implemented a practical strategies bundle to overcome common challenges to successfully engaging residents in clinical quality improvement. These strategies included QI work integrated into routine clinical assignments, just-in-time didactics, experiential learning with clear expectations and strategic project selection, timely and pertinent data from the residents' own practice, and real-time faculty coaching.

Domain-adaptive neural networks improve cross-species prediction of transcription factor binding.

The intrinsic DNA sequence preferences and cell type-specific cooperative partners of transcription factors (TFs) are typically highly conserved. Hence, despite the rapid evolutionary turnover of individual TF binding sites, predictive sequence models of cell type-specific genomic occupancy of a TF in one species should generalize to closely matched cell types in a related species. To assess the viability of cross-species TF binding prediction, we train neural networks to discriminate ChIP-seq peak locations from genomic background and evaluate their performance within and across species. Cross-species predictive performance is consistently worse than within-species performance, which we show is caused in part by species-specific repeats. To account for this domain shift, we use an augmented network architecture to automatically discourage learning of training species-specific sequence features. This domain adaptation approach corrects for prediction errors on species-specific repeats and improves overall cross-species model performance. Our results show that cross-species TF binding prediction is feasible when models account for domain shifts driven by species-specific repeats.

Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.

The Enigma of CRB1 and CRB1 Retinopathies.

Mutations in the gene Crumbs homolog 1 (CRB1) are responsible for several retinopathies that are diverse in severity and phenotype. Thus, there is considerable incentive to determine how disruption of this gene causes disease. Progress on this front will aid in developing molecular diagnostics that can predict disease severity with the ultimate goal of developing therapies for CRB1 retinopathies via gene replacement. The purpose of this review is to summarize what is known regarding CRB1 and highlights information outstanding. Doing so will provide a framework toward a thorough understanding of CRB1 at the molecular and protein level with the ultimate goal of deciphering how it contributes to the disease.

Providing Comprehensive Medication Management in Telehealth.

The goal of this white paper is to provide direction for clinical pharmacists providing telehealth. Telehealth care is rapidly emerging to improve patient access to health care and optimize patient health outcomes. With the increasing ability to access electronic health record portals, as many as 75 million telehealth encounters are expected in North America annually. Although electronic "point of access" undoubtedly increases the use of medical and pharmacy services, the real value of telehealth lies in improved access to patients in remote areas lacking adequate medical and pharmacy services and to high-risk patients requiring frequent monitoring. This document is intended to serve as a guide for those interested in or already using telehealth to provide direct patient care. Specifically, it focuses on general concepts of telehealth and demonstrates how the delivery of comprehensive medication management (CMM) by telehealth aligns with the Standards of Practice for Clinical Pharmacists set forth by the American College of Clinical Pharmacy. Although clinical pharmacists must be appropriately credentialed and privileged to provide CMM, their process of care must also be adapted to suit the remote patient. Patient assessment, evaluation of medication therapy, development and implementation of a plan of care, follow-up, monitoring, and documentation of all processes of care are influenced by the technology available, the collaborations established, and the applicable regulations and requirements for telehealth practice.

Nicotinamide and WLDS Act Together to Prevent Neurodegeneration in Glaucoma.

Glaucoma is a complex neurodegenerative disease characterized by progressive visual dysfunction leading to vision loss. Retinal ganglion cells are the primary affected neuronal population, with a critical insult damaging their axons in the optic nerve head. This insult is typically secondary to harmfully high levels of intraocular pressure (IOP). We have previously determined that early mitochondrial abnormalities within retinal ganglion cells lead to neuronal dysfunction, with age-related declines in NAD (NAD+ and NADH) rendering retinal ganglion cell mitochondria vulnerable to IOP-dependent stresses. The Wallerian degeneration slow allele, WldS , decreases the vulnerability of retinal ganglion cells in eyes with elevated IOP, but the exact mechanism(s) of protection from glaucoma are not determined. Here, we demonstrate that WldS increases retinal NAD levels. Coupled with nicotinamide administration (an NAD precursor), it robustly protects from glaucomatous neurodegeneration in a mouse model of glaucoma (94% of eyes having no glaucoma, more than WldS or nicotinamide alone). Importantly, nicotinamide and WldS protect somal, synaptic, and axonal compartments, prevent loss of anterograde axoplasmic transport, and protect from visual dysfunction as assessed by pattern electroretinogram. Boosting NAD production generally benefits major compartments of retinal ganglion cells, and may be of value in other complex, age-related, axonopathies where multiple neuronal compartments are ultimately affected.

GlyCAM1 negatively regulates monocyte entry into the optic nerve head and contributes to radiation-based protection in glaucoma.

BACKGROUND: We previously reported a profound long-term neuroprotection subsequent to a single radiation-therapy in the DBA/2J mouse model of glaucoma. This neuroprotection prevents entry of monocyte-like immune cells into the optic nerve head during glaucoma. Gene expression studies in radiation-treated mice implicated Glycam1 in this protection. Glycam1 encodes a proteoglycan ligand for L-selectin and is an excellent candidate to modulate immune cell entry into the eye. Here, we experimentally test the hypothesis that radiation-induced over-expression of Glycam1 is a key component of the neuroprotection. METHODS: We generated a null allele of Glycam1 on a DBA/2J background. Gene and protein expression of Glycam1, monocyte entry into the optic nerve head, retinal ganglion cell death, and axon loss in the optic nerve were assessed. RESULTS: Radiation therapy potently inhibits monocyte entry into the optic nerve head and prevents retinal ganglion cell death and axon loss. DBA/2J mice carrying a null allele of Glycam1 show increased monocyte entry and increased retinal ganglion cell death and axon loss following radiation therapy, but the majority of optic nerves were still protected by radiation therapy. CONCLUSIONS: Although GlyCAM1 is an L-selectin ligand, its roles in immunity are not yet fully defined. The current study demonstrates a partial role for GlyCAM1 in radiation-mediated protection. Furthermore, our results clearly show that GlyCAM1 levels modulate immune cell entry from the vasculature into neural tissues. As Glycam1 deficiency has a more profound effect on cell entry than on neurodegeneration, further experiments are needed to precisely define the role of monocyte entry in DBA/2J glaucoma. Nevertheless, GlyCAM1's function as a negative regulator of extravasation may lead to novel therapeutic strategies for an array of common conditions involving inflammation.

Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice.

Glaucomas are neurodegenerative diseases that cause vision loss, especially in the elderly. The mechanisms initiating glaucoma and driving neuronal vulnerability during normal aging are unknown. Studying glaucoma-prone mice, we show that mitochondrial abnormalities are an early driver of neuronal dysfunction, occurring before detectable degeneration. Retinal levels of nicotinamide adenine dinucleotide (NAD+, a key molecule in energy and redox metabolism) decrease with age and render aging neurons vulnerable to disease-related insults. Oral administration of the NAD+ precursor nicotinamide (vitamin B3), and/or gene therapy (driving expression of Nmnat1, a key NAD+-producing enzyme), was protective both prophylactically and as an intervention. At the highest dose tested, 93% of eyes did not develop glaucoma. This supports therapeutic use of vitamin B3 in glaucoma and potentially other age-related neurodegenerations.

Is It Painful to Manage Chronic Pain? A Cross-Sectional Study of Physicians In-Training in a University Program.

BACKGROUND: Prescribing opioids for chronic non-cancer pain (CNCP) is a challenge due to associated risks from abuse, addiction and adverse effects. We surveyed resident physicians on their knowledge, attitude and practices in opioid prescription practices in the ambulatory setting and conducted an educational module to address their knowledge gaps. METHODS: A phase 1 survey assessed knowledge, attitudes and practices of residents in the out-patient management of CNCP with opioids. Demographics, numbers of patients seen, those with concerns for risky behaviors, adverse effects and the reasons for concern were also recorded. In Phase 2, an educational module in the form of didactics and case based discussions addressed the perceived deficiencies noted from results of phase 1 survey. Pre and post module surveys assessed the effectiveness of the educational module. RESULTS: In the phase 1 study (45/49, 92% response rate, M:F = 30:15) 33.3% (15/45) were in Post-Graduate Year (PGY) 1, 35.6% (16/45) PGY2s and 31.1% (14/45) PGY3s; 80% (36/45) saw more than one patient with CNCP in the previous 3 months; 62.2% (28/45) had at least one patient with concerns for misuse and addiction; 77.8% (35/45) and 86.7% (39/45) reported a lack of training and consistent documentation respectively, and 82.2% (37/45) were uncomfortable to refill for other provider's patients. All (100%, 45/45) consulted the clinical pharmacist; 86.7% (39/45) believed that either focused education would be beneficial. In the phase 2 study (44/49, 89.7% response rate, M: F = 29: 15), the pre- and post-module responses showed that > 90% of the residents perceived improvement in knowledge and confidence in management of CNCP with opioids after the educational module. CONCLUSIONS: Internal medicine residents perceived deficits in their ability to manage CNCP. Following a focused educational training, residents' knowledge and confidence in prescription of opioids improved, demonstrating the need to include management of CNCP with opioids into their curriculum.

Implementation and evaluation of activities to foster a sense of community among pharmacy students.

INTRODUCTION: The purpose of this study was to investigate the association of a series of team-based instructional activities on students' sense of community for pharmacy students assigned to the main and distance program sites. MATERIALS AND METHODS: In conjunction with a teaching objective structured clinical examination (OSCE) conducted at the program's distance site, several team-based sense of community activities were completed. The classroom and school community inventory (CSCI) was adapted and administered to students prior to and following all learning activities to measure course and program sense of community. RESULTS: Pre- and post-surveys were completed by 116 students. Course and program sense of community significantly increased among students at the distance site (p = 0.007 and p = 0.008, respectively). No significant changes were found for students assigned to the main site. CONCLUSION: For programs with multiple sites, activities designed to enhance sense of community and conducted at the distance site are effective for students assigned to the distance site.

Programmed evolution for optimization of orthogonal metabolic output in bacteria.

Current use of microbes for metabolic engineering suffers from loss of metabolic output due to natural selection. Rather than combat the evolution of bacterial populations, we chose to embrace what makes biological engineering unique among engineering fields - evolving materials. We harnessed bacteria to compute solutions to the biological problem of metabolic pathway optimization. Our approach is called Programmed Evolution to capture two concepts. First, a population of cells is programmed with DNA code to enable it to compute solutions to a chosen optimization problem. As analog computers, bacteria process known and unknown inputs and direct the output of their biochemical hardware. Second, the system employs the evolution of bacteria toward an optimal metabolic solution by imposing fitness defined by metabolic output. The current study is a proof-of-concept for Programmed Evolution applied to the optimization of a metabolic pathway for the conversion of caffeine to theophylline in E. coli. Introduced genotype variations included strength of the promoter and ribosome binding site, plasmid copy number, and chaperone proteins. We constructed 24 strains using all combinations of the genetic variables. We used a theophylline riboswitch and a tetracycline resistance gene to link theophylline production to fitness. After subjecting the mixed population to selection, we measured a change in the distribution of genotypes in the population and an increased conversion of caffeine to theophylline among the most fit strains, demonstrating Programmed Evolution. Programmed Evolution inverts the standard paradigm in metabolic engineering by harnessing evolution instead of fighting it. Our modular system enables researchers to program bacteria and use evolution to determine the combination of genetic control elements that optimizes catabolic or anabolic output and to maintain it in a population of cells. Programmed Evolution could be used for applications in energy, pharmaceuticals, chemical commodities, biomining, and bioremediation.

The CHADS2versus the new CHA2DS2-VASc scoring systems for guiding antithrombotic treatment of patients with atrial fibrillation: review of the literature and recommendations for use.

The association of atrial fibrillation and resultant thromboembolic stroke is readily recognized in the published literature. However, the identification and weight of other risk factors that increase stroke risk are varied. To predict which patients are at greatest risk for thromboembolic stroke, numerous risk stratification schemas have been developed to guide thromboprophylactic treatment decisions. The well-known CHADS(2) scoring system incorporates risk factors such as congestive heart failure, hypertension, age 75 years or older, diabetes mellitus, and previous stroke or transient ischemic attack. Recently, a novel risk stratification model, CHA2DS2 -VASc, has entered the literature and international guidelines, prompting further review of newly added risk factors-age 65-74 years, presence of vascular disease, and female sex-and the increased allotment of 2 points (vs 1 point in CHADS2) for age 75 years or older. The rationale for CHA2DS2 -VASc, as put forth by its authors, is that other risk assessment models omit important risk factors, have low predictive ability, and categorize too many patients as intermediate risk, leaving the choice of anticoagulant or antiplatelet therapy to the discretion of the clinician. Although CHA2DS2 -VASc readily identifies those patients truly at low risk, it classifies more patients as high risk who would then receive anticoagulation therapy. Therefore, implementation of this risk schema warrants further evaluation, especially when weighing the risk for bleeding and the risk for stroke. This critical review provides practitioners with an understanding of the literature that prompted the inclusion of these new risk factors and increased point allocations, compares and contrasts the risk schemas, and reviews national and international guidelines, thereby equipping the health care provider with the knowledge to aid clinical decision-making.

Bleeding incidence with concomitant use of antidepressants and warfarin.

INTRODUCTION: Bleeding is the major complication associated with warfarin therapy. Some antidepressants are also associated with increased bleeding risk. Warfarin and antidepressants are used frequently in combination, but it is unclear whether concomitant use increases the risk of bleeding beyond that with warfarin alone. The primary goal of this study was to determine whether the use of warfarin and an antidepressant increases the risk for bleeding outcomes compared with the use of warfarin alone. The secondary goal was to characterize the risk of bleeding in warfarin-treated patients taking one specific class of antidepressant, selective serotonin reuptake inhibitors (SSRIs). MATERIALS AND METHODS: This was a retrospective, single-center, study of warfarin-treated patients prescribed (n = 46) and not prescribed (n = 54) an antidepressant. Medical records over 6 months were reviewed for international normalized ratio values, medical history, bleeding type and incidence, and hospitalization due to bleeding. Patients were included in the antidepressant group if they were taking concomitant warfarin and antidepressant therapy consistently for a period of 6 months and in the control group if they were not taking an antidepressant with warfarin. RESULTS: The use of any antidepressant with warfarin was not associated with the incidence of any bleeding or major bleeding during the 6-month period. However, the use of an SSRI with warfarin was associated with an increase in any bleeding event (odds ratio 2.6, 95% confidence interval, 1.01-6.4 P = 0.04). The use of an SSRI remained a significant predictor of bleeding after accounting for other factors associated with bleeding risk. CONCLUSIONS: Based on these data, it is important to clarify the interaction between warfarin and SSRIs in regard to bleeding risk given the high frequency of their concomitant use.

Interpreting nurses' responses to clinical documentation alerts.

OBJECTIVE: To understand how nurses respond to alerts that detect attempts to enter into electronic health records patient weights that vary significantly from previously recorded weights. METHODS: Examination of subsequent patient weights to determine if the alerts were true positive (TP) or false positive (FP), and whether nurses overrode alerts, changed their entry or quit without storing a value. RESULTS: Alerts occurred 2.74%, with 41.9% TP and 58.1% FP. Nurses overrode 30.3% of TP and 97.3% of FP alerts. CONCLUSIONS: The alert has an acceptable FP rate and does not appear to cause nurses to change entries to satisfy the alert. The alert improves recording of patient weights.