Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins.

Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

The Short Isoform of BRD4 Promotes HIV-1 Latency by Engaging Repressive SWI/SNF Chromatin-Remodeling Complexes.

BET proteins commonly activate cellular gene expression, yet inhibiting their recruitment paradoxically reactivates latent HIV-1 transcription. Here we identify the short isoform of BET family member BRD4 (BRD4S) as a corepressor of HIV-1 transcription. We found that BRD4S was enriched in chromatin fractions of latently infected T cells, and it was more rapidly displaced from chromatin upon BET inhibition than the long isoform. BET inhibition induced marked nucleosome remodeling at the latent HIV-1 promoter, which was dependent on the activity of BRG1-associated factors (BAF), an SWI/SNF chromatin-remodeling complex with known repressive functions in HIV-1 transcription. BRD4S directly bound BRG1, a catalytic subunit of BAF, via its bromodomain and extraterminal (ET) domain, and this isoform was necessary for BRG1 recruitment to latent HIV-1 chromatin. Using chromatin immunoprecipitation sequencing (ChIP-seq) combined with assay for transposase-accessible chromatin coupled to high-throughput sequencing (ATAC-seq) data, we found that the latent HIV-1 promoter phenotypically resembles endogenous long terminal repeat (LTR) sequences, pointing to a select role of BRD4S-BRG1 complexes in genomic silencing of invasive retroelements.

Lysine Acetylation Goes Global: From Epigenetics to Metabolism and Therapeutics.

Post-translational acetylation of lysine residues has emerged as a key regulatory mechanism in all eukaryotic organisms. Originally discovered in 1963 as a unique modification of histones, acetylation marks are now found on thousands of nonhistone proteins located in virtually every cellular compartment. Here we summarize key findings in the field of protein acetylation over the past 20 years with a focus on recent discoveries in nuclear, cytoplasmic, and mitochondrial compartments. Collectively, these findings have elevated protein acetylation as a major post-translational modification, underscoring its physiological relevance in gene regulation, cell signaling, metabolism, and disease.

Burden, timing, and relationship of cardiovascular hospitalization to mortality among Medicare beneficiaries with newly diagnosed atrial fibrillation.

BACKGROUND: Limited data exist on the burden and relationship of cardiovascular (CV) hospitalization to mortality after newly diagnosed with atrial fibrillation (AF). METHODS: Using a 20% sample of nationwide Medicare Part A and B claims data, we performed a retrospective cohort study of Medicare beneficiaries with newly diagnosed AF (2004-2008). Cox proportional hazards time-varying exposures were used to determine the risk of death among patients with CV hospitalization after AF diagnosis. RESULTS: Of 228,295 patients (mean age 79.6 ± 7.4 years, 56% female), 57% had a CV hospitalization after diagnosis of AF (41% in the first year). The most common primary CV hospitalization diagnoses were AF/supraventricular arrhythmias (21%), heart failure (19%), myocardial infarction (11%), and stroke/transient ischemic attack (7.7%). Incidence rates per 1,000 person-years among patients with and without CV hospitalization were 114 and 87, respectively, for all-cause mortality. After adjustment for covariates and time to CV hospitalization, the hazard of mortality among newly diagnosed AF patients with CV hospitalization, compared with those without CV hospitalization, was higher (hazard ratio 1.22, 95% CI 1.20-1.24). CONCLUSIONS: Cardiovascular hospitalization is common in the first year after AF diagnosis. Atrial fibrillation, heart failure, myocardial infarction, and stroke/transient ischemic attack account for half of primary hospitalization diagnosis. Cardiovascular hospitalization is independently associated with mortality, irrespective of time from diagnosis to first hospitalization, and represents a critical inflection point in survival trajectory. These findings highlight the importance of CV hospitalization as a marker of disease progression and poor outcomes. Efforts to clarify the determinants of hospitalization could inform interventions to reduce admissions and improve survival.

Instance segmentation of mitochondria in electron microscopy images with a generalist deep learning model trained on a diverse dataset.

Mitochondria are extremely pleomorphic organelles. Automatically annotating each one accurately and precisely in any 2D or volume electron microscopy (EM) image is an unsolved computational challenge. Current deep learning-based approaches train models on images that provide limited cellular contexts, precluding generality. To address this, we amassed a highly heterogeneous ∼1.5 × 106 image 2D unlabeled cellular EM dataset and segmented ∼135,000 mitochondrial instances therein. MitoNet, a model trained on these resources, performs well on challenging benchmarks and on previously unseen volume EM datasets containing tens of thousands of mitochondria. We release a Python package and napari plugin, empanada, to rapidly run inference, visualize, and proofread instance segmentations. A record of this paper's transparent peer review process is included in the supplemental information.

Effects of the atypical antipsychotic and D3/D2 dopamine partial agonist cariprazine on effort-based choice behavior: implications for modeling avolition.

RATIONALE: Cariprazine is an atypical antipsychotic that acts as a D3/D2 receptor partial agonist. In addition to treating positive symptoms of schizophrenia, cariprazine may have utility for treating negative symptoms. Rodent studies have focused on the effects of cariprazine on cognitive functions and behaviors thought to be related to anhedonia. Avolition, which is characterized by reduced initiation and persistence of goal-directed behavior, is another important negative symptom. OBJECTIVES: Effort-related choice tasks have been used as animal models of avolition. In these studies, cariprazine was assessed for its effects on effort-based choice in both rats and mice. Previous work has shown that D2 antagonists such as haloperidol and eticlopride produce a low-effort bias in rodents tested on effort-based choice tasks. RESULTS: Low doses of cariprazine produced a low-effort bias in rats tested on the fixed ratio 5/chow feeding choice task, decreasing lever pressing for high carbohydrate pellets but increasing chow intake. Cariprazine did not alter preference or intake of these foods in free-feeding tests. The effort-related effects of cariprazine were reversed by co-administration of the adenosine A2A antagonist istradefylline, and cariprazine failed to reverse the effort-related effects of the dopamine-depleting agent tetrabenazine. In mouse touchscreen choice tests, low doses of cariprazine also produced a low-effort bias, shifting behavior away from panel pressing. CONCLUSIONS: These results demonstrate that with these rodent models of avolition, cariprazine appears to act like a D2-family antagonist even at very low doses. Furthermore, the pharmacological regulation of avolition may differ from that of other negative symptoms.

Current Progress and Challenges in Large-Scale 3D Mitochondria Instance Segmentation.

In this paper, we present the results of the MitoEM challenge on mitochondria 3D instance segmentation from electron microscopy images, organized in conjunction with the IEEE-ISBI 2021 conference. Our benchmark dataset consists of two large-scale 3D volumes, one from human and one from rat cortex tissue, which are 1,986 times larger than previously used datasets. At the time of paper submission, 257 participants had registered for the challenge, 14 teams had submitted their results, and six teams participated in the challenge workshop. Here, we present eight top-performing approaches from the challenge participants, along with our own baseline strategies. Posterior to the challenge, annotation errors in the ground truth were corrected without altering the final ranking. Additionally, we present a retrospective evaluation of the scoring system which revealed that: 1) challenge metric was permissive with the false positive predictions; and 2) size-based grouping of instances did not correctly categorize mitochondria of interest. Thus, we propose a new scoring system that better reflects the correctness of the segmentation results. Although several of the top methods are compared favorably to our own baselines, substantial errors remain unsolved for mitochondria with challenging morphologies. Thus, the challenge remains open for submission and automatic evaluation, with all volumes available for download.

CEM500K, a large-scale heterogeneous unlabeled cellular electron microscopy image dataset for deep learning.

Automated segmentation of cellular electron microscopy (EM) datasets remains a challenge. Supervised deep learning (DL) methods that rely on region-of-interest (ROI) annotations yield models that fail to generalize to unrelated datasets. Newer unsupervised DL algorithms require relevant pre-training images, however, pre-training on currently available EM datasets is computationally expensive and shows little value for unseen biological contexts, as these datasets are large and homogeneous. To address this issue, we present CEM500K, a nimble 25 GB dataset of 0.5 × 106 unique 2D cellular EM images curated from nearly 600 three-dimensional (3D) and 10,000 two-dimensional (2D) images from >100 unrelated imaging projects. We show that models pre-trained on CEM500K learn features that are biologically relevant and resilient to meaningful image augmentations. Critically, we evaluate transfer learning from these pre-trained models on six publicly available and one newly derived benchmark segmentation task and report state-of-the-art results on each. We release the CEM500K dataset, pre-trained models and curation pipeline for model building and further expansion by the EM community. Data and code are available at https://www.ebi.ac.uk/pdbe/emdb/empiar/entry/10592/ and https://git.io/JLLTz.

Masking for COVID-19 Is Associated with Decreased Emergency Department Utilization for Non-COVID Viral Illnesses and Respiratory Conditions in Maryland.

BACKGROUND: Masking, which is known to decrease the transmission of respiratory viruses, was not widely practiced in the United States until the coronavirus disease 2019 (COVID-19) pandemic. This provides a natural experiment to determine whether the percentage of community masking was associated with decreases in emergency department (ED) visits due to non-COVID viral illnesses (NCVIs) and related respiratory conditions. METHODS: In this observational study of ED encounters in a 11-hospital system in Maryland during 2019-2020, year-on-year ratios for all complaints were calculated to account for "lockdowns" and the global drop in ED visits due to the pandemic. Encounters for specific complaints were identified using the International Classification of Diseases, version 10. Encounters with a positive COVID test were excluded. Linear regression was used to determine the association of publicly available masking data with ED visits for NCVI and exacerbations of asthma and chronic obstructive pulmonary disease (COPD), after adjusting for patient age, sex, and medical history. RESULTS: There were 285,967 and 252,598 ED visits across the hospital system in 2019 and 2020, respectively. There was a trend toward an association between the year-on-year ratio for all ED visits and the Maryland stay-at-home order (parameter estimate = -0.0804, P = .10). A 10% percent increase in the prevalence of community masking was associated with a 17.0%, 8.8%, and 9.4% decrease in ED visits for NCVI and exacerbations of asthma exacerbations and chronic obstructive pulmonary disease, respectively (P < .001 for all). CONCLUSIONS: Increasing the prevalence of masking is associated with a decrease in ED visits for viral illnesses and exacerbations of asthma and COPD. These findings may be valuable for future public health responses, particularly in future pandemics with respiratory transmission or in severe influenza seasons.

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist.

The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric "volume electron microscopy" or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a "Goldilocks zone" in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.

Breakthrough Therapy Designation: CDER Analysis of Requests 4 Years Into the Program.

BACKGROUND: A breakthrough therapy designation is intended to facilitate and expedite development and review of new drugs to address unmet medical need in the treatment of a serious condition. METHODS: The Center for Drug Evaluation and Research (CDER) at Food and Drug Administration (FDA) analyzed 364 breakthrough therapy designation requests received from program inception on July 9, 2012, through June 30, 2016. RESULTS: Of the 364 requests received during this time, CDER granted 133 (37%), denied 182 (50%), and the sponsor withdrew 49 (13%) before CDER made a decision. CONCLUSION: This analysis provides information on the distinguishing characteristics of the drugs seeking this designation and the decisional factors used by CDER to either grant or deny breakthrough therapy designation requests. This paper provides greater transparency into the CDER decision process, so the public can better understand how breakthrough therapy designations are determined.

Therapeutics Targeting Protein Acetylation Perturb Latency of Human Viruses.

Persistent viral infections are widespread and represent significant public health burdens. Some viruses endure in a latent state by co-opting the host epigenetic machinery to manipulate viral gene expression. Small molecules targeting epigenetic pathways are now in the clinic for certain cancers and are considered as potential treatment strategies to reverse latency in HIV-infected individuals. In this review, we discuss how drugs interfering with one epigenetic pathway, protein acetylation, perturb latency of three families of pathogenic human viruses-retroviruses, herpesviruses, and papillomaviruses.

Retrovirus Integration: Some Assembly Required?

Integration is a key feature of the retroviral life cycle. This process involves packaging of the viral genome into chromatin, which is often assumed to occur as a post-integration step. In this issue of Cell Host & Microbe, Wang and colleagues (Wang et al., 2016) show that chromatinization occurs before integration, raising new questions about the role of histones in retroviral integration and transcription.

The mTOR Complex Controls HIV Latency.

A population of CD4 T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and in vitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

Do strict formularies replicate failure for patients with schizophrenia?

OBJECTIVES: We measure the impact of Medicaid formulary restrictions (FRs) on the rate at which patients who previously failed a drug therapy for schizophrenia are returned to that therapy. STUDY DESIGN: We collect drug-level information on FRs in state Medicaid programs and examine claims of noninstitutionalized Medicaid enrollees with schizophrenia. METHODS: A difference-in-differences technique is used to compute the change in the probability of adverse outcomes before and after a state adopts an FR. This change is compared with the change in failure probabilities in states with no FRs. RESULTS: Regardless of FRs, patients tend to resume the same drug after an adverse medical event. In 2005, 69% of inpatient mental health-related admissions resulted in patients resuming the same therapy within 6 months of the event, and 63% of patients resumed the same drug after a mental health-related emergency department admission. In states where FRs limit access to all atypicals, the likelihood of a patient resuming the same atypical after having ceased treatment for at least 30 days increases by 20.1% relative to patients in states without restrictions. Additionally, patients in states that impose FRs on all atypicals are 11.6% more likely to discontinue all treatments. CONCLUSIONS: FR may increase the likelihood that patients will return to failed treatments or cease treatment altogether. Although formularies are designed to reduce drug spending, an unintended consequence may be an increase in the use of other services needed to treat patients with schizophrenia.

Alcohol Control and Foster Care.

Parental alcohol consumption is often associated with an increased likelihood of child abuse. As consumption is related to price, the purpose of this paper is to investigate the propensity for increases in the full price of alcohol to influence entry rates and the length of time spent in foster care. Using alcoholic beverage prices and a measure of availability in combination with data on foster care cases, we find that higher alcohol prices are not effective in reducing foster care entry rates; however, once in foster care, the duration of stay may be shortened by higher prices and reduced availability.

Medicaid prior authorization policies and imprisonment among patients with schizophrenia.

OBJECTIVE: To examine the impact of Medicaid prior authorization for atypical antipsychotics on the prevalence of schizophrenia among the prison population. Study DESIGN: We collected drug-level information on prior authorization restrictions from Medicaid programs in 30 states to determine which states had prior authorization requirements before 2004. We linked the regulatory data to a survey of prison inmates conducted in 2004. METHODS: We used a sample of 16,844 inmates from a nationally representative survey and analyzed the data using cross-sectional regression. To capture the impact of prior authorization, we estimated 2 models: the first included an indicator variable for states requiring prior authorization, and a second model used per capita atypical usage. RESULTS: Evidence indicated that prior authorization restrictions on atypical antipsychotics are associated with an increase in the odds of a schizophrenic resident being imprisoned in a state. State-level prior authorization requirements for atypical antipsychotics are associated with a 2.7% increase in the likelihood that an imprisoned inmate displays psychotic symptoms, and a 1.25 increase in the likelihood that an inmate was previously diagnosed with schizophrenia by a physician. Higher state-level atypical prescriptions per capita are also associated with lower likelihood of psychotic symptoms and of prior schizophrenia diagnosis among prisoners. CONCLUSIONS: Prior authorization requirements for atypical antipsychotics, which are designed to reduce healthcare costs, are associated with greater prevalence of mental illness within the criminal justice system.This association raises important questions about whether increased costs to the criminal justice system might mitigate or offset prescription drug savings created by prior authorization requirements.

Bromodomain proteins in HIV infection.

Bromodomains are conserved protein modules of ~110 amino acids that bind acetylated lysine residues in histone and non-histone proteins. Bromodomains are present in many chromatin-associated transcriptional regulators and have been linked to diverse aspects of the HIV life cycle, including transcription and integration. Here, we review the role of bromodomain-containing proteins in HIV infection. We begin with a focus on acetylated viral factors, followed by a discussion of structural and biological studies defining the involvement of bromodomain proteins in the HIV life cycle. We end with an overview of promising new studies of bromodomain inhibitory compounds for the treatment of HIV latency.

The impact of insulin type on severe hypoglycaemia events requiring inpatient and emergency department care in patients with type 2 diabetes.

AIMS: To evaluate the risk from different insulin types on severe hypoglycaemia (SHG) events requiring inpatient (IP) or emergency department (ED) care in patients with type 2 diabetes. METHODS: Type 2 diabetes patients newly started on insulin in a large commercial claims database were evaluated for SHG events. Patients were classified into an insulin group based on their most frequently used insulin type. Multivariable Cox models assessed the association between insulin type and the risk of SHG events. RESULTS: We identified 8626 patients (mean age 53.5 years; 55% female) with type 2 diabetes followed for an average of 4.0 years after insulin initiation. Of these, 161 (1.9%) had a SHG event at an average of 3.1y after insulin initiation. Patients with SHG events were slightly older (56.4 vs. 53.4 years), used a similar number of OADs (1.1 vs. 1.2) but had more co-morbidities compared with those without SHG events. In multivariate Cox models, premixed insulin (HR 2.12; p<0.01), isophane insulin (NPH) (HR 2.02; p<0.01), and rapid acting insulin (HR 2.75; p<0.01) had significantly higher risks of SHG events compared with glargine. No statistically significant difference in SHG events was seen with detemir (HR 1.20; p=0.73). CONCLUSIONS: Among patients with type 2 diabetes, the use of newer basal insulin analogues was associated with lower rates of SHG events requiring IP or ED care compared with users of other insulin formulations. Future research should examine the impact of hypoglycaemia events of different severity levels.