Search

Abstract A balanced activity of cGMP signaling contributes to the maintenance of cardiovascular homeostasis. Vascular smooth muscle cells (VSMCs) can generate cGMP via three ligand-activated guanylyl cyclases, the NO-sensitive guanylyl cyclase, the atrial natriuretic peptide (ANP)-activated GC-A, and the C-type natriuretic peptide (CNP)-stimulated GC-B. Here, we study natriuretic peptide signaling in murine VSMCs and atherosclerotic lesions. Correlative profiling of pathway activity and VSMC phenotype at the single-cell level shows that phenotypic modulation of contractile VSMCs to chondrocyte-like plaque cells during atherogenesis is associated with a switch from ANP/GC‑A to CNP/GC‑B signaling. Silencing of the CNP/GC-B axis in VSMCs results in an increase of chondrocyte-like plaque cells. These findings indicate that the CNP/GC-B/cGMP pathway is a marker and atheroprotective regulator of modulated VSMCs, limiting their transition to chondrocyte-like cells. Overall, this study highlights the plasticity of cGMP signaling in VSMCs and suggests analogies between CNP-dependent remodeling of bone and blood vessels.

Abstract BackgroundThe COVID-19 pandemic revealed major gaps in public health agencies’ (PHAs’) data and reporting infrastructure, which limited the ability of public health officials to conduct disease surveillance, particularly among racial or ethnic minorities disproportionally affected by the pandemic. Leveraging existing health information exchange organizations (HIOs) is one possible mechanism to close these technical gaps, as HIOs facilitate health information sharing across organizational boundaries. ObjectiveThe aim of the study is to survey all HIOs that are currently operational in the United States to assess HIO connectivity with PHAs and HIOs’ capabilities to support public health data exchange. MethodsDrawing on multiple sources, we identified all potential local, regional, and state HIOs that were operational in the United States as of March 1, 2022. We defined operational as HIOs that facilitated exchange between at least 2 independent entities. We fielded a survey among our census list of 135 HIOs in January-July 2023. The survey confirmed HIO status as well as captured organizational demographics and current and potential support for PHAs. We report descriptive statistics on HIO demographics and connectivity with PHAs. We also include results on services and data available to support PHAs, funding sources to support public health reporting, and barriers to public health reporting. Of the 135 potential HIOs that received the survey, 90 met our definition of an HIO, and 77 completed the survey, yielding an 86% response rate. ResultsWe found that 66 (86%) of HIOs in 45 states were electronically connected to at least 1 PHA, yielding 187 HIO-PHA connections across all HIOs. Among HIOs connected to PHAs, the most common type of public health reporting supported by HIOs was immunization registry (n=39, 64%), electronic laboratory result (n=37, 63%), and syndromic surveillance (n=34, 61%). In total, 58% (n=38) of HIOs connected to PHAs provided data to address COVID-19 information gaps, and an additional 30% (n=20) could do so. The most common types of data provided to PHAs were hospitalization information (n=54, 93%), other demographic data (n=53, 91%), health information (eg, chronic health conditions; n=51, 88%), and hospital laboratory results (n=51, 88%). A total of 64% (n=42) of HIOs provided at least 1 type of data analytic service to PHAs to support COVID-19 pandemic response efforts. Top HIO reported barriers to support PHA activities included limited PHA funding (n=21, 32%) and PHAs’ competing priorities (n=15, 23%). ConclusionsOur results show that many HIOs are already connected to PHAs and that they are assuming an emerging role to facilitate public health reporting. HIOs are well-positioned to provide value-added support for public health data exchange and address PHAs’ information gaps, as ongoing federal efforts to modernize public health data infrastructure and interoperability continue.

While thousands of health systems have begun to implement the Age-Friendly Health System’s 4Ms Framework to improve care for older patients, an important phase of work is achieving consistent adherence to 4Ms care processes. Identifying mechanisms that may lead to higher versus lower adherence serves to guide efforts to achieve consistent, equitable adherence. Drawing from prior literature, we identified three mechanisms that may influence 4Ms adherence. We then conducted a 3-year retrospective, observational study of inpatient encounters (n = 28 833) at UCSF Health System with patients aged 65+. We used least squares regression models to assess associations between hospital encounter-level measures of 4Ms adherence and proxy measures of patient and encounter characteristics for each hypothesized mechanism along with control variables. Encounter-level adherence to the 4Ms was 65.5% (SD = 14.3%). We found support for all three mechanisms. Negative implicit biases were associated with lower adherence for patients who were obese [0.79 percentage points (PP) lower; P

A group of intrinsically disordered proteins (IDPs) are subject to 20S proteasomal degradation in a ubiquitin-independent manner. Recently, we have reported that many IDPs/IDRs are targeted to the 20S proteasome via interaction with the C-terminus of the PSMA3 subunit, termed the PSMA3 Trapper. In this study, we investigated the biological significance of the IDP–Trapper interaction using the IDP p21. Using a split luciferase reporter assay and conducting detailed p21 mutagenesis, we first identified the p21 RRLIF box, localized at the C-terminus, as mediating the Trapper interaction in cells. To demonstrate the role of this box in p21 degradation, we edited the genome of HEK293 and HeLa cell lines using a CRISPR strategy. We found that the p21 half-life increased in cells with either a deleted or mutated p21 RRLIF box. The edited cell lines displayed an aberrant cell cycle pattern under normal conditions and in response to DNA damage. Remarkably, these cells highly expressed senescence hallmark genes in response to DNA damage, highlighting that the increased p21 half-life, not its actual level, regulates senescence. Our findings suggest that the p21 RRLIF box, which mediates interactions with the PSMA3 Trapper, acts as a ubiquitin-independent degron. This degron is positioned adjacent to the previously identified ubiquitin-dependent degron, forming a dual degron module that functionally regulates p21 degradation and its physiological outcomes.

Preparing patients for posthospital care may improve readmission risk. Alternative payment models (APMs) incent hospitals to reduce readmissions by tying payment to outcomes. The impact of APMs on preparation for discharge is not well understood. We assessed whether patient-reported preparation for posthospital care was associated with reduced readmissions, and whether APM participation was associated with improved preparation for posthospital care. We used mixed-effects regression on retrospective (2013–2017) observational data for 2685 U.S. hospitals. We measured patient-reported preparation for posthospital care using the 3-Item Care Transition Measure and readmission using 30-day all-cause risk-adjusted readmissions from Hospital Compare. Participation in accountable care organizations (ACOs), Medical Homes, and Medicare's Bundled Payments for Care Improvement program was obtained from Medicare, the American Hospital Association's Annual Survey, and Leavitt Partner's ACO database. We found that APMs are not associated with improved preparation for posthospital care, even though it was associated with reduced readmissions (Marginal Effect: −0.012 percentage points). This may be because hospitals are not investing in patient engagement. This study has limited insight into causality and reduced generalizability among smaller, rural, and non-teaching hospitals.

Abstract Background Proteasome inhibitors are in use in treating certain types of cancers. These drugs inhibit the catalytic activity of the 20S proteasome, shared by all the different proteasome complexes. Inhibitors of the 26S‐associated deubiquitinating activity explicitly inhibit the 26S proteasomal degradation of ubiquitinylated substrates. We have previously reported an alternative strategy that is based on reducing the 26S/20S ratio by depleting PSMD1, 6, and 11, the subunits of the 19S proteasome regulatory complex. Given the addiction of the many cancer types to a high 26S/20S ratio, the depletion strategy is highly effective in killing many aggressive cancer cell lines but not mouse and human immortalized and normal cells. Methods We used two aggressive cell lines, MDA‐MB‐231, a triple‐negative breast tumor cell line, and OVCAR8, a high‐grade ovary adenocarcinoma. Cell culture, mouse MDA‐MB‐231, OVCAR8 xenografts, and patient‐derived ovarian cancer xenograft (PDX) models were transduced with lentivectors expressing PSMD1 shRNA. Tumor size was measured to follow treatment efficacy. Results Using different experimental strategies of expressing shRNA, we found that PSMD1 depletion, either by expressing PSMD1 shRNA in an inducible manner or in a constitutive manner, robustly inhibited MDA‐MB‐231, and OVCAR8 xenograft tumor growth. Furthermore, the PSMD1 depletion strategy compromised the growth of the PDX of primary ovarian cancer. Conclusion Our results suggest that reducing the 26S/20S ratio might be a valuable strategy for treating drug‐resistant aggressive types of cancers.

Abstract The rapid adoption of electronic health records (EHRs) has created extensive repositories of digitized data that can be used to inform improvements in care delivery, processes, and patient outcomes. While the clinical data captured in EHRs are widely used for such efforts, EHRs also capture audit log data that reflect how users interact with the EHR to deliver care. Automatically collected audit log data provide a unique opportunity for new insights into EHR user behavior and decision‐making processes. Here, we provide an overview of audit log data and examples that could be used to improve oncology care and outcomes in four domains: diagnostic reasoning and consumption, care team collaboration and communication, patient outcomes and experience, and provider burnout/fatigue. This data source could identify gaps in performance and care, physician uptake of EHR features that enhance decision‐making, and integration of data trends for oncology. Ensuring researchers and oncologists are familiar with the data's potential and developing the data engineering capacity to utilize this rich data source, will expand the breadth of research to improve cancer care.

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. More than 274 million individuals have suffered from COVID-19 and over five million people have died from this disease so far. Therefore, there is an urgent need for therapeutic drugs. Repurposing FDA approved drugs should be favored since evaluation of safety and efficacy of de-novo drug design are both costly and time consuming. We report that imatinib, an Abl tyrosine kinase inhibitor, robustly decreases SARS-CoV-2 infection and uncover a mechanism of action. We show that imatinib inhibits the infection of SARS-CoV-2 and its surrogate lentivector pseudotype. In latter, imatinib inhibited both routes of viral entry, endocytosis and membrane-fusion. We utilized a system to quantify in real-time cell–cell membrane fusion mediated by the SARS-CoV-2 surface protein, Spike, and its receptor, hACE2, to demonstrate that imatinib inhibits this process in an Abl1 and Abl2 independent manner. Furthermore, cellular thermal shift assay revealed a direct imatinib-Spike interaction that affects Spike susceptibility to trypsin digest. Collectively, our data suggest that imatinib inhibits Spike mediated viral entry by an off-target mechanism. These findings mark imatinib as a promising therapeutic drug in inhibiting the early steps of SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Via the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus transduction, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2’s cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.

Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multi-subunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generates the 26S proteasome, which is the major proteasomal complex in cells. Native gel protocols are used to investigate the 26S/20S ratio. However, a simple method for detecting these proteasome complexes in cells is missing. To this end, using CRISPR technology, we YFP-tagged the endogenous PSMB6 (β1) gene, a 20S CP subunit, and co-tagged endogenous PSMD6 (Rpn7), a 19S RP subunit, with the mScarlet fluorescent protein. We observed the colocalization of the YFP and mScarlet fluorescent proteins in the cells, with higher nuclear accumulation. Nuclear proteasomal granules are formed under osmotic stress, and all were positive for YFP and mScarlet. Previously, we have reported that PSMD1 knockdown, one of the 19 RP subunits, gives rise to a high level of “free” 20S CPs. Intriguingly, under this condition, the 20S-YFP remained nuclear, whereas the PSMD6-mScarlet was mostly in cytoplasm, demonstrating the distinct subcellular distribution of uncapped 20S CPs. Lately, we have shown that the PSMA3 (α7) C-terminus, a 20S CP subunit, binds multiple intrinsically disordered proteins (IDPs). Remarkably, the truncation of the PSMA3 C-terminus is phenotypically reminiscent of PSMD1 knockdown. These data suggest that the PSMA3 C-terminal region is critical for 26S proteasome integrity.

The COVID-19 pandemic resulted from the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since its first appearance in 2019, new SARS-CoV-2 variants of concern (VOCs) have emerged frequently, changing the infection’s dynamic. SARS-CoV-2 infects cells via two distinct entry routes; receptor-mediated endocytosis or membrane fusion, depending on the absence or presence of transmembrane serine protease 2 (TMPRSS2), respectively. In laboratory conditions, the Omicron SARS-CoV-2 strain inefficiently infects cells predominantly via endocytosis and is phenotypically characterized by decreased syncytia formation compared to the earlier Delta variant. Thus, it is important to characterize Omicron’s unique mutations and their phenotypic manifestations. Here, by utilizing SARS-CoV-2 pseudovirions, we report that the specific Omicron Spike F375 residue decreases infectivity, and its conversion to the Delta S375 sequence significantly increases Omicron infectivity. Further, we identified that residue Y655 decreases Omicron’s TMPRSS2 dependency and entry via membrane fusion. The Y655H, K764N, K856N and K969N Omicron revertant mutations, bearing the Delta variant sequence, increased the cytopathic effect of cell–cell fusion, suggesting these Omicron-specific residues reduced the severity of SARS-CoV-2. This study of the correlation of the mutational profile with the phenotypic outcome should sensitize our alertness towards emerging VOCs.

The degradation of intrinsically disordered proteins (IDPs) by a non-26S proteasome process does not require proteasomal targeting by polyubiquitin. However, whether and how IDPs are recognized by the non-26S proteasome, including the 20S complex, remains unknown. Analyses of protein interactome datasets revealed that the 20S proteasome subunit, PSMA3, preferentially interacts with many IDPs. In vivo and cell-free experiments revealed that the C-terminus of PSMA3, a 69-amino-acids-long fragment, is an IDP trapper. A recombinant trapper is sufficient to interact with many IDPs, and blocks IDP degradation in vitro by the 20S proteasome, possibly by competing with the native trapper. In addition, over a third of the PSMA3 trapper-binding proteins have previously been identified as 20S proteasome substrates and, based on published datasets, many of the trapper-binding proteins are associated with the intracellular proteasomes. The PSMA3-trapped IDPs that are proteasome substrates have the unique features previously recognized as characteristic 20S proteasome substrates in vitro. We propose a model whereby the PSMA3 C-terminal region traps a subset of IDPs to facilitate their proteasomal degradation.

BackgroundThere is widespread agreement on the promise of patient-facing digital health tools to transform health care. Yet, few tools are in widespread use or have documented clinical effectiveness. ObjectiveThe aim of this study was to gain insight into the gap between the potential of patient-facing digital health tools and real-world uptake. MethodsWe interviewed and surveyed experts (in total, n=24) across key digital health stakeholder groups—venture capitalists, digital health companies, payers, and health care system providers or leaders—guided by the Consolidated Framework for Implementation Research. ResultsOur findings revealed that external policy, regulatory demands, internal organizational workflow, and integration needs often take priority over patient needs and patient preferences for digital health tools, which lowers patient acceptance rates. We discovered alignment, across all 4 stakeholder groups, in the desire to engage both patients and frontline health care providers in broader dissemination and evaluation of digital health tools. However, major areas of misalignment between stakeholder groups have stymied the progress of digital health tool uptake—venture capitalists and companies focused on external policy and regulatory demands, while payers and providers focused on internal organizational workflow and integration needs. ConclusionsMisalignment of the priorities of digital health companies and their funders with those of providers and payers requires direct attention to improve uptake of patient-facing digital health tools and platforms.

Summary: The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD+-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD+ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD+, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD+ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

The hepatitis B virus (HBV) is one of the smallest but most highly infectious human pathogens. With a DNA genome of only 3.2 kb and only four genes, HBV successfully completes its life cycle by using intricate processes to hijack the host machinery. HBV infects non-dividing liver cells in which dNTPs are limited. As a DNA virus, HBV requires dNTPs for its replication. HBV induces the ATR-mediated cellular DNA damage response pathway to overcome this constraint. This pathway upregulates R2 (RRM2) expression in generating an active RNR holoenzyme catalyzing de novo dNTP synthesis. Previously we reported that ERE, a small RNA fragment within the HBx ORF, is sufficient to induce R2 upregulation. Interestingly, there is high sequence similarity between ERE and a region within the R2 5′UTR that we named R2-box. Here, we established a mutant cell line in the R2-box region of the R2 gene using CRISPR-Cas9 technology to investigate the R2 regulation by ERE. This cell line expresses a much lower R2 level than the parental cell line. Interestingly, the HBV infection and life cycle were severely impaired. These cells became permissive to HBV infection upon ectopically R2 expression. These results validate the requirement of the R2 gene expression for HBV replication. Remarkably, the R2-box mutated cells became ERE refractory, suggesting that the homology region between ERE and R2 gene is critical for ERE-mediated R2 upregulation. Thus, along with the induction of the ATR pathway of the DNA damage response, ERE might also directly target the R2 gene via the R2-box.

OBJECTIVES/GOALS: The objective of this study is to examine the associated changes in the EHR use patterns after the widespread implementation of telehealth in the ambulatory care setting after the COVID pandemic. METHODS/STUDY POPULATION: The study sample will be all attending ambulatory care physicians at UCSF Health. Signal measures captured by Epic Systems are markers of EHR use pattern that characterize EHR use at the individual provider level in terms of time spent performing certain activities, time spent at particular times of the day, and the number of EHR tools being used. We will use the Single Interrupted Time Series framework to analyze the changes in Signal measures that occur after the widespread implementation of telehealth with pre-telehealth time period defined as Jan 2018 – Feb 2020 and post-telehealth time period defined as March 2020 – present. RESULTS/ANTICIPATED RESULTS: The outcomes of this study will reveal how the increased use of telehealth following the COVID pandemic has changed the way providers utilize various functions within EHR (e.g. time in EHR at particular time of day, documentation, medication and non-medication orders, chart review, etc). These results can, in turn, inform us potential impacts of increased telehealth use on physician burnout given that a number of markers of EHR use pattern (i.e. Signal measures) in previous studies have been associated with burnout. In addition, a stratified version of Interrupted Time Series by specialty and clinical work volume may inform us how different subgroups of providers exhibit varying EHR use patterns in response to the increased use of telehealth. DISCUSSION/SIGNIFICANCE: The use of telehealth will likely remain a strong presence in health care delivery in the post-COVID era. This study can serve as a baseline study on the influence of telehealth on EHR use. Future studies may focus on potential targeted interventions to best support the usage of telehealth.

Vascular smooth muscle cells (VSMCs) can switch from their contractile state to a synthetic phenotype resulting in high migratory and proliferative capacity and driving atherosclerotic lesion formation. The cysteine-rich LIM-only protein 4 (CRP4) reportedly modulates VSM-like transcriptional signatures, which are perturbed in VSMCs undergoing phenotypic switching. Thus, we hypothesized that CRP4 contributes to adverse VSMC behaviours and thereby to atherogenesis in vivo. The atherogenic properties of CRP4 were investigated in plaque-prone apolipoprotein E (ApoE) and CRP4 double-knockout (dKO) as well as ApoE-deficient CRP4 wildtype mice. dKO mice exhibited lower plaque numbers and lesion areas as well as a reduced content of α-smooth muscle actin positive cells in the lesion area, while lesion-associated cell proliferation was elevated in vessels lacking CRP4. Reduced plaque volumes in dKO correlated with significantly less intra-plaque oxidized low-density lipoprotein (oxLDL), presumably due to upregulation of the antioxidant factor peroxiredoxin-4 (PRDX4). This study identifies CRP4 as a novel pro-atherogenic factor that facilitates plaque oxLDL deposition and identifies the invasion of atherosclerotic lesions by VSMCs as important determinants of plaque vulnerability. Thus, targeting of VSMC CRP4 should be considered in plaque-stabilizing pharmacological strategies.

Abstract Background Health Information Organizations (HIOs) are third party organizations that facilitate electronic health information exchange (HIE) between providers in a geographic area. Despite benefits from HIE, HIOs have struggled to form and subsequently gain broad provider participation. We sought to assess whether market-level hospital and EHR vendor dynamics are associated with presence and level of hospital participation in HIOs. Methods 2014 data on 4523 hospitals and their EHR vendors were aggregated to the market level. We used multivariate OLS regression to analyze the relationship between hospital and vendor dynamics and (1) probability of HIO presence and (2) percent of hospitals participating in an HIO. Results 298 of 469 markets (64%) had HIO presence, and in those markets, 47% of hospitals participated in an HIO on average. In multivariate analysis, four characteristics were associated with HIO presence. Markets with more hospitals, markets with more EHR vendors, and markets with an EHR vendor-led HIE approach were more likely to have an HIO. Compared to markets with low hospital competition, markets with high hospital competition had a 25 percentage point lower probability of HIO presence. Two characteristics were associated with level of hospital HIO participation. Markets with more hospitals as well as markets with high vendor competition (compared to low competition) had lower participation. Conclusion Both hospital and EHR vendor dynamics are associated with whether a market has an HIO as well as the level of hospital participation in HIOs.

DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells.

The 26S proteasome is the endpoint of the ubiquitin- and ATP-dependent degradation pathway. Over the years, ATP was regarded as completely essential for 26S proteasome function due to its role in ubiquitin-signaling, substrate unfolding and ensuring its structural integrity. We have previously reported that physiological concentrations of NADH are efficient in replacing ATP to maintain the integrity of an enzymatically functional 26S PC. However, the substrate specificity of the NADH-stabilized 26S proteasome complex (26S PC) was never assessed. Here, we show that the binding of NADH to the 26S PC inhibits the ATP-dependent and ubiquitin-independent degradation of the structured ODC enzyme. Moreover, the NADH-stabilized 26S PC is efficient in degrading intrinsically disordered protein (IDP) substrates that might not require ATP-dependent unfolding, such as p27, Tau, c-Fos and more. In some cases, NADH-26S proteasomes were more efficient in processing IDPs than the ATP-26S PC. These results indicate that in vitro, physiological concentrations of NADH can alter the processivity of ATP-dependent 26S PC substrates such as ODC and, more importantly, the NADH-stabilized 26S PCs promote the efficient degradation of many IDPs. Thus, ATP-independent, NADH-dependent 26S proteasome activity exemplifies a new principle of how mitochondria might directly regulate 26S proteasome substrate specificity.

Federal investment spurred health information exchange organization (HIO) development and maturation to provide third-party approaches to electronic health information exchange across disparate electronic health record (EHR) systems. By creating opportunities for data aggregation across multiple medical institutions, HIOs also spur research. Using data from a 2015 national web-based survey of HIOs (N = 64), we identified HIOs supporting or not supporting research, and compared characteristics of the 2 groups. We found that 15 (23%) of the 64 HIOs reported supporting research, 30 (47%) reported planning to support research, and 19 (30%) did not support research. Research-supporting HIOs were more likely than nonresearch supporting HIOs to offer advanced functionality, such as allowing users to query and retrieve data from multiple sources. Our study offers encouraging preliminary evidence that HIOs are supporting research, which could offer a solution to current challenges in creating comprehensive longitudinal clinical data sources for research.

CRISPR/Cas9 is a powerful tool for genome editing in cells and organisms. Nevertheless, introducing directed templated changes by homology-directed repair (HDR) requires the cellular DNA repair machinery, such as the MRN complex (Mre11/Rad50/Nbs1). To improve the process, we tailored chimeric constructs of Cas9, in which SpCas9 was fused at its N- or C-terminus to a 126aa intrinsically disordered domain from HSV-1 alkaline nuclease (UL12) that recruits the MRN complex. The chimeric Cas9 constructs were two times more efficient in homology-directed editing of endogenous loci in tissue culture cells. This effect was dependent upon the MRN-recruiting activity of the domain and required lower amounts of the chimeric Cas9 in comparison with unmodified Cas9. The new constructs improved the yield of edited cells when making endogenous point mutations or inserting small tags encoded by oligonucleotide donor DNA (ssODN), and also with larger insertions encoded by plasmid DNA donor templates. Improved editing was achieved with both transfected plasmid-encoded Cas9 constructs as well as recombinant Cas9 protein transfected as ribonucleoprotein complexes. Our strategy was highly efficient in restoring a genetic defect in a cell line, exemplifying the possible implementation of our strategy in gene therapy. These constructs provide a simple approach to improve directed editing.

BACKGROUND: Disembodiment is a core feature of depersonalization disorder (DPD). Given the narratives of DPD patients about their disembodiment and emotional numbing and neurobiological findings of an inhibition of insular activity, DPD may be considered as a mental disorder with specific impairments of interoceptive awareness and body perception. METHODS: We investigated cardioceptive accuracy (CA) of DPD patients (n=24) as compared to healthy controls (n=26) with two different heartbeat detection tasks ("Schandry heartbeat counting task" and "Whitehead heartbeat discrimination task"). Self-rated clearness of body perception was measured by questionnaire. RESULTS: Contrary to our hypothesis, DPD patients performed similarly to healthy controls on the two different heartbeat detection tasks, and they had equal scores regarding their self-rated clearness of body perception. There was no correlation of the severity of "anomalous body experiences" and depersonalization with measures of interoceptive accuracy. Only among healthy controls CA in the Schandry task was positively correlated with self-rated clearness of body perception. Depersonalization was unrelated to severity of depression or anxiety, while depression and anxiety were highly correlated. Anxiety and depression did not modify the associations of depersonalization with interoceptive accuracy. CONCLUSIONS: Our main findings highlight a striking discrepancy of normal interoception with overwhelming experiences of disembodiment in DPD. This may reflect difficulties of DPD patients to integrate their visceral and bodily perceptions into a sense of their selves. This problem may be considered an important target for psychotherapeutic treatment approaches.

BACKGROUND: Patients with depersonalization disorder (DPD) typically complain about emotional detachment. Previous studies found reduced autonomic responsiveness to emotional stimuli for DPD patients as compared to patients with anxiety disorders. We aimed to investigate autonomic responsiveness to emotional auditory stimuli of DPD patients as compared to patient controls. Furthermore, we examined the modulatory effect of mindful breathing on these responses as well as on depersonalization intensity. METHODS: 22 DPD patients and 15 patient controls balanced for severity of depression and anxiety, age, sex and education, were compared regarding 1) electrodermal and heart rate data during a resting period, and 2) autonomic responses and cognitive appraisal of standardized acoustic affective stimuli in two conditions (normal listening and mindful breathing). RESULTS: DPD patients rated the emotional sounds as significantly more neutral as compared to patient controls and standardized norm ratings. At the same time, however, they responded more strongly to acoustic emotional stimuli and their electrodermal response pattern was more modulated by valence and arousal as compared to patient controls. Mindful breathing reduced severity of depersonalization in DPD patients and increased the arousal modulation of electrodermal responses in the whole sample. Finally, DPD patients showed an increased electrodermal lability in the rest period as compared to patient controls. CONCLUSIONS: These findings demonstrated that the cognitive evaluation of emotional sounds in DPD patients is disconnected from their autonomic responses to those emotional stimuli. The increased electrodermal lability in DPD may reflect increased introversion and cognitive control of emotional impulses. The findings have important psychotherapeutic implications.

BACKGROUND: The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically. METHODOLOGY/PRINCIPAL FINDINGS: We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization. CONCLUSIONS: Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.