The Silent Threat: Bartonella quintana Endocarditis Unveiling Heart Failure and Severe Pulmonary Hypertension.

BACKGROUND Bartonella quintana is a slow-growing gram-negative bacterium that can cause severe culture-negative endocarditis. In many cases, its insidious onset can be difficult to diagnose given the variable symptoms in the early phases of the disease. This delay in detection and thus treatment can cause advanced consequences of the disease, including heart failure and severe pulmonary hypertension. CASE REPORT A 51-year-old man presented to the Emergency Department with signs and symptoms indicating an acute stroke. Further investigation showed that the source was cardioembolic, and despite negative blood cultures, endocarditis was suspected due to echocardiogram findings. Bartonella endocarditis was diagnosed based on serology results. Further testing indicated severe pulmonary hypertension, a sequelae of chronic heart failure in the setting of endocarditis. This caused a significant delay in valvular repair surgery. This case illustrates the progression from acute to chronic infection, the sequelae of this disease process, and the considerations involved in management. CONCLUSIONS Bartonella is an under-appreciated cause of endocarditis and can evolve into chronic disease with clinical consequences requiring nuanced management. We described a case of chronic culture-negative endocarditis that presented with acute embolic stroke and the sequelae of severe multi-valvular disease in a patient with recent incarceration and unstable housing. This case provides clinicians with valuable insight into the recognition of Bartonella endocarditis, the variable clinical presentations of this pathology, the nuanced and multifactorial approaches to medical management, and the indications for surgery.

Rapid Multiplexed Flow Cytometric Validation of CRISPRi sgRNAs in Tissue Culture.

Genome-wide CRISPR-based screening is a powerful tool in forward genetics, enabling biologic discovery by linking a desired phenotype to a specific genetic perturbation. However, hits from a genome-wide screen require individual validation to reproduce and accurately quantify their effects outside of a pooled experiment. Here, we describe a step-by-step protocol to rapidly assess the effects of individual sgRNAs from CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems. All steps, including cloning, lentivirus generation, cell transduction, and phenotypic readout, can be performed entirely in 96-well plates. The system is highly flexible in both cell type and selection system, requiring only that the phenotype(s) of interest be read out via flow cytometry. We expect that this protocol will provide researchers with a rapid way to sift through potential screening hits, and prioritize them for deeper analysis in more complex in vitro or even in vivo systems. Graphical abstract.

Heparin Does Not Regulate Circulating Human PCSK9 (Proprotein Convertase Subtilisin-Kexin Type 9) in a General Population-Brief Report.

BACKGROUND: PCSK9 (proprotein convertase subtilisin-kexin type 9) chaperones the hepatic LDLR (low-density lipoprotein receptor) for lysosomal degradation, elevating serum LDL (low-density lipoprotein) cholesterol and promoting atherosclerotic heart disease. Though the major effect on the hepatic LDLR comes from secreted PCSK9, the details of PCSK9 reuptake into the hepatocyte remain unclear. In both tissue culture and animal models, HSPGs (heparan sulfate proteoglycans) on hepatocytes act as co-receptors to promote PCSK9 reuptake. We hypothesized that if this PCSK9:HSPG interaction is important in humans, disrupting it with unfractionated heparin (UFH) would acutely displace PCSK9 from the liver and increase plasma PCSK9. METHODS: We obtained remnant plasma samples from 160 subjects undergoing cardiac catheterization before and after administration of intravenous UFH. PCSK9 levels were determined using a commercial enzyme-linked immunosorbent assay. RESULTS: Median plasma PCSK9 was 113 ng/mL prior to UFH and 119 ng/mL afterward. This difference was not significant (P=0.83 [95% CI, -6.23 to 6.31 ng/mL]). Equivalence testing provided 95% confidence that UFH would not raise plasma PCSK9 by > 4.7%. Among all subgroups, only subjects with the lowest baseline PCSK9 concentrations exhibited a response to UFH (8.8% increase, adj. P=0.044). A modest correlation was observed between baseline plasma PCSK9 and the change in plasma PCSK9 due to UFH (RS=-0.3634; P<0.0001). CONCLUSIONS: Administration of UFH does not result in a clinically meaningful effect on circulating PCSK9 among an unselected population of humans. The results cast doubt on the clinical utility of disrupting the PCSK9:HSPG interaction as a general therapeutic strategy for PCSK9 inhibition. However, the observations suggest that in selected populations, disrupting the PCSK9:HSPG interaction could still affect PCSK9 reuptake and offer a therapeutic benefit.

Cold shock domain-containing protein E1 is a posttranscriptional regulator of the LDL receptor.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.

Deep Learning Algorithm for Automated Cardiac Murmur Detection via a Digital Stethoscope Platform.

Background Clinicians vary markedly in their ability to detect murmurs during cardiac auscultation and identify the underlying pathological features. Deep learning approaches have shown promise in medicine by transforming collected data into clinically significant information. The objective of this research is to assess the performance of a deep learning algorithm to detect murmurs and clinically significant valvular heart disease using recordings from a commercial digital stethoscope platform. Methods and Results Using >34 hours of previously acquired and annotated heart sound recordings, we trained a deep neural network to detect murmurs. To test the algorithm, we enrolled 962 patients in a clinical study and collected recordings at the 4 primary auscultation locations. Ground truth was established using patient echocardiograms and annotations by 3 expert cardiologists. Algorithm performance for detecting murmurs has sensitivity and specificity of 76.3% and 91.4%, respectively. By omitting softer murmurs, those with grade 1 intensity, sensitivity increased to 90.0%. Application of the algorithm at the appropriate anatomic auscultation location detected moderate-to-severe or greater aortic stenosis, with sensitivity of 93.2% and specificity of 86.0%, and moderate-to-severe or greater mitral regurgitation, with sensitivity of 66.2% and specificity of 94.6%. Conclusions The deep learning algorithm's ability to detect murmurs and clinically significant aortic stenosis and mitral regurgitation is comparable to expert cardiologists based on the annotated subset of our database. The findings suggest that such algorithms would have utility as front-line clinical support tools to aid clinicians in screening for cardiac murmurs caused by valvular heart disease. Registration URL: https://clinicaltrials.gov; Unique Identifier: NCT03458806.

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption 1,2 . There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

Cell-associated heparin-like molecules modulate the ability of LDL to regulate PCSK9 uptake.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets the LDL receptor (LDLR) for degradation, increasing plasma LDL and, consequently, cardiovascular risk. Uptake of secreted PCSK9 is required for its effect on the LDLR, and LDL itself inhibits this uptake, though how it does so remains unclear. In this study, we investigated the relationship between LDL, the PCSK9:LDLR interaction, and PCSK9 uptake. We show that LDL inhibits binding of PCSK9 to the LDLR in vitro more impressively than it inhibits PCSK9 uptake in cells. Furthermore, cell-surface heparin-like molecules (HLMs) can partly explain this difference, consistent with heparan sulfate proteoglycans (HSPGs) acting as coreceptors for PCSK9. We also show that HLMs can interact with either PCSK9 or LDL to modulate the inhibitory activity of LDL on PCSK9 uptake, with such inhibition rescued by competition with the entire PCSK9 prodomain, but not its truncated variants. Additionally, we show that the gain-of-function PCSK9 variant, S127R, located in the prodomain near the HSPG binding site, exhibits increased affinity for HLMs, potentially explaining its phenotype. Overall, our findings suggest a model where LDL acts as a negative regulator of PCSK9 function by decreasing its uptake via direct interactions with either the LDLR or HLMs.

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

Stepwise processing analyses of the single-turnover PCSK9 protease reveal its substrate sequence specificity and link clinical genotype to lipid phenotype.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) down-regulates the low-density lipoprotein (LDL) receptor, elevating LDL cholesterol and accelerating atherosclerotic heart disease, making it a promising cardiovascular drug target. To achieve its maximal effect on the LDL receptor, PCSK9 requires autoproteolysis. After cleavage, PCSK9 retains its prodomain in the active site as a self-inhibitor. Unlike other proprotein convertases, however, this retention is permanent, inhibiting any further protease activity for the remainder of its life cycle. Such inhibition has proven a major challenge toward a complete biochemical characterization of PCSK9's proteolytic function, which could inform therapeutic approaches against its hypercholesterolemic effects. To address this challenge, we employed a cell-based, high-throughput method using a luciferase readout to evaluate the single-turnover PCSK9 proteolytic event. We combined this method with saturation mutagenesis libraries to interrogate the sequence specificities of PCSK9 cleavage and proteolysis-independent secretion. Our results highlight several key differences in sequence identity between these two steps, complement known structural data, and suggest that PCSK9 self-proteolysis is the rate-limiting step of secretion. Additionally, we found that for missense SNPs within PCSK9, alterations in both proteolysis and secretion are common. Last, we show that some SNPs allosterically modulate PCSK9's substrate sequence specificity. Our findings indicate that PCSK9 proteolysis acts as a commonly perturbed but critical switch in controlling lipid homeostasis and provide a new hope for the development of small-molecule PCSK9 inhibitors.

The proprotein convertase subtilisin/kexin type 9 (PCSK9) active site and cleavage sequence differentially regulate protein secretion from proteolysis.

Biologic-based strategies to inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9) show promise as anti-hypercholesterolemic and, therefore, anti-atherosclerotic therapies. Despite substantial effort, no small molecule strategy to inhibit PCSK9 has demonstrated feasibility. In this study we interrogated the chemistry of the PCSK9 active site and its adjacent residues to identify a foothold with which to drug the PCSK9 processing pathway and ultimately disrupt the interaction with the LDL receptor. Here, we develop a system in which we amplify the readout of PCSK9 proteolysis with a highly specific substrate in cells, showing that the PCSK9 catalytic domain is capable of proteolysis in trans. We use this system to show that the substrate specificity for PCSK9 proteolysis is distinct from the specificity for PCSK9 secretion, demonstrating that PCSK9 processing occurs in two separate sequential steps: that of proteolysis followed by secretion. We show that specific residues in the protease recognition sequence can differentially modulate the effects on proteolysis and secretion. Additionally, we demonstrate that the clinically described, dominant negative Q152H mutation restricts proteolysis and secretion independently. Our results suggest that the PCSK9 active site and its adjacent residues serve as an allosteric modulator of protein secretion independent of its role in proteolysis, revealing a new strategy for intracellular PCSK9 inhibition.

Vesicular stomatitis viruses resistant to the methylase inhibitor sinefungin upregulate RNA synthesis and reveal mutations that affect mRNA cap methylation.

Sinefungin (SIN), a natural S-adenosyl-L-methionine analog produced by Streptomyces griseolus, is a potent inhibitor of methyltransferases. We evaluated the effect of SIN on replication of vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses. The 241-kDa large polymerase (L) protein of VSV methylates viral mRNA cap structures at the guanine-N-7 (G-N-7) and ribose-2'-O (2'-O) positions. By performing transcription reactions in vitro, we show that both methylations are inhibited by SIN and that methylation was more sensitive at the G-N-7 than at 2'-O position. We further show that SIN inhibited growth of VSV in cell culture, reducing viral yield by 50-fold and diminishing plaque size. We isolated eight mutants that were resistant to SIN as judged by their growth characteristics. The SIN-resistant (SINR) viruses contained mutations in the L gene, the promoter for L gene expression provided by the conserved sequence elements of the G-L gene junction and the M gene. Five mutations resulted in amino acid substitutions to conserved regions II/III and VI of the L protein. For each mutant, we examined viral gene expression in cells and cap methylation in vitro. SINR mutants upregulated RNA synthesis in the presence of SIN, which may be responsible for their resistance. We also found that some SINR viruses with L gene mutations were defective in cap methylation in vitro, yet their methylases were less sensitive to SIN inhibition than those of the wild-type parent. These studies show that the VSV methylases are inhibited by SIN, and they define new regions of L protein that affect cap methylation. These studies also provide experimental evidence that inhibition of cap methylases is a potential strategy for development of antiviral therapeutics against nonsegmented negative-strand RNA viruses.