Single-cell RNA-sequencing of PBMCs from SAVI patients reveals disease-associated monocytes with elevated integrated stress response.

Gain-of-function mutations in stimulator of interferon gene 1 (STING1) result in STING-associated vasculopathy with onset in infancy (SAVI), a severe autoinflammatory disease. Although elevated type I interferon (IFN) production is thought to be the leading cause of the symptoms observed in patients, STING can induce a set of pathways, which have roles in the onset and severity of SAVI and remain to be elucidated. To this end, we performed a multi-omics comparative analysis of peripheral blood mononuclear cells (PBMCs) and plasma from SAVI patients and healthy controls, combined with a dataset of healthy PBMCs treated with IFN-ß. Our data reveal a subset of disease-associated monocyte, expressing elevated CCL3, CCL4, and IL-6, as well as a strong integrated stress response, which we suggest is the result of direct PERK activation by STING. Cell-to-cell communication inference indicates that these monocytes lead to T cell early activation, resulting in their senescence and apoptosis. Last, we propose a transcriptomic signature of STING activation, independent of type I IFN response.

TEAD Inhibitors Sensitize KRASG12C Inhibitors via Dual Cell Cycle Arrest in KRASG12C-Mutant NSCLC.

KRASG12C is one of the most common mutations detected in non-small cell lung cancer (NSCLC) patients, and it is a marker of poor prognosis. The first FDA-approved KRASG12C inhibitors, sotorasib and adagrasib, have been an enormous breakthrough for patients with KRASG12C mutant NSCLC; however, resistance to therapy is emerging. The transcriptional coactivators YAP1/TAZ and the family of transcription factors TEAD1-4 are the downstream effectors of the Hippo pathway and regulate essential cellular processes such as cell proliferation and cell survival. YAP1/TAZ-TEAD activity has further been implicated as a mechanism of resistance to targeted therapies. Here, we investigate the effect of combining TEAD inhibitors with KRASG12C inhibitors in KRASG12C mutant NSCLC tumor models. We show that TEAD inhibitors, while being inactive as single agents in KRASG12C-driven NSCLC cells, enhance KRASG12C inhibitor-mediated anti-tumor efficacy in vitro and in vivo. Mechanistically, the dual inhibition of KRASG12C and TEAD results in the downregulation of MYC and E2F signatures and in the alteration of the G2/M checkpoint, converging in an increase in G1 and a decrease in G2/M cell cycle phases. Our data suggest that the co-inhibition of KRASG12C and TEAD leads to a specific dual cell cycle arrest in KRASG12C NSCLC cells.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5.

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 µM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

Notch activation shifts the fate decision of senescent progenitors toward myofibrogenesis in human adipose tissue.

Senescence is a key event in the impairment of adipose tissue (AT) function with obesity and aging but the underlying molecular and cellular players remain to be fully defined, particularly with respect to the human AT progenitors. We have found distinct profiles of senescent progenitors based on AT location between stroma from visceral versus subcutaneous AT. In addition to flow cytometry, we characterized the location differences with transcriptomic and proteomic approaches, uncovering the genes and developmental pathways that are underlying replicative senescence. We identified key components to include INBHA as well as SFRP4 and GREM1, antagonists for the WNT and BMP pathways, in the senescence-associated secretory phenotype and NOTCH3 in the senescence-associated intrinsic phenotype. Notch activation in AT progenitors inhibits adipogenesis and promotes myofibrogenesis independently of TGFß. In addition, we demonstrate that NOTCH3 is enriched in the premyofibroblast progenitor subset, which preferentially accumulates in the visceral AT of patients with an early obesity trajectory. Herein, we reveal that NOTCH3 plays a role in the balance of progenitor fate determination preferring myofibrogenesis at the expense of adipogenesis. Progenitor NOTCH3 may constitute a tool to monitor replicative senescence and to limit AT dysfunction in obesity and aging.

A Versatile Class of 1,4,4-Trisubstituted Piperidines Block Coronavirus Replication In Vitro.

There is a clear need for novel antiviral concepts to control SARS-CoV-2 infection. Based on the promising anti-coronavirus activity observed for a class of 1,4,4-trisubstituted piperidines, we here conducted a detailed analysis of the structure-activity relationship of these structurally unique inhibitors. Despite the presence of five points of diversity, the synthesis of an extensive series of analogues was readily achieved by Ugi four-component reaction from commercially available reagents. After evaluating 63 analogues against human coronavirus 229E, four of the best molecules were selected and shown to have micromolar activity against SARS-CoV-2. Since the action point was situated post virus entry and lying at the stage of viral polyprotein processing and the start of RNA synthesis, enzymatic assays were performed with CoV proteins involved in these processes. While no inhibition was observed for SARS-CoV-2 nsp12-nsp7-nsp8 polymerase, nsp14 N7-methyltransferase and nsp16/nsp10 2'-O-methyltransferase, nor the nsp3 papain-like protease, the compounds clearly inhibited the nsp5 main protease (Mpro). Although the inhibitory activity was quite modest, the plausibility of binding to the catalytic site of Mpro was established by in silico studies. Therefore, the 1,4,4-trisubstituted piperidines appear to represent a novel class of non-covalent CoV Mpro inhibitors that warrants further optimization and development.

A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase.

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.

Synthesis, Structure-Activity Relationships, and Antiviral Profiling of 1-Heteroaryl-2-Alkoxyphenyl Analogs as Inhibitors of SARS-CoV-2 Replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure-activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity.

Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV.

Peptidomics of Haemonchus contortus.

The nematode Haemonchus contortus (the barber's pole worm) is an endoparasite infecting wild and domesticated ruminants worldwide. Widespread anthelmintic resistance of H. contortus requires alternative strategies to control this parasite. Neuropeptide signaling represents a promising target for anthelmintic drugs. Identification and relative quantification of nematode neuropeptides are, therefore, required for the development of such therapeutic targets. In this work, we undertook the profiling of the whole H. contortus larvae at different stages for the direct sequencing of the neuropeptides expressed at low levels in these tissues. We set out a peptide extraction protocol and a peptidomic workflow to biochemically characterize bioactive peptides from both first-stage (L1) and third-stage larvae (L3) of H. contortus. This work led to the identification and quantification at the peptidomic level of more than 180 mature neuropeptides, including amidated and nonamidated peptides, arising from 55 precursors of H. contortus. The differential peptidomic approach provided evidence that both life stages express most FMRFamide-like peptides (FLPs) and neuropeptide-like proteins (NLPs). The H. contortus peptidome resource, established in this work, could add the discovery of neuropeptide system-targeting drugs for ruminants.

Obesity of mice lacking VAP-1/SSAO by Aoc3 gene deletion is reproduced in mice expressing a mutated vascular adhesion protein-1 (VAP-1) devoid of amine oxidase activity.

The product of Aoc3 gene is known as vascular adhesion protein-1 (VAP-1), a glycoprotein contributing to leukocyte extravasation and exhibiting semicarbazide-sensitive amine oxidase activity (SSAO). Regarding the immune functions of VAP-1/SSAO, it is known that mice bearing Aoc3 gene knock-out (AOC3KO) exhibit defects in leukocyte migration similar to those of mice expressing a mutated VAP-1 lacking functional SSAO activity (knock-in, AOC3KI). However, it has not been reported whether these models differ regarding other disturbances. Thus, we further compared endocrine-metabolic phenotypes of AOC3KO and AOC3KI mice to their respective control. Special attention was paid on adiposity, glucose and lipid handling, since VAP-1/SSAO is highly expressed in adipose tissue (AT). In both mouse lines, no tissue SSAO activity was found, while Aoc3 mRNA was absent in AOC3KO only. Although food consumption was unchanged, both AOC3KO and AOC3KI mice were heavier and fatter than their respective controls. Other alterations commonly found in adipocytes from both lines were loss of benzylamine insulin-like action with unchanged insulin lipogenic responsiveness and adiponectin expression. A similar downregulation of inflammatory markers (CD45, IL6) was found in AT. Glucose handling and liver mass remained unchanged, while circulating lipid profile was distinctly altered, with increased cholesterol in AOC3KO only. These results suggest that the lack of oxidase activity found in AOC3KI is sufficient to reproduce the metabolic disturbances observed in AOC3KO mice, save those related with cholesterol transport. Modulation of SSAO activity therefore constitutes a potential target for the treatment of cardiometabolic diseases, especially obesity when complicated by low-grade inflammation.

A fluorescence-based high throughput-screening assay for the SARS-CoV RNA synthesis complex.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emergence in 2003 introduced the first serious human coronavirus pathogen to an unprepared world. To control emerging viruses, existing successful anti(retro)viral therapies can inspire antiviral strategies, as conserved viral enzymes (eg., viral proteases and RNA-dependent RNA polymerases) represent targets of choice. Since 2003, much effort has been expended in the characterization of the SARS-CoV replication/transcription machinery. Until recently, a pure and highly active preparation of SARS-CoV recombinant RNA synthesis machinery was not available, impeding target-based high throughput screening of drug candidates against this viral family. The current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic revealed a new pathogen whose RNA synthesis machinery is highly (>96 % aa identity) homologous to SARS-CoV. This phylogenetic relatedness highlights the potential use of conserved replication enzymes to discover inhibitors against this significant pathogen, which in turn, contributes to scientific preparedness against emerging viruses. Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. The screening of a small (1520 compounds) chemical library of FDA-approved drugs demonstrates the robustness of our assay and will allow to speed-up drug discovery against the SARS-CoV-2.

High-Throughput Cellular RNA Sequencing (HiCAR-Seq): Cost-Effective, High-Throughput 3' mRNA-Seq Method Enabling Individual Sample Quality Control.

High-throughput screening is one of the pillars of drug development. Unbiased transcriptome profiling is now widely used for a deeper understanding of a drug's mechanisms of action, off target effects, and cytotoxicity. Although currently available high-throughput RNA-Seq (HT RNA-Seq) methods such as PLATE-Seq, DRUG-Seq, and BRB-Seq serve these purposes, the inherent nature of these methods does not allow sample-wise sequencing library quality control. Here, we describe an HTR method called High-throughput CellulAr RNA Sequencing (HiCAR-Seq). HiCAR-Seq was optimized to work directly on cultured cells (as little as 1,000 cells) or 10 ng of total RNA. HiCAR-Seq involves reverse transcription from cultured cells or total RNA using oligo-dT primers followed by the PCR amplification of full-length cDNAs using sample-specific barcode primers in individual plate wells. Amplification of cDNA from every sample can be verified using Bioanalyzer. This step not only reveals cDNA amplification but also provides greater precision for pooling equal concentrations of cDNA from different samples. A single pooled cDNA library is made suitable for sequencing on Illumina sequencers using a tagmentation kit. Because HiCAR-Seq targets a small region at the 3' of the mRNAs, as little as 3 to 4 million reads/sample are enough to infer changes in gene expression in human or mouse cells. We believe that HiCAR-Seq represents a robust and competitive addition to the existing set of transcriptome-based high-throughput screening methods. © 2020 Wiley Periodicals LLC. Basic Protocol 1: cDNA synthesis and barcoding/enrichment PCR Basic Protocol 2: Nextera tagmentation/amplification, quantification, and sequencing.

Drugs against SARS-CoV-2: What do we know about their mode of action?

The health emergency caused by the recent Covid-19 pandemic highlights the need to identify effective treatments against the virus causing this disease (SARS-CoV-2). The first clinical trials have been testing repurposed drugs that show promising anti-SARS-CoV-2 effects in cultured cells. Although more than 2400 clinical trials are already under way, the actual number of tested compounds is still limited to approximately 20, alone or in combination. In addition, knowledge on their mode of action (MoA) is currently insufficient. Their first results reveal some inconsistencies and contradictory results and suggest that cohort size and quality of the control arm are two key issues for obtaining rigorous and conclusive results. Moreover, the observed discrepancies might also result from differences in the clinical inclusion criteria, including the possibility of early treatment that may be essential for therapy efficacy in patients with Covid-19. Importantly, efforts should also be made to test new compounds with a documented MoA against SARS-CoV-2 in clinical trials. Successful treatment will probably be based on multitherapies with antiviral compounds that target different steps of the virus life cycle. Moreover, a multidisciplinary approach that combines artificial intelligence, compound docking, and robust in vitro and in vivo assays will accelerate the development of new antiviral molecules. Finally, large retrospective studies on hospitalized patients are needed to evaluate the different treatments with robust statistical tools and to identify the best treatment for each Covid-19 stage. This review describes different candidate antiviral strategies for Covid-19, by focusing on their mechanism of action.

Carneic Acids from an Endophytic Phomopsis sp. as Dengue Virus Polymerase Inhibitors.

Thirteen carneic acids were isolated from the fungal endophyte Phomopsis sp. SNB-LAP1-7-32. Their structures were identified by mass spectrometry and extensive one- and two-dimensional NMR spectroscopy and through comparison with data reported in the literature. Compounds 1-13 were investigated for their antipolymerase activities against DENV polymerase and Zika NS5. Five of them exhibited significant inhibition of dengue polymerase with IC50 values in the 10 to 20 µM range without cytotoxicity. None inhibited Zika virus NS5 protein.

ABHD11, a new diacylglycerol lipase involved in weight gain regulation.

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.

Remdesivir and SARS-CoV-2: Structural requirements at both nsp12 RdRp and nsp14 Exonuclease active-sites.

The rapid global emergence of SARS-CoV-2 has been the cause of significant health concern, highlighting the immediate need for antivirals. Viral RNA-dependent RNA polymerases (RdRp) play essential roles in viral RNA synthesis, and thus remains the target of choice for the prophylactic or curative treatment of several viral diseases, due to high sequence and structural conservation. To date, the most promising broad-spectrum class of viral RdRp inhibitors are nucleoside analogues (NAs), with over 25 approved for the treatment of several medically important viral diseases. However, Coronaviruses stand out as a particularly challenging case for NA drug design due to the presence of an exonuclease (ExoN) domain capable of excising incorporated NAs and thus providing resistance to many of these available antivirals. Here we use the available structures of the SARS-CoV RdRp and ExoN proteins, as well as Lassa virus N exonuclease to derive models of catalytically competent SARS-CoV-2 enzymes. We then map a promising NA candidate, GS-441524 (the active metabolite of Remdesivir) to the nucleoside active site of both proteins, identifying the residues important for nucleotide recognition, discrimination, and excision. Interestingly, GS-441524 addresses both enzyme active sites in a manner consistent with significant incorporation, delayed chain termination, and altered excision due to the ribose 1'-CN group, which may account for the increased antiviral effect compared to other available analogues. Additionally, we propose structural and function implications of two previously identified RdRp resistance mutations in relation to resistance against Remdesivir. This study highlights the importance of considering the balance between incorporation and excision properties of NAs between the RdRp and ExoN.

Incretin combination therapy for the treatment of non-alcoholic steatohepatitis.

AIMS: To test specific mono-agonists to the glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic peptide receptor (GIPR), individually and in combination, in a mouse model of diet-induced non-alcoholic steatohepatitis (NASH) and fibrosis in order to decipher the contribution of their activities and potential additive effects to improving systemic and hepatic metabolism. MATERIALS AND METHODS: We induced NASH by pre-feeding C57BL/6J mice a diet rich in fat, fructose and cholesterol for 36 weeks. This was followed by 8 weeks of treatment with the receptor-specific agonists 1-GCG (20 µg/kg twice daily), 2-GLP1 (3 µg/kg twice daily) or 3-GIP (30 µg/kg twice daily), or the dual (1 + 2) or triple (1 + 2 + 3) combinations thereof. A dual GLP-1R/GCGR agonistic peptide, 4-dual-GLP1/GCGR (30 µg/kg twice daily), and liraglutide (100 µg/kg twice daily) were included as references. RESULTS: Whereas low-dose 1-GCG or 3-GIP alone did not influence body weight, liver lipids and histology, their combination with 2-GLP1 provided additional weight loss, reduction in liver triglycerides and improvement in histological disease activity score. Notably, 4-dual-GLP-1R/GCGR and the triple combination of selective mono-agonists led to a significantly stronger reduction in the histological non-alcoholic fatty liver disease activity score compared to high-dose liraglutide, at the same extent of body weight loss. CONCLUSIONS: GCGR and GIPR agonism provide additional, body weight-independent improvements on top of GLP-1R agonism in a murine model of manifest NASH with fibrosis.

A 3D Human Liver Model of Nonalcoholic Steatohepatitis.

Background and Aims: To better understand nonalcoholic steatohepatitis (NASH) disease progression and to evaluate drug targets and compound activity, we undertook the development of an in vitro 3D model to mimic liver architecture and the NASH environment. Methods: We have developed an in vitro preclinical 3D NASH model by coculturing primary human hepatocytes, human stellate cells, liver endothelial cells and Kupffer cells embedded in a hydrogel of rat collagen on a 96-well plate. A NASH-like environment was induced by addition of medium containing free fatty acids and tumor necrosis factor-α. This model was then characterized by biochemical, imaging and transcriptomics analyses. Results: We succeeded in defining suitable culture conditions to maintain the 3D coculture for up to 10 days in vitro, with the lowest level of steatosis and reproducible low level of inflammation and fibrosis. NASH disease was induced with a custom medium mimicking NASH features. The cell model exhibited the key NASH disease phenotypes of hepatocyte injury, steatosis, inflammation, and fibrosis. Hepatocyte injury was highlighted by a decrease of CYP3A4 expression and activity, without loss of viability up to day 10. Moreover, the model was able to stimulate a stable inflammatory and early fibrotic environment, with expression and secretion of several cytokines. A global gene expression analysis confirmed the NASH induction. Conclusions: This is a new in vitro model of NASH disease consisting of four human primary cell-types that exhibits most features of the disease. The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets.

RNA Fragmentation and Sequencing (RF-Seq): Cost-Effective, Time-Efficient, and High-Throughput 3' mRNA Sequencing Library Construction in a Single Tube.

Over the past decade, transcriptomic studies using next-generation sequencing (NGS)-based RNA sequencing (RNA-Seq) have greatly contributed to characterizing biochemical and physiological changes in cells and tissues across organisms and experimental conditions. Critical steps in RNA-Seq include the preparation of the sequencing library from extracted RNA. Currently, a large panoply of RNA-Seq kits are commercially available. In these kits, conversion of RNA into a sequencing library involves multiple steps, which are labor-intensive, and cost per sample for library preparation may limit routine use of RNA-Seq. Here we describe a simple method for RNA-Seq library construction, referred to as RNA Fragmentation and Sequencing (RF-Seq). RF-Seq requires as little as 10 ng of total RNA and facilitates the sequencing of the 3' end of mRNAs. RF-Seq involves the fragmentation of total RNA followed by reverse transcription in presence of the oligo(dT) primer/template switch oligonucleotide and a sample barcoding/enrichment within a single PCR tube/well. The sample barcoding/enrichment step provides more flexibility for individual sample handling. The use of just twenty orthogonal Illumina TruSeq HT barcoding primers facilitates the preparation of 96 uniquely labeled RF-Seq libraries in a single 96-well PCR plate. Twelve RF-Seq libraries can be prepared within 4 hr, with an approximate cost of $10/sample. We provide an example of using RF-Seq to measure gene expression upon activation of an innate immune pathway using STING activator in human blood cells, highlighting the potential usefulness of the proposed method in routine transcriptomic applications such as high-throughput drug screening and/or preclinical toxicity assays. © 2019 by John Wiley & Sons, Inc. Basic Protocol: RNA fragmentation and sequencing (RF-Seq): Cost-effective, time-efficient, and high-throughput 3' mRNA sequencing library construction in a single tube.

A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology.

Here, we have used patient-derived induced pluripotent stem cell (iPSC) and gene-editing technology to study the cardiac-related molecular and functional consequences of mutations in GLA causing the lysosomal storage disorder Fabry disease (FD), for which heart dysfunction is a major cause of mortality. Our in vitro model recapitulated clinical data with FD cardiomyocytes accumulating GL-3 and displaying an increased excitability, with altered electrophysiology and calcium handling. Quantitative proteomics enabled the identification of >5,500 proteins in the cardiomyocyte proteome and secretome, and revealed accumulation of the lysosomal protein LIMP-2 and secretion of cathepsin F and HSPA2/HSP70-2 in FD. Genetic correction reversed these changes. Overexpression of LIMP-2 directly induced the secretion of cathepsin F and HSPA2/HSP70-2, implying causative relationship, and led to massive vacuole accumulation. In summary, our study has revealed potential new cardiac biomarkers for FD, and provides valuable mechanistic insight into the earliest pathological events in FD cardiomyocytes.