Discovery of RXFP2 genetic association in resistant hypertensive men and RXFP2 antagonists for the treatment of resistant hypertension.

Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women. This study shows that adrenal gland gene expression of RXFP2 is increased in men with hypertension and the RXFP2 natural ligand, INSL3, increases adrenal steroidogenesis and corticosteroid secretion in human adrenal cells. To address the hypothesis that RXFP2 activation is an important mechanism in rHTN, we discovered and characterized small molecule and monoclonal antibody (mAb) blockers of RXFP2. The novel chemical entities and mAbs show potent, selective inhibition of RXFP2 and reduce aldosterone and cortisol synthesis and release. The RXFP2 mAbs have suitable rat pharmacokinetic profiles to evaluate the role of RXFP2 in the development and maintenance of rHTN. Overall, we identified RXFP2 activity as a potential new mechanism in rHTN and discovered RXFP2 antagonists for the future interrogation of RXFP2 in cardiovascular and renal diseases.

Cost-effective DNA methylation profiling by FML-seq.

Current methods for profiling DNA methylation require costly reagents, sequencing, and labor time. We introduce fragmentation at methylated loci and sequencing (FML-seq), a sequencing library protocol that greatly reduces all these costs. Relative to other techniques tested on the same human cell lines, FML-seq produces similar measurements of absolute and differential cytosine methylation at a fraction of the price. FML-seq enables inexpensive, high-throughput experimental designs for large-scale epigenetics research projects.

Cost-effective DNA methylation profiling by FML-seq.

Current methods for profiling DNA methylation require costly reagents, sequencing, or labor time. We introduce FML-seq, a sequencing library protocol that greatly reduces all these costs. Relative to other techniques tested on the same human cell lines, FML-seq produces similar measurements of absolute and differential cytosine methylation at a fraction of the price. FML-seq enables inexpensive, high-throughput experimental designs for large-scale epigenetics research projects.

The microdissected gene expression landscape of nasopharyngeal cancer reveals vulnerabilities in FGF and noncanonical NF-κB signaling.

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)-positive epithelial malignancy with an extensive inflammatory infiltrate. Traditional RNA-sequencing techniques uncovered only microenvironment signatures, while the gene expression of the tumor epithelial compartment has remained a mystery. Here, we use Smart-3SEQ to prepare transcriptome-wide gene expression profiles from microdissected NPC tumors, dysplasia, and normal controls. We describe changes in biological pathways across the normal to tumor spectrum and show that fibroblast growth factor (FGF) ligands are overexpressed in NPC tumors, while negative regulators of FGF signaling, including SPRY1, SPRY2, and LGALS3, are down-regulated early in carcinogenesis. Within the NF-κB signaling pathway, the critical noncanonical transcription factors, RELB and NFKB2, are enriched in the majority of NPC tumors. We confirm the responsiveness of EBV-positive NPC cell lines to targeted inhibition of these pathways, reflecting the heterogeneity in NPC patient tumors. Our data comprehensively describe the gene expression landscape of NPC and unravel the mysteries of receptor tyrosine kinase and NF-κB pathways in NPC.

Transcriptome and genome evolution during HER2-amplified breast neoplasia.

BACKGROUND: The acquisition of oncogenic drivers is a critical feature of cancer progression. For some carcinomas, it is clear that certain genetic drivers occur early in neoplasia and others late. Why these drivers are selected and how these changes alter the neoplasia's fitness is less understood. METHODS: Here we use spatially oriented genomic approaches to identify transcriptomic and genetic changes at the single-duct level within precursor neoplasia associated with invasive breast cancer. We study HER2 amplification in ductal carcinoma in situ (DCIS) as an event that can be both quantified and spatially located via fluorescence in situ hybridization (FISH) and immunohistochemistry on fixed paraffin-embedded tissue. RESULTS: By combining the HER2-FISH with the laser capture microdissection (LCM) Smart-3SEQ method, we found that HER2 amplification in DCIS alters the transcriptomic profiles and increases diversity of copy number variations (CNVs). Particularly, interferon signaling pathway is activated by HER2 amplification in DCIS, which may provide a prolonged interferon signaling activation in HER2-positive breast cancer. Multiple subclones of HER2-amplified DCIS with distinct CNV profiles are observed, suggesting that multiple events occurred for the acquisition of HER2 amplification. Notably, DCIS acquires key transcriptomic changes and CNV events prior to HER2 amplification, suggesting that pre-amplified DCIS may create a cellular state primed to gain HER2 amplification for growth advantage. CONCLUSION: By using genomic methods that are spatially oriented, this study identifies several features that appear to generate insights into neoplastic progression in precancer lesions at a single-duct level.

The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin.

Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.

Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ.

RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.

Genomic analysis of benign prostatic hyperplasia implicates cellular re-landscaping in disease pathogenesis.

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.

Rapid Isolation and Concentration of Pathogenic Fungi Using Inertial Focusing on a Chip-Based Platform.

Systemic Candida infections remain a leading cause of nosocomial infections in the United States and worldwide. Many challenges remain in achieving rapid, direct diagnosis of fungal bloodstream infections due to limitations of conventional diagnostic methods that continue to demonstrate poor sensitivity, prolonged culture times that lead to delayed treatment, and detection variability between tests that compromises result reproducibility. Despite advancements in technology, mortality, and cost of care presented by blood stream infection with Candida spp. (candidemia) continues to rise and there is an urgent need for the development of novel methods to accurately detect Candida species present within the blood. This is especially true when patients are infected with drug resistant strains of Candida where accurate and immediate therapeutic treatment is of the importance. This study presents a method of separating fungal cells from lysed blood using inertial forces applied through microfluidics in order to abbreviate the time required to achieve a diagnosis by mitigating the need to grow blood cultures. We found that C. albicans can segregate into a focused stream distinct from white blood cells isolated within the Inertial Fungal Focuser (IFF) after red blood cell lysis. As a result of the focusing process, the collected cells are also concentrated 2.86 times. The same IFF device is applicable to non-albicans species: Candida parapsilosis, Candida glabrata, and Candida tropicalis, providing both isolation from lysed blood and a reduction in solution volume. Thus, the devised platform provides a means to isolate medically significant fungal cells from blood and concentrate the cells for further interrogation.

Discovery of a crystalline sulforaphane analog with good solid-state stability and engagement of the Nrf2 pathway in vitro and in vivo.

The antioxidant natural product sulforaphane (SFN) is an oil with poor aqueous and thermal stability. Recent work with SFN has sought to optimize methods of formulation for oral and topical administration. Herein we report the design of new analogs of SFN with the goal of improving stability and drug-like properties. Lead compounds were selected based on potency in a cellular screen and physicochemical properties. Among these, 12 had good aqueous solubility, permeability and long-term solid-state stability at 23 °C. Compound 12 also displayed comparable or better efficacy in cellular assays relative to SFN and had in vivo activity in a mouse cigarette smoke challenge model of acute oxidative stress.

Monolithic Chip for High-throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells.

Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm-50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15-20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.

Characterization of the Potent, Selective Nrf2 Activator, 3-(Pyridin-3-Ylsulfonyl)-5-(Trifluoromethyl)-2H-Chromen-2-One, in Cellular and In Vivo Models of Pulmonary Oxidative Stress.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2H-chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant (tert-butyl hydroperoxide)-induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)-induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke-exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke-induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1ß-induced nuclear factor-κB translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models.

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.

Hyperpolarized gas diffusion MRI of biphasic lung inflation in short- and long-term emphysema models.

During lung inflation, airspace dimensions are affected nonlinearly by both alveolar expansion and recruitment, potentially confounding the identification of emphysematous lung by hyperpolarized helium-3 diffusion magnetic resonance imaging (HP MRI). This study aimed to characterize lung inflation over a broad range of inflation volume and pressure values in two different models of emphysema, as well as in normal lungs. Elastase-treated rats (n = 7) and healthy controls (n = 7) were imaged with HP MRI. Gradual inflation was achieved by incremental changes to both inflation volume and airway pressure. The apparent diffusion coefficient (ADC) was measured at each level of inflation and fitted to the corresponding airway pressures as the second-order response equation, with minimizing residue (χ2 < 0.001). A biphasic ADC response was detected, with an initial ADC increase followed by a decrease at airway pressures >18 cmH2O. Discrimination between treated and control rats was optimal when airway pressure was intermediate (between 10 and 11 cmH2O). Similar findings were confirmed in mice following long-term exposure to cigarette smoke, where optimal discrimination between treated and healthy mice occurred at a similar airway pressure as in the rats. We subsequently explored the evolution of ADC measured at the intermediate inflation level in mice after prolonged smoke exposure and found a significant increase (P < 0.01) in ADC over time. Our results demonstrate that measuring ADC at intermediate inflation enhances the distinction between healthy and diseased lungs, thereby establishing a model that may improve the diagnostic accuracy of future HP gas diffusion studies.

Effects of airborne toxicants on pulmonary function and mitochondrial DNA damage in rodent lungs.

Inhalation of airborne toxicants such as cigarette smoke and ozone is a shared health risk among the world's populations. The use of toxic herbicides like paraquat (PQ) is restricted by many countries, yet in the developing world PQ has demonstrable ill effects. The present study examined changes in pulmonary function, mitochondrial DNA (mtDNA) integrity and markers of DNA repair induced by acute or repeated exposure of PQ to rats. Similar to cigarette smoke and ozone, PQ promotes oxidative stress, and the impact of PQ on mtDNA was compared with that obtained with these agents. Tracheal instillation (i.t.) of PQ (0.01-0.075 mg/kg) dose dependently increased Penh (dyspnoea) by 48 h while body weight and temperature declined. Lung wet weight and the wet/dry weight ratio rose; for the latter, by as much as 52%. At low doses (0.02 and 0.03 mg/kg), PQ increased Penh by about 7.5-fold at 72 h. It quickly waned to near baseline levels. The lung wet/dry weight ratio remained elevated 7 days after administration coincident with marked inflammatory cell infiltrate. Repeated administration of PQ (1 per week for 8 weeks) resulted in a similar rise in Penh on the first instillation, but the magnitude of this response was markedly attenuated upon subsequent exposures. Pulmonary [lactate] and catalase activity, [8-oxodG] and histone fragmentation (cell death) were significantly increased. Repeated PQ instillation downregulated the expression of the mitochondrial-encoded genes, mtATP8, mtNd2 and mtcyB and nuclear ones for the DNA glycosylases, Ogg1, Neil1, Neil2 and Neil3. Ogg1 protein content decreased after acute and repeated PQ administration. mtDNA damage or changes in mtDNA copy number were evident in lungs of PQ-, cigarette smoke- and ozone-exposed animals. Taken together, these data indicate that loss of pulmonary function and inflammation are coupled to the loss of mtDNA integrity and DNA repair capability following exposure to airborne toxicants.

Widespread Shortening of 3' Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection.

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3' UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3' UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3' UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.

Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery.

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch-NRF2 interaction. X-ray crystallographic screening identified three distinct "hot-spots" for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and in vivo models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1-NRF2 interaction.

HEB associates with PRC2 and SMAD2/3 to regulate developmental fates.

In embryonic stem cells, extracellular signals are required to derepress developmental promoters to drive lineage specification, but the proteins involved in connecting extrinsic cues to relaxation of chromatin remain unknown. We demonstrate that the helix-loop-helix (HLH) protein, HEB, directly associates with the Polycomb repressive complex 2 (PRC2) at a subset of developmental promoters, including at genes involved in mesoderm and endoderm specification and at the Hox and Fox gene families. While we show that depletion of HEB does not affect mouse ESCs, it does cause premature differentiation after exposure to Activin. Further, we find that HEB deposition at developmental promoters is dependent upon PRC2 and independent of Nodal, whereas HEB association with SMAD2/3 elements is dependent of Nodal, but independent of PRC2. We suggest that HEB is a fundamental link between Nodal signalling, the derepression of a specific class of poised promoters during differentiation, and lineage specification in mouse ESCs.

Collapsed state of polyglutamic acid results in amyloid spherulite formation.

Self-assembly of proteins and peptides into amyloid fibrils involves multiple distinct intermediates and late-stage fibrillar polymorphs. Understanding the conditions and mechanisms that promote the formation of one type of intermediate and polymorph over the other represents a fundamental challenge. Answers to this question are also of immediate biomedical relevance since different amyloid aggregate species have been shown to have distinct pathogenic potencies. One amyloid polymorph that has received comparatively little attention are amyloid spherulites. Here we report that self-assembly of the intrinsically disordered polymer poly(L-glutamic) acid (PLE) can generate amyloid spherulites. We characterize spherulite growth kinetics, as well as the morphological, optical and tinctorial features of this amyloid polymorph previously unreported for PLE. We find that PLE spherulites share both tinctorial and structural characteristics with their amyloid fibril counterparts. Differences in PLE's molecular weight, polydispersity or chemistry could not explain the selective propensity toward either fibril or spherulite formation. Instead, we provide evidence that PLE polymers can exist in either a collapsed globule or an extended random coil conformation. The collapsed globule consistently produces spherulites while the extended coil assembles into disordered fibril bundles. This results suggests that these 2 PLE conformers directly affect the morphology of the resulting macroscopic amyloid assembly.