Survey of extracellular communication of systemic and organ-specific inflammatory responses through cell free messenger RNA profiling in mice.

BACKGROUND: Inflammatory and immune responses are essential and dynamic biological processes that protect the body against acute and chronic adverse stimuli. While conventional protein markers have been used to evaluate systemic inflammatory response, the immunological response to stimulation is complex and involves modulation of a large set of genes and interacting signalling pathways of innate and adaptive immune systems. There is a need for a non-invasive tool that can comprehensively evaluate and monitor molecular dysregulations associated with inflammatory and immune responses in circulation and in inaccessible solid organs. METHODS: Here we utilized cell-free messenger RNA (cf-mRNA) RNA-Seq whole transcriptome profiling and computational biology to temporally assess lipopolysaccharide (LPS) induced and JAK inhibitor modulated inflammatory and immune responses in mouse plasma samples. FINDINGS: Cf-mRNA profiling displayed a pattern of systemic immune responses elicited by LPS and dysregulation of associated pathways. Moreover, attenuation of several inflammatory pathways, including STAT and interferon pathways, were observed following the treatment of JAK inhibitor. We further identified the dysregulation of liver-specific transcripts in cf-mRNA which reflected changes in the gene-expression pattern in this generally inaccessible biological compartment. INTERPRETATION: Using a preclinical mouse model, we demonstrated the potential of plasma cf-mRNA profiling for systemic and organ-specific characterization of drug-induced molecular alterations that are associated with inflammatory and immune responses. FUNDING: Molecular Stethoscope.

Noninvasive stratification of nonalcoholic fatty liver disease by whole transcriptome cell-free mRNA characterization.

Hepatic fibrosis stage is the most important determinant of outcomes in patients with nonalcoholic fatty liver disease (NAFLD). There is an urgent need for noninvasive tests that can accurately stage fibrosis and determine efficacy of interventions. Here, we describe a novel cell-free (cf)-mRNA sequencing approach that can accurately and reproducibly profile low levels of circulating mRNAs and evaluate the feasibility of developing a cf-mRNA-based NAFLD fibrosis classifier. Using separate discovery and validation cohorts with biopsy-confirmed NAFLD (n = 176 and 59, respectively) and healthy subjects (n = 23), we performed serum cf-mRNA RNA-Seq profiling. Differential expression analysis identified 2,498 dysregulated genes between patients with NAFLD and healthy subjects and 134 fibrosis-associated genes in patients with NAFLD. Comparison between cf-mRNA and liver tissue transcripts revealed significant overlap of fibrosis-associated genes and pathways indicating that the circulating cf-mRNA transcriptome reflects molecular changes in the livers of patients with NAFLD. In particular, metabolic and immune pathways reflective of known underlying steatosis and inflammation were highly dysregulated in the cf-mRNA profile of patients with advanced fibrosis. Finally, we used an elastic net ordinal logistic model to develop a classifier that predicts clinically significant fibrosis (F2-F4). In an independent cohort, the cf-mRNA classifier was able to identify 50% of patients with at least 90% probability of clinically significant fibrosis. We demonstrate a novel and robust cf-mRNA-based RNA-Seq platform for noninvasive identification of diverse hepatic molecular disruptions and for fibrosis staging with promising potential for clinical trials and clinical practice.NEW & NOTEWORTHY This work is the first study, to our knowledge, to utilize circulating cell-free mRNA sequencing to develop an NAFLD diagnostic classifier.

Author Correction: PGBD5 promotes site-specific oncogenic mutations in human tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Non-invasive characterization of human bone marrow stimulation and reconstitution by cell-free messenger RNA sequencing.

Circulating cell-free mRNA (cf-mRNA) holds great promise as a non-invasive diagnostic biomarker. However, cf-mRNA composition and its potential clinical applications remain largely unexplored. Here we show, using Next Generation Sequencing-based profiling, that cf-mRNA is enriched in transcripts derived from the bone marrow compared to circulating cells. Further, longitudinal studies involving bone marrow ablation followed by hematopoietic stem cell transplantation in multiple myeloma and acute myeloid leukemia patients indicate that cf-mRNA levels reflect the transcriptional activity of bone marrow-resident hematopoietic lineages during bone marrow reconstitution. Mechanistically, stimulation of specific bone marrow cell populations in vivo using growth factor pharmacotherapy show that cf-mRNA reflects dynamic functional changes over time associated with cellular activity. Our results shed light on the biology of the circulating transcriptome and highlight the potential utility of cf-mRNA to non-invasively monitor bone marrow involved pathologies.

PGBD5 promotes site-specific oncogenic mutations in human tumors.

Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

Local sequence assembly reveals a high-resolution profile of somatic structural variations in 97 cancer genomes.

Genomic structural variations (SVs) are pervasive in many types of cancers. Characterizing their underlying mechanisms and potential molecular consequences is crucial for understanding the basic biology of tumorigenesis. Here, we engineered a local assembly-based algorithm (laSV) that detects SVs with high accuracy from paired-end high-throughput genomic sequencing data and pinpoints their breakpoints at single base-pair resolution. By applying laSV to 97 tumor-normal paired genomic sequencing datasets across six cancer types produced by The Cancer Genome Atlas Research Network, we discovered that non-allelic homologous recombination is the primary mechanism for generating somatic SVs in acute myeloid leukemia. This finding contrasts with results for the other five types of solid tumors, in which non-homologous end joining and microhomology end joining are the predominant mechanisms. We also found that the genes recursively mutated by single nucleotide alterations differed from the genes recursively mutated by SVs, suggesting that these two types of genetic alterations play different roles during cancer progression. We further characterized how the gene structures of the oncogene JAK1 and the tumor suppressors KDM6A and RB1 are affected by somatic SVs and discussed the potential functional implications of intergenic SVs.

The antidepressant citalopram inhibits delayed rectifier outward K⁺ current in mouse cortical neurons.

Citalopram, a selective serotonin (5-HT) reuptake inhibitor (SSRI) as well as an antidepressant, is thought to exert its effects by increasing synaptic 5-HT levels. However, few studies have addressed the possibility that citalopram has other molecular mechanisms of action. We examined the effects of citalopram on delayed rectifier outward K(+) current (I(K) ) in mouse cortical neurons. Extracellular citalopram reversibly inhibited I(K) in a dose-dependent manner and significantly shifted both steady-state activation and inactivation curves toward hyperpolarization. Neither 5-HT itself nor antagonists of 5-HT and dopamine receptors could abolish citalopram-induced inhibition of I(K) . In addition, intracellular application of GTPγ-S similarly failed to prevent the inhibition of I(K) by citalopram. When applied intracellularly, citalopram had no effect on I(K) and did not influence the reduction of I(K) induced by extracellular citalopram. The effect of citalopram was use dependent, but not frequency dependent, and it did not require channel opening. Electrophysiological recordings in acute cortical slice showed that citalopram significantly reduced the action potential (AP) firing frequency of cortical neurons and increased action potential duration (APD). The selective Kv2.1 subunit blocker Jingzhaotoxin-III (JZTX-III) did not abolish citalopram-induced I(K) inhibition. Transfection of HEK293 cells with Kv2.1 or Kv2.2 constructs indicated that citalopram mainly inhibited Kv2.2 current. We suggest that citalopram-induced inhibition of I(K) in mouse cortical neurons is independent of G-protein-coupled receptors and might exert its antidepressant effects by enhancing presynaptic efficiency. Our results may help to explain some of the unknown therapeutic effects of citalopram.

Brain natriuretic peptide modulates the delayed rectifier outward K(+) current and promotes the proliferation of mouse Schwann cells.

Brain natriuretic peptide (BNP) may act as a neuromodulator via its associated receptors (natriuretic peptide receptors, NPRs) in the central nervous system (CNS), but few studies have reported its activity in the peripheral nervous system (PNS). In this study, we observed that BNP increased the tetraethylammonium chloride (TEA)-sensitive delayed rectifier outward potassium current (I(K)) in mouse Schwann cells (SCs) using whole-cell recording techniques. At concentrations of 1-100 nM, BNP reversibly activated I(K) in a dose-dependent manner, with modulating its steady-state activation and inactivation properties. The effect of BNP on I(K) was abolished by preincubation with the specific antagonist of NPR-A, and could not be mimicked by application of NPR-C agonist. These results were supported by immunocytochemical findings indicating that NPR-A was expressed in SCs. The application of 8-Br-guanosine 3',5'-monophosphate (8-Br-cGMP) mimicked the effect of BNP on I(K), but BNP was unable to further increase I(K) after the application of cyclic guanosine monophosphate (cGMP). Genistein blocked I(K) and also completely eliminated the effects of BNP and cGMP on I(K). The selective K(V)2.1 subunit blocker, Jingzhaotoxin-III (JZTX-III), reduced I(K) amplitude by 30%, but did not abolish the increase effect of BNP on I(K) amplitude. In addition, BNP significantly stimulated SCs proliferation and this effect could be partly inhibited by TEA. Together these results suggest that BNP modulated I(K) probably via cGMP- and tyrosine kinase-dependent pathways by activation of NPR-A. This effect of BNP on I(K) in SCs might partly explain its effect on cell proliferation.