Mitochondrial sequencing identifies long noncoding RNA features that promote binding to PNPase.

Extranuclear localization of long noncoding RNAs (lncRNAs) is poorly understood. Based on machine learning evaluations, we propose a lncRNA-mitochondrial interaction pathway where polynucleotide phosphorylase (PNPase), through domains that provide specificity for primary sequence and secondary structure, binds nuclear-encoded lncRNAs to facilitate mitochondrial import. Using FVB/NJ mouse and human cardiac tissues, RNA from isolated subcellular compartments (cytoplasmic and mitochondrial) and cross-linked immunoprecipitate (CLIP) with PNPase within the mitochondrion were sequenced on the Illumina HiSeq and MiSeq, respectively. lncRNA sequence and structure were evaluated through supervised [classification and regression trees (CART) and support vector machines (SVM)] machine learning algorithms. In HL-1 cells, quantitative PCR of PNPase CLIP knockout mutants (KH and S1) was performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. One hundred twelve (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most abundant. Most noncoding RNAs binding PNPase were lncRNAs, including Malat1. lncRNA fragments bound to PNPase compared against randomly generated sequences of similar length showed stratification with SVM and CART algorithms. The lncRNAs bound to PNPase were used to create a criterion for binding, with experimental validation revealing increased binding affinity of RNA designed to bind PNPase compared to control RNA. The binding of lncRNAs to PNPase was decreased through the knockout of RNA binding domains KH and S1. In conclusion, sequence and secondary structural features identified by machine learning enhance the likelihood of nuclear-encoded lncRNAs binding to PNPase and undergoing import into the mitochondrion.NEW & NOTEWORTHY Long noncoding RNAs (lncRNAs) are relatively novel RNAs with increasingly prominent roles in regulating genetic expression, mainly in the nucleus but more recently in regions such as the mitochondrion. This study explores how lncRNAs interact with polynucleotide phosphorylase (PNPase), a protein that regulates RNA import into the mitochondrion. Machine learning identified several RNA structural features that improved lncRNA binding to PNPase, which may be useful in targeting RNA therapeutics to the mitochondrion.

Comparative analysis of lung and blood transcriptomes in mice exposed to multi-walled carbon nanotubes.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNT) causes inflammation, fibroproliferation, immunotoxicity, and systemic responses in rodents. However, the search for representative biomarkers of exposure is an ongoing endeavor. Whole blood gene expression profiling is a promising new approach for the identification of novel disease biomarkers. We asked if the whole blood transcriptome reflects pathology-specific changes in lung gene expression caused by MWCNT. To answer this question, we performed mRNA sequencing analysis of the whole blood and lung in mice administered MWCNT or vehicle solution via pharyngeal aspiration and sacrificed 56 days later. The pattern of lung mRNA expression as determined using Ingenuity Pathway Analysis (IPA) was indicative of continued inflammation, immune cell trafficking, phagocytosis, and adaptive immune responses. Simultaneously, innate immunity-related transcripts (Plunc, Bpifb1, Reg3g) and cancer-related pathways were downregulated. IPA analysis of the differentially expressed genes in the whole blood suggested increased hematopoiesis, predicted activation of cancer/tumor development pathways, and atopy. There were several common upregulated genes between whole blood and lungs, important for adaptive immune responses: Cxcr1, Cd72, Sharpin, and Slc11a1. Trim24, important for TH2 cell effector function, was downregulated in both datasets. Hla-dqa1 mRNA was upregulated in the lungs and downregulated in the blood, as was Lilrb4, which controls the reactivity of immune response. "Cancer" disease category had opposing activation status in the two datasets, while the only commonality was "Hypersensitivity". Transcriptome changes occurring in the lungs did not produce a completely replicable pattern in whole blood; however, specific systemic responses may be shared between transcriptomic profiles.

Transcriptome profiling reveals novel BMI- and sex-specific gene expression signatures for human cardiac hypertrophy.

How obesity or sex may affect the gene expression profiles of human cardiac hypertrophy is unknown. We hypothesized that body-mass index (BMI) and sex can affect gene expression profiles of cardiac hypertrophy. Human heart tissues were grouped according to sex (male, female), BMI (lean<25 kg/m2, obese>30 kg/m2), or left ventricular hypertrophy (LVH) and non-LVH nonfailed controls (NF). We identified 24 differentially expressed (DE) genes comparing female with male samples. In obese subgroup, there were 236 DE genes comparing LVH with NF; in lean subgroup, there were seven DE genes comparing LVH with NF. In female subgroup, we identified 1,320 significant genes comparing LVH with NF; in male subgroup, there were 1,383 significant genes comparing LVH with NF. There were seven significant genes comparing obese LVH with lean NF; comparing male obese LVH with male lean NF samples we found 106 significant genes; comparing female obese LVH with male lean NF, we found no significant genes. Using absolute value of log2 fold-change > 2 or extremely small P value (10-20) as a criterion, we identified nine significant genes (HBA1, HBB, HIST1H2AC, GSTT1, MYL7, NPPA, NPPB, PDK4, PLA2G2A) in LVH, also found in published data set for ischemic and dilated cardiomyopathy in heart failure. We identified a potential gene expression signature that distinguishes between patients with high BMI or between men and women with cardiac hypertrophy. Expression of established biomarkers atrial natriuretic peptide A (NPPA) and B (NPPB) were already significantly increased in hypertrophy compared with controls.

Chronic, Recreational Chloroform-Induced Liver Injury.

Historically used as an anesthetic, chloroform is a halogenated hydrocarbon that is associated with central nervous system depression, arrhythmias, and hepatotoxicity. It is no longer used clinically, but accidental and intentional poisonings still occur. We report a case of chronic chloroform abuse leading to severe hepatotoxicity in a 26-year-old male graduate student. The patient presented to the emergency department with a three-day history of abdominal pain, dehydration, and scleral icterus. He drank several beers the night before the onset of symptoms, but denied taking acetaminophen, ibuprofen, or other drugs. An extensive work-up revealed an aspartate aminotransferase (AST) of 13,527 U/L and alanine aminotransferase (ALT) of 8,745 U/L, but the cause of his liver injury could not be determined. It was not until many months later that the patient admitted to inhaling chloroform in the weeks leading up to his illness.

The preliminary study of effects of tolfenamic Acid on cell proliferation, cell apoptosis, and intracellular collagen deposition in keloid fibroblasts in vitro.

Keloid scarring is a fibroproliferative disorder due to the accumulation of collagen type I. Tolfenamic acid (TA), a nonsteroidal anti-inflammatory drug, has been found to potentially affect the synthesis of collagen in rats. In this preliminary study, we aimed to test the effects of TA on cell proliferation, cell apoptosis, and the deposition of intracellular collagen in keloid fibroblasts. Normal fibroblasts (NFs) and keloid fibroblasts (KFs) were obtained from human dermis tissue. Within the dose range 10(-3)-10(-6) M and exposure times 24 h, 48 h, and 72 h, we found that 0.55 × 10(-3) M TA at 48 h exposure exhibited significantly decreased cell proliferation in both NFs and KFs. Under these experimental conditions, we demonstrated that (1) TA treatment induced a remarkable apoptotic rate in KFs compared to NFs; (2) TA treatment reduced collagen production in KFs versus NFs; (3) TA treatment decreased collagen type I expression in KFs comparing to that of NFs. In summary, our data suggest that TA decreases cell proliferation, induces cell apoptosis, and inhibits collagen accumulation in KFs.