Paternal immune activation by Poly I:C modulates sperm noncoding RNA profiles and causes transgenerational changes in offspring behavior.

Paternal pre-conceptual environmental experiences, such as stress and diet, can affect offspring brain and behavioral phenotypes via epigenetic modifications in sperm. Furthermore, maternal immune activation due to infection during gestation can reprogram offspring behavior and brain functioning in adulthood. However, the effects of paternal pre-conceptual exposure to immune activation on the behavior and physiology of offspring (F1) and grand-offspring (F2) are not currently known. We explored effects of paternal pre-conceptual exposure to viral-like immune activation on F1 and F2 behavioral and physiological phenotypes using a C57BL/6J mouse model. Males were treated with a single injection (intraperitoneal) of the viral mimetic polyinosinic:polycytidylic acid (Poly I:C: 12 mg/kg) then bred with naïve female mice four weeks after the Poly I:C (or 0.9% saline control) injection. The F1 offspring of Poly I:C treated fathers displayed increased depression-like behavior in the Porsolt swim test, an altered stress response in the novelty-suppressed feeding test, and significant transcriptomic changes in their hippocampus. Additionally, the F1 male offspring of Poly I:C treated F0 males showed significantly increased immune responsivity after a Poly I:C immune challenge (12 mg/kg). Furthermore, the F2 male grand-offspring took longer to enter and travelled significantly shorter distances in the light zone of the light/dark box. An analysis of the small noncoding RNA profiles in sperm from Poly I:C treated males and their male offspring revealed significant effects of Poly I:C on the sperm microRNA content at the time of conception and on the sperm PIWI-interacting RNA content of the male offspring. Notably, eight miRNAs with an FDR < 0.05 (miR-141-3p, miR-126b-5p, miR-669o-5p, miR-10b-3p, miR-471-5p, miR-463-5p, miR-148b-3p, and miR-181c-5p) were found to be significantly downregulated in the sperm of Poly I:C treated males. Collectively, we demonstrate that paternal pre-conceptual exposure to a viral immune challenge results in both intergenerational and transgenerational effects on brain and behavior that may be mediated by alterations in the sperm small noncoding RNA content.

Regulation of Carbapenemase Gene Conjugation in Escherichia coli Clinical Isolates.

Background: The purpose of this study is to raise awareness of the hazards of carbapenemase epidemics and provide theoretical support for preventing the spread of carbapenemase-producing organisms. Methods: A total of 893 non-duplicate E. coil strains were recruited from three major local hospitals. The carbapenemase genotype of each imipenem-resistant strain was analyzed. Molecular typing and homology analysis of the main carbapenemase-producing strains reveal the transmission mode of resistance genes. Through the conjugation experiment, the potential spreading risk of carbapenemase genes was analyzed. Extended-spectrum beta-lactamase genes and replicon detection of the conjugant carrying plasmid were performed. The unannotated Escherichia coli bacterial small non-coding RNAs (sRNAs) interacting with sdiA were predicted through a bioinformatics tool. The sRNAs overexpression and knockout strains were constructed, and the effect of sRNA on conjugation was analyzed. Results: A total of 8 carbapenemase-producing strains were detected (0.90%, 8/893). The main carbapenemase genotype was blaKPC -2 (7 strains). Multilocus sequence typing indicated that 7 E. coli isolates belonged to ST-10, ST-101, ST-131, ST-405, ST-410, and ST-1193, ST-2562, respectively. Homologous cluster analysis revealed that the sequence types among the 7 E. coli were high diversity. The blaKPC -2 genes were successfully transferred from these isolates to EC600 by conjugation. All transconjugant cells exhibited significantly reduced susceptibility to the imipenem. IncFII was the most common conjugative plasmid type (85.7%, 6/7). Bioinformatics predicted the interaction between RydB and sdiA. Further experiments found that the interaction between RydB and sdiA improved the bacterial conjugation rate between MG1655 and EC600. The regulation effect of RydB on E. coli conjugation was not affected by the replicon type and/or harboring resistance coding genotype in conjugative plasmids. Conclusion: Our findings emphasized the epidemiological characteristics of carbapenemase-resistant E. coli. A functional phenotype of the new sRNA RydB was identified, and the regulation effect of RydB on E. coli conjugation was improved.

Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice.

Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, we demonstrate that sRNA produced in the putamen of HD patients (HD-sRNA-PT) are sufficient to induce HD pathology in vivo. Mice injected with HD-sRNA-PT show motor abnormalities, decreased levels of striatal HD-related proteins, disruption of the indirect pathway, and strong transcriptional abnormalities, paralleling human HD pathology. Importantly, we show that the specific blockage of sCAG mitigates HD-sRNA-PT neurotoxicity only to a limited extent. This observation prompted us to identify other sRNA species enriched in HD putamen with neurotoxic potential. We detected high levels of tRNA fragments (tRFs) in HD putamen, and we validated the neurotoxic potential of an Alanine derived tRF in vitro. These results highlight that HD-sRNA-PT are neurotoxic, and suggest that multiple sRNA species contribute to striatal dysfunction and general transcriptomic changes, favoring therapeutic strategies based on the blockage of sRNA-mediated toxicity.

Mushroom small RNAs as potential anticancer agents: a closer look at Cantharellus cibarius proapoptotic and antiproliferative effects in colon cancer cells.

Screening aimed at the evaluation of the presence of small RNAs with anticancer properties in three mushrooms species, besides Boletus edulis, namely Boletus spretus (current name Baorangia emilei), Boletus pinophilus and Cantharellus cibarius, was conducted. All mushrooms yielded an ethanol insoluble and water soluble small RNA fraction purified from co-extracted polysaccharides by anion-exchange chromatography. Small RNAs from B. spretus and C. cibarius showed strong antiproliferative activity against human colon adenocarcinoma cell lines (IC50 of 5.6 µg mL-1 and 11.1 µg mL-1 for LS180 and 1.9 µg mL-1 and 12.6 µg mL-1 for HT-29 cell lines, respectively) while those isolated from B. pinophilus showed a much lower antiproliferative activity in these cells. All RNA fractions were nontoxic against CCD841 CoTr human colon epithelial cells. A detailed study of the anticancer mechanism of C. cibarius small RNAs showed that their antiproliferative activity was due to p53-dependent cell cycle arrest mediated by p21, while the proapoptotic effect was mostly dependent on the enhancement of p53 expression. Overall, small RNA fractions isolated from some edible mushrooms, namely C. cibarius, show potent antiproliferative activity without cytotoxicity to normal cells, being a potential new anticancer agent naturally present in mushrooms that we eat.

Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer.

Liver diseases are a growing epidemic worldwide. If unresolved, liver fibrosis develops and can lead to cirrhosis and clinical decompensation. Around 5% of cirrhotic liver diseased patients develop hepatocellular carcinoma (HCC), which in its advanced stages has limited therapeutic options and negative survival outcomes. CEPBA is a master regulator of hepatic function where its expression is known to be suppressed in many forms of liver disease including HCC. Injection of MTL-CEBPA, a small activating RNA oligonucleotide therapy (CEBPA-51) formulated in liposomal nanoparticles (NOV340- SMARTICLES) upregulates hepatic CEBPA expression. Here we show how MTL-CEBPA therapy promotes disease reversal in rodent models of cirrhosis, fibrosis, hepatosteatosis, and significantly reduces tumor burden in cirrhotic HCC. Restoration of liver function markers were observed in a carbon-tetrachloride-induced rat model of fibrosis following 2 weeks of MTL-CEBPA therapy. At 14 weeks, animals showed reduction in ascites and enhanced survival rates. MTL-CEBPA reversed changes associated with hepatosteatosis in non-alcoholic methionine and cholic-deficient diet-induced steaotic liver disease. In diethylnitrosamine induced cirrhotic HCC rats, MTL-CEBPA treatment led to a significant reduction in tumor burden. The data included here and the rapid adoption of MTL-CEBPA into a Phase 1 study may lead to new therapeutic oligonucleotides for undruggable diseases.

Epstein-Barr virus-encoded small non-coding RNAs induce cancer cell chemoresistance and migration.

Epstein-Barr virus (EBV) encoded small, non-coding, non-polyadenylated RNAs, known as EBERs are the most abundantly expressed viral transcripts in latently EBV infected cells. We found the specific role of EBERs in cell cycle progression, resistance against chemotherapeutic drug and cellular invasion in gastric cancer cells in vitro. Ectopic expression of EBERs upregulates the expression of IL-6 and activate its downstream STAT3, which is significantly involved in downregulating the expression of cell cycle inhibitor genes p21 and p27. Stable expression of EBERs regulates the activation of pFAK and pPAK1 and the expression of anti-metastatic genes RhoGDI and KAI-1 in gastric cancer cells. In addition, administration of neu-IL-6 antibody and dominant negative STAT3ß reduces chemoresistance and inhibits invasion of EBERs-expressing gastric cancer cells. Our results thus revealed a novel role of EBERs in the coordination of IL-6-STAT3 signaling pathway to chemoresistance and cellular migration.

Targeting ribosome assembly on the HCV RNA using a small RNA molecule.

Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome Entry Site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.