Intron-derived small RNAs for silencing viral RNAs in mosquito cells

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.     

Thyrotropin-releasing hormone inreases prolactin mRNA activity in the cytoplasm of GH-cells as measured by translation in a wheat germ cell-free system

A wheat germ embryo extract was used to translate cytoplasmic RNA isolated from rat pituitary tumor cells (GH-cells). This RNA directed the synthesis of a radioactive product which was precipitated with antiserum specific for rat prolactin. The molecular weight of this immunoprecipitated product was 24,500 as determined by electrophoresis in denaturing gels. Prolactin secreted by intact GH-cells had a molecular weight identical to standard pituitary prolactin, reported to be about 22,500. Our finding that a larger form of prolactin is made by the wheat germ system is similar to results recently described by Maurer, Stone and Gorski (J. Biol. Chem., in press). Thyrotropin-releasing hormone (TRH) stimulates prolactin synthesis in GH-cells, and cytoplasmic RNA isolated from cells treated with TRH directed the synthesis in wheat germ extracts of larger amounts of prolactin than RNA isolated from control cells. The increase in translatable cytoplasmic mRNA for prolactin corresponded to the increase in prolactin synthesis which suggests that the increase in prolactin synthesis in TRH-treated cells is a result of the accumulation of cytoplasmic mRNA for prolactin.     

Epidermal keratin 5 expression and distribution is under dermal influence

Human skin melanin pigmentation is regulated by systemic and local factors. According to the type of melanin produced by melanocytes, the transfer and degradation of melanosomes differ, thus accounting for most variations between ethnicities. We made the surprising observation that in a drastically changed environment, white and black phenotypes are reversible since Caucasian skin grafted onto nude mice can become black with all black phenotypic characteristics. Black xenografts differed essentially from other grafts by the levels of epidermal FGF‐2 and keratin 5. In vitro analysis confirmed that FGF‐2 directly regulates keratin 5. Interestingly, this phenomenon may be involved in human pathology. Keratin 5 mutations in Dowling–Degos Disease (DDD) have already been associated with the pheomelanosome–eumelanosome transition. In a DDD patient, keratin 5 was expressed in the basal and spinous layers, as observed in black xenografts. Furthermore, in a common age‐related hyperpigmentation disorder like senile lentigo (SL), keratin 5 distribution is also altered. In conclusion, modulation of keratin 5 expression and distribution either due to mutations or factors may account for the development of pigmentary disorders.     

Association of age with class VI composite restoration

Dental caries is the major oral health problem in most of the countries, affecting 60-90% of school children and a vast majority of adults. Therefore, it is of interest to evaluate the association of age with Class VI defects restored with composite restorations. We used 102 cases with data regarding Class VI composite restorations in a datasheet of 86,000 records at Saveetha Dental College, India for this study. Data shows that Class VI restorations were commonly seen in upper anterior teeth in the age group of 51 and above. The cavities prepared to receive Class VI restoration followed a conservative design of caries removal and used direct restoration techniques for reconstruction of the lost tooth structure.     

TRK-Fused Gene (TFG), a protein involved in protein secretion pathways,is an essential component of the antiviral innate immune response

Antiviral innate immune response to RNA virus infection is supported by Pattern-Recognition Receptors (PRR) including RIG-I-Like Receptors (RLR), which lead to type I interferons (IFNs) and IFN-stimulated genes (ISG) production. Upon sensing of viral RNA, the E3 ubiquitin ligase TNF Receptor-Associated Factor-3 (TRAF3) is recruited along with its substrate TANK-Binding Kinase (TBK1), to MAVS-containing subcellular compartments, including mitochondria, peroxisomes, and the mitochondria-associated endoplasmic reticulum membrane (MAM). However, the regulation of such events remains largely unresolved. Here, we identify TRK-Fused Gene (TFG), a protein involved in the transport of newly synthesized proteins to the endomembrane system via the Coat Protein complex II (COPII) transport vesicles, as a new TRAF3-interacting protein allowing the efficient recruitment of TRAF3 to MAVS and TBK1 following Sendai virus (SeV) infection. Using siRNA and shRNA approaches, we show that TFG is required for virus-induced TBK1 activation resulting in C-terminal IRF3 phosphorylation and dimerization. We further show that the ability of the TRAF3-TFG complex to engage mTOR following SeV infection allows TBK1 to phosphorylate mTOR on serine 2159, a post-translational modification shown to promote mTORC1 signaling. We demonstrate that the activation of mTORC1 signaling during SeV infection plays a positive role in the expression of Viperin, IRF7 and IFN-induced proteins with tetratricopeptide repeats (IFITs) proteins, and that depleting TFG resulted in a compromised antiviral state. Our study, therefore, identifies TFG as an essential component of the RLR-dependent type I IFN antiviral response.     

Inflammatory intestinal damage induced by 5-fluorouracil requires IL-4

Background5-Fluorouracil (5-FU) induces intestinal mucositis, which is characterized by epithelial ulcerations in the mucosa and clinical manifestations, such as pain and dyspeptic symptoms. Cytokines participate in the inflammatory and functional events of intestinal mucositis. IL-4 is an important mediator of intestinal inflammation, with either anti-inflammatory or pro-inflammatory functions, depending on the model of intestinal inflammation. This study aimed to evaluate the role of IL-4 in 5-FU-induced intestinal mucositis.MethodsIL-4^+/+ or IL-4^?/? mice (25–30 g) were intraperitoneally injected with 5-FU (450 mg/Kg) or saline (C). After 3 days, the mice were sacrificed and the duodenum was evaluated for epithelial damage, MPO activity and cytokine concentration.Results5-FU induced significant damage in the intestinal epithelium of IL-4^+/+ mice (reduction in the villus/crypt ratio: control = 3.31 ± 0.21 μm, 5-FU = 0.99 ± 0.10 μm). However, the same treatment did not induce significant damage in IL-4^?/? mice (5-FU = 2.87 ± 0.19 μm) compared to wild-type mice. 5-FU-induced epithelial damage increased the MPO activity (neutrophil number) and the level of pro-inflammatory cytokines (IL-4, TNF-α, IL-1β and CXCL-8) in the duodenum. These results were not observed in IL-4^?/? mice treated with 5-FU.ConclusionOur data suggest that IL-4 participates as a pro-inflammatory cytokine in a 5-FU-induced intestinal damage model and suggests that IL-4 antagonists may be novel therapeutics for this condition.     

Forecasting youth adjustment at age 15 from school readiness profiles at 54 months

ABSTRACT

A person-oriented approach examined the extent to which patterns of school readiness across social and cognitive domains in 944 typically-developing 54-month-old children forecast academic achievement, social-emotional development, risk taking, and executive functioning at age 15. Prior work identified six distinct profiles of school readiness at 54 months that predicted group differences in achievement in first grade, as well as achievement and social-emotional outcomes in fifth grade. After controlling for demographics, early language skills, and home and school factors, the 54-month readiness profiles demonstrated different performance on risk-taking and executive function behaviors assessed at age 15. Children with attention problems at 54 months were most likely to believe that peers were engaging in risky behaviors and to have smoked more than 2 cigarettes by age 15. Children with low working memory and low to average social skills at 54 months were outperformed by their peers on working memory and executive function tasks at age 15. Results are discussed in terms of continuity in forms of developmental function.