Finite mixture clustering of human tissues with different levels of IGF-1 splice variants mRNA transcripts

BACKGROUND: This study addresses a recurrent biological problem, that is to define a formal clustering structure for a set of tissues on the basis of the relative abundance of multiple alternatively spliced isoforms mRNAs generated by the same gene. To this aim, we have used a model-based clustering approach, based on a finite mixture of multivariate Gaussian densities. However, given we had more technical replicates from the same tissue for each quantitative measurement, we also employed a finite mixture of linear mixed models, with tissue-specific random effects. RESULTS: A panel of human tissues was analysed through quantitative real-time PCR methods, to quantify the relative amount of mRNA encoding different IGF-1 alternative splicing variants. After an appropriate, preliminary, equalization of the quantitative data, we provided an estimate of the distribution of the observed concentrations for the different IGF-1 mRNA splice variants in the cohort of tissues by employing suitable kernel density estimators. We observed that the analysed IGF-1 mRNA splice variants were characterized by multimodal distributions, which could be interpreted as describing the presence of several sub-population, i.e. potential tissue clusters. In this context, a formal clustering approach based on a finite mixture model (FMM) with Gaussian components is proposed. Due to the presence of potential dependence between the technical replicates (originated by repeated quantitative measurements of the same mRNA splice isoform in the same tissue) we have also employed the finite mixture of linear mixed models (FMLMM), which allowed to take into account this kind of within-tissue dependence. CONCLUSIONS: The FMM and the FMLMM provided a convenient yet formal setting for a model-based clustering of the human tissues in sub-populations, characterized by homogeneous values of concentrations of the mRNAs for one or multiple IGF-1 alternative splicing isoforms. The proposed approaches can be applied to any cohort of tissues expressing several alternatively spliced mRNAs generated by the same gene, and can overcome the limitations of clustering methods based on simple comparisons between splice isoform expression levels

Duplex DNA from Sites of Helicase-Polymerase Uncoupling Links Non-B DNA Structure Formation to Replicative Stress

BACKGROUND: Replication impediments can produce helicase-polymerase uncoupling allowing lagging strand synthesis to continue for as much as 6 kb from the site of the impediment. MATERIALS AND METHODS: We developed a cloning procedure designed to recover fragments from lagging strand near the helicase halt site. RESULTS: A total of 62% of clones from a p53-deficient tumor cell line (PC3) and 33% of the clones from a primary cell line (HPS-19I) were within 5 kb of a G-quadruplex forming sequence. Analyses of a RACK7 gene sequence, that was cloned multiple times from the PC3 line, revealed multiple deletions in region about 1 kb from the cloned region that was present in a non-B conformation. Sequences from the region formed G-quadruplex and i-motif structures under physiological conditions. CONCLUSION: Defects in components of non-B structure suppression systems (e.g. p53 helicase targeting) promote replication-linked damage selectively targeted to sequences prone to G-quadruplex and i-motif formation

The endocannabinoid 2-AG controls skeletal muscle cell differentiation via CB1 receptor-dependent inhibition of Kv7 channels.

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C2C12 myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP2 levels in C2C12 cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels

Molecular analysis of endocrine disruption in hornyhead turbot at wastewater outfalls in southern california using a second generation multi-species microarray.

Sentinel fish hornyhead turbot (Pleuronichthysverticalis) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences

Detection and Molecular Characterization of a Canine Norovirus

We identified a novel calicivirus in a pup with enteritis. The isolate was related genetically (90.1% aa identity in the capsid protein) to a lion norovirus strain

Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium

Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction

Identification of Novel Astroviruses in the Gastrointestinal Tract of Domestic Cats

Astroviruses, isolated from numerous avian and mammalian species including humans, are commonly associated with enteritis and encephalitis. Two astroviruses have previously been identified in cats, and while definitive evidence is lacking, an association with enteritis is suggested. Using metagenomic next-generation sequencing of viral nucleic acids from faecal samples, we identified two novel feline astroviruses termed Feline astrovirus 3 and 4. These viruses were isolated from healthy shelter-housed kittens (Feline astrovirus 3; 6448 bp) and from a kitten with diarrhoea that was co-infected with Feline parvovirus (Feline astrovirus 4, 6549 bp). Both novel astroviruses shared a genome arrangement of three open reading frames (ORFs) comparable to that of other astroviruses. Phylogenetic analysis of the concatenated ORFs, ORF1a, ORF1b and capsid protein revealed that both viruses were phylogenetically distinct from other feline astroviruses, although their precise evolutionary history could not be accurately determined due to a lack of resolution at key nodes. Large-scale molecular surveillance studies of healthy and diseased cats are needed to determine the pathogenicity of feline astroviruses as single virus infections or in co-infections with other enteric viruses

Discovery and Genomic Characterization of Noroviruses from a Gastroenteritis Outbreak in Domestic Cats in the US

Norovirus (NoV) RNA was detected in the stools of 6 out 14 (42.8%) 8–12-week-old cats with enteritis from a feline shelter, in New York State. Upon sequence analysis of the complete capsid, the six NoVs were found to be identical, suggesting the spread of a unique NoV strain in the shelter. The full-length genomic sequence (7839 nt) of one feline NoV, CU081210E/2010/US, was determined. In the capsid protein VP1 region, the virus displayed the highest amino acid identity to animal genogroup IV genotype 2 (GIV.2) NoVs: lion/Pistoia-387/06/IT (97.9%) and dog/Bari-170/07/IT (90.4%). These findings document the discovery of a novel feline calicivirus, different from vesiviruses, and extend the spectrum of NoV host range. Epidemiological studies using feline NoV-specific diagnostic tools and experimental infection of cats are required to understand whether NoVs have a pathogenic role in this species

Genomic characterization of a novel group A lamb rotavirus isolated in Zaragoza, Spain

An ovine rotavirus (OVR) strain, 762, was isolated from a 30-day-old lamb affected with severe gastroenteritis, in Zaragoza, Spain, and the VP4, VP7, VP6, NSP4, and NSP5/NSP6 genes were subsequently characterized molecularly. Strain OVR762 was classified as a P[14] rotavirus, as the VP4 and VP8* trypsin-cleavage product of the VP4 protein revealed the highest amino acid (aa) identity (94% and 97%, respectively) with that of the P11[14] human rotavirus (HRV) strain PA169, isolated in Italy. Analysis of the VP7 gene product revealed that OVR762 possessed G8 serotype specificity, a type common in ruminants, with the highest degree of aa identity(95–98%) shared with serotype G8 HRV, bovine rotavirus, and guanaco (Lama guanicoe) rotavirus strains. Moreover, strain OVR762 displayed a bovine-like NSP4 (genotype E2) and NSP5/NSP6 (genotype H3), and a VP6 genotype I2, as well as a long electropherotype pattern. This is the first report of a lamb rotavirus with P[14] and G8 specificities, providing additional evidence for the wide genetic and antigenic diversity of group A rotaviruses

Photoresponsive Polymer-Based Biomimetic Contractile Units as Building Block for Artificial Muscles

Loss of muscular mechanical function occurs in several diseases affecting millions of people worldwide, including heart failure, stroke, and neuromuscular disorders. To date, no medical or surgical treatments can restore muscular contractility, and the development of artificial muscles is of extreme interest. Mimicking biological muscles, which are optimized systems displaying quick reaction times, is not trivial; only few examples are reported, mainly focused on the use of biomimetic smart materials. Among them, liquid crystalline elastomers (LCEs) can be biocompatible, show contraction parameters comparable to those of native striated muscles, and are able to effectively potentiate cardiac contraction in vitro. To go further and develop in vivo implantable devices, the integration of the stimulation system with the LCE material represents an essential step. Here, a light-stimulated biomimetic contractile unit (BCU), combining ultra-thin photoresponsive LCE films and mini-LED (mLED) matrixes is described. BCU performance (in terms of extent and kinetics of contractile force and shortening) can be fine-tuned by modulating both mLED light power and spatial stimulation patterns, allowing to reproduce mechanical dynamics of native muscles. These results pave the way for the development of novel LCE-based contraction assist devices for cardiac, skeletal, or smooth muscle support by assembling multiple BCUs