Deletion in the EVC2 Gene Causes Chondrodysplastic Dwarfism in Tyrolean Grey Cattle

During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle.     

The Autonomic Signature of Guilt in Children: A Thermal Infrared Imaging Study

So far inferences on early moral development and higher order self conscious emotions have mostly been based on behavioral data. Emotions though, as far as arguments support, are multidimensional notions. Not only do they involve behavioral actions upon perception of an event, but they also carry autonomic physiological markers. The current study aimed to examine and characterise physiological signs that underlie self-conscious emotions in early childhood, while grounding them on behavioral analyses. For this purpose, the “mishap paradigm” was used as the most reliable method for evoking feelings of “guilt” in children and autonomic facial temperature variation were detected by functional Infrared Imaging (fIRI). Fifteen children (age: 39–42 months) participated in the study. They were asked to play with a toy, falsely informed that it was the experimenter''s “favourite”, while being unaware that it was pre-planned to break. Mishap of the toy during engagement caused sympathetic arousal as shown by peripheral nasal vasoconstriction leading to a marked temperature drop, compared to baseline. Soothing after the mishap phase induced an increase in nose temperature, associated with parasympathetic activity suggesting that the child''s distress was neutralized, or even overcompensated. Behavioral analyses reported signs of distress evoked by the paradigm, backing up the thermal observation. The results suggest that the integration of physiological elements should be crucial in research concerning socio-emotional development. fIRI is a non invasive and non contact method providing a powerful tool for inferring early moral emotional signs based on physiological observations of peripheral vasoconstriction, while preserving an ecological and natural context.     

Soluble Products of Escherichia coli Induce Mitochondrial Dysfunction-Related Sperm Membrane Lipid Peroxidation Which Is Prevented by Lactobacilli

Unidentified soluble factors secreted by E. coli, a frequently isolated microorganism in genitourinary infections, have been reported to inhibit mitochondrial membrane potential (ΔΨm), motility and vitality of human spermatozoa. Here we explore the mechanisms involved in the adverse impact of E. coli on sperm motility, focusing mainly on sperm mitochondrial function and possible membrane damage induced by mitochondrial-generated reactive oxygen species (ROS). Furthermore, as lactobacilli, which dominate the vaginal ecosystem of healthy women, have been shown to exert anti-oxidant protective effects on spermatozoa, we also evaluated whether soluble products from these microorganisms could protect spermatozoa against the effects of E. coli. We assessed motility (by computer-aided semen analysis), ΔΨm (with JC-1 dye by flow cytometry), mitochondrial ROS generation (with MitoSOX red dye by flow cytometry) and membrane lipid-peroxidation (with the fluorophore BODIPY C₁₁ by flow cytometry) of sperm suspensions exposed to E. coli in the presence and in the absence of a combination of 3 selected strains of lactobacilli (L. brevis, L. salivarius, L. plantarum). A Transwell system was used to avoid direct contact between spermatozoa and microorganisms. Soluble products of E. coli induced ΔΨm loss, mitochondrial generation of ROS and membrane lipid-peroxidation, resulting in motility loss. Soluble factors of lactobacilli prevented membrane lipid-peroxidation of E. coli-exposed spermatozoa, thus preserving their motility. In conclusion, sperm motility loss by soluble products of E. coli reflects a mitochondrial dysfunction-related membrane lipid-peroxidation. Lactobacilli could protect spermatozoa in the presence of vaginal disorders, by preventing ROS-induced membrane damage.     

Actin remodeling driven by circLIMA1: sperm cell as an intriguing cellular model

CircRNA cargo in spermatozoa (SPZ) participates in setting cell quality, in terms of morphology and motility. Cannabinoid receptor CB1 activity is correlated with a proper spermatogenesis and epididymal sperm maturation. Despite CB1 promotes endogenous skill to circularize mRNAs in SPZ, few notions are reported regarding the functional link between endocannabinoids and spermatic circRNA cargo. In CB1 knock-out male mice, we performed a complete dataset of spermatic circRNA content by microarray strategy.Differentially expressed (DE)-circRNAs, as a function of genotype, were identified. Within DE-circRNAs, we focused the attention on circLIMA1, as putative actin-cytoskeleton architecture regulator. The validation of circLIMA1 dependent-competitive endogenous RNA (ceRNA) network (ceRNET) in in vitro cell line confirmed its activity in the regulation of the cytoskeletal actin. Interestingly, a dynamic actin regulation in SPZ nuclei was found during their epididymal maturation.In this scenario, we showed for the first time an intriguing sperm nuclear actin remodeling, regulated via a ceRNET-independent pathway, consisting in the nuclear shuttling of circLIMA1-QKI interactome and downstream in Gelsolin regulation. In particular, the increased levels of circLIMA1 in CB1 knock-out SPZ, associated with an inefficient depolymerization of nuclear actin, specifically illustrate how endocannabinoids, by regulating circRNA cargo, may contribute to sperm morpho-cellular maturation.     

SIMD parallelization of the WORDUP algorithm for detecting statistically significant patterns in DNA sequences

The development of new techniques in sequencing nucleic acidshas produced a great amount of sequence data and has led tothe discovery of new relationships. In this paper, we studya method for parallelizing the algorithm WORDUP, which detectsthe presence of statistically significant patterns in DNA sequences.WORDUP implements an efficient method to identify the presenceof statistically significant oligomers in a nonhomologous groupof sequences. It is based on a modified version of the Boyer-Moorealgorithm, which is one of the fastest algorithms for stringmatching available in the literature. The aim of the parallelversion of WORDUP presented here is to speed up the computationaltime and allow the analysis of a greater set of longer nucleotidesequences, which is usually impractical with sequential algorithms.     

Trimethyltin Modulates Reelin Expression and Endogenous Neurogenesis in the Hippocampus of Developing Rats

Reelin is an extracellular matrix glycoprotein involved in the modulation of synaptic plasticity and essential for the proper radial migration of cortical neurons during development and for the integration and positioning of dentate granular cell progenitors; its expression is down-regulated as brain maturation is completed. Trimethyltin (TMT) is a potent neurotoxicant which causes selective neuronal death mainly localised in the CA1-CA3/hilus hippocampal regions. In the present study we analysed the expression of reelin and the modulation of endogenous neurogenesis in the postnatal rat hippocampus during TMT-induced neurodegeneration (TMT 6 mg/kg). Our results show that TMT administration induces changes in the physiological postnatal decrease of reelin expression in the hippocampus of developing rats. In particular, quantitative analysis of reelin-positive cells evidenced, in TMT-treated animals, a persistent reelin expression in the stratum lacunosum moleculare of Cornu Ammonis and in the molecular layer of Dentate Gyrus. In addition, a significant decrease in the number of bromodeoxyuridine (BrdU)-labeled newly-generated cells was also detectable in the subgranular zone of P21 TMT-treated rats compared with P21 control animals; no differences between P28 TMT-treated rats and age-matched control group were observed. In addition the neuronal commitment of BrdU-positive cells appeared reduced in P21 TMT-treated rats compared with P28 TMT-treated animals. Thus TMT treatment, administrated during development, induces an early reduction of endogenous neurogenesis and influences the hippocampal pattern of reelin expression in a temporally and regionally specific manner, altering the physiological decrease of this protein.     

Technological properties of bakers’ yeasts in durum wheat semolina dough

Properties of 13 Saccharomyces cerevisiae strains isolated from different sources (traditional sourdoughs, industrial baking yeasts etc.) were studied in dough produced with durum wheat (Sicilian semolina, variety Mongibello). Durum wheat semolina and durum wheat flour are products prepared from grain of durum wheat (Triticum durum Desf.) by grinding or milling processes in which the bran and germ are essentially removed and the remainder is comminuted to a suitable degree of fineness. Acidification and leavening properties of the dough were evaluated. Strains isolated from traditional sourdoughs (DSM PST18864, DSM PST18865 and DSM PST18866) showed higher leavening power, valuable after the first and second hours of fermentation, than commercial baking yeasts. In particular the strain DSM PST 18865 has also been successfully tested in bakery companies for the improvement of production processes. Baking and staling tests were carried out on five yeast strains to evaluate their fermentation ability directly and their resistance to the staling process. Amplified fragment length polymorphism (fAFLP) was used to investigate genetic variations in the yeast strains. This study showed an appreciable biodiversity in the microbial populations of both wild and commercial yeast strains.     

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G > A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.     

Genetic typing of bovine viral diarrhoea virus: evidence of an increasing number of variants in Italy

Bovine Viral Diarrhoea Virus (BVDV) is responsible worldwide for severe economic losses on cattle farms. BVDV is an RNA virus with a high genome variability having practical consequences on epidemiology, diagnosis and disease control. Genetic monitoring was suggested as the first step in BVDV control. Thirty-seven Bovine Viral Diarrhoea Viruses were identified in persistently infected cattle, mucosal disease-affected animals and in bulk milk, and were characterised genetically. The 5'UTR region was amplified and sequenced, and a phylogenetic analysis was carried out comparing all the Italian sequences of BVDV available from the Genbank database. An unusual number of persistent infected animals was evidenced on more than one farm. Phylogenetic analysis attributed all our viruses to BVDV type I and distinguished four different subgroups inside this genotype. Analysis of old and new viruses revealed the circulation of viruses classified in subgroups BVDV Ia and Ij never reported in Italy.     

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations

The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous Epstein-Barr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fluorescent LDL followed by flow cytometry analysis. Residual LDLR activity was calculated normalizing fluorescence for the mean fluorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes.