Dario Pediconi

Mosquitoes can harbour yeasts of clinical significance and contribute to their environmental dissemination

There is still a lack of studies on fungal microbiota in mosquitoes, compared with the number available on bacterial microbiota. This study reports the identification of yeasts of clinical significance in laboratory mosquito species: Anopheles gambiae, Anopheles stephensi, Culex quinquefasciatus, Aedes albopictus and Aedes aegypti. Among the yeasts isolated, they focused on the opportunistic pathogen Candida parapsilosis, since there is a need to better understand breakthrough candidaemia with resistance to the usual antifungals, which requires careful consideration in the broad-spectrum therapy, as documented in many clinical reports. C. parapsilosis occurs widely and has been isolated from diverse sources, including insects, which may contribute to its dissemination. In this study, it was isolated from the gut of An. gambiae and its presence in developmental stages and organs of different mosquito species was studied. Our results indicated that there was a stable association between C. parapsilosis and reared mosquitoes during the entire life cycle, and in adult male and female gut and gonads. A wide occurrence of C. parapsilosis was also documented in several populations of wild mosquitoes. Based on these findings, it can be said that mosquitoes might participate in the spreading of this opportunistic pathogen, not only as a carrier.

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.

Evidence of post-transcriptional readthrough regulation in FGF5 gene of alpaca

Two different phenotypes are described in alpaca, identified as suri and huacaya, which differ in the type of fleece. The huacaya fleece is characterized by compact, soft and highly crimped fibers, while the suri fleece is longer, straight, less-crimped and lustrous. In our study, the Fibroblast growth factor 5 (FGF5) was investigated as a possible candidate gene for hair length in alpaca (Vicugna pacos). As previously identified in other mammals, our results show that the alpaca FGF5 gene gives rise to a short (FGF5S) and a long (FGF5) isoform. Interestingly, in the long isoform, we observed a point mutation (i.e., a transition C>T at position 499 downstream of the ATG codon) that is able to generate a premature termination codon (PTC). The highly conserved nucleotide and amino acid sequence after PTC suggested a readthrough event (RT) that was confirmed by western blot analysis. The analysis of cDNA sequence revealed motifs and structures of mRNA undergoing RT. In fact, the event is positively influenced by particular signals harbored by the transcript. To the best of our knowledge, this is the first case of a readthrough event on PTC reported for the FGF5 gene and the first case of this translational mechanism in alpaca.

MOLECULAR CHARACTERIZATION OF FIBROBLAST GROWTH FACTOR 5 (FGF5) GENE IN ALPACA

Carbohydrates are not the only macronutrients triggering insulin release following food intake. Besides other components, leucine (Leu) has been demonstrated to induce insulin secretion in humans and in laboratory rodents. We hypothesized that Leu stimulates the release of insulin in dairy cows, and thus our objective was to test the effects of a single-dose of Leu infused intraduodenally on the concentrations of insulin, glucose and free amino acids (AA) in blood plasma as compared to infusions with either glucose or saline. Six duodenum-fistulated Holstein cows were studied in a replicated 3 × 3 Latin square design with 3 periods of 7 d, in which the treatments were applied at the end of each period. The treatments were duodenal bolus infusions of Leu (DIL; 0.15 g/kg BW), glucose (DIG; at Leu equimolar dosage) or saline (SAL). Blood samples were taken at -15, 0, 10, 20, 30, 40, 50, 60, 75, 90, 120, 180, 210, 240 and 300 min relative to the infusion and the concentrations of insulin, glucose and AA were determined in plasma. In DIG, insulin and glucose concentrations peaked at 30-40 and 40-50 min after the infusion, respectively. Insulin concentrations were greater (P<0.05) from 30-50 min in DIG than DIL and SAL. In DIG, glucose concentrations were greater (P≤0.01) from 30-75 and 40-50 min than in DIL and SAL, respectively. In DIL, Leu concentrations peaked 50-60 min after infusion, reaching 20 and 15-fold greater values than that in DIG and SAL, respectively. The plasma concentrations of total AA minus Leu were affected by treatment (P<0.0001), resulting in lowest mean concentrations of total AA minus Leu in DIL, followed by DIG and SAL. The data suggest that Leu infusion did not elicit an apparent insulin response, but may stimulate the tissue uptake of AA by mechanisms yet to be elucidated.Breeding goals of Holstein dairy cattle in New Zealand (NZL) focus on traits reflecting adaptation to pasture-based systems. Due to high prices for concentrates, and advantages for cattle health and welfare, low input grazing systems become important in regions of North-West Germany. The present study addresses the hypothesis that progeny of NZL-Holstein sires are superior in 30 German grassland farms compared to offspring of Holstein sires being progeny tested in German indoor systems. A research design was implemented to create three different genetic groups (F1 generation) within herds on the basis of a German Holstein cow (GHC) population: Group 1=GHC x NZL sires, group 2=GHC x GH sires, group 3=GHC x GH_pasture sires. GH_pasture sires represent bulls of German origin with high breeding values for the traits being important in NZL. Group comparison focused on the full set of production and functional traits from official recording systems, and in addition on further novel traits reflecting cow health and welfare: Scores for body condition (BCS), locomotion (LOCS), hock lesions (HLS), and total hygiene (THS). Linear and generalized linear mixed models were applied to estimate group differences for a longitudinal data structure (2998 observations). In parity 1, daughters of NZL sires had the highest LSMean for BCS (2.56), and lowest LSMean for LOCS (0.10), HLS (0.25) and THS (3.67), reflecting an enhanced robustness of HF strains from NZL. Within breed genetic analyses simultaneously considering all genetic groups revealed a moderate genetic background with the following heritabilities: BCS (0.25), LOCS (0.12), HLS (0.04), and THS (0.17). Genotype by environment interactions between pasture-based and indoor systems were studied by applying multiple trait models. For all traits, rg were smaller than 0.80, especially indicating GxE interactions for low heritability functional traits.