Whole genome sequencing analysis of alpaca suggests TRPV3 as a candidate gene for the suri phenotype.

BACKGROUND: Alpaca is a domestic South American camelid probably arising from the domestication of two wild camelids, the vicugna and the guanaco. Two phenotypes are described for alpaca, known as huacaya and suri. Huacaya fleece is characterized by compact, soft, and highly crimped fibers, while suri fleece is longer, straight, less crimped, and lustrous. The gene variants determining these phenotypes are still unknown, although previous studies suggested a dominant inheritance of the suri. Based on that, the aim of this study was the identification of the gene variants determining alpaca coat phenotypes through whole genome sequencing (WGS) analysis. RESULTS: The sample used includes two test-cross alpaca families, suri × huacaya, which produced two offspring, one with the suri phenotype and one with the huacaya phenotype. The analyzed sample was expanded through the addition of WGS data from six vicugnas and six guanacos; this because we assumed the absence of the gene variants linked to the suri phenotype in these wild species. The analysis of gene variant segregation with the suri phenotype, coupled with the filtering of gene variants present in the wild species, disclosed the presence in all the suri samples of a premature termination codon (PTC) in TRPV3 (transient receptor potential cation channel subfamily V member 3), a gene known to be involved in hair growth and cycling, thermal sensation, cold tolerance and adaptation in several species. Mutations in TRPV3 were previously associated with the alteration of hair structure leading to an impaired formation of the hair canal and the hair shaft in mouse. This PTC in TRPV3, due to a G > T substitution (p.Glu475*), results in a loss of 290 amino acids from the canonical translated protein, plausibly leading to a physiological dysfunction. CONCLUSION: The present results suggest that the suri phenotype may arise from a TRPV3 gene variant which may explain some of the suri features such as its longer hair fibre with lower number of cuticular scales compared to huacaya.

Phenotypic Correlation between Guard Hair and Down Hair in Chinese Alashan Left Banner White Cashmere Goat: A Preliminary Study.

In cashmere production studies, few trials have considered the guard hair features and their correlation with down fiber attributes. In this preliminary work, early observations on 158 one year old Chinese Alashan Left Banner White Cashmere goats were carried out. The aim was to describe the phenotypic correlation between the guard hair length and other fiber traits. The guard hair length was positively correlated with guard hair diameter and the down fiber length. Negative correlations were found between guard hair length and the coefficient of variation of guard hair diameter, between the guard hair diameter and its coefficient of variation, and between the down fiber diameter and the coefficient of variation of down fiber diameter. The body weight at first combing was not correlated with any of the other traits.

Exon-1 skipping and intron-1 retaining by alternative splicing of the c-KIT gene encodes a novel splice variant in the skin of Merino sheep (Ovis aries).

c-KIT, a type III receptor protein tyrosine kinase, plays an essential role in melanocyte development, migration, and survival. Mutations within the c-KIT gene are previously shown to cause the white coat color phenotypes in pigs, mice, goats, and humans. However, up so far, the splicing isoform(s), genomic architecture of c-KIT have not been characterized well in merino sheep. Reverse transcriptase (RT)-PCR analysis with molecular prediction identified two basic splice variants: Transcript Variant-1, 2 for 12 bp insertion coding sequences (CDS) corresponding to the four amino acids 'GNSK', respectively. Using 5' RACE, here we report for the first time a novel c-KIT 'Transcript Variant-3' from the skin of merino sheep by comparative genome analyses at exon(1)-intron(1)-exon(2) boundaries. In contrast, a single product of 795 bp was characterized by 3' RACE. We also demonstrated that the c-KIT gene expression at the transcript level is not mediated via an intron-9 splicing event. Overall, beyond what was observed in other mammals, our data provide novel insights into the molecular structure of the c-KIT gene in sheep.

PDGFA in Cashmere Goat: A Motivation for the Hair Follicle Stem Cells to Activate.

The cashmere hair follicle (HF) perpetually goes through cycles of growth, involution and rest. The photoperiod is the main factor in the control of seasonal coat change in cashmere goats while stem cells play a crucial role in the HF growth. Several factors, including Platelet-Derived Growth Factor A (PDGFA), Bone Morphogenetic Protein 2 (BMP2) and Lim-Homeobox gene 2 (LHX2) are implicated in HF morphogenesis and cycle. In this work, the mentioned molecules were investigated to evaluate their role in follicular cycle activation. The study was performed on skin samples collected at different periods of HF cycle and the molecular expression of PDGFA, BMP2 and LHX2 was evaluated by Real-Time PCR (qPCR) at each time point. Since PDGFA showed the most variation, the goat PDGFA gene was sequenced and the protein localization was investigated by immunohistochemistry together with PDGF receptor α (PDGFRα). PDGFA immunostaining was observed in the basal layer of the HF outer root sheath and the immunoreaction appeared stronger in the regressive HFs compared to those in the anagen phase according to qPCR analysis. PDGFRα was observed in the HF epithelium, proving the effect of PDGFA on the follicular structure. The data obtained suggest that PDGFA and BMP2 are both implicated in HF cycle in goat. In particular, PDGFA secreted by the HF is involved in the anagen activation.

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.

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.

Alternative splicing of the sheep MITF gene: novel transcripts detectable in skin.

Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor, which regulates the differentiation and development of melanocytes and pigment cell-specific transcription of the melanogenesis enzyme genes. Though multiple splice variants of MITF have been reported in humans, mice and other vertebrate species, in merino sheep (Ovis aries), MITF gene splicing has not yet been investigated until now. To investigate the sheep MITF isoforms, the full length mRNA/cDNAs from the skin of merino sheep were cloned, sequenced and characterized. Reverse transcriptase (RT)-PCR analysis and molecular prediction revealed two basic splice variants with (+) and without (-) an 18 bp insertion viz. CGTGTATTTTCCCCACAG, in the coding region (CDS) for the amino acids 'ACIFPT'. It was further confirmed by the complete nucleotide sequencing of splice junction covering intron-6 (2463 bp), wherein an 18bp intronic sequence is retained into the CDS of MITF (+) isoform. Further, full-length cDNA libraries were enriched by the method of 5' and 3' rapid amplification of cDNA ends (RACE-PCR). A total of seven sheep MITF splice variants, with distinct N-terminus sequences such as MITF-A, B, E, H, and M, the counterparts of human and mouse MITF, were identified by 5' RACE. The other two 5' RACE products were found to be novel splice variants of MITF and represented as 'MITF truncated form (Trn)-1, 2'. These alternative splice (AS) variants were illustrated using comparative genome analysis. By means of 3' RACE three different MITF 3' UTRs (625, 1083, 3167bp) were identified and characterized. We also demonstrated that the MITF gene expression determined at transcript level is mediated via an intron-6 splicing event. Here we summarize for the first time, the expression of seven MITF splice variants with three distinct 3' UTRs in the skin of merino sheep. Our data refine the structure of the MITF gene in sheep beyond what was previously known in humans, mice, dogs and other mammals.

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 D(175)G 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.

Effects of losartan and irbesartan administration on brain angiotensinogen mRNA levels.

Losartan, 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'(1H-tetrazol-5-yl)-biphenil-4-yl)methyl]imidazole, and Irbesartan, 2-n-butyl-3-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl)methyl]-1,3-diaza-spiro[4,4]non-1-en-4-one, are two angiotensin AT1 receptor antagonists largely used in human health care as antihypertensive agents. Their ability to cross the blood-brain barrier and to influence the central renin-angiotensin system are widely investigated, but how this brain system responds to the subchronic and chronic block of the angiotensin AT1 receptor is still unknown. Normotensive rats were intragastrically implanted for 7- and 30-day administration, with a dose of 3 and 30 mg/kg body weight. Treatments were shown to influence, in a dose-, time- and brain-area-dependent manner, angiotensinogen mRNA levels in scanned areas. This study showed a general up-regulation of angiotensinogen mRNA expression after 7 days and a widespread down-regulation or basal level of expression after a 30-day administration of two angiotensin AT1 receptor antagonists.

Preprotachykinin A gene expression after administration of 3,4-methylene dioxymethamphetamine (Ecstasy).

This study tested the effects of 8 days of subchronic administration of 3,4-methylene dioxymethamphetamine (MDMA) (5 mg/kg b.w.) on preprotachykinin A mRNA levels in discrete rat brain regions. In situ hybridization examined preprotachykinin A mRNA levels in the core and shell of the nucleus accumbens, the islands of Calleja, the olfactory tubercle, the dorsal and ventral caudate-putamen, the bed nucleus of the stria terminalis, the medial preoptic area, the medial habenular nucleus and in the postero-dorsal part of the medial amygdala. Higher levels of preprotachykinin A mRNA were found in the core and shell of the nucleus accumbens, in the islands of calleja, in the olfactory tubercle, in the bed nucleus of the stria terminalis, in the medial habenular nucleus and the postero-dorsal part of the medial amygdala, compared to control animals. Conversely, increased preprotachykinin A mRNA levels were observed in the dorsal and ventral caudate-putamen in MDMA treated when compared to control rats. In the social memory test, MDMA significantly impaired rats' short-term working memory. These results show that chronic exposure to MDMA strongly affects preprotachykinin A mRNA levels in discrete rat brain regions. These changes occur in experimental conditions in which working memory is markedly reduced, suggesting that changes in gene expression of tachykinin mechanisms may contribute to the effects of MDMA on memory function.

The vanadate-tolerant yeast Hansenula polymorpha undergoes cellular reorganization during growth in, and recovery from, the presence of vanadate.

When present at intracellular concentrations above micromolar, vanadate becomes toxic to most organisms. However, the yeast Hansenula polymorpha is able to grow on vanadate concentrations in the millimolar range, showing at the same time modifications in cellular ultrastructure and polyphosphate metabolism. Here, the development of the ultrastructural changes, and of vacuolar and secretory activities, during exponential growth on vanadate and upon a return to vanadate-free conditions was investigated. External invertase secretion was inhibited by vanadate, as shown by a decrease in external invertase activity, an intracellular accumulation of small vesicles and a cytoplasmic accumulation of internal invertase. An aberrant appearance of the cell wall and defects in cellular surface growth, possibly linked to defects in secretion, were also observed. However, inhibition of the secretory pathway was not complete since the activity of another secreted enzyme, exoglucanase, increased in the presence of vanadate. Growth on vanadate was also accompanied by an enhancement of vacuolar proteolysis, as indicated by an increase in carboxypeptidase Y activity. However, these modifications were all reversible upon return to vanadate-free conditions, with the normalization process being complex and involving new and dramatic ultrastructural changes and activation of an autophagic mechanism. This mechanism is involved in the elimination/resorption of the observed vanadate-induced aberrant cell structures and/or sites involved in vanadate accumulation, a necessary prerequisite for restoration of conventional ultrastructure and metabolic functions.