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1.
Reproduction ; 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38063339

RESUMO

MicroRNAs (miRNAs), which can be carried inside extracellular vesicles (EVs), play a crucial role in regulating embryo development up to the blastocyst stage. Yet, the molecular mechanisms underlying blastocyst development and quality are largely unknown. Recently, our group identified 69 differentially expressed miRNAs in extracellular vesicles (EVs) isolated from culture medium conditioned by bovine embryos that either developed to the blastocyst stage or did not (non-blastocysts). We found miR-146b to be more abundant in the EVs derived from media conditioned by non-blastocyst embryos. Using RT-qPCR, we here confirmed the upregulation of miR-146b in non-blastocyst (arrested at 2-4 cell and morula stage) embryos compared to blastocysts (p<0.005), which coincides with the upregulation of miR-146b in EVs derived from the medium of these non-blastocysts. To evaluate a functional effect, bovine embryo culture media were supplemented with miR-146b mimics, resulting in significantly decreased embryo quality, with lower blastocyst rates at day 7 and lower total cell numbers, while the opposite was found after supplementation with miR-146b inhibitors, which resulted in reduced apoptosis rates (P < 0.01). Transcriptomic analysis of embryos treated with miR-146b mimics or inhibitors showed differential expression (P < 0.01) of genes associated with apoptosis, cell differentiation, and the RNA Pol II transcription complex, including WDR36, MBNL2, ERCC6l2, PYGO1, and SNIP1. Overall, miR-146b is overexpressed in non-blastocyst embryos and in EVs secreted by these embryos, and it regulates genes involved in embryo development and apoptosis, resulting in decreased embryo quality.

2.
J Vet Intern Med ; 36(5): 1597-1606, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36054182

RESUMO

BACKGROUND: Primary congenital hypothyroidism (CH) is a rare endocrine disorder in cats with a largely unknown genetic cause. OBJECTIVES: Describe the clinical presentation of CH in 11 affected cats and identify the causal genetic variant. ANIMALS: Eleven CH-cats from 10 unrelated families, 11 CH-free family members, 21 unrelated CH-free cats, and 155 unrelated nondiagnosed cats from different breeds. METHODS: Case control study of CH-cats and their siblings (2019-2021). Diagnosis was based on low to low-normal serum thyroxine (T4) concentrations, high thyroid-stimulating hormone (TSH) concentrations and clinical signs compatible with CH. We identified the causal variant using Sanger sequencing, genotyping via PCR-RFLP and variant interpretation using ACMG/AMP guidelines. RESULTS: All CH-cats (5 weeks-8 years) had disproportionate dwarfism. A goiter was not palpable in all. Thyroid scintigraphy with radiopertechnetate showed abnormally high uptake by thyroid glands, whereas scintigraphy with radioiodine showed abnormally low uptake, compatible with a defect in iodine organification by thyroid peroxidase (TPO). All cases were homozygous for TPO variant XM_006930524.4:c.430G>A(p.(Gly144Arg)), while none of the CH-free cats were. All sampled parents were heterozygous for this recessive variant. This variant was found in 15 cat breeds with an estimated allele frequency of 9%. CONCLUSIONS AND CLINICAL IMPORTANCE: Disproportionate dwarfism, abnormally high TSH and abnormally low to low-normal T4 concentrations are diagnostic for CH in cats. All cases had dyshormonogenesis demonstrated by thyroid scintigraphy. This novel TPO missense variant (not described in humans) causes CH in cats and awareness of it can assist in diagnosis and breeding.


Assuntos
Doenças do Gato , Hipotireoidismo Congênito , Animais , Gatos , Estudos de Casos e Controles , Doenças do Gato/diagnóstico , Doenças do Gato/genética , Hipotireoidismo Congênito/diagnóstico , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/veterinária , Iodeto Peroxidase/genética , Radioisótopos do Iodo , Tireotropina , Tiroxina
3.
Animals (Basel) ; 12(15)2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35953997

RESUMO

Corneal sequestra are ophthalmic lesions that are remarkably common in Persian, Himalayan and exotic cats. In this study, the genetic aspects of this disease were investigated in a population of cats originating from a single cattery. Odds ratios were calculated for parents with affected offspring. The heritability of (owner-reported) corneal sequestra was estimated with a Markov chain Monte Carlo procedure. Well-phenotyped cases and controls were used for a genome-wide association study. Data from 692 cats originating from the cattery, of which 61 were affected, were used. Cats from two specific mothers had significantly higher odds of developing corneal sequestra, but no significant effect of the fathers was found (after correction for the mothers). The heritability of corneal sequestra was estimated to be 0.96. A genome-wide association study with 14 cases and 10 controls did not reveal an associated chromosomal region. The large effect that genetic factors had on the development of corneal sequestra in this study suggests that selective breeding could be an effective way to reduce the prevalence of this condition in these cat breeds.

4.
Anim Genet ; 53(4): 526-529, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35634705

RESUMO

Hypertrophic cardiomyopathy (HCM) is a common and potentially fatal heart disease in many cat breeds. An intronic variant in TNNT2, c.95-108G>A, was recently reported as the cause of HCM in the Maine Coon. The aim of this study was to determine this variant's allele frequency in different populations and its possible association with HCM. Based on 160 Maine Coon samples collected in Belgium, Italy, Sweden and the USA, the variant's allele frequency was estimated to be 0.32. Analysis of the 99 Lives feline whole genome sequencing database showed that the TNNT2 variant also occurs in other breeds, as well as mixed-breed cats. Comparison of 31 affected and 58 healthy cats did not reveal significantly increased odds for HCM in homozygotes. Based on the combined evidence and in agreement with the standards and guidelines for the interpretation of sequence variants, this variant is currently classified as a variant of unknown significance and should not be used for breeding decisions regarding HCM.


Assuntos
Cardiomiopatia Hipertrófica , Doenças do Gato , Animais , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/veterinária , Proteínas de Transporte/genética , Gatos , Homozigoto , Mutação , Sequenciamento Completo do Genoma
5.
Eur J Hum Genet ; 27(11): 1724-1730, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31164718

RESUMO

Hypertrophic cardiomyopathy (HCM) is the most common inherited human heart disease. The same disease has a high prevalence in cats, where it is also suspected to be inherited. More than 1500 variants in MYBPC3, MYH7 and other sarcomeric genes are associated with human HCM, while in cats, only two causative variants in MYBPC3 are currently known. Here, we describe an adult Domestic Shorthair cat with arterial thromboembolism and heart failure that was diagnosed with HCM on necropsy. Sequencing of the coding regions of MYBPC3 and MYH7 revealed 21 variants, of which the MYH7 c.5647G>A (p.(Glu1883Lys)) variant was further analysed, because its orthologous variant had already been reported in a human patient with HCM, but with limited causal evidence. This variant affects the highly conserved assembly competence domain, is predicted in silico to be damaging and was found only once in population databases. Recently, functional studies have confirmed its predicted damaging effect and a paralogous variant in MYH6 has been associated with cardiac disease in humans as well. This report of an orthologous variant in a cat with HCM and its absence in 200 additional cats provides further evidence for its disease-causing nature. As the first report of feline HCM caused by a variant in MYH7, this study also emphasises this gene as a candidate gene for future studies in cats and highlights the similarity between human and feline HCM.


Assuntos
Doenças dos Animais/genética , Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/genética , Gatos/genética , Predisposição Genética para Doença/genética , Cadeias Pesadas de Miosina/genética , Animais , Proteínas de Transporte/genética , Humanos , Masculino
6.
Eur J Hum Genet ; 27(10): 1561-1568, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31160700

RESUMO

Clinical, pathological, and genetic findings of a primary hereditary ataxia found in a Malinois dog family are described and compared with its human counterpart. Based on the family history and the phenotype/genotype relationships already described in humans and dogs, a causal variant was expected to be found in KCNJ10. Rather surprisingly, whole-exome sequencing identified the SLC12A6 NC_006612.3(XM_014109414.2): c.178_181delinsCATCTCACTCAT (p.(Met60Hisfs*14)) truncating variant. This loss-of-function variant perfectly segregated within the affected Malinois family in an autosomal recessive way and was not found in 562 additional reference dogs from 18 different breeds, including Malinois. In humans, SLC12A6 variants cause "agenesis of the corpus callosum with peripheral neuropathy" (ACCPN, alias Andermann syndrome), owing to a dysfunction of this K+-Cl- cotransporter. However, depending on the variant (including truncating variants), different clinical features are observed within ACCPN. The variant in dogs encodes the shortest isoform described so far and its resultant phenotype is quite different from humans, as no signs of peripheral neuropathy, agenesis of the corpus callosum nor obvious mental retardation have been observed in dogs. On the other hand, progressive spinocerebellar ataxia, which is the most important feature of the canine phenotype, hindlimb paresis, and myokymia-like muscle contractions have not been described in humans with ACCPN so far. As this is the first report of a naturally occurring disease-causing SLC12A6 variant in a non-human species, the canine model will be highly valuable to better understand the complex molecular pathophysiology of SLC12A6-related neurological disorders and to evaluate novel treatment strategies.


Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Fenótipo , Simportadores/genética , Animais , Biomarcadores , Cães , Eletromiografia , Feminino , Estudos de Associação Genética/métodos , Testes Genéticos , Humanos , Mutação INDEL , Masculino , Condução Nervosa , Degenerações Espinocerebelares/diagnóstico , Degenerações Espinocerebelares/genética , Degenerações Espinocerebelares/metabolismo
7.
Eur J Hum Genet ; 25(2): 222-226, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27966545

RESUMO

SeSAME/EAST syndrome is a multisystemic disorder in humans, characterised by seizures, sensorineural deafness, ataxia, developmental delay and electrolyte imbalance. It is exclusively caused by homozygous or compound heterozygous variations in the KCNJ10 gene. Here we describe a similar syndrome in two families belonging to the Malinois dog breed, based on clinical, neurological, electrodiagnostic and histopathological examination. Genetic analysis detected a novel pathogenic KCNJ10 c.986T>C (p.(Leu329Pro)) variant that is inherited in an autosomal recessive way. This variant has an allele frequency of 2.9% in the Belgian Malinois population, but is not found in closely related dog breeds or in dog breeds where similar symptoms have been already described. The canine phenotype is remarkably similar to humans, including ataxia and seizures. In addition, in half of the dogs clinical and electrophysiological signs of neuromyotonia were observed. Because there is currently no cure and treatment is nonspecific and unsatisfactory, this canine translational model could be used for further elucidating the genotype/phenotype correlation of this monogenic multisystem disorder and as an excellent intermediate step for drug safety testing and efficacy evaluations before initiating human studies.


Assuntos
Perda Auditiva Neurossensorial/genética , Homozigoto , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Canais de Potássio Corretores do Fluxo de Internalização/genética , Convulsões/genética , Animais , Encéfalo/patologia , Cães , Feminino , Genes Recessivos , Perda Auditiva Neurossensorial/veterinária , Deficiência Intelectual/veterinária , Masculino , Convulsões/veterinária
8.
FASEB J ; 30(2): 863-73, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26514167

RESUMO

Neonates with intrauterine growth restriction (IUGR) show lower efficiency of nutrient utilization compared to normal birth weight (NBW) newborns. This study was conducted using neonatal piglets as a model to test the hypothesis that IUGR affects the intestinal barrier function, intestinal structure, and antioxidant system development during the suckling period. The small intestinal mucosae were obtained from IUGR and NBW littermates in the suckling period (d 0, 3, 8, and 19 postnatal). The epithelial barrier function was assessed by FITC-dextran 4 (FD4) and horseradish peroxidase (HRP) fluxes across the epithelium, histomorphologic measurements, and expression of tight-junction proteins. Redox status represented by the glutathione disulfide/glutathione ratio and malondialdehyde concentrations was determined, whereas mRNA expressions of some redox-sensitive proteins were quantified. Results showed that IUGR piglets exhibited a 2-fold higher intestinal permeability in the proximal small intestine on d 0 (P < 0.05), and this difference between IUGR and NBW piglets was widened to 3 and 4 times for FD4 and HRP, respectively (P < 0.05), on d 3. In accordance, expression of occludin was down-regulated at the transcriptional level in IUGR piglets at d 0 and 19 (P < 0.01). Furthermore, the transcription of heme oxygenase 1, catalase, and thioredoxin reductase genes was down-regulated in IUGR piglets, mainly on postnatal d 0 and 19 (P < 0.01). It appears that IUGR subjects have a lower capacity to mount an antioxidant response in the early postnatal period. Collectively, these results add to our understanding of the mechanisms responsible for intestinal dysfunction in IUGR neonates.


Assuntos
Retardo do Crescimento Fetal/metabolismo , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , RNA Mensageiro/biossíntese , Animais , Antioxidantes/metabolismo , Feminino , Retardo do Crescimento Fetal/patologia , Heme Oxigenase-1/biossíntese , Mucosa Intestinal/patologia , Oxirredução , Permeabilidade , Gravidez , Suínos , Tiorredoxina Dissulfeto Redutase/biossíntese
9.
Eur J Hum Genet ; 24(6): 852-6, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26486469

RESUMO

Alexander disease (AxD) is a fatal neurodegenerative disorder of astrocyte dysfunction in man, for which already a number of causal variants are described, mostly de novo dominant missense variants in the glial fibrillary acidic protein (GFAP). A similar disorder was already phenotypically described in animals but without the identification of causal variants. We diagnosed a Labrador retriever with a juvenile form of AxD based on clinical (tetraparesis with spastic front limbs mimicking 'swimming puppy syndrome') and pathological (the detection of GFAP containing Rosenthal fibers in astrocytes) features. In order to identify a causal variant, the coding sequences of the four detected GFAP transcript variants (orthologues from human transcript variants α, γ, δ/ɛ and κ) were sequenced. From the five detected variants, a heterozygous c.719G>A nucleotide substitution resulting in a p.Arg240His substitution was considered to be causal, because it is orthologous to the heterozygous de novo dominant c.716G>A (p.Arg239His) hotspot variant in man, proven to cause a severe phenotype. In addition, the variant was not found in 50 unrelated healthy Labrador retrievers. Because the condition in dogs is morphologically similar to man, it could be a promising animal model for further elucidating the genotype/phenotype correlation in order to treat or prevent this disease.


Assuntos
Doença de Alexander/veterinária , Proteína Glial Fibrilar Ácida/genética , Mutação de Sentido Incorreto , Doença de Alexander/genética , Doença de Alexander/patologia , Animais , Cães , Fenótipo
10.
Reprod Fertil Dev ; 28(7): 948-959, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25515369

RESUMO

Retrotransposons are transposable elements that insert extra copies of themselves throughout the genome via an RNA intermediate using a 'copy and paste' mechanism. They account for more than 44% of the bovine genome and have been reported to be functional, especially during preimplantation embryo development. In the present study, we tested whether high oxygen tension (20% O2) influences global DNA methylation analysed by immunofluorescence staining of developing bovine embryos and whether this has an effect on the expression of some selected retrotransposon families. High oxygen tension significantly increased global DNA methylation in 4-cell embryos and blastocysts. A significant expression difference was observed for ERV1-1-I_BT in female blastocysts, but no significant changes were observed for the other retrotransposon families tested. Therefore, the study indicates that global DNA methylation is not necessarily correlated with retrotransposon expression in bovine preimplantation embryos.


Assuntos
Blastocisto/fisiologia , Bovinos , Metilação de DNA , Oxigênio/fisiologia , Retroelementos , Animais , Desenvolvimento Embrionário , Feminino , Gravidez
11.
Mol Biol Rep ; 42(8): 1257-64, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25749814

RESUMO

Eukaryotic translation elongation factor 1 alpha (EEF1A) plays a key role in protein synthesis. In higher vertebrates EEF1A occurs in two isoforms, EEF1A1 and EEF1A2, encoded by distinct genes. The purpose of this study was to compare the two porcine genes as for the genomic sequence, gene organization and mRNA expression in different tissues, as well as to search for polymorphism and chromosomal assignment. Standard methods of DNA and mRNA analysis were used. We determined the complete genomic sequence of the porcine EEF1A1 and EEF1A2 genes. The two genes differ in the lengths of transcription units (3102 and 8588 bp, respectively), but have similar genomic organization and their coding sequences are highly similar (78% identity of coding sequences and 92.4% identity of amino acid sequences). Several polymorphisms in the two genes were detected. EEF1A1 and EEF1A2 were mapped to SSC1p11.1 and SSC17q23.3, respectively. mRNA of EEF1A1 was expressed in all studied tissues (the highest expression was in 44-day fetal muscle and low expression in adult liver and brain), while EEF1A2 was expressed only in skeletal-muscle, tongue, heart, diaphragm and brain tissues. EEF1A2 was not expressed in fetal muscle tissue (44 days). In this paper results are provided on genomic sequences, genomic organization, polymorphism, chromosomal assignment and spatial and temporal expressions of the porcine EEF1A1 and EEF1A2 genes. Novel polymorphisms were described in both genes. Porcine EEF1A2 was studied for the first time.


Assuntos
Fator 1 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/genética , Polimorfismo Genético , Sus scrofa/genética , Animais , Sequência de Bases , Expressão Gênica , Perfilação da Expressão Gênica , Genômica , Dados de Sequência Molecular , Especificidade de Órgãos , Análise de Sequência de DNA , Sus scrofa/metabolismo
12.
BMC Genet ; 15: 103, 2014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-25277275

RESUMO

BACKGROUND: Glycosphingolipids (GSLs) are important membrane components composed of a carbohydrate structure attached to a hydrophobic ceramide. They can serve as specific membrane receptors for microbes and microbial products, such as F4 Escherichia coli (F4 ETEC) and isolated F4 fimbriae. The aim of this study was to investigate the hypothesis that variation in genes involved in the assembly of the F4 binding carbohydrate moiety of GSLs (i.e. ARSA, B4GALT6, GAL3ST1, GALC, GBA, GLA, GLB1, GLB1L, NEU1, NEU2, UGCG, UGT8) could account for differential binding of F4 ETEC and their fimbriae. RESULTS: RT-PCR could not reveal any differential expression of the 12 genes in the jejunum of F4 receptor-positive (F4R(+)) and F4 receptor-negative (F4R(-)) pigs. Sequencing the complete open reading frame of the 11 expressed genes (NEU2 was not expressed) identified 72 mutations. Although some of them might have a structural effect, none of them could be associated with a F4R phenotype. CONCLUSION: We conclude that no regulatory or structural variation in any of the investigated genes is responsible for the genetic susceptibility of pigs towards F4 ETEC.


Assuntos
Escherichia coli/fisiologia , Fímbrias Bacterianas/metabolismo , Glicoesfingolipídeos/biossíntese , Sus scrofa/genética , Animais , Aderência Bacteriana , Glicosilação , Glicosiltransferases/genética , Microvilosidades/microbiologia
13.
PLoS One ; 9(8): e105013, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25137053

RESUMO

F4 enterotoxigenic Escherichia coli (F4 ETEC) are an important cause of diarrhea in neonatal and newly-weaned pigs. Based on the predicted differential O-glycosylation patterns of the 2 MUC13 variants (MUC13A and MUC13B) in F4ac ETEC susceptible and F4ac ETEC resistant pigs, the MUC13 gene was recently proposed as the causal gene for F4ac ETEC susceptibility. Because the absence of MUC13 on Western blot from brush border membrane vesicles of F4ab/acR+ pigs and the absence of F4ac attachment to immunoprecipitated MUC13 could not support this hypothesis, a new GWAS study was performed using 52 non-adhesive and 68 strong adhesive pigs for F4ab/ac ETEC originating from 5 Belgian farms. A refined candidate region (chr13: 144,810,100-144,993,222) for F4ab/ac ETEC susceptibility was identified with MUC13 adjacent to the distal part of the region. This candidate region lacks annotated genes and contains a sequence gap based on the sequence of the porcine GenomeBuild 10.2. We hypothesize that a porcine orphan gene or trans-acting element present in the identified candidate region has an effect on the glycosylation of F4 binding proteins and therefore determines the F4ab/ac ETEC susceptibility in pigs.


Assuntos
Escherichia coli Enterotoxigênica/fisiologia , Infecções por Escherichia coli/veterinária , Mucinas/genética , Doenças dos Suínos/genética , Animais , Aderência Bacteriana , Infecções por Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Estudos de Associação Genética , Predisposição Genética para Doença , Ligação Proteica , Sus scrofa/genética , Sus scrofa/microbiologia , Suínos/genética , Suínos/microbiologia , Doenças dos Suínos/microbiologia
14.
BMC Vet Res ; 9: 240, 2013 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-24295090

RESUMO

BACKGROUND: In humans, adipose tissue (AT) originating from different depots shows varying gene expression profiles. In horses, the risk of certain metabolic disorders may also be influenced by the impact of specific AT depots. Macrophage infiltration in human and rat AT is considered to be a source of inflammatory changes. In horses, this relationship has not been extensively studied yet. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a useful method to evaluate differences in mRNA expression across different tissues, can be used to evaluate differences between equine AT depots. For a correct interpretation of the RT-qPCR results, expression data have to be normalized by the use of validated reference genes. The main objectives of this study were to compare mRNA expression of inflammation-related genes, as well as adipocyte morphology and number between different equine AT depots; and in addition, to investigate the presence of antigen presenting cells in equine AT and any potential relationship with adipokine mRNA expression. RESULTS: In this study, the mRNA expression of inflammation-related genes (leptin, chemokine ligand 5, interleukin 1ß, interleukin 6, interleukin 10, adiponectin, matrix metalloproteinase 2, and superoxide dismutase 2) and candidate reference gene stability was investigated in 8 different AT depots collected from the nuchal, abdominal (mesenteric, retroperitoneal, and peri-renal) and subcutaneous (tail head and loin) AT region. By using GeNorm analysis, HPRT1, RPL32, and GAPDH were found to be the most stable genes in equine AT. The mRNA expression of leptin, chemokine ligand 5, interleukin 10, interleukin 1ß, adiponectin, and matrix metalloproteinase 2 significantly differed across AT depots (P < 0.05). No significant AT depot effect was found for interleukin 6 and superoxide dismutase 2 (P > 0.05). Adipocyte area and number of antigen presenting cells per adipocyte significantly differed between AT depots (P < 0.05). CONCLUSIONS: Adipose tissue location was associated with differences in mRNA expression of inflammation-related genes. This depot-specific difference in mRNA expression suggests that the overall inflammatory status of horses could be partially determined by the relative proportion of the different AT depots.


Assuntos
Tecido Adiposo/metabolismo , Doenças dos Cavalos/genética , Inflamação/veterinária , Adipócitos/metabolismo , Animais , Regulação da Expressão Gênica , Doenças dos Cavalos/metabolismo , Cavalos/genética , Cavalos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Transcriptoma/genética
15.
J Invest Dermatol ; 133(1): 201-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22895360

RESUMO

The current treatments for hyperpigmentation are often associated with a lack of efficacy and adverse side effects. We hypothesized that microRNA (miRNA)-based treatments may offer an attractive alternative by specifically targeting key genes in melanogenesis. The aim of this study was to identify miRNAs interfering with the pigmentary process and to assess their functional role. miRNA profiling was performed on mouse melanocytes after three consecutive treatments involving forskolin and solar-simulated UV (ssUV) irradiation. Sixteen miRNAs were identified as differentially expressed in treated melan-a cells versus untreated cells. Remarkably, a 15-fold downregulation of miR-145 was detected. Overexpression or downregulation of miR-145 in melan-a cells revealed reduced or increased expression of Sox9, Mitf, Tyr, Trp1, Myo5a, Rab27a, and Fscn1, respectively. Moreover, a luciferase reporter assay demonstrated direct targeting of Myo5a by miR-145 in mouse and human melanocytes. Immunofluorescence tagging of melanosomes in miR-145-transfected human melanocytes displayed perinuclear accumulation of melanosomes with additional hypopigmentation of harvested cell pellets. In conclusion, this study has established an miRNA signature associated with forskolin and ssUV treatment. The significant down- or upregulation of major pigmentation genes, after modulating miR-145 expression, suggests a key role for miR-145 in regulating melanogenesis.


Assuntos
MicroRNAs/biossíntese , Pigmentação/fisiologia , Animais , Células Cultivadas , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Antígeno MART-1/análise , Antígeno MART-1/genética , Antígeno MART-1/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Melanossomas/genética , Melanossomas/metabolismo , Camundongos , MicroRNAs/genética , Fator de Transcrição Associado à Microftalmia/biossíntese , Monofenol Mono-Oxigenase/biossíntese , Cadeias Pesadas de Miosina/biossíntese , Miosina Tipo V/biossíntese , Pigmentação/genética , Fatores de Transcrição SOX9/biossíntese , Transfecção , Tripsina/biossíntese , Raios Ultravioleta , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab27 de Ligação ao GTP
16.
Reprod Fertil Dev ; 24(5): 691-703, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22697119

RESUMO

During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions.


Assuntos
Biomarcadores/análise , Blastocisto/metabolismo , Gatos/genética , Diferenciação Celular/genética , Oócitos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Biomarcadores/metabolismo , Gatos/embriologia , Gatos/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica no Desenvolvimento , Genes Controladores do Desenvolvimento , Gravidez , Padrões de Referência
17.
Neuromuscul Disord ; 22(6): 558-65, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22342001

RESUMO

KCNA1, KCNA2, KCNA6 and KCNQ2 are associated with peripheral nerve hyperexcitability in humans. In order to determine if these genes are also involved in Jack Russell Terriers with a similar syndrome characterized by myokymia and neuromyotonia, their predicted canine orthologs were first validated experimentally. They were found either incompletely or even incorrectly annotated, mainly due to gaps in the canine genomic sequence and insufficient transcript data. Canine KCNQ2 was found to contain 20 coding exons, of which three are not described in humans. It encodes for at least 14 different transcript variants in the frontal cortex of a single dog, of which only four are also described in humans. Mutation detection in Jack Russell Terriers diagnosed with peripheral nerve hyperexcitability revealed no pathogenetic relevant structural mutations. However, the four missense sequence variations and the 14 transcript variants of KCNQ2 will contribute to the study of the functional diversity of voltage-gated potassium channels.


Assuntos
Doenças do Cão/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.6/genética , Doenças do Sistema Nervoso Periférico/veterinária , Animais , Cães , Estudos de Associação Genética , Síndrome de Isaacs/genética , Síndrome de Isaacs/veterinária , Mutação , Mioquimia/genética , Mioquimia/veterinária , Doenças do Sistema Nervoso Periférico/genética
19.
Reproduction ; 143(2): 173-81, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22089531

RESUMO

The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.


Assuntos
Embrião de Mamíferos/citologia , Fertilização in vitro , Cavalos/embriologia , Útero/fisiologia , Animais , Células Cultivadas , Microambiente Celular/fisiologia , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/fisiologia , Técnicas de Cultura Embrionária/métodos , Transferência Embrionária/veterinária , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização in vitro/veterinária , Cavalos/fisiologia , Lipocalinas/farmacologia , Gravidez , Proteínas Recombinantes/farmacologia
20.
Vet Res ; 42: 15, 2011 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-21314974

RESUMO

The objective of the current study was to investigate (i) the outcome of experimentally induced Escherichia coli mastitis in primiparous cows during early lactation in relation with production of eicosanoids and inflammatory indicators, and (ii) the validity of thermography to evaluate temperature changes on udder skin surface after experimentally induced E. coli mastitis. Nine primiparous Holstein Friesian cows were inoculated 24 ± 6 days (d) after parturition in both left quarters with E. coli P4 serotype O32:H37. Blood and milk samples were collected before and after challenge with E. coli. The infrared images were taken from the caudal view of the udder following challenge with E. coli. No relationship was detected between severity of mastitis and changes of thromboxane B2 (TXB2), leukotriene B4 (LTB4) and lipoxin A4 (LXA4). However, prostaglandin E2 (PGE2) was related to systemic disease severity during E. coli mastitis. Moreover, reduced somatic cell count (SCC), fewer circulating basophils, increased concentration of tumor necrosis factor-α (TNF-α) and higher milk sodium and lower milk potassium concentrations were related to systemic disease severity. The thermal camera was capable of detecting 2-3 °C temperature changes on udder skin surface of cows inoculated with E. coli. Peak of udder skin temperature occurred after peak of rectal temperature and appearance of local signs of induced E. coli mastitis. Although infrared thermography was a successful method for detecting the changes in udder skin surface temperature following intramammary challenge with E. coli, it did not show to be a promising tool for early detection of mastitis.


Assuntos
Doenças dos Bovinos/imunologia , Citocinas/metabolismo , Eicosanoides/metabolismo , Infecções por Escherichia coli/veterinária , Escherichia coli/fisiologia , Glândulas Mamárias Animais/imunologia , Termografia/métodos , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Citocinas/sangue , Eicosanoides/sangue , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Lactação , Glândulas Mamárias Animais/microbiologia , Leite/química , Leite/microbiologia , Paridade , Temperatura
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