Investigation of Egg Weight, Ovarian Follicles Morphology and Growth Differentiation Factor 9 mRNA Expression in Potchefstroom Koekoek Chicken Breed

The growth differentiation factor 9 (GDF9) gene plays a vital role in the growth and maturation of ovarian follicles in laying hens. However, its messenger ribonucleic acid (mRNA) expression levels in preovulatory ovarian follicles of indigenous chickens remain poorly understood. The study aimed to identify the association between egg weight and egg quality traits, ovarian follicles morphology, and mRNA expression levels of the GDF9 gene in pre-ovulatory ovarian follicles of the South African Potchefstroom Koekoek chicken breed. The correlation results showed that egg weight (EW) had a positively high significant correlation (p<0.01) with egg width (EWD), yolk weight (YW), shell surface area (SSA), albumen weight (AW), albumen ratio (AR) and egg volume (EV), and a positive significant correlation (p<0.05) with egg length (EL). The Student's T-test results revealed that the numbers of large yellow follicles were significantly lower (p<0.05) than those of small yellow follicles. ANOVA findings showed that there was a significant difference (p<0.05) in the average weight of the large yellow follicles. The quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) findings indicated that there were significant differences (p<0.05) in the mRNA expression levels of the GDF9 gene in preovulatory ovarian follicles of the Potchefstroom Koekoek chicken breed. The mRNA expression was more abundant in F1 and F4 than in other ovarian follicles.(AU)

Comparative study on the distribution and expression of Neuroglobin and Hypoxia-inducible factor-1α in the telencephalon of yak and cattle / Estudo comparativo da distribuição e expressão de fator-1 indutível por neuroglobina e hipóxia no telencéfalo de iaques e bovinos

Abstract The telencephalon refers to the most highly developed and anterior part of the forebrain, consisting mainly of the cerebral hemispheres. The study determined Neuroglobin (Ngb) and Hypoxia-inducible factor (HIF-1α) expression in the telencephalon of yak and cattle, and compare the expression and distribution pattern of Ngb and HIF-1α in the two animals. Immunohistochemistry (IHC), quantitative real-time Polymerase Chain Reaction (qRT-PCR), and Western blot (WB) were employed to investigate Ngb and Hif-1α expression in the telencephalon of yak and cattle. mRNA and protein expressions of Ngb and HIF-1α showed positive in different tissues of the yak and cattle telencephalon. Ngb expression in tissues of the yak recorded higher as compare to cattle while HIF-1α expression was found higher in cattle than yak. The HIF-1α expression in some tissues of yak telencephalon was consistent with the cattle. The results documented that HIF-1α may have a direct or indirect synergistic effect on Ngb expression in the yak telencephalon to improve hypoxia adaptation. It is suggested that yak may need more Ngb expression for adaptation, but the expression of HIF-1α seems to be down-regulated during long-term adaptation, and the specific causes of this phenomenon needs to be further verified.

Resumo O telencéfalo refere-se à parte anterior e mais desenvolvida do prosencéfalo, consistindo principalmente dos hemisférios cerebrais. O estudo determinou a expressão de neuroglobina (Ngb) e fator indutível por hipóxia (HIF-1α) no telencéfalo de iaques e bovinos e comparou a expressão e o padrão de distribuição de Ngb e HIF-1α nos dois animais. Imuno-histoquímica (IHC), reação em cadeia da polimerase quantitativa em tempo real (qRT-PCR) e Western blot (WB) foram empregados para investigar a expressão de Ngb e Hif-1α no telencéfalo de iaques e bovinos. As expressões de mRNA e proteínas de Ngb e HIF-1α mostraram-se positivas em diferentes tecidos do telencéfalo de iaque e bovino. A expressão de Ngb nos tecidos do iaque foi registrada mais alta em comparação com o gado, enquanto a expressão do HIF-1α foi encontrada mais alta no gado do que no iaque. A expressão de HIF-1α em alguns tecidos do telencéfalo de iaque foi consistente com o gado. Os resultados documentaram que o HIF-1α pode ter um efeito sinérgico direto ou indireto na expressão de Ngb no telencéfalo de iaque para melhorar a adaptação à hipóxia. É sugerido que o iaque pode precisar de mais expressão de Ngb para adaptação, mas a expressão de HIF-1α parece ser regulada para baixo durante a adaptação de longo prazo, e as causas específicas desse fenômeno precisam ser verificadas.

Comparative study of the distribution and expression of Neuroglobin and Hypoxia-inducible factor-1α in the adult and young Yak Brain / Estudo comparativo da distribuição e expressão de neuroglobina e fator-1α indutível por hipóxia em adultos e jovens iaques (Yak Brain)

Abstract Background The brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. Aim The study examined the expression of Neuroglobin (Ngb) and Hypoxia-inducible factor-1α (Hif-1α) in adult and young yak brain tissues, and provided researchers with meaningful insight into the anatomy, physiology, and biochemistry of this mammal. Method The study employed immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR), and Western blot (WB) to obtain the results. Results Ngb and Hif-1α were significantly (P<0.05) expressed in the cerebellar cortex, piriform lobe, medulla, and corpus callosum of the adult yak while in the young yak brain tissues, the protein expressions were significantly found in the white matter of the cerebellum, pineal gland, corpus callosum, and cerebellar cortex. The Ngb and Hif-1α expression showed similarities and differences. This may have resulted from similar animal species, source of nutrition, age factors, brain size, emotional activities, and communication. The findings documented that Ngb and Hif-1α are commonly expressed in various adult and young yak brain tissues. Multiple roles in the brain tissues of the adult and young yaks are involved in the expression and distribution and are proposed to play a significant role in the adaptation of the yak to the high altitude environment. Conclusion This study provides meaningful data to understand the adaptive mechanism to hypoxia and recommended researchers to expand on the adaptive mechanism and brain tissues that are not recorded.

Resumo Contexto O cérebro é um órgão que funciona como o centro do sistema nervoso em todos os animais vertebrados e na maioria dos invertebrados. Objetivo O estudo examinou a expressão de neuroglobina (Ngb) e fator-1α indutível por hipóxia (Hif-1α) em tecidos cerebrais de iaques adultos e jovens e forneceu aos pesquisadores uma visão significativa da anatomia, fisiologia e bioquímica desse mamífero. Método O estudo utilizou imuno-histoquímica (IHC), PCR quantitativo em tempo real (qRT-PCR) e western blot (WB) para a obtenção dos resultados. Resultados Ngb e Hif-1α foram significativamente (P < 0,05) expressos no córtex cerebelar, lobo piriforme, medula e corpo caloso do iaque adulto, enquanto nos tecidos cerebrais do iaque jovem as expressões proteicas foram encontradas significativamente na substância branca do cerebelo, glândula pineal, corpo caloso e córtex cerebelar. A expressão de Ngb e Hif-1α apresentou semelhanças e diferenças. Isso pode ter resultado de espécies animais semelhantes, fonte de nutrição, fatores de idade, tamanho do cérebro, atividades emocionais e comunicação. Os resultados documentaram que o Ngb e o Hif-1α são comumente expressos em vários tecidos cerebrais de iaques adultos e jovens. Múltiplos papéis nos tecidos cerebrais de iaques adultos e jovens estão envolvidos na expressão e distribuição e são propostos para desempenhar um papel significativo na adaptação do iaque ao ambiente de alta altitude. Conclusão Este estudo fornece dados significativos para compreender o mecanismo adaptativo à hipóxia e recomendou que os pesquisadores expandissem o mecanismo adaptativo e os tecidos cerebrais que não foram registrados.

Expression analysis of m6A-related genes in various tissues of Meishan pigs at different developmental stages

To characterize the N6-methyladenosine (m6A)-related gene expression profiles in various tissues of Meishan pigs at different stages, m6A modification-related genes (METTL3, METTL14, METTL16, WTAP, RBM15, and FTO) were detected from newborn to physical maturity of Meishan pigs at eight important developmental stages (1, 7, 14, 21, 28, 35, 134, and 158 days old). The expression of m6A-related genes was tissue-specific. Furthermore, the level of METTL3 messenger RNA (mRNA) was higher on day 35 than in other stages in most tissues, and the expression of METTL14 increased after day 35, and FTO exhibited a peak on day 14 in muscle, intestine, lymph nodes, thymus, and kidney. This study provided a reference for an in-depth study of the expression patterns of m6A modification-related genes in Meishan pigs.

Molecular characterization and association of lactoferrin gene to subclinical mastitis in goats (Capra hircus)

The study characterized the lactoferrin (Lf) mRNA gene in different goat breeds in the Philippines and determined its association with subclinical mastitis (SCM). The study involved collection of milk at second week of lactation (n=75) and blood samples (n=5) to obtain extracted RNA and using cDNA to amplify Lf gene through polymerase chain reaction. The nucleotide and amino acid sequences were determined and used as reference in the evaluation of phylogenetic relationship. Amplified products were utilized for RFLP analysis before determining the association of the gene with SCM. Results of the study demonstrated that Lf gene in goats registered a molecular weight of 2135. Nucleotide and amino acid sequence of Lf gene revealed high similarity (99%) in Saanen, Anglo-Nubian and Philippine native goats with that of Capra hircus (U53857) Lf gene submitted to GenBank. Phylogenetic studies showed that Lf gene of Anglo-Nubian, Saanen and Native goats clade together with Lf gene of C. hircus (U53857). Three genotypes in goats were documented using the restriction enzymes AluI and HaeIII. Based on the Statistical analysis, association (comp 5.65, p = 0.0308) has been established between the Lf genes of goats with genotype BB to SCM using HaeIII restriction enzyme.(AU)

Effects of Dietary Fiber on Growth Performance, Fat Deposition, Fat Metabolism, and Expression of Lipoprotein Lipase Mrna in Two Breeds of Geese

ABSTRACT The present study was conducted to investigate the effects of dietary fiber on growth performance, fat deposition, serum lipids, fat metabolism, and mRNA (messenger RNA) expression of lipoprotein lipase (LPL) in Jilin white and Carlos geese. Sixty Jilin white and sixty Carlos geese aged six-weeks and of similar health and weight (average weight 313.11g) were selected. Geese of each breed were randomly divided into two groups (n=30), and with each group containing three replicate subgroups of 10 geese. The diet was supplemented with 8% or 11% fiber (corn straw powder). The Jilin white geese are divided into A1 (8%) and A2 (11%) groups, and Carlos geese are divided into B1 (8%) and B2 (11%) groups. The experiment lasted 35 days. The results showed that high dietary fiber can significantly (p 0.05) increase average daily feed intake (ADFI), significantly (p 0.05) reduce final weight (FW) and average daily gain (ADG) of both varieties, and increase LPL mRNA expression levels in abdominal fat, liver, sebum, and urethral glands. High dietary fiber accelerates intestinal peristalsis, affects the absorption of other nutrients, reduces the available energy value of the absorbed feed, and increases fat loss. Compared with the to Carlos geese, high dietary fiber content had a more significant effect on the live, slaughter, and sebum weights and sebum percentage of the Jilin white geese, indicating that the Carlos geese have higher requirements for dietary fiber content. High fiber content will reduce the growth performance, slaughter performance, and fat deposition of geese.

Evaluation and Validation of the Six Housekeeping Genes for Normalizing Mrna Expression in the Ovarian Follicles and Several Tissues in Chicken

Expression of housekeeping genes is relatively constant in different tissues and cells by RT-qPCR analysis. Housekeeping genes (HGs) are usually utilized as the reference to evaluate and compare mRNA expression abundances of target genes in different cells or tissues sampled. However, the expression stabilities of different HGs in diverse samples may appear divergence. Currently, there is no exact reference data of HGs in hen ovarian follicular tissues during egg-laying period available yet. In this study, we detected the expression of 18SrRNA, ACTB, HOXC8, GAPDH, alpha-A, and alpha-D mRNA in the varied-size ovarian follicles (1-8 mm in diameter and F5), hearts, livers, spleens, lungs, and breast muscles of the laying hens by RT-qPCR, to analyze the results via Ct value, geNorm, Normfinder, and Bestkeeper. The data showed that the expression levels of 18SrRNA, alpha-A, and alpha-D transcripts were more significantly stable than the other three genes for normalizing mRNA expression in the hen ovarian follicles examination. Moreover, alpha-D, 18SrRNA, and alpha-A were also most suitable for the expression normalization in the tissues of the heart, liver, spleen, lung and breast muscle. In contrast, 18SrRNA has the most stable mRNA expression levels in all tissues sampled, so it can serve as an excellent inner control for the evaluation of the transcription levels in chickens. It is a remarkable fact that HOXC8 as a candidate reference should be avoided. Our study establishes a set of stably expressed candidate inner references in the hen ovarian follicles and several tissues, it firstly provided an exact data for validation of the inner references in normalizing transcription levels of a target gene in chickens.(AU)

Evaluation and Validation of the Six Housekeeping Genes for Normalizing Mrna Expression in the Ovarian Follicles and Several Tissues in Chicken

Expression of housekeeping genes is relatively constant in different tissues and cells by RT-qPCR analysis. Housekeeping genes (HGs) are usually utilized as the reference to evaluate and compare mRNA expression abundances of target genes in different cells or tissues sampled. However, the expression stabilities of different HGs in diverse samples may appear divergence. Currently, there is no exact reference data of HGs in hen ovarian follicular tissues during egg-laying period available yet. In this study, we detected the expression of 18SrRNA, ACTB, HOXC8, GAPDH, alpha-A, and alpha-D mRNA in the varied-size ovarian follicles (1-8 mm in diameter and F5), hearts, livers, spleens, lungs, and breast muscles of the laying hens by RT-qPCR, to analyze the results via Ct value, geNorm, Normfinder, and Bestkeeper. The data showed that the expression levels of 18SrRNA, alpha-A, and alpha-D transcripts were more significantly stable than the other three genes for normalizing mRNA expression in the hen ovarian follicles examination. Moreover, alpha-D, 18SrRNA, and alpha-A were also most suitable for the expression normalization in the tissues of the heart, liver, spleen, lung and breast muscle. In contrast, 18SrRNA has the most stable mRNA expression levels in all tissues sampled, so it can serve as an excellent inner control for the evaluation of the transcription levels in chickens. It is a remarkable fact that HOXC8 as a candidate reference should be avoided. Our study establishes a set of stably expressed candidate inner references in the hen ovarian follicles and several tissues, it firstly provided an exact data for validation of the inner references in normalizing transcription levels of a target gene in chickens.

HMGB1 and inflammatory cytokines in experimental acute lung injury induced in rabbits / [HMGB1 e citocinas inflamatórias em lesão pulmonar aguda experimental induzida em coelhos]

The aim of this work was to measure HMGB1, TNF-alpha, and IL-8 in bronchoalveolar lavage (BAL), serum and TLR2 and TLR4mRNA expression in lung tissue of rabbits with two grades of acute lung injury (ALI). The animals were randomly assigned to groups with severe (S) and mild/moderate (MM) ALI, induced with warm saline, and a control group. HMGB1, TNF-alpha, IL-8, TLR2mRNA and TLR4mRNA were measured after ALI induction. The results showed increased levels of IL-8, TNF-alpha, HMGB1 and TLR4mRNA in the ALI groups. HMGB1, IL-8 and TNF-alpha concentrations in BAL were higher in S compared MM. Increased TLR4mRNA was observed in S and MM versus control. The results suggest an early participation of HMGB1 in ALI together with IL-8 and TNF-alpha and association with severity. TLR4 has early expression and role in ALI pathophysiology but is not associated with severity.(AU)

O objetivo deste trabalho é determinar os níveis de HMGB1, TNF-alfa e IL-8 no lavado broncoalveolar (BAL), bem como quantificar a expressão sérica de TLR2 e TLR4 mRNA em tecido pulmonar de coelhos com dois graus de lesão pulmonar aguda (LPA). Os animais foram distribuídos aleatoriamente em grupos com LPA grave (S) e leve / moderada (MM), induzidas com solução salina morna, e um grupo controle. HMGB1, TNF-alfa, IL-8, TLR2mRNA e TLR4mRNA foram medidos após a indução de LPA e quatro horas de ventilação mecânica. Os resultados mostraram níveis aumentados de IL-8, TNF-alfa, HMGB1 e TLR4mRNA nos grupos com LPA. As concentrações de HMGB1, IL-8 e TNF-alfa no LBA foram maiores no S comparado ao MM. Aumento de TLR4mRNA foi observado em S e MM versus controle. Os resultados sugerem uma participação precoce da HMGB1 na LPA em conjunto com IL-8 e TNF-alfa e associação com a gravidade da LPA. O TLR4 foi expresso na ALI e possivelmente possui papel precoce na fisiopatologia da LPA, mas sem associação com a gravidade.(AU)

HMGB1 and inflammatory cytokines in experimental acute lung injury induced in rabbits / [HMGB1 e citocinas inflamatórias em lesão pulmonar aguda experimental induzida em coelhos]

The aim of this work was to measure HMGB1, TNF-alpha, and IL-8 in bronchoalveolar lavage (BAL), serum and TLR2 and TLR4mRNA expression in lung tissue of rabbits with two grades of acute lung injury (ALI). The animals were randomly assigned to groups with severe (S) and mild/moderate (MM) ALI, induced with warm saline, and a control group. HMGB1, TNF-alpha, IL-8, TLR2mRNA and TLR4mRNA were measured after ALI induction. The results showed increased levels of IL-8, TNF-alpha, HMGB1 and TLR4mRNA in the ALI groups. HMGB1, IL-8 and TNF-alpha concentrations in BAL were higher in S compared MM. Increased TLR4mRNA was observed in S and MM versus control. The results suggest an early participation of HMGB1 in ALI together with IL-8 and TNF-alpha and association with severity. TLR4 has early expression and role in ALI pathophysiology but is not associated with severity.(AU)

O objetivo deste trabalho é determinar os níveis de HMGB1, TNF-alfa e IL-8 no lavado broncoalveolar (BAL), bem como quantificar a expressão sérica de TLR2 e TLR4 mRNA em tecido pulmonar de coelhos com dois graus de lesão pulmonar aguda (LPA). Os animais foram distribuídos aleatoriamente em grupos com LPA grave (S) e leve / moderada (MM), induzidas com solução salina morna, e um grupo controle. HMGB1, TNF-alfa, IL-8, TLR2mRNA e TLR4mRNA foram medidos após a indução de LPA e quatro horas de ventilação mecânica. Os resultados mostraram níveis aumentados de IL-8, TNF-alfa, HMGB1 e TLR4mRNA nos grupos com LPA. As concentrações de HMGB1, IL-8 e TNF-alfa no LBA foram maiores no S comparado ao MM. Aumento de TLR4mRNA foi observado em S e MM versus controle. Os resultados sugerem uma participação precoce da HMGB1 na LPA em conjunto com IL-8 e TNF-alfa e associação com a gravidade da LPA. O TLR4 foi expresso na ALI e possivelmente possui papel precoce na fisiopatologia da LPA, mas sem associação com a gravidade.(AU)

Expression analysis of long non-coding RNAs in a renal ischemia-reperfusion injury model

Purpose: To investigate the long non-coding RNAs (lncRNAs) profile on renal ischemia reperfusion in a mouse model. Methods: Microarray analysis was used to study the expression of misregulated lncRNA in a mouse model of renal ischemia reperfusion (I/R) with long ischemia time. Quantitative real-time PCR (qPCR) was used to verify the expression of selected lncRNAs and mRNAs. The potential functions of the lncRNA was analyzed by bioinformatics tools and databases. Results: Kidney function was impaired in I/R group compared to the normal group. Analysis showed that a total of 2267 lncRNAs and 2341 messenger RNAs (mRNAs) were significantly expressed in I/R group (≥2.0-fold, p < 0.05). The qPCR result showed that lncRNAs and mRNAs expression were consistent with the microarray analysis. The co-expression network profile analysis based on five validated lncRNAs and 203 interacted mRNAs showed it existed a total of 208 nodes and 333 connections. The GO and KEEG pathway analysis results showed that multiple lncRNAs are involved the mechanism of I/R. Conclusion: Multiple lncRNAs are involved in the mechanism of I/R. These analysis results will help us to further understand the mechanism of I/R and promote the new methods targeted at lncRNA to improve I/R injury.(AU)

O papel das nucleases no laboratório de biologia molecular: vilãs ou aliadas? / The role of nucleases in the molecular biology laboratory: villains or allies?

Em laboratório de biologia molecular existem normas para prevenir que nucleases destruam os ácidos nucleicos em análise. Rígida adesão a estas normas é primordial, principalmente em laboratórios de análises clínicas e ao se lidar com amostras com número restrito de cópias do genoma-alvo. Em contraposição, diversas nucleases têm tido importância fundamental, por exemplo, na identificação do ácido nucleico de vírus, investigação de RNA mensageiro, purificação de vírus em abordagem metagenômica, edição de genomas com o sistema CRISPR/Cas e descoberta de enzimas. O conhecimento de como nucleases podem ser tanto vilãs quanto aliadas é essencial na formação de todos que trabalham no campo de biologia molecular.

In a molecular biology laboratory there are standards to prevent nucleases from destroying the nucleic acids under analysis. Strict adherence to these standards is paramount, mainly in clinical analysis laboratories and when dealing with samples with a limited number of copies of the target genome. In contrast, several nucleases have been of fundamental importance, for example, in the identification of the type of viral nucleic acid, investigation of messenger RNA, virus purification in metagenomic approach, genome editing with the CRISPR/Cas system, and enzyme discovery. Knowledge of how nucleases can be both villains and allies is essential in the training of all working in the field of molecular biology.

O papel das nucleases no laboratório de biologia molecular: vilãs ou aliadas? / The role of nucleases in the molecular biology laboratory: villains or allies?

Em laboratório de biologia molecular existem normas para prevenir que nucleases destruam os ácidos nucleicos em análise. Rígida adesão a estas normas é primordial, principalmente em laboratórios de análises clínicas e ao se lidar com amostras com número restrito de cópias do genoma-alvo. Em contraposição, diversas nucleases têm tido importância fundamental, por exemplo, na identificação do ácido nucleico de vírus, investigação de RNA mensageiro, purificação de vírus em abordagem metagenômica, edição de genomas com o sistema CRISPR/Cas e descoberta de enzimas. O conhecimento de como nucleases podem ser tanto vilãs quanto aliadas é essencial na formação de todos que trabalham no campo de biologia molecular.(AU)

In a molecular biology laboratory there are standards to prevent nucleases from destroying the nucleic acids under analysis. Strict adherence to these standards is paramount, mainly in clinical analysis laboratories and when dealing with samples with a limited number of copies of the target genome. In contrast, several nucleases have been of fundamental importance, for example, in the identification of the type of viral nucleic acid, investigation of messenger RNA, virus purification in metagenomic approach, genome editing with the CRISPR/Cas system, and enzyme discovery. Knowledge of how nucleases can be both villains and allies is essential in the training of all working in the field of molecular biology.(AU)

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA

The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.(AU)

Expressão do mRNA para IGF-2 em oócitos e células do cumulus extraídos de folículos antrais e pré-antrais de ovelhas nativas do Estado de Pernambuco / IGF-2 mRNA expression in oocytes and cumulus cells of antral and preantral native sheep follicles in Pernambuco State, Brazil

Objetivou-se avaliar a expressão do mRNA para o gene do fator de crescimento IGF-2 em oócitos e células do cumulus de ovelhas em diferentes estágios do desenvolvimento folicular. Os folículos classificados morfologicamente como antrais (terciários e pré-ovulatórios) foram aspirados manualmente para obtenção dos oócitos e células do cumulus. Os folículos pré-antrais (secundários) foram extraídos do córtex ovariano, por microdissecção, e os oócitos retirados. Nos dois grupos, os oócitos foram desnudados e agrupados em pools de dez células cada (Grupo A, n=10; Grupo B, n=10) e dez amostras com grupos de células do cumulus (Grupo A1, n=10, B1, n=10). O mRNA foi extraído e convertido em cDNA utilizando a técnica da RT-PCR, utilizando Oligo DT randômico para o mRNA. A análise da expressão confirmou a expressão gênica para IGF-2 nos grupos de oócitos e células do cumulus. Houve um aumento da expressão relativa do mRNA para IGF-2 nos grupos de oócitos durante a fase mais tardia do desenvolvimento folicular e as diferenças foram consideradas significantes (p<0,05). Não houve variação significante da expressão de IGF2 entre os grupos de células do cumulus. Conclui-se que o fator de crescimento IGF-2 tem níveis mais elevados de expressão em oócitos ovinos, na segunda fase do desenvolvimento folicular, mas expressão semelhante em células do cumulus durante as fases estudadas do desenvolvimento folicular.(AU)

The aim of this study was to analyze the mRNA expression of IGF-2 growth factor in oocytes and cumulus cells from native sheep follicles at different stages of follicular development. The classified morphologically as antral follicles (tertiary preovulatory) were aspirated manually to obtain the oocyte and the cumulus cells. The preantral follicles (secondary) were extracted from the ovarian cortex by microdissection, and oocytes were removed. In both groups, oocytes were denuded and grouped into pools of ten cells each (Group A, n=10, Group B, n=10) and ten samples with groups of cumulus cells (Group A1, n=10; B1, n=10). The mRNA was extracted and converted to cDNA using the RT-PCR technique. The expression analysis confirmed the expression of IGF-2 gene for groups of oocyte and the cumulus cells. There was an increase in the relative expression of mRNA for IGF-2 for groups of oocytes during the later stage of follicular development and differences were considered significant (p<0.05). There was no significant variation in the expression of IGF2 between groups of cumulus cells. It is concluded that the growth factor IGF-2 has higher levels of expression in sheep oocytes in the second stage of follicular development in the conditions adopted and similar expression in cumulus cells during various stages of follicular development.(AU)

Expressão do mRNA para IGF-2 em oócitos e células do cumulus extraídos de folículos antrais e pré-antrais de ovelhas nativas do Estado de Pernambuco / IGF-2 mRNA expression in oocytes and cumulus cells of antral and preantral native sheep follicles in Pernambuco State, Brazil

Objetivou-se avaliar a expressão do mRNA para o gene do fator de crescimento IGF-2 em oócitos e células do cumulus de ovelhas em diferentes estágios do desenvolvimento folicular. Os folículos classificados morfologicamente como antrais (terciários e pré-ovulatórios) foram aspirados manualmente para obtenção dos oócitos e células do cumulus. Os folículos pré-antrais (secundários) foram extraídos do córtex ovariano, por microdissecção, e os oócitos retirados. Nos dois grupos, os oócitos foram desnudados e agrupados em "pools" de dez células cada (Grupo A, n=10; Grupo B, n=10) e dez amostras com grupos de células do cumulus (Grupo A1, n=10, B1, n=10). O mRNA foi extraído e convertido em cDNA utilizando a técnica da RT-PCR, utilizando Oligo DT randômico para o mRNA. A análise da expressão confirmou a expressão gênica para IGF-2 nos grupos de oócitos e células do cumulus. Houve um aumento da expressão relativa do mRNA para IGF-2 nos grupos de oócitos durante a fase mais tardia do desenvolvimento folicular e as diferenças foram consideradas significantes (p<0,05). Não houve variação significante da expressão de IGF2 entre os grupos de células do cumulus. Conclui-se que o fator de crescimento IGF-2 tem níveis mais elevados de expressão em oócitos ovinos, na segunda fase do desenvolvimento folicular, mas expressão semelhante em células do cumulus durante as fases estudadas do desenvolvimento folicular.(AU)

The aim of this study was to analyze the mRNA expression of IGF-2 growth factor in oocytes and cumulus cells from native sheep follicles at different stages of follicular development. The classified morphologically as antral follicles (tertiary preovulatory) were aspirated manually to obtain the oocyte and the cumulus cells. The preantral follicles (secondary) were extracted from the ovarian cortex by microdissection, and oocytes were removed. In both groups, oocytes were denuded and grouped into "pools" of ten cells each (Group A, n=10, Group B, n=10) and ten samples with groups of cumulus cells (Group A1, n=10; B1, n=10). The mRNA was extracted and converted to cDNA using the RT-PCR technique. The expression analysis confirmed the expression of IGF-2 gene for groups of oocyte and the cumulus cells. There was an increase in the relative expression of mRNA for IGF-2 for groups of oocytes during the later stage of follicular development and differences were considered significant (p<0.05). There was no significant variation in the expression of IGF2 between groups of cumulus cells. It is concluded that the growth factor IGF-2 has higher levels of expression in sheep oocytes in the second stage of follicular development in the conditions adopted and similar expression in cumulus cells during various stages of follicular development.(AU)

Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus

Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13 × 3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13 × 3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.(AU)

Effect of Heat Stress on the Expression of GABA Receptor mRNA in the HPG Axis of Wenchang Chickens

We investigated the effect of heat stress (HS) on the expression of the GABA receptor in the hypothalamic-pituitary-gonadal (HPG) axis of Wenchang chickens. Real-time quantitative RT-PCR (qRT-PCR) was used to quantify the GABA receptor mRNA levels along the HPG axis of chickens under HS (40±0.5 °C) for 1-6 weeks. Our results showed that the expression of GABAA and GABAB receptor at the mRNAs levels in the tissues of HPG axis exhibited fluctuation and variability. After HS, the mRNA level of GABAA receptor was significantly reduced in the hypothalamus of 1-week-old and in the pituitary of 3-week-old chickens, but significantly increased in the pituitary of 1-, 4-, and 5-week-old chickens. The GABAB receptor mRNA level significantly declined in the hypothalamus of 1-week-old and in the pituitary of 3-week-old chickens, but was significantly upregulated in the pituitary and testis of 1- and 2-week-old chickens. At other time points, the expressions of GABAA receptor and GABAB receptor showed no significant differences compared with control group. These results indicated that the levels of GABAA receptor and GABAB receptor mRNAs varied in different tissues of the HPG axis in chickens of different ages, displaying temporal and spatial variations. GABA receptor behaved as a positively-regulated gene by HS, i.e., its mRNA was increased by HS; similarly, it was a negatively-regulated gene by HS, when its expression was reduced by HS.

Effect of Heat Stress on the Expression of GABA Receptor mRNA in the HPG Axis of Wenchang Chickens

We investigated the effect of heat stress (HS) on the expression of the GABA receptor in the hypothalamic-pituitary-gonadal (HPG) axis of Wenchang chickens. Real-time quantitative RT-PCR (qRT-PCR) was used to quantify the GABA receptor mRNA levels along the HPG axis of chickens under HS (40±0.5 °C) for 1-6 weeks. Our results showed that the expression of GABAA and GABAB receptor at the mRNAs levels in the tissues of HPG axis exhibited fluctuation and variability. After HS, the mRNA level of GABAA receptor was significantly reduced in the hypothalamus of 1-week-old and in the pituitary of 3-week-old chickens, but significantly increased in the pituitary of 1-, 4-, and 5-week-old chickens. The GABAB receptor mRNA level significantly declined in the hypothalamus of 1-week-old and in the pituitary of 3-week-old chickens, but was significantly upregulated in the pituitary and testis of 1- and 2-week-old chickens. At other time points, the expressions of GABAA receptor and GABAB receptor showed no significant differences compared with control group. These results indicated that the levels of GABAA receptor and GABAB receptor mRNAs varied in different tissues of the HPG axis in chickens of different ages, displaying temporal and spatial variations. GABA receptor behaved as a positively-regulated gene by HS, i.e., its mRNA was increased by HS; similarly, it was a negatively-regulated gene by HS, when its expression was reduced by HS.(AU)

Expresión génica: una visión general de los métodos y aplicaciones en la pesquisa de cáncer / Gene expression: an overview of methods and applications for cancer research / Expressão gênica: uma visão geral dos métodos e aplicações na pesquisa do câncer

La expresión génica es el estudio de cómo el genotipo da lugar al fenotipo mediante la investigación de la cantidad de RNAm transcrito en un sistema biológico. Una gran cantidad de métodos fueron estandarizados para identificar variaciones en la expresión génica, incluyendo la hibridación sustractiva, differential display, análisis en serie de la expresión génica, la hibridación de microarrays, y la secuenciación por RNA-seq. La mayoría de las técnicas se han centrado en la investigación y diagnóstico del cáncer, produciendo una gran cantidad de datos, lo que permitió a entender la progresión del cáncer y las vías, descubrir y evaluar nuevas intervenciones de tratamiento, nuevas herramientas moleculares para el diagnóstico y el pronóstico, y analizar el tiempo de sobrevivencia en pacientes humanos y animales. De esta manera, las técnicas de expresión génica trajeron nuevas perspectivas importantes para el campo de la medicina veterinaria, y nuevas investigaciones centradas en oncología proporcionarán mucho más conocimiento acerca de las vías y la interacción de las células sanas y tumorales, mejorando las intervenciones diarias por los oncólogos y los clínicos.(AU)

Gene expression is the study of how the genotype gives rise to the phenotype by investigating the amount of transcribed mRNA in a biological system. A lot of methods have been standardized to identify the variation in gene expression, including subtractive hybridization, differential display, serial analysis of gene expression, microarray hybridization, and RNAseq sequencing. Most of techniques have been focused in cancer research and diagnosis, producing a huge amount of data, which allowed to understand the cancer progression and pathways, discover and evaluate new treatment interventions, new molecular tools for diagnosis and prognosis, and analyze the survival time in human and animal patients. In this way, gene expression techniques brought new important perspectives for the medical and veterinary fields, and further researches focusing oncology will provide much more knowledge concerning the pathways and interaction of healthy and tumor cells, improving the perspectives of the daily interventions by the oncologists and clinicians.(AU)

A expressão genética é o estudo de como o genótipo dá origem ao fenótipo a partir da investigação da quantidade de RNAm transcrito em um sistema biológico. Vários métodos já foram padronizados para identificar variações na expressão gênica, dentre eles a hibridização subtrativa, differential display, análise em série da expressão genética, hibridização de microarranjo, e sequenciamento por RNA-seq. A maioria das técnicas tem focado na pesquisa e diagnóstico do câncer, gerando enorme quantidade de dados, o que permitiu compreender a progressão do câncer e suas vias, descobrir e analisar novas intervenções terapêuticas, novas ferramentas moleculares para o diagnóstico e prognóstico, e analisar o tempo de sobrevivência em pacientes humanos e animais. Desta forma, as diferentes técnicas de expressão gênica trouxeram novas e importantes perspectivas para a área médica e veterinária, e novas pesquisas focadas em oncologia fornecerão muito mais conhecimento sobre as vias e interações entre células saudáveis e tumorais, melhorando as perspectivas das intervenções diárias pelos oncologistas e clínicos.(AU)