Phagocytotic activity and gene expression of leukocytes isolated from Astyanax lacustris / Atividade fagocítica e expressão gênica de leucócitos isolados de Astyanax lacustris

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris­an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research­using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1ß, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1ß and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

A constante intensificação da aquicultura tem aumentado consideravelmente os níveis de estresse dos animais cultivados e, consequentemente, o número e a intensidade dos surtos de doenças. Logo, estudos sobre a resposta imune dos peixes, especialmente relacionados com a interação dos leucócitos de peixes com potenciais patógenos e xenobióticos, são de grande importância para o desenvolvimento de novas estratégias profiláticas e curativas. No presente trabalho, nós obtivemos sucesso ao isolar leucócitos oriundos do rim cranial de Astyanax lacustris ­ uma importante espécie de peixe Neotropical para a aquicultura e um modelo em potencial para pesquisas em aquicultura Neotropical ­ usando um protocolo de centrifugação com Percoll. Os leucócitos isolados foram incubados com lipossacarídeo (LPS) e, a expressão dos genes IL-1ß, IL-8, LysC, e LysG foi avaliada. Ainda, um novo protocolo para avaliação da atividade fagocítica dos leucócitos utilizando leveduras coradas com Vermelho Congo foi estabelecido. Os leucócitos isolados responderam à indução com LPS, exibindo up regulation dos genes IL-1ß e IL-8, duas das interleucinas pró-inflamatórias mais importantes para a resposta imune de vertebrados. Além do mais, a concentração ótima de leveduras para a avaliação da fagocitose foi de 106 células mL-1, resultando em uma capacidade fagocítica (PC) aceitável, mas sem excesso de leveduras durante o processo de contagem, garantindo maior precisão e eficácia do método. Até o presente momento, o presente estudo é o primeiro a investigar a expressão gênica e atividade fagocítica de leucócitos isolados de A. lacustris através da abordagem in vitro. Ainda, nossos resultados servirão de referência para futuros estudos em imunologia e toxicologia de peixes Neotropicais.

Bioinformatics and expression analysis of the Xeroderma Pigmentosum complementation group C (XPC) of Trypanosoma evansi in Trypanosoma cruzi cells / Análises de bioinformática e da expressão do gene Xeroderma Pigmentosum complementation group C (XPC) de Trypanosoma evansi em células de Trypanosoma cruzi

Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T. cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.

O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T. cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.

Bioinformatics and expression analysis of the Xeroderma Pigmentosum complementation group C (XPC) of Trypanosoma evansi in Trypanosoma cruzi cells / Análises de bioinformática e da expressão do gene Xeroderma Pigmentosum complementation group C (XPC) de Trypanosoma evansi em células de Trypanosoma cruzi

Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T. cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.(AU)

O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T. cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.(AU)

Expression of calpain system transcripts responds inversely to beef tenderization after vitamin D3 supplementation in Nellore cattle / Expressão de transcritos do sistema calpaína responde de forma inversa ao amaciamento após suplementação com vitamina D3 em bovinos Nelore

Uma vez que o sistema calpaína é central para o amaciamento da carne e dada a importância da atividade de calpastatina na determinação da maciez de bifes de bovinos Bos taurus indicus, o objetivo do presente estudo foi determinar se a expressão do gene de µ-calpaína (CAPN1), calpastatina total (CAST T), e suas variantes (CAST I e II) foi induzido pela inclusão de vitamina D3 na dieta. Os animais receberam nenhuma ou 2 × 106 UI dose de vitamina D3 por 2 ou 8 dias antes do abate, e foram submetidos a diferentes condições durante o confinamento: exposição solar ou sombreamento artificial. Bifes do Longissimus lumborum foram fabricados e submetidos a maturação por 1, 7, e 21 dias post-mortem e posteriormente usados para determinação da força de cisalhamento e do índice de fragmentação miofibrilar. Vitamina D3 não influenciou a abundância de RNAm, exceto para maior abundância de transcritos de CAST II em animais que foram suplementados por 8 dias antes do abate. Foi encontrada associação negativa entre a abundância de CAST II e a força de cisalhamento. Essa contradição revela uma importante modulação da expressão do sistema calpaína resultado da suplementação com vitamina D que pode ser importante na determinação de estratégias para melhorar a maciez da carne.(AU)

The calpain system is the central player for meat tenderization and the calpastatin activity plays an important role in beef tenderness of Bos taurus indicus cattle. This study investigated whether dietary vitamin D3 induced gene expression of µ-calpain (CAPN1), total calpastatin (CAST T), and their variants (CAST I and II). Animals received none or 2 × 106 IU of vitamin D3 for either 2 or 8 days before slaughter and were submitted to different conditions during feedlot: sun exposure or artificial shade. Steaks from Longissimus lumborum were fabricated, aged for 1, 7, and 21 days post-mortem, and later used for the analyses of shear force and the myofibrillar fragmentation index. Vitamin D3 did not influence mRNA abundance; however, it induced a greater CAST II transcript in animals supplemented 8 days before slaughter. There was a negative association between CAST II abundance and the shear force, which revealed an important modulation of the calpain system expression due to vitamin D supplementation. This result is an important tool for strategies to improve beef tenderness.(AU)

Innate immunity gene expression profiles in conjunctival membrane biopsies from Amazonian buffalo / Perfis de expressão de genes da imunidade inata em biopsias de membrana conjuntival de búfalos da Amazônia

Domestic buffalo production plays an economically important role in the Brazilian Amazon, but they are susceptible to many diseases favored by the tropical climate and annually flooded habitats, including ocular diseases. In this context, it is important to select genotypes that maximize innate ocular immunity in Amazonian herds. We aimed to characterise, for the first time, gene expression profiles of the innate immune system in the conjunctival membrane of buffalo. Ocular conjunctival tissue samples were collected from 60 clinically healthy slaughtered animals in the northern Brazilian state of Amapá. The samples were histologically processed for classification into three groups according to the quantitative degree of lymphoid tissue associated with the conjunctiva (discrete, G1; slight, G2; and moderate, G3 presence of lymphoid tissue). RT-PCR was used to quantify gene expression of inflammatory cytokine (IL6, IL10, TNFA, IFNG), Toll-like receptor 4 (TLR4), and Defensin beta 110 (DEFB110), relative to the endogenous GAPDH gene. G1 animals presented low expression for IL6, IL10, TNFA, and DEFB110, while G2 exhibited high expression for IL6, IL10, IFNG, and TLR4. All G3 animals showed high expression for all tested genes. These results suggest a greater resistance to pathogenic microorganisms of buffalos in the G3 group, and the proportion of lymphoid tissue associated with the conjunctiva may be related to the immune resistance of individuals.(AU)

A produção de búfalos domésticos desempenha um papel economicamente importante na Amazônia brasileira, mas eles são suscetíveis a muitas doenças favorecidas pelo clima tropical e habitats inundados anualmente, incluindo doenças oculares. Nesse contexto, é importante selecionar genótipos que maximizem a imunidade ocular inata em rebanhos amazônicos. Objetivamos caracterizar, pela primeira vez, perfis de expressão gênica do sistema imune inato na membrana conjuntival de búfalos. Amostras de tecido conjuntival ocular foram coletadas de 60 animais clinicamente saudáveis abatidos no estado do Amapá, norte do Brasil. As amostras foram processadas histologicamente para classificação em três grupos de acordo com o grau quantitativo de tecido linfoide associado à conjuntiva (discreta, G1; leve, G2; e moderada, G3 presença de tecido linfoide). RT-PCR foi utilizado para quantificar a expressão gênica de citocinas inflamatórias (IL6, IL10, TNFA, IFNG), receptor Toll-like 4 (TLR4) e Defensina beta 110 (DEFB110), em relação ao gene GAPDH endógeno. Os animais do G1 apresentaram baixa expressão para IL6, IL10, TNFA e DEFB110, enquanto G2 exibiu alta expressão para IL6, IL10, IFNG e TLR4. Todos os animais do G3 apresentaram alta expressão para todos os genes testados. Esses resultados sugerem maior resistência aos microrganismos patogênicos dos búfalos do grupo G3, e a proporção de tecido linfoide associado à conjuntiva pode estar relacionada à resistência imunológica dos indivíduos.(AU)

Era-like GTP protein gene expression in rice / Expressão do gene da proteína GTP semelhante à era no arroz

The mutations are genetic changes in the genome sequences and have a significant role in biotechnology, genetics, and molecular biology even to find out the genome sequences of a cell DNA along with the viral RNA sequencing. The mutations are the alterations in DNA that may be natural or spontaneous and induced due to biochemical reactions or radiations which damage cell DNA. There is another cause of mutations which is known as transposons or jumping genes which can change their position in the genome during meiosis or DNA replication. The transposable elements can induce by self in the genome due to cellular and molecular mechanisms including hypermutation which caused the localization of transposable elements to move within the genome. The use of induced mutations for studying the mutagenesis in crop plants is very common as well as a promising method for screening crop plants with new and enhanced traits for the improvement of yield and production. The utilization of insertional mutations through transposons or jumping genes usually generates stable mutant alleles which are mostly tagged for the presence or absence of jumping genes or transposable elements. The transposable elements may be used for the identification of mutated genes in crop plants and even for the stable insertion of transposable elements in mutated crop plants. The guanine nucleotide-binding (GTP) proteins have an important role in inducing tolerance in rice plants to combat abiotic stress conditions.

Mutações são alterações genéticas nas sequências do genoma e têm papel significativo na biotecnologia, genética e biologia molecular, até mesmo para descobrir as sequências do genoma de um DNA celular junto com o sequenciamento do RNA viral. As mutações são alterações no DNA que podem ser naturais ou espontâneas e induzidas devido a reações bioquímicas ou radiações que danificam o DNA celular. Há outra causa de mutações, conhecida como transposons ou genes saltadores, que podem mudar sua posição no genoma durante a meiose ou a replicação do DNA. Os elementos transponíveis podem induzir por si próprios no genoma devido a mecanismos celulares e moleculares, incluindo hipermutação que causou a localização dos elementos transponíveis para se moverem dentro do genoma. O uso de mutações induzidas para estudar a mutagênese em plantas cultivadas é muito comum, bem como um método promissor para a triagem de plantas cultivadas com características novas e aprimoradas para a melhoria da produtividade e da produção. A utilização de mutações de inserção por meio de transposons ou genes saltadores geralmente gera alelos mutantes estáveis que são marcados quanto à presença ou ausência de genes saltadores ou elementos transponíveis. Os elementos transponíveis podem ser usados para a identificação de genes mutados em plantas de cultivo e até mesmo para a inserção estável de elementos transponíveis em plantas de cultivo mutadas. As proteínas de ligação ao nucleotídeo guanina (GTP) têm papel importante na indução de tolerância em plantas de arroz para combater as condições de estresse abiótico.

Expression pattern of CRYAB and CTGF genes in two pig breeds at different altitudes / [Padrão de expressão dos genes CRYAB e CTGF em duas raças de suínos em altitudes diferentes]

Tibetan pigs are characterized by significant phenotypic differences relative to lowland pigs. Our previous study demonstrated that the genes CRYAB and CTGF were differentially expressed in heart tissues between Tibetan (highland breed) and Yorkshire (lowland breed) pigs, indicating that they might participate in hypoxia adaptation. CRYAB (ɑB-crystallin) and CTGF (connective tissue growth factor) have also been reported to be associated with lung development. However, the expression patterns of CRYAB and CTGF in lung tissues at different altitudes and their genetic characterization are not well understood. In this study, qRT-PCR and western blot of lung tissue revealed higher CRYAB expression levels in highland and middle-highland Tibetan and Yorkshire pigs than in their lowland counterparts. With an increase in altitude, the expression level of CTGF increased in Tibetan pigs, whereas it decreased in Yorkshire pigs. Furthermore, two novel single-nucleotide polymorphism were identified in the 5' flanking region of CRYAB (g.39644482C>T and g.39644132T>C) and CTGF (g.31671748A>G and g.31671773T>G). The polymorphism may partially contribute to the differences in expression levels between groups at the same altitude. These findings provide novel insights into the high-altitude hypoxia adaptations of Tibetan pigs.

Porcos tibetanos são caracterizados por diferenças fenotípicas significativas em relação aos porcos de planície. Nosso estudo anterior demonstrou que os genes CRYAB e CTGF eram expressos diferentemente nos tecidos do coração entre os porcos tibetanos (raça das terras altas) e Yorkshire (raça das terras baixas), indicando que eles poderiam participar da adaptação à hipoxia. CRYAB (ɑB-crystallin) e CTGF (fator de crescimento do tecido conjuntivo) também foram relatados como estando associados ao desenvolvimento pulmonar. Entretanto, os padrões de expressão do CRYAB e CTGF nos tecidos pulmonares em diferentes altitudes e sua caracterização genética não são bem compreendidos. Neste estudo, o qRT-PCR e a mancha ocidental de tecido pulmonar revelou níveis de expressão de CRYAB mais elevados em porcos tibetanos e Yorkshire de altitude e média altitude do que em seus pares de planície. Com um aumento na altitude, o nível de expressão do CTGF aumentou nos porcos tibetanos, enquanto diminuiu nos porcos Yorkshire. Além disso, foram identificados dois novos polimorfismos de um único nucleotídeo na região flanqueadora de CRYAB (g.39644482C>T e g.39644132T>C) e CTGF (g.31671748A>G e g.31671773T>G). O polimorfismo pode contribuir parcialmente com as diferenças nos níveis de expressão entre grupos a uma mesma altitude. Estas descobertas proporcionam novos conhecimentos sobre as adaptações de hipoxia a alta altitude dos porcos tibetanos.

The Effects of Saccharomyces Cerevisiae and Citric Acid on Productive Performance, Egg Quality Parameters, Small Intestinal Morphology, and Immune-Related Gene Expression in Laying Japanese Quails

This experiment evaluated the effects of Saccharomyces cerevisiae (S. cerevisiae) and citric acid on production performance, egg quality, intestine histomorphology, and avian ß-defensin 1 and 2 (AvBD 1 and 2) gene expressions in laying Japanese quails. A total of 400 48-day-old quails were randomly assigned to a 2×2×2 factorial arrangement of treatments with 5 replicates (each containing 10 quails) for 7 weeks. Variable factors consisted of S. cerevisiae (0 and 100 mg/kg diet), citric acid (0 and 5 g/kg diet), and Virginiamycin (0 and 50 mg/kg diet). At the completion of the trial, one bird per replicate was randomly killed, and jejunal tissue samples were removed to evaluate intestinal morphometric characteristics. Samples were taken from the midpoint of the jejunum to measure the gene expression of AvBD 1 and 2. Dietary inclusion of both S. cerevisiae and citric acid resulted in increased egg weight, egg mass, reduced feed intake, and improved FCR (p<0.05). The addition of S. cerevisiae to diets containing citric acid reduced feed intake, increased egg weight, and improved FCR (p<0.05). Shell weight and shell thickness were increased in birds fed each of S. cerevisiae and citric acid supplements (p<0.05). Dietary S. cerevisiae and citric acid similarly increased intestinal villus height, width, surface area, and the villus height to crypt depth ratio (p<0.0001). Results showed that AvBD 1 and 2 genes expression were up-regulated on quails fed S. cerevisiae-supplemented diets (p<0.0001). In conclusion, these results suggest that supplementation of S. cerevisiae and citric acid as functional feed additives either alone or in combination could be a potential alternative to antibiotics in the diet of Japanese laying quails.(AU)

Supering Apis mellifera (Hymenoptera, Apidae) beehives impairs honey production and biomarker genes

For honey production, beekeepers add one or more supers to the hives to allow honeybees to store their products. However, the increase in hive space can affect the social and health organization in the colony, promoting stress. This study assessed the management of honey production, physicochemical honey properties, population development, and forages immune system gene expression patterns to be used as biomarker for monitoring beekeeping welfare. The treatments comprised 40 beehives divided in four treatments. Treatment 1 - control, supers added according to storage necessity. Treatments 2, 3, and 4 presented two, three, and four supers at the beginning of the experiment, respectively. T1 presented greater honey production (39.4 % increased). No difference in open brood area in the colonies was observed and honey properties and only T2 showed closed brood area higher than the other treatments. Foragers from T4 showed higher catalase and defensin gene expression at the middle-end experiment. Thus, the increasing internal space at the beginning of honey season can affect honey production and immune system of foragers. Catalase and defensin can be used as biomarkers for monitoring honey production welfare.

Cell culture and variations in the comet assay do not affect the genomic integrity of adipose-derived stem cells

The application of quality control tests, such as the comet assay, are essential when adipose-derived stem cells are cultured for therapeutic purposes. However, the steps involved in the development of this assay should be investigated, in order to reduce their influence on genomic damage in cells. The study aimed to evaluate if the cell culture process causes DNA damage, and if variations in the lysis time and pH of the electrophoresis buffer interfere in the genotoxicity results. Four different comet assay protocols were evaluated, and the effects of lysis time and pH conditions of the electrophoresis buffer solution were stated as follows: 2 hours and pH 12; 24 hours and pH 12; 2 hours and pH ≥ 13 and 24 hours and pH ≥ 13. The tail moment was analyzed, and results indicated that at the time cells were detached from the flasks, there was little damage to the DNA in the adipose-derived stem cells, which was confirmed by evaluation of the expression of mRNA genes involved in damage and repair processes of genetic material. Also, the tail moment values ​​did not show significant differences among the four evaluated protocols (p < 0.05), with no indication of damage when compared to the positive control (p < 0.05). Thus, any of the tested protocols can be applied in genotoxicity tests with adipose-derived stem cells, without causing damage to them.(AU)

Perinatal exposure to a high-fat diet alters proopiomelanocortin, neuropeptide Y and dopaminergic receptors gene expression and the food preference in offspring adult rats / Exposição perinatal a uma dieta hiperlipídica altera a expressão gênica de proopiomelanocortina, neuropepitídeo Y e receptores dopaminérgicos e a preferência alimentar em ratos adultos descendentes

Exposure to the hight-fat diet may alter the control of food intake promoting hyperphagia and obesity. The objective of this study was to investigate the effects of this diet on dopamine receptors (drd1 and drd2), proopiomelanocortin (pomc), neuropeptideY (npy) genes expression, and preference food in adult rats. Wistar female rats were fed a hight-fat or control diet during pregnancy and lactation. The offspring were allocated into groups: Lactation - Control (C) and High-fat (H). Post-weaning ­ Control Control (CC), offspring of mothers C, fed a control diet after weaning; Control Hight-fat (CH), offspring of mothers C, fed a hight-fat diet after weaning; Hight-fat Control (HC), offspring of mothers H, fed with control diet after weaning; and Hight-fat Hight-fat (HH), offspring of mothers H, fed a H diet after weaning. The groups CH and HH presented greater expression of drd1 in comparison to the CC. The drd2 of CH and HC presented higher gene expression than did CC. HH presented higher pomc expression in comparison to the other groups. HC also presented greater expression in comparison to CH. The npy of HH presented greater expression in relation to CH and HC. HH and HC have had a higher preference for a high-fat diet at 102º life's day. The high-fat diet altered the gene expression of the drd1, drd2, pomc and npy, and influencing the food preference for high-fat diet.

A exposição à dieta hiperlipídica pode alterar o controle da ingestão de alimentos, promovendo hiperfagia e obesidade. O objetivo deste estudo foi investigar os efeitos dessa dieta sobre a expressão gênica dos receptores de dopamina (drd1 e drd2), da proopiomelanocortina (pomc) e neuropeptídeo Y (npy), e preferência alimentar em ratos adultos. Ratas Wistar foram alimentadas com uma dieta hiperlipídica ou controle durante a gestação e lactação. Os descendentes foram alocados em grupos: Lactação ­ Controle (C) e Hiperlipídica (H). Pós-desmame - Controle Controle (CC), descendentes das genitoras do grupo controle e alimentados com dieta controle após o desmame; Controle Hiperlipídica (CH), descendentes das genitoras do grupo controle e alimentados com dieta hiperlipídica após o desmame; Hiperlipídica Controle (HC), descendentes das genitoras do grupo hiperlipídica e alimentados com dieta controle após o desmame; Hiperlipídica Hiperlipídica (HH), descendentes das genitoras do grupo hiperlipídica e alimentados com dieta hiperlipídica após o desmame. Os grupos CH e HH apresentaram maior expressão de drd1 em comparação ao CC. O drd2 de CH e HC apresentou maior expressão gênica que o CC. HH apresentou maior expressão de pomc em comparação com os outros grupos. O HC também apresentou maior expressão de pomc em comparação ao CH. O npy do HH apresentou maior expressão em relação ao CH e HC. HH e HC tiveram uma preferência maior por uma dieta rica em gordura no 102º dia de vida. A dieta hiperlipídica alterou a expressão gênica dos drd1, drd2, pomc e npy e influenciou na preferência alimentar pela dieta hiperlipídica.

Comparative toxicity of fipronil, malathion, and thiamethoxam on the stingless bee Tetragonisca fiebrigi (Schwarz, 1938)

Stingless bees are important pollinators for various plant crops. We investigated the susceptibility of Tetragonisca fiebrigito sublethal concentrations of insecticides fipronil, malathion, and thiamethoxam (administered through contact and ingestion) by determining the LC50values after 24hoursof exposure and analyzing changes in the activity of esterase isoenzymes and the chromatin in brain cells. The LC50values showed that all three insecticides were highly toxic through contact and ingestion. Electrophoretic analysis revealed that the relative EST-4 (carboxylesterase) activity in T. fiebrigi was partially inhibited by malathion and fipronil ingestion. Moreover, the EST-4 band intensity was increased following high-concentration thiamethoxam (contact) exposure, indicating the increased relative activity of this isoenzyme to detoxify the compound. In the cytochemical analysis of brain cells, the critical electrolyte concentration (CEC) points for the control stingless bees and malathion ingestion-exposed and thiamethoxam-exposed (contact and ingestion) stingless bees were in the range of 0.20-0.30 M MgCl2, whereas that for malathion contact-exposed bees was 0.15 M MgCl2, indicating chromatin relaxation and suggesting an increase in gene expression. In conclusion, T. fiebrigistingless bees are susceptible to the insecticides tested, and the parameters analyzed may be used as biomarkers to detect the presence of these compounds.(AU)

Expression of TGF-ß/Smads in Cecum and Spleen of Chicken Infected with E. Tenella

Chicken coccidiosis is a common and severe parasitic disease caused by infection from Eimeria spp., which affects the integrity of the intestinal mucosa. TGF-ß has been shown to play an important role in the healing of intestinal mucosas, immunity, and the maintenance of bowel mucosa integrity. Very little is known about the presence of the components of TGF-ß/Smads signaling pathway of chicken at different times following coccidian infection. In the present study, we measured the levels of TGF-ß2, 3, 4, receptor TßRI, II, down-stream Smad 2, 3, 7 in cecum and spleen of chicken at different times after inoculation with Eimeria tenella using quantitative real-time PCR. The results showed that the TGF-ß/Smads signaling pathway was not activated in cecum in the early stage of infection. However, on the 8th day, the expression of TGF-ß2, 4, down-stream protein Smad 2, 7 were significant up-regulated in the spleen, which indicated that the TGF-ß/Smads signaling was changed in the E. tenella infection and was differentially expressed in various tissues in the early stages of infection.(AU)

Reference gene stability in the Longissimus thoracis et lumborum muscle of Colombian Creole sheep / Estabilidade de genes de referência no músculo Longissimus thoracis et lumborum de ovelhas crioulas colombianas

The Colombian Creole sheep breed has a high economic and social importance for Colombia. Both males and females of this breed are multipurpose animals, and evaluating the production and meat quality of both sexes is important for small farmers in Colombia. This requires the use of tools that help to evaluate critical production points, such as Real-time quantitative polymerase chain reaction (RT-qPCR), which is a widely used molecular tool for the relative quantification of candidate genes in various tissues. For its correct use, the use of housekeeping genes with stable expression, so-called "reference genes", is required. However, recent studies have shown that the expression of these reference genes can vary among tissues and can be modulated by breed, sex, or external stimuli. Likewise, there is little information regarding the expression of these genes in the Longissimus thoracis et lumborum muscle of male and female Colombian Creole sheep. In this study, the stability in the expression of seven reference genes (ACTB, YWHAZ, SDHA, GAPDH, TUBB2A, B2M, and PGK1) in the Longissimus thoracis et lumborum muscle of male and female Colombian Creole sheep was compared since they are used in RT-qPCR studies to determine the most stable ones for this breed. Twelve animals, six males and six females, with a body weight of 26 ± 4 kg and 12 ± 3 months of age, were used under grazing conditions. Biopsies of the Longissimus thoracis et lumborum muscle were taken, from which RNA was extracted and cDNA was synthesized. Expression was determined using RT-qPCR, and its stability was analyzed by computational algorithms using the geNorm, Normfinder, and BestKeeper software packages, which were integrated using the RefFinder software package. The results indicate that GAPDH, ACTB, and SDHA have the highest stability, whereas the most variable expression was found for B2M. These data provide the basis for more precise results in RT-qPCR studies of gene expression in the muscle of Colombian Creole sheep.(AU)

A raça de ovinos Crioula Colombiana tem grande importância econômica e social para a Colômbia. Avaliar a produção e a qualidade da carne de machos e fêmeas é importante para pequenos produtores do país e, assim, faz necessário o uso ferramentas que ajudam a avaliar os pontos críticos de produção, como a reação em cadeia da polimerase em tempo real (Real-time quantitative polymerase chain reaction [RT-qPCR]). Esta é uma ferramenta molecular amplamente usada para a quantificação relativa de genes candidatos em vários tecidos. Para o seu uso correto, é necessário o uso de genes com expressão estável denominados genes de referência. No entanto, estudos recentes têm mostrado que a expressão desses genes de referência pode variar entre os tecidos e pode ser modulada por raça, sexo ou estímulos externos. Da mesma forma, existem poucas informações sobre a expressão desses genes no músculo Longissimus thoracis et lumborum de ovinos machos e fêmeas da raça Crioula Colombiana. Neste estudo foi comparada a estabilidade na expressão de sete genes de referência (ACTB, YWHAZ, SDHA, GAPDH, TUBB2A, B2M e PGK1) no músculo Longissimus thoracis et lumborum de ovinos Crioulo Colombiano machos e fêmeas, por serem genes utilizados em estudos de RT-qPCR visando determinar os mais estáveis para esta raça. Doze animais com peso corporal de 26 ± 4 kg e 12 ± 3 meses de idade foram utilizados em condições de pastejo. Foram realizadas biópsias do músculo Longissimus thoracis et lumborum, de onde o RNA foi extraído e o cDNA foi sintetizado. A expressão foi determinada usando RT-qPCR e sua estabilidade foi analisada por algoritmos computacionais usando geNorm, Normfinder e BestKeeper pacote de software, os quais foram integrados usando RefFinder pacote de software. Os resultados indicam que GAPDH, ACTB e SDHA apresentam maior estabilidade, enquanto a expressão mais variável foi para B2M. Esses dados fornecem a base para resultados mais precisos em estudos de RT-qPCR de expressão gênica em músculos defeminino ovinos da raça Crioula Colombiana.(AU)

Starch synthesis and gelatinization properties of potato tubers / Síntese de amido e propriedades de gelatinização de tubérculos de batata com diferentes teores de amido

Biosynthesis is the only source of potato starch which is an important raw material for food processing, modified starch and biomass energy. However, it is not clear about the evolution of starch synthesis with tuber development in potato. The present study evaluated the differences of starch synthesis and gelatinization properties of potato tubers with different starch content. Relative to cultivars of medium and low starch content, cultivars of high starch content showed significantly higher SBEII gene expression, AGPase and SSS enzyme activity, and total starch content after middle stage of starch accumulation, and had smaller average starch granule size during whole process of tuber development, and had higher pasting temperature before late stages of tuber growth, and had lower pasting temperature after middle stage of starch accumulation. Path analysis showed that, after middle stage of starch accumulation, effects on starch gelatinization of cultivars with high, medium and low starch content represented starch synthesis enzyme activity > starch accumulation > starch granule distribution > starch synthesis enzyme gene expression, starch synthesis enzyme gene expression > starch synthesis enzyme activity > starch accumulation > starch granule distribution, starch synthesis enzyme gene expression > starch granule distribution > starch synthesis enzyme activity > starch accumulation, respectively. In the study, phases existed in the starch biosynthesis of potato tuber, and the starch quality and its formation process were different among varieties with different starch content. The findings might contribute to starch application and potato industries.

A biossíntese é a única fonte de amido de batata que é uma importante matéria-prima para o processamento de alimentos, amido modificado e energia de biomassa. No entanto, não está claro sobre a evolução da síntese do amido com o desenvolvimento do tubérculo na batata. O presente estudo teve como objetivo avaliar as diferenças nas propriedades de síntese e gelatinização do amido de tubérculos de batata com diferentes teores de amido. Em relação às cultivares de médio e baixo teor de amido, as cultivares de alto teor de amido apresentaram expressão do gene SBEII, atividade enzimática AGPase e SSS e teor de amido total significativamente maiores após o estágio intermediário de acúmulo de amido, bem como menor tamanho médio dos grânulos de amido durante todo o processo de desenvolvimento do tubérculo, maior temperatura de colagem antes dos estágios finais de crescimento do tubérculo e menor temperatura de colagem após o estágio intermediário de acúmulo de amido. A análise de trilha mostrou que, após o estágio intermediário de acúmulo de amido, os efeitos na gelatinização do amido de cultivares com alto, médio e baixo teor de amido representaram a atividade da enzima de síntese de amido> acúmulo de amido> distribuição de grânulos de amido> expressão gênica de enzima de síntese de amido; expressão gênica de enzima de síntese de amido > atividade da enzima de síntese de amido> acúmulo de amido> distribuição de grânulos de amido; expressão gênica da enzima de síntese de amido> distribuição de grânulos de amido> atividade de síntese de amido> acúmulo de amido, respectivamente. No estudo, as fases existentes na biossíntese do amido do tubérculo de batata, e a qualidade do amido e seu processo de formação foram diferentes entre as variedades com diferentes teores de amido. As descobertas podem contribuir para a aplicação de amido e as indústrias de batata.

Effects of ß-glucan from S. cerevisiae on the expression of Casp9, Ccna2 and Sod1 genes in MCF-7 cells

ß-Glucans (ßG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. ßG was not cytotoxic. The mRNA levels of CCNA2of cells exposed to ß-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9suggest that ßG could inhibit promotion and progression steps of carcinogenesis, eliminatingneoplastic cells. The upregulation of CCNA2gene in combined treatment could be occurred due to ability of ßG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1suggests that ßG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of ßG during chemotherapy.Keywords:caspase-9; cyclin A2; superoxide dismutase 1; cell cycle; antioxidant.Received on July 2, 2020.Accepted on February 7, 2022.IntroductionGlucans are polysaccharides widely distributed in nature and oftenstudied due to chemopreventive properties. They are constituent of the cell wall of plants (oats and barley), algae, bacteria and fungi. ß-glucans (ßG)have a common structure comprising a main chain of ß-(1,3) and/or ß-(1,4) D-glucopyranosyl unit and they differ in length and branching structures. ßG of Saccharomyces cerevisiaehave 1→6 side branches while those of bacteria have 1→4 side branches (Chan, Chan, & Sze, 2009). ßGcan prevent DNA damage induced by chemical and physical agents (Ghavami,Goliaei, Taghizadeh, & Nikoofar, 2014). Some authors showed its significant efficacy in preventing mutagenic effects caused by doxorubicin, cyclophosphamide and cisplatin (Tohamy, El-Ghor, El-Nahas, & Noshy, 2003), methyl methanesulfonate (Oliveira et al., 2007)and hydrogen peroxide (Slamenová, 2003). Moreover, some studies have related the antioxidant ability of ßGagainst reactive free radicals formed by endogenous metabolic processes or exogenous chemicals (Tsiapali et al., 2001; Slamenová,2003; Sener, Eksioglu-Demiralp, Cetiner, Ercan, & Yegen, 2006; Guerra Dore et al., 2007; Kofuji et al., 2012; Lei et al., 2015). Yeast-derived ßGhave modulating action of humoral and cellular immune responses (Vetvicka et al., 2007).This activity provides protection to the organism against infections and cancer development (Samuelsen, Schrezenmeir, & Knutsen, 2014; Roudbary, Daneshmand, Hajimorad, Roudbarmohammadip, & Hassan, 2015). Despite postulated modes of action by which ß-glucan works are lacking information about the molecular mechanisms involved in the chemopreventive activity of this polysaccharide. In addition, compounds with chemopreventive properties can contribute to reduce side effects and toxicity during the chemotherapeutic treatment. Therefore, the aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on the expression of genes related with apoptosis (CASP9), cell cycle control (CCNA2)and antioxidant defense (SOD1)in human breast cancer MCF-7 cells. Doxorubicin (Dox) was chosen because it is one of the most used chemotherapeutic agent for cancer treatment. The limitation on the use of Dox in cancer treatment is the lack of selectivity against cancer cells and, consequently, its toxicity to patients.(AU)

Characterization and expression of the gene glucose transporter 2 (GLUT2) in embryonic, larval and adult Bay snook Petenia splendida (Cichliformes: Cichlidae)

Bay snook (Petenia splendida) is a carnivorous cichlid species with excellent economic value in Southeast Mexico. Although this species presents an excellent potential for commercial aquaculture, the information about its nutritional, physiological, and reproductive metabolic pathways is meager. The current study focuses on the expression of glucose transporter 2 (glut2) in embryos and larvae at 5, 10, 15-, 20-, 25-, and 30-days post-hatch (dph) and in the liver, intestine, kidney, muscle, heart, testicle, gill, stomach, pancreas, and brain of adult fish. The partial sequence of glut2 was obtained, and specific qPCR primers were designed. In embryos, the expression was lower compared to larvae at 5, 15, and 20 dph. The highest expression in larvae occurred at 20 dph and the lowest at 25 and 30 dph. Maximum expression levels in adults occurred in the liver and intestine. Our results show that glut2 is expressed differentially across tissues of adult bay snook, and it fluctuates during larval development.(AU)

La mojarra tenguayaca (Petenia splendida) es una especie de cíclido carnívoro con excelente valor comercial en el sureste de México. A pesar de su potencial para la acuicultura, existe muy poca información sobre sus rutas metabólicas relacionadas con su nutrición, fisiología y reproducción. El presente estudio se enfoca en la expresión del transportador de glucosa (glut2) en embriones y larvas de 5, 10, 15, 20, 25 y 30 días post eclosión (dph) y en el hígado, intestino, riñón, músculo, corazón, testículo, branquias, estómago, páncreas y cerebro en peces adultos. Se diseñaron cebadores de qPCR específicos para glut2. La expresión en los embriones fue menor que en larvas a los 5, 15 y 20 dph. La expresión máxima en larvas se observó a los 20 dph y la mínima a los 25 y 30 dph. La expresión más alta en los adultos ocurrió en el hígado y el intestino. Nuestros resultados muestran que el gen glut2 se expresa de manera diferencial en los tejidos de adultos de la mojarra tenguayaca y su expresión fluctúa durante el desarrollo larvario.(AU)

Expression of the NSE, SP, NFH and DbetaH in normal and cryptorchid testes of Bactrian camel

Neuroendocrine substances play essential roles in regulating the normal physiological functions of testicles. The purpose of this study is to explore the localization and effects of four neuroendocrine markers (NSE, SP, NFH and DβH) in normal and cryptorchid testes of Bactrian camels using western blotting, transmission electron microscopy, immunohistochemistry, and immunofluorescence methods. The results showed that cryptorchidism caused a reduction in layers of spermatogenic epithelium and decreased glycogen positivity in the basement membrane. The ultrastructure revealed that macrophages were always found around the Leydig cells, crowded with swelling mitochondria in cryptorchidism. Expression of NSE in the Leydig cells of cryptorchidism was significantly weakened compared to that in the normal group(p<0.01). We found that SP was always distributed along the nerve fibers in normal testes and was expressed in the Leydig cells of cryptorchidism. However, expression of NFH in the cryptorchidic tissue was strongly positive in the spermatogenic epithelium, with limited expression in Leydig cells and no expression in peritubular myoid cells. Therefore, the expression of DβH in the Sertoli cells was comparatively strong in both the normal and cryptorchidism groups. NFH and DβH expression was significantly increased in the cryptorchidism group compared with the normal group (p<0.01). These findings indicated that the underdeveloped seminiferous epithelium and pathological changes in cryptorchid tissue in Bactrian camels were potentially related to a disorder in glycoprotein metabolism. Our results suggest that NSE and SP could help judge the pathological changes of cryptorchidism. The present study provides the first evidence at the protein level for the existence of NFH and DβH in Sertoli and Leydig cells in Bactrian camel cryptorchidism and provides a more in-depth understanding of neuroendocrine regulation is crucial for animal cryptorchidism.(AU)

Supering Apis mellifera (Hymenoptera, Apidae) beehives impairs honey production and biomarker genes

For honey production, beekeepers add one or more supers to the hives to allow honeybees to store their products. However, the increase in hive space can affect the social and health organization in the colony, promoting stress. This study assessed the management of honey production, physicochemical honey properties, population development, and forages immune system gene expression patterns to be used as biomarker for monitoring beekeeping welfare. The treatments comprised 40 beehives divided in four treatments. Treatment 1 - control, supers added according to storage necessity. Treatments 2, 3, and 4 presented two, three, and four supers at the beginning of the experiment, respectively. T1 presented greater honey production (39.4 % increased). No difference in open brood area in the colonies was observed and honey properties and only T2 showed closed brood area higher than the other treatments. Foragers from T4 showed higher catalase and defensin gene expression at the middle-end experiment. Thus, the increasing internal space at the beginning of honey season can affect honey production and immune system of foragers. Catalase and defensin can be used as biomarkers for monitoring honey production welfare.

Profiling and Functional Analysis of long non-coding RNAs in yak healthy and atretic follicles

Yak is the livestock on which people live in plateau areas, but its fecundity is low. Follicular development plays a decisive role in yak reproductive performance. As an important regulatory factor, the expression of long non-coding RNA (lncRNAs) in yak follicular development and its regulatory mechanism remains unclear. To explore the differentially expressed lncRNAs between healthy and atretic follicular in yaks. We used RNA-seq to construct lncRNA, miRNA, and mRNA expression profiles in yak atretic and healthy follicles, and the RNA sequence results were identified by qPCR. In addition, the correlation of lncRNA and targeted mRNA was also analyzed by Starbase software. Moreover, lncRNA/miRNA/mRNA networks were constructed by Cytoscape software, and the network was verified by dual-luciferase analysis. A total of 682 novel lncRNAs, 259 bta-miRNAs, and 1704 mRNAs were identified as differentially expressed between healthy and atretic follicles. Among them, 135 mRNAs were positively correlated with lncRNA expression and 97 were negatively correlated, which may be involved in the yak follicular development. In addition, pathway enrichment analysis of differentially expressed lncRNA host genes by Kyoto Genome Encyclopedia (KEGG) showed that host genes were mainly involved in hormone secretion, granulosa cell apoptosis, and follicular development. In conclusion, we identified a series of novel lncRNAs, constructed the lncRNA ceRNA regulatory network, and provided comprehensive resources for exploring the role of lncRNAs in yak ovarian follicular development.(AU)