Obesidade genética não sindrômica: histórico, fisiopatologia e principais genes / Non-syndromic genetic obesity: history, pathophysiology and key genes

A obesidade é definida pelo excesso de gordura corporal acumulada no tecido adiposo quando o indivíduo atinge valores de IMC igual ou superior a 30 Kg/m2. Constitui um dos principais fatores de risco para várias doenças não transmissíveis (DNTs) como por exemplo, diabetes mellitus tipo 2 (DM2), doenças cardiovasculares, hipertensão arterial, acidente vascular cerebral e até mesmo o câncer. Embora a obesidade esteja diretamente relacionada com o consumo calórico excessivo em relação ao gasto energético diário, sua etiologia pode estar associada aos baixos níveis de atividade física, às alterações neuroendócrinas e aos fatores genéticos. Considerando o componente genético, esta pode ser classificada como sindrômicas e estar associada às alterações cromossômicas estruturais ou numéricas, ou como não sindrômica, quando relacionada, principalmente, com os polimorfismos de nucleotídeos simples (SNPs) em alelos que atuam como herança monogênica, ou ainda com a interação vários genes (poligênica multifatorial). Apesar de existirem muitas etiologias diferentes, normalmente a obesidade é tratada a partir da mesma abordagem, desconsiderando a fisiologia que a desencadeou. Dessa forma, o objetivo do presente trabalho foi abordar a obesidade genética não sindrômica por meio a) da descrição breve de perspectiva histórica sobre seu entendimento; b) da exposição dos principais mecanismos moleculares envolvidos com o controle de peso; c) da compilação dos principais genes e SNPs relacionados; d) da definição dos principais genes; e e) da abordagem das principais perspectivas de intervenção.

Obesity is defined as excess body fat accumulated in the adipose tissue when the individual reaches BMI values equal to or greater than 30 kg/m2. It is one of the main risk factors for several non-communicable diseases (NCDs), such as Type 2 Diabetes mellitus (T2D), cardiovascular diseases, high blood pressure, stroke and even cancer. Although obesity is directly related to excessive calorie intake in relation to daily energy expenditure, its etiology may be associated with low levels of physical activity, neuroendocrine changes, and genetic factors. Considering the genetic component, it can be classified as syndromic and be associated with chromosomal or numerical changes, or as non-syndromic and being related mainly to single nucleotide polymorphisms (SNPs) in alleles that act as monogenic inheritance, or with an interaction of several genes (multifactorial polygenic). Although there are many different etiologies, obesity is usually treated using the same approach, disregarding the physiology that triggered it. Thus, the aim of this study was to address non-syndromic genetic obesity through a) a brief description of a historical perspective on its understanding; b) the exposure of the main molecular mechanisms involved in weight control, c) the compilation of the key genes and related SNPs, d) the definition of the key genes and e) the approach of the main intervention representations.

Expressão e localização celular da diguanilato ciclase DgcP em Pseudomonas aeruginosa / Expression and cell localization of the diguanylate cyclase DgcP in Pseudomonas aeruginosa

A bactéria Gram-negativa Pseudomonas aeruginosa é um patógeno oportunista frequentemente associado a vítimas de queimaduras graves ou indivíduos com fibrose cística, sendo os isolados resistentes a carbepenêmicos dessa espécie considerados pela OMS como uma das maiores ameaças ao controle de infecções. O estabelecimento da infecção por esse patógeno é dependente de uma série de fatores de virulência, entre eles o pilus tipo IV (T4P), que possui papel importante na adesão a superfícies e motilidade do tipo twitching, essenciais para a colonização do hospedeiro. Uma das moléculas importantes na diferenciação entre as formas séssil e planctônica de P. aeruginosa é o segundo mensageiro bis-(3,5)-di-guanosina monofosfato cíclico (c-di-GMP), cuja síntese é feita enzimaticamente por diguanilato ciclases (DGCs). DgcP é uma DGC localizada nos polos da célula, que tem sua atividade de síntese de c-di-GMP aumentada na presença da proteína FimV, essencial para a montagem do T4P em P. aeruginosa. Neste trabalho, ensaios de microscopia de fluorescência, organização e expressão gênica foram realizados com o objetivo de aumentar a compreensão sobre o papel de DgcP em relação a sua expressão e aos fatores que regulam o T4P de P. aeruginosa. A proteína DgcP em fusão com mNeonGreen no C-terminal, expressa a partir do locus cromossômico, se localiza de maneira predominantemente bipolar tanto na linhagem selvagem quanto nos mutantes ΔpilA, ΔpilR e ΔchpA, evidenciando que seu padrão de localização não depende dos sistemas de regulação Pil-Chp e PilS-PilR. Ensaios de RT-PCRmostraram que dgcP se encontra em operon com PA14_72430 e dsbA1, indicando um papel celular conjunto entre esses genes, até o momento, desconhecido. Por fim, ensaios de qRT-PCR revelaram que os níveis de mRNA de dgcP são invariáveis nas linhagens WT, ΔpilA, ΔpilR, ΔchpA e ΔfimV, cultivadas em meio líquido ou meio sólido. Os resultados aqui mostrados, combinados com trabalhos prévios do nosso e de outros grupos, sugerem que DgcP é uma diguanilato ciclase responsável por geração constante de c-di-GMP nos polos da célula, possivelmente, atuando na sinalização local dependente do dinucleotídeo cíclico, cuja localização e atividade não são dependentes dos sistemas de regulação que atuam sobre o T4P

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe burn victims or individuals with cystic fibrosis, which carbapenem-resistant isolates were classified by th World Health Organization classified one of the greatest threats to infection control. The establishment of infection by this pathogen is dependent on a series of virulence factors, including the type IV pilus (T4P), which plays an important role in adhesion to surfaces and twitching motility, essential features for host colonization. Bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger that involved in processes of biofilm formation, motility, and virulence. The diguanylate cyclase DgcP synthetizes cdi-GMP and it is located at the cell poles, and its activity depends on the scaffold protein FimV, essential for T4P assembly in P. aeruginosa. By increasing c-di-GMP levels, DgcP decreases flagellum-dependent motility and increases biofilm formation. In this work, fluorescence microscopy, gene organization and expression assays were performed to understand the whether DgcP localization and expression are under the control of T4P regulatory proteins. Fluorescence microscopy analysis showed that DgcP localizes predominantly at both cell poles in ΔpilA, ΔpilR, and ΔchpA mutants, showing that its localization pattern does not depend on the Pil-Chp and PilS-PilR systems. Furthermore, RT-PCR assays showed that dgcP is found in an operon with PA14_72430 and dsbA1, indicating an unknown putative related cellular role for these genes. Finally, qRT-PCR assays indicated that DgcP expression is invariant in ΔpilA, ΔpilR, ΔchpA, and ΔfimV mutants, either in liquid or solid medium. The results shownhere, combined with previous work by ours and other groups, suggest that DgcP is a diguanylate cyclase responsible for constant generation of c-di-GMP at the cell poles, possibly acting in local signaling dependent on the cyclic dinucleotide, but that is not under the control of the known T4P regulatory systems

Estudo imuno-histoquímico e classificação molecular dos adenocarcinomas de endométrio com expressão heterogênea dos genes de reparo do DNA / Immunohistochemical study and molecular classification of endometrial adenocarcinomas with heterogeneous expression of DNA repair genes

Introdução: O carcinoma endometrial (CE) foi classificado pelo sistema de Bokhman em tipos I e II com base em observações clínicas e epidemiológicas. O tipo I corresponde aos tumores de baixo grau e o tipo II aos tumores de alto grau. Adicionalmente, estudos recentes propuseram que a classificação também fosse baseada em aspectos histológicos e moleculares com base nos dados do TCGA (The Cancer Genome Atlas). Foram identificados quatro grupos moleculares distintos de CE: (1) com mutações no POLE (fenótipo "ultramutado"), (2) "alto número de cópias" (mutações em TP53), (3) !baixo número de cópias" (em que os tumores não apresentam nenhuma das alterações descritas nos outros tipos) e (4) tumores com predomínio de instabilidade de microssatélites. A imunohistoquímica (IHC) para proteínas do gene de reparo é usada para identificar a deficiência de genes de reparo do DNA (Mismatch Repair ­ MMR) associada à instabilidade de microssatélites(MSI). A coloração nuclear positiva representa a expressão retida de proteínas MMR, enquanto a perda completa representa deficiência de MMR. O padrão de expressão heterogênea (HEP), ou seja, concomitância em um mesmo espécime de áreas positivas e totalmente negativas tem sido observada em CE. No presente momento, as principais diretrizes determinam que a presença de HEP seja interpretada como expressão retida de proteínas MMR. Não há, porém, consenso quanto à classificação e interpretação de HEP, nem conhecimento do impacto da classificação de HEP como subtipo molecular diferente em relação às características clínicas e prognósticas. Objetivos: realizar a classificação molecular dos casos de CE com HEP das proteínas relacionadas aos genes de reparo do DNA e comparação do perfil molecular entre áreas positivas e negativas no estudo imunohistoquímico. Materiais e Métodos: De janeiro/2007 a dezembro/2017 foram identificados 356 casos de CE, 16 deles com HEP. A classificação molecular foi feita com base no protocolo PROMISE para CE. Cada área (expressão retida ou perdida) foi macrodissecada e o status molecular foi avaliado separadamente quanto ao status MSI (Idylla), metilação do promotor MLH1 (NGS - ponto de corte para positividade ≥ 15%), status POLE (NGS) e status p53 (IHC). Variáveis clínicas e patológicas também foram avaliadas e correlacionadas com cada caso. Resultados: A histologia endometrioide foi predominante (15 casos), bem como ausência de invasão linfovascular (11 casos), ausência de padrão MELF (10 casos), graus FIGO 1 e 2 (13 casos), invasão miometrial < 50% (13 casos) e estadiamento T1 (13 casos). Todos os pacientes estavam vivos e sem evidência de doença no último acompanhamento, exceto por um caso, cujo status de sobrevida era desconhecido. Dois casos que seriam descritos como apresentando expressão retida de proteínas relacionadas a genes de reparo do DNA por IHC apresentaram-se na análise molecular com instabilidade de microssatélites(MSI-H). Nos casos de HEP, a proteína MSH6 foi a maisfrequentemente envolvida (9 casos, 7 isolados). A proteína MLH1 apresentou-se alterada em 6 casos, sendo a única proteína associada a co-alterações (com MSH6 e PMS2). Seis casos apresentaram-se metilados por MLH1, padrão encontrado tanto em áreas com perda quanto em áreas com retenção das proteínas relacionadas a MMR por IHC e dois casos apresentaram metilação em apenas uma das áreas. Em relação ao status de POLE, 6 casos apresentaram mutação, 2 com mutações tanto em áreas com perda quanto em áreas com retenção de expressão, 3 apenas na área com perda e 1 apenas na área com retenção. Dois casos apresentam padrão aberrante de p53 (MSH6 alterados) em ambas as áreas. Conclusão: em pacientes portadoras CE e com tumores apresentando HEP a correlação entre a IHC e os achados moleculares é heterogênea e o diagnóstico entre casos com retenção ou das proteínas relacionadas a MMR não é factível apenas com realização de IHC. A análise molecular deve ser realizada em todos os casos de CE com HEP para determinar adequadamente as característicasintrínsecas de cada tumor. Devido à raridade desse achado, esta proposta é financeiramente viável e tem o potencial de mudar a prática clínica em um subconjunto de pacientes, permitindo tratamentos inovadores. HEP deve ser relatado como um padrão distinto e não considerado como uma expressão sinônimo de expressão retida de proteínas MMR em CE.

Introduction: Endometrial adenocarcinoma is classified by the Bokhman system in type I and II based on clinical and epidemiological observations, whereas the type I represents low grade tumors and type II high grade tumors. Additionally, a classification based on histological aspects and molecular profile has been proposed. The TCGA (The Cancer Genome Atlas) identified four molecular groups of endometrial adenocarcinomas: (1) mutations in POLE ("ultramutated" phenotype), (2) "high copy number" (mutations in TP53), (3) "low number of copies " (in which the tumors do not exhibit any of the changes described in the other types) and (4) tumors with predominance of microsatellite instability. In a small number of patients, heterogeneous staining is observed in the evaluation protein expression for mismatch repair genes. Objectives: to evaluate and perform the molecular classifications of cases of endometrial carcinoma with heterogeneous staining by IHC of proteins related to mismatch repair genes and comparison of the molecular profile of positive and negative areas in the IHC study. Cases and Methods: From January/2007 to December/2017 354 cases with EC were identified, 16 of those with HEP. Molecular classification was made based on the PROMISE protocol for EC. Each area (retained and lost expression) was macrodissected and molecular status was evaluated separately regarding MSI status (Idylla), MLH1 promoter methylation (NGS - cutoff for positivity ≥ 15%), POLE status (NGS) and p53 status (IHC). Clinical and pathologic variables were also evaluated and correlated with each case. Results: Endometrioid histology was predominant (15 cases), as absent lymphovascular invasion (11 cases), absence of MELF pattern (10 cases), FIGO Grade 1 and 2 (13 cases), and T1 stage (13 cases). All patients were alive and disease-free at the last follow-up. Two cases that would be described as retained by IHC presented in the molecular analysis as MSI-H. In HEP cases MSH6 was more frequent (9 cases, 7 isolated). MLH1 was altered in 6 cases, and wasthe only protein associated with co-alterations (with MSH6 and PMS2). Six cases were MLH1 methylated, found both in lost and retained areas. As POLE status, there were 6 mutated cases, 2 of those with mutations both in lost and retained areas, and 3 the lost area. Two cases had p53 aberrant pattern (MSH6 altered), that was seen both in the retained and in the lost areas. Conclusion: Correlation between IHC and molecular findings is heterogeneous, and determination between retained or lost expression of MMR proteins by IHC when HEP occurs, however feasible, does not represent the actual molecular alterations. Thus, molecular analysis should be performed every case to adequately determine the intrinsic features of each tumor. Due to the rarity of this finding, this is financially viable and has the potential to change clinical practice in a subset of patients. HEP should be reported as a distinct pattern, and not considered as a synonym expression of retained expression of MMR proteins in EC.

Chronic Lymphocytic Leukemia (CLL): evaluation of AKT protein kinase and microRNA gene expression related to disease pathogenesis

Abstract The present study evaluated 56 patients diagnosed with Chronic Lymphocytic Leukemia (CLL) and a control group of 44 clinically healthy subjects with no previous history of leukemia. Genetic expressions of AKT and microRNAs were evaluated by quantitative PCR (qPCR). A significant increase in AKT gene expression in patients when compared to controls was observed (p = 0.017). When the patients were stratified according to Binet subgroups, a significant difference was observed between the subgroups, with this protein kinase appearing more expressed in the B+C subgroup (p = 0.013). Regarding miRNA expression, miR-let-7b and miR-26a were reduced in CLL patients, when compared to controls. However, no significant differences were observed in these microRNA expressions between the Binet subgroups (A versus B+C). By contrast, miR-21 to miR-27a oncogenes showed no expression difference between CLL patients and controls. AKT protein kinase is involved in the signaling cascade that occurs with BCR receptor activation, leading to increased lymphocyte survival and protection against the induction of cell death in CLL. Thus, increased AKT protein kinase expression and the reduction of miR-let-7b and miR-26a, both tumor suppressors, may explain increased lymphocyte survival in CLL patients and may be promising markers for the prognostic evaluation of this disease.

Genotypic evaluation of antimicrobial resistance in Staphylococcus spp. isolated from bovine clinical mastitis / [Avaliação genotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite clínica bovina]

Bovine clinical mastitis caused by Staphylococcus spp. is a serious and widespread disease in the world of dairy farming. Antimicrobial therapy is of fundamental importance in the prevention and treatment of infectious mastitis, but the indiscriminate use of antimicrobials acts as a determining factor for the spread of the disease. The present study evaluated the resistance profiles of 57 Staphylococcus spp. isolated from bovine clinical mastitis to beta-lactams and gentamicin, relating characteristics of phenotype (in vitro susceptibility tests) and genotype (detection and expression of genes encoding resistance - mecA, mecALGA251, blaZ, femA, femB, and aacA-aphD - using PCR and RT-PCR, respectively). One or more genes coding for resistance to different antimicrobials were detected in 50 Staphylococcus spp. isolates. The femA and femB genes were the most frequent (75.4% for both). The observed expression of the genes was as follows: blaZ (60%), femA (39.5%), aacA-aphD (50%), femB (32.6%), mecA (8.3%), and mecALGA251 (0%). Considering the relevance of the genus Staphylococcus to bovine mastitis, this study aimed to elucidate aspects regarding the genotypic and phenotypic profiles of these microorganisms so as to contribute to the development of effective strategies for mastitis control.(AU)

A mastite clínica bovina causada por Staphylococcus spp. é uma doença grave e generalizada no mundo da pecuária leiteira. A terapia antimicrobiana é de fundamental importância na prevenção e no tratamento da mastite infecciosa, mas o uso indiscriminado de antimicrobianos atua como fator determinante para a disseminação da doença. O presente estudo avaliou os perfis de resistência de 57 Staphylococcus spp. isolados de mastite clínica bovina em relação ao uso de betalactâmicos e gentamicina, relacionando características do fenótipo (testes de suscetibilidade in vitro) e genótipo (detecção e expressão de genes que codificam resistência - mecA, mecALGA251, blaZ, femA, femB, e aacA-aphD - usando PCR e RT-PCR, respectivamente). Um ou mais genes que codificam resistência a diferentes antimicrobianos foram detectados em 50 Staphylococcus spp. isolados. Os genes femA e femB foram os mais frequentes (75,4% para ambos). A expressão observada dos genes foi a seguinte: blaZ (60%), femA (39,5%), aacA-aphD (50%), femB (32,6%), mecA (8,3%) e mecALGA251 (0%). Considerando-se a relevância do gênero Staphylococcus para a mastite bovina, este estudo teve como objetivo elucidar aspectos referentes aos perfis genotípico e fenotípico desses microrganismos, a fim de contribuir para o desenvolvimento de estratégias eficazes para o controle da mastite.(AU)

Influência da nutrição na expressão de genes relacionados à obesidade / Influence of nutrition on the expression of genes related to obesity

A obesidade possui vários prejuízos para a saúde e está associada à inúmeras patologias e baixa expectativa de vida. O desequilíbrio alimentar é um fator que necessita de atenção especial, pois é capaz de alterar as interações entre nutrientes e genes. O objetivo deste trabalho foi verificar as principais linhas de pesquisa associadas à nutrigenômica, e evidenciar a relação da influência da nutrição na expressão de genes relacionados à obesidade. Realizou-se o levantamento bibliográfico e a análise cienciométrica por meio do banco de dados publicados na Biblioteca Virtual em Saúde (BVS) e do Centro Latino-Americano de Informação em Ciências da Saúde (BIREME). Identificou-se 118 artigos originais, os quais foram agrupados em cinco classes: restrição calórica, expressão gênica, alimentos, intervenção dietética e diversos. Os resultados evidenciaram que a restrição calórica possui relação direta da expressão gênica com o controle das células cancerígenas e a diminuição do excesso de tecido adiposo. Além disso, a análise cienciométrica relacionou a importância das fibras alimentares na redução do colesterol e sensibilidade à insulina, bem como a ação do jejum na regulação negativa de genes que contribuem para o crescimento do tecido adiposo. Dessa forma, este artigo fornece princípios ideológicos para auxiliar especialistas na aplicabilidade de estratégias para atingir a redução de peso sustentável por meio da expressão gênica.

Obesity has several health risks and is associated with numerous pathologies and low life expectancy. Food imbalance is a factor that needs special attention, as it is able to change the interactions between nutrients and genes. This study aimed at verifying the main lines of research associated with nutrigenomics, and at showing the relationship of the influence of nutrition on the expression of genes related to obesity. The bibliographic survey and scientometric analysis were carried out through the database published in the Biblioteca Virtual em Saúde (BVS) and the Centro Latino-Americano de Informação em Ciências da Saúde (BIREME). A total of 118 articles of original research were identified, and were grouped into five categories: caloric restriction; gene expression; food; dietary intervention; and miscellaneous. The results showed that caloric restriction has a direct relationship between gene expression and the control of cancer cells and the reduction of excess adipose tissue. Furthermore, the scientometric analysis related the importance of dietary fibers in reducing cholesterol and insulin sensitivity as well as the action of fasting in the negative regulation of genes that contribute to the growth of adipose tissue. Thus, this paper provides ideological principles to assist specialists in the applicability of strategies to achieve sustainable weight reduction through gene expression.

Estudo da expressão gênica dos principais metabolizadores de fase II de xenobióticos: GSTM1, GSTP1 e GSTT1 em carcinoma de células escamosas bucal / Study of the gene expression of the main xenobiotic phase II metabolizers: GSTM1, GSTP1 and GSTT1 in squamous cell carcinoma of the oral cavity

Tabaco e álcool são considerados os principais fatores de risco para o carcinoma de células escamosas (CCE) bucal contribuindo de maneira desfavorável para o tratamento e desfecho clínico. Seus carcinógenos são metabolizados em duas fases, sendo a segunda fase realizada pelas Glutationa S-transferases (GSTs). O objetivo do presente estudo foi avaliar a expressão gênica da forma selvagem dos genes GSTM1, GSTP1 e GSTT1 por qPCR em 33 amostras de CCE bucal de fumantes, ex-fumantes e não fumantes, e 15 controles em busca de uma correlação clínica com consumo de tabaco, álcool e estadiamento clínico. A dependência nicotínica foi avaliada pelo Teste de Fagerström pra Dependência a Cigarros (TFDC) e para consumo de etílicos o Teste AUDIT. Foi observado aumento da expressão de GSTM1 no Grupo CCE fumante em relação ao Grupo Controle (p=0,0161). Contrariamente, foi encontrada uma menor expressão de GSTT1 no Grupo CCE fumante em relação ao Grupo Controle fumante (p=0,0183). No grupo CCE fumante não foi encontrada uma correlação entre a expressão dos genes estudados e fatores ligados ao tabagismo, etilismo e estadiamento clinico. No grupo Controle fumante, houve correlação entre teste AUDIT e a expressão de GSTM1 (p=0,0000). Para GSTP1 e GSTT1 houve correlação entre a expressão quando comparada a idade do paciente (p=0,0008; p=0,0095), idade de inicio do tabagismo (p=0,0033; p=0,0081), TFDC (p=0,0102; p=0,0085) e AUDIT (p=0,0052; p=0,0219) respectivamente. Para GSTT1 foi encontrada uma correlação entre a expressão e número de cigarros/dia (p=0,0175). Concluímos que as formas selvagens das GSTs estudadas apresentaram uma alta expressão nas amostras de CCE bucal, entretanto, quantitativamente essa expressão foi baixa, com grande variabilidade interindividual. Outrossim, não houve uma correlação direta entre níveis de expressão, carga tabágica, TFDC, teste AUDIT e estadiamento clínico. O aumento da expressão de GSTM1 e GSTP1 parece não ter tido um efeito protetor. A baixa expressão de GSTT1 em pacientes fumantes com CCE bucal se mostrou um potencial marcador a ser avaliado em pacientes fumantes que ainda não desenvolveram uma neoplasia maligna(AU)

Tobacco and alcohol are considered to be the main risk factors for oral squamous cell carcinoma (SCC), contributing to treatment and clinical outcome. Its carcinogens are metabolized in two phases, being the second phase carried out by Glutathione Stransferases (GSTs). The objective of the present study was to evaluate the wild-type gene expression of the GSTM1, GSTP1 and GSTT1 genes by qPCR in 33 samples of oral SCC from smokers, former smokers and nonsmokers, and 15 controls looking for a clinical correlation with tobacco and alcohol consumption and clinical staging. Nicotinic dependence was assessed by the Fagerström Test for Cigarette Dependence (TFCD) and alcohol consumption by the AUDIT Test. Increased expression of GSTM1 in the Smoker SCC Group was observed in relation to the Control Group (p=0.0161). Conversely, a lower expression of GSTT1 was found in the smoker SCC group compared to the Smoker Control Group (p=0.0183). In the smoker SCC group, no correlation was found between the genes expression studied and factors related to smoking, alcoholism and clinical staging. In the Smoker Control Group, there was a correlation between the AUDIT test and the GSTM1 expression (p=0.0000). For GSTP1 and GSTT1, there was a correlation between the expression compared with the patient's age (p=0.0008, p=0.0095), age of starting smoking (p=0.0033, p=0.0081), FTCD (p=0.0102, p=0.0085) and AUDIT (p=0.0052, p=0.0219) respectively. For GSTT1 a correlation was found between expression and number of cigarettes/day (p=0.0175). We concluded that the wild forms of the GSTs studied presented a high expression in the samples of oral SCC; however, quantitatively this expression was low, with great interindividual variability. Also, there was no direct correlation between levels of expression, pack-years, FTCD, AUDIT Test and clinical stage. Increased expression of GSTM1 and GSTP1 appears to have had no protective effect. The low GSTT1 expression in smokers with oral SCC was shown to be a potential marker to be evaluated in smoker patients who have not yet developed a malignant neoplasm(AU)

A broad pH range and processive chitinase from a metagenome library

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

MicroRNA-122 regulates caspase-8 and promotes the apoptosis of mouse cardiomyocytes

Cardiomyocyte apoptosis plays key roles in the pathogenesis of heart diseases such as myocardial infarction. MicroRNAs are important regulators of gene expression, which are also involved in the regulation of cardiomyocyte apoptosis. However, cardiomyocyte apoptosis regulated by microRNA (miR)-122 is largely unexplored. The aim of this study focused on the role of miR-122 in cardiomyocyte apoptosis. Cardiomyocytes were isolated from neonatal mice and primarily cultured. MiR-122 mimic and inhibitor were transfected to cardiomyocytes and verified by qRT-PCR. Cell viability and apoptosis post-transfection were assessed by MTT assay and flow cytometry, respectively. Changes in expression of caspase-8 were quantified by qRT-PCR and western blot. Results showed that miR-122 mimic and inhibitor successfully induced changes in miR-122 levels in cultured cardiomyocytes (P<0.01). MiR-122 overexpression suppressed viability and promoted apoptosis of cardiomyocytes (P<0.05), and miR-122 knockdown promoted cell viability and inhibited apoptosis (P<0.05). The mRNA and protein levels of caspase-8 were elevated by miR-122 overexpression (P<0.01) and reduced by miR-122 knockdown (P<0.001). These results suggest an inductive role of miR-122 in cardiomyocyte apoptosis, which may be related to its regulation on caspase-8.

Relationship between matrix metalloproteinases and the occurrence and development of ovarian cancer

Ovarian cancer is one of the most malignant genital cancers, with a high mortality rate. Many researchers have suggested that matrix metalloproteinases (MMPs) have remarkably high expression in ovarian cancer tissues. MMPs are considered to be related to the occurrence, development, invasion and metastasis of ovarian cancer. Moreover, some studies have discovered that the unbalance between MMPs and tissue inhibitor of metalloproteinases (TIMPs) are associated with the malignant phenotype of tumors. This review summarizes the latest research progress of MMPs in ovarian cancer. The investigation of MMP mechanism in ovarian cancer will facilitate the development of effective anti-tumor drugs, and thereby improve the survival rate of patients with ovarian cancer.

Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients

ABSTRACT PURPOSE: To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. METHODS: Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. RESULTS: Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. CONCLUSION: There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

Three new shuttle vectors for heterologous expression in Zymomonas mobilis

Background: Zymomonas mobilis, as a novel platform for bio-ethanol production, has been attracted more attention and it is very important to construct vectors for the efficient expression of foreign genes in this bacterium. Results: Three shuttle vectors ( pSUZM 1, pSUZM2 and pSUZM3 ) were first constructed with the origins of replication from the chromosome and two native plasmids (pZZM401 and pZZM402) of Z. mobilis ZM4, respectively. The three shuttle vectors were stable in Z. mobilis ZM4 and have 3,32 and 27 copies, respectively. The promoter Ppdc (a), from the pyruvate decarboxylase gene, was clonedinto the shuttle vectors, generatingthe expressionvectors pSUZM1(2, 3)a. The codon-optimized glucoamylase gene from Aspergillus awamori combined with the signal peptide sequence from the alkaline phosphatase gene of Z. mobilis was cloned into pSUZM1(2, 3)a, resulting in the plasmids pSUZM1a-GA, pSUZM2a-GA and pSUZM3a-GA, respectively. After transforming these plasmids into Z. mobilis ZM4, the host was endowed with glucoamylase activity for starch hydrolysis. Both pSUZM2a-GA and pSUZM3a-GA were more efficientatproducingglucoamylase thanpSUZM1a-GA. Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

Regulação epigenética da expressão gênica de Schistosoma mansoni induzida por inibidor de histona deacetilase / Epigenetic regulation of gene expression in Schistosoma mansoni induced by histone deacetilase inhibitor

A esquistossomose é um grave problema de saúde pública, com alta mortalidade e morbidade em países endêmicos, causada pelo verme trematódeo do gênero Schistosoma. O praziquantel é a única droga disponível para tratamento da doença, é usada em larga escala para tratamento de populações de áreas endêmicas, porém não previne a reinfecção e tem efeito somente em vermes adultos. Drogas estudadas em câncer como inibidores de histona deacetilases (iHDACs) modificam o padrão epigenético da célula desencadeando a morte celular, e em Schistosoma mansoni já foi mostrado que a inibição de HDACs além de aumentar a acetilação de histonas alterou o fenótipo de miracídios e provocou morte em esquistossômulos e vermes adultos. O presente estudo investigou o efeito do iHDAC Trichostatin A (TSA) na regulação da transcrição gênica em esquistossômulos, detectando por meio de ensaios de microarray centenas de genes diferencialmente expressos, relacionados a replicação de DNA, metabolismo e complexos modificadores de histonas. A inibição de HDAC em vermes adultos levou a um aumento da acetilação nas marcas de histonas H3K9ac, H3K14ac e H4K5ac relacionadas à indução de transcrição. Com imunoprecipitação de cromatina seguida de PCR (ChIP-qPCR) detectou-se o aumento de deposição de H3K9ac e H3K14ac na região promotora de genes com expressão aumentada ou diminuída, porém a marca de repressão H3K27me3 não sofreu alteração na região promotora de nenhum gene analisado. Análises adicionais indicaram um conjunto de genes diferencialmente expressos que codificam proteínas histone readers, que fazem parte de complexos modificadores de histonas, como EED capaz de identificar a marca de repressão H3K27me3 e regular a atividade de EZH2, apontando um novo alvo terapêutico. O efeito sinérgico entre iHDAC e um iEZH2 foi testado e detectou-se o aumento da mortalidade de esquistossômulos. A estrutura de SmEZH2 foi modelada por homologia e usada para análises computacionais que sugeriram uma alta afinidade de ligação de SmEZH2 com o iEZH2, abrindo uma perspectiva de desenvolvimento de novas drogas específicas para tratamento da esquistossomose

Schistosomiasis is a serious public health problem, with high mortality and morbidity in endemic countries, caused by trematode worms of the genus Schistosoma. Praziquantel is the only available drug for treatment of the disease; it is used extensively to treat populations in endemic areas, but does not prevent reinfection and is effective only in adult worms. Drugs studied in cancer as histone deacetylase inhibitors (iHDACs) modify the epigenetic status of the cell, triggering cell death, and it has been shown in Schistosoma mansoni that inhibition of HDACs increase histone acetylation, alter the phenotype of miracidia and cause death in schistosomules and adult worms. The present study investigated the effect of iHDAC Trichostatin A (TSA) on the regulation of gene transcription in schistosomules, detecting by means of microarray assays hundreds of differentially expressed genes related to DNA replication, metabolism and histone remodeling complexes. Inhibition of HDAC in adult worms led to an increase in histone acetylation marks H3K9ac, and H3K14ac H4K5ac related to transcriptional induction. With chromatin immunoprecipitation followed PCR (ChIP-qPCR) we detected an increased deposition of H3K9ac and H3K14ac at the promoter region of genes with increased or decreased expression, but the repressive mark H3K27me3 was not changed at all analyzed gene promoter regions. Additional analysis indicated a set of differentially expressed genes that encode histone reader proteins that are part of histone modifier complexes such as EED, which is able to identify the repression mark H3K27me3 and to regulate EZH2 activity, pointing to a new therapeutic target. The synergistic effect between iHDAC and one iEZH2 has been tested and found to cause an increase in schistosomules mortality. The SmEZH2 structure was modeled by homology and used for computational analyses, which suggested a high affinity binding of SmEZH2 with iEZH2, opening the opportunity for development of new specific drugs for treatment of schistosomiasis

Expressão gênica de metaloproteinases e de seus reguladores em neoplasias mieloproliferativas: associação com biomarcadores de angiogênese e status mutacional / Gene expression of metalloproteinases and theirs regulators myeloproliferative neoplasms: association with angiogenesis markers and mutational status

As neoplasias mieloproliferativas (NMPs) BCR-ABL1 negativas compreendem a mielofibrose primária (PMF), trombocitemia essencial (TE) e a policitemia vera (PV). A patogênese e progressão dessas NMPs não estão completamente elucidadas. As metaloproteinases de matriz (MMPs) degradam a matriz extracelular, ativando citocinas e fatores de crescimento que, por sua vez, participam da tumorigênese e angiogênese. O objetivo deste estudo foi avaliar a relação da expressão gênica das MMPs, TIMPs, HIF1-α e SPARC com os marcadores angiogênicos bFGF e VEGFA em pacientes com MF e TE, considerando o status mutacional; bem como avaliar a regulação desses genes em camundongos submetidos à hipóxia, e em modelos HIF1-α(-/-) e VHL(-/-). Foram incluídos 21 pacientes com MF, 21 com MF pós-TE, 6 com MF pós-PV, 23 com TE e 78 indivíduos controle. As análises realizadas foram: dosagem sérica e expressão de RNAm de MMP2, MMP9, TIMP1, TIMP2 e SPARC, hemograma, determinação da proteína C reativa ultrassensível, determinação das concentrações de VEGFA e bFGF e avaliação das mutações nos genes JAK2, cMPL e CALR. A avaliação da densidade microvascular da medula óssea foi feita em 30 dos pacientes incluídos. Os pacientes com MFP, MFPTE e TE apresentaram maior expressão de MMP2, SPARC, TIMP1, TIMP2 e bFGF quando comparados aos seus controles (P<0,05), enquanto MMP9 foi mais expressa nos pacientes com MFPTE e TE (P= 0,011 e P=0,047, respectivamente). Os pacientes com TE apresentaram maior expressão de HIF1-α e VEGFA em relação ao grupo controle (P<0,05). Pacientes com MF JAK2V617F positivos apresentaram maiores concentrações de MMP9, TIMP2, bFGF e VEGFA quando comparados aos pacientes portadores de mutações na CALR (P<0,05). Os pacientes com TE JAK2V617F positivos apresentaram maiores concentrações de MMP2 e TIMP2 (P=0,049 e P=0,020, respectivamente). As concentrações das proteínas estudadas não apresentaram correlação com a carga alélica de JAK2V617F e nem com a densidade microvascular da medula óssea. Células de medula óssea de camundongos submetidos à hipóxia apresentaram maior expressão de MMP2 e TIMP1 comparados aos camundongos em normóxia. Camundongos VHL(-/-) apresentaram aumento na expressão dos genes MMP2, MMP9, TIMP1, TIMP2 e VEGFA. Diferentemente, embriões HIF1-α(-/-) não foram considerados um bom modelo para este estudo devido ao envolvimento das MMPs na embriogênese/organogênese. Frente aos resultados encontrados, pode-se sugerir que a maior expressão de MMP2, SPARC e de bFGF estão associadas às NMPs. A mutação JAK2V617F foi associada a maiores concentrações de MMPs, TIMP2 VEGFA e bFGF. HIF1-α foi mais expresso na PV e na TE, sugerindo uma possível regulação da expressão das MMPs e TIMPs nessas doenças

Myeloproliferative neoplasms (MPNs) BCR-ABL1-negative include primary myelofibrosis (PMF), essential thrombocythemia (ET) and polycythemia vera (PV). The mechanisms underlying the pathology and disease progression in MPN are not completely elucidated. The matrix metalloproteinases (MMPs) cleave extracellular matrix, activating cytokines and growth factors that, in turn, regulate tumorigenesis and angiogenesis. The aim of this study was to evaluate the relationship of MMPs, TIMPs, HIF1-α and SPARC gene expression with angiogenic markers bFGF and VEGFA in patients with MPN considering their mutational status; as well as to assess the regulation of these genes in animal models HIF1-α and VHL knockouts. Twenty-one MF, 21 MF post-ET, 6 MF post-PV, 23 ET patients and 78 controls were enrolled. The analysis performed in peripheral blood were: serum and mRNA expression of MMP2, MMP9, TIMP1, TIMP2 and SPARC, blood count, high-sensitivity C-reactive protein determination and VEGFA and bFGF measurements in plasma. We also evaluate mutations in JAK2, MPL and CALR. The assessment of microvascular density (MVD) in bone marrow was performed in 30 patients. Patients with MFP, MFPET and ET presented higher expression of MMP2, SPARC, TIMP1, TIMP2 and bFGF compared to their controls (P <0.05), while MMP9 expression was higher in patients with MFPET and ET (P=0.011 and P=0.047, respectively). Higher expression of HIF1-α and VEGFA was found in ET patients compared to the controls (P <0.05). PMF JAK2V617F patients had higher concentrations of MMP9, TIMP2, bFGF and VEGFA compared to CALR mutated ones (P <0.05). ET patients JAK2V617F positive had higher levels of MMP2 and TIMP2 (P=0.049 and P=0.020, respectively). The JAK2V617F allele burden was not associated with MVD in the bone marrow. Bone marrow cells from mice in hypoxia condition showed higher MMP2 and TIMP1 expression compared to the control. VHL(-/-) mice exhibited increased expression of MMP2, MMP9, TIMP1, TIMP2 and VEGFA. In contrast, the HIF1-α(-/-) embryos were not considered an applicable model for this study due to MMPs role in embryogenesis/organogenesis. In view of these findings, we can conclude that increased expression of MMP2, SPARC and bFGF are associated with MPN. The JAK2V617F mutation was associated with higher concentrations of MMPs, TIMP2 VEGFA and bFGF. HIF1-α is upregulated in PV and ET and perhaps regulate the MMPs and TIMPs expression in these diseases

Lipídios estruturados obtidos por interesterificação da tributirina com óleo de linhaça e seu potencial quimiopreventivo durante a fase de promoção inicial da hepatocarcinogênese experimental em ratos / Structured lipids obtained from intersterification of tributyrin and flax seed oil and their chemopreventive potential during early promotion phase of experimental hepatocarcinogenesis in rats

A combinação de agentes quimiopreventivos com diferentes mecanismos de ação tem sido considerada uma estratégia promissora para a prevenção do câncer. Dentre os diversos compostos bioativos em alimentos, destacam-se a tributirina, um pró-fármaco do ácido butírico presente em laticínios e produzido pela fermentação de fibras dietéticas, e o óleo de linhaça, fonte de ácido alfa linolênico. Nesse contexto, foi avaliada a atividade quimiopreventiva de lipídios estruturados obtidos a partir da interesterificação enzimática de tributirina e óleo de linhaça durante a fase de promoção inicial da hepatocarcinogênese experimental. Ratos Wistar machos submetidos ao modelo do hepatócito resistente receberam diariamente, por via intragástrica (i.g), maltodextrina, óleo de linhaça, tributirina, a mistura não esterificada ou lipídios estruturados durante a fase de promoção inicial. O tratamento com lipídios estruturados demonstrou atividade quimiopreventiva comparável à da tributirina, mesmo resultando em menor concentração hepática de ácido butírico. Tanto a tributirina quanto os lipídios estruturados não inibiram a proliferação celular em lesões preneoplásicas, mas induziram a apoptose naquelas em remodelação. Os efeitos inibitórios da tributirina em fases iniciais da hepatocarcinogênese experimental estão relacionados ao aumento da acetilação de histonas e à modulação de processos de translocação nuclear da p53. No presente estudo, foi observado aumento substancial da razão nuclear/citoplasmática de p53 e importina-alfa em fígados de animais submetidos ao modelo e tratados com tributirina, mas não nos tratados com lipídios estruturados. Por outro lado, o tratamento com lipídios estruturados reduziu a expressão dos oncogenes Bcl2, Ccnd2, Pdgfa, Vegfa e aumentou a expressão dos genes supressores de tumor Cdh13, Fhit e Socs3. Assim, embora o potencial quimiopreventivo dos lipídios estruturados seja comparável ao da tributirina, os resultados sugerem que o novo composto não exibe atividade de HDACi, e que seus efeitos inibitórios na hepatocarcinogênese possam ser atribuídos à modulação da expressão de oncogenes e genes supressores de tumor

Combination of chemopreventive agents with different mechanisms of action has been considered a promising strategy to cancer prevention. Among several bioactive food compounds, tributyrin, a butyric acid prodrug obtained from dairy products and dietetic fiber fermentation, and flax seed oil, a rich source of alpha linolenic acid have shown chemopreventive potential. Here, we evaluated the chemopreventive activity of structured lipids obtained by enzymatic interesterification of tributyrin and flax seed oil during the early promotion phase of experimental hepatocarcinogenesis. Male Wistar rats subjected to the resistant hepatocyte model were treated daily, i.g, with maltodextrin, flax seed oil, tributyrin, non-sterified blend, or structured lipids. Treatment structured lipids showed similar chemopreventive activity compared to tributyrin, even when structured lipids yielded lower concentrations of butyric in the liver. Tributyrin and structured lipids did not inhibit cell proliferation in preneoplastic lesions, but both of them induced apoptosis in remodeling preneoplastic lesions. In addition, histone acetylation and p21 restored expression tributyrin molecular mechanisms were related to modulation of p53 nuclear shuttling mechanisms. In the present study, it was observed a substantial increase in p53 nuclear/cytoplasmic ratio and importin-alpha in preneoplastic livers of tributyrin treated rats, but not in those treated with structured lipids. In contrast, treatment structured lipids downregulated expression of major oncogenes Bcl2, Ccnd2, Pdgfa, and Vegfa; and upregulated expression of critical tumor suppressor genes, Cdh13, Socs3 and Fhit. Hence, although structured lipids and tributyrin show similar chemopreventive potential, the results suggest that the new compound does not exhibit HDACi activity, and that its inhibitory effects may be attributed to the modulation of oncogenes and tumor suppressor genes expression

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway^® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.

Phytase production through response surface methodology and molecular characterization of Aspergillus fumigatus NF191.

Phytase play an important role in phytic acid catalysis that act as a food inhibitor in cereals. Here, we isolated high phytase producing isolates NF191 closely related to Aspergillus fumigatus sp. from piggery soil. DNA was isolated from the fungal culture and amplified the ITS region using ITS1 and ITS4 primer using PCR. The 400-900 bp amplicon was gel eluted and subjected to sequencing. The sequencing results were assembled and compared with NCBI data base which showed the 99% identity of Aspergilllus fumigatus. Different carbon sources viz., fructose, galactose, lactose, dextrose, sucrose, maltose and different nitrogen sources (organic & inorganic) NH4Cl, NH4NO3, (NH4)2SO4, KNO3, NaNO3, urea, yeast extract, peptone, beef extract were tested for optimal production. The 0.3% dextrose, 0.5% NH4NO3 and 96 h incubation time showed the best production and enzyme activity at 45 ºC incubation temperature. The selected parameters, dextrose, ammonium sulphate and incubation time, when employed with statistical optimization approach involving response surface optimization using Box Behnken Design, gave a 1.3 fold increase in phytase production compared to unoptimized condition.

Isolation and gene expression analysis of Phospholipase C in response to abiotic stresses from Vigna radiata (L.) Wilczek.

Phosphatidylinositol (PtdIns) is a major phospholipid in eukaryotic cells. Many studies have revealed that the phosphoinositide (PI) signaling pathway plays an important role in plant growth and development. Phospholipase C (PLC) is reported to have a crucial role in the PI pathway. This work focuses on the isolation and investigation of PLC in response to abiotic stress factors in green gram. The PLC cDNA, designated VrPLC, encoding a protein of 591 amino acids was cloned and expressed in E. coli.The predicted isoelectric point (pI) and molecular weight were 5.96 and 67.3 kDa, respectively. The tertiary structure of the PLC was also predicted and found to be mainly composed of random coils. In addition, VrPLC expression analysis was performed under environmental stress and the results showed that the expression of VrPLC was rapidly induced in an abscisic acid independent manner in response to drought and salt stress. PLC expression was found to be up-regulated by SA and down-regulated by wound in leaf tissues; however, there was no significant difference in the expression of PLC in plants subjected to high temperature and H2O2. Our results suggest that a close link/relationship between PLC expression and stress responses in green gram.

Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis

We measured circulating endothelial precursor cells (EPCs), activated circulating endothelial cells (aCECs), and mature circulating endothelial cells (mCECs) using four-color multiparametric flow cytometry in the peripheral blood of 84 chronic myeloid leukemia (CML) patients and 65 healthy controls; and vascular endothelial growth factor (VEGF) by quantitative real-time PCR in 50 CML patients and 32 healthy controls. Because of an increase in mCECs, the median percentage of CECs in CML blast crisis (0.0146%) was significantly higher than in healthy subjects (0.0059%, P<0.01) and in the accelerated phase (0.0059%, P=0.01). There were no significant differences in the percentages of CECs in chronic- or active-phase patients and healthy subjects (P>0.05). In addition, VEGF gene expression was significantly higher in all phases of CML: 0.245 in blast crisis, 0.320 in the active phase, and 0.330 in chronic phase patients than it was in healthy subjects (0.145). In conclusion, CML in blast crisis had increased levels of CECs and VEGF gene expression, which may serve as markers of disease progression and may become targets for the management of CML.