A role for mammalian target of rapamycin (mTOR) pathway in non alcoholic steatohepatitis related-cirrhosis

Non-alcoholic fatty liver disease (NAFLD) encompasses the whole spectrum of steatosis, nonalcoholic steatohepatitis (NASH), and NASH-related cirrhosis (NASH/Cir). Although molecular advances have been made in this field, the pathogenesis of NAFLD is not completely understood. The gene expression profiling associated to NASH/Cir was assessed, in an attempt to better characterize the pathways involved in its etiopathogenesis. Methods: In the first step, we used cDNA microarray to evaluate the gene expression profiles in normal liver (n=3) and NASH/Cir samples (n=3) by GeneSifter (TM) analysis to identify differentially expressed genes and biological pathways. Second, tissue microarray was used to determine immunohistochemical expression of phosphorylated mTOR and 4E-BP1 in 11 normal liver samples, 10 NASH/Cir samples and in 37 samples of cirrhosis of other etiologies to further explore the involvement of the mTOR pathway evidenced by the gene expression analysis. Results: 138 and 106 genes were, respectively, up and down regulated in NASH/Cir in comparison to normal liver. Among the 9 pathways identified as significantly modulated in NASH/Cir, the participation of the mTOR pathway was confirmed, since expression of cytoplasmic and membrane phospho-mTOR were higher in NASH/Cir in comparison to cirrhosis of other etiologies and to normal liver. Conclusions: Recent findings have suggested a role for the cellular ""nutrient sensor"" mTOR in NAFLD and the present study corroborates the participation of this pathway in NASH/Cir. Phospho-mTOR evaluation might be of clinical utility as a potential marker for identification of NASH/Cir in cases mistakenly considered as cryptogenic cirrhosis owing to paucity of clinical data.

Suppression subtractive hybridization analysis reveals expression of conserved and novel genes in male accessory glands of the ant Leptothorax gredleri

Background: During mating, insect males eject accessory gland proteins (Acps) into the female genital tract. These substances are known to affect female post-mating behavior and physiology. In addition, they may harm the female, e. g., in reducing its lifespan. This is interpreted as a consequence of sexual antagonistic co-evolution. Whereas sexual conflict abounds in non-social species, the peculiar life history of social insects (ants, bees, wasps) with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes. Harming the female during mating would negatively affect male fitness and sexual antagonism is therefore not expected. Indeed, mating appears to increase female longevity in at least one ant species. Acps are presumed to play a role in this phenomenon, but the underlying mechanisms are unknown. In this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant Leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid. Results: By a suppression subtractive hybridization protocol we obtained 20 unique sequences (USs). Twelve had mutual best matches with genes predicted for Apis mellifera and Nasonia vitripennis. Functional information (Gene Ontology) was available only for seven of these, including intracellular signaling, energy-dependent transport and metabolic enzyme activities. The remaining eight USs did not match sequences from other species. Six genes were further analyzed by quantitative RT-PCR in three life cycle stages of male ants. A gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males. Conclusions: Our study is the first one to investigate differential gene expression in ants in a context related to mating. Our findings indicate that male accessory glands of L. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. Identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial Hymenoptera. While the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females.

Expression of eight genes of nuclear factor-kappa B pathway in multiple myeloma using bone marrow aspirates obtained at diagnosis

Purpose: To evaluate the expression of NF-kappa B pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival. Material and methods: Expression of eight genes related to NF-kappa B pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples. Results: The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p = 0.04). Conclusion: We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.

Genetic polymorphisms in TLR4, CR1 and Duffy genes are not associated with malaria resistance in patients from Baixo Amazonas region, Brazil

The main purpose of this research was to analyze the relation of the genetic polymorphisms frequently expressed by antigen-presenting cells, erythrocytes and malaria susceptibility/resistance with the human malaria infection cases. The sample used consisted of 23 Plasmodium vivax ( Pv)- and P. falciparum ( Pf)-infected patients, and 21 healthy individuals as a control group, from the Baixo Amazonas population in Para, Brazil. The Asp299Gly polymorphisms in the Toll-like receptor 4 ( TLR4), and Gly42Asp, Arg89Cys, Ala100Thr, and T-33C in the Duffy gene ( FY) were analyzed by restriction fragment length polymorphism-polymerase chain reaction. The Lys1590Glu and Arg1601Gly polymorphisms in the complement receptor type 1 (CR1) were analyzed by DNA sequencing. According to the results obtained and statistical analysis considering a significance level or alpha = 0.01, we conclude that the low heterozygote frequency (2.27%) for the Asp299Gly mutation, detected in the TLR4 gene, is not related to the Pv and Pf infections in the patients analyzed. Also, the promoter region GATA-1 analysis of the FY gene in the Pv-infected patients showed that the heterozygote frequency for the T-33C mutation (11.36% of the infected patients and 20.45% of the control patients) is not related to infection resistance. Regarding the CR1 gene, the observed heterozygote frequency (9.09%) for the Arg1601Gly mutation in Pf-infected patients when compared to heterozygote frequency in the control group (18.18%) suggests that there is no correlation with infection resistance.

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

The cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Functional microarray analysis suggests repressed cell-cell signaling and cell survival-related modules inhibit progression of head and neck squamous cell carcinoma

Background: Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease. Methods: Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results: The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions: The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.

Searching for Moniliophthora perniciosa pathogenicity genes

The basidiomycete Moniliophthora perniciosa is the causal agent of witches` broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as ""Defence genes, Virulence, and Cell response"" presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen`s own survival in the hostile environment. (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Age-related association of rDNA and telomeres with the nuclear matrix in mouse hepatocytes

Transcribed sequences have been suggested to be associated with the nuclear matrix, differing from non-transcribing sequences, which have been reported to be contained in DNA loops. However, although a dozen of genes have their expression level affected by aging, data on chromatin-nuclear matrix interactions under this physiological condition are still scarce. In the present study, liver imprints from young, adult and old mice were subjected to FISH (fluorescence in situ hybridization) for 45S rDNA and telomeric sequences, with or without a lysis treatment to produce extended chromatin fibres. There was an increased amount of 45S rDNA sequences located in DNA loops as the animals grow older, while telomeric sequences were always observed in DNA loops irrespective of the animal age. We assume that active rRNA genes associate with the nuclear matrix, while DNA loops contain silent sequences. Transcription of each 45S rDNA repeat unit is suggested to be dependent on its interaction with the nuclear matrix.

In silico analysis of candidate genes involved in light sensing and signal transduction pathways in soybean

Several aspects of photoperception and light signal transduction have been elucidated by studies with model plants. However, the information available for economically important crops, such as Fabaceae species, is scarce. In order to incorporate the existing genomic tools into a strategy to advance soybean research, we have investigated publicly available expressed sequence tag ( EST) sequence databases in order to identify Glycine max sequences related to genes involved in light-regulated developmental control in model plants. Approximately 38,000 sequences from open-access databases were investigated, and all bona fide and putative photoreceptor gene families were found in soybean sequence databases. We have identified G. max orthologs for several families of transcriptional regulators and cytoplasmic proteins mediating photoreceptor-induced responses, although some important Arabidopsis phytochrome-signaling components are absent. Moreover, soybean and Arabidopsis gene-family homologs appear to have undergone a distinct expansion process in some cases. We propose a working model of light perception, signal transduction and response-eliciting in G. max, based on the identified key components from Arabidopsis. These results demonstrate the power of comparative genomics between model systems and crop species to elucidate several aspects of plant physiology and metabolism.

Genetic diversity and plant-growth related features of Burkholderia spp. from sugarcane roots

Brazil is the largest sugarcane producer in the world, mainly due to the development of different management strategies. Recently, microbial-plant related studies revealed that bacterial isolates belonging to the genus Burkholderia are mainly associated with this plant and are responsible for a range of physiological activity. In this study, we properly evaluate the physiological activity and genetic diversity of endophytic and rhizospheric Burkholderia spp. isolates from sugarcane roots grown in the field in Brazil. In total, 39 isolates previously identified as Burkholderia spp. were firstly evaluated for the capability to fix nitrogen, produce siderophores, solubilise inorganic phosphates, produce indole-acetic acid and inhibit sugarcane phytopathogens in vitro. These results revealed that all isolates present at least two positive evaluated activities. Furthermore, a phylogenetic study was carried out using 16S rRNA and gyrB genes revealing that most of the isolates were affiliated with the Burkholderia cepacia complex. Hence, a clear separation given by endophytic or rhizospheric niche occupation was not observed. These results presented an overview about Burkholderia spp. isolates from sugarcane roots and supply information about the physiological activity and genetic diversity of this genus, given direction for further studies related to achieve more sustainable cultivation of sugarcane.

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of Methylobacterium in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism-plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR-DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.

Enteroinvasive Escherichia coli vs. Shigella flexneri : how different patterns of gene expression affect virulence

Important features of the enteroinvasive Escherichia coli (EIEC) phenotype and gene expression likely to confer EIEC with a lower ability to cause disease than Shigella flexneri were described here for the first time. To confirm the lower pathogenicity of EIEC, we have analyzed the keratoconjunctivitis developed in guinea-pigs with EIEC or S. flexneri. Shigella flexneri induced a more pronounced proinflammatory response, whereas EIEC induced a mild form of the disease. EIEC showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Plaques formed by EIEC during intercellular spreading were four times smaller than those formed by S. flexneri. At the molecular level, the lower expression of virulence genes by EIEC during infection of Caco-2 cells highlighted the importance of effective gene transcription for bacterial pathogenicity.

Evaluation of the Experimental Inoculation of Cryptococcus albidus and Cryptococcus laurentii in Normal Mice: Virulence Factors and Molecular Profile Before and After Animal Passage

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37A degrees C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.

Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc

A nostocalean nitrogen-fixing cyanobacterium isolated from an eutrophic freshwater reservoir located in Piracicaba, Sao Paulo, Brazil, was evaluated for the production of hepatotoxic cyclic heptapeptides, microcystins. Morphologically this new cyanobacterium strain appears closest to Nostoc, however, in the phylogenetic analysis of 165 rRNA gene it falls into a highly stable cluster distantly only related to the typical Nostoc cluster. Extracts of Nostoc sp. CENA88 cultured cells, investigated using ELISA assay, gave positive results and the microcystin profile revealed by ESI-Q-TOF/MS/MS analysis confirmed the production of [Dha(7)]MCYST-YR. Further, Nostoc sp. CENA88 genomic DNA was analyzed by PCR for sequences of mcyD, mcyE and mcyG genes of microcystin synthetase (mcy) cluster. The result revealed the presence of mcyD, mcyE and mcyG genes with similarities to those from mcy of Nostoc sp. strains 152 and IO-102-I and other cyanobacterial genera. The phylogenetic tree based on concatenated McyG, McyD and McyE amino acids clustered the sequences according to cyanobacterial genera, with exception of the Nostoc sp. CENA88 sequence, which was placed in a clade distantly related from other Nostoc strains, as previously observed also in the 165 rRNA phylogenetic analysis. The present study describes for the first time a Brazilian Nostoc microcystin producer and also the occurrence of demethyl MCYST-YR variant in this genus. The sequenced Nostoc genes involved in the microcystin synthesis can contribute to a better understanding of the toxigenicity and evolution of this cyanotoxin. (C) 2009 Elsevier Ltd. All rights reserved.