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Despite significant advances in the diagnosis and treatment of cardiovascular diseases, recent calls have emphasized the unmet need to improve precision-based approaches in cardiovascular disease. Although some studies provide preliminary evidence of the diagnostic and prognostic potential of circulating coding and non-coding RNAs, the complex RNA biology and lack of standardization have hampered the translation of these markers into clinical practice. In this position paper of the CardioRNA COST action CA17129, we provide recommendations to standardize the RNA development process in order to catalyse efforts to investigate novel RNAs for clinical use. We list the unmet clinical needs in cardiovascular disease, such as the identification of high-risk patients with ischaemic heart disease or heart failure who require more intensive therapies. The advantages and pitfalls of the different sample types, including RNAs from plasma, extracellular vesicles, and whole blood, are discussed in the sample matrix, together with their respective analytical methods. The effect of patient demographics and highly prevalent comorbidities, such as metabolic disorders, on the expression of the candidate RNA is presented and should be reported in biomarker studies. We discuss the statistical and regulatory aspects to translate a candidate RNA from a research use only assay to an in-vitro diagnostic test for clinical use. Optimal planning of this development track is required, with input from the researcher, statistician, industry, and regulatory partners., peer-reviewed

Phenotypic plasticity, the ability to produce multiple phenotypes from a single genotype, represents an excellent model with which to examine the relationship between gene expression and phenotypes. Analyses of the molecular foundations of phenotypic plasticity are challenging, however, especially in the case of complex social phenotypes. Here we apply a machine learning approach to tackle this challenge by analyzing individual-level gene expression profiles of Polistes dominula paper wasps following the loss of a queen. We find that caste-associated gene expression profiles respond strongly to queen loss, and that this change is partly explained by attributes such as age but occurs even in individuals that appear phenotypically unaffected. These results demonstrate that large changes in gene expression may occur in the absence of outwardly detectable phenotypic changes, resulting here in a socially mediated de-differentiation of individuals at the transcriptomic level but not at the levels of ovarian development or behavior., Connecting genotypes to complex social behaviour is challenging. Taylor et al. use machine learning to show a strong response of caste-associated gene expression to queen loss, wherein individual wasp’s expression profiles become intermediate between queen and worker states, even in the absence of behavioural changes.

To study the possible roles of type-2C protein phosphatases (PP2Cs) which have been confirmed to play roles in the response to diverse abiotic stresses in paper mulberry, we launched a series of genomic and functional studies of BpPP2Cs. Sixty-three PP2C proteins in paper mulberry (Broussonetia papyrifera) were classified into 13 clades. Four BpPP2Cs with kinase domains were verified to be highly conserved in organisms ranging from algae to dicots. Seven pairs of BpPP2C genes were found to be expanding, and 18 BpPP2C genes had orthologues in Arabidopsis. BpPP2Cs showed broad expression in different tissues; the expression levels of 18 BpPP2Cs were changed and the phosphorylation levels of seven BpPP2C proteins increased at low temperature. Cold-response elements were found in the promoter region of 31 BpPP2Cs. Finally, Bp01g0320 was found to act as a hub protein and Bp01g0512 and Bp09g1278 played key roles in the ABA-signaling pathway and MAPK cascades, respectively. These results suggest that the PP2C gene family of paper mulberry is evolutionarily conserved and participates the regulation of the response to cold stress, which will play a vital role in further research on phosphatases in paper mulberry.

Phenotypic plasticity, the ability to produce multiple phenotypes from a single genotype, represents an excellent model with which to examine the relationship between gene expression and phenotypes. Despite this, analyses of the molecular bases of plasticity have been limited by the challenges of linking individual phenotypes with individual-level gene expression profiles, especially in the case of complex social phenotypes. Here, we tackle this challenge by analysing the individual-level gene expression profiles of Polistes dominula paper wasps following the loss of a queen, a perturbation that induces some individuals to undergo a significant phenotypic shift and become replacement reproductives. Using a machine learning approach, we find a strong response of caste-associated gene expression to queen loss, wherein individuals’ expression profiles become intermediate between queen and worker states. Importantly, this change occurs even in individuals that appear phenotypically unaffected. Part of this response is explained by individual attributes, most prominently age. These results demonstrate that large changes in gene expression may occur in the absence of detectable phenotypic changes, resulting here in a socially mediated de-differentiation of individuals at the transcriptomic but not the phenotypic level. Our findings also highlight the complexity of the relationship between gene expression and phenotype, where transcriptomes are neither a direct reflection of the genotype nor a proxy for the molecular underpinnings of the external phenotype.

Social interactions have large effects on individual physiology and fitness. In the immediate sense, social stimuli are often highly salient and engaging. Over longer time scales, competitive interactions often lead to distinct social ranks and differences in physiology and behavior. Understanding how initial responses lead to longer-term effects of social interactions requires examining the changes in responses over time. Here we examined the effects of social interactions on transcriptomic signatures at two times, at the end of a 45-minute interaction and 4 hours later, in female Polistes fuscatus paper wasp foundresses. Female P. fuscatus have variable facial patterns that are used for visual individual recognition, so we separately examined the transcriptional dynamics in the optic lobe and the non-visual brain. Results demonstrate much stronger transcriptional responses to social interactions in the non-visual brain compared to the optic lobe. Differentially regulated genes in response to social interactions are enriched for memory-related transcripts. Comparisons between winners and losers of the encounters revealed similar overall transcriptional profiles at the end of an interaction, which significantly diverged over the course of 4 hours, with losers showing changes in expression levels of genes associated with aggression and reproduction in paper wasps. On nests, subordinate foundresses are less aggressive, do more foraging and lay fewer eggs compared to dominant foundresses and we find losers shift expression of many genes in the non-visual brain, including vitellogenin, related to aggression, worker behavior, and reproduction within hours of losing an encounter. These results highlight the early neurogenomic changes that likely contribute to behavioral and physiological effects of social status changes in a social insect., Author summary Aggressive interactions often create inequalities–some individuals win while others lose. Winning versus losing can lead to large physiological differences between individuals, including different neurogenomic profiles between winners and losers. How this information about contest outcome leads to distinct neurogenomic profiles is poorly understood. Here we examine gene expression in response to aggressive social encounters in paper wasps, which naturally form dominance hierarchies on their nests in the wild. Shortly following encounters winners and losers have similar expression profiles, likely because similar mechanisms are engaged by social experiences. Four hours later, we find divergent neurogenomic profiles between winners and losers, with losers showing larger shifts in expression compared to winners. Many of the most dynamically expressed genes have been previously associated with dominance and caste differences in paper wasps showing how a single interaction can engage many of the same genomic networks that are involved in mediating more dramatic differences in queen-worker behavioral differences are also involved in responses shortly following social interactions.

open access article Food contact materials (FCM) are any type of item intended to come into contact with foods and thus represent a potential source for human exposure to chemicals. Regarding FCMs made of paper and board, information pertaining to their chemical constituents and the potential impacts on human health remains scarce, which hampers safety evaluation. We describe an effect-directed strategy to identify and characterize emerging chemicals in paper and board FCMs. Twenty FCMs were tested in eight reporter gene assays, including assays for the AR, ER, AhR, PPARγ, Nrf2 and p53, as well as mutagenicity. All FCMs exhibited activities in at least one assay. As proof-of-principle, FCM samples obtained from a sandwich wrapper and a pizza box were carried through a complete step-by-step multi-tiered approach. The pizza box exhibited ER activity, likely caused by the presence of bisphenol A, dibutyl phthalate, and benzylbutyl phthalate. The sandwich wrapper exhibited AR antagonism, likely caused by abietic acid and dehydroabietic acid. Migration studies confirmed that the active chemicals can transfer from FCMs to food simulants. In conclusion, we report an effect-directed strategy that can identify hazards posed by FCMs made from paper and board, including the identification of the chemical(s) responsible for the observed activity.

La duplicació completa del genoma (WGD) en càncer està associada amb inestabilitat genòmica, alteracions recurrents en gens de control del cicle cel·lular, nivells alts d'alteracions en el nombre de còpies i mal pronòstic clínic, però es desconeix l'efecte global que té sobre els programes transcripcionals. El treball consisteix en un anàlisi d'expressió gènica diferencial entre tumors de càncer colorectal i adenocarcinoma de pulmó amb i sense WGD a partir de dades públiques de seqüenciació d'ARN, a partir del qual s'han identificat mecanismes moleculars i processos cel·lulars associats, posant especial èmfasi en mecanismes d'evasió del sistema immunitari. En els dos tipus de càncer s'ha observat una correlació negativa entre WGD i resposta immunitària antitumoral. A més, en càncer colorectal s'han observat diferències metabòliques i en càncer de pulmó diferències en vies de divisió cel·lular i replicació. En conjunt, aquests resultats podrien explicar l'agressivitat i mal pronòstic associats als tumors que han patit WGD. Whole genome doubling (WGD) in cancer is associated to genomic instability, recurrent alterations in cell cycle control genes, high levels of copy number alterations and a poor prognosis, but its global effect on the transcriptional programs is unknown. This work consists of a differential gene expression analysis between colorectal and lung adenocarcinoma tumors with and without WGD using RNA-sequencing public data. We have identified the molecular mechanisms and biological processes associated, putting special attention to immune evasion strategies. We show a negative correlation between WGD and antitumor immune response in both colorectal and lung adenocarcinoma tumors. In addition, we find metabolic changes in colorectal tumors and changes in cell division pathways and DNA replication in lung tumors. Overall, these results potentially explain the aggressiveness and poor prognosis associated with tumors that have undergone WGD. La duplicación completa del genoma (WGD) en cáncer está asociada con inestabilidad genómica, alteraciones recurrentes en genes de control del ciclo celular, niveles altos de alteraciones en el nombre de copias y mal pronóstico clínico, pero es desconoce el efecto global que té sobre los programes transcripcionales. El trabajo consiste en un análisis de expresión génica diferencial entre tumores de cáncer colorrectal y adenocarcinoma de pulmón con y sin WGD a partir de datos públicos de secuenciación de ARN, a partir del cual se han identificado mecanismos moleculares y procesos celulares asociados, poniendo especial énfasis en mecanismos de evasión del sistema inmunitario. En los dos tipos de cáncer se ha observado una correlación negativa entre WGD y respuesta inmunitaria antitumoral. Además, en cáncer colorrectal se han observado diferencias metabólicas y en cáncer de pulmón diferencias en vías de división celular y replicación. En conjunto, estos resultados podrían explicar la agresividad y mal pronóstico asociados a los tumores que han sufrido WGD.

Pathotyping and subtyping of influenza A virus were performed with a packaged paper fluidic-based analytical microdevice (PFAM) after one-step reverse transcription-polymerase chain reaction (RT-PCR). The PFAM contains two test lines: one for detecting M gene to identify the influenza A virus and another for haemagglutinin subtyping to determine the viral strain among H1N1, H3N2, and H5N1. The M gene and the haemagglutinin gene (H1, H3, and H5 genes) were amplified by using the Digoxigenin and the Texas Red modified primers, respectively, in the multiplex RT-PCR. The amplicon products were loaded in the conjugate pad of the PFAM in which the streptavidin coated gold nanoparticles were linked with the biotin moieties that were incorporated in the middle of the DNA strands, and then captured by the anti-Digoxigenin and anti-Texas Red immobilized on the test lines. Influenza A H1N1, H3N2, and H5N1 could be identified with a limit of detection of 10(2) copies of RNA templates in 10 min. Pathotyping and subtyping of the clinical nasopharyngeal swab samples were also analyzed whose results were confirmed by real-time RT-PCR.

Juvenile whitefish (Coregonus lavaretus L. s.l.) were exposed by caging in the field to diluted effluents from three operating pulp, paper, and paperboard mills in Southern Lake Saimaa, Finland. The expression of the vitellogenin gene, used as a biomarker of estrogenic contamination of effluents, was measured using a Northern blotting method. Increased mRNA levels, the most specific and reliable evidence for estrogen receptor-mediated actions in vivo, were found in fish caged in the vicinity of one of three mills studied. This mill was found to discharge wood-derived compounds, such as sterols and resin acids, into Lake Saimaa in amounts considerably exceeding those from the other two mills. The increased vitellogenin gene expression suggests that the effluent is a source of estrogenic contaminants.

Many studies link pulp and paper mill effluent (PPME) exposure to adverse effects in fish populations present in the mill receiving environments. These impacts are often characteristic of endocrine disruption and may include impaired reproduction, development and survival. While these physiological endpoints are well-characterized, the molecular mechanisms causing them are not yet understood. To investigate changes in gene transcription induced by exposure to a PPME at several stages of treatment, male and female fathead minnows (FHMs) were exposed for 6 days to 25% (v/v) secondary (biologically) treated kraft effluent (TK) or 100% (v/v) combined mill outfall (CMO) from a mill producing both kraft pulp and newsprint. The gene expression changes in the livers of these fish were analyzed using a 22 K oligonucleotide microarray. Exposure to TK or CMO resulted in significant changes in the expression levels of 105 and 238 targets in male FHMs and 296 and 133 targets in females, respectively. Targets were then functionally analyzed using gene ontology tools to identify the biological processes in fish hepatocytes that were affected by exposure to PPME after its secondary treatment. Proteolysis was affected in female FHMs exposed to both TK and CMO. In male FHMs, no processes were affected by TK exposure, while sterol, isoprenoid, steroid and cholesterol biosynthesis and electron transport were up-regulated by CMO exposure. The results presented in this study indicate that short-term exposure to PPMEs affects the expression of reproduction-related genes in the livers of both male and female FHMs, and that secondary treatment of PPMEs may not neutralize all of their metabolic effects in fish. Gene ontology analysis of microarray data may enable identification of biological processes altered by toxicant exposure and thus provide an additional tool for monitoring the impact of PPMEs on fish populations.

We evaluated plasma testosterone (T) and 17β-estradiol (E2) levels and ovarian aromatase CYP19a gene expression following a single intraperitoneal injection of Chilean pulp and paper mill effluent extracts into juvenile triploid rainbow trout. Fish injected with untreated effluent extracts had increased plasma T after 4 days, while plasma E2 concentration was increased in fish injected with both primary and secondary treated effluent extracts at the same sampling period. Ovarian CYP19a gene expression as measured by qRT-PCR was significantly induced in fish injected with the untreated, primary and secondary treated pulp and paper mill effluent extracts. Similar induction of CYP19a expression was found in fish injected with the androgens androstenedione (ADD) and T. A Principal Component Analysis (PCA) was conducted in order to identify structure in relationships between all measured variables and identifying which factors were most responsible for the variance observed within the plasma steroid levels, upregulation of ovarian CYP19a gene expression and the final estrogenic effect of increased plasma VTG levels. This analysis indicated a cluster correlation between plasma T levels and CYP19a gene expression (Factor 1, explaining 27.2% of total variance), a cluster including condition factor and liver somatic index (Factor 2, explaining 17.3%) and an additional cluster including plasma E2 and vitellogenin levels (Factor 3, explaining an additional 15.8%). The present results indicate that Chilean pulp and paper mill effluent extracts cause estrogenic effects in triploid rainbow trout. These effects could be related to the compounds present in the effluent that act as estrogen receptor agonists, or that induce changes leading to increased amounts of endogenous estrogens, reflected by increased E2 levels and induced aromatase expression/activity.

The toxicity of pulp and paper mill effluents (PPMEs) has been greatly decreased, yet some continue to negatively affect fish reproduction. We hypothesized that PPMEs are affecting the brain resulting in decreased reproductive performance. Our goal was to use gene expression profiling to test the hypothesis that PPMEs are having an effect on neural systems in the fathead minnow (FHM; Pimephales promelas) in vivo. Sexually mature male and female FHM were exposed to 100% final biotreated PPMEs from 5 different sources for 5 days. Using an oligo-array (15K genes) we examined the effect of PPMEs on gene expression in the hypothalamus of female fish. We validated selected genes (cholecystokinin, RevErbbeta2, and urotensin I) that were identified by microarray analysis using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We compared the FHM microarray dataset to multiple microarray datasets from experiments conducted with goldfish injected with different dopaminergic pharmaceuticals to examine whether PPMEs could be affecting the dopamine system. Exposure of FHM to PPMEs resulted in varying degrees of spawning inhibition. Microarray analysis revealed surprisingly few genes in the brain that were commonly affected by the different PPMEs. Real-time RT-PCR confirmed the changes in expression for cholecystokinin, RevErbbeta2, and urotensin I. Comparison of the FHM and goldfish microarray datasets suggest that some PPMEs may be acting on the dopamine system. We show that PPMEs are neuroactive in fish and may be acting through some of the pathways in a manner similar to dopamine.

A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of paper and pulp mill showed high β-1, 4-endoglucanase (2.5 IU/ml) but low activity of β-1, 4-exoglucanase (0.8 IU/ml) and β-glucosidase (0.084 IU/ml). The β-1, 4-endoglucanase gene of IARI-SP-1 was amplified using degenerate primers designed based on sequences already available in NCBI GenBank. A full length gene of β-1, 4-endonuclease consisting of 1499 nucleotides was identified through sequence analysis of the amplified product. The ORF encoded for a protein of 500 amino acids with a predicted molecular weight of 55 kDa. The gene was cloned in pET-28a and over expressed in Escherichia coli BL21 (DE3). In comparison to wild strain ( B. subtilis ), the transformed E. coli exhibited four times increase in cellulase production. Higher enzyme activity was observed in supernatant (8.2 IU/ml) than cell pellet (2.8 IU/ml) suggesting more extracellular production of β-1, 4-endoglucanase. SDS-PAGE and CMC plate assay also confirmed the overproduction by the transformed E. coli . The pH and temperature optima of expressed β-1, 4-endoglucanase enzyme was identical to that of wild strain and was 8 and 50–60 °C, respectively.

[eng] HDAC11 is the newest member of the histone deacetylase (HDAC) family and one of the less studied. Its expression was described to be enriched in skeletal muscle tissues from the first moment of its discovery, yet now after 15 years, its roles in myogenesis remain unknown. We started this thesis by analyzing the expression changes of all HDAC’s’ members between proliferation and early differentiation conditions, which constitutes a crucial cell fate point in which cells have to decide whether to continue proliferating or enter to irreversible G0 arrest state to differentiate. With this analysis, we found HDAC11 as the HDAC family member the most upregulated in the skeletal muscle differentiation process. By CRISPR/Cas9 knock-in tagging of endogenous HDAC11, we show that HDAC11 protein levels are absent in proliferating cells and increased through differentiation. The silencing of HDAC11 in proliferation conditions is mediated, at least in part, by class I HDAC’s deacetylation of MYOD. In differentiation conditions, acetylated MYOD and myogenin, the two master regulators of muscle differentiation, bind to HDAC11 promoter regions and trigger its expression. HDAC11 deficient myoblasts did not present major alterations in cell proliferation or differentiation capacities but show reduced fusion ability. Genome-wide transcriptomic analysis of differentiating HDAC11 deficient myoblasts revealed an upregulation of genes involved in proliferation and a decreased expression of genes involved in muscle contraction, suggesting a delayed entry in G0 irreversible arrest state. Our ChIP results suggest that HDAC11 would mediate repression of proliferation related genes by deacetylation of H3 in their promoter regions. Moreover, HDAC11 expression is also highly expressed in additional G0 states, like in reversible arrested quiescent satellite cells. In skeletal muscle tissues, HDAC11 is higher expressed in fast muscles than slow ones, especially in males. The analysis of HDAC11 deficient mice concludes that HDAC11 absence do not cause major alterations in muscle development, adult myofiber growth or fiber type composition in basal conditions. In regeneration conditions, HDAC11 deficient mice show advanced regeneration capacity at 7 days post injury, probably mediated at least in part, by an increased expression of Il-10 by HDAC11 deficient macrophages. Finally, we show that HDAC11 upregulation through differentiation is conserved in human myoblast and its expression is reduced in rhabdomyosarcoma cells, which present impaired differentiation capabilities. Altogether, our results place HDAC11 as a new epigenetic regulator in in vitro an in vivo myogenesis., [cat] HDAC11 és el membre més recentment descobert de la família de deacetilases d'histones (HDAC) i un dels menys estudiats. En el moment del seu descobriment es va veure que estava altament expressada en teixits de múscul esquelètic encara que després de 15 anys, les seves funcions en la miogènesi resten encara desconegudes. Vam començar aquesta tesi analitzant els canvis d'expressió de tots els membres de la família HDAC entre les condicions de proliferació i diferenciació primerenca, un moment crucial on les cèl·lules han de decidir si continuen dividint-se o entren en l'estat d'aturada irreversible G0 per diferenciar-se. Amb aquesta anàlisi vam trobar HDAC11 com el membre de la família HDAC que augmentava més la seva expressió en aquest procés. Amb la tècnica d'enginyeria genètica CRISPR/Cas9 vam aconseguir inserir un epítop en el locus genòmic de HDAC11, que ens permeté demostrar que la proteïna HDAC11 està absent en condicions de proliferació i augmenta amb la diferenciació. El silenciament de l'expressió de HDAC11 durant la proliferació està mitjançada, almenys en part, per la deacetilació de MYOD per part de la classe I de HDACs. Durant la diferenciació, MYOD acetilat i miogenina, els dos reguladors responsables d’iniciar la diferenciació muscular, s'uneixen al promotor de HDAC11 i activen la seva expressió. Els mioblasts deficients en HDAC11 no presenten greus alteracions en la proliferació cel·lular o en la seva capacitat de diferenciar-se però mostren una reduïda capacitat de fusió. L'estudi transcriptòmic a escala global de mioblasts deficients en HDAC11 va revelar una sobreexpressió de gens involucrats en la proliferació cel·lular i una reducció en l'expressió de gens amb funcions en la contracció muscular, suggerint una entrada més tardana en la fase G0 d'aturada irreversible. Els nostres resultats de ChIP suggereixen que HDAC11 podria intervindre en la repressió dels gens involucrats en la proliferació cel·lular mitjançant la deacetilació de les histones H3 dels seus promotors. A més, l'expressió d'HDAC11 també és alta en estats addicionals de G0 com l'arrest reversible de les cèl·lules satèl·lit quiescents. En teixits de múscul esquelètic, HDAC11 està més expressada en músculs ràpids que lents, especialment en mascles. L'anàlisi de ratolins deficients en HDAC11 conclogué que l'absència de HDAC11 no causa greus alteracions en el desenvolupament muscular, el creixement de miofibres adultes o en la composició en tipus de fibres en condicions basals. Durant la regeneració muscular, els ratolins deficients en HDAC11 mostren un avanç en la seva capacitat de regeneració 7 dies després de la lesió, probablement mitjançat en part per un increment en l’expressió de Il-10 per part dels macròfags deficients en HDAC11. Finalment, mostrem que l’augment d’expressió de HDAC11 està conservat en la diferenciació de mioblasts humans i que la seva expressió està reduïda en rabdomiosarcoma, patologia tumoral que presenta un impediment en la diferenciació muscular. En conjunt, els nostres resultats situen HDAC11 com un nou regulador epigenètic en la miogènesi in vitro i in vivo.

Gene expression responses of paper birch ( Betula papyrifera ) leaves to elevated concentrations of CO 2 and O 3 were studied with microarray analyses from three time points during the summer of 2004 at Aspen FACE. Microarray data were analyzed with clustering techniques, self-organizing maps, K -means clustering and Sammon's mappings, to detect similar gene expression patterns within sampling times and treatments. Most of the alterations in gene expression were caused by O 3 , alone or in combination with CO 2 . O 3 induced defensive reactions to oxidative stress and earlier leaf senescence, seen as decreased expression of photosynthesis- and carbon fixation-related genes, and increased expression of senescence-associated genes. The effects of elevated CO 2 reflected surplus of carbon that was directed to synthesis of secondary compounds. The combined CO 2 + O 3 treatment resulted in differential gene expression than with individual gas treatments or in changes similar to O 3 treatment, indicating that CO 2 cannot totally alleviate the harmful effects of O 3 .

Made available in DSpace on 2017-05-12T14:47:05Z (GMT). No. of bitstreams: 1 JULIANA_ MOCO CORREA (2).pdf: 1734110 bytes, checksum: 24beceb790c634d766ff59c9de448991 (MD5) Previous issue date: 2014-11-27 PAPER 1 Depletion of the xynB2 gene upregulates β-Xylosidase expression in C. crescentus. Caulobacter crescentus is able to express several enzymes involved in the utilization of lignocellulosic biomasses. Five genes, xynB1-5, that encode β-xylosidases are present in the genome of this bacterium. In this study, the xynB2 gene, which encodes - xylosidase II (CCNA_02442), was cloned under the control of the PxylX promoter to generate the O-xynB2 strain, which overexpresses the enzyme in the presence of xylose. In addition, a null mutant strain, -xynB2, was created by two homologous recombination events where the chromosomal xynB2 gene was replaced by a copy that was disrupted by the spectinomycin-resistant cassette. It was demonstrated that C. crescentus cells lacking -xylosidase II up-regulates the xynB genes inducing β- xylosidase activity. Transcriptional analysis revealed that xynB1 (RT-PCR analysis) and xynB2 (lacZ transcription fusion) gene expression was induced in the -xynB2 cells, and high -xylosidase activity was observed in the presence of different agroindustrial residues in the null mutant strain, a characteristic that can be explored and applied in biotechnological processes. In contrast, overexpression of the xynB2 gene caused down-regulation of the expression and activity of the -xylosidase. For example, the β-xylosidase activity that was obtained in the presence of sugar cane bagasse was 7-fold and 16-fold higher than the activity measured in the C. crescentus parental and O-xynB2 cells, respectively. Our results suggest that -xylosidase II may have a role in controlling the expression of the xynB1 and xynB2 genes in C.crescentus. PAPER 2 - OPTIMIZATION OF THE PRODUCTION β-XYLOSIDASE: A NEW Thermomyces lanuginosus ISOLATED FROM ATLANTIC FOREST BIOME. The successful production of enzymes for the deconstruction of plant biomass depends not only on the isolation and identification of new microorganism producers of hemicellulases, but also on the implementation and improvement of experimental strategies that lead to maximal induction of enzymatic activities. In this work, a new strain of Thermomyces lanuginosus (T. lanuginosus) was isolated from the Atlantic Forest biome in Brazil, and its β-xylosidase activity in response to agro-industrial residues was tested. Using the (CCRD) statistical approach as a strategy for optimization, the induction of β-xylosidase activity was evaluated in residual corn straw, which was used as a carbon source, and improved so that the optimum condition achieved high β-xylosidase activity (1,003 U ml -1; specific activity = 1.683 U mg-1) with 214 U ml -1. The optimal conditions for the crude enzyme extract were pH 5.5 and 60° C showing better thermostability at 55° C. The saccharification ability of β-xylosidase in the presence of hemicellulose obtained from corn straw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50° C. These data suggest that β-xylosidase from T. lanuginosus isolated from the Atlantic Forest can be used for the saccharification of hemicellulose derived from corn straw, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production that relies on the conversion of plant biomass. RESUMO GERAL As Beta-D-Xilosidases (1,4-β-D-xilano xilohidrolase; EC 3.2.1.37) são glicosídeo hidrolases que tem papel crucial em catalizar a liberação de unidades de xilose a partir de xilo-oligossacarídeos derivados da degradação do xilano. A completa degradação do xilano é um passo chave do ciclo do carbono na natureza e é um processo também realizado por microrganismos. A bioconversão de materiais lignocelulósicos é vantajosa não somente do ponto de vista ambiental mais também econômico o que é recebido nos setores produtivos com um considerável interesse, pois esses materiais representam vasta fonte de carbono, que podem ser empregados no desenvolvimento de bioprocessos que resultam em produtos de alto valor agregado; entre os quais estão os açúcares fermentáveis, combustíveis, fármacos, enzimas e substâncias de interesse industrial, além de fazer uma gestão integrada do efluente que por não haver um desenvolvimento biotecnológico adequado é descartado e acumulado na natureza. Em face disso, o presente trabalho teve por objetivos estudar a regulação gênica do gene xynB2 da bactéria Caulobacter crescentus que codifica para a Beta-xilosidase II através de abordagens moleculares e otimizar a produção enzimática de Betaxilosidases de Thermomyces lanuginosus na presença de diferentes resíduos de biomassa vegetal por delineamento experimental. No primeiro artigo exploramos a bactéria aquática Caulobacter crescentus por possuir várias enzimas envolvidas na utilização de biomassas lignocelulósicas; contendo em seu genoma cinco genes que codificam β-xilosidases. A partir do gene xynB2, que codifica para enzima -xylosidase II (CCNA_02442), desenvolvemos duas linhagens mutantes denominadas O-xynB2, que super-expressa a enzima na presença de xilose e -xynB2 que tem o gene xynB2 interrompido, o que possibilitou avaliar que a ausência da enzima -xylosidase II em células de C. crescentus regula positivamente os genes xynB, induzindo a atividade global de β-xilosidases, revelando um papel regulatório para a mesma. No segundo trabalho um fungo da linhagem Thermomyces lanuginosus isolado de bioma de Mata Atlântica foi identificado e analisado quanto à capacidade de produzir Beta-xilosidases na presença de diferentes resíduos vegetais; em decorrência disso foi otimizado a produção enzimática com delineamento experimental DCCR, o que permitiu alcançar altos níveis de atividade enzimática beta-xilosidásica na presença de palha de milho.

Altered expression of histone deacetylases (HDACs) is a common feature in several human malignancies and may represent an interesting target for cancer treatment, including haematological malignancies. We evaluated the mRNA gene expression profile of 12 HDAC genes by quantitative real-time polymerase chain reaction in 94 consecutive childhood acute lymphoblastic leukaemia (ALL) samples and its association with clinical/biological features and survival. ALL samples showed higher expression levels of HDAC2, HDAC3, HDAC8, HDAC6 and HDAC7 when compared to normal bone marrow samples. HDAC1 and HDAC4 showed high expression in T-ALL and HDAC5 was highly expressed in B-lineage ALL. Higher than median expression levels of HDAC3 were associated with a significantly lower 5-year event-free survival (EFS) in the overall group of patients (P = 0·03) and in T-ALL patients (P = 0.01). HDAC7 and HADC9 expression levels higher than median were associated with a lower 5-year EFS in the overall group (P = 0.04 and P = 0.003, respectively) and in B-lineage CD10-positive patients (P = 0.009 and P = 0·005, respectively). Our data suggest that higher expression of HDAC7 and HDAC9 is associated with poor prognosis in childhood ALL and could be promising therapeutic targets for the treatment of refractory childhood ALL.

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β-thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β-globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ-globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β-thalassaemia severity although they had the same ΗBA- and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans-acting repressor factor of HBG expression and modulates the HPFH phenotype.

The paper mulberry is one of the multifunctional tree species in agroforestry systems and is also commonly utilized in traditional medicine in China and other Asian countries. However, little is known about its molecular genetics, which hinders research on and exploitation of this valuable resource. To discern the correlation between gene expression and the essential properties of the paper mulberry, we performed a transcriptomics analysis, assembling a total of 37,725 unigenes from 54,638,676 reads generated by RNA-seq. Among these, 22,692 unigenes showed greater than 60% similarity with genes from other species. The lengths of 13,566 annotated unigenes were longer than 1,000 bp. Functional clustering analysis with COG (Cluster of Orthologous Groups) revealed that 17,184 unigenes are primarily involved in transcription, translation, signal transduction, carbohydrate metabolism, secondary metabolism, and energy metabolism. GO (Gene Ontology) annotation suggests enrichment of genes encoding antioxidant activity, transporter activity, biosynthesis, metabolism and stress response, with a total of 30,659 unigenes falling in these categories. KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway analysis showed that 7,199 unigenes are associated with 119 metabolic pathways. In addition to the basic metabolism, these genes are enriched for plant pathogen interaction, flavonoid metabolism and other secondary metabolic processes. Furthermore, differences in the transcriptomes of leaf, stem and root tissues were analyzed and 7,233 specifically expressed unigenes were identified. This global expression analysis provided novel insights about the molecular mechanisms of the biosynthesis of flavonoid, lignin and cellulose, as well as on the response to biotic and abiotic stresses including the remediation of contaminated soil by the paper mulberry.

The gene encoding factor C (facC), an extracellular signal protein involved in cellular differentiation, was cloned from Streptomyces griseus 45H, and the complete nucleotide sequence was determined. The deduced amino acid sequence was confirmed by HPLC/electrospray ionization-mass spectrometry analysis. The full-length protein consists of 324 amino acids and has a predicted molecular mass of 34523 Da. The mature extracellular 286 amino acid protein (31038 Da) is probably produced by cleaving off a 38 amino acid secretion signal sequence. Southern hybridization detected facC in several other Streptomyces strains, but database searches failed to identify a protein with significant homology to factor C. Expression of facC from a low-copy-number vector in S. griseus 52-1 resulted in a phenotypic effect similar to that given by exogenously added factor C protein.

Omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) have been shown to be of major importance in the promotion of cardiovascular health, proper human development and the prevention of some cancers. A high proportion of bacterial isolates from low-temperature and high-pressure marine environments produce EPA or DHA. This paper presents the sequence of a 33 kbp locus from the deep-sea bacterium Photobacterium profundum strain SS9 which includes four of the five genes required for EPA biosynthesis. As with other bacterial pfa (polyunsaturated fatty acid) genes, the deduced amino acid sequences encoded by the SS9 genes reveal large multidomain proteins that are likely to catalyse EPA biosynthesis by a novel polyketide synthesis mechanism. RNase protection experiments separated the SS9 pfa genes into two transcriptional units, pfaA-C and pfaD. The pfaA transcriptional start site was identified. Cultivation at elevated hydrostatic pressure or reduced temperature did not increase pfa gene expression despite the resulting increase in percentage composition of EPA under these conditions. However, a regulatory mutant was characterized which showed both increased expression of pfaA-D and elevated EPA percentage composition. This result suggests that a regulatory factor exists which coordinates pfaA-D transcription. Additional consideration regarding the activities required for PUFA synthesis is provided together with comparative analyses of bacterial pfa genes and gene products.