Trichoderma reesei CRE1-mediated Carbon Catabolite Repression in Re-sponse to Sophorose Through RNA Sequencing Analysis.

Carbon catabolite repression (CCR) mediated by CRE1 in Trichoderma reesei emerged as a mechanism by which the fungus could adapt to new environments. In the presence of readily available carbon sources such as glucose, the fungus activates this mechanism and inhibits the production of cellulolytic complex enzymes to avoid unnecessary energy expenditure. CCR has been well described for the growth of T. reesei in cellulose and glucose, however, little is known about this process when the carbon source is sophorose, one of the most potent inducers of cellulase production. Thus, we performed high-throughput RNA sequencing to better understand CCR during cellulase formation in the presence of sophorose, by comparing the mutant ∆cre1 with its parental strain, QM9414. Of the 9129 genes present in the genome of T. reesei, 184 were upregulated and 344 downregulated in the mutant strain ∆cre1 compared to QM9414. Genes belonging to the CAZy database, and those encoding transcription factors and transporters are among the gene classes that were repressed by CRE1 in the presence of sophorose; most were possible indirectly regulated by CRE1. We also observed that CRE1 activity is carbon-dependent. A recent study from our group showed that in cellulose, CRE1 repress different groups of genes when compared to sophorose. CCR differences between these carbon sources may be due to the release of cellodextrins in the cellulose polymer, resulting in different targets of CRE1 in both carbon sources. These results contribute to a better understanding of CRE1-mediated CCR in T. reesei when glucose comes from a potent inducer of cellulase production such as sophorose, which could prove useful in improving cellulase production by the biotechnology sector.

The impact of chromatin remodelling on cellulase expression in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging. RESULTS: Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1. CONCLUSIONS: The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Defining the genome-wide role of CRE1 during carbon catabolite repression in Trichoderma reesei using RNA-Seq analysis.

The ascomycete Trichoderma reesei is one of the most well-studied cellulolytic fungi and is widely used by the biotechnology industry in the production of second generation bioethanol. The carbon catabolite repression (CCR) mechanism adopted by T. reesei is mediated by the transcription factor CRE1. CCR represses genes related to cellulase production when a carbon source is readily available in the medium. Using RNA sequencing, we investigated CCR during the synthesis of cellulases, comparing the T. reesei Δcre1 mutant strain with its parental strain, QM9414. Of 9129 genes in the T. reesei genome, 268 genes were upregulated and 85 were downregulated in the presence of cellulose (Avicel). In addition, 251 genes were upregulated and 230 were downregulated in the presence of a high concentration of glucose. Genes encoding cellulolytic enzymes and transcription factors and genes related to the transport of nutrients and oxidative metabolism were also targets of CCR, mediated by CRE1 in a carbon source-dependent manner. Our results also suggested that CRE1 regulates the expression of genes related to the use of copper and iron as final electron acceptors or as cofactors of enzymes that participate in biomass degradation. As a result, the final effect of CRE1-mediated transcriptional regulation is to modulate the access of cellulolytic enzymes to cellulose polymers or blocks the entry of cellulase inducers into the cell, depending on the glucose content in the medium. These results will contribute to a better understanding of the mechanism of carbon catabolite repression in T. reesei, thereby enhancing its application in several biotechnology fields.

[Image-guided percutaneous biopsy of gastric lesions with suspicion of malignancy and negative endoscopic biopsies: report of our experience and revision of the literature]. / Biopsia percutánea guiada por imágenes de lesiones gástricas sospechosas de malignidad y biopsias endoscópicas negativas: reporte de nuestra experiencia y revisión de la literatura.

BACKGROUND: Endoscopic biopsy is the most efficient way to obtain a biopsy specimen from neoplastic lesions located in the stomach. Therefore, it is the procedure of choice to obtain a histologic diagnosis of gastric cancer. Image guided percutaneous biopsy is an alternative method to obtain histologic material for definitive diagnosis when the biopsies are negative for neoplastic cells in several endoscopies. OBJECTIVE: The purpose of this study was to evaluate the efficacy, complications, and histologic results of image guided percutaneous biopsies of gastric lesions in our center. MATERIALS AND METHODS: Between March 2004 and March 2007, 6 patients with strong suspicion of gastric cancer and negative endoscopic biopsies were referred for image guided percutaneous biopsy of gastric lesions (5 were guided by ultrasonography and 1 by CT scan). RESULTS: A histologic diagnosis was made in 5 patients: poorly differentated signet-ring cell carcinoma 3, B-cell non-Hodgkin's lymphoma 2. One patient suffered a perforation during the procedure and the final histologic diagnosis was obtained after the consequent surgery (B-cell non-Hodgkin's lymphoma). CONCLUSION: Despite of the scant published series, image guided percutaneous biopsy of gastric lesions is an useful, safe and effective technique to obtain a histologic diagnosis in patients with strong suspicion of gastric cancer and negative endoscopic biopsies.

Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa.

Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry.

Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis.

We defined the role of the transcriptional factor-XYR1-in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the Δxyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the Δxyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields.

Expression pattern of cellulolytic and xylanolytic genes regulated by transcriptional factors XYR1 and CRE1 are affected by carbon source in Trichoderma reesei.

Trichoderma reesei is the most important fungus for the industrial production of enzymes to biomass deconstruction. Most of the genes encoding cellulases and hemicellulases are regulated by the transcription factors CRE1 and XYR1. In this work, the regulation of 22 genes of cellulases and xylanases by these transcription factors was investigated under three different carbon sources. Analysis of gene expression and enzymatic profiles of CMCase, ß-glucosidase, and xylanases showed different regulation that was depended of the carbon source in both Δxyr1 and Δcre1 mutants. In the presence of glucose, the majority of genes evaluated (82%) showed increased expression levels in the Δcre1 mutant compared to the parental QM9414 strain. In the Δxyr1 mutant, it was observed that expression of cellulase and xylanase genes was reduced compared to the parental QM9414 strain, when cultured in the presence of cellulose or sophorose. Interesting, in the presence of glucose, approximately 60% of the analyzed genes had increased expression in the Δxyr1 mutant compared to parental strain. Furthermore, no correlation between gene expression and the number of putative binding sites of XYR1 and CRE1 to promoter region of cellulolytic and xylanolytic studied genes was observed. Therefore, these results demonstrated that the regulation of cellulase and xylanase by the transcription factors CRE1 and XYR1 is influenced by different carbon sources.

Deciphering the cis-regulatory elements for XYR1 and CRE1 regulators in Trichoderma reesei.

In this work, we report the in silico identification of the cis-regulatory elements for XYR1 and CRE1 proteins in the filamentous fungus Trichoderma reesei, two regulators that play a central role in the expression of cellulase genes. Using four datasets of condition-dependent genes from RNA-seq and RT-qPCR experiments, we performed unsupervised motif discovery and found two short motifs resembling the proposed binding consensus for XYR1 and CRE1. Using these motifs, we analysed the presence and arrangement of putative cis-regulatory elements recognized by both regulators and found that shortly spaced sites were more associated with XYR1- and CRE1-dependent promoters than single, high-score sites. Furthermore, the approach used here allowed the identification of the previously reported XYR1-binding sites from cel7a and xyn1 promoters, and we also mapped the potential target sequence for this regulator at the cel6a promoter that has been suggested but not identified previously. Additionally, seven other promoters (for cel7b, cel61a, cel61b, cel3c, cel3d, xyn3 and swo genes) presented a putative XYR1-binding site, and strong sites for CRE1 were found at the xyr1 and cel7b promoters. Using the cis-regulatory architectures nearly defined for XYR1 and CRE1, we performed genome-wide identification of potential targets for direct regulation by both proteins and important differences on their functional regulons were elucidated. Finally, we performed binding site mapping on the promoters of differentially expressed genes found in T. reesei mutant strains lacking xyr1 or cre1 and found that indirect regulation plays a key role on their signalling pathways. Taken together, the data provided here sheds new light on the mechanisms for signal integration mediated by XYR1 and CRE1 at cellulase promoters.

Comparative metabolism of cellulose, sophorose and glucose in Trichoderma reesei using high-throughput genomic and proteomic analyses.

BACKGROUND: The filamentous fungus Trichoderma reesei is a major producer of lignocellulolytic enzymes utilized by bioethanol industries. However, to achieve low cost second generation bioethanol production on an industrial scale an efficient mix of hydrolytic enzymes is required for the deconstruction of plant biomass. In this study, we investigated the molecular basis for lignocellulose-degrading enzyme production T. reesei during growth in cellulose, sophorose, and glucose. RESULTS: We examined and compared the transcriptome and differential secretome (2D-DIGE) of T. reesei grown in cellulose, sophorose, or glucose as the sole carbon sources. By applying a stringent cut-off threshold 2,060 genes were identified as being differentially expressed in at least one of the respective carbon source comparisons. Hierarchical clustering of the differentially expressed genes identified three possible regulons, representing 123 genes controlled by cellulose, 154 genes controlled by sophorose and 402 genes controlled by glucose. Gene regulatory network analyses of the 692 genes differentially expressed between cellulose and sophorose, identified only 75 and 107 genes as being specific to growth in sophorose and cellulose, respectively. 2D-DIGE analyses identified 30 proteins exclusive to sophorose and 37 exclusive to cellulose. A correlation of 70.17% was obtained between transcription and secreted protein profiles. CONCLUSIONS: Our data revealed new players in cellulose degradation such as accessory proteins with non-catalytic functions secreted in different carbon sources, transporters, transcription factors, and CAZymes, that specifically respond in response to either cellulose or sophorose.

Adaptación de la escala integral de calidad de vida en personas con discapacidad intelectual y del desarrollo en población Chilena / Adjustment of the integral scale of life quality in persons with intellectual disability and the development in Chilean population

El objetivo de esta investigación fue examinar la confiabilidad y validez de la Escala Integral de Calidad de Vida para población con discapacidad intelectual y del desarrollo, en población chilena. Este es uno de los principales instrumentos empleados para evaluar la calidad de vida en esta población desde una doble perspectiva objetiva, completada por los profesionales a cargo, y subjetiva administrada a los propios sujetos. Para ello, han participado 751 estudiantes y 82 profesionales. Los principales resultados demuestran que existe una adecuada fiabilidad del instrumento en población chilena y la estructura factorial es totalmente congruente con la versión española. Los resultados confirman que la Escala Integral es un instrumento válido y con una óptima fiabilidad en población chilena para evaluar la calidad de vida de personas con discapacidad intelectual y del desarrollo.

This paper aims to evaluate the reliability and validity of the Integral Quality of Life Scale in people with intellectual and developmental disability in the Chilean people. That is a principal instrument to evaluate the quality of life in this collective from subjective and objective vision. For this, 751 students have participated and 82 professionals. The main results show that there is adequate instrument reliability and factorial Chilean population structure is fully consistent with the Spanish version. The results confirm that the Integral Scale is a valid and optimum reliability instrument in Chilean population to assess the quality of life for people with intellectual and developmental disabilities.

Perfil do aleitamento materno em menores de um ano no Município de Londrina, Paraná / Breastfeeding profile in babies below one year old in the municipality of Londrina, State of Parana, Brazil

OBJETIVOS: descrever e analisar dados de um inquérito realizado em Londrina, Paraná, sobre práticas alimentares no primeiro ano de vida. MÉTODOS: os dados foram obtidos durante a Campanha Nacional de Vacinacão, realizada em agosto de 2002. As criancas foram selecionadas por plano de amostragem por conglomerado. Aplicou-se um questionário com questões sobre alimentacão da crianca nas 24 horas precedentes. Para obtencão das freqüências empregou-se análise de probitos. Pela análise de regressão logística, foram analisados os fatores de risco para interrupcão da amamentacão exclusiva em menores de quatro meses e desmame em menores de um ano. RESULTADOS: em menores de quatro meses, a prevalência do aleitamento materno exclusivo (AME) foi 29,3 por cento e, para aleitamento materno predominante (AMP) 20,7 por cento. Nos menores de seis meses, a prevalência do AME foi 21,0 por cento e do AMP 16,5 por cento. Para o aleitamento materno (AM) em menores de um ano, a prevalência foi 62,4 por cento. As medianas em dias foram 11,9 para AME e 257,3 para AM. Entre os fatores de risco para interrupcão do AME e do AM destacam-se: uso de chupeta, primiparidade, acompanhamento ambulatorial em servico público, idade e trabalho materno. CONCLUSÕES: as taxas de aleitamento foram consideradas baixas, indicando a necessidade de se estabelecerem políticas públicas adequadas para alimentacão da crianca.

Perfil do aleitamento materno em menores de um ano no Município de Londrina, Paraná / Breastfeeding profile in babies below one year old in the municipality of Londrina, State of Parana, Brazil

OBJETIVOS: descrever e analisar dados de um inquérito realizado em Londrina, Paraná, sobre práticas alimentares no primeiro ano de vida. MÉTODOS: os dados foram obtidos durante a "Campanha Nacional de Vacinacäo, realizada em agosto de 2002. As criancas foram selecionadas por plano de amostragem por conglomerado. Aplicou-se um questionário com questöes sobre alimentacäo da crianca nas 24 horas precedentes. Para obtencäo das freqüências empregou-se análise de probitos. Pela análise de regressäo logística, foram analisados os fatores de risco para interrupcäo da amamentacäo exclusiva em menores de quatro meses e desmame em menores de um ano. RESULTADOS: em menores de quatro meses, a prevalência do aleitamento materno exclusivo (AME) foi 29,3 por cento e, para aleitamento materno predominante (AMP) 20,7 por cento. Nos menores de seis meses, a prevalência do AME foi 21,0 por cento e do AMP 16,5 por cento. Para o aleitamento materno (AM) em menores de um ano, a prevalência foi 62,4 por cento. As medianas em dias foram 11,9 para AME e 257,3 para AM. Entre os fatores de risco para interrupcäo do AME e do AM destacam-se: uso de chupeta, primiparidade, acompanhamento ambulatorial em servico público, idade e trabalho materno. CONCLUSÖES: as taxas de aleitamento foram consideradas baixas, indicando a necessidade de se estabelecerem políticas públicas adequadas para alimentacäo da crianca. (AU)

Esclerosis tuberosa asociada a carcinoma de células renales con metástasis hepática / Tuberous sclerosis and renal adenocarcinoma with liver metastasis

La esclerosis tuberosa, enfermedad de Bourneville, Epiloia de Sherlock, enfermedad de Brushfield-Wyatt, escleroma de Virchow o adenomas sebáceos de Pringle, es un síndrome neurocutáneo que presenta gran variedad de manifestaciones clínicas, patológicas y radiológicas debidas al resultado de malformaciones hemartomatosas multiorgánicas a nivel de la piel, sistema nerviosos central, riñones, aparato cardiovascular, pulmones, ojos, huesos y aparato digestivo. Presentamos una paciente de 33 años con un cuadro de saciedad precoz, disfagia con una masa palpable en hipocondrio y flanco izquierdo. Comparamos los hallazgos en la literatura y concluimos que si bien es infrecuente la asociación de la esclerosis tuberosa con el carcinoma de células renales, existe una participación genética en el desarrollo tumoral renal, siendo aún más rara, la presencia de metástasis hepática

Esclerosis tuberosa asociada a carcinoma de células renales con metástasis hepática / Tuberous sclerosis and renal adenocarcinoma with liver metastasis

La esclerosis tuberosa, enfermedad de Bourneville, Epiloia de Sherlock, enfermedad de Brushfield-Wyatt, escleroma de Virchow o adenomas sebáceos de Pringle, es un síndrome neurocutáneo que presenta gran variedad de manifestaciones clínicas, patológicas y radiológicas debidas al resultado de malformaciones hemartomatosas multiorgánicas a nivel de la piel, sistema nerviosos central, riñones, aparato cardiovascular, pulmones, ojos, huesos y aparato digestivo. Presentamos una paciente de 33 años con un cuadro de saciedad precoz, disfagia con una masa palpable en hipocondrio y flanco izquierdo. Comparamos los hallazgos en la literatura y concluimos que si bien es infrecuente la asociación de la esclerosis tuberosa con el carcinoma de células renales, existe una participación genética en el desarrollo tumoral renal, siendo aún más rara, la presencia de metástasis hepática (AU)