A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.Peer Reviewe

Tuberculose em doentes com Sida. Tratamento completo sob observação directa## Projecto de investigação subsidiado pela Comissão Nacional de Luta Contra a SIDA

RESUMO: A co-infecção VIH-tuberculose tem um enorme impacto em termos de saúde pública. A deficiente adesão ao tratamento antibacilar, particularmente em toxicodependentes infectados por VIH, contribui para a elevada morbilidade e mortalidade registadas e, bem assim, para o recrudescimento da multirresistência da bacilo da tuberculose e para a sua disseminação na comunidade.Tendo em conta resultados obtidos em doentes observados entre 1986 e 1997, os autores iniciaram um projecto de investigação cujo objectivo principal era o de melhorar a adesão à terapêutica da tuberculose em toxicodependentes infectados por VIH. Esse projecto, que teve a duração de dois anos, previa que todos os doentes tomassem, sob observação directa, a terapêutica antibacilar conjuntamente com metadona.Apresentam-se os resultados desse trabalho, salientando-se que a comparação estatística de diferentes parâmetros observados nos doentes de 1986-97 e nos doentes envolvidos neste projecto, apontam para a prossecução desta metodologia e para a sua generalização.REV PORT PNEUMOL 2001; VII (3): SUMMARY: HIV and tuberculosis co-infection has an enormous impact on public health. Inadequate patient compliance for tuberculosis therapy, especially in intravenous drug-abusers (IVDA), has contributed to increased mortality and morbidity rates, as well as a renewed activity of multi-resistant tuberculosis and its dissemination in the community.Taken into consideration results obtained between 1986 and 1997, the authors´ main objective was to initiate a program whose main purpose was to improve patient compliance in HIV-positive IVDA undergoing treatment for tuberculosis. This two-year program stipulated a daily directly observed tuberculosis treatment in conjunction with methadone.Through various observed parameters, we present the results obtained in this study, and hope to point out that the comparison of the results registered in the patients treated between 1986-97 and those of the present study, suggest a continuation of these methodologies and their general usage.REV PORT PNEUMOL 2001; VII (3)

A defective TLR4 signaling for IFN-β expression is responsible for the innately lower ability of BALB/c macrophages to produce NO in response to LPS as compared to C57BL/6.

C57BL/6 mice macrophages innately produce higher levels of NO than BALB/c cells when stimulated with LPS. Here, we investigated the molecular events that account for this intrinsic differential production of NO. We found that the lower production of NO in BALB/c is not due to a subtraction of L-arginine by arginase, and correlates with a lower iNOS accumulation, which is independent of its degradation rate. Instead, the lower accumulation of iNOS is due to the lower levels of iNOS mRNA, previously shown to be also independent of its stability, suggesting that iNOS transcription is less efficient in BALB/c than in C57BL/6 macrophages. Activation of NFκB is more efficient in BALB/c, thus not correlating with iNOS expression. Conversely, activation of STAT-1 does correlate with iNOS expression, being more prominent in C57BL/6 than in BALB/c macrophages. IFN-β and IL-10 are more highly expressed in C57BL/6 than in BALB/c macrophages, and the opposite is true for TNF-α. Whereas IL-10 and TNF-α do not seem to participate in their differential production of NO, IFN-β has a determinant role since 1) anti-IFN-β neutralizing antibodies abolish STAT-1 activation reducing NO production in C57BL/6 macrophages to levels as low as in BALB/c cells and 2) exogenous rIFN-β confers to LPS-stimulated BALB/c macrophages the ability to phosphorylate STAT-1 and to produce NO as efficiently as C57BL/6 cells. We demonstrate, for the first time, that BALB/c macrophages are innately lower NO producers than C57BL/6 cells because they are defective in the TLR-4-induced IFN-β-mediated STAT-1 activation pathway

IFN-β mRNA expression and effect of IFN-β neutralization on NO production and STAT-1 activation in BALB/c and C57BL/6 macrophages.

<p>Peritoneal macrophages (5×10⁶) were stimulated with 1 µg/mL LPS for the indicated periods of time. Total RNA was extracted and IFN-β mRNA levels were determined by Real Time RT-PCR. The relative levels of mRNA expression were calculated by reference to the β-actin expression in each sample, using the 2^−ΔΔCt method (A). Alternatively, cells (1×10⁵) were stimulated for 24 h (B) or for the indicated periods of time (C–E) with 1 µg/mL LPS in the presence or absence of the indicated concentrations (B) or with 200 U/mL of polyclonal anti-IFN-β (C–E). Culture supernatants were analyzed for NO⁻₂ levels using the Griess reaction, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#s2" target="_blank">Materials and Methods</a>. Purified normal rabbit IgG was used as an isotype control. (B) represents the dose-response of one experiment and (C) represents the time course of a second one, out of three independent experiments. Values represent the mean of samples assayed in triplicate. (D and E) represents STAT-1 activation as analyzed by Western blot of total protein extracts of C57BL/6 peritoneal macrophages (3×10⁵) using antibodies specific for pSTAT-1 (Y701) and β-actin. The levels of pSTAT-1 (E) were expressed as ratios of the signal intensity of the bands normalized to that of β-actin. Data are representative of three independent and reproducible experiments. Additional experiments to illustrate the variability in the results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#pone.0098913.s004" target="_blank">Fig. S4</a>.</p

Effect of exogenous IFN-β on NO production and STAT-1 activation in BALB/c and C57BL/6 macrophages.

<p>Peritoneal macrophages (1×10⁶) were stimulated with 1 µg/mL LPS for the indicated periods of time. After 8 hs, supernatants of C57BL/6 and BALB/c cells, still devoid of NO, were swapped and, cells were cultured in the presence (dotted lines) or absence (dashed lines) of anti- IFN-β for the indicated periods of time, when supernatants were analyzed for NO⁻₂ levels using the Griess reaction, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#s2" target="_blank">Materials and Methods</a> (A). Alternatively, 1×10⁶ (B and C) or 3×10⁶ (D and E) were stimulated for 48 h (B) or for the indicated periods of time (C–E) with 1 µg/mL LPS in the presence or absence of the indicated concentrations (B) or with 10 U/mL rIFN-β (C–E). Culture supernatants were analyzed for NO⁻₂ levels as above (B and C). (B) represents the dose-response of one experiment and (C) represents the time course of a second one, out of three independent experiments. Values represent the mean of samples assayed in triplicate. D and E represent STAT-1 activation as analyzed by Western blot of total protein extracts of C57BL/6 peritoneal macrophages (3×10⁵) using antibodies specific for pSTAT-1 (Y701) and β-actin. The levels of pSTAT-1 (F) were expressed as ratios of the signal intensity of the bands normalized to that of β-actin. Data are representative of three independent and reproducible experiments. Additional experiments to illustrate the variability in the results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#pone.0098913.s005" target="_blank">Fig. S5</a>.</p

STAT-1 and NF-κB expression and activation in BALB/c and C57BL/6 macrophages.

<p>Total protein extracts of C57BL/6 or BALB/c peritoneal macrophages (1×10⁶) stimulated with 1 µg/mL LPS for the indicated periods of time were analyzed by Western blot using specific anti-STAT-1, anti-pSTAT-1 (Y701) (A) or anti-pp65 (Ser536), anti-IκB-α (B) and anti-β-actin antibodies (A and B). The levels of STAT-1 (C), p-STAT-1 (E), p-p65 (D) or IκB-α (F) were expressed as ratios of the signal intensity of the bands normalized to that of β-actin. Data are representative of three independent and reproducible experiments.</p