1,8-cineol potentiates IRF3-mediated antiviral response in human stem cells and an ex vivo model of rhinosinusitis

Common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial super-infections resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential of 1,8-cineole for treating primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates Poly(I:C)-induced activity of the anti-viral transcription factor Interferon Regulatory Factor 3, while simultaneously reducing pro-inflammatory NF-κB-activity in human cell lines, inferior turbinate stem cells (ITSCs) and ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with Poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared to Poly(I:C) alone, whereas NF-κB-activity was reduced. Accordingly, 1,8-cineole- and Poly(I:C)-treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared to the Poly(I:C)-treated approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with LPS and 1,8-cineole compared to the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on pro-inflammatory NF-κB-signalling and may thus broaden its field of application.

Genomic Analysis of Wild Tomato Introgressions Determining Metabolism- and Yield-Associated Traits

With the aim of determining the genetic basis of metabolic regulation in tomato fruit, we constructed a detailed physical map of genomic regions spanning previously described metabolic quantitative trait loci of a Solanum pennellii introgression line population. Two genomic libraries from S. pennellii were screened with 104 colocated markers from five selected genomic regions, and a total of 614 bacterial artificial chromosome (BAC)/cosmids were identified as seed clones. Integration of sequence data with the genetic and physical maps of Solanum lycopersicum facilitated the anchoring of 374 of these BAC/cosmid clones. The analysis of this information resulted in a genome-wide map of a nondomesticated plant species and covers 10% of the physical distance of the selected regions corresponding to approximately 1% of the wild tomato genome. Comparative analyses revealed that S. pennellii and domesticated tomato genomes can be considered as largely colinear. A total of 1,238,705 bp from both BAC/cosmid ends and nine large insert clones were sequenced, annotated, and functionally categorized. The sequence data allowed the evaluation of the level of polymorphism between the wild and cultivated tomato species. An exhaustive microsynteny analysis allowed us to estimate the divergence date of S. pennellii and S. lycopersicum at 2.7 million years ago. The combined results serve as a reference for comparative studies both at the macrosyntenic and microsyntenic levels. They also provide a valuable tool for fine-mapping of quantitative trait loci in tomato. Furthermore, they will contribute to a deeper understanding of the regulatory factors underpinning metabolism and hence defining crop chemical composition.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortus Delta wbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. Delta wbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence. (C) 2010 Elsevier Ltd. All rights reserved.

Inovação tecnológica nas empresas no setor de biotecnologia no Brasil

O objetivo deste trabalho é contribuir para o entendimento do processo de inovação tecnológica nas empresas brasileiras de biotecnologia. Para isso, procurou-se: identificar os fatores mais críticos ao processo de inovação das empresas do setor; analisar a dinâmica do setor e a contribuição das universidades e incubadoras para o processo de inovação e; avaliar como esses elementos afetam o processo de gestão da inovação tecnológica dentro e fora da organização. Este estudo exploratório de natureza qualitativa baseou-se na metodologia de estudo de casos múltiplos e para interpretação dos resultados foi utilizada a técnica da análise de conteúdo, bem como com a análise de dados secundários. O estudo indicou que entre os muitos obstáculos à inovação, o acesso ao financiamento é o mais crítico. As parcerias, apontadas como uma alternativa para esse e outros problemas por proporcionar a redução do investimento e do risco e o acesso à mão-de-obra qualificada, equipamentos e laboratórios sofisticados, têm sido prejudicadas em função de uma série de fatores institucionais e regulatórios – a começar pela falta de leis e de regras claras relativas à propriedade intelectual. Somando-se a isso uma incipiente cultura colaborativa entre empresas. Os resultados do estudo indicam que as empresas de biotecnologia brasileiras buscam compensar essas barreiras conferindo agilidade aos seus processos internos, criando estruturas organizacionais flexíveis e um ambiente organizacional favorável à inovação, que é internalizada, como prática, de maneira tácita. O processo de gestão da inovação ocorre de maneira informal e pouco sistematizada. Por fim, os resultados mostram que o processo de inovação nas empresas brasileiras de biotecnologia beneficia-se de uma postura pró-ativa adotada por elas de gerenciar e aprender com as adversidades.

Differential morphofunctional characteristics and gene expression in fast and slow muscle of rats with monocrotaline-induced heart failure

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MyoD, myogenin and proliferating cell nuclear antigen expression in growing Nile tilapia (Oreochromis niloticus L.)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Growth hormone attenuates skeletal muscle changes in experimental chronic heart failure

Objective: This study evaluated the effects of growth hormone (GH) on morphology and myogenic regulatory factors (MRF) gene expression in skeletal muscle of rats with ascending aortic stenosis (AAS) induced chronic heart failure.Design: Male 90-100 g Wistar rats were subjected to thoracotomy. AAS was created by placing a stainless-steel clip on the ascending aorta. Twenty five weeks after surgery, rats were treated with daily subcutaneous injections of recombinant human GH (2 mg/kg/day; AAS-GH group) or saline (AAS group) for 14 days. Sham-operated animals served as controls. Left ventricular (LV) function was assessed before and after treatment. IGF-1 serum levels were measured by ELISA. After anesthesia, soleus muscle was frozen in liquid nitrogen. Histological sections were stained with HE and picrosirius red to calculate muscle fiber cross-sectional area and collagen fractional area, respectively. MRF myogenin and MyoD expression was analyzed by reverse transcription PCR.Results: Body weight was similar between groups. AAS and AAS-GH groups presented dilated left atrium, left ventricular (LV) hypertrophy (LV mass index: Control 1.90 +/- 0.15; AAS 3.11 +/- 0.44; AAS-GH 2.94 +/- 0.47 g/kg; p < 0.05 AAS and AAS-GH vs. Control), and reduced LV posterior wall shortening velocity. Soleus muscle fiber area was significantly lower in AAS than in Control and AAS-GH groups; there was no difference between AAS-GH and Control groups. Collagen fractional area was significantly higher in MS than Control; AAS-GH did not differ from both Control and AAS groups. Serum IGF-1 levels decreased in AAS compared to Control. MyoD mRNA was significantly higher in AAS-GH than AAS; there was no difference between AAS-GH and Control groups. Myogenin mRNA levels were similar between groups.Conclusion: In rats with aortic stenosis-induced heart failure, growth hormone administration increases MyoD gene expression above non-treated animal levels, preserves muscular trophism and attenuates interstitial fibrosis. These results suggest that growth hormone may have a potential role as an adjuvant therapy for chronic heart failure. (C) 2009 Elsevier Ltd. All rights reserved.

Nandrolone Stimulates Myod Expression During Muscle Regeneration in the Condition of Myonecrosis Induced by Bothrops jararacussu Venom Poisoning

Myonecrosis with permanent loss of muscle mass is a relevant local toxic effect following envenomation with Bothrops jararacussu snake venom. Regeneration of adult skeletal muscle involves the activation of satellite cells, a process regulated by myogenic regulatory factors (MRF). MyoD is an MRF involved in both proliferation and differentiation of satellite cells. Androgens are modulators of skeletal muscle, known to increase muscle mass and strength. This study examined the hypothesis that anabolic androgens improve the muscle regeneration process in mice following envenomation by Bothrops jararacussu snake venom. Myonecrosis was induced by venom injection (30 g/50 l in physiological solution) over the extensor digitorum longus (EDL) muscles of mice. Nandrolone (ND) (6 mg/kg, sc) was administered after 12 h, 7 d, and 14 d following venom injection. The histological changes in EDL muscle at 1, 3, 7, and 21 d after muscle injury were analyzed by light microscopy. Cross-sectional areas of fibers were measured. MyoD was evaluated by immunofluorescence technique. Histological examination revealed the presence of a regeneration process in ND-treated animals, characterized by the appearance of some myotubes at 3 d, and numerous myotubes at 7 d from venom injection. Nandrolone treatment reduced the frequency of small fibers at 7 and 21 d after venom administration, and increased the frequency of large fibers at 7 d postinjury. Nandrolone also significantly augmented the expression of MyoD-positive cells at 7 and 21 d after envenomation. These results suggest that ND accelerates muscle regeneration and indicate the involvement of MyoD in this process.

Genomic Strategy Identifies a Missense Mutation in WD-Repeat Domain 65 (WDR65) in an Individual With Van der Woude Syndrome

Genetic variation in the transcription factor interferon regulatory factor 6 (IRF6) causes and contributes risk for oral clefting disorders. We hypothesized that genes regulated by IRF6 are also involved in oral clefting disorders. We used five criteria to identify potential IRF6 target genes; differential gene expression in skin taken from wild-type and Irf6-deficient murine embryos, localization to the Van der Woude syndrome 2 (VWS2) locus at 1p36-1p32, overlapping expression with Irf6, presence of a conserved predicted-binding site in the promoter region, and a mutant murine phenotype that was similar to the Irf6 mutant mouse. Previously, we observed altered expression for 573 genes; 13 were located in the murine region syntenic to the VWS2 locus. Two of these genes, Wdr65 and Stratifin, met 4 of 5 criteria. Wdr65 was a novel gene that encoded a predicted protein of 1,250 amino acids with two WD domains. As potential targets for Irf6 regulation, we hypothesized that disease-causing mutations will be found in WDR65 and Stratifin in individuals with VWS or VWS-like syndromes. We identified a potentially etiologic missense mutation in WDR65 in a person with VWS who does not have an exonic mutation in IRF6. The expression and mutation data were consistent with the hypothesis that WDR65 was a novel gene involved in oral clefting. (C) 2011 Wiley-Liss, Inc.

Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Down-regulation of MyoD gene expression in rat diaphragm muscle with heart failure

Diaphragm myopathy has been described in patients with heart failure (HF), with alterations in myosin heavy chains (MHC) expression. The pathways that regulate MHC expression during HF have not been described, and myogenic regulatory factors (MRFs) may be involved. The purpose of this investigation was to determine MRF mRNA expression levels in the diaphragm. Diaphragm muscle from both HF and control Wistar rats was studied when overt HF had developed, 22 days after monocrotaline administration. MyoD, myogenin and MRF4 gene expression were determined by RT-PCR and MHC isoforms by polyacrylamide gel electrophoresis. Heart failure animals presented decreased MHC IIa/IIx protein isoform and MyoD gene expression, without altering MHC I, IIb, myogenin and MRF4. Our results show that in HF, MyoD is selectively down-regulated, which might be associated with alterations in MHC IIa/IIx content. These changes are likely to contribute to the diaphragm myopathy caused by HF.

Maternal protein restriction induce skeletal muscle changes without altering the MRFs MyoD and myogenin expression in offspring

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological aspects of neuromuscular junctions and gene expression of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle of rats with heart failure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mecanismos celulares e moleculares que controlam o desenvolvimento e o crescimento muscular

O músculo estriado esquelético é formado pela associação de fibras musculares com a matriz extracelular. Esse tecido possui alta plasticidade e o conhecimento das características morfológicas, da miogênese, e da dinâmica do crescimento é importante para o entendimento da morfofisiologia bem como para a seleção de animais visando a melhoria na produção de carne. A maioria dos músculos estriados originam-se de células precursoras do mesoderma a partir dos somitos do embrião e o controle da diferenciação ocorre pela ação de fatores indutores ou inibidores. Um grupo de fatores transcricionais, pertencentes à família MyoD tem um papel central na diferenciação muscular. Coletivamente chamados de Fatores de Regulação Miogênica (MRFs), são conhecidos quatro tipos: MyoD, myf-5, miogenina e MRF4. Esses fatores ligam-se à seqüências de DNA conhecidas como Ebox (CANNTG) na região promotora de vários genes músculo-específicos, levando à expressão dos mesmos. As células embrionárias com potencial para diferenciação em células musculares (células precursoras miogênicas) expressam MyoD e Myf-5 e são denominadas de mioblastos. Essas células proliferam, saem do ciclo celular, expressam miogenina e MRF4, que regulam a fusão e a diferenciação da fibra muscular. Uma população de mioblastos que se diferencia mais tardiamente, as células miossatélites, são responsáveis pelo crescimento muscular no período pós natal, que pode ocorrer por hiperplasia e hipertrofia das fibras. As células satélites quiescentes não expressam os MRFs, porém, sob a ação de estímulos como fatores de crescimento ou citocinas, ocorre a ativação desse tipo celular que prolifera e expressa os MRFs de maneira similar ao que ocorre com as células precursoras miogênicas durante a miogênese. Os mecanismos de crescimento muscular são regulados pela expressão temporal dos (MRFs), que controlam a expressão dos genes relacionados com o crescimento muscular.

Heart failure alters MyoD and MRF4 expressions in rat skeletal muscle

Heart failure (HF) is characterized by a skeletal muscle myopathy with increased expression of fast myosin heavy chains (MHCs). The skeletal muscle-specific molecular regulatory mechanisms controlling MHC expression during HF have not been described. Myogenic regulatory factors (MRFs), a family of transcriptional factors that control the expression of several skeletal muscle-specific genes, may be related to these alterations. This investigation was undertaken in order to examine potential relationships between MRF mRNA expression and MHC protein isoforms in Wistar rat skeletal muscle with monocrotaline-induced HF. We studied soleus (Sol) and extensor digitorum longus (EDL) muscles from both HF and control Wistar rats. MyoD, myogenin and MRF4 contents were determined using reverse transcription-polymerase chain reaction while MHC isoforms were separated using polyacrylamide gel electrophoresis. Despite no change in MHC composition of Wistar rat skeletal muscles with HF, the mRNA relative expression of MyoD in Sol and EDL muscles and that of MRF4 in Sol muscle were significantly reduced, whereas myogenin was not changed in both muscles. This down-regulation in the mRNA relative expression of MRF4 in Sol was associated with atrophy in response to HF while these alterations were not present in EDL muscle. Taken together, our results show a potential role for MRFs in skeletal muscle myopathy during HF. © 2006 Blackwell Science Ltd.