Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆ zface1 Strain

Abstract The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

Analysis of the muscle tissue of Wistar rats submitted to the sciatic nerve compression model and cryotherapy.

OBJECTIVE: To evaluate the effects of right sciatic nerve compression and cryotherapy on muscle tissue. METHODS: We used 42 male Wistar rats, subdivided in the following Groups Control, Injury 3, Injury 8 and Injury 15 submitted to nerve compression and euthanized in the 3rd, 8th and 15th day after surgery. The Cryotherapy Injury 3 was entailed treatment with cryotherapy by immersion of the animal in recipient for 20 minutes during 1 day, then animals were euthanized at the 3rd day after surgery, and the Cryotherapy Injury 8 and the Cryotherapy Injury 15 was treated for 6 days, and euthanized at the 8th and 15th day after surgery. Functional evaluation was performed by the grasping strength of the right pelvic limb. The right tibialis anterior muscles were evaluated for mass, smaller diameter and cross-sectional area. In the Cryotherapy Injury 8 and the Cryotherapy Injury 15 groups, the hydroxyproline was dosed in the right soles. RESULTS: In the compression there was a significant difference in the Injury Groups compared with the Control Group (p<0.05). In the smaller diameter, the compression in Control Group was higher than Injury 8 (p=0.0094), Injury 15 (p=0.002) and Cryotherapy Injury 15 (p<0.001) groups. The comparison between groups with euthanasia in the same post-operative period, a significant difference (p=0.0363) was seen in day 8th after surgery, and this result in Cryotherapy Injury Group was greater than Injury Group. In the fiber area, Control Group was also higher than the Injury 8 (p=0.0018), the Injury 15 (p<0.001) and the Cryotherapy Injury 15 (p<0.001). In hydroxyproline, no significant difference was seen between groups. CONCLUSION: Nerve damage resulted in decreased muscle strength and trophism, the cryotherapy delayed hypotrophy, but this effect did not persist after cessation of treatment.

Neurotrophin expression and histomorphometric evaluation in Wistar rats subjected to neural mobilization after compression of the median nerve.

OBJECTIVE: To evaluate the neurotrophin mRNA expression and axon count in the median nerve of Wistar rats submitted to neural mobilization (NM) after nerve compression. METHODS: Eighteen animals were randomly divided into G1 (nerve compression only), G2 (NM for 1 min), and G3 (NM for 3 min). For NM, the animals were anesthetized and the right scapula received the mobilization, adapted as indicated for humans, on alternate days, from the third to the 13th postoperative (PO) day, totaling six days of therapy. On the 14th PO day, animals were anesthetized and euthanized. Fragments of the median nerve, distal to the compression procedure, were removed for histomorphometric analysis and expression of neurotrophins, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) by RT-PCR. RESULTS: Histomorphometric analysis revealed differences in the number of axons in the injured side, which was significantly lower in the injured limb nerve compared to the control limb, whereas the RT-PCR analysis showed no significant differences in the expression of NGF or BDNF. CONCLUSION: NM treatment did not affect median nerve regeneration, which maintained normal recovery rates.

OBJETIVO: Avaliar a expressão de RNAm de neurotrofinas e a contagem de axônios no nervo mediano de ratos Wistar submetidos à mobilização neural (MN) após compressão nervosa. MÉTODOS: Foram divididos aleatoriamente 18 animais em G1 (apenas compressão nervosa), G2 (MN por 1 minuto) e G3 (MN por 3 minutos). Para a MN, os animais foram anestesiados e o membro escapular direito recebeu a mobilização, adaptada da forma indicada para humanos, em dias alternados, do terceiro ao 13° dia de pós-operatório (PO), em seis dias de terapia. No 14° dia PO, os animais foram anestesiados e eutanasiados. Fragmentos do nervo mediano, distais ao procedimento de compressão, foram retirados para análise histomorfométrica e de expressão das neutrotrofinas, fator de crescimento do nervo (NGF) e fator de crescimento derivado do cérebro (BNDF) por RT-PCR. RESULTADOS: A análise histomorfométrica evidenciou diferenças no número de axônios nos lados lesionados, que foi significativamente menor no nervo do membro lesado comparado com o membro controle; por sua vez, a análise por RT-PCR não apontou diferenças significativas na expressão de NGF e nem de BNDF. CONCLUSÃO: O tratamento de MN não afetou a regeneração do nervo mediano, que manteve índices normais de recuperação.

Neurotrophin expression and histomorphometric evaluation in Wistar rats subjected to neural mobilization after compression of the median nerve / Expressão de neurotrofinas e avaliação histomorfométrica em ratos Wistar submetidos à mobilização neural após compressão do nervo mediano

ABSTRACT Objective: To evaluate the neurotrophin mRNA expression and axon count in the median nerve of Wistar rats submitted to neural mobilization (NM) after nerve compression. Methods: Eighteen animals were randomly divided into G1 (nerve compression only), G2 (NM for 1 min), and G3 (NM for 3 min). For NM, the animals were anesthetized and the right scapula received the mobilization, adapted as indicated for humans, on alternate days, from the third to the 13th postoperative (PO) day, totaling six days of therapy. On the 14th PO day, animals were anesthetized and euthanized. Fragments of the median nerve, distal to the compression procedure, were removed for histomorphometric analysis and expression of neurotrophins, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) by RT-PCR. Results: Histomorphometric analysis revealed differences in the number of axons in the injured side, which was significantly lower in the injured limb nerve compared to the control limb, whereas the RT-PCR analysis showed no significant differences in the expression of NGF or BDNF. Conclusion: NM treatment did not affect median nerve regeneration, which maintained normal recovery rates.

RESUMO Objetivo: Avaliar a expressão de RNAm de neurotrofinas e a contagem de axônios no nervo mediano de ratos Wistar submetidos à mobilização neural (MN) após compressão nervosa. Métodos: Foram divididos aleatoriamente 18 animais em G1 (apenas compressão nervosa), G2 (MN por 1 minuto) e G3 (MN por 3 minutos). Para a MN, os animais foram anestesiados e o membro escapular direito recebeu a mobilização, adaptada da forma indicada para humanos, em dias alternados, do terceiro ao 13° dia de pós-operatório (PO), em seis dias de terapia. No 14° dia PO, os animais foram anestesiados e eutanasiados. Fragmentos do nervo mediano, distais ao procedimento de compressão, foram retirados para análise histomorfométrica e de expressão das neutrotrofinas, fator de crescimento do nervo (NGF) e fator de crescimento derivado do cérebro (BNDF) por RT-PCR. Resultados: A análise histomorfométrica evidenciou diferenças no número de axônios nos lados lesionados, que foi significativamente menor no nervo do membro lesado comparado com o membro controle; por sua vez, a análise por RT-PCR não apontou diferenças significativas na expressão de NGF e nem de BNDF. Conclusão: O tratamento de MN não afetou a regeneração do nervo mediano, que manteve índices normais de recuperação.

Analysis of the muscle tissue of Wistar rats submitted to the sciatic nerve compression model and cryotherapy / Análise do tecido muscular de ratos Wistar submetidos ao modelo de compressão do nervo isquiático e à crioterapia

ABSTRACT Objective: To evaluate the effects of right sciatic nerve compression and cryotherapy on muscle tissue. Methods: We used 42 male Wistar rats, subdivided in the following Groups Control, Injury 3, Injury 8 and Injury 15 submitted to nerve compression and euthanized in the 3rd, 8th and 15th day after surgery. The Cryotherapy Injury 3 was entailed treatment with cryotherapy by immersion of the animal in recipient for 20 minutes during 1 day, then animals were euthanized at the 3rd day after surgery, and the Cryotherapy Injury 8 and the Cryotherapy Injury 15 was treated for 6 days, and euthanized at the 8th and 15th day after surgery. Functional evaluation was performed by the grasping strength of the right pelvic limb. The right tibialis anterior muscles were evaluated for mass, smaller diameter and cross-sectional area. In the Cryotherapy Injury 8 and the Cryotherapy Injury 15 groups, the hydroxyproline was dosed in the right soles. Results: In the compression there was a significant difference in the Injury Groups compared with the Control Group (p<0.05). In the smaller diameter, the compression in Control Group was higher than Injury 8 (p=0.0094), Injury 15 (p=0.002) and Cryotherapy Injury 15 (p<0.001) groups. The comparison between groups with euthanasia in the same post-operative period, a significant difference (p=0.0363) was seen in day 8th after surgery, and this result in Cryotherapy Injury Group was greater than Injury Group. In the fiber area, Control Group was also higher than the Injury 8 (p=0.0018), the Injury 15 (p<0.001) and the Cryotherapy Injury 15 (p<0.001). In hydroxyproline, no significant difference was seen between groups. Conclusion: Nerve damage resulted in decreased muscle strength and trophism, the cryotherapy delayed hypotrophy, but this effect did not persist after cessation of treatment.

RESUMO Objetivo: Avaliar os efeitos da compressão nervosa do isquiático direito e da crioterapia no tecido muscular. Métodos: Foram utilizados 42 ratos Wistar machos, subdivididos nos Grupos Controle, Lesão 3, Lesão 8 e Lesão 15, submetidos a compressão nervosa e eutanasiados, respectivamente, no 3°, 8° e 15° dias pós-operatório; Lesão Crioterapia 3, tratado com crioterapia, por imersão durante 20 minutos, por 1 dia, e eutanasiados no 3° dia pós-operatório; e Lesão Crioterapia 8 e Lesão Crioterapia 15, tratados durante 6 dias e eutanasiados no 8° e 15° dias pós-operatório. A avaliação funcional foi realizada pela força de preensão do membro pélvico direito. Os músculos tibiais anteriores direitos foram avaliados quanto a massa, menor diâmetro e área de secção transversa. Em Lesão Crioterapia 8 e Lesão Crioterapia 15, foi dosada a hidroxiprolina nos sóleos direitos. Resultados: Na preensão, houve diferença significativa nos Grupos Lesão quando comparados ao Grupo Controle (p<0,05). No menor diâmetro, o Grupo Controle foi maior que Lesão 8 (p=0,0094), Lesão 15 (p = 0,002) e Lesão Crioterapia 15 (p<0,001). Na comparação entre os grupos com eutanásia no mesmo pós-operatório, houve diferença significativa (p=0,0363) no 8° pós-operatório, sendo Lesão Crioterapia maior que Lesão. Na área das fibras, o Grupo Controle também foi maior que Lesão 8 (p=0,0018), Lesão 15 (p<0,001) e Lesão Crioterapia 15 (p<0,001). Na hidroxiprolina, não houve diferença significativa entre os grupos. Conclusão: A lesão nervosa resultou na diminuição da força e em trofismo muscular, e a crioterapia retardou a hipotrofia, porém este efeito não se manteve após o tratamento cessar.

Effects of the platelet-rich fibrin associated with physical exercise in a model of median nerve compression

AIMS: Platelet-Rich Fibrin (PRF) is a new and promising technique for tissue repair, however, there is still a gap about its action on peripheral nerve injury, as well as its association with physical exercise. Thus, the aim of this study was to evaluate the effects of the PRF associated with physical exercise in a model of median nerve compression. METHODS: 42 rats, divided into: Control, non-injured limb, and other six groups with nerve lesion (Control lesion; Treated with PRF; Treadmill walking; Free swimming; PRF and treadmill; PRF and swimming). The lesion model was performed with median nerve compression. To obtain the PRF, 1.5 mL of blood was centrifuged and the fibrin clot positioned directly over the compression region. The exercise protocols were performed during 2 weeks. The evaluations performed were grip strength tests and histologic analysis. RESULTS: both, grip strength and histomorphometric evaluations (fiber numbers and axon density) did not present significant differences between the treated and lesion groups, however, in the morphological evaluation it was possible to distinguish characteristics of the repair process for the treated groups. CONCLUSION: a slight qualitative improvement was observed for the treated groups, especially when it was associated PRF with free swimming.(AU)

Avaliação nociceptiva da associação entre exercício físico e fibrina rica em plaquetas em ratos Wistar submetidos ao modelo de compressão de nervo mediano / Nociceptive evaluation of the association between physical exercises and platelet-rich fibrin in Wistar rats submitted to median nerve compression

RESUMO JUSTIFICATIVA E OBJETIVOS: Fibrina rica em plaquetas é uma técnica nova e promissora na aceleração do reparo, com possíveis efeitos analgésicos, contudo, ainda há uma lacuna com relação à lesão nervosa periférica, bem como com a associação com exercícios físicos. Assim, o objetivo deste estudo foi avaliar os efeitos da fibrina rica em plaquetas associada a exercício físico sobre a nocicepção e o edema, em modelo experimental de compressão do nervo mediano. MÉTODOS: Foram utilizados 36 ratos, todos submetidos a compressão do nervo mediano e divididos em seis grupos: G1: sem manipulações adicionais; G2: compressão e tratado com fibrina rica em plaquetas; G3: compressão e tratado com natação livre; G4: compressão e exercício de caminhada em esteira; G5: natação livre + fibrina rica em plaquetas; G6: caminhada em esteira + fibrina rica em plaquetas. O modelo de lesão foi realizado com amarria do nervo mediano, com fio catgut 4.0 cromado. Para obtenção da fibrina rica em plaquetas, 1,5mL de sangue foi centrifugado e o coágulo de fibrina foi posicionado diretamente sobre a região da compressão. Os protocolos de exercício foram realizados durante 2 semanas, entre o 3º e 14º dias de pós-operatório. As avaliações nociceptivas e de edema ocorreram, respectivamente, pelo limiar de retirada de pata e pletismometria, nos momentos prévios à lesão, no 3º, 7º e 15º dias de pós-operatório. RESULTADOS: Não houve diferenças entre os grupos, apenas entre as avaliações, denotando que houve aumento da nocicepção e do edema, o qual perdurou ou foi decaindo, respectivamente, com o passar do tempo. CONCLUSÃO: O uso isolado ou associado da fibrina rica em plaquetas com exercícios físicos não produziu alterações na nocicepção e edema.

ABSTRACT BACKGROUND AND OBJECTIVES: Platelet-rich fibrin is a new and promising technique to accelerate repair, with possible analgesic effects; however, there is still a gap with regard to peripheral nerve injury and the association with physical exercises. So, this study aimed at evaluating the effects of platelet-rich fibrin associated to physical exercises on nociception and edema in experimental median nerve compression model. METHODS: Thirty-six rats, all submitted to median nerve compression, were divided in six groups: G1: without additional manipulation; G2: compression and treated with platelet-rich fibrin; G3: compression and treated with free swimming; G4: compression and walking on a treadmill; G5: free swimming + platelet-rich fibrin; G6: walking on a treadmill + platelet-rich fibrin. Injury was induced by tying the median nerve with chrome plated catgut 4.0. Platelet-rich fibrin was obtained by centrifuging 1.5 mL of blood and positioning the fibrin clot directly on the compression region. Exercises were carried out during two weeks, between the 3rd and 14th postoperative days. Nociception and edema were evaluated, respectively, by flinch threshold and plethysmometer, in moments before injury and in the 3rd, 7th and 15th postoperative days. RESULTS: There have been no differences among groups, only among evaluations, showing increased nociception and edema, which has lasted or improved, respectively, over time. CONCLUSION: Platelet-rich fibrin alone or associated to physical exercises has not changed nociception and edema.

The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus ß-Xylosidase I.

The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved ß-Xylosidase and α-L-Arabinofuranosidase (ß-Xyl I-α-L-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant ß-Xyl I-α-L-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant ß-Xyl I-α-L-Ara exhibited a specific ß-Xylosidase I activity of 1.25 U mg(-1) to oNPX and a specific α-L-Arabinofuranosidase activity of 0.47 U mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its ß-Xylosidase I activity, and the enzymatic characterization was focused on it. The ß-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, ß-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89 ± 0.13 mM and 1.4 ± 0.04 µM min(-1) to oNPX, respectively. The modeled structure of ß-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.

Sequence and analysis of the mitochondrial DNA control region in the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae)

This study aimed at the sequence and analysis of the mtDNA control region (CR) of the Diatraea saccharalis. The genome PCR amplification was performed using the complementary primers to the flanking regions of Bombyx mori CR mitochondrial segment. The sequencing revealed that the amplified product was 568 bp long, which was smaller than that observed for B. mori (725 bp). Within the amplified segment, a sequence with 338 nucleotides was identified as the control region, which displayed a high AT content (93.5%). The D. saccharalis mtDNA CR multiple sequence alignment analysis showed that this region had high similarity with the Lepidoptera Cydia pomonella.

A broca da cana, Diatraea saccharalis pertence à família dos lepidópteros. A presença da larva pode ser extremamente destrutiva, chegando a inviabilizar a atividade canavieira, causando prejuízos consideráveis à agroindústria sucro-alcooleira. Atualmente a broca da cana vem sendo extinta da plantação por métodos de controle biológico, entretanto a evolução desses programas depende de maiores conhecimentos básicos da biologia molecular deste inseto. O estudo do segmento do genoma mitocondrial denominado região controle é amplamente utilizado em análises genéticas e filogenéticas em insetos. O objetivo desse trabalho foi sequenciar e analisar a região controle do genoma mitocondrial de Diatraea saccharalis. Esse segmento apresentou 338 nucleotídeos, menor que o observado em Bombyx mori, com conteúdo de 93,5% de A/T. As analises realizadas mostraram que Diatraea saccharalis apresenta 76% de similaridade com Cydia pomonella.

Intrinsic bent DNA colocalizes with the sequence involved in the Nd-sD mutation in the Bombyx mori fibroin light chain gene.

Multiple sequence alignments of the Bombyx mori fibroin light chain gene (fib-L) from hybrids and from Chinese and Japanese strains demonstrated that 51.6% of the fib-L third intron is conserved. One of these conserved segments, 41 bp long, contains the sequence CGTTATTATACATATT, which is duplicated in the B. mori Nd-s(D) mutant. In the present work, electrophoretic mobility assays and computational analyses revealed a major peak of intrinsic bent DNA within the segment that undergoes breakage in the previously-described Nd-s(D) mutation. This result suggested that this intrinsically-curved region might mediate DNA cleavage and enhance recombination events in the third intron of the Bombyx mori fib-L gene.

Nuclear halo from Bradysia hygida (Diptera: Sciaridae) salivary gland polytene cells

Observações à microscopia eletrônica e estudos bioquímicos de cromossomos e núcleos sem histonas tem suportado a hipótese que o DNA de eucariotos é organizado em alças associadas com o esqueleto cromossômico ou à matriz nuclear. A observação da matriz nuclear sem a remoção do DNA, através da digestão com enzimas de restrição, apresenta uma figura em halo que representa a liberação das alças de DNA. Um protocolo para a obtenção de halos nucleares de núcleos politênicos de insetos, através da extração de proteínas usando o detergente LIS, é reportado nesse trabalho. Foram realizadas análises utilizando-se microscopia de fluorescência e microscopia de varredura confocal a laser. A extração de halos nucleares foi possível somente com o isolamento da fração nuclear em tampão sem espermina e espermidina. A obtenção de halos nucleares de núcleos politênicos de glândula salivar de Bradysia hygida contribui significativamente para o estudo da estrutura e função dessas organelas tão especiais.