Crystal Structure of a Sucrose-6-phosphate Hydrolase from Lactobacillus gasseri with Potential Applications in Fructan Production and the Food Industry.

Fructooligosaccharides (FOSs) are polymers of fructose with a prebiotic activity because of their production and fermentation by bacteria that inhabit the gastrointestinal tract and are widely used in the industry and new functional foods. Lactobacillus gasseri stands out as an important homofermentative microorganism related to FOS production, and its potential applications in the industry are undeniable. In this study, we report the production and characterization of a sucrose-6-phosphate hydrolase from L. gasseri belonging to the GH32 family. Apo-LgAs32 and LgAs32 complexed with ß-d-fructose structures were determined at a resolution of 1.94 and 1.84 Å, respectively. The production of FOS, fructans, 1-kestose, and nystose by the recombinant LgAs32, using sucrose as a substrate, shown in this study is very promising. When compared to its homologous enzyme from Lactobacillus reuteri, the production of 1-kestose by LgAs32 is increased; thus, LgAs32 can be considered as an alternative in fructan production and other industrial applications.

Insights into the dual cleavage activity of the GH16 laminarinase enzyme class on ß-1,3 and ß-1,4 glycosidic bonds.

Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including ß-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-ß-1,3(4)-glucanase (EC 3.2.1.6), can cleave both ß-1,3 and ß-1,4 glycosidic bonds in glucans, such as laminarin, barley ß-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-ß-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of ß-1,3 and ß-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.

Functional and structural characterization of an α-ʟ-arabinofuranosidase from Thermothielavioides terrestris and its exquisite domain-swapped ß-propeller fold crystal packing.

The fungus Thermothielavioides terrestris plays an important role in the global carbon cycle with enzymes capable of degrading polysaccharides from biomass, therefore an attractive source of proteins to be investigated and understood. From cloning to a three-dimensional structure, we foster a deeper characterization of an α-ʟ-arabinofuranosidase, a glycoside hydrolase from the family 62 (TtAbf62), responsible to release arabinofuranose from non-reducing ends of polysaccharides. TtAbf62 was tested with synthetic (pNP-Araf) and polymeric substrates (arabinan and arabinoxylan), showing optimal temperature and pH (for pNP-Araf) of 30 °C and 4.5-5.0, respectively. Kinetic parameters revealed different specific activity for the three substrates, with a higher affinity for pNP-Araf (KM: 4 ± 1 mM). The hydrolyzing activity of TtAbf62 on sugarcane bagasse suggests high efficiency in the decomposition of arabinoxylan, abundant hemicellulose presented in the sugarcane cell wall. The crystal packing of TtAbf62 reveals an exquisite domain swapping, located at the supramolecular arrangement through a disulfide bond. All crystallographic behaviors go against its monomeric state in solution, indicating a crystal-induced artifact. Structural information will form the basis for further studies aiming the development of optimized enzymatic properties to be used in biotechnological applications.

Low-resolution structure, oligomerization and its role on the enzymatic activity of a sucrose-6-phosphate hydrolase from Bacillus licheniformis.

Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.

Política Nacional de Humanização na Atenção Primária à Saúde: estrategias de implantação e fortalecimento da diretriz "participação social" / National Humanization Policy in Primary Health Care: strategies for implementing and strengthening the guideline "social participation"

A participação social, diretriz da Política Nacional de Humanização (PNH), se constitui como importante mecanismo de resgate ao "SUS que da certo", trazendo o usuário para o centro do debate das políticas de saúde e buscando uma atenção mais humanizada e acolhedora. Por sua vez, a Atenção Primária à Saúde (APS) que representa a porta de entrada do Sistema Único de Saúde (SUS), se torna cenário ideal para a efetivação da participação da sociedade nos processos de gestão e fiscalização das políticas públicas. Através da análise bibliográfica, o objetivo do estudo foi buscar contextualizar mecanismos de fortalecimento e efetivação da diretriz da PNH participação social na APS, passando pelo contexto histórico da criação do SUS, a inserção e participação de diversos atores sociais nessa trajetória de democratização das políticas de saúde, e sem deixar de mencionar os desafios enfrentados mediante o descaso com a saúde pública

The social participation, a guideline of the National Humanization Policy (PNH), constitutes an important mechanism for rescuing the "SUS that is right", bringing the user to the center of the health policy debate and seeking a more humanized and atendence. In turn, Primary Health Care (APS) , which represents the entry point of the Brazilian Unified Health System (SUS), becomes an ideal scenario for the effective participation of the society in the processes of management and oversight of public polices. Through the bibliographic analysis, the aim of the study was to contextualize mechanisms for strengthening and effective PNH guideline social participation in APS, going through the historical context of the creantion of SUS, the insertion and participation of several social actors in this path of democratization of health polices, and the challenges with disregard for public health

Rheumatoid arthritis seems to have DMARD treatment decision influenced by fibromyalgia.

OBJECTIVE: To compare DMARD use in patients with and without FM over time, including overtreatment and undertreatment rates in both groups. METHODS: A prospective cohort study with patients attending an RA outpatient clinic was conducted. Participants were consecutively recruited between March 2006 and June 2007 and were followed through December 2013. Data on DMARD use (prevalences, doses and escalation rates), DAS28, HAQ and radiographic progression were compared among RA patients with FM and without FM. Mistreatment clinical scenarios were allegedly identified and compared between groups. RESULTS: 256 RA patients (32 with FM) were followed for 6.2±2.0 (mean±SD) years comprising 2986 visits. At baseline, RA duration was 11.1±7.4 years. DAS28 and HAQ were greater in RA with FM group, and were closer to RA without FM group towards the end. RA patients with FM used higher doses of tricyclic antidepressants, leflunomide and prednisone, and lower doses of methotrexate. When compared to RA patients without FM, participants with RA and FM used more often tricyclic antidepressants, leflunomide, prednisone, continuous analgesics and less often methotrexate. Groups presented similar 7-year biologic-free survival, and radiographic progression-free survival in Cox regression. RA patients with FM had greater proportions of visits in mistreatment scenarios when compared to RA patients without FM (28.4 vs. 19.8%, p<0.001). CONCLUSIONS: RA patients with FM used more leflunomide and prednisone, and RA mistreatment was more frequent in FM patients. Certainly, RA patients with FM will benefit from a personalized T2T strategy, including ultrasound (when suitable) and proper FM treatment.

Rheumatoid arthritis seems to have DMARD treatment decision influenced by fibromyalgia / As decisões de tratamento com DMARD na artrite reumatoide parecem ser influenciadas pela fibromialgia

Abstract Objective: To compare DMARD use in patients with and without FM over time, including overtreatment and undertreatment rates in both groups. Methods: A prospective cohort study with patients attending an RA outpatient clinic was conducted. Participants were consecutively recruited between March 2006 and June 2007 and were followed through December 2013. Data on DMARD use (prevalences, doses and escalation rates), DAS28, HAQ and radiographic progression were compared among RA patients with FM and without FM. Mistreatment clinical scenarios were allegedly identified and compared between groups. Results: 256 RA patients (32 with FM) were followed for 6.2 ± 2.0 (mean ± SD) years comprising 2986 visits. At baseline, RA duration was 11.1 ± 7.4 years. DAS28 and HAQ were greater in RA with FM group, and were closer to RA without FM group towards the end. RA patients with FM used higher doses of tricyclic antidepressants, leflunomide and prednisone, and lower doses of methotrexate. When compared to RA patients without FM, participants with RA and FM used more often tricyclic antidepressants, leflunomide, prednisone, continuous analgesics and less often methotrexate. Groups presented similar 7-year biologic-free survival, and radiographic progression-free survival in Cox regression. RA patients with FM had greater proportions of visits in mistreatment scenarios when compared to RA patients without FM (28.4 vs. 19.8%, p < 0.001). Conclusions: RA patients with FM used more leflunomide and prednisone, and RA mistreatment was more frequent in FM patients. Certainly, RA patients with FM will benefit from a personalized T2T strategy, including ultrasound (when suitable) and proper FM treatment.

Resumo Objetivo: Comparar o uso de fármacos antirreumáticos modificadores da doença (DMARD) em pacientes com e sem fibromialgia (FM) ao longo do tempo, incluindo as taxas de tratamento excessivo e subtratamento em ambos os grupos. Métodos: Estudo de coorte prospectiva com pacientes atendidos em um ambulatório de artrite reumatoide (AR). Os participantes foram recrutados consecutivamente entre março de 2006 e junho de 2007 e foram seguidos até dezembro de 2013. Compararam-se os dados de uso de DMARD (prevalências, doses e taxas de escalonamento), 28-Joint Disease Activity Score (DAS28), Health Assessment Questionnaire (HAQ) e progressão radiográfica entre pacientes com e sem FM. Os cenários clínicos de tratamento supostamente incorreto foram identificados e comparados entre os grupos. Resultados: Seguiram-se 256 pacientes com AR (32 com FM) por 6,2 ± 2,0 (média ± DP) anos, período que abrangeu 2.986 consultas. No início do estudo, a duração da AR era de 11,1 ± 7,4 anos. O DAS28 e o HAQ foram maiores no grupo AR com FM e estavam mais próximos do grupo AR sem FM no fim do estudo. Os pacientes com AR com FM usaram doses mais altas de antidepressivos tricíclicos, leflunomida e prednisona e doses mais baixas de metotrexato. Quando comparados com os pacientes com AR sem FM, os participantes com AR e FM usaram mais frequentemente antidepressivos tricíclicos, leflunomida, prednisona e analgésicos contínuos e menos frequentemente metotrexato. Os grupos apresentaram sobrevida em sete anos sem agentes biológicos e livres de progressão radiográfica semelhantes na regressão Cox. Os pacientes com AR com FM apresentaram uma maior proporção de consultas em cenários de tratamento supostamente incorreto quando comparados com os pacientes com AR sem FM (28,4 vs. 19,8%, p < 0,001). Conclusões: Os pacientes com AR e FM usaram mais leflunomida e prednisona e o tratamento supostamente incorreto na AR foi mais frequente em pacientes com FM. Os pacientes com AR com FM certamente se beneficiarão de uma estratégia personalizada de tratamento por metas (T2 T), incluindo ultrassonografia (quando apropriado) e controle da FM.

Solução estética após falha de planejamento protético / Esthetic solution after prosthesis design failure

A manutenção de uma auto imagem positiva tem relação direta a dentição do indivíduo. A utilização de próteses sejam elas parciais ou totais, tem papel fundamental tanto na saúde oral, quanto na saúde sistêmica do paciente. O restabelecimento da função oclusal, a preservação de estruturas orais remanescentes dependem diretamente de um planejamento protético rigoroso. A satisfação do paciente é fator determinante do sucesso ou insucesso de uma reabilitação protética, bem como o restabelecimento do bem-estar físico, mental e social. Paciente sexo feminino, 32 anos, relata insatisfação estética 120 dias após reabilitação estética com prótese parcial removível. A oroscopia observou-se, inclinação para distal dos elementos 12 e 21 bem como excesso de material restaurador. Observou-se ainda lesão séssil de cor avermelhada em gengiva inserida na região do elemento 11 (ausente), bordas delimitadas, apresentando dor provocada ao toque, sem sangramento e de surgimento a aproximadamente 12 meses, sendo anterior a instalação da PPR. Realizado biópsia excisional, com histopatologia compatível ao granuloma. Tratamento endodôntico do elemento 21. Remoção de carie dos elementos acometidos, inclusive pilar protético. Faceta direta em Resina Fotopolimerizável nos elementos 12 e 21. Recuperação da estética dentaria e saúde dos tecidos de suporte. O objetivo do presente está em destacar aspectos relacionados à falhas de PPR se não corretamente diagnosticadas, avaliadas e prevenidas.(AU)

Influência da reabilitação protética imediata na saúde periodontal: relato de caso / Influence immediate prosthetic rehabilitation on periodontal health: Case Report

A busca por uma composição agradável no sorriso, tornou-se uma demanda mundial, porém para produzir sorrisos harmoniosos, é essencial uma abordagem multidisciplinar. Paciente sexo masculino, leucoderma, 48 anos, gênero masculino, não fumante, não etilista, queixou-se de ausências de dentes, observou-se doença periodontal crônica agressiva com presença de sangramento, exsudato purulento, halitose, abfrações e lesão periapical. Foram feitas múltiplas raspagens periodontais associadas a terapia periodontal cirúrgica, com consequente avulsão dentária juntamente com protocolo medicamentoso e acompanhamento semanal por um período de 06 meses. Noventa dias após remissão da doença periodontal indicou-se exodontia de múltiplos elementos e iniciou-se tratamento restaurador protético imediato. Paciente encontra-se em proservação já há 12 meses, mantendo níveis ótimos de higiene oral e satisfação funcional e estética. Na diária clínica, a associação de procedimentos periodontais, cirúrgicos e protéticos, bem como a colaborativa do paciente tornam-se de extrema importância, para harmonizar a relação entre os aspectos biológicos e estéticos, a fim de restabelecer a saúde bucal e a composição do sorrisonfluência da reabilitação protética imediata na saúde periodontal: relato de caso (AU)

The search for a pleasant composition in the smile has become a worldwide demand, but to produce harmonious smiles, a multidisciplinary approach is essential. Male patient, leucoderma, 48 years old, male, non-smoker, non-alcoholic, complained of absence of teeth, aggressive chronic periodontal disease with bleeding, purulent exudate, halitosis, periapical lesions and periapical lesions. Multiple periodontal scrapes were associated with periodontal surgical therapy, with consequent dental avulsion along with drug protocol and weekly followup for a period of 06 months. Ninety days after remission of periodontal disease, multiple element extraction was indicated and immediate prosthetic restorative treatment was initiated. Patient has been in proservation for 6 months, maintaining optimal levels of oral hygiene and functional and aesthetic satisfaction. In the clinical daily routine, the association of periodontal, surgical and prosthetic procedures, as well as the patient's collaborative, become extremely important, in order to harmonize the relationship between biological and aesthetic aspects, in order to restore oral health and smile composition .(AU)

Manifestações Orais e Sistêmicas de carcinoma epidermóide de orofaringe em estádio avançado / Oral and systemic manifestations of advanced oropharyngeal squamous cell carcinoma

Racional ­ Tumor maligno pode comprometer a cavidade oral e promover alterações sistêmicas muito graves que podem levar à morte. Objetivo ­ Descrever como um tumor pode atingir a cavidade oral de um indivíduo de forma a modificar sua qualidade de vida até culminar com seu óbito. Material e Métodos ­ Adulto do sexo masculino, desempregado, usuário de drogas como: maconha, cocaína, crack, álcool e tabaco, apresentou, durante a fase aguda, pequeno nódulo em região de hemiarco superior esquerdo, na altura dos pré-molares. No exame físico, apresentava dor à palpação com pequeno nódulo não flutuante, localizada em região cervical esquerda. A biópsia evidenciou células tumorais malignas. Resultados ­ Observado progressão rápida, com debilidade adstrita do paciente; resultando em óbito. Conclusão ­ Conclui-se com base no caso acima relatado e tendo a literatura como aliada, que os tumores avançados de orofaringe podem progredir de forma rápida e devastadora, sendo sua taxa de sobrevida muito baixa, além de causar ao paciente constrangimento sócio emocional.(AU)

Rheumatoid arthritis seems to have DMARD treatment decision influenced by fibromyalgia. / As decisões de tratamento com DMARD na artrite reumatoide parecem ser influenciadas pela fibromialgia.

OBJECTIVE: To compare DMARD use in patients with and without FM over time, including overtreatment and undertreatment rates in both groups. METHODS: A prospective cohort study with patients attending an RA outpatient clinic was conducted. Participants were consecutively recruited between March 2006 and June 2007 and were followed through December 2013. Data on DMARD use (prevalences, doses and escalation rates), DAS28, HAQ and radiographic progression were compared among RA patients with FM and without FM. Mistreatment clinical scenarios were allegedly identified and compared between groups. RESULTS: 256 RA patients (32 with FM) were followed for 6.2±2.0 (mean±SD) years comprising 2,986 visits. At baseline, RA duration was 11.1±7.4 years. DAS28 and HAQ were greater in RA with FM group, and were closer to RA without FM group towards the end. RA patients with FM used higher doses of tricyclic antidepressants, leflunomide and prednisone, and lower doses of methotrexate. When compared to RA patients without FM, participants with RA and FM used more often tricyclic antidepressants, leflunomide, prednisone, continuous analgesics and less often methotrexate. Groups presented similar 7-year biologic-free survival, and radiographic progression-free survival in Cox regression. RA patients with FM had greater proportions of visits in mistreatment scenarios when compared to RA patients without FM (28.4 vs. 19.8%, p<0.001). CONCLUSIONS: RA patients with FM used more leflunomide and prednisone, and RA mistreatment was more frequent in FM patients. Certainly, RA patients with FM will benefit from a personalized T2T strategy, including ultrasound (when suitable) and proper FM treatment.

Mechanisms of peroxisome proliferator activated receptor γ regulation by non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) display anti-inflammatory, antipyretic and analgesic properties by inhibiting cyclooxygenases and blocking prostaglandin production. Previous studies, however, suggested that some NSAIDs also modulate peroxisome proliferator activated receptors (PPARs), raising the possibility that such off target effects contribute to the spectrum of clinically relevant NSAID actions. In this study, we set out to understand how peroxisome proliferator activated receptor-γ (PPARγ/PPARG) interacts with NSAIDs using X-ray crystallography and to relate ligand binding modes to effects on receptor activity. We find that several NSAIDs (sulindac sulfide, diclofenac, indomethacin and ibuprofen) bind PPARγ and modulate PPARγ activity at pharmacologically relevant concentrations. Diclofenac acts as a partial agonist and binds to the PPARγ ligand binding pocket (LBP) in typical partial agonist mode, near the ß-sheets and helix 3. By contrast, two copies of indomethacin and sulindac sulfide bind the LBP and, in aggregate, these ligands engage in LBP contacts that resemble agonists. Accordingly, both compounds, and ibuprofen, act as strong partial agonists. Assessment of NSAID activities in PPARγ-dependent 3T3-L1 cells reveals that NSAIDs display adipogenic activities and exclusively regulate PPARγ-dependent target genes in a manner that is consistent with their observed binding modes. Further, PPARγ knockdown eliminates indomethacin activities at selected endogenous genes, confirming receptor-dependence of observed effects. We propose that it is important to consider how individual NSAIDs interact with PPARγ to understand their activities, and that it will be interesting to determine whether high dose NSAID therapies result in PPAR activation.

A Novel Member of GH16 Family Derived from Sugarcane Soil Metagenome.

Glycoside hydrolases (GHs) are enzymes found in all living kingdoms that are involved in multiple physiological functions. Due to their multiple enzymatic activities, GHs are broadly applied in bioethanol, food, and paper industry. In order to increase the productivity of these industrial processes, a constant search for novel and efficient enzymes has been proved to be necessary. In this context, metagenomics is a powerful approach to achieve this demand. In the current study, we describe the discovery and characterization of a novel member of GH16 family derived from the sugarcane soil metagenome. The enzyme, named SCLam, has 286 amino acid residues and displays sequence homology and activity properties that resemble known laminarases. SCLam is active against barley beta-glucan, laminarin, and lichenan (72, 33, and 10 U mg(-1), respectively). The optimal reaction conditions were identified as 40 °C and pH 6.5. The low-resolution structure was determined using the small-angle X-ray scattering technique, revealing that SCLam is a monomer in solution with a radius of gyration equal to 19.6 Å. To the best of our knowledge, SCLam is the first nonspecific (1,3/1,3:1,4)-ß-D-glucan endohydrolase (EC 3.2.1.6) recovered by metagenomic approach to be characterized.

Different binding and recognition modes of GL479, a dual agonist of Peroxisome Proliferator-Activated Receptor α/γ.

Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-dependent transcription factors that control various functions in human organism, including the control of glucose and lipid metabolism. PPARγ is a target of TZD agonists, clinically used to improve insulin sensitivity whereas fibrates, PPARα ligands, lower serum triglyceride levels. We report here the structural studies of GL479, a synthetic dual PPARα/γ agonist, designed by a combination of clofibric acid skeleton and a phenyldiazenyl moiety, as bioisosteric replacement of stilbene group, in complex with both PPARα and PPARγ receptors. GL479 was previously reported as a partial agonist of PPARγ and a full agonist of PPARα with high affinity for both PPARs. Our structural studies reveal different binding modes of GL479 to PPARα and PPARγ, which may explain the distinct activation behaviors observed for each receptor. In both cases the ligand interacts with a Tyr located at helix 12 (H12), resulting in the receptor active conformation. In the complex with PPARα, GL479 occupies the same region of the ligand-binding pocket (LBP) observed for other full agonists, whereas GL479 bound to PPARγ displays a new binding mode. Our results indicate a novel region of PPARs LBP that may be explored for the design of partial agonists as well dual PPARα/γ agonists that combine, simultaneously, the therapeutic effects of the treatment of insulin resistance and dyslipidemia.

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for ß-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl ß-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

Tratamento videolaparoscópico para retirada de dispositivo intrauterino em fossa ilíaca direita / Laparoscopic treatment for removal of intrauterine device in the right iliac fossa

Dispositivo intrauterino pode sofrer expulsão da cavidade uterina. Objetivo: Descrever um caso de tratamento videolaparoscópico para retirada de dispositivo intrauterino de fossa ilíaca direita. Material e Métodos: Jovem do sexo feminino apresentou dor em fossa ilíaca direita de quatro dias de duração. No exame físico, apresentava dor abdominal localizada em fossa ilíaca direita, com discreta resistência de parede e dor à percussão e descompressão brusca na região. Em tomografia computadorizada, evidenciou-se dispositivo intrauterino fora do útero, na fossa ilíaca direita. Foram utilizadas as bases de dados SciELO, MedLine, Sobracil e PubMed no período compreendido de Abril a Junho de 2014. Vinte e dois artigos foram relacionados, entretanto somente os 10 artigos que compõem as referências bibliográficas foram selecionados por conter informações relevantes acerca do tema. Resultados: Na videolaparoscopia, pôde ser observado DIU em fossa ilíaca direita, próximo ao intestino, fora do útero, sem aderência, sem perfurações evidentes ou maiores complicações. Conclusão: A videolaparoscopia permite a retirada adequada de dispositivo intrauterino localizado indevidamente fora da cavidade uterina; permite inclusive avaliação adequada de toda a região pélvica para excluir lesões associadas.(AU)

Intrauterine device may suffer expulsion of the uterine cavity. Objective: To describe a laparoscopic treatment of the case for removal of intrauterine device right iliac fossa. Material and Methods: Young female presented pain in the right iliac fossa of four days. On physical examination, showed abdominal pain localized in the right iliac fossa, with thin wall of resistance and pain on percussion and rebound in the region. In CT scan it was seen an intrauterine device outside the uterus, in the right iliac fossa. The SciELO, MedLine, Sobracil and PubMed databases were used in the period April to June 2014. Twenty-two articles were related, though only 10 articles that make up the references were selected because they contain important information about the theme. Results: In the laparoscopy it could be observed the IUD in the right iliac fossa, near the intestine, outside the uterus, without grip, with no obvious or larger perforations complications.Conclusion: Laparoscopy allows adequate removal of intrauterine device located improperly outside the uterine cavity; even allows proper evaluation of the entire pelvic region to rule out associated injuries.(AU)

Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa.

Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage.

The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on ß-glucan.

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-ß-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on ß-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on ß-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using ß-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Molecular mechanism of peroxisome proliferator-activated receptor α activation by WY14643: a new mode of ligand recognition and receptor stabilization.

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase.

Xylose isomerase (EC 5.3.1.5) is a key enzyme in xylose metabolism which is industrially important for the transformation of glucose and xylose into fructose and xylulose, respectively. The Bifidobacterium adolescentis xylA gene (NC_008618.1) encoding xylose isomerase (XI) was cloned and the enzyme was overexpressed in Escherichia coli. Purified recombinant XI was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as the precipitating agent. A complete native data set was collected to 1.7 Šresolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 88.78, b = 123.98, c = 78.63 Å.