Methanosarcina acetivorans C2A Topoisomerase IIIα, an Archaeal Enzyme with Promiscuity in Divalent Cation Dependence

Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases. Here we report Methanosarcina acetivorans TopoIIIα (MacTopoIIIα) as the first biochemically characterized mesophilic archaeal topoisomerase. Maximal activity for MacTopoIIIα was elicited at 30–35°C and 100 mM NaCl. As little as 10 fmol of the enzyme initiated DNA relaxation, and NaCl concentrations above 250 mM inhibited this activity. The present study also provides the first evidence that a type IA Topoisomerase has activity in the presence of all divalent cations tested (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+). Activity profiles were, however, specific to each metal. Known type I (ssDNA and camptothecin) and type II (etoposide, novobiocin and nalidixic acid) inhibitors with different mechanisms of action were used to demonstrate that MacTopoIIIα is a type IA topoisomerase. Alignment of MacTopoIIIα with characterized topoisomerases identified Y317 as the putative catalytic residue, and a Y317F mutation ablated DNA relaxation activity, demonstrating that Y317 is essential for catalysis. As the role of Domain V (C-terminal domain) is unclear, MacTopoIIIα was aligned with the canonical E. coli TopoI 67 kDa fragment in order to construct an N-terminal (1–586) and a C-terminal (587–752) fragment for analysis. Activity could neither be elicited from the fragments individually nor reconstituted from a mixture of the fragments, suggesting that native folding is impaired when the two fragments are expressed separately. Evidence that each of the split domains plays a role in Zn2+ binding of the enzyme is also provided

Mtb32 is a promising tuberculosis antigen for DNA vaccination in pre-and post-exposure mouse models

Identification of antigens that provide protective immunity via prophylactic and therapeutic vaccination against Mycobacterium tuberculosis is critical for the development of subunit vaccines for tuberculosis (TB). In this study, we performed a head-to-head comparison of seven well-known TB antigens delivered by DNA vaccine, and evaluated their respective immunogenicities and protective efficacies in pre- and post-exposure mouse models. All TB antigens were designed as a chimeric fusion with Flt3-L to enhance antigen-specific T-cell immunity upon vaccination. Prophylactic vaccination with the Flt3L (F)-Mtb32 DNA vaccine elicited significant protection in both the spleen and lungs against M. tuberculosis challenge, comparable to the Bacillus Calmette-Guerin vaccine. F-Ag85A and F-Mtb32 DNA vaccines, in combination with chemotherapy, reduced the bacterial burden to undetectable levels in the lungs of all mice infected with M. tuberculosis. These data collectively indicate that the F-Mtb32 DNA vaccine confers the most efficient protective immunity that suppresses bacterial growth in the active or latent status of M. tuberculosis. Gene Therapy (2012) 19, 570-575; doi:10.1038/gt.2011.140; published online 29 September 2011X1110sciescopu

Electrical and optical properties of Ti doped ZnO films grown on glass substrate by atomic layer deposition

Titanium doped zinc oxide (Ti doped ZnO) films were prepared by atomic layer deposition methods at a deposition temperature of 200 °C. The Ti content in Ti doped ZnO films was varied from 5.08 at.% to 15.02 at.%. X-ray diffraction results indicated that the crystallinity of the Ti doped ZnO films had degraded with increasing Ti content. Transmission electron microscopy was used to investigate the microstructural evolution of the Ti doped ZnO films, showing that both the grain size and crystallinity reduced with increasing Ti content. The electrical resistivity of the Ti doped ZnO films showed a minimum value of 1.6 × 10-3 Ω cm with the Ti content of 6.20 at.%. Furthermore, the Ti doped ZnO films exhibited excellent transmittance. © 2014 Elsevier Ltd.

Fluxograma diferencial entre a artrite séptica e sinovite transitória do quadril em crianças Flowchart distinguishing between septic arthritis and transitory synovitis of the hip in children

O diagnóstico diferencial entre a artrite séptica e a sinovite transitória do quadril não é fácil de ser realizado, pois não há um exame que seja simples, nem satisfatoriamente sensível e específico para diferenciá-las. Sendo assim, é muitas vezes utilizada uma propedêutica armada de exames que demanda maior custo e requer melhor infra-estrutura da instituição de saúde. Perante isso, torna-se evidente a necessidade de uma análise multifatorial dos dados clínicos e laboratoriais envolvidos para a proposição de um fluxograma em que se possa racionalizar exames visando a correta abordagem e evitar a indicação de procedimentos, muitas vezes, desnecessários como a ressonância magnética ou mais invasivos como a artrocentese e a própria drenagem cirúrgica. Realizamos uma ampla revisão da literatura nas bases de dados do Pubmed e Cochrane até maio de 2009 em que foi analisada a importância do exame clínico, dos testes laboratoriais e de imagem para a diferenciação entre as duas afecções. Mediante o cruzamento dos dados foi elaborado um fluxograma para o diagnóstico e conduta na criança e no adolescente, com idade de seis meses a dezoito anos, com sintoma de dor no quadril, na suspeita de quadro inflamatório. Nivel de Evidência III, estudos diagnósticos, investigação de um exame para diagnóstico.<br>The distinguishing diagnosis between the septic arthritis and the transitory synovitis of a hip is not easy to be carried through; therefore, there is not a simple, nor sensible and satisfactorily specific examination to differentiate them. Thus, the use of a propedeutic set of examinations becomes necessary to demand greater cost and depend on a bigger infrastructure of the health institution. First of all, the necessity of a multifactorial analysis on a flowchart is evident, so it can rationalize the indication of unnecessary or more invasive procedures as artrocentesis, magnetic resonance and the surgical draining. A revision of literature on the databases of Pubmed and Cochrane until May of 2009 was carried through, and the importance of the clinical examination, laboratorial tests and imaging exams was analyzed. By studies on the data, it was elaborated a flowchart for the diagnosis and the conduction of the management on the patient, between the ages of six months and eighteen years old, with complaint of pain on the hip under suspicion of inflammatory picture. Level of Evidence: Level III, diagnostic studies - investigating a diagnostic test