Francisco Canindé de Sousa Júnior

Recovery and purification of recombinant 503 antigen of Leishmania infantum chagasi using expanded bed adsorption chromatography

Visceral leishmaniasis, a disease caused by Leishmania infantum chagasi, represents a major public health problem in many areas of the world. However, there is currently no vaccine for human use. The aim of this work was to purify the 503 antigen of Leishmania i. chagasi directly from unclarified Escherichia coli feedstock through expanded bed adsorption (EBA) chromatography. Batch experiments were performed to optimize the adsorption and elution conditions of the antigen onto a STREAMLINE Chelating resin using two central composite rotatable designs (CCRD). The results showed that the optimal binding conditions of the 503 antigen were pH 8.0 in the presence of 2.4 M NaCl. For the elution of the target protein, the optimized conditions included the presence of 600.0 mM imidazole. The adsorption isothermal data of the 503 antigen were fitted to the Langmuir adsorption isotherm. The EBA experiment successfully recovered 59.2% of the 503 antigen from the unclarified E. coli homogenate with a purification factor of 6.0.

Influence of culture medium on the production of eif antigen from Leishmania chagasi in recombinant Escherichia coli

With the advent of recombinant DNA technology, recombinant protein expression has become an important tool in the study of the structure, function and identification of new proteins, especially those with therapeutic functions. Escherichia coli has been the predominant prokaryote used in genetic engineering studies due to the abundance of information about its metabolism. Despite significant advances in molecular biology and immunology of infections, there are as yet no prophylactic drugs capable of preventing visceral leishmaniasis. It is therefore important to identify specific antigens in order to develop vaccines and diagnostic kits against this disease. The objective of this study was to evaluate the influence of culture medium on the production of eIF antigen from Leishmania chagasi in recombinant Escherichia coli. An induction procedure using IPTG was carried out in a series of trials, to observe the influence of culture medium (2xTY, TB) under expression of the recombinant eIF protein. Results showed that recombinant protein expression was associated to growth and that the highest eIF antigen expression was obtained in the 2xTY medium.

Evaluation of different methods for detecting methicillin resistance in Staphylococcus aureus isolates in a university hospital located in the Northeast of Brazil

Many methods have been described for the detection of methicillin-resistant Staphylococcus aureus (MRSA), but the heterogeneous expression of methicillin resistance affects the reliability of these methods. The aim of the present study was to evaluate some methods for detecting methicillin resistance in Staphylococcus aureus isolates in a university hospital located in the Northeast of Brazil. Among the isolates, 15 were methicillin-susceptible and 45 were methicillin-resistant, including low-level heterogeneous resistance strains. Both the 30 ηg-cefoxitin disk and PBP2a test had 100% sensibility/specificity and appear to be good options for the detection of MRSA in the clinical laboratory.

Enhancing enzymatic hydrolysis of coconut husk through Pseudomonas aeruginosa AP 029/GLVIIA rhamnolipid preparation

This work investigated the influence of chemical (Triton X-100) and biological surfactant preparation (rhamnolipids) in coconut husk hydrolysis that was subjected to pretreatment with acid-alkali or alkaline hydrogen peroxide. The natural and pretreated biomass was characterized using the National Renewable Energy Laboratory protocol analysis as well as X-ray diffraction and scanning electron microscopy. The results demonstrated that in terms of the total reducing sugars, there was no significant difference between the hydrolysis using Triton X-100 and rhamnolipids, regardless of the pretreatment. A cellulosic conversion value as high as 33.0% was obtained in experiments with rhamnolipids. The coconut husk was observed to be a potential biomass that could produce second generation ethanol, and the rhamnolipid preparation can be used to support for the enzymatic hydrolysis, enhancing the advantage of cellulose conversion into glucose over chemical surfactants because it is an environmentally friendly approach.

A 7-year survey of superficial and cutaneous mycoses in a public hospital in Natal, Northeast Brazil

In the present study, we determined the frequency of superficial and cutaneous mycoses and their etiologic agents during a 7-year period (2002–2008) in Natal, Brazil. A total of 1,717 specimens of skin, nail, and hair were collected from 1,382 patients with suspected mycoses lesions and were then subjected to direct microscopy and culture.

Prevalence of Candida species in umbilical catheters implanted in newborns in Natal, Brazil

All the newborns with umbilical venous catheters, hospitalized in the neonatal intensive care unit at Januario Cicco Maternity Hospital in Natal, Brazil, between January, 2003 and December, 2004 were studied. The prevalence of Candida species in the tips of intra-venous catheters was assessed, as well as the coexisting exposures that the patients were subjected to. Catheter tips were cultivated in blood agar and when yeast culture occurred, the colony was subcultivated for species identification through morphologic and biochemical assays. From a total of 240 catheters, 41 were positive for yeasts, and 34 were submitted to identification. The following agents were isolated: 13 C. albicans (38%), 10 Candida parapsilosis (29%), 8 C. tropicalis (20%), one C. guilliermondii (3%), one C. famata (3%) and one Trichosporon spp. (3%). Three patients among those with positive catheter tip fungal cultures were also hemoculture positive, with the same fungal species at both sites. Among the coexisting exposures, it should be pointed out that all the patients underwent broad spectrum antibiotic therapy, used a nasogastric probe, in addition to undergoing other invasive procedures such as mechanical ventilation and umbilical catheter implantation.

Simultaneous recombinant 503 antigen recovery and endotoxin removal from E. coli M15 homogenate using expanded bed adsorption chromatography

ABSTRACT This study evaluated the simultaneous recovery of recombinant 503 antigen and endotoxin removal from E. coli homogenate by immobilized metal affinity- expanded bed adsorption chromatography (EBAC), using the non-ionic surfactant Triton X-114 during the washing step. The strategy employed was able to remove more than 99% of endotoxin in all conditions tested, and the lowest residual concentration obtained was 10.22 EU/mL, with a recovery of 503 antigen ranging from 41.4% to 61.1%. Therefore, the procedure of integrating the clarification, recovery of protein, and endotoxin removal into a unit operation exploiting the EBAC was successful.

Production of recombinant 503 antigen of Leishmania infantum chagasi using cultivation in batch and fed-batch

Background Visceral leishmaniasis is an infecto-parasitarian disease caused by obligatory intracellular protozoa belonging to the genus Leishmania, and might be lethal since there is no precocious diagnosis and proper treatment. Despite the considerable effort, there is no effective and safe vaccine for human use [1]. Genetically modified microorganisms are used industrially in general for production of hormone, antibiotics and proteins. In the production of heterologous molecules, both the expression and the production step are important [2]. Therefore, the study of cultivation conditions in bioreactor plays an important role for the process viability of batch and fed-batch [3]. Thus, the aim of this work was to study strategies for production of the 503 antigen of Leishmania infantum chagasi using cultivation in batch and fed-batch.