José L. Lima-Filho

Association between HLA-G 3'UTR 14-bp polymorphism and HIV vertical transmission in Brazilian children.

Objectives:The aim of our study was to verify the possible association between an HLA-G 14-bp deletion/insertion polymorphism and perinatal HIV transmission in Brazilian children. Design:We analyzed the 14-bp deletion/insertion polymorphisms in seronegative (i.e., exposed uninfected, N = 71) and seropositive (exposed infected, N = 175) Brazilian children born from HIV-positive mothers and in healthy controls (n = 175). Methods:HLA-G 14-bp deletion/insertion polymorphism (rs16375) was detected by PCR amplification of the target sequence followed by agarose gel electrophoresis. All the samples were also analyzed by direct sequencing in order to validate the genotyping results. Results:HIV-exposed uninfected children showed significant differences in their allele and genotype frequencies of the HLA-G 14-bp polymorphism when compared to both seropositive children and healthy controls. The 14-bp-deleted (D) allele was more frequent in exposed uninfected children (79%) than in healthy controls (60%) and HIV-positive children (58%); the higher percentage of the D allele found in the exposed uninfected children with respect to HIV-positive individuals was significantly associated with a reduced risk of vertical transmission. This effect was ascribable to the presence of the D/D homozygous genotype. Conclusion:Our findings support the possible role for the HLA-G 14-bp deletion/insertion polymorphism in the HIV vertical transmission in Brazilian children. The presence of the D allele and D/D genotype is associated with a protective effect toward HIV perinatal infection.

Electrochemical DNA biosensor for human papillomavirus 16 detection in real samples

An electrochemical DNA biosensor for human papillomavirus (HPV) 16 detection has been developed. For this proposed biosensor, L-cysteine was first electrodeposited on the gold electrode surface to form L-cysteine film (CYSFILM). Subsequently, HPV16-specific probe was immobilized on the electrode surface with CYSFILM. Electrochemistry measurement was studied by differential pulse voltammetry method (DPV). The measurement was based on the reduction signals of methylene blue (MB) before and after hybridization either between probe and synthetic target or extracted DNA from clinical samples. The effect of probe concentration was analyzed and the best results were seen at 1000 nM. The hybridization detection presented high sensitivity and broad linear response to the synthetic-target concentration comprised between 18.75 nM and 250 nM as well as to a detection limit of 18.13 nM. The performance of this biosensor was also investigated by checking probe-modified electrode hybridization with extracted DNA from samples. The results showed that the biosensor was successfully developed and exhibited high sensitivity and satisfactory selectivity to HPV16. These results allow for the possibility of developing a new portable detection system for HPVs and for providing help in making an effective diagnosis in the early stages of infection.

Electrochemical DNA biosensor for bovine papillomavirus detection using polymeric film on screen-printed electrode

A new electrochemical DNA biosensor for bovine papillomavirus (BPV) detection that was based on screen-printed electrodes was comprehensively studied by electrochemical methods of cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A BPV probe was immobilised on a working electrode (gold) modified with a polymeric film of poly-L-lysine (PLL) and chitosan. The experimental design was carried out to evaluate the influence of polymers, probe concentration (BPV probe) and immobilisation time on the electrochemical reduction of methylene blue (MB). The polymer poly-L-lysine (PLL), a probe concentration of 1 μM and an immobilisation time of 60 min showed the best result for the BPV probe immobilisation. With the hybridisation of a complementary target sequence (BPV target), the electrochemical signal decreased compared to a BPV probe immobilised on the modified PLL-gold electrode. Viral DNA that was extracted from cattle with papillomatosis also showed a decrease in the MB electrochemical reduction, which suggested that the decreased electrochemical signal corresponded to a bovine papillomavirus infection. The hybridisation specificity experiments further indicated that the biosensor could discriminate the complementary sequence from the non-complementary sequence. Thus, the results showed that the development of analytical devices, such as a biosensor, could assist in the rapid and efficient detection of bovine papillomavirus DNA and help in the prevention and treatment of papillomatosis in cattle.

Extraction of amylase from fermentation broth in poly (Ethylene Glycol) salt aqueous two-phase system

Studies were carried out on the partition of amylase from Bacillus subtilis in a minimal medium at 37 oC and 110 rpm. Enzyme recovery was carried out in aqueous two-phase system PEG-Phosphate salt were carried out. The best purification factor (5.4) was obtained in system PEG 1000 (16.7% w/w) with potassium phosphate (14.8% w/w), at pH 6.0, resulting in a recovery of 45.2% activity enzymatic in the salt-rich phase.

A Sensitive and Selective Label-Free Electrochemical DNA Biosensor for the Detection of Specific Dengue Virus Serotype 3 Sequences

Dengue fever is the most prevalent vector-borne disease in the world, with nearly 100 million people infected every year. Early diagnosis and identification of the pathogen are crucial steps for the treatment and for prevention of the disease, mainly in areas where the co-circulation of different serotypes is common, increasing the outcome of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Due to the lack of fast and inexpensive methods available for the identification of dengue serotypes, herein we report the development of an electrochemical DNA biosensor for the detection of sequences of dengue virus serotype 3 (DENV-3). DENV-3 probe was designed using bioinformatics software and differential pulse voltammetry (DPV) was used for electrochemical analysis. The results showed that a 22-m sequence was the best DNA probe for the identification of DENV-3. The optimum concentration of the DNA probe immobilized onto the electrode surface is 500 nM and a low detection limit of the system (3.09 nM). Moreover, this system allows selective detection of DENV-3 sequences in buffer and human serum solutions. Therefore, the application of DNA biosensors for diagnostics at the molecular level may contribute to future advances in the implementation of specific, effective and rapid detection methods for the diagnosis dengue viruses.

Label-Free Electrochemical Detection of the Specific Oligonucleotide Sequence of Dengue Virus Type 1 on Pencil Graphite Electrodes

A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing between the DNA probe (TS-1P) immobilized on the modified electrode, and the target (TS-1T) was achieved. The target could be quantified in a range from 1 to 40 nM with good linearity and a detection limit of 0.92 nM. The specificity of the electrochemical biosensor was tested using non-complementary sequences of dengue virus 2 and 3.

Recovery of ascorbic oxidoreductase from crude extract with an aqueous two-phase system in a perforated rotating disc contactor

A continuous perforated rotating disc contactor was used to extract the enzyme ascorbic oxidoreductase (E.C.1.10.3.3) from crude extract of Curcubita maxima with an aqueous two-phase system of poly (ethylene glycol) and phosphate salts. The effect of dispersed phase velocity on either protein mass transfer coefficients or separation efficiency at 1, 2 and 3 mL/min was studied. An increase of the mass transfer coefficients was observed with the dispersed phase velocity, while the separation efficiency showed a small decrease with the increase of this parameter. The experimental results obtained during continuous extraction showed that the ascorbic oxidoreductase activity was partitioned preferentially into the salt-rich phase in all conditions studied. The best recovery of enzyme activity was 236%, with a purification factor of 34 in flow rates of 1 mL/min for dispersed phase.

Studies on growth kinetics and plasmid stability of a recombinant Escherichia coli expressing a Schistosoma mansoni antigen

Abstract A 13 kDa Schistosoma mansoni tegumental antigen (Sm13) was cloned and expressed in Escherichia coli. The fermentation product of 55 kDa corresponds to the maltose binding protein-recombinant Sm13 fusion protein (MBP-rSm13). The growth conditions for maximum IPTG inducible MBP-rSm13 production was investigated by examining the following parameters: innoculum size, agitation, temperature of induction and concentration of the inducer, IPTG. The maximum MBP-rSm13 production was achieved by two steps-fermentation. First, the recombinant strain was cultivated in a 37 °C orbital shaker, at 160 rpm, for 3 hours. The MBP-rSm13 was then induced by adding 0.3 mM IPTG and placing the culture in a 28 °C orbital shaker for 2 hours. Under these conditions the MBP-rSm13 production yield was approximately 50% of total intracellular proteins and the degradation by bacterial intracellular proteases seemed to be minimized. The plasmid stability was also studied during the fermentation of the Sm13-expressing E. coli in non induced and induced conditions. In both conditions there was a slightly plasmid loss before induction, however after the addition of IPTG the loss was increased. The rate of plasmid loss was 5.5 and 13.5 before and after IPTG induction respectively.

Quantitative expression analysis of Bodhesin genes in the buck (Capra hircus) reproductive tract by real-time polymerase chain reaction (qRT-PCR)

Knowledge of the different patterns of gene expression along the male reproductive tract can assist in understanding the physiological processes of species-specific reproduction in mammals. In the present work, expression profiles of buck spermadhesin (bodhesin) genes along the reproductive tract by qRT-PCR were investigated. Total RNA from the seminal vesicle, testis, epididymis, bulbourethral gland and ductus deferens were reverse transcribed and the cDNA produced was submitted to qRT-PCR. For each homologous bodhesin gene, namely Bdh-1, Bdh-2 and Bdh-3, sets of specific primers and recombinant plasmids were prepared for gene quantification. In buck seminal vesicles, Bdh-2 is the homologue predominantly expressed, with a copy number on the order of millions of times more than Bdh-1 and thousand times more than Bdh-3. The copy number of Bdh-3 mRNA is only 10-fold greater than that of Bdh-1. Bodhesin transcripts were detected in all tissues examined, except in ductus deferens. The quantitative analysis also demonstrated clearly the differential gene expression of spermadhesin in bulbourethral gland. The striking differences in bodhesin gene expression indicate that each isoform could have a specific biological function in the buck genital tract, which deserves further detailed studies.

Expression of mRNAs coding for VAP1/crotastatin-like metalloproteases in the venom glands of three South American pit vipers assessed by quantitative real-time PCR.

Snake venom metalloproteases encompass a large family of toxins, with approximately 200 members already catalogued, which exhibit a diversity of structures and biological functions. From this relatively large number, only a dozen examples of apoptosis-inducing metalloproteases, like VAP1 and 2 from the venom of Crotalus atrox, are known. Since most VAP1-like toxins ever characterized were purified from the venom of Viperidae species inhabiting diverse places on earth, we investigate the expression of VAP-like metalloproteases in the venom gland of three representative pit vipers of the Brazilian territory. By molecular cloning and quantitative real-time polymerase chain reaction, using as calibrator gene the Crotalus durissus terrificus homolog of VAP1, named crotastatin, it is reported here that VAP1/crotastatin-like homologues in the venom gland of Bothrops atrox, C. d. cascavella and Lachesis m. rhombeata are expressed at different levels. Hence, batroxstatins, the crotastatin-like precursors from B. atrox, are expressed 87 times more than crotastatin-1, from C. d. cascavella, and 7.5-fold that lachestatins, from L. m. rhombeata. Moreover, in silico structural analysis of amino acid sequences indicates that batroxstatin-2, crotastatins and lachestatin-1 and -2 which share the archetypal motifs and metal- binding sites of VAP1, are subgrouped in a branch that comprises some apoptosis-inducing toxins.