Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera

BackgroundEnzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform.Methodology/Principal findingsDAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV.Conclusions/SignificanceDAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.     

Closure of rRNA related gaps in the Chromobacterium violaceum genome with the PCR-assisted contig extension (PACE) protocol

In the finishing phase of the Chromobacterium violaceum genome project, the shotgun sequences were assembled into 57 contigs that were then organized into 19 scaffolds, using the information from shotgun and cosmid clones. Among the 38 ends resulting from the 19 scaffolds, 10 ended with sequences corresponding to rRNA genes (seven ended with the 5S rRNA gene and three ended with the 16S rRNA gene). The 28 non-ribosomal ends were extended using the PCR-assisted contig extension (PACE) methodology, which immediately closed 15 real gaps. We then applied PACE to the 16S rRNA gene containing ends, resulting in eight different sequences that were correctly assembled within the C. violaceum genome by combinatory PCR strategy, with primers derived from the non-repetitive genomic region flanking the 16S and 5S rRNA gene. An oriented combinatory PCR was used to correctly position the two versions (copy A and copy B, which differ by the presence or absence of a 100-bp insert); it revealed six copies corresponding to copy A, and two to copy B. We estimate that the use of PACE, followed by combinatory PCR, accelerated the finishing phase of the C. violaceum genome project by at least 40%.     

Bacterial sulfate reduction and salinity: two controls on dolomite precipitation in Lagoa Vermelha and Brejo do Espinho (Brazil)

The hydrological system of Lagoa Vermelha, a dolomite-precipitating lagoon in Brazil, was investigated using hydrogen and oxygen stable isotopic composition of the water collected during an annual cycle (1996–1997). These data demonstrated that dolomite formed in May–June during high saline conditions. High salinity apparently provides the ions and saturation state necessary for dolomite precipitation. Ion concentrations in the lagoon water indicated an identical timing of dolomite precipitation and demonstrated that dolomite formed at decreased sulfate concentrations. In Brejo do Espinho, a neighbouring lagoon, the ion concentrations in the water column revealed that dolomite precipitates throughout the year, most likely due to its higher salinity than Lagoa Vermelha during the measured period. In Lagoa Vermelha, high ³⁴S of pore water sulfate and high sulfide concentrations correlated with dolomitic horizons, demonstrating the association of bacterial sulfate reduction with dolomite formation. In Brejo do Espinho high ³⁴S of pore water sulfate and high sulfide concentrations occurred throughout the dolomitic sedimentary column. We conclude that elevated salinity and sulfate reduction are the main factors inducing dolomite precipitation in these lagoons, confirming the microbial dolomite formation theory. These results suggest that there may be other settings where sulfate-reducing bacteria induce dolomite precipitation under saline conditions, such as deep-sea sediments or sabkhas, and imply that microbial dolomite may significantly contribute to the sedimentary carbonate budget, particularly in the earliest Earth's history when anoxic conditions were more prevalent.     

Effects of haloperidol on rat behavior and density of dopaminergic D2-like receptors

The present work shows the effects of a typical neuroleptic drug (haloperidol, HAL) on rat behavior (catalepsy and locomotor activity) and dopaminergic D2-like receptor densities in the hippocampus and striatum. Male Wistar rats (2-3 months old) were treated daily for 30 days with HAL (0.2 or 1mg/kg, intraperitoneally (i.p.)). At the end of treatment and 1h or 1, 3, 7 and 15 days after drug withdrawal, animals were subjected to behavioral tests and sacrificed afterwards for binding assays. The results showed that behavioral effects with both doses were significant only 1h and 1 day after withdrawal, and similar to controls at the third day. An up-regulation of D2 receptors was observed in the striatum (28% increase) but not in the hippocampus after 24h HAL (1mg/kg) withdrawal. However, an up-regulation was seen in both areas (1mg/kg) 3 days after drug withdrawal (58 and 42% increases in the hippocampus and striatum, respectively), and continued after 7 days of withdrawal only in the striatum (43 and 49% for the doses of 0.2 and 1mg/kg, respectively), suggesting the influence of dose, age, and time of drug withdrawal on these parameters. The up-regulation disappeared after 15 days of haloperidol withdrawal. Increases (72 and 140%) in constant dissociation values (K(d)) values were also observed 7 days after withdrawal. Results show differences on a time-basis between behavioral alterations and dopaminergic D2 receptors up-regulation.     

Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisation

Continuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l^–1 h^–1 and a 84% (w/w) glycerol consumption were attained when cultures were grown at pH 6 and D = 0.05 h^–1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.     

Expression of Parkin,APC, APE1, and Bcl-xL in Colorectal Polyps

Colorectal cancer can develop through molecular, chromosomal, and epigenetic cumulative changes that transform the normal intestinal epithelium into the colorectal polyps, called conventional adenomas (CAs) or serrated polyps (SPs), recognized as precursors of invasive colorectal neoplasia. These benign lesions need to explore the morphology, histological diagnosis, and biomarkers profile to accurately characterize lesions with potential for evolution to cancer. This study aimed to correlate the immunohistochemical expression of Parkin and Adenomatous Polyposis Coli (APC; tumor suppressors), Human Apurinic/Apyrimidinic endonuclease 1 (APE1), and B-cell lymphoma-extra-large (Bcl-xL; oncogenic proteins) in sporadic colorectal polyps with clinical, endoscopic, and diagnostic data. Immunohistochemical analysis was performed on tissue microarray samples of 306 polyps. Based on the Allred score, the expressions were graduated in the cytoplasm and nucleus of superficial and cryptic cells. There was higher Parkin nuclear expression (p=0.006 and 0.010) and APC cytoplasmic expression in cryptic cells (p<0.001) in SPs. CAs, APE1 (p<0.001) and Bcl-xL (p<0.001) were more expressed in the nuclei and cytoplasms, respectively. These results are related to the biological role proposed for these proteins in cellular functions. They can contribute to the diagnosis criteria for polyps and improve the knowledge of biomarkers that could predict cancer development:     

Length–weight relationships for fishes caught by shrimp trawl in Santa Catarina coast,south Atlantic,Brazil

Length–weight relationships (TW = a TL^b) were estimated for fish species caught by bottom shrimp trawl north of Santa Catarina Island (Brazil) from October 2003 to September 2004. For three of the species this is the first LWR data in Brazil.