HSP60 mimetic peptides from Mycobacterium leprae as new antigens for immunodiagnosis of Leprosy.

The early diagnosis of leprosy serves as an important tool to reduce the incidence of this disease in the world. Phage display (PD) technology can be used for mapping new antigens to the development of immunodiagnostic platforms. Our objective was to identify peptides that mimic Mycobacterium leprae proteins as serological markers using phage display technology. The phages were obtained in the biopanning using negative and positive serum from household contacts and leprosy patients, respectively. Then, the peptides were synthesized and validated in silico and in vitro for detection of IgG from patients and contacts. To characterize the native protein of M. leprae, scFv antibodies were selected against the synthetic peptides by PD. The scFv binding protein was obtained by immunocapture and confirmed using mass spectrometry. We selected two phase-fused peptides, MPML12 and MPML14, which mimic the HSP60 protein from M. leprae. The peptides MPML12 and MPML14 obtained 100% and 92.85% positivity in lepromatous patients. MPML12 and MPM14 detect IgG, especially in the multibacillary forms. The MPML12 and MPML14 peptides had positivity of 11.1% and 16.6% in household contacts, respectively. There was no cross-reaction in patient's samples with visceral leishmaniasis, tuberculosis and other mycobacteriosis for both peptides. Given these results and the easy obtainment of mimetic antigens, our peptides are promising markers for application in the diagnosis of leprosy, especially in endemic and hyperendemic regions.

A novel peptide-based electrochemical biosensor for breast cancer characterization over a poly 3-(3-aminophenyl) propionic acid matrix.

In this work, a new electrochemical biosensor was developed using peptides selected by Phage Display as biorecognition phase to Breast Cancer (BC) characterization. Phage clones were selected against MCF-7 (ER-positive BC) proteins, in order to characterize patients with aggressive luminal BC. Biotin-C3 and biotin-H2 peptides were chemically synthesized and validated by flow cytometry, immunofluorescence assays, and ELISA assays, being more reactive to the MCF-7 lineage. Furthermore, a new matrix for the coupling of biomolecules on the surface of graphite electrodes was generated, through electrochemical modification with a new material derived from 3-(3-aminophenyl)propionic acid (3-3-APPA). Electrochemical and morphological characterizations were carried out, and the mechanism of electropolymerization of poly(3-3-APPA) was proposed, in which the carboxylate groups are kept in the structure of the formed polymer. Then, a biosensor was developed by immobilizing the biotin-C3 and biotin-H2 peptides in the SPE/poly(3-3-APPA)/avidin system for the detection of BC tumor markers in serological samples. Finally, peptides were validated using samples from patients with BC and Benign Breast Disease. Biotin-C3 peptide characterized luminal BC according to p53 status and to HER2 expression, being the biosensor a better strategy when compared to ELISA test. This new biosensor will open a new perspective for a rapid and electrochemical platform for the characterization of BC and its molecular subtypes.

Glibenclamide treatment modulates the expression and localization of myosin-IIB in diabetic rat brain.

BACKGROUND: Myosin-IIB is a non-muscle isoform in the brain with increased expression in the brains of diabetic rats. Chronic hyperglycemia caused by diabetes can impair learning and memory. Oral hypoglycemic agents such as glibenclamide have been used to control hyperglycemia. We report changes in the expression and distribution of myosin-IIB in the frontal cortex and hippocampus of diabetic rats treated with glibenclamide. METHODS: The brains were removed after 43 days of treatment with glibenclamide (6 mg/kg bw orally), homogenized and analyzed by Western blotting, qRT-PCR and immunohistochemistry. RESULTS: Myosin-IIB expression increased in the brains of diabetic rats. However, protein expression returned to control levels when treated with glibenclamide. In addition, the expression of MYH10 gene encoding non-muscle myosin heavy chain-B decreased in diabetic rats treated with glibenclamide. Moreover, we found weak myosin-IIB labeling in the hippocampus and frontal cortex of rats treated with glibenclamide. Therefore, the expression of myosin-IIB is affected by diabetes mellitus and may be modulated by glibenclamide treatment in rats. Structural changes in the hippocampus and prefrontal cortex are reversible, and glibenclamide treatment may reduce the patho-physiological changes in the brain. CONCLUSIONS: Our findings can contribute to the understanding of the regulation of myosins in the brains of diabetic rats.