Influence of anti-sclerostin monoclonal antibody in the repair of post-extraction sockets of ovariectomized rats.

OBJECTIVE: This study assessed the impact of an anti-sclerostin monoclonal antibody (Scl-Ab)-based osteoporosis drug on the post-extraction alveolar repair of ovariectomized rats. DESIGN: Fifteen female rats were randomly distributed into three groups: CTR (healthy animals), OST (osteoporosis induced by ovariectomy), and OST+Scl-Ab (osteoporosis induction followed by Scl-Ab treatment). Ovariectomy or sham surgery was performed 30 days before baseline, and Scl-Ab or a vehicle was administered accordingly in the groups. After seven days, all rats underwent the first lower molar extraction and were euthanized 15 days later. Computed microtomography, histological analysis, and collagen content measurement were performed on post-extraction sockets and intact mandibular and maxillary bone areas. RESULTS: Microtomographic analyses of the sockets and mandibles did not reveal significant differences between groups on bone morphometric parameters (p > 0.05), while maxillary bone analyses resulted in better maintenance of bone architecture in OST+Scl-Ab, compared to OST (p < 0.05). Descriptive histological analysis and polarization microscopy indicated better post-extraction socket repair characteristics and collagen content in OST+Scl-Ab compared to OST (p < 0.05). CONCLUSIONS: Scl-Ab-based medication did not accelerate alveolar bone formation but exhibited better post-extraction repair characteristics, and collagen content compared to ovariectomized animals only.

Zinc-Based Nanoparticles Reduce Bacterial Biofilm Formation.

Biofilm formation is important for microbial survival in hostile environments and a phenotype that provides microorganisms with antimicrobial resistance. Zinc oxide (ZnO) and Zinc sulfide (ZnS) nanoparticles (NPs) present potential antimicrobial properties for biomedical and food industry applications. Here, we aimed to analyze, for the first time, the bactericidal and antibiofilm activity of ZnS NPs against Staphylococcus aureus, Klebsiella oxytoca, and Pseudomonas aeruginosa, all medically important bacteria in developed countries. We compared ZnS NPs antimicrobial activity to ZnO NPs, which have been extensively studied. Using the colorimetric XTT reduction assay to observe the metabolic activity of bacterial cells and the crystal violet assay to measure biofilm mass, we demonstrated that ZnS and ZnO had similar efficacy in killing planktonic bacterial cells and reducing biofilm formation, with S. aureus being more susceptible to both therapeutics than K. oxytoca and P. aeruginosa. Crystal violet staining and confocal microscopy validated that Zn NPs inhibit biofilm formation and cause architectural damage. Our findings provide proof of principle that ZnS NPs have antibiofilm activity, and can be potentially used in medical and food industry applications, such as treatment of wound infections or package coating for food preservation. IMPORTANCE Zinc (Zn)-based nanoparticles (NPs) can be potentially used in medical and food preservation applications. As proof of principle, we investigated the bactericidal and antibiofilm activity of zinc oxide (ZnO) and zinc sulfide (ZnS) NPs against medically important bacteria. Zn-based NPs were similarly effective in killing planktonic and biofilm-associated Staphylococcus aureus, Klebsiella oxytoca, and Pseudomonas aeruginosa cells. However, S. aureus was more susceptible to these investigational therapeutics. Although further studies are warranted, our findings suggest the possibility of future use of Zn-based NPs in the treatment of skin infections or preservation of food.

ZnO@ZIF-8 Nanoparticles as Nanocarrier of Ciprofloxacin for Antimicrobial Activity.

Numerous antimicrobial drugs have been prescribed to kill or inhibit the growth of microbes such as bacteria, fungi, and viruses. Despite the known therapeutic efficacy of these drugs, inefficient delivery could result in an inadequate therapeutic index and several side effects. In order to overcome this adversity, the present study investigated antibiotic drug loading in zeolitic imidazolate frameworks (ZIFs), in association with ZnO nanoparticles with known antimicrobial properties. In an economic synthesis method, the ZnO surface was first converted to ZIF-8 with 2-methylimidazole as a ligand, resulting in a ZnO@ZIF-8 structure. This system enables the high drug-loading efficiency (46%) of an antimicrobial drug, ciprofloxacin, within the pores of the ZIF-8. This association provides a control of the release of the active moieties, in simulated body-fluid conditions, with a maximum of 67% released in 96 h. The antibacterial activities of ZnO@ZIF-8 and CIP-ZnO@ZIF-8 were tested against the Gram-positive Staphylococcus aureus strain and the Gram-negative Pseudomonas aeruginosa strain, showing good growth inhibition. This result was obtained by combining ZnO@ZIF-8 with ciprofloxacin in a minimal inhibitory concentration (MIC) that was 10 times lower than ZnO@ZIF-8 for S. aureus and 200 times lower for P. aeruginosa, suggesting that CIP-ZnO@ZIF-8 may have potential application in prolonged antimicrobial treatment.

The Emergence of the New P.4 Lineage of SARS-CoV-2 With Spike L452R Mutation in Brazil.

The emergence of several SARS-CoV-2 lineages presenting adaptive mutations is a matter of concern worldwide due to their potential ability to increase transmission and/or evade the immune response. While performing epidemiological and genomic surveillance of SARS-CoV-2 in samples from Porto Ferreira-São Paulo-Brazil, we identified sequences classified by pangolin as B.1.1.28 harboring Spike L452R mutation, in the RBD region. Phylogenetic analysis revealed that these sequences grouped into a monophyletic branch, with others from Brazil, mainly from the state of São Paulo. The sequences had a set of 15 clade defining amino acid mutations, of which six were in the Spike protein. A new lineage was proposed to Pango and it was accepted and designated P.4. In samples from the city of Porto Ferreira, P.4 lineage has been increasing in frequency since it was first detected in March 2021, corresponding to 34.7% of the samples sequenced in June, the second in prevalence after P.1. Also, it is circulating in 30 cities from the state of São Paulo, and it was also detected in one sample from the state of Sergipe and two from the state of Rio de Janeiro. Further studies are needed to understand whether P.4 should be considered a new threat.

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors.

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

Development of diagnostic methods and study of the immunoreactivity of a mixture of recombinant core and E2 proteins fused to GST with control serum positive for hepatitis C.

The hepatitis C virus (HCV) is an enveloped virus that is about 50-70 nm in diameter, has positive-strand RNA, and belongs to the genus Hepacivirus and the family Flaviridae. The detection and quantification of the core antigen, HCV nucleocapsid protein, has been successful in many trials and is considered a marker of viral replication since it presents a sequence of highly conserved amino acids, giving it high sensitivity and specificity. The E2 protein is an envelope glycoprotein of HCV with 11 glycosylation sites; most of these are well-conserved, making it a target antigen. The aim of this study is to develop high-sensitivity, low-cost diagnostic methods for HCV, which could be used for serological screening. The genomic regions encoding the core (part 136 aa) and E2 proteins of HCV were expressed in Escherichia coli Rosetta strain, cloned in expression vector pET-42a, and induced with 0.4 m mol L(-1) IPTG, producing recombinant proteins that were fused to glutathione S-transferase (GST) protein, which was then purified by affinity chromatography. The immunoreactivity was assessed by Western blot, Slot Blot, and developed and improved diagnostic methods (capture, indirect, and immunoblotting enzyme-linked immunosorbent assay (ELISA)). After applying the results to the formulas for determining the quality parameters, obtained for immunoblotting method 100% sensitivity and specificity and for ELISA 100% sensitivity and 87.5% specificity. The methods developed were more sensitive and specific using the mixture of the recombinant proteins fused to GST (core+E2).

Development of a rapid one-step immunochromatographic assay for HCV core antigen detection.

The hepatitis C virus (HCV) core protein possesses amino acid sequences highly conserved among HCV isolates and has proved useful for various diagnostic tests. To date, no information has been published regarding the development of an immunochromatographic test for HCV core antigen detection. Therefore, the aim of this research was to develop a rapid, easily performed, highly sensitive and specific test for detection of the HCV core antigen, based on the immunochromatographic strip. The genomic region encoding the core protein (amino acids 1-136) of the hepatitis C virus was expressed in Escherichia coli as a recombinant fusion protein with glutathione S-transferase (GST) cloned into the prokaryotic expression vector pET42a and was confirmed by immunological detection with HCV positive serum. Positive reactions were detected weakly at a 1:15 dilution of the serum and more strongly in 1:10, 1:5, 1:2 and 1:1 dilutions, by the immunochromatographic test. In addition, the test was capable of detecting 0.25-12.0 microg of the recombinant protein. This immunochromatographic technique opens new perspectives for the diagnosis of hepatitis C during the early seroconversion phase and for a rapid core antigen detection.

Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus.

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45V versus Ag/AgCl using H(2)O(2) and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease.

The beta-chemokines MIP-1alpha and RANTES and lipoprotein metabolism in HIV-infected Brazilian patients.

HIV patients are predisposed to the development of hypertriglyceridemia and hypercholesterolemia as a result of both viral infection and HIV infection therapy, especially the protease inhibitors. Chemokines and cytokines are present at sites of inflammation and can influence the nature of the inflammatory response in atherosclerosis. We investigated the correlation between biochemical variables and beta-chemokines (MIP-1alpha and RANTES) and the apolipoprotein E genotype in HIV-infected individuals. The apolipoproteins were measured by nephelometry. Triglycerides and total cholesterol were determined by standard enzymatic procedures. The beta-chemokines were detected by ELISA. The genetic category of CCR5 and apolipoprotein E were determined by PCR amplification and restriction enzymes. Immunological and virological profiles were assessed by TCD(4)+ and TCD(8)+ lymphocyte counts and viral load quantification. Positive correlations were found between apo E and CD(8)+ (p = 0.035), apo E and viral load (p = 0.018), MIP-1alpha and triglycerides (p = 0.039) and MIP-1a and VLDL (p = 0.040). Negative correlations were found between viral load and CD(4)+ (p = 0.05) and RANTES and CD(4)+ (p = 0.029). The beta-chemokine levels may influence lipid metabolism in HIV-infected individuals.

Immunosensor for the diagnosis of Chagas' disease.

Trypanosoma cruzi proteins from epimastigote membranes, herein referred as antigens, have been used for the construction of an amperometric immunosensor for serological diagnosis of Chagas' disease. The proteins used had a molecular mass ranging from 30 to 100 kDa. The gold electrode was treated with cysteamine and glutaraldehyde prior to antigen immobilization. Antibodies present in the serum of patients with Chagas' disease were captured by the immobilized antigens and the affinity interaction was monitored by chronoamperometry at a potential of -400 mV (versus Ag pseudo-reference electrode) using peroxidase-labeled IgG conjugate and hydrogen peroxide, iodide substrate. The incubation time to allow maximum antigen-antibody and antibody-peroxidase-labeled IgG interactions was 20 min with a reactivity threshold at -0.104 microA.

Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C virus.

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of hepatitis C virus (HCV) in serum. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). Piezoelectric biosensors were used in a flow-through setup for direct monitoring of DNA resulting from the reverse transcriptase-linked polymerase chain reaction (RT-PCR) amplification of the original viral RNA. The samples of patients with hepatitis C were analyzed and the results were compared with the standard RT-PCR procedure (Amplicor test kit of Roche, microwell format with spectrophotometric evaluation). The piezoelectric hybridization assay was completed in 10 min and the same sensing surface was suitable for repeated use.

[Evaluation of the polymorphisms in methylenetetrahydrofolate reductase gene and the levels of folate and B12 in HIV-infected patients under antiretroviral therapy]. / Avaliação do polimorfismo no gene da metilenotetrahidrofolato redutase e concentração de folato e vitamina B12 em pacientes portadores do HIV-1 em tratamento com anti-retrovirais.

In this study we sought to investigate the B12 and folate levels regarding the influence of methylenetetrahydrofolate reductase genotypes, immunological profile and antiretroviral therapy in the Brazilian HIV-infected population. The study group comprised 89 HIV-infected individuals and 29 blood donors. There was a decrease in the B12 levels in the HIV-infected group with higher TCD4+ lymphocyte counts. No differences in the genotype distribution for methylenetetrahydrofolate reductase polymorphisms between the HIV-infected individuals and the controls were found. HIV-infected individuals carrying the C677C genotype presented lower B12 levels (313.91 +/- 154.05) than those with the same genotype in the control group (408.27 +/- 207.69). Also, the antiretroviral therapy was not a source of variation of the folate and B12 serum levels. Further studies are needed to reanalyze the prevalence of low levels of folate and B12 and hyperhomocysteinemia among HIV-infected patients with regard to the use of HAART and the increased life expectancy of such patients.