A multiple genome analysis of Mycobacterium tuberculosis reveals specific novel genes and mutations associated with pyrazinamide resistance.

BACKGROUND: Tuberculosis (TB) is a major global health problem and drug resistance compromises the efforts to control this disease. Pyrazinamide (PZA) is an important drug used in both first and second line treatment regimes. However, its complete mechanism of action and resistance remains unclear. RESULTS: We genotyped and sequenced the complete genomes of 68 M. tuberculosis strains isolated from unrelated TB patients in Peru. No clustering pattern of the strains was verified based on spoligotyping. We analyzed the association between PZA resistance with non-synonymous mutations and specific genes. We found mutations in pncA and novel genes significantly associated with PZA resistance in strains without pncA mutations. These included genes related to transportation of metal ions, pH regulation and immune system evasion. CONCLUSIONS: These results suggest potential alternate mechanisms of PZA resistance that have not been found in other populations, supporting that the antibacterial activity of PZA may hit multiple targets

Isolation of adipose derived mesenchymal stem cells by using anti-CD105 VHH-magnetic beads

Background: Human adipose tissue-derived stem cells (ASCs) are widely used in regenerative medicine and tissue engineering. Magnetic-activated cell sorting (MACS) with monoclonal antibodies (mAbs) that bind surface markers of stem cells such as CD105, CD73, and CD90 is currently applied for the enrichment and isolation of ASCs. Alternatives to mAbs are the variable domains of heavy chain antibodies (VHHs), which are naturally produced in camelids. We report the application of an anti-CD105 VHH conjugated to magnetic beads in the isolation of ASCs in vitro. Results: Two identical anti-CD105 VHHs (EngVHH17) were screened by phage display from a VHH cDNA library constructed from the PBMCs of an alpaca immunized with a lysate of human bladder cancer cell line (T24 cells). EngVHH17 was cloned in pET22b(+), expressed in Escherichia coli BL21 and purified in Ni-NTA chromatography resulting in a ~15 kDa VHH antibody. EngVHH17 binds CD105 with high affinity (Kd = 3.9 × 10-10 M). The specific binding of EngVHH17 to CD105 was visualized by fluorescence inmunolabeling of phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 cells using FITC labeled anti-6 × His Tag IgG1 mouse mAb. EngVHH17-magnetic beads selectively sorted human ASCs from osteoblasts in a mixed culture in vitro. The selective recovery of ASC cells using different ASC/Osteoblast ratios was higher than 85%. Conclusions: EngVHH17 coupled to magnetic beads binds CD105 expressed on the cell surface of ASCs and isolates them from osteoblasts in mixed cultures in vitro by application of an external magnetic field. EngVHH17 can be further evaluated for the isolation of MSCs by MACS. How to cite: Gushiken E, Quintana M, Perez L, et al. Isolation of adipose derived mesenchymal stem cells by using anti-CD105 VHH-magnetic beads. Electron J Biotechnol 2023;66. https://doi.org/10.1016/j.ejbt.2023.06.004

Efeito da metoclopramida, da ondansetrona ou do placebo na qualidade da recuperação após a administração de morfina intratecal em pacientes submetidos à correção cirúrgica de fraturas de membros inferiores

Introdução: A administração da morfina pela via intratecal é reconhecida como uma das melhores formas de prevenir a ocorrência de dor pós-operatória em pacientes submetidos à cirurgia ortopédica nos membros inferiores. No entanto, para muitos indivíduos os benefícios observados são acompanhados de efeitos indesejáveis causados pela presença desse opióide no espaço liquórico. Algumas intervenções podem reduzir a ocorrência de tais complicações, como o emprego da metoclopramida e da ondansetrona. Objetivos: O estudo tem como objetivo avaliar e comparar os efeitos da metoclopramida, da ondasterona e do placebo como possíveis agentes benéficos na recuperação dos pacientes submetidos à correção cirúrgica de fratura de membros inferiores após a utilização de morfina no procedimento Metodologia: O estudo envolve a análise de informações coletadas a partir dos questionários que serão aplicados aos pacientes submetidos à cirurgia ortopédica nos membros inferiores no Hospital Santa Lucinda e no Hospital Regional de Sorocaba. Tais questionários serão específicos ao medicamento que for administrado, sendo a escolha de tal medicamento feita de forma randomizada

Prediction of Mycobacterium tuberculosis pyrazinamidase function based on structural stability, physicochemical and geometrical descriptors.

BackgroundPyrazinamide is an important drug against the latent stage of tuberculosis and is used in both first- and second-line treatment regimens. Pyrazinamide-susceptibility test usually takes a week to have a diagnosis to guide initial therapy, implying a delay in receiving appropriate therapy. The continued increase in multi-drug resistant tuberculosis and the prevalence of pyrazinamide resistance in several countries makes the development of assays for prompt identification of resistance necessary. The main cause of pyrazinamide resistance is the impairment of pyrazinamidase function attributed to mutations in the promoter and/or pncA coding gene. However, not all pncA mutations necessarily affect the pyrazinamidase function.ObjectiveTo develop a methodology to predict pyrazinamidase function from detected mutations in the pncA gene.MethodsWe measured the catalytic constant (kcat), KM, enzymatic efficiency, and enzymatic activity of 35 recombinant mutated pyrazinamidase and the wild type (Protein Data Bank ID = 3pl1). From all the 3D modeled structures, we extracted several predictors based on three categories: structural stability (estimated by normal mode analysis and molecular dynamics), physicochemical, and geometrical characteristics. We used a stepwise Akaike's information criterion forward multiple log-linear regression to model each kinetic parameter with each category of predictors. We also developed weighted models combining the three categories of predictive models for each kinetic parameter. We tested the robustness of the predictive ability of each model by 6-fold cross-validation against random models.ResultsThe stability, physicochemical, and geometrical descriptors explained most of the variability (R2) of the kinetic parameters. Our models are best suited to predict kcat, efficiency, and activity based on the root-mean-square error of prediction of the 6-fold cross-validation.ConclusionsThis study shows a quick approach to predict the pyrazinamidase function only from the pncA sequence when point mutations are present. This can be an important tool to detect pyrazinamide resistance

Lupeol, a Dietary Triterpene, Enhances Wound Healing in Streptozotocin-Induced Hyperglycemic Rats with Modulatory Effects on Inflammation, Oxidative Stress, and Angiogenesis

Impaired wound healing is a debilitating complication of diabetes that leads to significant morbidity, particularly foot ulcers. Natural products have shown to be effective in treating skin wounds. Lupeol is known to stimulate angiogenesis, fibroblast proliferation, and expressions of cytokines and growth factors involved in wound healing. The study is performed to evaluate the wound healing activity of lupeol in streptozotocin-induced hyperglycemic rats by macroscopical, histological, immunohistochemical, immunoenzymatic, and molecular methods. Percentage of wound closure and contraction was increased in the lupeol-treated group when compared to the Lanette group. Histopathological observation revealed decreased inflammatory cell infiltration and increased proliferation of fibroblasts, vascularization, and deposition of collagen fibers after lupeol treatment. Immunohistochemical analyses showed decreased intensity of NF-κB and increased intensity of FGF-2, TGF-β1, and collagen III. ELISA results revealed downregulated IL-6 levels and upregulated IL-10 levels in response to lupeol. The mRNA expression levels of Hif-1α, Sod-2, and Ho-1 were significantly increased in response to lupeol as compared to Lanette whereas Nf-κb and Vegf-A levels were decreased in relation to insulin and lupeol treatment. These findings indicate that lupeol possesses wound healing potential in hyperglycemic conditions and may be useful as a treatment for chronic wounds in diabetic patients

N, N ', N ''-trisubstituted guanidines: Synthesis, characterization and evaluation of their leishmanicidal activity

Leishmaniasis is a group of diseases caused by protozoan parasites from the genus Leishmania. There are estimated 1.3 million new cases annually with a mortality of 20,000-30,000 per year, when patients are left untreated. Current chemotherapeutic drugs available present high toxicity and low efficacy, the latter mainly due to the emergence of drug-resistant parasites, which makes discovery of novel, safe, and efficacious antileishmanial drugs mandatory. The present work reports the synthesis, characterization by ESI-MS, H-1 and C-13 NMR, and FTIR techniques as well as in vitro and in vivo evaluation of leishmanicidal activity of guanidines derivatives presenting lower toxicity. Among ten investigated compounds, all being guanidines containing a benzoyl, a benzyl, and a substituted phenyl moiety, LQOF-G2 (IC50-ama 5.6 mu M: SI = 131.8) and LQOF-G7 (IC50-ama 7.1 mu M; SI = 87.1) were the most active against L. amazonensis intracellular amastigote, showing low cytotoxicity to the host cells according to their selectivity index. The most promising compound, LQOF-G2, was further evaluated in an in vivo model and was able to decrease 60% of the parasite load in foot lesions at a dose of 0.25 mg/kg/day. Moreover, this guanidine derivative demonstrated reduced hepatotoxicity compared to other leishmanicidal compounds and did not show nephrotoxicity, as determined by the analyses of biomarkers of hepatic damage and renal function, which make this compound a potential new hit for therapy against leishmaniasis. (C) 2019 Elsevier Masson SAS. All rights reserved171116128COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2013/24487-6; 2013/08248-1; 2016/19289-9; 2017/03552-

Additional file 4: Table S4. of A multiple genome analysis of Mycobacterium tuberculosis reveals specific novel genes and mutations associated with pyrazinamide resistance

Table of the 110 genes that were significantly associated with PZA-resistance at gene level, which also have mutations significantly associated with PZA-resistance at the mutation level. The association p-value at mutation level was re-calculated excluding the strains harboring mutations strongly related with PZA resistance. These genes were annotated with their molecular function. (DOCX 297 kb