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1.
Microb Pathog ; 141: 103991, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31978426

RESUMO

Mycobacterium tuberculosis is capable of escaping the clearance of immune system mainly due to its complex constituents of cell wall. Certain studies show that glycoproteins are involved in immune evasion and act as virulence factors. Peptidoglycan deacetylase Rv1096 is a member of mannosylated proteins. Previously, we reported Rv1096 protein contributed to the resistance of Mycobacterium smegmatis (M. smegmatis) to lysozyme, but more characterization of this protein is required where further intracellular function is unknown. Here, Rv1096 was heterologously over-expressed in the fast-growing and nonpathogenic M. smegmatis (named as M. smegmatis/Rv1096). We observed the morphological alterations in M. smegmatis/Rv1096 including an elongated rod-like shape and increased amounts of Z-rings, which implied that Rv1096 facilitated the cell growth and division. Moreover, a series of assays concerning the interaction between M. smegmatis/Rv1096 and host were carried out. The results showed that M. smegmatis/Rv1096 evaded the killing of macrophages due to the inhibition of phagosome-lysosome fusion, nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. The secretion of interleukin-12 and tumor necrosis factor-α was also impaired by Rv1096. In addition, five putative interaction partners of Rv1096 were identified, which possibly cooperated with Rv1096 in cell division and immune regulation. These results suggested that Rv1096 had effects on mycobacterial division and might act as a virulence factor to mediate the immune evasion in macrophage during mycobacterial infection.

2.
Anal Chem ; 92(1): 1424-1430, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31813221

RESUMO

A facile and rapid postsynthetic modification strategy for functionalization of covalent organic framework (COF) was developed to synthesize a tailor-made pH-responsive COF called TpPa-1@Au@GSH for highly efficient extraction of N1-methyladenosine (m1A). Glutathione (GSH) was judiciously designed as the functional group for extracting and releasing m1A by pH variations. With the aid of gold nanoparticles (Au NPs) as linkers, GSH was successfully introduced to the robust substrate TpPa-1 in only one step spending only 1 h. Owing to the several-to-one immobilization of GSH on Au NPs and the large surface area of TpPa-1, this functional COF was constructed with abundant m1A binding sites. TpPa-1@Au@GSH showed excellent selectivity for m1A extraction by capturing m1A from a mixture of 14 nucleoside analogues followed by mass spectrometry analysis. It was proved to have ultrafast adsorption ability (only 1 min incubation time), high binding capacity (5 mg g-1, m1A/TpPa-1@Au@GSH), good reusability (at least 5 times), and good storage stability (at least 8 months at room temperature). Great performance was also achieved in extracting m1A from both animal and plant biological samples. The adsorption mechanism was demonstrated to be based on the electrostatic interaction. This work proposed a new approach for m1A extraction, demonstrated the high potential of COFs in biological sample pretreatment, and offered an effective and versatile route for functionalization of COFs.

3.
Int J Biol Macromol ; 143: 393-400, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31830456

RESUMO

In previous researches, the results showed that selenium Hericium erinaceus polysaccharide and Hericium erinaceus polysaccharide-loaded poly (lactic-co-glycolic acid) nanoparticles enhanced immune responses. In order to further enhance the immune adjuvant activity and phagocytosis of the nanoparticles, two way of combination (selenium-HEP loaded PLGA nanoparticles and selenium modified HEP-PLGA nanoparticles) were prepared to investigate the effects on macrophages in vitro. After treatment with the nanoparticles, the effects of phagocytosis, co-stimulatory molecules expression, nitric oxide (NO), and cytokines secretion were evaluated. The results showed that the particle size, PDI and zeta potential of the selenium-HEP loaded PLGA nanoparticles (Se-HEP-PLGA) and selenium modifified HEP-PLGA nanoparticles (HEP-PLGA-Se) were presented. Se-HEP-PLGA and HEP-PLGA-Se nanoparticles significantly stimulated phagocytic activity, CD40 and CD86 expression of macrophages. In addition, the levels of NO, TNF-α, IL-1ß and IL-6 were enhanced in the peritoneal macrophages by stimulation with Se-HEP-PLGA and HEP-PLGA-Se nanoparticles. Among them, Se-HEP-PLGA showed the best effects on the expression of co-stimulatory molecules, secretions of NO and cytokines. These results indicated that Se-HEP-PLGA could enhance the activation of macrophages, and it could be potentially used as an HEP delivery system for the induction of strong immune responses.

4.
J Cell Biol ; 219(1)2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31816056

RESUMO

The development of cerebral cortex requires spatially and temporally orchestrated proliferation, migration, and differentiation of neural progenitor cells (NPCs). The molecular mechanisms underlying cortical development are, however, not fully understood. The neural cell adhesion molecule (NCAM) has been suggested to play a role in corticogenesis. Here we show that NCAM is dynamically expressed in the developing cortex. NCAM expression in NPCs is highest in the neurogenic period and declines during the gliogenic period. In mice bearing an NPC-specific NCAM deletion, proliferation of NPCs is reduced, and production of cortical neurons is delayed, while formation of cortical glia is advanced. Mechanistically, NCAM enhances actin polymerization in NPCs by interacting with actin-associated protein profilin2. NCAM-dependent regulation of NPCs is blocked by mutations in the profilin2 binding site. Thus, NCAM plays an essential role in NPC proliferation and fate decision during cortical development by regulating profilin2-dependent actin polymerization.

5.
Int J Biol Macromol ; 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31758997

RESUMO

In present study, the optimal condition of prepared drug was determined by response surface methodology. In addition, their physicochemical properties, drug release and uptake ability of CS-MWCNT-HEP were studied, and the distribution of the drug in ICR mice and the sites of action were further evaluated. Under the optimal condition, the mean experimental loaded efficiency 68.55 ± 1.47% was corresponded well with the predicted value of 68.28%. The results of in vitro experiments proved that a release of the drug in a pH-dependent behavior. Flow cytometry and inverted microscope showed that the uptake of CS-MWCNT-HEP in Raw264.7 cells increased significantly as the time increased. In vivo experiment proved that the HEP and CS-MWCNT-HEP were mainly accumulated in the kidney, shown the characteristics of kidney metabolism. On the other hand, the extended retention of CS-MWCNT-HEP in the mice could enhance the immune function. CS-MWCNT-HEP has high loaded efficiency and pH-responsive drug released, which could significantly improved the body's immunity and enhance the body's ability to absorbed drugs. These findings proposed a well characterized novel CS-MWCNT-HEP formulation as drug delivery system, and its mechanism and application will be further investigated in our undergoing studies.

6.
Front Microbiol ; 10: 1799, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31481936

RESUMO

Mycobacterium tuberculosis is one of most pathogenic microorganisms in the world. Previously, the bifunctional enzyme GlmU with glucosamine-1-phosphate acetyltransferase activity and N-acetylglucosamine-1-phosphate uridyltransferase activity has been suggested as a potential drug target; therefore, discovering compounds targeting GlmU acetyltransferase is necessary. The natural products were tested for inhibition of GlmU acetyltransferase activity. We found that dicumarol exhibited inhibitory effects on GlmU acetyltransferase, with a concentration achieving a 50% inhibition (IC50) value of 4.608 µg/ml (13.7 µM). The inhibition kinetics indicated that dicumarol uncompetitively inhibited acetyl CoA and showed mixed-type inhibition for glucosamine-1-phosphate (GlcN-1-P). The activity of dicumarol against M. tuberculosis H37Ra was evaluated with a minimum inhibitory concentration (MIC) value of 6.25 µg/ml (18.55 µM) in the Alamar blue assay. Dicumarol also exhibited inhibitory effects on several clinically sensitive M. tuberculosis strains and drug-resistant strains, with a range of MIC value of 6.25 to >100 µg/ml. Dicumarol increased the sensitivity of anti-tuberculosis drugs (isoniazid and rifampicin) when dicumarol was present at a low concentration. The transcriptome and proteome data of M. tuberculosis H37Ra treated by dicumarol showed that the affected genes were associated with cell wall synthesis, DNA damage and repair, metabolic processes, and signal transduction. These results provided the mechanism of dicumarol inhibition against GlmU acetyltransferase and M. tuberculosis and also suggested that dicumarol is a potential candidate for TB treatment.

7.
Artigo em Inglês | MEDLINE | ID: mdl-31380295

RESUMO

Mycobacterium tuberculosis bifunctional enzyme GlmU is a novel target for anti-TB drugs and is involved in glycosyl donor UDP-N-acetylglucosamine biosynthesis. Here, we found that TPSA (2-[5-(2-{[4-(2-thienyl)-2-pyrimidinyl]sulfanyl}acetyl)-2-thienyl]acetic acid) was a novel inhibitor for GlmU acetyltransferase activity (IC50: 5.3 µM). The interaction sites of GlmU and TPSA by molecular docking were confirmed by site-directed mutagenesis. TPSA showed an inhibitory effect on Mtb H37Ra growth and intracellular H37Ra in macrophage cells (MIC: 66.5 µM). To investigate why TPSA at a higher concentration (66.5 µM) was able to inhibit H37Ra growth, proteome and transcriptome of H37Ra treated with TPSA were analyzed. The expression of two methyltransferases MRA_0565 (Rv0558) and MRA_0567 (Rv0560c) were markedly increased. TPSA was pre-incubated with purified Rv0558 and Rv0560c in the presence of S-adenosylmethionine (methyl donor) respectively, resulting in its decreased inhibitory effect of GlmU on acetyltransferase activity. The inhibition of TPSA on growth of H37Ra with overexpressed Rv0558 and Rv0560c was reduced. These implied that methyltransferases could modify TPSA. The methylation of TPSA catalyzed by Rv0560c was subsequently confirmed by LC-MS. Therefore, TPSA as a GlmU acetyltransferase activity inhibitor may offer a structural basis for new anti-tuberculosis drugs. TPSA needs to be modified further by some groups to prevent its methylation by methyltransferases.

8.
Mikrochim Acta ; 186(8): 542, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31317336

RESUMO

Polyacrylonitrile fibers with and without magnetic nanoparticles (Fe3O4 NPs) were prepared by electrospinning. The pure polyacrylonitrile (PAN) fibers and the composited polyacrylonitrile (PAN/Fe3O4) fibers were studied with respect to their capability for enrichment of glycoproteins. Specifically, the glycoproteins ovalbumin (OB) and transferrin (Trf) were studied and compared to the non-glycoproteins bovine serum albumin and lysozyme. Following adsorption and subsequent protein elution with 0.1 wt% of CTAB solution, the glycoproteins were analyzed by SDS polyacrylamide gel electrophoresis. The strong interaction between PAN or PAN/Fe3O4 fibers and glycoproteins is attributed to the synergistic effects of hydrophilic and hydrogen bond interactions. The PAN/Fe3O4 fibers have an attractive additional feature of allowing magnetic separation. The PAN and PAN/Fe3O4 fibers have a high adsorption capacity toward OB and Trf. The treated PAN/Fe3O4 fibers display good selectivity, fast adsorption kinetics, and were applied to extractions of mixed protein samples. The detection limits of OB and Trf are 0.32 and 0.22 µg·mL-1, respectively. The PAN/Fe3O4 fibers offered an alternative solution for adsorption of glycoproteins from biological samples. Graphical abstract The pure polyacrylonitrile (PAN) fibers and the composited polyacrylonitrile (PAN/Fe3O4) fibers were studied with respect to their capability for enrichment of glycoproteins: glycoproteins ovalbumin (OB) and transferrin (Trf). The treated PAN/Fe3O4 fibers showed fast adsorption kinetics, were applied in a physiological state, mixed and real samples.

9.
Environ Sci Pollut Res Int ; 26(26): 27516-27533, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31338758

RESUMO

With rapid industrialization and urbanization, regional water shortages and water quality deterioration have posed great challenges for the sustainable development of cities in North China, especially those with a large demand for agricultural irrigation water. Based on an input-output analysis, this paper develops a dynamic optimization model consisting of three sub-models and multiple constraint conditions to solve the water crisis of Baoding, a typical city experiencing water shortages and serious water pollution in North China. The water resource carrying capacity (WRCC) indicator is introduced in the analysis of the results to comprehensively assess the effect of integrated water environmental policies (IWEPs) from 2013 to 2025. In the optimal scenario, the annual chemical oxygen demand (COD) discharge and annual water demand in Baoding can be reduced by 2.6% and 0.6%, respectively, with an annual gross regional product (GRP) growth rate of 7.52%. The WRCC can be improved from moderately overloaded to weakly unsaturated, which indicates that water resources can meet the socioeconomic development requirements. The results demonstrate the effectiveness of the linear optimization model with input-output analysis in coordinating the relationships among water demand, water environment protection, and economic development, and the IWEPs provide an applicable reference for decision-makers in Baoding and other similar cities in North China to address deteriorating water systems.


Assuntos
Conservação dos Recursos Hídricos/métodos , Política Ambiental , Modelos Teóricos , Análise da Demanda Biológica de Oxigênio , China , Cidades , Simulação por Computador , Desenvolvimento Econômico , Alocação de Recursos , Desenvolvimento Sustentável , Urbanização , Poluição da Água , Qualidade da Água , Abastecimento de Água
10.
Microb Pathog ; 131: 218-226, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974158

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital-acquired infective pathogen that has developed resistance to many antibiotics. It is imperious to develop novel anti-MRSA drugs to control the emergence of drug resistance. The biosynthesis of cysteine in bacteria is catalyzed by CysE and CysK. CysE was predicted to be important for bacterial viability, it could be a potential drug target. The serine acetyltransferase activity of CysE was detected and its catalytic properties were also determined. CysE homology model was built to investigate interaction sites between CysE and substrate L-Ser or inhibitors by molecular docking. Docking data showed that residues Asp94 and His95 were essential for serine acetyltransferase activity of CysE, which were confirmed by site-directed mutagenesis. Colorimetric assay was used to screen natural products and six compounds which inhibited CysE activity (IC50 ranging from 29.83 µM to 203.13 µM) were found. Inhibition types of two compounds 4 (11-oxo-ebracteolatanolide B) and 30 ((4R,4aR)-dihydroxy-3-hydroxymethyl-7,7,10a-trimethyl-2,4,4a,5,6,6a,7,8,9,10,10a,l0b-dodecahydrophenanthro[3,2-b]furan-2-one) on CysE were determined. Compounds 4 and 30 showed inhibitory effect on MRSA growth (MIC at 12.5 µg/ml and 25 µg/ml) and mature biofilm. The established colorimetric assay will facilitate further high-throughput screening of CysE inhibitors from different compound libraries. The compounds 4 and 30 may offer structural basis for developing new anti-MRSA drugs.


Assuntos
Produtos Biológicos/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Serina O-Acetiltransferase/efeitos dos fármacos , Serina O-Acetiltransferase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biofilmes/efeitos dos fármacos , Domínio Catalítico , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Cinética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Serina O-Acetiltransferase/genética
11.
Nanoscale ; 11(12): 5526-5534, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30860530

RESUMO

The development of facilely synthetic materials for highly efficient enrichment of N-linked glycopeptides is essential in glycoproteome analysis. In this work, by utilizing the self-assembling of glutathione (GSH) on silver nanoparticles (Ag NPs), and the formation and dispersion of Ag NPs on a robust TpPa-1 substrate, a newly functionalized covalent organic framework (COF) called TpPa-1@Ag@GSH was synthesized via a simple two step post-synthetic modification. TpPa-1@Ag@GSH and intermediate products were confirmed and evaluated by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, transmission electron microscopy, scanning electron microscopy-energy dispersive spectroscopy, Brunauer-Emmett-Teller and thermogravimetric analyses. Benefiting from the judicious selection of the substrate, the abundance of binding sites, relatively high affinity between GSH and N-linked glycopeptides, and the multivalent interactions between N-linked glycopeptides and unoccupied surfaces of Ag NPs, this porous material showed great performance in N-linked glycopeptide enrichment. By enriching N-linked glycopeptides in tryptic digests of human serum immunoglobulin G (human IgG) followed by mass spectrometry analysis, our method was proved to have good sensitivity (1 fmol), high selectivity (1 : 1500, human IgG to bovine serum albumin), high binding capacity (160 mg g-1, IgG/TpPa-1@Ag@GSH), ultra-fast capture ability (only 1 min incubation time), and good reusability (at least 5 times). It was also successfully applied to the enrichment of N-linked glycopeptides from complex biological samples. Our work improved the enrichment selectivity of COFs, reached the most rapid capture ability among off-column enrichment materials, and provided a very facile and easily popularized post-synthetic modification route for COFs in glycoproteome analysis.


Assuntos
Glutationa/química , Glicopeptídeos/química , Nanopartículas Metálicas/química , Estruturas Metalorgânicas/química , Prata/química , Cromatografia Líquida de Alta Pressão , Glicopeptídeos/análise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/metabolismo , Microscopia Eletrônica de Transmissão , Porosidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier
12.
Int Immunopharmacol ; 70: 56-66, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30785091

RESUMO

The purpose of the present study is to investigate the immunological activities of EPS-1 in the non-specific immune response and specific immune response of chickens. In vitro, the results showed that EPS-1 could increase the proliferation and cytokine secretion (IL-2, IL-4, IFN-γ and TNF-α) of spleen lymphocytes, expression of key surface molecules (MHC II, CD11c, CD40 and CD86) and cytokine secretion (TNF-α and IL-10) of matured chBM-DCs, phagocytic rate of matured chBM-DCs, and enhance the maturation and stimulating capacity of chBM-DCs. In vivo, EPS-1 could also prompt the HI antibody titer, boost the peripheral lymphocyte proliferation, enhance the release of cytokine products in blood (IFN-γ, IL-4 and IL-2) and duodenum (IL-17 and sIgA) of chickens. These results indicated that EPS-1 may have the potential as a powerful immune adjuvant in the treatment of chicken diseases.


Assuntos
Galinhas/imunologia , Células Dendríticas/imunologia , Linfócitos/imunologia , Polissacarídeos/imunologia , Adjuvantes Imunológicos , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Medicamentos de Ervas Chinesas , Epimedium/imunologia , Imunidade Humoral , Imunomodulação , Ativação Linfocitária
13.
Int J Biol Macromol ; 125: 534-543, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30521910

RESUMO

Codonopsis pilosula polysaccharides (CPPS) has been shown to possess a variety of biological activities. In previous study, CPPS was successfully modified to obtain its best selenizing Codonopsis pilosula polysaccharides (sCPPS5). The purpose of this study was to investigate the protective effect of the selenizing derivative of CPPS (sCPPS5) from H2O2-induced oxidative damage in RAW264.7 murine macrophages and the possible mechanism of this protection. Results showed that the sCPPS5 was significantly stronger than that of the corresponding unmodified polysaccharide, CPPS. Meanwhile, sCPPS5 treatment could improve the production of reactive oxygen species (ROS), antioxidant enzyme, MMP, caspases-3 and apoptosis capacity of H2O2-induced RAW264.7 cells. Moreover, the mechanism might be elucidated that sCPPS5 could increase expression level of Nrf2 and its downstream ARE gene battery, promote production of corresponding antioxidative enzymes and protein, and enhance Keap1-Nrf2/ARE signaling pathway to avoid male reproductive dysfunction. Overall, these results showed that sCPPS5 as a potent antioxidant could reduce reproductive oxidative stress damage related to Keap1-Nrf2/ARE pathway.


Assuntos
Codonopsis/química , Peróxido de Hidrogênio/farmacologia , Polissacarídeos/farmacologia , Substâncias Protetoras/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
14.
Int J Biol Macromol ; 118(Pt A): 932-937, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29966670

RESUMO

In present study, HEP was successfully encapsulated into the Poly (lactic-coglycolicacid) (PLGA) to constitute the HEP-PLGA. The effects of three independent factors (the proper range of ratio of organic phase (o) to internal water phase (w1) (X1), ratio of external water phase (w2) to the primary emulsion (PE) (X2), and the concentration of PLGA (X3) on the extraction yield of encapsulation efficiency (EE) from the HEP was optimized using response surface methodology. The optimal extraction conditions for HEP-PLGA were determined as follows: X1: 8:1, X2: 7:1 and X3: 20 mg·mL-1. Under these optimal conditions, the mean experimental EE 90.86 ±â€¯0.576% was corresponded well with the predicted value of 91.81%. In addition, to investigate the transport properties of HEP and HEP-PLGA using a Caco-2 cell monolayer, and study the roles of the efflux transporters (P-gp) during the transport process. These results suggested that HEP can be absorbed more efficiently when encapsulated within the PLGA. These findings highlight the potential to the application of HEP in the formulation of functional foods. These results provide strategies in designing high absorbed polysaccharides with bioactive benefits.


Assuntos
Basidiomycota/química , Polissacarídeos Fúngicos , Ácido Láctico , Nanopartículas/química , Ácido Poliglicólico , Células CACO-2 , Polissacarídeos Fúngicos/química , Polissacarídeos Fúngicos/farmacocinética , Polissacarídeos Fúngicos/farmacologia , Humanos , Ácido Láctico/química , Ácido Láctico/farmacocinética , Ácido Láctico/farmacologia , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacocinética , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
15.
Biochem Mol Biol Educ ; 46(4): 354-360, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29717550

RESUMO

Here, we developed an integrated course based on two dimensional-electrophoresis and spectrometry mass (2DE-MS) technique for undergraduate students to help them learn proteomic techniques. The soluble proteins in wild type and gene knockout bacteria were separated by 2DE and the differently expressed proteins were identified by MS analysis. The proteomic data was finally confirmed by RT-PCR detection. The separated experiments of 2DE, MS, RNA isolation, RT-PCR, as well as essential bioinformatic analysis, were integrated into a one-week course, which provided students an opportunity to systematically understand the proteomic techniques and their applications in current scientific research. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:354-360, 2018.


Assuntos
Eletroforese em Gel Bidimensional , Espectrometria de Massas , Aprendizagem Baseada em Problemas , Proteômica/educação , Proteômica/métodos , Estudantes/psicologia , Ensino , Humanos , Laboratórios , Proteínas/análise , Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Universidades
16.
Molecules ; 23(2)2018 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-29401644

RESUMO

(1) Background: d-alanine-d-alanine ligase (DdlA), an effective target for drug development to combat against Mycobacterium tuberculosis (Mtb), which threatens human health globally, supplies a substrate of d-alanyl-d-alanine for peptidoglycan crosslinking by catalyzing the dimerization of two d-alanines. To obtain a better understanding of DdlA profiles and develop a colorimetric assay for high-throughput inhibitor screening, we focused on explicating and characterizing Tb-DdlA. (2) Methods and Results: Rv2981c (ddlA) was expressed in Escherichia coli, and the purified Tb-DdlA was identified using (anti)-polyhistidine antibody followed by DdlA activity confirmation by measuring the released orthophosphate via colorimetric assay and the yielded d-alanyl-d-alanine through high performance thin layer chromatography (HP-TLC). The kinetic assays on Tb-DdlA indicated that Tb-DdlA exhibited a higher affinity to ATP (KmATP: 50.327 ± 4.652 µmol/L) than alanine (KmAla: 1.011 ± 0.094 mmol/L). A colorimetric assay for Tb-DdlA activity was developed for high-throughput screening of DdlA inhibitors in this study. In addition, we presented an analysis on Tb-DdlA interaction partners by pull-down assay and MS/MS. Eight putative interaction partners of Tb-DdlA were identified. (3) Conclusions: Our dataset provided a valuable resource for exploring Tb-DdlA biology, and developed an easy colorimetric assay for screening of Tb-DdlA inhibitors.


Assuntos
Trifosfato de Adenosina/metabolismo , Alanina/metabolismo , Proteínas de Bactérias/metabolismo , Dipeptídeos/metabolismo , Mycobacterium tuberculosis/enzimologia , Peptídeo Sintases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Bioensaio , Clonagem Molecular , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Mycobacterium tuberculosis/genética , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/genética , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
17.
Oncotarget ; 8(53): 90879-90896, 2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29207610

RESUMO

Long-term exposure to arsenic has been linked to tumorigenesis in different organs and tissues, such as skin; however, the detailed mechanism remains unclear. In this present study, we integrated "omics" including microRNAome, proteomics and metabolomics to investigate the potential molecular mechanisms. Compared with non-malignant human keratinocytes (HaCaT), twenty-six miRNAs were significantly altered in arsenic-induced transformed cells. Among these miRNAs, the differential expression of six miRNAs was confirmed using Q-RT-PCR, representing potential oxidative stress genes. Two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MS) were performed to identify the differential expression of proteins in arsenic-induced transformed cells, and twelve proteins were significantly changed. Several proteins were associated with oxidative stress and carcinogenesis including heat shock protein beta-1 (HSPB1), peroxiredoxin-2 (PRDX2). Using ultra-performance liquid chromatography and Q-TOF mass spectrometry (UPLC/Q-TOF MS), 68 metabolites including glutathione, fumaric acid, citric acid, phenylalanine, and tyrosine, related to redox metabolism, glutathione metabolism, citrate cycle, met cycle, phenylalanine and tyrosine metabolism were identified and quantified. Taken together, these results indicated that arsenic-induced transformed cells exhibit alterations in miRNA, protein and metabolite profiles providing novel insights into arsenic-induced cell malignant transformation and identifying early potential biomarkers for cutaneous squamous cell carcinoma induced by arsenic.

18.
Stem Cell Reports ; 9(6): 1948-1960, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29198824

RESUMO

Accumulating evidence suggests that cancer cells with stem cell-like phenotypes drive disease progression and therapeutic resistance in glioblastoma (GBM). NOTCH regulates self-renewal and resistance to chemoradiotherapy in GBM stem cells. However, NOTCH-targeted γ-secretase inhibitors (GSIs) exhibited limited efficacy in GBM patients. We found that farnesyltransferase inhibitors (FTIs) significantly improved sensitivity to GSIs. This combination showed significant antineoplastic and radiosensitizing activities in GBM stem cells, whereas non-stem GBM cells were resistant. These combinatorial effects were mediated, at least partially, through inhibition of AKT and cell-cycle progression. Using subcutaneous and orthotopic GBM models, we showed that the combination of FTIs and GSIs, but not either agent alone, significantly reduced tumor growth. With concurrent radiation, this combination induced a durable response in a subset of orthotopic tumors. These findings collectively suggest that the combination of FTIs and GSIs is a promising therapeutic strategy for GBM through selectively targeting the cancer stem cell subpopulation.


Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Inibidores Enzimáticos/administração & dosagem , Farnesiltranstransferase/genética , Glioblastoma/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Autorrenovação Celular/efeitos dos fármacos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Farnesiltranstransferase/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Camundongos , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Tolerância a Radiação/efeitos dos fármacos , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Biomed Rep ; 7(6): 567-572, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29188061

RESUMO

Coriolus versicolor (CV) contains high levels of bioactive compounds, including the glucan (1→6)-α-D-glucopyranosyl. However, there is a lack of data regarding the potential effect of this CV glucan (CVG) on the stimulation of cytokine production. The present study evaluated the effect of CVG on the stimulation of cytokine production in sarcoma-180-bearing mice. Mice were treated with three doses of CVG (40, 100 or 200 mg/kg body weight) for nine days, after which serum levels of cytokines, namely interleukin (IL)-2, -4, -6, -10, -17A and interferon (IFN)-α and -γ, were investigated by ELISA. CVG significantly promoted the secretion of IL-2, -4, -6, -10, -17A and IFN-α and -γ at the doses of 100 (P<0.05) and 200 (P<0.01) mg/kg, but not at 40 mg/kg (P>0.05), when compared with cyclophosphamide treatment, as a positive control. Additionally, cytokine production associated with T helper (Th)2 and Th17 cells was enhanced compared with that of Th1 cytokines, and the immunomodulatory function of CVG appeared to be IL-10-dependent. These results demonstrate that CVG may stimulate the production of cytokines and serve as a Th2/IL-10-dependent immunomodulator, and thus has promise in supporting cancer therapies.

20.
Artigo em Inglês | MEDLINE | ID: mdl-28884091

RESUMO

Introduction: Human gut microbiota is believed to be directly or indirectly involved in cardiovascular diseases and hypertension. However, the identification and functional status of the hypertension-related gut microbe(s) have not yet been surveyed in a comprehensive manner. Methods: Here we characterized the gut microbiome in hypertension status by comparing fecal samples of 60 patients with primary hypertension and 60 gender-, age-, and body weight-matched healthy controls based on whole-metagenome shotgun sequencing. Results: Hypertension implicated a remarkable gut dysbiosis with significant reduction in within-sample diversity and shift in microbial composition. Metagenome-wide association study (MGWAS) revealed 53,953 microbial genes that differ in distribution between the patients and healthy controls (false discovery rate, 0.05) and can be grouped into 68 clusters representing bacterial species. Opportunistic pathogenic taxa, such as, Klebsiella spp., Streptococcus spp., and Parabacteroides merdae were frequently distributed in hypertensive gut microbiome, whereas the short-chain fatty acid producer, such as, Roseburia spp. and Faecalibacterium prausnitzii, were higher in controls. The number of hypertension-associated species also showed stronger correlation to the severity of disease. Functionally, the hypertensive gut microbiome exhibited higher membrane transport, lipopolysaccharide biosynthesis and steroid degradation, while in controls the metabolism of amino acid, cofactors and vitamins was found to be higher. We further provided the microbial markers for disease discrimination and achieved an area under the receiver operator characteristic curve (AUC) of 0.78, demonstrating the potential of gut microbiota in prediction of hypertension. Conclusion: These findings represent specific alterations in microbial diversity, genes, species and functions of the hypertensive gut microbiome. Further studies on the causality relationship between hypertension and gut microbiota will offer new prospects for treating and preventing the hypertension and its associated diseases.


Assuntos
Bactérias/classificação , Disbiose/microbiologia , Microbioma Gastrointestinal/genética , Trato Gastrointestinal/microbiologia , Hipertensão/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Fezes/microbiologia , Feminino , Humanos , Masculino , Metagenoma/genética , Pessoa de Meia-Idade , Sequenciamento Completo do Genoma
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