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1.
Pharmacol Res ; : 104884, 2020 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-32428667

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible lung disease with limited therapeutic strategies. Lycorine (LYC), an alkaloid isolated from Amaryllidaceae family plants, exhibits effective anti-inflammatory, antiviral, and anti-tumor activities. In this study, we attempted to determine the effect of LYC on bleomycin (BLM)-induced IPF and NLRP3 inflammasome activation. Our results demonstrated that the LYC treatment ameliorated BLM-induced pulmonary fibrosis and inflammation in mice. LYC inhibited active Caspase-1 expression and lactate dehydrogenase (LDH) release during BLM-induced acute lung injury (ALI) in mice. Furthermore, our in vitro assay showed that LYC inhibited LPS/Nigericin- or LPS/ATP-induced NACHT, LRP and PYD domains-containing protein 3 (NLRP3) inflammasome activation, and pyroptosis in bone marrow-derived macrophages (BMDMs). Mechanically, LYC could disturb the interaction of NLRP3 with apoptosis-associated speck-like protein containing a CARD (ASC) by targeting the pyrin domain (PYD) on Leu9, Leu50, and Thr53. Our findings indicate that LYC ameliorated BLM-induced pulmonary fibrosis by inhibiting NLRP3 inflammasome activation and pyroptosis through targeting the PYD domain of ASC. Thus, LYC might be a potential therapeutic agent for pulmonary inflammation and fibrosis.

2.
Microb Pathog ; 145: 104240, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32360522

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen that is highly resistant to antibiotics, especially when it grows in biofilms. As an alternative to antibiotic intervention, antimicrobial antibody drugs have drawn attention in recent years due to their immunotherapeutic functions. In this study, we designed a monoclonal scFv-Fc-form antibody, MFb, targeting P. aeruginosa antigen alginate and investigated its function against this bacterium in vitro. MFb was generated by transient gene expression in HEK293 cells and purified by one-step protein A affinity chromatography. Experiments showed that MFb could recognize alginate specifically based on enzyme-linked immunosorbent assays. Its KD value of 8.31 nM was determined by surface plasmon resonance, demonstrating its high affinity for alginate. Further detailed studies revealed that the antibody exerted antibacterial effects by three mechanisms: 1) MFb inhibited P. aeruginosa biofilm formation with an IC50 of 0.58 µg/mL; 2) MFb reduced P. aeruginosa adhesion to HeLa cells, and successfully prevented its invasion on epithelial cells; 3) based on an in vitro macrophage phagocytosis assay, MFb could enhance the phagocytotic capacity of macrophages for P. aeruginosa in a concentration-dependent manner. Taken together, our work demonstrated that the antimicrobial monoclonal antibody MFb has a protective effect on HeLa cells, and it may be a promising novel strategy to treat P. aeruginosa infection.

3.
Biomed Res Int ; 2020: 3548618, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32382546

RESUMO

Worldwide interest in the use of functional foods containing probiotic bacteria such as Lactobacillus and Bifidobacterium for health promotion and disease prevention has increased significantly. Probiotics have demonstrated beneficial properties including strengthening the body's natural defense system, inhibiting the growth of pathogenic bacteria, and regulating mental activity, but their effects on the human vagina have not been fully elucidated. The primary purpose of our study was to isolate Lactobacillus strains from old yogurt, a traditional dairy product, and investigate their probiotic potential with respect to the human vaginal system. Four Lactobacillus plantarum (L. plantarum) strains, named ZX1, ZX2, ZX27, and ZX69, were isolated from the yogurt samples. Simultaneously, we used a commercial Lactobacillus strain (Lactobacillus delbrueckii DM8909) as a control strain. We tested the antimicrobial activity of Lactobacillus isolates against Escherichia coli and Gardnerella vaginalis by agar spot and well diffusion tests. Then, we tested the antibiotic susceptibility of the 5 strains by using the minimal inhibitory concentration method. We attempted to detect possible bacteriocin genes by PCR sequencing technique. Using a chemically defined medium simulating genital tract secretions, we found that the selected Lactobacillus strains could alter the expression of known virulence genes in Gardnerella vaginalis. Bacteriocins derived from these isolated strains had potent antibacterial activity against G. vaginalis and E. coli, with the most effective activity observed in the case of ZX27. In addition, all strains including the L. delbrueckii DM8909 were positive for the presence of the plantaricin cluster of genes described in L. plantarum C11. The tested stains possessed the pln gene indicating that one of the antibacterial agents was plantaricin. We assume that the production of antimicrobial substances such as bacteriocins induce G. vaginalis to upregulate antimicrobial resistance genes. The new isolated strains have bacteriocin-related genes and can change the antimicrobial resistance gene transcription of G. vaginalis.

4.
Org Biomol Chem ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32412572

RESUMO

Nosiheptide (NOS) is a member of bicyclic thiopeptides possessing a biologically important indolic acid (IA) moiety appended onto the family-characteristic core system. The IA formation relies primarily on NosL, a radical S-adenosylmethionine (SAM) protein that catalyzes a complex rearrangement of the carbon side chain of l-tryptophan, leading to the generation of 3-methyl-2-indolic acid (MIA). Here, we establish an efficient mutational biosynthesis strategy for the structural expansion of the side-ring system of NOS. The nosL-deficient mutant Streptomyces actuosus SL4005 complemented by chemically feeding 6-fluoro-MIA is capable of accumulating two new products. The target product 6'-fluoro-NOS contains an additional fluorine atom at C6 of the IA moiety, in contrast with an unexpected product 6'-fluoro-NOSint that features an open side ring and a bis-dehydroalanine (Dha) tail. The newly obtained 6'-fluoro-NOS displayed equivalent or slightly reduced activities against the tested drug-resistant pathogens compared with NOS, but dramatically decreased water solubility compared with NOS. Our results indicate that the modification of the IA moiety of NOS not only affects its biological activity but also affects its activity which will be key considerations for further modification.

5.
mSphere ; 5(2)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238571

RESUMO

Colistin is used as the "last resort" to treat infections caused by multidrug-resistant Acinetobacter baumannii, which is at the top of the World Health Organization's list of the most dangerous bacterial species that threaten human health. Unfortunately, colistin resistance has emerged in A. baumannii To broaden the study of the resistance mechanism of colistin in A. baumannii, we obtained colistin-resistant mutants via two methods: (i) screening and isolation from a mariner-based A. baumannii ATCC 19606 transposon mutant library; (ii) selection from challenge of ATCC 19606 with successively increasing concentrations of colistin. A total of 41 mutants with colistin MIC of 4 µg/ml to 64 µg/ml were obtained by transposon mutant library screening. Five highly resistant mutants with colistin MICs ranging from 256 µg/ml to 512 µg/ml were selected from successive colistin challenges. Genotypic complementation and remodeling of the transposon mutants revealed that the genes inactivated by the transposon insertion were not responsible for resistance. Whole-genome sequence analysis of the colistin-resistant strains revealed that the main causes of the resistance to colistin were mutations in the pmrA-pmrB genes, including pmrA P102R, pmrB P233S, and pmrB T235N and the novel alleles pmrA I13M and pmrB Q270P Interestingly, we found that miaA I221V mutation of A. baumannii strain ATCC 19606 (pmrA P102R) resulted in 4-fold increases in the colistin MIC, which rose from 32 µg/ml to 128 µg/ml. But miaA I221V itself had little effect on the colistin susceptibility of ATCC 19606. These data broaden knowledge of the scope of chromosomally encoded mechanisms of resistance to colistin.IMPORTANCE Acinetobacter baumannii is an important Gram-negative opportunistic pathogen commonly infecting critically ill patients. It possesses a remarkable ability to survive in the hospital environment and acquires resistance determinants corresponding to a wide range of antibacterial agents. Given that the current treatment options for multidrug resistant A. baumannii are extremely limited, colistin administration has become the treatment of last resort. However, colistin-resistant A. baumannii strains have recently been reported. The mechanism of resistance to colistin in A. baumannii has rarely been reported. Here, we found two novel mutations in pmrA (I13M) and pmrB (Q270P) that caused colistin resistance. It is also first reported here that the presence of miaA with a I221V mutation enhanced the colistin resistance of pmrA P102R.

6.
J Environ Manage ; 263: 110323, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32174515

RESUMO

Metal tailings are potential sources of strong environmental pollution. In situ remediation involves the installation of a plant cover to stabilize materials and pollutants. Whether metal(loid)s are effectively immobilized in remediated tailing ponds submitted to heavy rainfall remains uncertain. In this study, tailing materials were collected from bare tailings (control), grass-planted (G) and grass-shrub planted (GS) areas on a former Pb/Zn mine site. Batch column experiments were performed with three rainfall intensities of 0.36, 0.48, and 0.50 mL min-1 for 18 d in the lab. The pH, Eh, Cd, Pb, Zn and As concentration in leachate were recorded. Selected leached tailing materials were finally characterized. Results showed that leachates from control were strongly acidic (pH 3.11-4.65), and that Cd, Pb, Zn and As were quickly released at high rate (e.g., 945 mg L-1 Zn). During the experiment up to 4% Cd present in the material was released and almost 1% Zn. With material collected from the G area, leachates were even more acidic (2.16-2.84) with a rainfall intensity of 0.50 mL min-1 and exhibited a high redox potential (588-639 mV). However, concentrations of metals in leachates were much lower than that in the control, except for Zn (e.g., 433 mg L-1), and they tended to decrease with time. Cumulative leaching rate was still relatively high (e.g., 0.68% Cd; 0.75% Zn) during the first eight days (stage I). However, with the GS treatment, leachate pH gradually raised from acid to alkaline values (3.9-8.2) during stage I, then remained high until the end of the experiment (stage II). Also, amounts of elements released during the 18 d were low in general. The releasing ratios of Cd (R2 > 0.95), Pb (R2 > 0.95), As (R2 > 0.87), and Zn (R2 > 0.90) fitted well with a two-constant model. In conclusion, under subtropical climate with heavy rainfall, phytostabilization is effective but immobilization of metals is higher with a combination of grass and shrub than with only grass to reduce leaching of As and Zn.

7.
Angew Chem Int Ed Engl ; 59(9): 3658-3664, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-31868285

RESUMO

With the ever-increasing threat posed by the multi-drug resistance of bacteria, the development of non-antibiotic agents for the broad-spectrum eradication of clinically prevalent superbugs remains a global challenge. Here, we demonstrate the simple supramolecular self-assembly of structurally defined graphene nanoribbons (GNRs) with a cationic porphyrin (Pp4N) to afford unique one-dimensional wire-like GNR superstructures coated with Pp4N nanoparticles. This Pp4N/GNR nanocomposite displays excellent dual-modal properties with significant reactive-oxygen-species (ROS) production (in photodynamic therapy) and temperature elevation (in photothermal therapy) upon light irradiation at 660 and 808 nm, respectively. This combined approach proved synergistic, providing an impressive antimicrobial effect that led to the complete annihilation of a wide spectrum of Gram-positive, Gram-negative, and drug-resistant bacteria both in vitro and in vivo. The study also unveils the promise of GNRs as a new platform to develop dual-modal antimicrobial agents that are able to overcome antibiotic resistance.

8.
Biol Trace Elem Res ; 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31845206

RESUMO

Selenium (Se) is one of the essential elements required to maintain human health. Although various kinds of Se supplements are now available on the market, their biological activities and toxicities vary based on the transportation characteristics of Se. In this study, we compared the absorption and distribution of Se in rats administered with different Se supplements: Se-enriched Bifidobacterium longum DD98 (Se-DD98), selenized yeast (Se-Y), and sodium selenite (Na2SeO3). Se-DD98, Se-Y, and Na2SeO3 were orally administered to rats. The plasma Se content at different time points after administration was determined within 72 h. Pharmacokinetic parameters were analyzed to reveal the absorption of Se. Se-DD98, Se-Y, and Na2SeO3 were also repeatedly administered by oral gavage for 30 days, and Se content of the heart, liver, spleen, lungs, kidneys, and muscle was determined to analyze the distribution of Se. The results showed that the organic Se supplements (Se-DD98 and Se-Y) were more easily absorbed into the blood and retained longer in the plasma than the inorganic Na2SeO3 was. Moreover, Se-DD98 induced better absorption of Se in plasma than Se-Y did. Furthermore, significantly higher concentrations of Se were found in the heart, liver, spleen, kidneys, and muscle of rats administered with organic Se supplements (Se-DD98 and Se-Y) than those administered the inorganic Na2SeO3. Rats administered Se-DD98 accumulated more Se in the spleen, lung, and kidney than those administered Se-Y, while Se-Y led to higher concentration of Se in the liver compared to Se-DD98. These results suggest that the organic form of Se was better absorbed and accumulated than the inorganic form was. Se-enriched B. longum DD98 induced greater absorption of Se in plasma and accumulation of Se in several organs than the selenized yeast did, which could suggest the potential superior nutritional function of Se-DD98.

9.
J Mater Chem B ; 7(45): 7141-7151, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31663577

RESUMO

Triple-negative breast cancer (TNBC) is characterized by a high metastatic rate, which can seriously threaten women's health. ROS play an important role in tumor development and metastasis. Excessive ROS can induce tumor cell apoptosis and inhibit tumor cell metastasis. This study investigated Fenton-reaction-stimulative nanoparticles (P@P/H NPs) containing ROS-responsive molecular switches for antitumor metastasis by amplifying the ROS and activating the cascade biological reaction of ROS in tumor cells. Spheroidal P@P/H NPs exhibited a uniform size of 68.18 ± 0.29 nm, high drug cumulative release of 97.59% in response to H2O2 at 24 h, and satisfactory cytotoxicity with the IC50 value of 0.50 ± 0.02 µg mL-1. The markedly elevated ROS level caused by P@P/H NPs generated an evident antitumor metastasis effect in vitro by facilitating the expressions of cytochrome c, caspase-9, and caspase-3 and blocking that of matrix metalloprotein 9 (MMP-9). Moreover, P@P/H NPs engendered an excellent tumor inhibition rate of 56.37% and antitumor metastasis effect in vivo. Therefore, P@P/H NPs could respond to H2O2 in tumor cells to rapidly disassemble and further increase the ROS to induce antitumor metastasis via the Fenton reaction.

10.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1174-1183, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328474

RESUMO

With the rapid development of antibody genetic engineering, bispecific antibody technology has been advanced. They are capable of binding two or more different epitopes simultaneously, thus offering specific advantages over natural monoclonal antibodies in immunotherapy. Bispecific antibodies have been successfully used in cancer therapy (e.g. melanoma, Hodgkin's lymphoma, liver cancer, and stomach cancer) and inflammation therapy (e.g. rheumatoid arthritis, psoriasis and Crohn's disease), but are still in their early stage for viral immunotherapy. In this study, we reviewed the research progress of bispecific antibodies for immunotherapy of virus infections, especially those with good effects in vivo and in vitro, to provide references for the research and development of bispecific antibodies for antivirus treatment.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Viroses , Anticorpos Monoclonais , Epitopos , Humanos , Imunoterapia
11.
Front Microbiol ; 10: 1389, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31316479

RESUMO

Bifidobacteria are considered one of the most important intestinal probiotics because of their significant health impact. However, this ability is usually limited by gastrointestinal fluid and temperature sensitivity. Emulsification and internal gelation is an encapsulation technique with great potential for probiotic protection during storage and the gastrointestinal transit process. This study prepared microcapsules using an emulsification and internal gelation encapsulation method with sodium alginate, chitosan, and Bifidobacterium longum as wall material, coating material, and experimental strain, respectively. Optical, scanning electron, and focal microscopes were used to observe the microcapsule surface morphology and internal viable cell distribution, and a laser particle size analyzer and zeta potentiometer were used to evaluate the chitosan-coating characteristics. In addition, microcapsule probiotic viability after storage, heat treatment, and simulated gastrointestinal fluid treatment were examined. Alginate microcapsules and chitosan-coated alginate microcapsules both had balling properties and uniform bacterial distribution. The latter kept its balling properties after freeze-drying, verified by scanning electronic microscopy (SEM), and had a clear external coating, observed by an optical microscope. The particle size of chitosan-coated alginate microcapsules was slightly larger than the uncoated microcapsules. The zeta potential of alginate and chitosan-coated alginate microcapsules was negative and positive, respectively. Heat, acid and bile salt tolerance, and stability tests revealed that the decrease of viable cells in the chitosan-coated alginate microcapsule group was significantly lower than that in uncoated microcapsules. These experimental results indicate that the chitosan-coated alginate microcapsules protect B. longum from gastrointestinal fluid and high-temperature conditions.

12.
Food Funct ; 10(8): 4975-4984, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31343650

RESUMO

The aim of this study was to investigate the characteristics of a novel selenium-enriched Bifidobacterium longum DD98 (Se-B. longum DD98) supplement food and its repairing effects on the intestinal ecology of mammals. We assessed the growth, Se accumulation, and Se biotransformation of B. longum DD98 and its effects on antibiotic-induced intestinal dysbacteriosis in mice. The viable bacterial count at the end of fermentation was not significantly affected by the presence of Se. Bifidobacterium longum DD98 took up inorganic Se from the medium and biotransformed it into Se-containing proteins and selenoamino acids. The dominant Se species was selenomethionine (SeMet), which comprised 87% of the total Se in Se-B. longum DD98. Furthermore, Se-B. longum DD98 showed better regulation of the disrupted intestinal microbiota back to normal levels and repaired damaged colon tissues compared to the natural recovery and B. longum DD98 treatments. These findings suggest that B. longum DD98 efficiently biotransformed inorganic Se into more bioactive organic Se forms and may have therapeutic potential for the restoration of antibiotic-induced intestinal dysbacteriosis.


Assuntos
Antibacterianos/efeitos adversos , Bifidobacterium longum/química , Disbiose/tratamento farmacológico , Intestinos/microbiologia , Probióticos/administração & dosagem , Selênio/análise , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bifidobacterium longum/crescimento & desenvolvimento , Bifidobacterium longum/metabolismo , Biotransformação , Disbiose/etiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Probióticos/análise , Selênio/metabolismo
13.
Reprod Domest Anim ; 54(6): 882-891, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974481

RESUMO

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus-induced mastitis. In the present study, we overexpressed and suppressed miR-145 to investigate the function of miR-145 in Mac-T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac-T cytokines and in cell proliferation. We found that overexpression of miR-145 in Mac-T cells significantly reduced the secretion of IL-12 and TNF-α, but increased the secretion of IFN-γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real-time PCR (qRT-PCR), Western blotting and luciferase multiplex verification techniques, we found that miR-145 targeted and regulated FSCN1. Knock-down of FSCN1 significantly increased the secretion of IL-12, while the secretion of TNF-α was significantly downregulated in Mac-T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta-miR-145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.


Assuntos
Proteínas de Transporte/metabolismo , Mastite Bovina/imunologia , MicroRNAs/metabolismo , Proteínas dos Microfilamentos/metabolismo , Infecções Estafilocócicas/veterinária , Animais , Proteínas de Transporte/genética , Bovinos , Linhagem Celular , Proliferação de Células , Citocinas/metabolismo , Células Epiteliais/fisiologia , Feminino , Proteínas dos Microfilamentos/genética , Infecções Estafilocócicas/imunologia , Staphylococcus aureus
14.
Artigo em Inglês | MEDLINE | ID: mdl-30745385

RESUMO

Colistin-based combination therapy has become an important strategy to combat the carbapenem-resistant Acinetobacter baumannii (CRAB). However, the optimal dosage regimen selection for the combination with maximum efficacy is challenging. Checkerboard assay was employed to evaluate the synergy of colistin in combination with meropenem, rifampin, fosfomycin, and minocycline against nine carbapenem-resistant A. baumannii isolates (MIC of meropenem [MICMEM], ≥32 mg/liter) isolated from Chinese hospital-acquired pneumonia (HAP) patients. A static time-kill assay, in vitro dynamic pharmacokinetic/pharmacodynamic (PK/PD) model, and semimechanistic PK/PD modeling were conducted to predict and validate the synergistic effect of the most efficacious combination. Both checkerboard and static time-kill assays demonstrated the superior synergistic effect of the colistin-meropenem combination against all CRAB isolates. In the in vitro PK/PD model, the dosage regimen of 2 g meropenem daily via 3-h infusion combined with steady-state 1 mg/liter colistin effectively suppressed the bacterial growth at 24 h with a 2-log10 decrease, compared with the initial inocula against two CRAB isolates. The semimechanistic PK/PD model predicted that more than 2 mg/liter colistin combined with meropenem (2 g, 3-h infusion) was required to achieve the killing below the limit of detection (

15.
Nanomedicine (Lond) ; 14(4): 447-464, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30694105

RESUMO

AIM: Enzyme CYP1B1 (CYP1B1) is usually overexpressed in multidrug resistance (MDR) breast cancer cells, which could metabolically inactivate docetaxel (DTX). MATERIALS & METHODS: The cationic core-shell nanoparticles (hyaluronic acid/polyethyleneimine nanoparticles [HA/PEI NPs]) modified with hyaluronic acid (HA) were developed and coloaded with DTX and α-napthtoflavone (ANF, a CYP1B1 inhibitor) to overcome MDR in breast cancer induced by CYP1B1. Physicochemical characterization, MDR reversing effect in vitro and pharmacokinetics in vivo of HA/PEI NPs were evaluated. RESULTS: The HA/PEI NPs exhibited spherical morphology with size of (193.6 ± 3.1) nm. The HA/PEI NPs could reverse MDR effectively by downregulating the expression of CYP1B1. The HA/PEI NPs improved the bioavailability of DTX. CONCLUSION: The HA/PEI NPs might be a promising strategy to overcome CYP1B1-mediated breast cancer MDR.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/uso terapêutico , Nanopartículas/química , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Microscopia Confocal , Ratos Sprague-Dawley
16.
Nat Commun ; 10(1): 258, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651565

RESUMO

Antimicrobial resistance is a public health emergency and warrants coordinated global efforts. Challenge is that no alternative molecular platform has been identified for discovery of abundant antimicrobial hit compounds. Xanthene libraries have been screened for bioactive compounds. However, the potentially accessible chemistry space of xanthene dyes is limited by the existing xanthene synthesis. Herein we report a mild one-step synthesis, which permits late-stage introduction of a xanthene moiety onto i.e. natural products, pharmaceuticals, and bioactive compounds and construction of a focused library of rhodamine dyes exhibiting facile functional, topographical and stereochemical diversity. In vitro screening yields 37 analogs with mid-to-high bactericidal activity against WHO priority drug-resistant pathogens. These findings suggest that synthetic dye libraries exhibiting high structural diversity is a feasible chemical space combating antibacterial resistance, to complement the natural sources.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Rodaminas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antibacterianos/síntese química , Bactérias/genética , Bactérias/ultraestrutura , Produtos Biológicos/síntese química , Produtos Biológicos/farmacologia , Técnicas de Química Combinatória , Desenvolvimento de Medicamentos/métodos , Farmacorresistência Bacteriana/genética , Eritrócitos , Estudos de Viabilidade , Genoma Bacteriano/efeitos dos fármacos , Genoma Bacteriano/genética , Células HeLa , Hemólise/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Estrutura Molecular , Rodaminas/síntese química , Ovinos , Bibliotecas de Moléculas Pequenas/síntese química , Sequenciamento Completo do Genoma
17.
Biotechnol J ; 13(9): e1700588, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30039929

RESUMO

Mycobacterium smegmatis is an important model strain of Mycobacterium for scientific study because it is non-pathogenic and grows rapidly. However, research is limited by the low efficiency and time-consuming nature of existing genome editing tools. Although the Streptococcus pyogenes CRISPR-Cas9 system is widely used in bacterial genome editing, it cannot be introduced into M. smegmatis because of its toxicity. The authors test 14 different Cas effector proteins in M. smegmatis. Cas9 (TdCas9_m) from Treponema denticola, Cas9 (NmCas9) from Neisseria meningitidis, and Corynebacterium glutamicum codon-optimized Cpf1 (FnCpf1_cg) from Francisella tularensis do not affect cell growth. The numbers of transformant plasmids expressing TdCas9_m, NmCas9, or FnCpf1_cg, and guide RNAs (gRNA) targeting ku(MSMEG_5580), ligD(MSMEG_6301), pta(MSMEG_0783), or ackA(MSMEG_0784) decreases by about 10-, 10-, or 100-fold, respectively, compared with plasmids expressing only the Cas effector proteins. Non-homologous end joining (NHEJ) is detected only in the CRISPR-FnCpf1_cg system. The one-plasmid-based, CRISPR-FnCpf1-assisted NHEJ system enables N iterative rounds of genome editing in 7N + 2 days, with an editing efficiency up to 70%; thus, this system should greatly reduce the necessary genome manipulation time for M. smegmatis.


Assuntos
Reparo do DNA por Junção de Extremidades , Edição de Genes/métodos , Mycobacterium smegmatis/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Corynebacterium glutamicum/genética , Genoma Bacteriano
18.
Front Immunol ; 9: 1503, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30008720

RESUMO

Induced expression of serum amyloid A (SAA) is a hallmark of many inflammatory diseases, but whether SAA exacerbates inflammation or protects tissues against injury remains unclear. In dextran sulfate sodium (DSS)-induced colitis, SAA3 is the predominant isoform of inducible SAA proteins that also include SAA1 and SAA2, and mice with genetic deletion of Saa3 exhibits increased production of proinflammatory cytokines, decreased expression of IL-22 along with aggravated epithelium disruption, and reduced colon length compared with wild-type littermates. Colonic neutrophils have been identified as a major source of IL-22 in these mice. Administration of exogenous SAA3 as recombinant protein to Saa3-/- mice improves neutrophil IL-22 production, colonic epithelial integrity, and secretion of the antimicrobial peptides Reg3ß and Reg3γ. Stimulation of mouse bone marrow neutrophils with mouse SAA3 or human SAA1 leads to expansion of IL-22-producing neutrophils. Unlike previously reported IL-22 induction through IL-23, the SAA3-induced neutrophil IL-22 expression utilizes a TLR2-dependent mechanism that does not depend on IL-23. Adoptive transfer of the SAA3-treated neutrophils to Saa3-/- mice ameliorates DSS-induced colitis and improves colonic epithelial integrity. These findings suggest that in the DSS-induced mouse colitis model, SAA isoforms are expressed to different extent in colon and deletion of Saa3 renders these mice more susceptible to DSS-induced injury. The presence of SAA3 in the inflamed colon mucosal serves to protect epithelial barrier in part through expansion of IL-22-producing neutrophils. It is speculated that SAA3 stimulation of autologous neutrophils may have therapeutic potential for inflammatory bowel disease.

19.
Free Radic Biol Med ; 123: 1-7, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29709704

RESUMO

Nitric oxide (NO) donors are valuable tools to probe the profound implications of NO in health and disease. The elusive nature of NO bio-relevance has largely limited the use of spontaneous NO donors and promoted the development of next generation NO donors, whose NO release is not only stimulated by a trigger, but also readily monitored via a judiciously built-in self-calibration mechanism. Light is without a doubt the most sensitive, versatile and biocompatible method of choice for both triggering and monitoring, for applications in complex biological matrices. Herein, we designed and synthesized an N-nitroso rhodamine derivative (NOD560) as a photo-triggered and photo-calibrated NO donor to address this need. NOD560 is essentially non-fluorescent. Upon irradiation by green light (532 nm), it efficiently release NO and a rhodamine dye, the dramatic fluorescence turn-on from which could be harnessed to conveniently monitor the localization, flux, and dose of NO release. The potentials of NOD560 for in vitro biological applications were also exemplified in in vitro biological models, i.e. mesenchymal stem cell (MSC) migration suppression. NOD560 is expected to complement the existing NO donors and find widespread applications in chemical biological studies.


Assuntos
Movimento Celular , Células-Tronco Mesenquimais/metabolismo , Doadores de Óxido Nítrico/química , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Processos Fotoquímicos , Calibragem , Células Cultivadas , Desenho de Fármacos , Fluorescência , Corantes Fluorescentes , Células HeLa , Humanos , Luz , Células-Tronco Mesenquimais/citologia
20.
Bioconjug Chem ; 29(4): 1194-1198, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29498825

RESUMO

Nitric oxide (NO) is a versatile endogenous molecule, involved in various physiological processes and implicated in the progression of many pathological conditions. Therefore, NO donors are valuable tools in NO related basic and applied applications. The traditional spontaneous NO donors are limited in scenarios where flux, localization, and dose of NO could be monitored. This has promoted the development of novel NO donors, whose NO release is not only under control, but also self-calibrated. Herein, we reported a phototriggered and photocalibrated NO donor (NOD565) with an N-nitroso group on a rhodamine dye. NOD565 is nonfluorescent and could release NO efficiently upon irradiation by green light. A bright rhodamine dye is generated as a side-product and its fluorescence can be used to monitor the NO release. The potentials of NOD565 in practical applications are showcased in in vitro studies, e.g., platelet aggregation inhibition and fungi growth suppression.


Assuntos
Doadores de Óxido Nítrico/química , Doadores de Óxido Nítrico/farmacologia , Processos Fotoquímicos , Raios Ultravioleta , Anti-Infecciosos/farmacologia , Calibragem , Fluorescência , Óxido Nítrico/química , Inibidores da Agregação de Plaquetas/farmacologia , Rodaminas/química , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Água/química
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