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1.
Wiad Lek ; 73(8): 1615-1619, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33055321

RESUMO

OBJECTIVE: The aim: To perform microbiological investigation and analytic mathematic prediction of clinical isolates of S. aureus to aminoglycosides in patients with severe burns. PATIENTS AND METHODS: Materials and methods: We analyzed resistance of 199 S. aureus strains to aminoglycosides (gentamicin, tobramycin and amikacin) and doxycycline from 435 patients treated in the regional hospital due to burns for the period from 2011-2017. RESULTS: Results: We created predictive curves for the prediction of susceptibility of S. aureus strains to aminoglycosides and doxycycline based on the changes in S. aureus resistance during the years of observation and expressed in mathematic equations. Susceptibility of S. aureus to gentamicin was 42.86 % at the end of observation and will decline in future. Despite tobramycin was efficient against 72.86 % of strains in 2017, mathematic modeling indicates rapid decline in its efficacy in future. Efficacy of amikacin was dropping during the last years, but according to the equation it efficacy will increase over 60 % in 2018. S. aureus susceptibility to doxycycline was 65.38 % in 2017 and mathematic modeling indicates its gradual decline in the nearest future. CONCLUSION: Conclusions: Predicitive values of S. aureus susceptibility indicates not sufficient efficacy of these drugs in patients with infectious complications of burns. Tendency of the slight decline of S. aureus susceptibility to doxycycline still indicates sufficient levels of its efficacy in the nearest future. This justify its use as a second-line therapy with the causative agent in patients with burns.


Assuntos
Aminoglicosídeos , Doxiciclina , Aminoglicosídeos/farmacologia , Doxiciclina/farmacologia , Doxiciclina/uso terapêutico , Resistência Microbiana a Medicamentos , Humanos , Prognóstico , Staphylococcus , Staphylococcus aureus
2.
Nat Commun ; 11(1): 4903, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994412

RESUMO

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.


Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas/métodos , Edição de Genes/métodos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína 9 Associada à CRISPR/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia/genética , Recombinação Genética/efeitos dos fármacos , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacos , Transfecção/métodos , Transgenes/genética
3.
Nat Commun ; 11(1): 4061, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792541

RESUMO

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.


Assuntos
Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Animais , Benzofenonas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Doxiciclina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Imunofluorescência , Gastroenteropatias/metabolismo , Gastroenteropatias/patologia , Humanos , Imuno-Histoquímica , Isoxazóis/farmacologia , Lipossomos/metabolismo , Camundongos , Células NIH 3T3 , Parabiose , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologia
4.
OMICS ; 24(10): 568-580, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32757981

RESUMO

Although the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is wreaking havoc and resulting in mortality and morbidity across the planet, novel treatments are urgently needed. Drug repurposing offers an innovative approach in this context. We report here new findings on the in silico potential of several antimalarial drugs for repurposing against COVID-19. We conducted analyses by docking the compounds against two SARS-CoV-2-specific targets: (1) the receptor binding domain spike protein and (2) the main protease of the virus (MPro) using the Schrödinger software. Importantly, the docking analysis revealed that doxycycline (DOX) showed the most effective binding to the spike protein of SARS-CoV-2, whereas halofantrine and mefloquine bound effectively with the main protease among the antimalarial drugs evaluated in the present study. The in silico approach reported here suggested that DOX could potentially be a good candidate for repurposing for COVID-19. In contrast, to decipher the actual potential of DOX and halofantrine against COVID-19, further in vitro and in vivo studies are called for. Drug repurposing warrants consideration as a viable research and innovation avenue as planetary health efforts to fight the COVID-19 continue.


Assuntos
Antimaláricos/farmacologia , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Pneumonia Viral/tratamento farmacológico , Antimaláricos/química , Antivirais/química , Betacoronavirus/química , Sítios de Ligação , Simulação por Computador , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/efeitos dos fármacos , Doxiciclina/química , Doxiciclina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacos , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/efeitos dos fármacos
6.
Infect Genet Evol ; 84: 104451, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32640381

RESUMO

WHO has declared the outbreak of COVID-19 as a public health emergency of international concern. The ever-growing new cases have called for an urgent emergency for specific anti-COVID-19 drugs. Three structural proteins (Membrane, Envelope and Nucleocapsid protein) play an essential role in the assembly and formation of the infectious virion particles. Thus, the present study was designed to identify potential drug candidates from the unique collection of 548 anti-viral compounds (natural and synthetic anti-viral), which target SARS-CoV-2 structural proteins. High-end molecular docking analysis was performed to characterize the binding affinity of the selected drugs-the ligand, with the SARS-CoV-2 structural proteins, while high-level Simulation studies analyzed the stability of drug-protein interactions. The present study identified rutin, a bioflavonoid and the antibiotic, doxycycline, as the most potent inhibitor of SARS-CoV-2 envelope protein. Caffeic acid and ferulic acid were found to inhibit SARS-CoV-2 membrane protein while the anti-viral agent's simeprevir and grazoprevir showed a high binding affinity for nucleocapsid protein. All these compounds not only showed excellent pharmacokinetic properties, absorption, metabolism, minimal toxicity and bioavailability but were also remain stabilized at the active site of proteins during the MD simulation. Thus, the identified lead compounds may act as potential molecules for the development of effective drugs against SARS-CoV-2 by inhibiting the envelope formation, virion assembly and viral pathogenesis.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Proteínas do Nucleocapsídeo/química , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Vírion/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Doxiciclina/química , Doxiciclina/farmacologia , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinoxalinas/química , Quinoxalinas/farmacologia , Rutina/química , Rutina/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Simeprevir/química , Simeprevir/farmacologia , Termodinâmica , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Vírion/genética , Vírion/metabolismo
7.
PLoS One ; 15(7): e0235633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628709

RESUMO

The antibacterial efficacy of the tetracycline antibiotics has been greatly reduced by the development of resistance, hence a decline in their clinical use. The hok/sok locus is a type I toxin/antitoxin plasmid stability element, often associated with multi-drug resistance plasmids, especially ESBL-encoding plasmids. It enhances host cell survivability and pathogenicity in stressful growth conditions, and increases bacterial tolerance to ß-lactam antibiotics. The hok/sok locus forms dsRNA by RNA:RNA interactions between the toxin encoding mRNA and antitoxin non-coding RNA, and doxycycline has been reported to bind dsRNA structures and inhibit their cleavage/processing by the dsRNase, RNase III. This study investigated the antibacterial activities of doxycycline in hok/sok host bacteria cells, the effects on hok/sok-induced changes in growth and the mechanism(s) involved. Diverse strains of E. coli were transformed with hok/sok plasmids and assessed for doxycycline susceptibility and growth changes. The results show that the hok/sok locus increases bacterial susceptibility to doxycycline, which is more apparent in strains with more pronounced hok/sok-induced growth effects. The increased doxycycline susceptibility occurs despite ß-lactam resistance imparted by hok/sok. Doxycycline was found to induce bacterial death in a manner phenotypically characteristic of Hok toxin expression, suggesting that it inhibits the toxin/antitoxin dsRNA degradation, leading to Hok toxin expression and cell death. In this way, doxycycline could counteract the multi-drug resistance plasmid maintenance/propagation, persistence and pathogenicity mechanisms associated with the hok/sok locus, which could potentially help in efforts to mitigate the rise of antimicrobial resistance.


Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Doxiciclina/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , RNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Bacteriano/metabolismo , RNA de Cadeia Dupla/metabolismo
8.
PLoS One ; 15(7): e0236758, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730301

RESUMO

Chlamydiosis is the most significant infectious disease of koalas (Phascolarctos cinereus). It is primarily a systemic sexually transmitted disease caused by Chlamydia pecorum and was responsible for 46% of the 2348 koala admissions to Australia Zoo Wildlife Hospital between 2013 and 2017. Treatment of chlamydiosis in koalas is complicated by three major factors. Firstly, koalas rely on bacterial fermentation of their high fibre diet making antibiotic therapy a risk. Secondly, they possess efficient metabolic pathways for the excretion of plant toxins and potentially of therapeutic agents. Thirdly, wild koalas, often present to rehabilitation facilities with chronic and severe disease. Traditional anti-chlamydial antibiotics used in other species may cause fatal dysbiosis in koalas or be excreted before they can be effective. We compared five anti-chlamydial antibiotics, azithromycin, chloramphenicol, doxycycline, enrofloxacin and florfenicol, which were used to treat 86 wild koalas with chlamydiosis presented to Australia Zoo Wildlife Hospital under consistent conditions of nutrition, housing, husbandry and climate. Response to treatment was assessed by recovery from clinical signs, and clearance of detectable Chlamydia via quantitative PCR. Doxycycline was the most effective anti-chlamydial antibiotic of the five, producing a 97% cure rate, followed by chloramphenicol (81%), enrofloxacin (75%), florfenicol (66%) and azithromycin (25%). The long-acting injectable preparation of doxycycline was well tolerated by koalas when administered via the subcutaneous route, and the weekly dosing requirement is a major advantage when treating wild animals. These findings indicate that doxycycline is the current drug of choice for the treatment of chlamydiosis in koalas, with chloramphenicol being the best alternative.


Assuntos
Antibacterianos/farmacologia , Infecções por Chlamydia/tratamento farmacológico , Chlamydia/efeitos dos fármacos , Phascolarctidae/microbiologia , Animais , Austrália , Azitromicina/farmacologia , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/patologia , Cloranfenicol/farmacologia , Doxiciclina/farmacologia , Enrofloxacina/farmacologia , Feminino , Masculino , Tianfenicol/análogos & derivados , Tianfenicol/farmacologia
9.
Nat Commun ; 11(1): 3015, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541784

RESUMO

The interplay between glioma stem cells (GSCs) and the tumor microenvironment plays crucial roles in promoting malignant growth of glioblastoma (GBM), the most lethal brain tumor. However, the molecular mechanisms underlying this crosstalk are incompletely understood. Here, we show that GSCs secrete the Wnt-induced signaling protein 1 (WISP1) to facilitate a pro-tumor microenvironment by promoting the survival of both GSCs and tumor-associated macrophages (TAMs). WISP1 is preferentially expressed and secreted by GSCs. Silencing WISP1 markedly disrupts GSC maintenance, reduces tumor-supportive TAMs (M2), and potently inhibits GBM growth. WISP1 signals through Integrin α6ß1-Akt to maintain GSCs by an autocrine mechanism and M2 TAMs through a paracrine manner. Importantly, inhibition of Wnt/ß-catenin-WISP1 signaling by carnosic acid (CA) suppresses GBM tumor growth. Collectively, these data demonstrate that WISP1 plays critical roles in maintaining GSCs and tumor-supportive TAMs in GBM, indicating that targeting Wnt/ß-catenin-WISP1 signaling may effectively improve GBM treatment and the patient survival.


Assuntos
Neoplasias Encefálicas/genética , Proteínas de Sinalização Intercelular CCN/genética , Glioma/genética , Macrófagos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Proteínas de Sinalização Intercelular CCN/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Doxiciclina/farmacologia , Glioma/metabolismo , Glioma/terapia , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais/genética , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Células U937 , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
PLoS One ; 15(6): e0220350, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32544163

RESUMO

Mycoplasma hyopneumoniae is the major pathogenic microorganism causing enzootic pneumonia in pigs. With increasing resistance of M. hyopneumoniae to conventional antibiotics, treatment is becoming complicated. Herein, we investigated the mutant selection window (MSW) of doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin for treating the M. hyopneumoniae type strain (ATCC 25934) to determine the likelihood of promoting resistance with continued use of these antibiotics. Minimum inhibitory concentration (MIC) values against M. hyopneumoniae were determined for each antimicrobial agent based on microdilution broth and agar dilution methods (bacterial numbers ranged from 105 colony-forming units (CFU)/mL to 109 CFU/mL). The minimal concentration inhibiting colony formation by 99% (MIC99) and the mutant prevention concentration (MPC) were determined by the agar dilution method with three inoculum sizes. Antimicrobial killing was determined based on MIC99 and MPC values for all five agents. MIC values ranged from 0.001 to 0.25 µg/mL based on the microdilution broth method, and from 0.008 to 1.0 µg/mL based on the agar dilution method. MPC values ranged from 0.0016 to 10.24 µg/mL. MPC/MIC99 values were ordered tylosin > doxycycline > danofloxacin > tiamulin > valnemulin. MPC achieved better bactericidal action than MIC99. Based on pharmacodynamic analyses, danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.


Assuntos
Antibacterianos/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Mutação , Mycoplasma hyopneumoniae/efeitos dos fármacos , Mycoplasma hyopneumoniae/genética , Diterpenos/farmacologia , Doxiciclina/farmacologia , Fluoroquinolonas/farmacologia , Cinética , Testes de Sensibilidade Microbiana , Mycoplasma hyopneumoniae/fisiologia , Tilosina/farmacologia
11.
Nat Commun ; 11(1): 2797, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493999

RESUMO

Fat distribution is an independent cardiometabolic risk factor. However, its molecular and cellular underpinnings remain obscure. Here we demonstrate that two independent GWAS signals at RSPO3, which are associated with increased body mass index-adjusted waist-to-hip ratio, act to specifically increase RSPO3 expression in subcutaneous adipocytes. These variants are also associated with reduced lower-body fat, enlarged gluteal adipocytes and insulin resistance. Based on human cellular studies RSPO3 may limit gluteofemoral adipose tissue (AT) expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis. RSPO3 may also promote upper-body fat distribution by stimulating abdominal adipose progenitor (AP) proliferation. The distinct biological responses elicited by RSPO3 in abdominal versus gluteal APs in vitro are associated with differential changes in WNT signalling. Zebrafish carrying a nonsense rspo3 mutation display altered fat distribution. Our study identifies RSPO3 as an important determinant of peripheral AT storage capacity.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Distribuição da Gordura Corporal , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Trombospondinas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Adipócitos/efeitos dos fármacos , Tecido Adiposo/metabolismo , Adiposidade/genética , Adulto , Alelos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Células-Tronco/metabolismo , Trombospondinas/genética , Relação Cintura-Quadril , Via de Sinalização Wnt/efeitos dos fármacos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
12.
Int J Infect Dis ; 97: 270-277, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32526389

RESUMO

OBJECTIVES: A standard treatment regimen against Mycobacteroides abscessus complex (MABC) infections has not yet been established, making MABC difficult to treat successfully. In this study, we sought to develop an active ingredient for the clinical treatment of MABC infections. METHODS: We screened 102 MABC strains isolated from clinical specimens using DNA sequence analysis with the housekeeping genes hsp65 and rpoB. Drug susceptibility testing was performed against two subspecies-Mycobacteroides abscessus subsp. abscessus (M. abscessus) and Mycobacteroides abscessus subsp. massiliense (M. massiliense)-using eight antimicrobial agents (clarithromycin, amikacin, doxycycline, imipenem, linezolid, moxifloxacin, faropenem, and rifampicin). The combined efficacy of the antimicrobial agents was investigated using a checkerboard method. RESULTS: We identified 51 isolates as M. abscessus, 46 as M. massiliense, and five as others. Most of the M. abscessus isolates (83.0 %) exhibited inducible resistance to clarithromycin via the expression of the erm(41) gene. Combinations of imipenem with linezolid, moxifloxacin, and rifampicin exhibited additive effects against 81.0 %, 40.7 %, and 26.9 % of M. abscessus, respectively, and against 54.5 %, 69.2 %, and 30.8 % of M. massiliense, respectively. CONCLUSIONS: These results demonstrated the potential efficacy of a regimen containing imipenem against M. abscessus and M. massiliense infections.


Assuntos
Antibacterianos/farmacologia , Mycobacteriaceae/efeitos dos fármacos , Infecções por Actinomycetales/microbiologia , Amicacina/farmacologia , Claritromicina/farmacologia , Doxiciclina/farmacologia , Humanos , Imipenem/farmacologia , Linezolida/farmacologia , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Mycobacteriaceae/classificação , Mycobacteriaceae/crescimento & desenvolvimento , Análise de Sequência de DNA , beta-Lactamas/farmacologia
13.
Mol Biol (Mosk) ; 54(2): 308-312, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392201

RESUMO

A synthetic genetic chain was created-a two-promoter integrator, the activity of the output signal of which is high only with the high activity of two promoters at the input of the chain. The integrator is shown to work by the example of cancer-specific promoters of the human survivin and telomerase genes that can be used for specific transcription of target genes in tumor cells. To create the integrator, we used the well-known TET-ON 3G transactivator, regulated by doxycycline.


Assuntos
Doxiciclina/farmacologia , Regiões Promotoras Genéticas , Transativadores , Humanos , Survivina/genética , Telomerase/genética , Tetraciclina
14.
Nat Commun ; 11(1): 2437, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415104

RESUMO

DNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly and leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within the Pol ε catalytic core influences both of these replication steps in budding yeast. Modeling cancer-associated mutations in this domain reveals its unexpected effect on incorporating Pol ε into the four-member pre-loading complex during replisome assembly. In addition, genetic and biochemical data suggest that the examined domain supports Pol ε catalytic activity and symmetric movement of replication forks. Contrary to previously characterized Pol ε cancer variants, the examined mutants cause genome hyper-rearrangement rather than hyper-mutation. Our work thus suggests a role of the Pol ε catalytic core in replisome formation, a reliance of Pol ε strand synthesis on a unique domain, and a potential tumor-suppressive effect of Pol ε in curbing genome re-arrangements.


Assuntos
DNA Polimerase II/fisiologia , Replicação do DNA , Regulação da Expressão Gênica , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular/genética , Estruturas Cromossômicas/genética , DNA Polimerase II/química , Doxiciclina/farmacologia , Genoma Humano , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
16.
PLoS One ; 15(3): e0222619, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32150577

RESUMO

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-ß1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-ß1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.


Assuntos
Eczema/genética , Epiderme/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Ceratose/genética , Pele/metabolismo , Transgenes , Acetamidas/farmacologia , Animais , Citocinas/metabolismo , Doxiciclina/farmacologia , Eczema/tratamento farmacológico , Feminino , Homeostase/genética , Hiperplasia/tratamento farmacológico , Hiperplasia/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Queratinócitos/metabolismo , Ceratose/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transativadores/metabolismo , Compostos de Tritil/farmacologia
17.
J Assoc Physicians India ; 68(3): 59-63, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32138486

RESUMO

Introduction: Doxycycline acts against a broad range of gram-positive, gramnegative and 'atypical' bacteria as well as some protozoan pathogens such as malaria. In this era of increasing multidrug-resistance, recycling of old antimicrobials should be considered and need more focus in this domain of research. We, therefore, aimed to assess the antimicrobial susceptibility patterns of commonly isolated pathogens against doxycycline, azithromycin, cefuroxime, and amoxicillin from common clinical specimens by using laboratory-based diagnostic data from western India. Materials and Methods: The non-interventional retrospective study was conducted on secondary data extracted from multi-center diagnostic laboratory based in Mumbai, India. Susceptibility data of bacteria isolated from blood, urine, pus, and sputum were used in the study and culture positive samples were segregated. Antimicrobial susceptibility status of doxycycline was checked and compared with azithromycin, cefuroxime, and amoxicillin. Chi-square tests of significance were carried out to assess significant differences in susceptibility patterns. Association between variables was considered statistically significant if the p-value was <0.05. Results: Percentage susceptibility of collective bacterial isolates was found to be highest for doxycycline in all four specimens (93.1%). Individual percentage susceptibility was observed to be highest for sputum isolates (97.5%) followed by blood (93.8%), pus (92.7%) and urine (70.0%). The activity of doxycycline was found to be 93.5% for the samples resistant to azithromycin. Doxycycline also showed good susceptibility for the isolates resistant to amoxicillin and cefuroxime which was 75.9% and 64.8%, respectively. Conclusion: Several bacterial isolates from all four sources were found to be susceptible to Doxycycline. It has an important role in the form of a better alternative of major antimicrobial agents like azithromycin, cefuroxime, and amoxicillin against gram-positive cocci. Doxycycline appeared to show better activity against isolates which were resistant to other three antimicrobials.


Assuntos
Antibacterianos/farmacologia , Amoxicilina/farmacologia , Azitromicina/farmacologia , Cefuroxima/farmacologia , Doxiciclina/farmacologia , Índia , Testes de Sensibilidade Microbiana , Estudos Retrospectivos
18.
J Vis Exp ; (157)2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32202533

RESUMO

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis and metabolism. Indeed, fluctuations in transcript expression could modify the translation efficiency and ultimately the cellular activity of a transcript. Several experimental approaches have been developed to investigate the half-life of mRNA although some of these methods have limitations that prevent the proper study of posttranscriptional modulation. A promoter induction system can express a gene of interest under the control of a synthetic tetracycline-regulated promoter. This method allows the half-life estimation of a given mRNA under any experimental condition without disturbing cell homeostasis. One major drawback of this method is the necessity to transfect cells, which limits the use of this technique in isolated primary cells that are highly resistant to conventional transfection techniques. Alveolar epithelial cells in primary culture have been used extensively to study the cellular and molecular biology of the alveolar epithelium. The unique characteristics and phenotype of primary alveolar cells make it essential to study the posttranscriptional modulations of genes of interest in these cells. Therefore, our aim was to develop a novel tool to investigate the posttranscriptional modulations of mRNAs of interest in alveolar epithelial cells in primary culture. We designed a fast and efficient transient transfection protocol to insert a transcriptionally controlled plasmid expression system into primary alveolar epithelial cells. This cloning strategy, using a viral epitope to tag the construct, allows for the easy discrimination of construct expression from that of endogenous mRNAs. Using a modified ΔΔ quantification cycle (Cq) method, the expression of the transcript can then be quantified at different time intervals to measure its half-life. Our data demonstrate the efficiency of this novel approach in studying posttranscriptional regulation in various pathophysiological conditions in primary alveolar epithelial cells.


Assuntos
Células Epiteliais Alveolares/metabolismo , Regulação da Expressão Gênica , Plasmídeos/genética , Estabilidade de RNA/genética , Transcrição Genética , Regiões 3' não Traduzidas/genética , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Células Cultivadas , Primers do DNA/metabolismo , Dactinomicina/farmacologia , Doxiciclina/farmacologia , Eletroporação , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/patologia , Cinética , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Transcrição Genética/efeitos dos fármacos , Transfecção
19.
Vet Parasitol ; 279: 109057, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32126342

RESUMO

Filarial nematodes are tissue-dwelling parasitic worms that can cause a range of disfiguring pathologies in humans and potentially lethal infections of companion animals. The bacterial endosymbiont, Wolbachia, is present within most human and veterinary filarial pathogens, including the causative agent of heartworm disease, Dirofilaria immitis. Doxycycline-mediated drug targeting of Wolbachia leads to sterility, clearance of microfilariae and gradual death of adult filariae. This mode of action is attractive in the treatment of filariasis because it avoids severe host inflammatory adverse reactions invoked by rapid-killing anthelmintic agents. However, doxycycline needs to be taken for four weeks to exert curative activity. In this review, we discuss the evidence that Wolbachia drug targeting is efficacious in blocking filarial larval development as well as in the treatment of chronic filarial disease. We present the current portfolio of next-generation anti-Wolbachia candidates discovered through phenotypic screening of chemical libraries and validated in a range of in vitro and in vivo filarial infection models. Several novel chemotypes have been identified with selected narrow-spectrum anti-Wolbachia specificity and superior time-to-kill kinetics compared with doxycycline. We discuss the opportunities of developing these novel anti-Wolbachia agents as either cures, adjunct therapies or new preventatives for the treatment of veterinary filariasis.


Assuntos
Antibacterianos , Dirofilaria immitis/efeitos dos fármacos , Dirofilaria repens/efeitos dos fármacos , Dirofilariose/prevenção & controle , Doxiciclina/farmacologia , Filaricidas/farmacologia , Animais , Wolbachia
20.
Int J Nanomedicine ; 15: 125-136, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021169

RESUMO

Background: Despite recent advancements in surgical techniques, the repair of tendon rupture remains a challenge for surgeons. The purpose of this study was to develop novel doxycycline-loaded biodegradable nanofibrous membranes and evaluate their efficacy for the repair of Achilles tendon rupture in a rat model. Materials and Methods: The drug-loaded nanofibers were prepared using the electrospinning process and drug release from the prepared membranes was investigated both in vitro and in vivo. Furthermore, the safety and efficacy of the drug-loaded nanofibrous membranes were evaluated in rats that underwent tendon surgeries. An animal behavior cage was employed to monitor the post-surgery activity of the animals. Results: The experimental results demonstrated that poly(D,L-lactide-co-glycolide) (PLGA) nanofibers released effective concentrations of doxycycline for more than 40 days post-surgery, and the systemic plasma drug concentration was low. Rats receiving implantation of doxycycline-loaded nanofibers also showed greater activities and stronger tendons post-operation. Conclusion: Nanofibers loaded with doxycycline may have great potential in the repair of Achilles tendon rupture.


Assuntos
Doxiciclina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Nanofibras/administração & dosagem , Traumatismos dos Tendões/terapia , Implantes Absorvíveis , Tendão do Calcâneo/lesões , Animais , Doxiciclina/administração & dosagem , Doxiciclina/farmacocinética , Liberação Controlada de Fármacos , Membranas Artificiais , Nanofibras/química , Nanofibras/uso terapêutico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Ratos , Ratos Sprague-Dawley , Cicatrização
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