Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.386
Filtrar
2.
Mol Med ; 27(1): 105, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34503440

RESUMO

BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.


Assuntos
Antivirais/farmacologia , Atorvastatina/farmacologia , COVID-19/tratamento farmacológico , Pulmão/efeitos dos fármacos , Organoides/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Atorvastatina/química , COVID-19/prevenção & controle , Linhagem Celular , Proteases 3C de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/química , Doxiciclina/farmacologia , Aprovação de Drogas , Reposicionamento de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/virologia , Modelos Biológicos , Simulação de Acoplamento Molecular , Organoides/virologia , Cloridrato de Raloxifeno/química , Cloridrato de Raloxifeno/farmacologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Trifluoperazina/química , Trifluoperazina/farmacologia , Estados Unidos , United States Food and Drug Administration , Vesiculovirus/genética , Internalização do Vírus/efeitos dos fármacos
3.
Viruses ; 13(9)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34578326

RESUMO

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.


Assuntos
COVID-19/virologia , Interações Hospedeiro-Patógeno , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Fenômenos Fisiológicos Virais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/metabolismo , Ciclo Celular , Chlorocebus aethiops , Doxiciclina/farmacologia , Células HEK293 , Humanos , Ligação Proteica , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus , Transdução Genética , Células Vero
4.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360785

RESUMO

Metabolic alteration is characteristic during tumour growth and therapy; however, targeting metabolic rewiring could overcome therapy resistance. mTOR hyperactivity, autophagy and other metabolic processes, including mitochondrial functions, could be targeted in breast cancer progression. We investigated the growth inhibitory mechanism of rapamycin + doxycycline treatment in human breast cancer model systems. Cell cycle and cell viability, including apoptotic and necrotic cell death, were analysed using flow cytometry, caspase activity measurements and caspase-3 immunostainings. mTOR-, autophagy-, necroptosis-related proteins and treatment-induced morphological alterations were analysed by WesTM, Western blot, immunostainings and transmission electron microscopy. The rapamycin + doxycycline combination decreased tumour proliferation in about 2/3rd of the investigated cell lines. The continuous treatment reduced tumour growth significantly both in vivo and in vitro. The effect after short-term treatment was reversible; however, autophagic vacuoles and degrading mitochondria were detected simultaneously, and the presence of mitophagy was also observed after the long-term rapamycin + doxycycline combination treatment. The rapamycin + doxycycline combination did not cause apoptosis or necrosis/necroptosis, but the alterations in autophagy- and mitochondria-related protein levels (LC3-B-II/I, p62, MitoTracker, TOM20 and certain co-stainings) were correlated to autophagy induction and mitophagy, without mitochondria repopulation. Based on these results, we suggest considering inducing metabolic stress and targeting mTOR hyperactivity and mitochondrial functions in combined anti-cancer treatments.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Doxiciclina/farmacologia , Feminino , Células HT29 , Humanos , Células MCF-7 , Mitocôndrias/patologia , Sirolimo/farmacologia
5.
J Clin Invest ; 131(15)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34338231

RESUMO

Pulmonary cavitation is a hallmark of Mycobacterium tuberculosis (Mtb) infection that provides an immune-privileged niche for extracellular bacillary replication, which associates with increased transmission rates, drug resistance, and chronic lung dysfunction following antituberculous therapy (ATT). Inhibitors of matrix metalloproteinases (MMPs), which are induced by Mtb infection, have shown efficacy in preclinical models and improved microbiologic and immunopathologic outcomes. In this issue of the JCI, Hao Miow et al. performed a double-blind, randomized controlled trial exploring host-directed effects of the MMP inhibitor doxycycline versus placebo when added to standard ATT for pulmonary tuberculosis. Doxycycline treatment over two weeks durably modulated host blood transcription profiles, including the resolution of inflammatory gene programs. Reduced immunopathology markers in doxycycline-treated participants also included improved lung cavity volumes and lower MMP levels in blood and sputum. These findings provide mechanistic insight and momentum for using experimental medicine trials to develop host-directed therapies for tuberculosis.


Assuntos
Mycobacterium tuberculosis , Tuberculose Pulmonar , Tuberculose , Método Duplo-Cego , Doxiciclina/farmacologia , Humanos , Tuberculose/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico
6.
Int J Mol Sci ; 22(13)2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34281250

RESUMO

Amelogenin comprises ~90% of enamel proteins; however, the involvement of Amelx transcriptional activation in regulating ameloblast differentiation from induced pluripotent stem cells (iPSCs) remains unknown. In this study, we generated doxycycline-inducible Amelx-expressing mouse iPSCs (Amelx-iPSCs). We then established a three-stage ameloblast induction strategy from Amelx-iPSCs, including induction of surface ectoderm (stage 1), dental epithelial cells (DECs; stage 2), and ameloblast lineage (stage 3) in sequence, by manipulating several signaling molecules. We found that adjunctive use of lithium chloride (LiCl) in addition to bone morphogenetic protein 4 and retinoic acid promoted concentration-dependent differentiation of DECs. The resulting cells had a cobblestone appearance and keratin14 positivity. Attenuation of LiCl at stage 3 together with transforming growth factor ß1 and epidermal growth factor resulted in an ameloblast lineage with elongated cell morphology, positivity for ameloblast markers, and calcium deposition. Although stage-specific activation of Amelx did not produce noticeable phenotypic changes in ameloblast differentiation, Amelx activation at stage 3 significantly enhanced cell adhesion as well as decreased proliferation and migration. These results suggest that the combination of inducible Amelx transcription and stage-specific ameloblast induction for iPSCs represents a powerful tool to highlight underlying mechanisms in ameloblast differentiation and function in association with Amelx expression.


Assuntos
Ameloblastos/citologia , Ameloblastos/metabolismo , Amelogenina/metabolismo , Ameloblastos/fisiologia , Amelogenina/genética , Animais , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Doxiciclina/farmacologia , Células Epiteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Transdução de Sinais , Ativação Transcricional/fisiologia
7.
Molecules ; 26(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299596

RESUMO

Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (-7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.


Assuntos
Doxiciclina/farmacologia , Fibroblastos/metabolismo , Pele/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Zika virus/fisiologia , Animais , Chlorocebus aethiops , Doxiciclina/química , Fibroblastos/virologia , Humanos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Pele/virologia , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Zika virus/química
8.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201124

RESUMO

BMP-7 has shown inductive potential for in vitro osteogenic differentiation of mesenchymal stem cells, which are an ideal resource for regenerative medicine. Externally applied, recombinant BMP-7 was able to induce the osteogenic differentiation of DPSCs but based on our previous results with BMP-2, we aimed to study the effect of the tetracyclin-inducible BMP-7 expression on these cells. DPSC, mock, and DPSC-BMP-7 cell lines were cultured in the presence or absence of doxycycline, then alkaline phosphatase (ALP) activity, mineralization, and mRNA levels of different osteogenic marker genes were measured. In the DPSC-BMP-7 cell line, the level of BMP-7 mRNA significantly increased in the media supplemented with doxycycline, however, the expression of Runx2 and noggin genes was upregulated only after 21 days of incubation in the osteogenic medium with doxycycline. Moreover, while the examination of ALP activity showed reduced activity in the control medium containing doxycycline, the accumulation of minerals remained unchanged in the cultures. We have found that the induced BMP-7 expression failed to induce osteogenic differentiation of DPSCs. We propose three different mechanisms that may worth investigating for the engineering of expression systems that can be used for the induction of differentiation of mesenchymal stem cells.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Diferenciação Celular , Polpa Dentária/citologia , Doxiciclina/farmacologia , Osteogênese , Células-Tronco/citologia , Fosfatase Alcalina/metabolismo , Antibacterianos/farmacologia , Proliferação de Células , Células Cultivadas , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Humanos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
9.
Cell Prolif ; 54(8): e13090, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34197016

RESUMO

OBJECTIVES: Derivation and maintenance of pluripotent stem cells (PSCs) generally require optimized and complex culture media, which hinders the derivation of PSCs from various species. Expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) can reprogram somatic cells into induced PSCs (iPSCs), even for species possessing no optimal culture condition. Herein, we explored whether expression of OSKM could induce and maintain pluripotency without PSC-specific growth factors and signaling inhibitors. METHODS: The culture medium of Tet-On-OSKM/Oct4-GFP mouse embryonic stem cells (ESCs) was switched from N2B27 with MEK inhibitor, GSK3ß inhibitor, and leukemia inhibitory factor (LIF) (2iL) to N2B27 with doxycycline. Tet-On-OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed in N2B27 with doxycycline. Cell proliferation was traced. Pluripotency was assessed by expression of ESC marker genes, teratoma, and chimera formation. RNA-Seq was conducted to analyze gene expression. RESULTS: Via continuous expression of OSKM, mouse ESCs (OSKM-ESCs) and the resulting iPSCs (OSKM-iPSCs) reprogrammed from MEF cells propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Transcriptional landscapes of OSKM-iPSCs resembled those of ESCs cultured in 2iL and were more similar to those of ESCs cultured in serum/LIF. Furthermore, OSKM-iPSCs contributed to germline transmission. CONCLUSIONS: Expression of OSKM could induce and maintain mouse pluripotency without specific culturing factors. Importantly, OSKM-iPSCs could produce gene-modified animals through germline transmission, with potential applications in other species.


Assuntos
Autorrenovação Celular , Reprogramação Celular , Fatores de Transcrição/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Edição de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Teratoma/metabolismo , Teratoma/patologia , Fatores de Transcrição/genética , Transcriptoma/efeitos dos fármacos
10.
Methods Mol Biol ; 2320: 261-281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302664

RESUMO

Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Sequência de Bases , Causalidade , Células Cultivadas , Cromatografia Líquida/métodos , DNA/isolamento & purificação , Doxiciclina/farmacologia , Citometria de Fluxo , Estudos de Associação Genética , Vetores Genéticos/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lentivirus/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RNA Guia/genética , Transfecção
11.
Biomed Pharmacother ; 142: 111956, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34332377

RESUMO

Novel coronavirus 2019 (COVID-19) is a zoonosis that revised the global economic and societal progress since early 2020. The SARS-CoV-2 has been recognized as the responsible pathogen for COVID-19 with high infection and mortality rate potential. It has spread in 192 countries and infected about 1.5% of the world population, and still, a proper therapeutic approach is not unveiled. COVID-19 indication starts with fever to shortness of breathing, leading to ICU admission with the ventilation support in severe conditions. Besides the symptomatic mainstay clinical therapeutic approach, only Remdesivir has been approved by the FDA. Several pharmaceutical companies claimed different vaccines with exceptionally high efficacy (90-95%) against COVID-19; how long these vaccines can protect and long-term safety with the new variants are unpredictable. After the worldwide spread of the COVID-19 pandemic, numerous clinical trials with different phases are being performed to find the most appropriate solution to this condition. Some of these trials with old FDA-approved drugs showed promising results. In this review, we have precisely compiled the efforts to curb the disease and discussed the clinical findings of Ivermectin, Doxycycline, Vitamin-D, Vitamin-C, Zinc, and cannabidiol and their combinations. Additionally, the correlation of these molecules on the prophylactic and diseased ministration against COVID-19 has been explored.


Assuntos
COVID-19/tratamento farmacológico , Canabidiol/farmacologia , SARS-CoV-2 , Antivirais/farmacologia , Ácido Ascórbico/farmacologia , COVID-19/epidemiologia , COVID-19/prevenção & controle , Suplementos Nutricionais , Doxiciclina/farmacologia , Reposicionamento de Medicamentos/métodos , Quimioterapia Combinada/métodos , Humanos , Ivermectina , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/isolamento & purificação , Resultado do Tratamento , Vitamina D/farmacologia , Zinco/farmacologia
12.
Nat Protoc ; 16(8): 4004-4030, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34244697

RESUMO

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system.


Assuntos
DNA/metabolismo , Análise de Célula Única , Sequência de Aminoácidos , Antibacterianos/farmacologia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Doxiciclina/farmacologia , Epigenoma , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma
13.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069024

RESUMO

Precise analysis of the genetic expression and functioning of proteins requires experimental approaches that, among others, enable tight control of gene expression at the transcriptional level. Doxycycline-induced Tet-On/Tet-Off expression systems provide such an opportunity, and are frequently used to regulate the activity of genes in eukaryotic cells. Since its development, the Tet-system has evolved tight gene control in mammalian cells; however, some challenges are still unaddressed. In the current set up, the establishment of the standard Tet-based system in target cells is time-consuming and laborious and has been shown to be inefficient, especially in a long-term perspective. In this work, we present an optimized inducible expression system, which enables rapid generation of doxycycline-responsive cells according to a one- or two-step protocol. The reported modifications of the Tet-On system expand the toolbox for regulated mammalian gene expression and provide high, stable, and homogenous expression of the Tet-On3G transactivator, which is of fundamental importance in the regulation of transgenes.


Assuntos
Antibacterianos/farmacologia , Regulação da Expressão Gênica , Técnicas Genéticas , Vetores Genéticos/genética , Animais , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Proteína Ribossômica L10/genética , Tetraciclina/farmacologia , Transativadores/genética , Transgenes
14.
Cell Death Dis ; 12(7): 657, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183648

RESUMO

Subcellular organelles communicate with each other to regulate function and coordinate responses to changing cellular conditions. The physical-functional coupling of the endoplasmic reticulum (ER) with mitochondria allows for the direct transfer of Ca2+ between organelles and is an important avenue for rapidly increasing mitochondrial metabolic activity. As such, increasing ER-mitochondrial coupling can boost the generation of ATP that is needed to restore homeostasis in the face of cellular stress. The mitochondrial unfolded protein response (mtUPR) is activated by the accumulation of unfolded proteins in mitochondria. Retrograde signaling from mitochondria to the nucleus promotes mtUPR transcriptional responses aimed at restoring protein homeostasis. It is currently unknown whether the changes in mitochondrial-ER coupling also play a role during mtUPR stress. We hypothesized that mitochondrial stress favors an expansion of functional contacts between mitochondria and ER, thereby increasing mitochondrial metabolism as part of a protective response. Hela cells were treated with doxycycline, an antibiotic that inhibits the translation of mitochondrial-encoded proteins to create protein disequilibrium. Treatment with doxycycline decreased the abundance of mitochondrial encoded proteins while increasing expression of CHOP, C/EBPß, ClpP, and mtHsp60, markers of the mtUPR. There was no change in either mitophagic activity or cell viability. Furthermore, ER UPR was not activated, suggesting focused activation of the mtUPR. Within 2 h of doxycycline treatment, there was a significant increase in physical contacts between mitochondria and ER that was distributed throughout the cell, along with an increase in the kinetics of mitochondrial Ca2+ uptake. This was followed by the rise in the rate of oxygen consumption at 4 h, indicating a boost in mitochondrial metabolic activity. In conclusion, an early phase of the response to doxycycline-induced mitochondrial stress is an increase in mitochondrial-ER coupling that potentiates mitochondrial metabolic activity as a means to support subsequent steps in the mtUPR pathway and sustain cellular adaptation.


Assuntos
Antibacterianos/farmacologia , Doxiciclina/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Células HeLa , Humanos , Cinética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/efeitos dos fármacos
15.
Endocrinology ; 162(7)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34019639

RESUMO

Tafazzin (TAZ) is a cardiolipin (CL) biosynthetic enzyme important for maintaining mitochondrial function. TAZ affects both the species and content of CL in the inner mitochondrial membrane, which are essential for normal cellular respiration. In pancreatic ß cells, mitochondrial function is closely associated with insulin secretion. However, the role of TAZ and CL in the secretion of insulin from pancreatic islets remains unknown. Male 4-month-old doxycycline-inducible TAZ knock-down (KD) mice and wild-type littermate controls were used. Immunohistochemistry was used to assess ß-cell morphology in whole pancreas sections, whereas ex vivo insulin secretion, CL content, RNA-sequencing analysis, and mitochondrial oxygen consumption were measured from isolated islet preparations. Ex vivo insulin secretion under nonstimulatory low-glucose concentrations was reduced ~52% from islets isolated from TAZ KD mice. Mitochondrial oxygen consumption under low-glucose conditions was also reduced ~58% in islets from TAZ KD animals. TAZ deficiency in pancreatic islets was associated with significant alteration in CL molecular species and elevated polyunsaturated fatty acid CL content. In addition, RNA-sequencing of isolated islets showed that TAZ KD increased expression of extracellular matrix genes, which are linked to pancreatic fibrosis, activated stellate cells, and impaired ß-cell function. These data indicate a novel role for TAZ in regulating pancreatic islet function, particularly under low-glucose conditions.


Assuntos
Aciltransferases/deficiência , Aciltransferases/fisiologia , Secreção de Insulina/fisiologia , Ilhotas Pancreáticas/fisiologia , Mitocôndrias/fisiologia , Aciltransferases/genética , Animais , Cardiolipinas/análise , Cardiolipinas/química , Doxiciclina/farmacologia , Ácidos Graxos Insaturados/análise , Feminino , Fibrose , Técnicas de Silenciamento de Genes , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredução , Consumo de Oxigênio/fisiologia , Pâncreas/patologia
16.
J Med Microbiol ; 70(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33999797

RESUMO

Introduction. Mycobacterium avium complex (MAC) has been reported as the most common aetiology of lung disease involving nontuberculous mycobacteria.Hypothesis. Antimicrobial susceptibility and clinical characteristics may differ between Mycobacterium avium and Mycobacterium intracellulare.Aim. We aimed to evaluate the differences in antimicrobial susceptibility profiles between two major MAC species (Mycobacterium avium and Mycobacterium intracellulare) from patients with pulmonary infections and to provide epidemiologic data with minimum inhibitory concentration (MIC) distributions.Methodology. Between January 2019 and May 2020, 45 M. avium and 242 M. intracellulare isolates were obtained from Shanghai Pulmonary Hospital. The demographic and clinical characteristics of patients were obtained from their medical records. The MICs of 13 antimicrobials were determined for the MAC isolates using commercial Sensititre SLOWMYCO MIC plates and the broth microdilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI; Standards M24-A2). MIC50 and MIC90 values were derived from the MIC distributions.Results. M. intracellulare had higher resistance rates than M. avium for most tested antimicrobials except clarithromycin, ethambutol, and ciprofloxacin. Clarithromycin was the most effective antimicrobial against both the M. avium (88.89 %) and M. intracellulare (91.32 %) isolates, with no significant difference between the species (P=0.601). The MIC90 of clarithromycin was higher for M. avium (32 µg ml-1) than M. intracellulare (8 µg ml-1). The MIC50 of rifabutin was more than four times higher for M. intracellulare (1 µg ml-1) than M. avium (≤0.25 µg ml-1). The percentages of patients aged >60 years and patients with sputum, cough, and cavitary lesions were significantly higher than among patients with M. intracellulare infection than M. avium infections.Conclusions. The pulmonary disease caused by distinct MAC species had different antimicrobial susceptibility, symptoms, and radiographic findings.


Assuntos
Antibacterianos/farmacologia , Pneumopatias/microbiologia , Complexo Mycobacterium avium/efeitos dos fármacos , Infecção por Mycobacterium avium-intracellulare/microbiologia , Mycobacterium avium/efeitos dos fármacos , Adulto , Idoso , China , Ciprofloxacina/farmacologia , Claritromicina/farmacologia , Tosse , Doxiciclina/farmacologia , Farmacorresistência Bacteriana , Feminino , Humanos , Pulmão/diagnóstico por imagem , Pneumopatias/fisiopatologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium avium/isolamento & purificação , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/diagnóstico , Infecção por Mycobacterium avium-intracellulare/fisiopatologia , Radiografia , Escarro
17.
Oxid Med Cell Longev ; 2021: 4681041, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33959214

RESUMO

The main objective of this study was to investigate the action of doxycycline hyclate (Dx) in the skin wound healing process in Wistar rats. We investigated the effect of Dx on inflammatory cell recruitment and production of inflammatory mediators via in vitro and in vivo analysis. In addition, we analyzed neovascularization, extracellular matrix deposition, and antioxidant potential of Dx on cutaneous repair in Wistar rats. Male animals (n = 15) were divided into three groups with five animals each (protocol: 72/2017), and three skin wounds (12 mm diameter) were created on the back of the animals. The groups were as follows: C, received distilled water (control); Dx1, doxycycline hyclate (10 mg/kg/day); and Dx2, doxycycline hyclate (30 mg/kg/day). The applications were carried out daily for up to 21 days, and tissues from different wounds were removed every 7 days. Our in vitro analysis demonstrated that Dx led to macrophage proliferation and increased N-acetyl-ß-D-glucosaminidase (NAG) production, besides decreased cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and metalloproteinases (MMP), which indicates that macrophage activation and COX-2 inhibition are possibly regulated by independent mechanisms. In vivo, our findings presented increased cellularity, blood vessels, and the number of mast cells. However, downregulation was observed in the COX-2 and PGE2 expression, which was limited to epidermal cells. Our results also showed that the downregulation of this pathway benefits the oxidative balance by reducing protein carbonyls, malondialdehyde, nitric oxide, and hydrogen peroxide (H2O2). In addition, there was an increase in the antioxidant enzymes (catalase and superoxide dismutase) after Dx exposure, which demonstrates its antioxidant potential. Finally, Dx increased the number of types I collagen and elastic fibers and reduced the levels of MMP, thus accelerating the closure of skin wounds. Our findings indicated that both doses of Dx can modulate the skin repair process, but the best effects were observed after exposure to the highest dose.


Assuntos
Antibacterianos/uso terapêutico , Antioxidantes/uso terapêutico , Ciclo-Oxigenase 2/metabolismo , Doxiciclina/uso terapêutico , Metaloproteinases da Matriz/metabolismo , Cicatrização/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Doxiciclina/farmacologia , Masculino , Ratos , Ratos Wistar
18.
Am J Physiol Renal Physiol ; 320(5): F883-F896, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33818128

RESUMO

Neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) regulates the expression of Kir4.1, thiazide-sensitive NaCl cotransporter (NCC), and epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN), and Nedd4-2 deletion causes salt-sensitive hypertension. We now examined whether Nedd4-2 deletion compromises the effect of high-salt (HS) diet on Kir4.1, NCC, ENaC, and renal K+ excretion. Immunoblot analysis showed that HS diet decreased the expression of Kir4.1, Ca2+-activated large-conductance K+ channel subunit-α (BKα), ENaCß, ENaCγ, total NCC, and phospho-NCC (at Thr53) in floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4lfl/fl) mice, whereas these effects were absent in kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. Renal clearance experiments also demonstrated that Nedd4-2 deletion abolished the inhibitory effect of HS diet on hydrochlorothiazide-induced natriuresis. Patch-clamp experiments showed that neither HS diet nor low-salt diet had an effect on Kir4.1/Kir5.1 currents of the distal convoluted tubule in Nedd4-2-deficient mice, whereas we confirmed that HS diet inhibited and low-salt diet increased Kir4.1/Kir5.1 activity in Nedd4lflox/flox mice. Nedd4-2 deletion increased ENaC currents in the ASDN, and this increase was more robust in the cortical collecting duct than in the distal convoluted tubule. Also, HS-induced inhibition of ENaC currents in the ASDN was absent in Nedd4-2-deficient mice. Renal clearance experiments showed that HS intake for 2 wk increased the basal level of renal K+ excretion and caused hypokalemia in Ks-Nedd4-2-KO mice but not in Nedd4lflox/flox mice. In contrast, plasma Na+ concentrations were similar in Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HS diet. We conclude that Nedd4-2 plays an important role in mediating the inhibitory effect of HS diet on Kir4.1, ENaC, and NCC and is essential for maintaining normal renal K+ excretion and plasma K+ ranges during long-term HS diet.NEW & NOTEWORTHY The present study suggests that Nedd4-2 is involved in mediating the inhibitory effect of high salt (HS) diet on Kir4.1/kir5.1 in the distal convoluted tubule, NaCl cotransporter function, and epithelial Na+ channel activity and that Nedd4-2 plays an essential role in maintaining K+ homeostasis in response to a long-term HS diet. This suggests the possibility that HS intake could lead to hypokalemia in subjects lacking proper Nedd4-2 E3 ubiquitin ligase activity in aldosterone-sensitive distal nephron.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Hipopotassemia/etiologia , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Sódio na Dieta/efeitos adversos , Animais , Antibacterianos/farmacologia , Transporte Biológico , Doxiciclina/farmacologia , Canais Epiteliais de Sódio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipopotassemia/induzido quimicamente , Hipopotassemia/genética , Transporte de Íons/fisiologia , Camundongos , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4/genética , Néfrons/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Sódio/metabolismo , Sódio na Dieta/administração & dosagem , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo
19.
J Vis Exp ; (170)2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33871464

RESUMO

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.


Assuntos
Fibroblastos/citologia , Mioblastos/citologia , Diferenciação Celular , Doxiciclina/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Lentivirus/genética , Fibras Musculares Esqueléticas/citologia , Proteína MyoD/genética , Doenças Neuromusculares/tratamento farmacológico , Pele/citologia , Telomerase/genética
20.
Drug Saf ; 44(6): 635-644, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33864232

RESUMO

INTRODUCTION AND OBJECTIVE: Ivermectin (IVM) and doxycycline (DOXY) have demonstrated in-vitro activity against SARS-CoV-2, and have a reasonable safety profile. The objective of this systematic review was to explore the evidence in the literature on the safety and efficacy of their use as monotherapy and combination therapy in COVID-19 management. METHODS: After prospectively registering the study protocol with the Open Science Framework, we searched PubMed, Google Scholar, clinicaltrials.gov, various pre-print servers and reference lists for relevant records published until 16 February, 2021 using appropriate search strategies. Baseline features and data pertaining to efficacy and safety outcomes were extracted separately for IVM monotherapy, DOXY monotherapy, and IVM + DOXY combination therapy. Methodological quality was assessed based on the study design. RESULTS: Out of 200 articles screened, 19 studies (six retrospective cohort studies, seven randomised controlled trials, five non-randomised trials, one case series) with 8754 unique patients with multiple stages of COVID-19 were included; four were pre-prints and one was an unpublished clinicaltrials.gov document. The comparator was standard care and 'hydroxychloroquine + azithromycin' in seven and three studies respectively, and two studies were placebo controlled; six studies did not have a comparator. IVM monotherapy, DOXY monotherapy and IVM + DOXY were explored in eight, five and five studies, respectively; one study compared IVM monotherapy and IVM + DOXY with placebo. While all studies described efficacy, the safety profile was described in only six studies. Efficacy outcomes were mixed with some studies concluding in favour of the intervention and some studies displaying no significant benefit; barring one study that described 9/183 patients with erosive esophagitis and non-ulcer dyspepsia with IVM + DOXY (without causality assessment details), there were no new safety signals of concern with any of the three interventions considered. The quality of studies varied widely, with five studies having a 'good' methodological quality. CONCLUSIONS: Evidence is not sufficiently strong to either promote or refute the efficacy of IVM, DOXY, or their combination in COVID-19 management. SYSTEMATIC REVIEW PROTOCOL REGISTRATION DETAILS: Open Science Framework: https://osf.io/n7r2j .


Assuntos
COVID-19/tratamento farmacológico , Doxiciclina/farmacologia , Ivermectina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Anti-Infecciosos/farmacologia , Quimioterapia Combinada/efeitos adversos , Quimioterapia Combinada/métodos , Humanos , Resultado do Tratamento
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...