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1.
Proc Natl Acad Sci U S A ; 119(49): e2207824119, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36454756

RESUMO

Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.


Assuntos
Reprogramação Celular , Doxiciclina , Animais , Camundongos , Camundongos Transgênicos , Reprogramação Celular/genética , Transgenes , Células Clonais , Doxiciclina/farmacologia
2.
Microb Pathog ; 173(Pt A): 105865, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36332790

RESUMO

A large number of evidence showed that regulatory sRNAs could modulate antibiotic resistance and sensitivity. In this study, we used RNA-sequencing to profile sRNAs in wild type and antibiotic-resistant PAO1 selected by four antibiotics (polymyxin B, ciprofloxacin, doxycycline, and ceftriaxone). Totally, we found 113, 25, 91 and 12 differentially expressed sRNAs in polymyxin B-, ciprofloxacin-, doxycycline-, and ceftriaxone-resistant P. aeruginosa, respectively. To elucidate functions of differentially expressed sRNA, we predicated their target genes and obtained pathways enriched by their target genes. In addition, our results indicated that the downregulated sRNA spae884.1, spae3443.1, and spae5681.1 might involve in polymyxin B resistance by enhancing their target genes arnA, arnD, and arnT expression in PAO1, respectively. The upregulated sRNA spae3443.1 and spae649.2 might implicate in ciprofloxacin resistance by promoting their target gene pslK expression to increase biofilm formation in PAO1. The upregulated spae1558.1 might increase oprJ expression, as well as spae3959.1 and spae3706.1 might increase mexC expression to modulate doxycycline resistance in PAO1. The sRNA novel-N714 might involve in virulence in ceftriaxone-resistant PAO1 by activating its target gene PA1429 expression. Our study might provide bases of the underlying mechanism of sRNA in regulating antibiotic resistance of PAO1 against different antibiotics.


Assuntos
Antibacterianos , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Polimixina B/farmacologia , Ceftriaxona , Doxiciclina/farmacologia , Ciprofloxacina/farmacologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica
3.
Int J Mol Sci ; 23(22)2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36430553

RESUMO

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.


Assuntos
Doxiciclina , Edição de Genes , Tamoxifeno , Animais , Feminino , Camundongos , Doxiciclina/farmacologia , Edição de Genes/métodos , Integrases/genética , Integrases/metabolismo , Camundongos Transgênicos , Tamoxifeno/farmacologia
4.
Biomed Res Int ; 2022: 2760744, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36408277

RESUMO

Objective: As a highly malignant tumour, malignant rhabdoid tumours of the kidney (MRTK) are prone to metastasis and invasion, while tumour metastasis and invasion are inseparable from matrix metalloproteinases (MMPs) and epithelial-mesenchymal transformation (EMT). Moreover, the key to EMT is remodelling of the cytoskeleton. Therefore, our study is aimed at investigating whether doxycycline hydrochloride (DCH), an inhibitor of MMPs, could reverse EMT in MRTK to exert an antitumour effect by regulating MMPs and the cytoskeleton. Methods: The existence of EMT in MRTK cells was verified by bioinformatics analysis, immunofluorescence, and western blotting (WB). In vitro, the proliferation, migration, and invasion abilities of G401 cells were examined by Cell Counting Kit-8 (CCK-8), scratch, and Transwell assays, respectively. The effect of DCH on tumour growth in tumour-bearing mice was explored in in vivo experiments, and the expression of MMP2 and MMP9 and EMT correlation indexes was measured by immunofluorescence and WB, and the changes in cytoskeletal F-actin and ß-tubulin were measured by fluorescence. Results: The altered extracellular matrix (ECM) composition, EMT, and high expression of MMP2 and MMP9 existed in MRTK. DCH inhibited the proliferation, migration, and invasion of G401 cells in vitro. In vivo, DCH inhibited tumour growth in mice, downregulated the expression of MMP2 and MMP9, and partially reversed EMT. Alternatively, DCH resulted in cytoskeletal rearrangements of G401 cells. Conclusions: DCH, as an MMP inhibitor, is used for the first time in MRTK research, showing good antitumour effects by reversing EMT and potentially providing new therapeutic measures for MRTK treatment.


Assuntos
Neoplasias Renais , Tumor Rabdoide , Camundongos , Animais , Transição Epitelial-Mesenquimal , Doxiciclina/farmacologia , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Invasividade Neoplásica , Movimento Celular , Linhagem Celular Tumoral , Citoesqueleto , Metaloproteinases da Matriz , Aberrações Cromossômicas
5.
Toxins (Basel) ; 14(11)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36355993

RESUMO

The presence of deoxynivalenol (DON) in feed may increase intestinal barrier permeability. Disturbance of the intestinal barrier integrity may affect the absorption of antibiotics used in animals. Since the bioavailability of orally administered antibiotics significantly affects their efficacy and safety, it was decided to evaluate how DON influences the absorption of the most commonly used antibiotics in pigs, i.e., amoxicillin (AMX) and doxycycline (DOX). The studies were conducted using jejunal explants from adult pigs. Explants were incubated in Ussing chambers, in which a buffer containing DON (30 µg/mL), AMX (50 µg/mL), DOX (30 µg/mL), a combination of AMX + DON, or a combination of DOX + DON was used. Changes in transepithelial electrical resistance (TEER), the flux of transcellular and intracellular transport markers, and the flux of antibiotics across explants were measured. DON increased the permeability of small intestine explants, expressed by a reduction in TEER and an intensification of transcellular marker transport. DON did not affect AMX transport, but it accelerated DOX transport by approximately five times. The results suggest that DON inhibits the efflux transport of DOX to the intestinal lumen, and thus significantly changes its absorption from the gastrointestinal tract.


Assuntos
Doxiciclina , Jejuno , Suínos , Animais , Doxiciclina/farmacologia , Amoxicilina , Mucosa Intestinal , Antibacterianos
6.
Sci Rep ; 12(1): 19519, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376366

RESUMO

The circadian rhythm is a biological oscillation of physiological activities with a period of approximately 24 h, that is driven by a cell-autonomous oscillator called the circadian clock. The current model of the mammalian circadian clock is based on a transcriptional-translational negative feedback loop in which the protein products of clock genes accumulate in a circadian manner and repress their own transcription. However, several studies have revealed that constitutively expressed clock genes can maintain circadian oscillations. To understand the underlying mechanism, we expressed Bmal1 in Bmal1-disrupted cells using a doxycycline-inducible promoter and monitored Bmal1 and Per2 promoter activity using luciferase reporters. Although the levels of BMAL1 and other clock proteins, REV-ERBα and CLOCK, showed no obvious rhythmicity, robust circadian oscillation in Bmal1 and Per2 promoter activities with the correct phase relationship was observed, which proceeded in a doxycycline-concentration-dependent manner. We applied transient response analysis to the Bmal1 promoter activity in the presence of various doxycycline concentrations. Based on the obtained transfer functions, we suggest that, at least in our experimental system, BMAL1 is not directly involved in the oscillatory process, but modulates the oscillation robustness by regulating basal clock gene promoter activity.


Assuntos
Fatores de Transcrição ARNTL , Relógios Circadianos , Animais , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Doxiciclina/farmacologia , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Mamíferos/metabolismo , Regulação da Expressão Gênica
7.
Int J Mol Sci ; 23(19)2022 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-36232578

RESUMO

A clear relationship between the tau assemblies and toxicity has still to be established. To correlate the tau conformation with its proteotoxic effect in vivo, we developed an innovative cell-worm-based approach. HEK293 cells expressing tau P301L under a tetracycline-inducible system (HEK T-Rex) were employed to produce different tau assemblies whose proteotoxic potential was evaluated using C. elegans. Lysates from cells induced for five days significantly reduced the worm's locomotor activity. This toxic effect was not related to the total amount of tau produced by cells or to its phosphorylation state but was related to the formation of multimeric tau assemblies, particularly tetrameric ones. We investigated the applicability of this approach for testing compounds acting against oligomeric tau toxicity, using doxycycline (Doxy) as a prototype drug. Doxy affected tau solubility and promoted the disassembly of already formed toxic aggregates in lysates of cells induced for five days. These effects translated into a dose-dependent protective action in C. elegans. These findings confirm the validity of the combined HEK T-Rex cells and the C. elegans-based approach as a platform for pharmacological screening.


Assuntos
Tauopatias , Animais , Caenorhabditis elegans , Doxiciclina/farmacologia , Células HEK293 , Humanos , Proteínas tau
8.
Cell Mol Life Sci ; 79(11): 544, 2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36219276

RESUMO

Human embryonic stem cells (hESCs) have unique characteristics, such as self-renewal and pluripotency, which are distinct from those of other cell types. These characteristics of hESCs are tightly regulated by complex signaling mechanisms. In this study, we demonstrate that yes-associated protein (YAP) functions in an hESC-specific manner to maintain self-renewal and survival in hESCs. hESCs were highly sensitive to YAP downregulation to promote cell survival. Interestingly, hESCs displayed dynamic changes in YAP expression in response to YAP downregulation. YAP was critical for the maintenance of self-renewal. Additionally, the function of YAP in maintenance of self-renewal and cell survival was hESC-specific. Doxycycline upregulated YAP in hESCs and attenuated the decreased cell survival induced by YAP downregulation. However, decreased expression of self-renewal markers triggered by YAP downregulation and neural/cardiac differentiation were affected by doxycycline treatment. Collectively, the results reveal the mechanism underlying the role of YAP and the novel function of doxycycline in hESCs.


Assuntos
Células-Tronco Embrionárias Humanas , Diferenciação Celular/fisiologia , Doxiciclina/metabolismo , Doxiciclina/farmacologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Transdução de Sinais , Proteínas de Sinalização YAP
9.
Phytomedicine ; 107: 154482, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36202057

RESUMO

BACKGROUND: CD4+Foxp3+ regulatory T cells (Tregs) represent the primary cellular mechanism of tumor immune evasion. Elimination of Treg activity by the pharmacological agent may enhance anti-tumor immune responses. However, Treg-eliminating agents, especially those with small molecules, are rarely reported. PURPOSE: To identify small molecule inhibitors of Treg cells from natural products. METHODS: Compounds from Diploclisia glaucescens were isolated by column chromatography, and structures were identified by spectroscopic evidence and quantum calculations. The tet-On system for Foxp3-GFP expression in Jurkat T cells was generated to screen Treg inhibitors based on Foxp3 expression. The effect of the compound on TNF-induced proliferative expansion of naturally occurring Tregs (nTregs) and TGF-ß-induced generation of Tregs (iTregs) from naive CD4+ Tcells was further examined. RESULTS: A novel dimeric proaporphine alkaloid, designated as distepharinamide (DSA) with a symmetric structure isolated from the stems of D. glaucescens, restrained the doxycycline (Doxy)-induced Foxp3-tGFP expression, decreased the half-life of Foxp3 mRNA as well as reduced the mRNA levels of chemokine receptors (CCR4, CCR8 and CCR10) in Jurkat T cells with inducible Foxp3-tGFP expression. In lymphocytes or purified Tregs from wild-type C57BL/6 mice or from C57BL/6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax mice, DSA markedly inhibited TNF-induced proliferative expansion of Tregs present in the unfractionated CD4+ T cells, accompanied by the down-regulation of TNFR2, CD25 and CTLA4 expression on Tregs. Furthermore, DSA potently inhibited TGF-ß-induced differentiation of Foxp3-expressing iTregs. Importantly, the expression of Foxp3 mRNA by both nTregs and iTregs was decreased by DSA treatment. Nevertheless, DSA at the same concentrations did not inhibit the proliferation of conventional CD4+ and CD8+ T cells stimulated by anti-CD3/CD28 antibodies. CONCLUSION: DSA, a novel dimeric proaporphine alkaloid, potently inhibited the expansion of nTregs and generation of iTregs. Therefore, DSA or its analogs may merit further investigation as novel immunotherapeutic agents.


Assuntos
Alcaloides , Antineoplásicos , Produtos Biológicos , Alcaloides/metabolismo , Alcaloides/farmacologia , Animais , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos , Antígeno CTLA-4/metabolismo , Doxiciclina/metabolismo , Doxiciclina/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/farmacologia , Linfócitos T Reguladores , Fator de Crescimento Transformador beta/metabolismo
10.
Nat Commun ; 13(1): 6442, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36307419

RESUMO

The experimental need to engineer the genome both in time and space, has led to the development of several photoactivatable Cre recombinase systems. However, the combination of inefficient and non-intentional background recombination has prevented thus far the wide application of these systems in biological and biomedical research. Here, we engineer an optimized photoactivatable Cre recombinase system that we refer to as doxycycline- and light-inducible Cre recombinase (DiLiCre). Following extensive characterization in cancer cell and organoid systems, we generate a DiLiCre mouse line, and illustrated the biological applicability of DiLiCre for light-induced mutagenesis in vivo and positional cell-tracing by intravital microscopy. These experiments illustrate how newly formed HrasV12 mutant cells follow an unnatural movement towards the interfollicular dermis. Together, we develop an efficient photoactivatable Cre recombinase mouse model and illustrate how this model is a powerful genome-editing tool for biological and biomedical research.


Assuntos
Doxiciclina , Optogenética , Camundongos , Animais , Doxiciclina/farmacologia , Camundongos Transgênicos , Edição de Genes , Integrases/genética , Integrases/metabolismo , Camundongos Endogâmicos
11.
Acta Biomater ; 152: 60-73, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36049625

RESUMO

Patients with diabetes suffer from a variety of complications and easily develop diabetic chronic wounds. The microenvironment of diabetic wounds is characterized by an excessive amount of reactive oxygen species (ROS) and an imbalance of proinflammatory and anti-inflammatory cells/factors, which hinder the regeneration of chronic wounds. In the present study, a wound dressing with immunomodulation and electroconductivity properties was prepared and assayed in vitro and in vivo. [2-(acryloyloxy) ethyl] Trimethylammonium chloride (Bio-IL) and gelatin methacrylate (GelMA) were 3D printed onto a doxycycline hydrochloride (DOXH)-loaded and ROS-degradable polyurethane (PFKU) nanofibrous membrane, followed by UV irradiation to obtain conductive hydrogel strips. DOXH was released more rapidly under a high ROS environment. The dressing promoted migration of endothelial cells and polarization of macrophages to the anti-inflammatory phenotype (M2) in vitro. In a diabetic rat wound healing test, the combination of conductivity and DOXH was most effective in accelerating wound healing, collagen deposition, revascularization, and re-epithelialization by downregulating ROS and inflammatory factor levels as well as by upregulating the M2 macrophage ratio. STATEMENT OF SIGNIFICANCE: The microenvironment of diabetic wounds is characterized by an excessive amount of reactive oxygen species (ROS) and an imbalance of proinflammatory and anti-inflammatory cells/factors, which hinder the regeneration of chronic wounds. Herein, a wound dressing composed of a DOXH-loaded ROS-responsive polyurethane membrane and 3D-printed conductive hydrogel strips was prepared, which effectively accelerated skin regeneration in diabetic wounds in vivo with better epithelialization, angiogenesis, and collagen deposition. DOXH regulated the dysfunctional wound microenvironment by ROS scavenging and polarizing macrophages to M2 phenotype, thereby playing a dominant role in diabetic wound regeneration. This design may have great potential for preparing other similar materials for the therapy of other diseases with excessive inflammation or damage to electrophysiological organs, such as nerve defect and myocardial infarction.


Assuntos
Diabetes Mellitus , Nanofibras , Animais , Cloretos/farmacologia , Colágeno/farmacologia , Doxiciclina/farmacologia , Células Endoteliais , Gelatina/farmacologia , Hidrogéis/farmacologia , Metacrilatos/farmacologia , Poliuretanos/farmacologia , Ratos , Espécies Reativas de Oxigênio , Cicatrização
12.
FEBS Open Bio ; 12(10): 1896-1908, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36062323

RESUMO

The tetracycline repressor (tetR)-regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA-binding protein 43 (TDP-43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP-43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA-approved drugs. While the primary screen identified several compounds that prevented TDP-43 toxicity, further experiments revealed that these chemicals abrogated the doxycycline-dependent TDP-43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet-On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome-wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase and TRIM28 as potential modifiers of TDP-43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline-dependent expression of TDP-43. In summary, we have identified new chemical and genetic regulators of the tetR system, thereby raising awareness of the limitations of this approach to conduct chemical or genetic screening in mammalian cells.


Assuntos
Doxiciclina , Proteínas Repressoras , Antibacterianos , Proteínas de Ligação a DNA/genética , Doxiciclina/farmacologia , Expressão Gênica , Testes Genéticos , Humanos , Metiltransferases/genética , Proteínas Repressoras/metabolismo , Tetraciclina/farmacologia , Fatores de Transcrição/genética
13.
Toxicol Lett ; 370: 1-6, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36100150

RESUMO

Exposure of humans to aflatoxin B1 (AFB1) via ingestion of contaminated agricultural products is a major concern for human health throughout the world because epoxidized AFB1, biotransformed from AFB1 by hepatic CYP3A4, is strongly hepatotoxic and hepatocarcinogenic. Intestinal epithelial cells serve as a physical and physiological barrier against xenobiotics via their intercellular tight junction (TJ) seals and the metabolizing enzyme CYP3A4. However, the effect of AFB1 on the intestinal barrier remains unclear. Here, we investigated the influence of AFB1 on these physical and physiological intestinal barriers by means of an in vitro human intestinal model utilizing doxycycline-inducible CYP3A4-expressing Caco-2 cells, in which CYP3A4 activity is comparable to that in the adult human intestine. Cellular toxicity of AFB1 in induced Caco-2 cells (i.e., cells in which expression of CYP3A4 is induced by doxycycline) was approximately 5 times that of uninduced Caco-2 cells. Exposure to 16 µM AFB1 did not decrease the transepithelial electric resistance (TEER; a measure of TJ barrier integrity) in monolayers of uninduced Caco-2 cells to 95.8 % of that in vehicle-treated cells; in contrast, in induced Caco-2 cells, TEER was reduced to 28.8 %. Exposure to 16 µM AFB1 increased paracellular permeation of 4- and 20-kDa dextrans (paracellular permeation markers) through monolayers of induced Caco-2 cells to 5.4 and 5.2 times that through uninduced Caco-2 cells. These results together show that ingested AFB1 can modulate the intestinal barrier, and that inducible CYP3A4-expressing Caco-2 cells are a promising tool for evaluating the safety of food contaminants in the human intestine.


Assuntos
Aflatoxina B1 , Citocromo P-450 CYP3A , Adulto , Aflatoxina B1/toxicidade , Células CACO-2 , Citocromo P-450 CYP3A/metabolismo , Dextranos/metabolismo , Dextranos/farmacologia , Doxiciclina/farmacologia , Humanos , Intestinos , Junções Íntimas
14.
Pathog Dis ; 80(1)2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36152595

RESUMO

Enterococci are important pathogens of nosocomial infections and are increasingly difficult to treat due to their intrinsic and acquired resistance to a range of antibiotics. Therefore, there is an urgent need to develop novel antibacterial agents, while drug repurposing is a promising approach to address this issue. Our study aimed to determine the antimicrobial efficacy of halicin against enterococci and found that the minimum inhibitory concentrations (MIC) of halicin against different strains of Enterococcus faecalis and Enterococcus faecium ranged from 4 to 8 µg/ml. In addition, the synergistic antibacterial effect between halicin and doxycycline (DOX) against Enterococcus was observed through the checkerboard method, and it was observed that halicin and DOX could significantly synergistically inhibit biofilm formation and eradicate preformed biofilms at sub-MICs. Moreover, the electron microscope results revealed that halicin could also disrupt the bacterial cell membrane at high concentrations. Furthermore, it is also confirmed that the combination of halicin and DOX has no significant cytotoxic effect on erythrocytes and other human-derived cells. In addition, the mouse subcutaneous model and H&E staining showed that the combination of halicin and DOX could effectively reduce the bacterial load and inflammatory infiltration without obvious side effects. In nutshell, these results demonstrate the potential of halicin in combination with DOX as a novel therapy against infections by Enterococcus.


Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Doxiciclina/farmacologia , Doxiciclina/uso terapêutico , Enterococcus , Enterococcus faecalis , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Tiadiazóis
15.
Cytokine ; 160: 156022, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36099756

RESUMO

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.


Assuntos
Doxiciclina , Ligante RANK/metabolismo , Transcriptoma , Aglutininas/metabolismo , Animais , Citocinas/metabolismo , Doxiciclina/farmacologia , Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Queratina-5/genética , Queratina-5/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Fenótipo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Timo/metabolismo
16.
FEMS Yeast Res ; 22(1)2022 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-36047937

RESUMO

Candida glabrata is an important pathogen causing superficial to invasive disease in human. Conditional expression systems are helpful in addressing the function of genes and especially when they can be applied to in vivo studies. Tetracycline-dependent regulation systems have been used in diverse fungi to turn-on (Tet-on) or turn-off (Tet-off) gene expression either in vitro but also in vivo in animal models. Up to now, only a Tet-off expression has been constructed for gene expression in C. glabrata. Here, we report a Tet-on gene expression system which can be used in vitro and in vivo in any C. glabrata genetic background. This system was used in a mice model of systemic infection to demonstrate that the general amino acid permease Gap1 is important for C. glabrata virulence.


Assuntos
Candida glabrata , Doxiciclina , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Candida glabrata/metabolismo , Doxiciclina/metabolismo , Doxiciclina/farmacologia , Humanos , Camundongos , Tetraciclina/metabolismo , Virulência
17.
Food Chem Toxicol ; 169: 113431, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36116547

RESUMO

Most of the studies on doxycycline (DOX) and florfenicol (FF) remain focused on the improvement of antimicrobial activity and antimicrobial spectrum, and there is no relevant report on whether there is interaction between the two drugs after the combination. This research study evaluated the effect of DOX on FF metabolism in vitro and its mechanisms. The findings of this study showed that DOX inhibits FF metabolism in two ways. Firstly, DOX significantly inhibits the expression of CYP3A29, leading to the slower metabolism of FF; secondly, DOX affects the binding of FF to R106 and R372 by competing for the R372 and/or by a "steric-like effect", thus slowing down FF metabolism, which may increase the residual concentration of FF in edible tissues of food producing animals.


Assuntos
Antibacterianos , Citocromo P-450 CYP3A , Doxiciclina , Tianfenicol , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Ligação Competitiva , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Doxiciclina/farmacologia , Interações Medicamentosas , Tianfenicol/análogos & derivados , Tianfenicol/metabolismo , Tianfenicol/farmacologia , Suínos , Mutação
18.
Dalton Trans ; 51(38): 14458-14465, 2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36069661

RESUMO

Tetracycline antibiotic residues have attracted worldwide attention due to the serious damage to human health and the environment. However, most of the reported fluorescent probes were based on a single fluorescence channel-based response, which often suffered from signal fluctuation-induced poor reproducibility. Herein, by taking advantage of the unique properties of zeolitic imidazolate frameworks (ZIFs), a novel europium-doped ZIF nanocomposite (ZIF-Eu) was reported for the detection of doxycycline (DOX) in a ratiometric fluorescence manner. The synthesized probe only showed blue fluorescence at 420 nm since the fluorescence of Eu was quenched by the coordinated water molecules. However, due to the strong coordination ability of DOX to Eu atoms, the probe solution demonstrated an obvious fluorescence enhancement at 615 nm in the presence of DOX, while the blue fluorescence signal remained unchanged, realizing a ratiometric fluorescence response to DOX with a good linear range from 1 to 9 µM and a detection limit of 49 nM. Interestingly, it is found that DOX could be discriminated from other tetracycline antibiotics using this ratiometric fluorescent probe. In addition, direct detection of DOX in soil samples, the DOX removal efficiency and the reusability of the proposed ZIF-Eu nanocomposite were demonstrated to evaluate its potential in environmental remediation application.


Assuntos
Antibacterianos , Corantes Fluorescentes , Antibacterianos/farmacologia , Doxiciclina/farmacologia , Európio/química , Corantes Fluorescentes/química , Humanos , Reprodutibilidade dos Testes , Solo , Água
19.
Ann Med ; 54(1): 2500-2510, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36120867

RESUMO

Objective: To determine the minimum inhibitory concentration (MIC) distribution of antibacterial drugs and the susceptibility of non-tuberculous mycobacterial (NTM) isolates to provide a reference basis for the clinical selection of an effective starting regimen.Methods: The common clinical isolates of NTM in the respiratory tract, which met the standards of the American Thoracic Society for NTM lung disease, were collected. The MICs of 81 isolates were determined using the microbroth dilution method (Thermo Fisher Scientific, USA), as recommended by the Clinical and Laboratory Standards Institute, USA.Results: Included were 43 Mycobacterium avium complex (MAC) strains, 24 M. abscessus complex (MAB) strains, and 14 M. kansasii strains. The sensitivity rates of MAC to clarithromycin and amikacin were 81.4% and 79.1%, respectively, while the sensitivity rates to linezolid and moxifloxacin were only 20.9% and 9.3%; the MIC of rifabutin was the lowest (MIC50% was just 2 µg/mL). After incubation for 3-5 days, the sensitivity rate of MAB to clarithromycin was 87.5%; this decreased to 50% after 14 days' incubation. Most of them were susceptible to amikacin (91.6%), and most were resistant to moxifloxacin (95.8%), ciprofloxacin (95.8%), imipenem (95.8%), amoxicillin/clavulanate (95.8%), tobramycin (79.1%), doxycycline (95.8%) and trimethoprim/sulfamethoxazole (95.8%). intermediate (83.3%) and resistant (16.7%) to cefoxitin. The susceptibility to linezolid was only 33.3%. The sensitivity and resistance breakpoints of tigecycline were set to ≤0.5 and ≥8 µg/mL, respectively, and the sensitivity and resistance rates were 50% and 0%, respectively. M. kansasii was susceptible to clarithromycin, amikacin, linezolid, moxifloxacin, rifampicin and rifabutin (100%).Discussion: In Wenzhou, clarithromycin, amikacin and rifabutin have good antibacterial activity against MAC, while linezolid and moxifloxacin have high resistance. Amikacin and tigecycline have strong antibacterial activity against MAB, while most other antibacterial drugs are resistant to varying degrees. Most antibacterial drugs are susceptible to M. kansasii and have good antibacterial activity.Conclusion: The identification of NTM species and the detection of their MICs have certain guiding values for the treatment of NTM lung disease.Key MessageThe three most common respiratory non-tuberculous mycobacterial (NTM) isolates with clinical significance in the Wenzhou area were tested for drug susceptibility. The broth microdilution method was used to determine the minimum inhibitory concentration distribution of antibacterial drugs and the susceptibility of NTM isolates to provide a reference basis for the clinical selection of an effective starting regimen.


Assuntos
Pneumopatias , Infecções por Mycobacterium não Tuberculosas , Amicacina/farmacologia , Amicacina/uso terapêutico , Amoxicilina , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cefoxitina/farmacologia , Cefoxitina/uso terapêutico , Ciprofloxacina/farmacologia , Ciprofloxacina/uso terapêutico , Claritromicina/farmacologia , Ácido Clavulânico/farmacologia , Ácido Clavulânico/uso terapêutico , Doxiciclina/farmacologia , Doxiciclina/uso terapêutico , Humanos , Imipenem/farmacologia , Imipenem/uso terapêutico , Linezolida/farmacologia , Linezolida/uso terapêutico , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Moxifloxacina/uso terapêutico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas , Sistema Respiratório , Rifabutina/farmacologia , Rifabutina/uso terapêutico , Rifampina/farmacologia , Rifampina/uso terapêutico , Sulfametoxazol/farmacologia , Sulfametoxazol/uso terapêutico , Tigeciclina/farmacologia , Tigeciclina/uso terapêutico , Tobramicina/farmacologia , Tobramicina/uso terapêutico , Trimetoprima/farmacologia , Trimetoprima/uso terapêutico
20.
Front Public Health ; 10: 988317, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36176509

RESUMO

Human non-typhoidal salmonellosis is among the leading cause of morbidity and mortality worldwide, resulting in huge economic losses and threatening the public health systems. To date, epidemiological characteristics of non-typhoidal Salmonella (NTS) implicated in human salmonellosis in China are still obscure. Herein, we investigate the antimicrobial resistance and genomic features of NTS isolated from outpatients in Shaoxing city in 2020. Eighty-seven Salmonella isolates were recovered and tested against 28 different antimicrobial agents, representing 12 categories. The results showed high resistance to cefazolin (86.21%), streptomycin (81.61%), ampicillin (77.01%), ampicillin-sulbactam (74.71%), doxycycline (72.41%), tetracycline (71.26%), and levofloxacin (70.11%). Moreover, 83.91% of isolates were resistant to ≥3 categories, which were considered multi-drug resistant (MDR). Whole-genome sequencing (WGS) combined with bioinformatic analysis was used to predict serovars, MLST types, plasmid replicons, antimicrobial resistance genes, and virulence genes, in addition to the construction of phylogenomic to determine the epidemiological relatedness between isolates. Fifteen serovars and 16 STs were identified, with the dominance of S. I 4, [5], 12:i:- ST34 (25.29%), S. Enteritidis ST11 (22.99%), and S. Typhimurium ST19. Additionally, 50 resistance genes representing ten categories were detected with a high prevalence of aac(6')-Iaa (100%), bla TEM-1B (65.52%), and tet(A) (52.87%), encoding resistance to aminoglycosides, ß-lactams, and tetracyclines, respectively; in addition to chromosomic mutations affecting gyrA gene. Moreover, we showed the detection of 18 different plasmids with the dominance of IncFIB(S) and IncFII(S) (39.08%). Interestingly, all isolates harbor the typical virulence genes implicated in the virulence mechanisms of Salmonella, while one isolate of S. Jangwani contains the cdtB gene encoding typhoid toxin production. Furthermore, the phylogenomic analysis showed that all isolates of the same serovar are very close to each other and clustered together in the same clade. Together, we showed a high incidence of MDR among the studied isolates which is alarming for public health services and is a major threat to the currently available treatments to deal with human salmonellosis; hence, efforts should be gathered to further introduce WGS in routinely monitoring of AMR Salmonella in the medical field in order to enhance the effectiveness of surveillance systems and to limit the spread of MDR clones.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Infecções por Salmonella , Salmonella , Aminoglicosídeos/farmacologia , Ampicilina/farmacologia , Antibacterianos/farmacologia , Cefazolina/farmacologia , Doxiciclina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Genômica , Humanos , Levofloxacino/farmacologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Pacientes Ambulatoriais , Salmonella/efeitos dos fármacos , Salmonella/genética , Salmonella/isolamento & purificação , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/microbiologia , Estreptomicina/farmacologia , Tetraciclina/farmacologia
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