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1.
Nat Commun ; 12(1): 1782, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741963

RESUMEN

Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.


Asunto(s)
Macrólidos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biocatálisis/efectos de los fármacos , Bovinos , Microscopía por Crioelectrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Macrólidos/química , Macrólidos/metabolismo , Modelos Moleculares , Estructura Molecular , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/ultraestructura
2.
Nat Commun ; 12(1): 1732, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741980

RESUMEN

Macrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides' macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.


Asunto(s)
Antibacterianos/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Esterasas/química , Esterasas/genética , Macrólidos/química , Antibacterianos/farmacología , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Eritromicina/química , Genes Bacterianos , Macrólidos/farmacología , Simulación del Acoplamiento Molecular , Conformación Proteica , Difracción de Rayos X
3.
Nat Commun ; 12(1): 1864, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767144

RESUMEN

Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.


Asunto(s)
Antígenos de Diferenciación/metabolismo , Transporte Biológico/fisiología , Comunicación Celular/fisiología , Vesículas Extracelulares/metabolismo , Línea Celular Tumoral , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Luciferasas/metabolismo , Macrólidos/farmacología , Fusión de Membrana/fisiología
4.
J Med Microbiol ; 70(3)2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33570485

RESUMEN

Introduction. Mycobacterium abscessus complex (MABC) is an infectious agent associated with macrolide resistance and treatment failure.Hypothesis/Gap Statement. Despite drug-susceptibility testing for MABC isolates including clarithromycin (CAM), long-term treatment with azithromycin (AZM) for MABC disease is recommended.Aim. We compared phenotypic and genotypic resistance to AZM and CAM in clinical isolates and evaluated the accumulation of intrinsic macrolide resistance (AIM) and morphological changes by macrolides exposure.Methodology. Forty-nine isolates were characterized regarding erm(41) sequevars. Sequencing data were compared to the nucleotide sequence of rrl and whiB7. The AIM MIC was performed in three reference strains and 15 isolates were randomized [each set of five isolates with M. abscessus subsp. abscessus (MAA) T28, MAA C28 and subsp. massiliense (MAM)].Results. The 49 isolates were distributed as 24 MAA T28, 5 MAA C28 and 20 MAM. The MIC50 values to CAM at day 3 in MAA T28, C28 and MAM were 1, 0.12 and 0.12 µg ml-1, while those at day 14 were 32, 0.5 and 0.12 µg ml-1, respectively. The AZM-MIC50 values at day 3 of the above isolates were 4, 0.25 and 0.5 µg ml-1, while those at day 14 were >64, 0.5 and 0.5 µg ml-1, respectively. Neither mutations in rrl of MAA T28 with acquired resistance nor deletions in whiB7 of MAA T28 without inducible resistance were observed . For AIM MIC, MAA T28 showed that the time-to-detection of AZM resistance was significantly faster over that of CAM (P<0.05). Morphological changes were not determined in all isolates.Conclusion. Our findings did not support the suggestion for the preferential use of AZM for, at least, MAA T28 disease due to the high-level MIC value and the increased AIM. The long duration of AZM-based treatment eventually may favour the emergence of isolates with a high-level of intrinsic resistance.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Macrólidos/farmacología , Mycobacterium abscessus/aislamiento & purificación , Azitromicina/farmacología , Azitromicina/uso terapéutico , Proteínas Bacterianas/genética , Claritromicina/farmacología , Claritromicina/uso terapéutico , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/genética , Fenotipo
5.
J Nat Prod ; 84(2): 537-543, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33631936

RESUMEN

A new bafilomycin derivative (1) and another seven known bafilomycins (2-8) were isolated from feces-derived Streptomyces sp. HTL16. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic analysis. Biological testing demonstrated that these bafilomycins exhibited potent antiviral activities against the influenza A and SARS-CoV-2 viruses, with IC50 values in the nanomolar range, by inhibiting the activity of endosomal ATP-driven proton pumps.


Asunto(s)
Antivirales/farmacología , Heces/microbiología , Macrólidos/farmacología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Streptomyces/metabolismo , Animales , Perros , Virus de la Influenza A/efectos de los fármacos , Células de Riñón Canino Madin Darby , /efectos de los fármacos
6.
Virol J ; 18(1): 46, 2021 02 27.
Artículo en Inglés | MEDLINE | ID: mdl-33639976

RESUMEN

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and broke out as a global pandemic in late 2019. The acidic pH environment of endosomes is believed to be essential for SARS-CoV-2 to be able to enter cells and begin replication. However, the clinical use of endosomal acidification inhibitors, typically chloroquine, has been controversial with this respect. METHODS: In this study, RT-qPCR method was used to detect the SARS-CoV-2N gene to evaluate viral replication. The CCK-8 assay was also used to evaluate the cytotoxic effect of SARS-CoV-2. In situ hybridization was used to examine the distribution of the SARS-CoV-2 gene in lung tissues. Hematoxylin and eosin staining was also used to evaluate virus-associated pathological changes in lung tissues. RESULTS: In this study, analysis showed that endosomal acidification inhibitors, including chloroquine, bafilomycin A1 and NH4CL, significantly reduced the viral yields of SARS-CoV-2 in Vero E6, Huh-7 and 293T-ACE2 cells. Chloroquine and bafilomycin A1 also improved the viability and proliferation of Vero E6 cells after SARS-CoV-2 infection. Moreover, in the hACE2 transgenic mice model of SARS-CoV-2 infection, chloroquine and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues, as well as improved structures of alveolar septum and pulmonary alveoli. CONCLUSIONS: Our research investigated the antiviral effects of endosomal acidification inhibitors against SARS-CoV-2 in several infection models and provides an experimental basis for further mechanistic studies and drug development.


Asunto(s)
Antivirales/farmacología , /virología , Endosomas/efectos de los fármacos , /fisiología , Replicación Viral/efectos de los fármacos , Cloruro de Amonio/farmacología , /metabolismo , Animales , /patología , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cloroquina/farmacología , Endosomas/metabolismo , Femenino , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Pulmón/patología , Macrólidos/farmacología , Ratones , Ratones Transgénicos , Distribución Aleatoria , Células Vero
7.
Carbohydr Polym ; 255: 117484, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33436244

RESUMEN

Wound dressing composed of chitosan, based crosslinked gelatin/ polyvinyl pyrrolidone, embedded silver nanoparticles were fabricated using solution casting method. The membrane was characterized by FTIR, SEM and TGA. Glutaraldehyde (0.5 %) was used for the crosslinking of membrane components and associated with 7-folds boosted mechanical performance, 28 % more hydrolytic stability, 3-folds thickness reduction and morphological roughness. Silver nanoparticles were characterized by UV-vis, XRD and TEM for an average size of 9.9 nm. The membrane with higher concentration of silver nanoparticles showed maximum antibacterial activity against human pathogenic bacteria; and the measured inhibition zones ranged from 1.5 to 3 cm. The activity of the particles ranged from severe to complete reduction in Penicillin, Erythromycin and Macrolide family's resistance genes expression such as ß-Lactamase, mecA and erm. This developed membrane can serve as promising and cost-effective system against severe diabetic and burn wound infections.


Asunto(s)
Antibacterianos/farmacología , Vendajes , Quitosano/química , Citrullus colocynthis/química , Gelatina/química , Povidona/química , Plata/farmacología , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Eritromicina/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Macrólidos/farmacología , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Metiltransferasas/genética , Metiltransferasas/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Penicilinas/farmacología , Cultivo Primario de Células , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Salmonella typhi/efectos de los fármacos , Salmonella typhi/crecimiento & desarrollo , Plata/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , beta-Lactamasas/genética , beta-Lactamasas/metabolismo
8.
Biochem Biophys Res Commun ; 534: 107-113, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33316543

RESUMEN

Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.


Asunto(s)
Autofagia/efectos de los fármacos , Encéfalo/efectos de los fármacos , Cloroquina/farmacología , Lisosomas/efectos de los fármacos , Animales , Evaluación Preclínica de Medicamentos/métodos , Femenino , Células HeLa , Humanos , Lisosomas/metabolismo , Macrólidos/farmacología , Masculino , Mefloquina/farmacología , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Promazina/farmacología , Quinacrina/farmacología , Trifluoperazina/farmacología
9.
Food Chem ; 339: 127580, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-32858380

RESUMEN

In this study, a microbiological inhibition method for rapidly screening antibiotics in swine urine was established with an easy sample pre-treatment. The microbiological system consisted of an agar medium mixed with nutrients, sensitizers, a test bacterium (Geobacillus stearothermophilus ATCC12980) and pH indicator (bromocresol purple). It was observed that the detection limits of the test kit for twenty-eight common antimicrobial residues in urine, including ß-lactams, aminoglycosides, tetracyclines, sulfonamides, macrolides, and lincosamides, were less than or equal to the maximum residue limits of the kidney, as determined by the EU and China. Moreover, the false negative rate and the false positive rate, along with other performance indexes such as interassay coefficients of variation and shelf life of the kit, all met the standard requirements of the ISO13969:2003 guidelines. Additionally, our results were consistent with those using the gold-standard physical chemistry method, which suggest the proposed method is suitable for screening antibiotic residues.


Asunto(s)
Antibacterianos/orina , Residuos de Medicamentos/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Drogas Veterinarias/orina , Aminoglicósidos/farmacología , Aminoglicósidos/orina , Animales , Antibacterianos/análisis , Antibacterianos/farmacología , Medios de Cultivo , Reacciones Falso Negativas , Reacciones Falso Positivas , Contaminación de Alimentos/análisis , Geobacillus stearothermophilus/efectos de los fármacos , Límite de Detección , Macrólidos/farmacología , Macrólidos/orina , Sensibilidad y Especificidad , Sulfonamidas/farmacología , Sulfonamidas/orina , Porcinos , Tetraciclinas/farmacología , Tetraciclinas/orina , Drogas Veterinarias/farmacología
10.
PLoS One ; 15(12): e0224953, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33370278

RESUMEN

The spliceosome assembles on pre-mRNA in a stepwise manner through five successive pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the spliceosome have been identified and they are cytotoxic. However, little is known about genetic determinants of cell sensitivity. Activating transcription factor 3 (ATF3) is a transcription factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, we used a genetic approach to determine if ATF3 was important in determining the sensitivity of mouse embryonic fibroblasts (MEFs) to two splicing inhibitors: pladienolide B (PB) and isoginkgetin (IGG), that target different pre-spliceosome complexes. Both compounds led to increased ATF3 expression and apoptosis in control MEFs while ATF3 null cells were significantly protected from the cytotoxic effects of these drugs. Similarly, ATF3 was induced in response to IGG and PB in the two human tumour cell lines tested while knockdown of ATF3 protected cells from both drugs. Taken together, ATF3 appears to contribute to the cytotoxicity elicited by these spliceosome inhibitors in both murine and human cells.


Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Biflavonoides/farmacología , Muerte Celular/efectos de los fármacos , Compuestos Epoxi/farmacología , Fibroblastos/efectos de los fármacos , Macrólidos/farmacología , Empalmosomas/metabolismo , Factor de Transcripción Activador 3/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Muerte Celular/fisiología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Ratones , ARN Interferente Pequeño
11.
Proc Natl Acad Sci U S A ; 117(52): 33519-33529, 2020 12 29.
Artículo en Inglés | MEDLINE | ID: mdl-33318204

RESUMEN

Pseudomonas aeruginosa causes severe multidrug-resistant infections that often lead to bacteremia and sepsis. Physiologically relevant conditions can increase the susceptibility of pathogens to antibiotics, such as azithromycin (AZM). When compared to minimal-inhibitory concentrations (MICs) in laboratory media, AZM had a 16-fold lower MIC in tissue culture medium with 5% Mueller Hinton broth (MHB) and a 64-fold lower MIC in this tissue culture medium with 20% human serum. AZM also demonstrated increased synergy in combination with synthetic host-defense peptides DJK-5 and IDR-1018 under host-like conditions and in a murine abscess model. To mechanistically study the altered effects of AZM under physiologically relevant conditions, global transcriptional analysis was performed on P. aeruginosa with and without effective concentrations of AZM. This revealed that the arn operon, mediating arabinosaminylation of lipopolysaccharides and related regulatory systems, was down-regulated in host-like media when compared to MHB. Inactivation of genes within the arn operon led to increased susceptibility of P. aeruginosa to AZM and great increases in synergy between AZM and other antimicrobial agents, indicating that dysregulation of the arn operon might explain increased AZM uptake and synergy in host-like media. Furthermore, genes involved in central and energy metabolism and ribosome biogenesis were dysregulated more in physiologically relevant conditions treated with AZM, likely due to general changes in cell physiology as a result of the increased effectiveness of AZM in these conditions. These data suggest that, in addition to the arn operon, there are multiple factors in host-like environments that are responsible for observed changes in susceptibility.


Asunto(s)
Azitromicina/farmacología , Medios de Cultivo/farmacología , Pseudomonas aeruginosa/crecimiento & desarrollo , Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Sinergismo Farmacológico , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Macrólidos/farmacología , Pruebas de Sensibilidad Microbiana , Operón/genética , Péptidos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Suero
12.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-33348896

RESUMEN

SF3B1 is a core component of the U2 spliceosome that is frequently mutated in cancer. We have previously shown that titrating the activity of SF3B1, using the inhibitor pladienolide B (PB), affects distinct steps of the heat shock response (HSR). Here, we identify other genes that are sensitive to different levels of SF3B1 (5 vs. 100 nM PB) using RNA sequencing. Significant changes to mRNA splicing were identified at both low PB and high PB concentrations. Changes in expression were also identified in the absence of alternative splicing, suggesting that SF3B1 influences other gene expression pathways. Surprisingly, gene expression changes identified in low PB are not predictive of changes in high PB. Specific pathways were identified with differential sensitivity to PB concentration, including nonsense-mediated decay and protein-folding homeostasis, both of which were validated using independent reporter constructs. Strikingly, cells exposed to low PB displayed enhanced protein-folding capacity relative to untreated cells. These data reveal that the transcriptome is exquisitely sensitive to SF3B1 and suggests that the activity of SF3B1 is finely regulated to coordinate mRNA splicing, gene expression and cellular physiology.


Asunto(s)
Empalme Alternativo , Fenómenos Fisiológicos Celulares , Regulación de la Expresión Génica/efectos de los fármacos , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/metabolismo , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma/efectos de los fármacos , Compuestos Epoxi/farmacología , Células HEK293 , Humanos , Macrólidos/farmacología , Degradación de ARNm Mediada por Codón sin Sentido , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , ARN Mensajero/genética
13.
N Engl J Med ; 383(20): 1941-1950, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33176084

RESUMEN

BACKGROUND: Mass distribution of azithromycin to preschool children twice yearly for 2 years has been shown to reduce childhood mortality in sub-Saharan Africa but at the cost of amplifying macrolide resistance. The effects on the gut resistome, a reservoir of antimicrobial resistance genes in the body, of twice-yearly administration of azithromycin for a longer period are unclear. METHODS: We investigated the gut resistome of children after they received twice-yearly distributions of azithromycin for 4 years. In the Niger site of the MORDOR trial, we enrolled 30 villages in a concurrent trial in which they were randomly assigned to receive mass distribution of either azithromycin or placebo, offered to all children 1 to 59 months of age every 6 months for 4 years. Rectal swabs were collected at baseline, 36 months, and 48 months for analysis of the participants' gut resistome. The primary outcome was the ratio of macrolide-resistance determinants in the azithromycin group to those in the placebo group at 48 months. RESULTS: Over the entire 48-month period, the mean (±SD) coverage was 86.6±12% in the villages that received placebo and 83.2±16.4% in the villages that received azithromycin. A total of 3232 samples were collected during the entire trial period; of the samples obtained at the 48-month monitoring visit, 546 samples from 15 villages that received placebo and 504 from 14 villages that received azithromycin were analyzed. Determinants of macrolide resistance were higher in the azithromycin group than in the placebo group: 7.4 times as high (95% confidence interval [CI], 4.0 to 16.7) at 36 months and 7.5 times as high (95% CI, 3.8 to 23.1) at 48 months. Continued mass azithromycin distributions also selected for determinants of nonmacrolide resistance, including resistance to beta-lactam antibiotics, an antibiotic class prescribed frequently in this region of Africa. CONCLUSIONS: Among villages assigned to receive mass distributions of azithromycin or placebo twice yearly for 4 years, antibiotic resistance was more common in the villages that received azithromycin than in those that received placebo. This trial showed that mass azithromycin distributions may propagate antibiotic resistance. (Funded by the Bill and Melinda Gates Foundation and others; ClinicalTrials.gov number, NCT02047981.).


Asunto(s)
Antibacterianos/administración & dosificación , Azitromicina/administración & dosificación , Farmacorresistencia Bacteriana/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Macrólidos/farmacología , Administración Masiva de Medicamentos , Antibacterianos/farmacología , Azitromicina/farmacología , Mortalidad del Niño , Preescolar , Farmacorresistencia Bacteriana/genética , Femenino , Humanos , Lactante , Macrólidos/uso terapéutico , Masculino , Metagenoma , Niger , Análisis de Secuencia de ADN
14.
Nat Commun ; 11(1): 5374, 2020 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-33097713

RESUMEN

The emergence of resistance to azithromycin complicates treatment of Neisseria gonorrhoeae, the etiologic agent of gonorrhea. Substantial azithromycin resistance remains unexplained after accounting for known resistance mutations. Bacterial genome-wide association studies (GWAS) can identify novel resistance genes but must control for genetic confounders while maintaining power. Here, we show that compared to single-locus GWAS, conducting GWAS conditioned on known resistance mutations reduces the number of false positives and identifies a G70D mutation in the RplD 50S ribosomal protein L4 as significantly associated with increased azithromycin resistance (p-value = 1.08 × 10-11). We experimentally confirm our GWAS results and demonstrate that RplD G70D and other macrolide binding site mutations are prevalent (present in 5.42% of 4850 isolates) and widespread (identified in 21/65 countries across two decades). Overall, our findings demonstrate the utility of conditional associations for improving the performance of microbial GWAS and advance our understanding of the genetic basis of macrolide resistance.


Asunto(s)
Farmacorresistencia Bacteriana/genética , Genoma Bacteriano , Estudio de Asociación del Genoma Completo , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Antibacterianos/farmacología , Azitromicina/farmacología , Sitios de Unión/genética , Gonorrea/tratamiento farmacológico , Gonorrea/microbiología , Humanos , Macrólidos/farmacología , Pruebas de Sensibilidad Microbiana , Mutación/efectos de los fármacos , ARN Ribosómico 23S/genética
16.
Anticancer Res ; 40(9): 5035-5041, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32878791

RESUMEN

BACKGROUND/AIM: Based on the cytotoxic agent (-)-zampanolide, N,N'-(arylmethylene)bisamides were designed and synthesized as candidate anti-cancer agents. Among them, N,N'-[(3,4-dimethoxyphenyl)methylene]biscinnamide (DPMBC) was identified as the most potent cytotoxic analog against cancer cells. In this study, we investigated the mechanisms underlying DPMBC-induced cell death in HL-60 human promyelocytic leukemia and PC-3 human prostate cancer cells. MATERIALS AND METHODS: Cell growth was assessed by the WST-8 assay. Induction of apoptosis was assessed by nuclear morphology, DNA ladder formation, and flow cytometry using Annexin V staining. Activation of factors in the apoptotic signaling pathway was assessed by western blot analyses. Knockdown of death receptor 5 (DR5) was performed using siRNA. RESULTS: DPMBC up-regulated expression levels of DR5 protein and induced apoptosis through the extrinsic apoptotic pathway mediated by DR5 and caspases. CONCLUSION: DPMBC is an extrinsic apoptosis inducer, which has potential as a therapeutic agent for cancer therapy.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Macrólidos/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Antineoplásicos/química , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Humanos , Macrólidos/química , Estructura Molecular , ARN Interferente Pequeño/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo
17.
PLoS One ; 15(9): e0239019, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32941497

RESUMEN

The melanosome is a specialized membrane-bound organelle that is involved in melanin synthesis, storage, and transportation. In contrast to melanosome biogenesis, the processes underlying melanosome degradation remain largely unknown. Autophagy is a process that promotes degradation of intracellular components' cooperative process between autophagosomes and lysosomes, and its role for process of melanosome degradation remains unclear. Here, we assessed the regulation of autophagy and its contributions to depigmentation associated with Melasolv (3,4,5-trimethoxycinnamate thymol ester). B16F1 cells-treated with Melasolv suppressed the α-MSH-stimulated increase of melanin content and resulted in the activation of autophagy. However, introduction of bafilomycin A1 strongly suppressed melanosome degradation in Melasolv-treated cells. Furthermore, inhibition of autophagy by ATG5 resulted in significant suppression of Melasolv-mediated depigmentation in α-MSH-treated cells. Taken together, our results suggest that treatment with Melasolv inhibits skin pigmentation by promoting melanosome degradation via autophagy activation.


Asunto(s)
Cinamatos/farmacología , Melanosomas/efectos de los fármacos , Melanosomas/metabolismo , Animales , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Cinamatos/metabolismo , Macrólidos/farmacología , Melaninas/metabolismo , Melanocitos/metabolismo , Ratones , Pigmentación/efectos de los fármacos , Trastornos de la Pigmentación/metabolismo , Pigmentación de la Piel/efectos de los fármacos , alfa-MSH/efectos de los fármacos , alfa-MSH/metabolismo
18.
Antivir Chem Chemother ; 28: 2040206620961712, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32972196

RESUMEN

Macrolides are a large group of antibiotics characterised by the presence of a macro-lactone ring of variable size. The prototype of macrolide antibiotics, erythromycin was first produced by Streptomyces and associated species more than half a century ago; other related drugs were developed. These drugs have been shown to have several pharmacological properties: in addition to their antibiotic activity, they possess some anti-inflammatory properties and have been also considered against non-bacterial infections. In this review, we analysed the available clinical evidences regarding the potential anti-viral activity of macrolides, by focusing on erythromycin, clarithromycin and azithromycin. Overall, there is no significant evidences so far that macrolides might have a direct benefit on most of viral infections considered in this review (RSV, Influenza, coronaviruses, Ebola and Zika viruses). However, their clinical benefit cannot be ruled out without further and focused clinical studies. Macrolides may improve the clinical course of viral respiratory infections somehow, at least through indirect mechanisms relying on some and variable anti-inflammatory and/or immunomodulatory effects, in addition to their well-known antibacterial activity.


Asunto(s)
Antibacterianos/farmacología , Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Macrólidos/farmacología , Neumonía Viral/tratamiento farmacológico , Animales , Antibacterianos/farmacocinética , Antibacterianos/uso terapéutico , Antivirales/farmacocinética , Antivirales/uso terapéutico , Betacoronavirus/efectos de los fármacos , Humanos , Macrólidos/farmacocinética , Macrólidos/uso terapéutico , Pandemias
19.
Proc Natl Acad Sci U S A ; 117(38): 23835-23846, 2020 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-32900948

RESUMEN

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.


Asunto(s)
VIH-1 , Interacciones Huésped-Patógeno , Macrólidos , Linfocitos T Citotóxicos , Células Cultivadas , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Macrólidos/inmunología , Macrólidos/farmacología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana
20.
Exp Parasitol ; 217: 107961, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32777223

RESUMEN

Scabies is considered one of the commonest dermatological diseases that has a global health burden. Current treatment with ivermectin (IVM) is insufficient and potential drug resistance was noticed. Moxidectin (MOX), with a better pharmacological profile may be a promising alternative. The efficacy of moxidectin against Sarcoptes scabiei was assessed both in vitro and in vivo in comparison with ivermectin. For the in vitro assay, both drugs were used in two concentrations (50 µg/ml and 100 µg/ml). For the in vivo assay, twenty rabbits infected with Sarcoptes scabiei were divided into three groups: untreated, moxidectin-treated and ivermectin-treated with the same dose of 0.3 mg/kg once. Another four rabbits were used as a normal control non-infected group. Treatment efficacy was evaluated by clinical assessment, parasitological evaluation and histopathological examination of skin samples using Hematoxylin and eosin and toluidine blue for mast cell staining. Immune response was also assessed by immunohistochemical staining of CD3 T cells in skin samples. Our results showed that moxidectin had a high efficacy (100%) in killing mites when used in both concentrations (50 µg/ml, 100 µg/ml) in the in vitro assay. Concerning the in vivo assay, on day 14 post-treatment, all MOX-treated rabbits were mite-free with full clinical cure by the end of the study (D21) showing (100%) reduction of mites count. Also, marked improvement in the epidermis with absence of mites in skin samples were shown. Poor clinical and parasitological improvements were noted in the ivermectin-treated rabbits, when given as a single dose with a percentage reduction (60.67%) in the 2nd week and progressive increase in lesions and mites count in the 3rd week post-treatment. Regarding the immune response, MOX-treated group showed mild infiltration with both mast cells and CD3 T cells in comparison to severe infiltration with both types of cells in the untreated and IVM-treated group. On conclusion, our results demonstrated that a single dose of MOX was more effective than IVM, supporting MOX as a valuable therapeutic approach for scabies therapy.


Asunto(s)
Acaricidas/farmacología , Macrólidos/farmacología , Sarcoptes scabiei/efectos de los fármacos , Escabiosis/tratamiento farmacológico , Acaricidas/uso terapéutico , Animales , Biopsia con Aguja , Oído Externo/efectos de los fármacos , Oído Externo/parasitología , Oído Externo/patología , Inmunohistoquímica , Ivermectina/farmacología , Ivermectina/uso terapéutico , Macrólidos/uso terapéutico , Masculino , Conejos , Piel/parasitología , Piel/patología
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