RESUMEN
Two strains of Streptococcus pneumoniae, one expressing the methyltransferase Erm(B) and the other negative for erm(B), were selected for solithromycin resistance in vitro either with direct drug selection or with chemical mutagenesis followed by drug selection. We obtained a series of mutants that we characterized by next-generation sequencing. We found mutations in various ribosomal proteins (L3, L4, L22, L32, and S4) and in the 23S rRNA. We also found mutations in subunits of the phosphate transporter, in the DEAD box helicase CshB, and in the erm(B)L leader peptide. All mutations were shown to decrease solithromycin susceptibility when transformed into sensitive isolates. Some of the genes derived from our in vitro screens were found to be mutated also in clinical isolates with decreased susceptibility to solithromycin. While many mutations were in coding sequences, some were found in regulatory regions. These included novel phenotypic mutations in the intergenic regions of the macrolide resistance locus mef(E)/mel and in the vicinity of the ribosome binding site of erm(B). Our screens highlighted that macrolide-resistant S. pneumoniae can easily acquire resistance to solithromycin, and they revealed many new phenotypic mutations.
Asunto(s)
Antibacterianos , Macrólidos , Macrólidos/farmacología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Streptococcus pneumoniae , Farmacorresistencia Bacteriana/genética , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
Antimicrobial resistance (AMR) is a public health threat worldwide. Next-generation sequencing (NGS) has opened unprecedented opportunities to accelerate AMR mechanism discovery and diagnostics. Here, we present an integrative approach to investigate trimethoprim (TMP) resistance in the key pathogen Streptococcus pneumoniae. We explored a collection of 662 S. pneumoniae genomes by conducting a genome-wide association study (GWAS), followed by functional validation using resistance reconstruction experiments, combined with machine learning (ML) approaches to predict TMP minimum inhibitory concentration (MIC). Our study showed that multiple additive mutations in the folA and sulA loci are responsible for TMP non-susceptibility in S. pneumoniae and can be used as key features to build ML models for digital MIC prediction, reaching an average accuracy within ±1 twofold dilution factor of 86.3%. Our roadmap of in silico analysis-wet-lab validation-diagnostic tool building could be adapted to explore AMR in other combinations of bacteria-antibiotic. IMPORTANCE: In the age of next-generation sequencing (NGS), while data-driven methods such as genome-wide association study (GWAS) and machine learning (ML) excel at finding patterns, functional validation can be challenging due to the high numbers of candidate variants. We designed an integrative approach combining a GWAS on S. pneumoniae clinical isolates, followed by whole-genome transformation coupled with NGS to functionally characterize a large set of GWAS candidates. Our study validated several phenotypic folA mutations beyond the standard Ile100Leu mutation, and showed that the overexpression of the sulA locus produces trimethoprim (TMP) resistance in Streptococcus pneumoniae. These validated loci, when used to build ML models, were found to be the best inputs for predicting TMP minimal inhibitory concentrations. Integrative approaches can bridge the genotype-phenotype gap by biological insights that can be incorporated in ML models for accurate prediction of drug susceptibility.
Asunto(s)
Antibacterianos , Estudio de Asociación del Genoma Completo , Aprendizaje Automático , Pruebas de Sensibilidad Microbiana , Streptococcus pneumoniae , Resistencia al Trimetoprim , Trimetoprim , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/efectos de los fármacos , Trimetoprim/farmacología , Antibacterianos/farmacología , Humanos , Resistencia al Trimetoprim/genética , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Infecciones Neumocócicas/microbiología , MutaciónRESUMEN
Fanconi anemia (FA) proteins are thought to play a role in chromosome stability and repair of DNA cross-links; however, these functions may not fully explain the developmental abnormalities and bone marrow failure that are characteristic of FA individuals. Here we associate the FA proteins with the Notch1 developmental pathway through a direct protein-protein interaction between the FA core complex and the hairy enhancer of split 1 (HES1). HES1 interaction with FA core complex members is dependent on a functional FA pathway. Cells depleted of HES1 exhibit an FA-like phenotype that includes cellular hypersensitivity to mitomycin C (MMC) and lack of FANCD2 monoubiquitination and foci formation. HES1 is also required for proper nuclear localization or stability of some members of the core complex. Our results suggest that HES1 is a novel interacting protein of the FA core complex.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteínas de Homeodominio/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Línea Celular Transformada , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/fisiología , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/deficiencia , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Células HeLa , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Noqueados , Mitomicina/farmacología , Complejos Multiproteicos , Unión Proteica , ARN Interferente Pequeño/genética , Receptor Notch1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factor de Transcripción HES-1 , Técnicas del Sistema de Dos Híbridos , UbiquitinaciónRESUMEN
To gain insight into presenilin-1 (PS1) structural aspects, we explored the structure-function relationship of its N- and C-terminal (NTF and CTF, respectively) complexes. We demonstrated that both NTF and CTF act as independent but inter-changing binding units capable of binding each other (NTF/CTF) or their homologues (NTF/NTF; CTF/CTF). The Alzheimer's disease-associated PS1 mutations Y115H and M146L do not affect their ability to hetero- and/or homodimerize, thus conserving their basic integrity and function(s). These results suggest that PS1 associates intra-molecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or gamma-secretase-associated activity.
Asunto(s)
Proteínas de la Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Dimerización , Humanos , Sustancias Macromoleculares , Proteínas de la Membrana/genética , Mutación , Fragmentos de Péptidos/genética , Presenilina-1 , Estructura Terciaria de Proteína/fisiología , Relación Estructura-Actividad , Técnicas del Sistema de Dos HíbridosRESUMEN
SUMMARY: It has been previously demonstrated that the Notch1 signalling pathway is impaired in presenilin-1 null cells. This observation suggests a role for presenilin-1 in the Notch1 developmental pathway, possibly through physical interaction. Here, we show that presenilin-1 and Notch1 do not interact directly with each other but are associated in the cell. These findings raise the possibility that the gamma-secretase cleavage occurs via a presenilin complex in association with a putative co-factor specific for the molecule that is being cleaved (e.g. Notch1, (beta-amyloid precursor protein, E-cadherin and ErbB-4, all of which are gamma-secretase substrates).
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular , Factores de Transcripción , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Línea Celular , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Hidrólisis , Proteínas de la Membrana/genética , Presenilina-1 , Receptor Notch1RESUMEN
OBJECTIVE: The goal of this study was to compare the effects of 2 doses of pioglitazone hydrochloride (a thiazolidinedione insulin sensitizer) with placebo on glycated hemoglobin (HbA(1c)), insulin sensitivity, and lipid profiles in patients with type 2 diabetes mellitus who had suboptimal glycemic control and mild dyslipidemia. METHODS: Patients with type 2 diabetes mellitus (HbA(1c) >/=6.5% and =9.8%) who had not previously received insulin or oral antihyperglycemic medications (OAMs) were randomized to treatment with placebo, pioglitazone 30 mg QD, or pioglitazone 45 mg QD in double-blind fashion for 16 weeks at 41 centers in Canada and Spain. RESULTS: A total of 297 patients were randomized (99 in each group). Overall, 286 (96.3%) were white. Mean (SD) age was 58.4 (10.9) years (range, 24-85 years), mean (SD) body mass index was 31.4 (4.8) kg/m(2), mean (SD) duration of type 2 diabetes mellitus was 20.0 (37.4) months, and 30.6% of patients were receiving medication for dyslipidemia. Treatment with pioglitazone 30 or 45 mg QD for 16 weeks reduced mean HbA(1c) by 0.8% and 0.9% from baseline, respectively (both P < 0.001 vs baseline and placebo). A reduction in HbA(1c) of 0.2% was observed in the placebo group (P = 0.025). In patients with medium (>/=7% to <8%) or high (>/=8% to =9.8%) baseline HbA(1c), both doses of pioglitazone significantly reduced HbA(1c) (both P < 0.001 vs placebo). Pioglitazone 30 and 45 mg significantly reduced fasting serum insulin versus placebo (P = 0.008 and P = 0.006, respectively) and increased insulin sensitivity by Homeostasis Model Assessment versus placebo (P = 0.039 and P = 0.001, respectively). Relative to placebo, pioglitazone 30 and 45 mg significantly increased high-density lipoprotein cholesterol (HDL-C [P = 0.028 and P < 0.001, respectively]) and lowered the atherogenic index of plasma (P = 0.018 and P < 0.001, respectively). Pioglitazone 45 mg also significantly reduced serum triglycerides, apolipoprotein B, and total cholesterol:HDL-C ratio versus placebo (P = 0.007, P = 0.015, and P = 0.005, respectively). Pioglitazone 30 and 45 mg were associated with a significant reduction in serum alanine aminotransferase relative to placebo (P = 0.036 and P = 0.005, respectively). Pioglitazone appeared to be safe and was well tolerated. CONCLUSIONS: In the present study, pioglitazone 30 and 45 mg produced significant improvements in HbA(1c), insulin sensitivity, and lipid profile in OAM-naive patients with type 2 diabetes mellitus with suboptimal glycemic control and mild dyslipidemia.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hiperlipidemias/complicaciones , Hipoglucemiantes/uso terapéutico , Tiazoles/uso terapéutico , Tiazolidinedionas , Adulto , Anciano , Anciano de 80 o más Años , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Hiperlipidemias/sangre , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/efectos adversos , Insulina/sangre , Lípidos/sangre , Masculino , Persona de Mediana Edad , Pioglitazona , Tiazoles/administración & dosificación , Tiazoles/efectos adversos , Resultado del TratamientoRESUMEN
The objectives of this study were (1) to investigate the effect of operator variability on the shear bond strength of adhesives to dentin and (2) to determine the effectiveness of education on bonding performance for different types of adhesives. Thirty general practitioners were recruited for a CE course by a regional mailing. They used bovine dentin as a substrate for bonding the adhesive system they routinely used in practice and two other materials (no previous experience). Each adhesive OptiBond FL, ScotchBond Multipurpose Plus, ScotchBond 1 and Clearfil SE was applied according to manufacturer's instructions and immediately tested using a shear bond strength test. Shear bond strengths between adhesives and dentin were compared before and after a 90 min lecture on bonding principles and materials. For dentists with and without previous experience with a material, there were no statistically significant differences seen before and after the lecture (paired t-tests, p < or = 0.05). However, in every case, the bond strengths after the lecture were higher than those before (range of improvement from 15 to 150%). For dentists with routine experience with a particular material, all materials were statistically equivalent after the lecture, although the OptiBond FL was the highest. For dentists who had no previous experience with a material, the ScotchBond 1 had lower bond strengths than the other materials after the lecture. There was a large range in the ability of dentists to manipulate adhesive systems correctly. However, if a dentist has sufficient experience and receives sufficient education, any of these materials can give reasonable results.
Asunto(s)
Recubrimiento Dental Adhesivo , Recubrimientos Dentinarios , Operatoria Dental/educación , Medicina Familiar y Comunitaria/educación , Cementos de Resina , Análisis de Varianza , Animales , Bovinos , Competencia Clínica , Resinas Compuestas , Dentina , Educación Continua en Odontología , Humanos , Dióxido de Silicio , Estadísticas no Paramétricas , CirconioRESUMEN
Light-emitting sources are currently used to activate the setting reaction of restorative composite resins. It is well known that the polymerization extent of the resins will highly influence their clinical behavior. The monomer-polymer transformation will mostly depend on the chemical nature of the photo-initiators used and on the luminous energy given by the curing device. The aims of this study were to characterize three light curing devices and to compare their efficiency to polymerize a composite resin (SureFil). For that purpose, the emission spectra and the irradiance of a plasma arc (Flipo), a QTH visible light (Elipar Trilight), and a LEDs curing device (Elipar Freelight) were measured. Then, the depth of cure and the Vickers hardness of composite samples were evaluated, for each curing device, as a function of the exposure time. The emission spectra obtained showed the irradiance of the visible light emitted as a function of the wavelength, which was different for each light-curing device. The maximum cured depth (approximately 4 mm) give only a qualitative indication of the extent of the polymerization, as the hardness of the composite samples diminished as a function of the depth. Moreover, it strongly depended on the exposure time. Hence, to obtain a hardness of approximately 100 HV 0.5 at 2 mm depth, the illumination time must be at least of 3 s with the plasma arc Flipo, 10 s with the LEDs Elipar Freelight and 20 s with the QTH Elipar Trilight. In order to efficiently polymerize a composite resin, the emission spectra of the luminous source shall correspond with the absorption spectra of the photo-initiators. The present study showed that the LEDs Elipar Freelight is the most efficient curing device when the camphorquinone is used, as 92% of the emitted energy will be absorbed by the initiator. However, the power of this source is relatively low, hence higher exposure time shall be used.
Asunto(s)
Resinas Compuestas/efectos de la radiación , Equipo Dental , Dureza/efectos de la radiación , Luz , Polímeros/efectos de la radiación , Semiconductores , Terpenos/química , Factores de Tiempo , XenónRESUMEN
The Fanconi anemia group C protein (FANCC) is one of the several proteins that comprise the Fanconi anemia (FA) network involved in genomic surveillance. FANCC is mainly cytoplasmic and has many functions, including apoptosis suppression through caspase-mediated proteolytic processing. Here, we examined the role of FANCC proteolytic fragments by identifying their binding partners. We performed a yeast two-hybrid screen with caspase-mediated FANCC cleavage products and identified the dependence receptor uncoordinated-5A (UNC5A) protein. Here, we show that FANCC physically interacts with UNC5A, a pro-apoptotic dependence receptor. FANCC interaction occurs through the UNC5A intracellular domain, specifically via its death domain. FANCC modulates cell sensitivity to UNC5A-mediated apoptosis; we observed reduced UNC5A-mediated apoptosis in the presence of FANCC and increased apoptosis in FANCC-depleted cells. Our results show that FANCC interferes with UNC5A's functions in apoptosis and suggest that FANCC may participate in developmental processes through association with the dependence receptor UNC5A.
Asunto(s)
Apoptosis , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Citoplasma , Humanos , Modelos Biológicos , Receptores de Netrina , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas/métodos , Transporte de Proteínas , Receptores de Superficie Celular/química , Técnicas del Sistema de Dos HíbridosRESUMEN
A growing body of evidence indicates that presenilins could exist and be active as oligomeric complexes. Using yeast two-hybrid and cell culture analysis, we provide evidence that presenilin-1 (PS1) may self-oligomerize giving rise to specific full-length/full-length homodimers. When expressed in N2A and HEK239T cultured cells, full-length PS1-wt and 5(')myc-PS1-wt form specific homodimers corresponding to twice their molecular weight. The Alzheimer's disease-associated PS1 mutations Y115H, M146L, L392V, deltaE10(PS1(1-289/320-467)), the gamma-secretase dominant negative mutant D257A, and the PS1 polymorphism mutant E318G do not affect their ability to self-oligomerize. Under non-denaturing conditions, endogenous PS1 forms specific homo-oligomers in human cultured cells. The results obtained herein suggest that PS1 associates intramolecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or gamma-secretase-associated activity.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Estructura Cuaternaria de Proteína , Animales , Línea Celular , Dimerización , Humanos , Sustancias Macromoleculares , Proteínas de la Membrana/genética , Ratones , Peso Molecular , Mutación , Presenilina-1 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques. The major constituent of these plaques, occurring largely in brain areas important for memory and cognition, is the 40-42 amyloid residues (Abeta). Abeta is derived from the amyloid protein precursor after cleavage by the recently identified beta-secretase (BACE1) and the putative gamma-secretase complex containing presenilin 1 (PS1). In an attempt to develop a functional secretase enzymatic assay in yeast we demonstrate a direct binding between BACE1 and PS1. This interaction was confirmed in vivo using coimmunoprecipitation and colocalization studies in human cultured cells. Our results show that PS1 preferably binds immature BACE1, thus possibly acting as a functional regulator of BACE1 maturation and/or activity.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos , Endopeptidasas , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Riñón/metabolismo , Pruebas de Precipitina , Presenilina-1 , Proteínas Recombinantes , Saccharomyces cerevisiae , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The function of the Fanconi anemia group C protein (FANCC) is still unknown, though many studies point to a role in damage response signaling. Unlike other known FA proteins, FANCC is mainly localized to the cytoplasm and is thought to act as a messenger of cellular damage rather than an effector of repair. FANCC has been shown to interact with several cytoplasmic and nuclear proteins and to delay the onset of apoptosis through redox regulation of GSTP1. We investigated the fate and function of FANCC during apoptosis. Here we show that FANCC undergoes proteolytic modification by a caspase into a predominant 47-kDa ubiquitinated protein fragment. Lack of proteolytic modification at the putative cleavage site delays apoptosis but does not affect MMC complementation. These results suggest that FANCC function is regulated through proteolytic processing.