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OBJECTIVE: Globally, there are more than six million deaths due to cerebrovascular disease, which is the second leading cause of death. Although the imaging findings of magnetic resonance imaging (MRI) are more accurate than computed tomography for acute ischemic stroke (AIS), it is uncommon in recombinant tissue plasminogen activator (rTPA) treatment. Alteplase is not only strongly recommended treatment for acute ischemic stroke within 4.5 hours, but also decreases the disability and mortality rate. Besides, low-dose rTPA was associated with significant reductions in symptomatic intracerebral hemorrhage (sICH), compared with standard one. However, the benefits of low-dose rTPA for the treatment of AIS without large vessel occlusion (LVO) have not been fully demonstrated. We evaluated whether the low-dose rTPA in AIS without LVO could improve prognosis in patients three months post-treatment. PATIENTS AND METHODS: This was a cross-sectional study on patients with AIS treated within 4.5 hours of symptom onset admitted to Can Tho S.I.S General Hospital between February 2019 and July 2021. The eligibility criteria were patients aged > 18 years treated with low-dose rTPA (0.6 mg/kg) and screened by 3T MRI. Patients with a pre-hospital modified Rankin score (mRS) ≥ 2 points, intracranial hemorrhage, LVO, or ≥ 3 microbleeds on brain MRI were excluded. The primary outcomes were the favorable outcome rate at three months and safety, which were evaluated by the rates of intracranial hemorrhage and mortality at three months. RESULTS: This study enrolled 92 eligible patients between February 2019 and July 2021. Their National Institute of Health Stroke Scale (NIHSS) scores were 7.5 ± 3.7 at admission, 3.3 ± 3.5 at discharge or seven days after discharge, and 2.2 ± 2.8 at three months. Their mRS were 2.9 ± 0.8 at admission, 1.4 ± 1.3 at discharge or seven days after discharge, and 1.1 ± 1.1 at three months. Elevated cardiac enzymes, age ≥ 75 years, and body mass index ≥ 25 were associated with increased poor outcomes at three months. While AIS was more common in men than women, a similar number of men (33.3%) and women had poor mRS. Three patients had complications associated with low-dose rTPA treatment: one (1.1%) had intracranial hemorrhage, one (1.1%) had new infarcts, and one (1.1%) had gastrointestinal bleeding. No deaths occurred within three months. CONCLUSIONS: Our study indicates the efficacy and safety of low-dose rTPA treatment for AIS without LVO within 4.5 hours. Patient selection for rTPA by 3T MRI decreased complications and mortality.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Femenino , Humanos , Masculino , Isquemia Encefálica/diagnóstico por imagen , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/complicaciones , Estudios Transversales , Fibrinolíticos , Hemorragias Intracraneales/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/diagnóstico por imagen , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Imagen por Resonancia Magnética , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/tratamiento farmacológico , Terapia Trombolítica , Activador de Tejido Plasminógeno , Resultado del TratamientoRESUMEN
Background: Acute mastoiditis is a suppurative infection of mastoid air cells and is the most common intratemporal complication of otitis media. Aim: This study aimed to evaluate the demographic and clinical characteristics and treatment outcomes of children with acute mastoiditis (AM). Patients and Methods: We retrospectively reviewed the medical records of hospitalized pediatric patients aged between 1 month and 18 years with a diagnosis of AM between May 2015 and December 2021. Results: A total of 28 hospitalized children with AM were enrolled in this study, of whom 22 (78.6%) were males and 6 (21.4%) were females with a mean ± standard deviation age of 93.5 ± 53.2 months (range = 6 months-16.1 years). The most common clinical symptoms were postauricular erythema (n = 17, 60.7%), tenderness (n = 16, 57.1%), swelling (n = 14, 50%), fever (n = 14, 50%), and auricular protrusion (n = 7, 25%). Mastoiditis complications occurred in 10 (35.7%) children. The most common extracranial complication was subperiosteal abscess (n = 8, 28.6%). The erythrocyte sedimentation rate (ESR) and the rate of antibiotic use before hospitalization were higher in patients with complicated mastoiditis (P = 0.006 and P = 0.039, respectively). Surgery was performed in 12 (42.9%) patients. Statistically, more surgical interventions were performed in patients who developed complications (P = 0.003). Conclusion: AM continues as an important disease of childhood. Successful results are obtained with systemic antibiotic therapy and additional surgical intervention as necessary. A careful evaluation of patients with a high ESR and those who received antibiotic therapy before hospitalization is appropriate due to the correlation between these factors and the risk of complication development.
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Mastoiditis , Masculino , Femenino , Niño , Humanos , Lactante , Mastoiditis/complicaciones , Mastoiditis/epidemiología , Mastoiditis/terapia , Estudios Retrospectivos , Centros de Atención Terciaria , Enfermedad Aguda , Antibacterianos/uso terapéuticoRESUMEN
Since interleukin-8 (IL-8/CXCL8) and its receptor, CXCR1 and CXCR2, were known in the early 1990s, biological pathways related to these proteins were proven to have high clinical value in cancer and inï¬ammatory/autoimmune conditions treatment. Recently, IL-8 has been identified as biomarker for severe COVID-19 patients and COVID-19 prognosis. Boyles et al. (mAbs 12 (2020), pp. 1831880) have published a high-resolution X-ray crystal structure of the LY3041658 Fab in a complex human CXCL8. They described the ability to bind to IL-8 and the blocking of IL-8/its receptors interaction by the LY3041658 monoclonal antibody. Therefore, the study has been designed to identify potential small molecules inhibiting interleukin-8 by targeting LY3041658/IL-8 complex structure using an in silico approach. A structurebased pharmacophore and molecular docking models of the protein active site cavity were generated to identify possible candidates, followed by virtual screening with the ZINC database. ADME analysis of hit compounds was also conducted. Molecular dynamics simulations were then performed to survey the behaviour and stability of the ligand-protein complexes. Furthermore, the MM/PBSA technique has been utilized to evaluate the free binding energy. The final data confirmed that one newly obtained compound, ZINC21882765, may serve as the best potential inhibitor for IL-8.
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Tratamiento Farmacológico de COVID-19 , Interleucina-8 , Humanos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad Cuantitativa , Simulación de Dinámica Molecular , LigandosRESUMEN
OBJECTIVE: In cases of acute ischemic stroke (AIS) caused by intracranial large vessel occlusion, rescue intracranial stenting (RIS) has recently emerged as a treatment option for achieving recanalization when mechanical thrombectomy (MT) fails. However, few studies to date have reported on the beneficial outcomes of RIS. Our goal was to analyze whether RIS use can improve prognosis in patients 3 months post-treatment. PATIENTS AND METHODS: A retrospective analysis was performed on a prospective cohort of patients with AIS treated with RIS at Can Tho S.I.S General Hospital. The study inclusion criteria were evidence of intracranial large vessel occlusion, absence of intracranial hemorrhage (ICH), and severe stenosis or reocclusion after MT. Patients with tandem occlusions, failure to follow up after discharge, or severe or fatal illness concomitant with AIS were excluded from the study. The primary outcome was the "non-poor" prognosis status rate at 3 months after RIS and post-procedural symptomatic ICH (sICH). RESULTS: The post-treatment outcomes of 85 eligible patients who received RIS between August 2019 and May 2021 were assessed. Of the 85 included patients, 82 (96.5%) achieved successful recanalization, and 4 (4.7%) experienced sICH. At 3-months post-treatment, 47 (55.3%) patients had "non-poor" outcomes, whereas 35 (41.2%) had good outcomes. The use of dual antiplatelet therapy was associated with new infarcts (relative risk [RR]: 0.1; 95% confidence interval [CI]: 0.01-0.7) and sICH occurrence (RR: 0.1; 95% CI: 0.01-0.9). CONCLUSIONS: Our study suggests that despite the occurrence of post-procedural sICH in a small proportion of cases, RIS could serve as a useful alternative or additional treatment in the event of MT failure.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Trombectomía/efectos adversos , Accidente Cerebrovascular Isquémico/cirugía , Estudios Retrospectivos , Accidente Cerebrovascular/cirugía , Accidente Cerebrovascular/etiología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Estudios Prospectivos , Resultado del Tratamiento , Pueblo Asiatico , Isquemia Encefálica/terapia , Isquemia Encefálica/complicacionesRESUMEN
OBJECTIVE: Intravenous (IV) recombinant tissue plasminogen activator is the standard of care for patients with acute ischemic stroke (AIS) who present to the hospital within 4.5 hours of symptom onset. However, IV thrombolysis, even bridging thrombolysis (combining intravenous thrombolysis and mechanical thrombectomy) has limited efficacy among patients who had occlusive lesions associated with high-grade arterial stenosis requiring revascularization to improve neurological deficits. We evaluated whether rescue stenting results in good outcomes among patients after the failure of intravenous thrombolysis and bridging thrombolysis. PATIENTS AND METHODS: We retrospectively analyzed patients with AIS who underwent rescue stenting for large vessel occlusion with severe atherosclerotic stenosis between May 2020 and August 2022 at Can Tho S.I.S General Hospital. Primary outcomes included the incidence of hemorrhagic transformation and the rate of good outcomes (modified Rankin Scale < 3) at 3-month follow-up. RESULTS: We identified 13 patients who received rescue stenting after the failure of IV alteplase and bridging thrombolysis, but only 11 patients met the inclusion criteria. All patients experienced successful recanalization, and 1 (9.1%) patient experienced new infarcts. Of these 11 patients, 10 (90.9%) had good outcomes 3 months after rescue stenting. Additionally, a loading dose of dual antiplatelet therapy (DAPT) applied concurrently with IV alteplase improved the recanalization rate for large target arteries but had no significant effect on the incidence of symptomatic intracranial hemorrhage. CONCLUSIONS: Rescue stenting appears to represent an additional therapeutic option in cases that fail to resolve with IV alteplase, which may improve clinical outcomes.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Isquemia Encefálica/tratamiento farmacológico , Constricción Patológica/complicaciones , Constricción Patológica/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Estudios Retrospectivos , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/etiología , Trombectomía/efectos adversos , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/uso terapéutico , Resultado del Tratamiento , VietnamRESUMEN
We developed a high-resolution and comprehensive typing method for swine leukocyte antigen 3 (SLA-3), an MHC class I gene, employing locus-specific genomic PCR followed by subsequent direct sequencing. A total of 292 individuals from nine pure, one cross-breed and six cell lines were successfully typed. A total of 21 SLA-3 alleles were identified, of which four were found to be novel alleles. However, the allelic diversity of SLA-3 was lower than that of previously reported class I genes, SLA-1 and -2. More SLA-3 alleles were observed in the Landrace and Yorkshire breeds than the other breeds. SLA-3*04:01 was identified in seven out of nine breeds and was the most widely distributed allele across all breeds. Therefore, the typing method reported in this study completes our efforts to develop high-resolution typing methods for major SLA molecules, facilitating the combined analysis of major SLA genes from field samples, which is important to understand the relationship between the adaptive immune responses against pathogens and the immunogenetic makeup of an individual.
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Técnicas de Genotipaje/veterinaria , Antígenos de Histocompatibilidad Clase I/genética , Análisis de Secuencia de ADN/veterinaria , Sus scrofa/genética , Animales , Análisis de Secuencia de ADN/métodosRESUMEN
Interleukin (IL)-33 is a new cytokine of the IL-1 family that is related to several inflammatory and autoimmune diseases. IL-33 binds to its ST2 receptor and leads to biological responses thereof. Currently, no drugs have been approved for the treatment of IL-33 related diseases. The aim of this study was to search for small molecules that inhibit the protein-protein interaction between IL-33 and ST2. A virtual screening was first performed to identify potential molecules that can bind IL-33. By analysing the interactions between key residues in the complex of IL-33/ST2, two pharmacophore hypotheses were then generated based on the 'mimicry' and 'pair-rule' principles. From a database of 62,074 compounds, 60 molecules satisfying the pharmacophore models were identified and docked to IL-33. Among 35 compounds successfully docked into the protein, 9 potential ligands in complex with IL-33 were selected for further analysis by molecular dynamics simulations. Based on the stability of the complexes and the interactions of each ligand with the key residues of IL-33, two compounds DB00158 and DB00642 were identified as the most potential inhibitors that can be further investigated as promising novel IL-33 inhibitory drugs.
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Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Relación Estructura-Actividad CuantitativaRESUMEN
Influenza A virus (IAV) has caused epidemic infections worldwide, with many strains resistant to inhibitors of a surface protein, neuraminidase (NA), due to point mutations on its structure. A novel NA inhibitor named peramivir was recently approved, but no exhaustive computational research regarding its binding affinity with wild-type and mutant NA has been conducted. In this study, a thorough investigation of IAV-NA PDB entries of 9 subtypes is described, providing a list of residues constituting the protein-ligand binding sites. The results of induced-fit docking approach point out key residues of wild-type NA participating in hydrogen bonds and/or ionic interactions with peramivir, among which Arg 368 is responsible for a peramivir-NA ionic interaction. Mutations on this residue greatly reduced the binding affinity of peramivir with NA, with 3 mutations R378Q, R378K and R378L (NA6) capable of deteriorating the docking performance of peramivir by over 50%. 200 compounds from 6-scaffolds were docked into these 3 mutant versions, revealing 18 compounds giving the most promising results. Among them, CMC-2012-7-1527-56 (benzoic acid scaffold, IC50 = 32 nM in inhibitory assays with IAV) is deemed the most potential inhibitor of mutant NA resisting both peramivir and zanamivir, and should be further investigated.
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Antivirales/química , Ciclopentanos/química , Inhibidores Enzimáticos/química , Guanidinas/química , Neuraminidasa/química , Proteínas Virales/química , Ácidos Carbocíclicos , Sitios de Unión , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Mutación , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Relación Estructura-Actividad Cuantitativa , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genéticaRESUMEN
BACKGROUND: The experiences of and reflections on interpersonal violence and victimisation among adolescents living in low- and middle-income countries are poorly understood. The aim was to describe Vietnamese adolescents' reflections on their experiences of victimisation. METHOD: A self-completed, cross-sectional survey investigating exposure to violence among high school students in Hanoi, Vietnam was conducted during 2013-2014. The last section invited participants to write about any of the matters covered in the questionnaire. Thematic analysis was conducted on these free-text comments. RESULTS: A total of 73/76 eligible students participated in the pilot and 1616/1745 in the main survey. Of these, a total of 239 records with free-text comments were analysed. Students described experiences of violence occurring at home, at school and in the community. Experiences of violence led to sadness, loneliness, having extremely negative thoughts about the value of life, and suicidal ideas. Adolescents' experiences occurred in the context of poor parent-adolescent and teacher-student relationships, particularly concerning dissatisfaction with academic performance. Adolescents wanted to be trusted, to be given more autonomy, and to improve their relationships with parents and teachers. CONCLUSIONS: Vietnamese adolescents experience various forms of victimisation, which are detrimental to their health and wellbeing. Understanding of their experiences of and perceptions of violence and its impact on their health and wellbeing is important in the prevention of violence against young people in Vietnam.
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The efflux pumps P-glycoprotein (P-gp) in humans and NorA in Staphylococcus aureus are of great interest for medicinal chemists because of their important roles in multidrug resistance (MDR). The high polyspecificity as well as the unavailability of high-resolution X-ray crystal structures of these transmembrane proteins lead us to combining ligand-based approaches, which in the case of this study were machine learning, perceptual mapping and pharmacophore modelling. For P-gp inhibitory activity, individual models were developed using different machine learning algorithms and subsequently combined into an ensemble model which showed a good discrimination between inhibitors and noninhibitors (acctrain-diverse = 84%; accinternal-test = 92% and accexternal-test = 100%). For ligand promiscuity between P-gp and NorA, perceptual maps and pharmacophore models were generated for the detection of rules and features. Based on these in silico tools, hit compounds for reversing MDR were discovered from the in-house and DrugBank databases through virtual screening in an attempt to restore drug sensitivity in cancer cells and bacteria.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Aprendizaje Automático , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Relación Estructura-Actividad Cuantitativa , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Algoritmos , Proteínas Bacterianas/química , Bases de Datos de Compuestos Químicos , Descubrimiento de Drogas , Humanos , Ligandos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Staphylococcus aureusRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0152310.].
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The miR-200 family promotes the epithelial state by suppressing the Zeb1/Zeb2 epithelial gene transcriptional repressors. To identify other miR-200-regulated genes, we isolated mRNAs bound to transfected biotinylated miR-200c in mouse breast cancer cells. In all, 520 mRNAs were significantly enriched in miR-200c binding at least twofold. Putative miR-200-regulated genes included Zeb2, enriched 3.5-fold in the pull down. However, Zeb2 knockdown does not fully recapitulate miR-200c overexpression, suggesting that regulating other miR-200 targets contributes to miR-200's enhancement of epithelial gene expression. Candidate genes were highly enriched for miR-200c seed pairing in their 3'UTR and coding sequence and for genes that were downregulated by miR-200c overexpression. Epidermal growth factor receptor and downstream MAPK signaling pathways were the most enriched pathways. Genes whose products mediate transforming growth factor (TGF)-ß signaling were also significantly overrepresented, and miR-200 counteracted the suppressive effects of TGF-ß and bone morphogenic protein 2 (BMP-2) on epithelial gene expression. miR-200c regulated the 3'UTRs of 12 of 14 putative miR-200c-binding mRNAs tested. The extent of mRNA binding to miR-200c strongly correlated with gene suppression. Twelve targets of miR-200c (Crtap, Fhod1, Smad2, Map3k1, Tob1, Ywhag/14-3-3γ, Ywhab/14-3-3ß, Smad5, Zfp36, Xbp1, Mapk12, Snail1) were experimentally validated by identifying their 3'UTR miR-200 recognition elements. Smad2 and Smad5 form a complex with Zeb2 and Ywhab/14-3-3ß and Ywhag/14-3-3γ form a complex with Snail1. These complexes that repress transcription assemble on epithelial gene promoters. miR-200 overexpression induced RNA polymerase II localization and reduced Zeb2 and Snail1 binding to epithelial gene promoters. Expression of miR-200-resistant Smad5 modestly, but significantly, reduced epithelial gene induction by miR-200. miR-200 expression and Zeb2 knockdown are known to inhibit cell invasion in in vitro assays. Knockdown of each of three novel miR-200 target genes identified here, Smad5, Ywhag and Crtap, also profoundly suppressed cell invasion. Thus, miR-200 suppresses TGF-ß/BMP signaling, promotes epithelial gene expression and suppresses cell invasion by regulating a network of genes.
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Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , Proteínas de Homeodominio/genética , MicroARNs/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Regiones no Traducidas 3' , Animales , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/farmacología , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Homeodominio/metabolismo , Ratones , Células 3T3 NIH , Proteínas Represoras/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
The genetic diversity of the major histocompatibility complex (MHC) class I molecules of pigs has not been well characterized. Therefore, the influence of MHC genetic diversity on the immune-related traits of pigs, including disease resistance and other MHC-dependent traits, is not well understood. Here, we attempted to develop an efficient method for systemic analysis of the polymorphisms in the epitope-binding region of swine leukocyte antigens (SLA) class I genes. We performed a comparative analysis of the last 92 bp of the 5' untranslated region (UTR) to the beginning of exon 4 of six SLA classical class I-related genes, SLA-1, -2, -3, -4, -5, and -9, from 36 different sequences. Based on this information, we developed a genomic polymerase chain reaction (PCR) and direct sequencing-based comprehensive typing method for SLA-2. We successfully typed SLA-2 from 400 pigs and 8 cell lines, consisting of 9 different pig breeds, and identified 49 SLA-2 alleles, including 31 previously reported alleles and 18 new alleles. We observed differences in the composition of SLA-2 alleles among different breeds. Our method can be used to study other SLA class I loci and to deepen our knowledge of MHC class I genes in pigs.
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Variación Genética , Genoma/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase I/genética , Porcinos/genética , Regiones no Traducidas 5' , Alelos , Animales , Secuencia de Bases , Cruzamiento , Línea Celular , Dermatoglifia del ADN , Exones , Sitios Genéticos , Técnicas de Genotipaje , Antígenos de Histocompatibilidad Clase I/clasificación , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/clasificación , Antígenos de Histocompatibilidad Clase II/inmunología , Intrones , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Porcinos/inmunologíaRESUMEN
BACKGROUND: Fructose intake is associated with non-alcoholic fatty liver disease (NAFLD) development. OBJECTIVE: The objective of this study was to measure fructose absorption/metabolism in paediatric NAFLD compared with obese and lean controls. METHODS: Children with histologically proven NAFLD, and obese and lean controls received oral fructose (1 g kg(-1) ideal body weight). Serum glucose, insulin, uric acid, and fructose, urine uric acid, urine fructose, and breath hydrogen levels were measured at baseline and multiple points until 360 min after fructose ingestion. RESULTS: Nine NAFLD (89% Hispanic, mean age 14.3 years, mean body mass index [BMI] 35.3 kg m(-2)), six obese controls (67% Hispanic, mean age 12.7 years, mean BMI 31.0 kg m(-2)) and nine lean controls (44% Hispanic, mean age 14.3 years, mean BMI 19.4 kg m(-2)) were enrolled. Following fructose ingestion, NAFLD vs. lean controls had elevated serum glucose, insulin and uric acid (P < 0.05), higher urine uric acid (P = 0.001), but lower fructose excretion (P = 0.002) and lower breath hydrogen 180-min AUC (P = 0.04). NAFLD vs. obese controls had similar post-fructose serum glucose, insulin, urine uric acid and breath hydrogen, but elevated serum uric acid (P < 0.05) and lower urine fructose excretion (P = 0.02). CONCLUSIONS: Children with NAFLD absorb and metabolize fructose more effectively than lean subjects, associated with an exacerbated metabolic profile following fructose ingestion.
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Fructosa/metabolismo , Hidrógeno/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Adolescente , Biomarcadores/metabolismo , Glucemia/metabolismo , Índice de Masa Corporal , Pruebas Respiratorias , Niño , Ingestión de Alimentos , Femenino , Humanos , Hidrógeno/química , Insulina/sangre , Resistencia a la Insulina , Masculino , Valor Predictivo de las Pruebas , Ácido Úrico/sangreRESUMEN
We previously reported the development of genomic-DNA-based high-resolution genotyping methods for SLA-DQB1 and DRB1. Here, we report the successful typing of SLA-DQA using similar methodological principles. We designed a method for comprehensive genotyping of SLA-DQA using intronic sequence information of SLA-DQA exon 2 that we had obtained from 12 animals with different SLA-DQB1 genotypes. We expanded our typing to 76 selected animals with diverse DQB1 and DRB1 genotypes, 140 random animals from 7 pig breeds, and 3 wild boars. This resulted in the identification of 17 DQA alleles with 49 genotypes. Two new alleles were identified from wild boars. Combine with SLA-DQB1, and DRB1 typing results, we identified 34 SLA class II haplotypes including 25 that were previously unreported.
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Genes MHC Clase II , Antígenos de Histocompatibilidad Clase II/genética , Porcinos/genética , Porcinos/inmunología , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/genética , Exones , Técnicas de Genotipaje/métodos , Haplotipos , Antígenos de Histocompatibilidad Clase I , Intrones , Datos de Secuencia Molecular , Filogenia , Polimorfismo Genético , Homología de Secuencia de Ácido NucleicoRESUMEN
The bacterial pathogen Campylobacter jejuni is the leading cause of foodborne gastroenteritis in the developed world, with the organism being transmitted by ingestion of contaminated and undercooked poultry. Exposure to acid is an inevitable stressor for C. jejuni during gastric passage, yet the effect of low pH on C. jejuni virulence is still poorly understood. Here, we investigate the effect of acid-shock on C. jejuni viability, gene expression and host-cell invasion. C. jejuni strain NCTC 11168 survived acid exposure at pH 3.5 and above for up to 30 min without a drop in viability, and this exposure induced the expression of flagellar genes transcribed from σ(54)-dependent promoters. Furthermore, acid-shock resulted in increased C. jejuni invasion of m-ICcl2 mouse small intestine crypt cells grown on transwells, but not when the cells were grown on flat-bottomed wells. This suggests that C. jejuni might be invading intestinal epithelial cells at the basolateral side, possibly after paracellular passage. We hypothesize that acid-shock prior to intestinal entry may serve as a signal that primes C. jejuni to express its virulence gene repertoire including flagellar motility genes, but this requires further study in the context of an appropriate colonization or disease model.
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BACKGROUND AND PURPOSE: Insulin-regulated aminopeptidase (IRAP) and the insulin-dependent glucose transporter GLUT4 colocalize in specific intracellular vesicles (that is, GLUT4 vesicles). These vesicles move slowly to the cell surface, but their translocation is markedly enhanced by insulin, resulting in higher glucose uptake. Previous studies of the insulin-mediated translocation of IRAP to the cell surface have been hampered by the laborious detection of IRAP at the cell surface. We aimed to develop a more direct and faster method to detect IRAP. To this end, we used model systems with well-characterized IRAP: CHO-K1 cells expressing endogenous IRAP and recombinant HEK293 cells expressing human IRAP. A more widespread application of the method was demonstrated by the use of 3T3-L1 adipocytes. EXPERIMENTAL APPROACH: After stimulation of the cells with insulin, internalization of IRAP was inhibited by the addition of phenyl arsine oxide (PAO). Then, cell-surface IRAP was detected by the high-affinity binding of radiolabelled angiotensin (Ang) IV (either 125I or 3H). KEY RESULTS: We monitored the time- and concentration dependence of insulin-mediated translocation of IRAP in both cell lines and 3T3-L1 adipocytes. A plateau was reached between 6 and 8 min, and 10(-7) M insulin led to the highest amount of IRAP at the cell surface. CONCLUSIONS AND IMPLICATIONS: Based on the capacity of the IRAP apoenzyme to display high affinity for radiolabelled Ang IV and on the ability of PAO to inhibit IRAP internalization, we developed a more direct and faster method to measure insulin-mediated translocation of IRAP to the cell surface.
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Cistinil Aminopeptidasa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Ensayo de Unión Radioligante/métodos , Células 3T3 , Adipocitos/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Insulina/administración & dosificación , Ratones , Transporte de Proteínas , Factores de TiempoRESUMEN
BACKGROUND AND PURPOSE: Whereas some angiotensin II (Ang II) type 1 receptor blockers (ARBs) produce surmountable antagonism of AT(1) receptors, others such as olmesartan and telmisartan display varying degrees of insurmountability. This study compared the molecular interactions of olmesartan and telmisartan with the human AT(1) receptor, using well characterised in vitro methods and model systems. EXPERIMENTAL APPROACH: CHO-K1 cells that stably express human AT(1) receptors (CHO-hAT(1) cells) were used in several pharmacological studies of olmesartan and telmisartan, including direct radioligand binding and inhibition of Ang II-induced inositol phosphate (IP) accumulation. KEY RESULTS: Both ARBs were found to be competitive antagonists that displayed high affinity, slow dissociation, and a high degree of insurmountability for the AT(1) receptor (the latter greater with olmesartan). Their receptor interactions could be described by a two-step process with the initial formation of a loose complex (IR) and subsequent transformation into a tight binding complex (IR*). In washout experiments, [(3)H] telmisartan dissociated from the receptor with a half-life of 29 min and the Ang II-mediated IP accumulation response was 50% maximally restored within 24 min, whereas values for [(3)H] olmesartan were 72 min and 76 min, respectively. CONCLUSIONS AND IMPLICATIONS: The high degree of insurmountability, slow dissociation, and high affinity of olmesartan for its receptor may relate to its ability to stabilise IR* via the carboxyl group of its imidazole core. In comparison, telmisartan displays a less potent interaction with the receptor.