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1.
J Mol Diagn ; 21(4): 580-592, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31026601

RESUMEN

A point-of-care HIV-1 genotypic resistance assay that could be performed during a clinic visit would enable care providers to make informed treatment decisions for patients starting therapy or experiencing virologic failure on therapy. The main challenge for such an assay is the genetic variability at and surrounding each drug-resistance mutation (DRM). We analyzed a database of diverse global HIV sequences and used thermodynamic simulations to design an array of surface-bound oligonucleotide probe sets with each set sharing distinct 5' and 3' flanking sequences but having different centrally located nucleotides complementary to six codons at HIV-1 DRM reverse transcriptase position 103: AAA, AAC, AAG, AAT, AGA, and AGC. We then performed in vitro experiments using 80-mer oligonucleotides and PCR-amplified DNA from clinical plasma HIV-1 samples and culture supernatants that contained subtype A, B, C, D, CRF01_AE, and CRF02_AG viruses. Multiplexed solid-phase melt curve analysis discriminated perfectly among each of the six reported reverse transcriptase position 103 codons in both 80-mers and clinical samples. The sensitivity and specificity for detecting targets that contained AAC mixed with targets that contained AAA were >98% when AAC was present at a proportion of ≥10%. Multiplexed solid-phase melt curve analysis is a promising approach for developing point-of-care assays to distinguish between different codons in genetically variable regions such as those surrounding HIV-1 DRMs.


Asunto(s)
Farmacorresistencia Viral , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , Pruebas en el Punto de Atención , Bases de Datos Genéticas , Genotipo , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Humanos , Mutación , ARN Viral
2.
Nat Biotechnol ; 36(8): 738-745, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30010676

RESUMEN

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.


Asunto(s)
Virus ADN/efectos de los fármacos , Genotipo , Mycobacterium tuberculosis/efectos de los fármacos , Virus ARN/efectos de los fármacos , Semiconductores , Recuento de Colonia Microbiana , Sondas de ADN , Virus ADN/genética , Virus ADN/aislamiento & purificación , ADN Viral/análisis , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Viral/genética , Estudios de Factibilidad , Genoma Bacteriano , Humanos , Miniaturización , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico , Virus ARN/genética , Virus ARN/aislamiento & purificación , ARN Viral/análisis
3.
Biol Methods Protoc ; 3(1): bpy005, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-32161799

RESUMEN

PCR-based techniques are widely used to identify disease causing bacterial and viral pathogens, especially in point-of-care or near-patient clinical settings that require rapid results and sample-to-answer workflows. However, such techniques often fail to differentiate between closely related species that have highly variable genomes. Here, a homogenous (closed-tube) pathogen identification and classification method is described that combines PCR amplification, array-based amplicon sequence verification, and real-time detection using an inverse fluorescence fluorescence-resonance energy transfer technique. The amplification is designed to satisfy the inclusivity criteria and create ssDNA amplicons, bearing a nonradiating quencher moiety at the 5'-terminus, for all the related species. The array includes fluorescent-labeled probes which preferentially capture the variants of the amplicons and classify them through solid-phase thermal denaturing (melt curve) analysis. Systematic primer and probe design algorithms and empirical validation methods are presented and successfully applied to the challenging example of identification of, and differentiation between, closely related human rhinovirus and human enterovirus strains.

4.
Tissue Eng Regen Med ; 14(5): 587-593, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30603512

RESUMEN

Human breast milk stem cells (hBSCs) contain a population of cells with the ability to differentiate into various cell lineages for cell therapy applications. The current study examined the differentiation potential of hBSCs into hepatocytes-like cells. The cells were isolated from the breast milk and were treated with hepatogenic medium containing hepatocyte growth factor, insulin-like growth factor and dexamethasone for 7 days subsequently; Oncostatin M was added to the culture media. RT-PCR and immunocytochemistry were performed to detect the hepatogenic markers. The glycogen storage and the ability of the cells to absorb and release indocynanin green were also tested. The data showed that most of the differentiated cells formed cell aggregates after the 30th day, with more cells accumulated to form spheroids. RT-PCR revealed the expression of the hepatic nuclear factor, albumin, cytokeratin 18 and 19, cytochrome P2B6, glucose-6-phospahtase and claudin. The functional assays also showed glycogen storage and omission of indicynine green. Our study demonstrated hBSCs are novel population that can differentiate into hepatocyte-like cells.

5.
J Huntingtons Dis ; 4(2): 109-18, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26397892

RESUMEN

Within the past decade, there has been increasing interest in the role of tryptophan (Trp) metabolites and the kynurenine pathway (KP) in diseases of the brain such as Huntington's disease (HD). Evidence is accumulating to suggest that this pathway is imbalanced in neurologic disease states. The KP diverges into two branches that can lead to production of either neuroprotective or neurotoxic metabolites. In one branch, kynurenine (Kyn) produced as a result of tryptophan (Trp) catabolism is further metabolized to neurotoxic metabolites such as 3-hydroxykunurenine (3-HK) and quinolinic acid (QA). In the other branch, Kyn is converted to the neuroprotective metabolite kynurenic acid (KA). The enzyme Indoleamine 2,3 dioxygenase (IDO1) catalyzes the conversion of Trp into Kyn, the first and rate-limiting enzymatic step of the KP. This reaction takes place throughout the body in multiple cell types as a required step in the degradation of the essential amino acid Trp. Studies of IDO1 in brain have focused primarily on a potential role in depression, immune tolerance associated with brain tumours, and multiple sclerosis; however the role of this enzyme in neurodegenerative disease has garnered significant attention in recent years. This review will provide a summary of the current understanding of the role of IDO1 in Huntington's disease and will assess this enzyme as a potential therapeutic target for HD.


Asunto(s)
Enfermedad de Huntington/enzimología , Enfermedad de Huntington/prevención & control , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Quinurenina/metabolismo , Enfermedad de Alzheimer/enzimología , Animales , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Parkinson/enzimología , Transducción de Señal , Triptófano/metabolismo
6.
J Neurochem ; 127(6): 852-67, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23786539

RESUMEN

Indoleamine 2,3 dioxygenase (Ido1), the first and rate-limiting enzyme of the kynurenine pathway (KP), is a striatally enriched gene with increased expression levels in the YAC128 mouse model of Huntington disease (HD). Our objective in this study was to delineate age-related KP alterations in this model. Three enzymes potentially catalyze the first step of the KP; Ido1 and Indoleamine 2,3 dioxygenase-2 were highly expressed in the striatum and Tryptophan 2,3 dioxygenase (Tdo2) in the cerebellum. During development, Ido1 mRNA expression is dynamically regulated and chronically up-regulated in YAC128 mice. Kynurenine (Kyn) to tryptophan (Trp) ratio, a measure of activity in the first step of the KP, was elevated in YAC128 striatum, but no change in Tdo2 mRNA levels or Kyn to Trp ratio was detected in the cerebellum. Ido1 induction was coincident with Trp depletion at 3 months and Kyn accumulation at 12 months of age in striatum. Changes in downstream KP metabolites of YAC128 mice generally followed a biphasic pattern with neurotoxic metabolites reduced at 3 months and increased at 12 months of age. Striatally specific induction of Ido1 and downstream KP alterations suggest involvement in HD pathogenesis, and should be taken into account in future therapeutic developments for HD.


Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Enfermedad de Huntington/metabolismo , Quinurenina/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Cuerpo Estriado/crecimiento & desarrollo , Cuerpo Estriado/metabolismo , Femenino , Genotipo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Masculino , Ratones , Ácido Quinolínico/metabolismo , Serotonina/metabolismo , Transducción de Señal , Triptófano/metabolismo , Triptófano Oxigenasa/metabolismo
7.
J Neurosci ; 30(43): 14318-29, 2010 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-20980587

RESUMEN

YAC transgenic mice expressing poly(Q)-expanded full-length huntingtin (mhtt) recapitulate many behavioral and neuropathological features of Huntington disease (HD). We have previously observed a reduction in phosphorylation of mhtt at S421 in the presence of the mutation for HD. In addition, phosphorylation of normal S421-htt is reduced after excitotoxic stimulation of NMDA receptors (NMDARs). To test whether NMDAR stimulation contributes to reduced pS421-htt levels in HD, we determined phosphorylation of htt at Ser421 after NMDA-induced excitotoxicity in neurons from YAC128 mice. Here, we report that the total level of pS421-htt is reduced in YAC128 primary neurons after excitotoxic NMDAR stimulation. Similarly, the total level of pS421-htt is reduced in YAC128 transgenic mice after quinolinic acid injection into the striatum. In contrast, loss of phosphorylation of pS421-htt is prevented in YAC mice that never develop clinical or neuropathological features of HD [the caspase 6-resistant YAC128 transgene (C6R)]. To gain insight into the mechanisms underlying these findings, we determined that the Ser/Thr protein phosphatases PP1 and PP2A dephosphorylate pS421-htt in situ and after excitotoxic stimulation of NMDARs in neurons. Furthermore, increasing the phosphorylation of htt at S421 by blocking PP1 and PP2A activity protects YAC128 striatal neurons from NMDA-induced cell death. These results, together with the observed modulation of pS421-htt levels by dopamine, the reduced expression of PP1 inhibitor Darpp-32 in the striatum of YAC128 mice, and the reduced phosphorylation of PP1 substrate CreB, point to altered regulation of phosphatase activity in HD and highlight enhancing phosphorylation of htt at S421 as a therapeutic target.


Asunto(s)
Agonistas de Aminoácidos Excitadores/toxicidad , N-Metilaspartato/antagonistas & inhibidores , N-Metilaspartato/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 1/fisiología , Proteína Fosfatasa 2/fisiología , Animales , Muerte Celular/fisiología , Células Cultivadas , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Maleato de Dizocilpina/farmacología , Fosfoproteína 32 Regulada por Dopamina y AMPc/biosíntesis , Fosfoproteína 32 Regulada por Dopamina y AMPc/genética , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Proteína Huntingtina , Masculino , Ratones , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Fosforilación , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 2/antagonistas & inhibidores , Ácido Quinolínico/farmacología , Receptores de Dopamina D1/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Técnicas Estereotáxicas
8.
Hum Mol Genet ; 19(4): 609-22, 2010 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-19934114

RESUMEN

Selective degeneration of striatal neurons is a pathologic hallmark of Huntington disease (HD). The exact mechanism(s) behind this specific neurodegeneration is still unknown. Expression studies of diseased human post-mortem brain, as well as different mouse models exhibiting striatal degeneration, have demonstrated changes in the expression of many important genes with a large proportion of changes being observed in the striatal-enriched genes. These investigations have raised questions about how enrichment of particular transcripts in the striatum can lead to its selective vulnerability to neurodegeneration. Monitoring the expression changes of striatal-enriched genes during the course of the disease may be informative about their potential involvement in selective degeneration. In this study, we analyzed a Serial Analysis of Gene Expression (SAGE) database (www.mouseatlas.org) and compared the mouse striatum to 18 other brain regions to generate a novel list of striatal-enriched transcripts. These novel striatal-enriched transcripts were subsequently evaluated for expression changes in the YAC128 mouse model of HD, and differentially expressed transcripts were further examined in human post-mortem caudate samples. We identified transcripts with altered expression in YAC128 mice, which also showed consistent expression changes in human post-mortem tissue. The identification of novel striatal-enriched genes with altered expression in HD offers new avenues of study, leading towards a better understanding of specific pathways involved in the selective degeneration of striatal neurons in HD.


Asunto(s)
Cuerpo Estriado/metabolismo , Perfilación de la Expresión Génica , Enfermedad de Huntington/genética , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedad de Huntington/metabolismo , Ratones
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