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1.
Bioanalysis ; 15(24): 1461-1468, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38044848

RESUMO

While using the cloud environment for various functions has become commonplace, relatively little attention has been given to considerations for the use of third-party cloud services for regulated bioanalytical workflow and data management. Little guidance has been provided as to how to utilize the cloud to support bioanalytical activities. It can be intimidating when considering how to go about using cloud services for data acquisition, but there are some general ideas to keep in mind when evaluating ways to accommodate regulated bioanalysis online. Determining how to incorporate the use of cloud storage with data that are generated from regulated bioanalytical analysis is an important step in maintaining the security of the data.


Assuntos
Computação em Nuvem
2.
Bioanalysis ; 13(17): 1313-1321, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34515519

RESUMO

Challenges for data storage during drug development have become increasingly complex as the pharmaceutical industry expands in an environment that requires on-demand availability of data and resources for users across the globe. While the efficiency and relative low cost of cloud services have become increasingly attractive, hesitancy toward the use of cloud services has decreased and there has been a significant shift toward real-world implementation. Within GxP laboratories, the considerations for cloud storage of data include data integrity and security, as well as access control and usage for users around the globe. In this review, challenges and considerations when using cloud storage options for the storage of laboratory-based GxP data are discussed and best practices are defined.


Assuntos
Computação em Nuvem/normas , Armazenamento e Recuperação da Informação/métodos , Laboratórios/normas , Humanos
3.
Physiol Rep ; 3(7)2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26229004

RESUMO

Despite established health benefits of regular exercise, the majority of Americans do not meet the recommended levels of physical activity. While it is known that voluntary activity levels are largely heritable, the genetic mechanisms that regulate activity are not well understood. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit transcription by binding to a target gene, inhibiting protein production. The purpose of this study was to investigate differential miRNA expression between inherently high- (C57L/J) and low- (C3H/HeJ) active inbred mice in soleus, extensor digitorum longus (EDL), and nucleus accumbens tissues. Expression was initially determined by miRNA microarray analysis, and selected miRNAs were validated by qRT-PCR. Expression of 13 miRNAs varied between strains in the nucleus accumbens, 20 in soleus, and eight in EDL, by microarray analysis. Two miRNAs were validated by qRT-PCR in the nucleus accumbens; miR-466 was downregulated (~4 fold; P < 0.0004), and miR-342-5p was upregulated (~115 fold; P < 0.0001) in high-active mice. MiR-466 was downregulated (~5 fold; P < 0.0001) in the soleus of high-active mice as well. Interestingly, miR-466 is one of several miRNA families with sequence located in intron 10 of Sfmbt2; miRNAs at this locus are thought to drive imprinting of this gene. "Pathways in cancer" and "TGFß signaling" were the most significant pathways of putative target genes in both the soleus and nucleus accumbens. Our results are the first to consider differential miRNA expression between high- and low-active mice, and suggest that miRNAs may play a role in regulation of physical activity.

4.
Biomed Res Int ; 2014: 361048, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24551844

RESUMO

Numerous candidate genes have been suggested in the recent literature with proposed roles in regulation of voluntary physical activity, with little evidence of these genes' functional roles. This study compared the haplotype structure and expression profile in skeletal muscle and brain of inherently high- (C57L/J) and low- (C3H/HeJ) active mice. Expression of nine candidate genes [Actn2, Actn3, Casq1, Drd2, Lepr, Mc4r, Mstn, Papss2, and Glut4 (a.k.a. Slc2a4)] was evaluated via RT-qPCR. SNPs were observed in regions of Actn2, Casq1, Drd2, Lepr, and Papss2; however, no SNPs were located in coding sequences or associated with any known regulatory sequences. In mice exposed to a running wheel, Casq1 (P = 0.0003) and Mstn (P = 0.002) transcript levels in the soleus were higher in the low-active mice. However, when these genes were evaluated in naïve animals, differential expression was not observed, demonstrating a training effect. Among naïve mice, no genes in either tissue exhibited differential expression between strains. Considering that no obvious SNP mechanisms were determined or differential expression was observed, our results indicate that genomic structural variation or gene expression data alone is not adequate to establish any of these genes' candidacy or causality in relation to regulation of physical activity.


Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Atividade Motora/genética , Músculo Esquelético/metabolismo , Animais , Perfilação da Expressão Gênica , Haplótipos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único
5.
Artigo em Inglês | MEDLINE | ID: mdl-23891912

RESUMO

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for a PEGylated adnectin therapeutic protein in cynomolgus monkey plasma. The validated method was performed using protein precipitation coupled with trypsin digestion, followed by LC-MS/MS detection of a surrogate peptide generated from the PEGylated adnectin protein. A tryptic peptide generated from a PEGylated adnectin protein analog was used as the internal standard to standardize the digestion, extraction, and quantitation processes. The protein precipitation extraction of the protein from cynomolgus plasma was performed using an acidic 2-propanol organic solution. Following the extraction, the supernatant was removed and a 45min trypsin digestion was performed at 60°C on the supernatant layer. The linear dynamic range of the assay was 50.0-25,000ng/mL. Chromatographic separation was performed with an Acquity BEH C18 (1.7µm particle size, 2.1mm×50mm) column using gradient elution. The assay proved to have robust accuracy, precision, and stability for the representative surrogate peptide of the PEGylated adnectin protein being evaluated. The validated method was implemented as a high throughput assay for a PEGylated adnectin protein using a similar PEGylated adnectin therapeutic protein as the internal standard that can be used for future monkey toxicokinetic (TK) studies.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fibronectinas/sangue , Macaca fascicularis/sangue , Polietilenoglicóis/química , Espectrometria de Massas em Tandem/métodos , Tripsina/química , Animais , Precipitação Química , Fibronectinas/química
6.
Mol Cancer Ther ; 12(9): 1701-14, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23804705

RESUMO

Angiosarcoma is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human angiosarcoma, we studied primary cells and tumorgrafts derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive extracellular signal-regulated kinase (ERK) activation. The mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multireceptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human angiosarcoma, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was upregulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human angiosarcoma. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human angiosarcoma, and it highlights the use of spontaneous canine cancers as a model of human disease.


Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Difenilamina/análogos & derivados , Hemangiossarcoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Animais , Difenilamina/farmacologia , Modelos Animais de Doenças , Cães , Ensaios de Seleção de Medicamentos Antitumorais , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Hemangiossarcoma/tratamento farmacológico , Hemangiossarcoma/metabolismo , Hemangiossarcoma/veterinária , Humanos , Camundongos , Camundongos Nus , Niacinamida/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Artigo em Inglês | MEDLINE | ID: mdl-22177787

RESUMO

A screening method was developed to extract and detect berberine and hydrastine alkaloids from goldenseal root powder and urine samples using HPLC with UV detection. The isocratic method was developed to detect alkaloids in 5 mL of urine prior to drug screening. Urine samples were spiked with the alkaloids at varying concentrations and extracted twice with 3:1 chloroform:2-propanol (CHCl(3):2-propanol). The extracts were combined, concentrated using nitrogen gas and the residue was then reconstituted with a mobile phase of acetonitrile:buffer (32:68). A 17 min isocratic run time was performed with a flow rate of 2.0 mL/min, and UV detection at 230 nm using a C(18) (250 mm × 4.6 mm) column at room temperature. The method showed good linearity for berberine (r(2)=0.9990) and hydrastine (r(2)=0.9983) over a range of 11.80 ng/mL to 17.64 µg/mL. The LOD for berberine in urine was 12.74 ng/mL and the LOD for hydrastine in urine was 54.48 ng/mL. Urine samples were spiked with goldenseal root powder and liquid extract as part of a blinded study to determine whether berberine and hydrastine alkaloids could also be extracted in vitro from goldenseal and show a presence in urine samples. Out of the 37 blinded urine samples extracted the two spiked samples were correctly identified based on the presence or absence of berberine and hydrastine. The results demonstrated that this method will enable laboratories to test for the herbal supplement in submitted urine samples prior to drug testing, avoiding false negative results.


Assuntos
Benzilisoquinolinas/urina , Berberina/urina , Cromatografia Líquida de Alta Pressão/métodos , Hydrastis/química , Humanos , Limite de Detecção , Modelos Lineares , Extratos Vegetais/química , Raízes de Plantas/química , Reprodutibilidade dos Testes , Espectrofotometria Ultravioleta/métodos , Detecção do Abuso de Substâncias
8.
Bioanalysis ; 4(10): 1151-3, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22651557

RESUMO

The 2011 Eastern Analytical Symposium (EAS) and Exposition was held at the Garden State Exhibit Center in Somerset NJ, USA, 14-17 November and marked the 50th anniversary of EAS, with a theme of 'Celebrating Innovation in Analysis'. The technical program was rich in presentations relevant to bioanalytical sciences, covering biomarkers, proteomics/metabolomics, small molecule and protein LC-MS bioanalysis, immunogenicity and biological sample preparation. This conference report highlights some of the lectures and short courses of interest to bioanalysts at the 2011 EAS.


Assuntos
Pesquisa Biomédica , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Biomarcadores/análise , Química Analítica/tendências , Humanos , Metabolômica/métodos , New Jersey , Preparações Farmacêuticas/análise , Proteínas/análise , Proteômica/métodos
9.
J Pharm Biomed Anal ; 70: 401-7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22776736

RESUMO

To support a first-in-human (FIH) clinical study in healthy volunteers, a human plasma assay, a 20-fold more sensitive method than the validated non-clinical LC-MS/MS assays, was requested. For the clinical assay, a LLOQ of 0.050 ng/mL for Compound A and 0.100 ng/mL for Compound B was desired to accurately determine the analyte concentrations in human plasma samples across all treatment groups. A design of experiment (DOE) investigation was performed in an effort to optimize the extraction procedure of the bioanalytical assay used to support the first in human study and future clinical studies. Three factors, extraction buffer pH (two pHs), volume ratio of organic solvent to plasma (two ratios), and extraction shake time (three times), were selected for the DOE. Both analytes were analyzed at a low concentration, 0.150 ng/mL, and a stable isotope label internal standard was used for each analyte. To estimate the recovery of each analyte from the extraction, the response ratio of each analyte over the respective internal standard was used, and to estimate matrix effects, the absolute response (peak area) of each analyte was used. The results of the DOE indicated that the three factors tested had a more significant effect on the extraction of the metabolite, Compound B, compared to that of the parent, Compound A. The extraction buffer pH had the greatest influence on Compound B and the volume of extraction solvent had an influence on both analytes. Unexpectedly, a longer extraction time caused an apparent decrease in the overall recovery for both analytes. This was presumably due to an increased extraction of interfering matrix components. Optimal conditions were achieved for the combined analysis of both compounds using the DOE approach.


Assuntos
Bioensaio/métodos , Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão , Projetos de Pesquisa , Espectrometria de Massas em Tandem , Bioensaio/normas , Soluções Tampão , Calibragem , Cromatografia Líquida de Alta Pressão/normas , Humanos , Concentração de Íons de Hidrogênio , Modelos Estatísticos , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Solventes/química , Espectrometria de Massas em Tandem/normas , Fatores de Tempo
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