731 research outputs found

    Functional Maturation of Induced Pluripotent Stem Cell Hepatocytes in Extracellular Matrix-A Comparative Analysis of Bioartificial Liver Microenvironments

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    Induced pluripotent stem cells (iPSCs) are new diagnostic and potentially therapeutic tools to model disease and assess the toxicity of pharmaceutical medications. A common limitation of cell lineages derived from iPSCs is a blunted phenotype compared with fully developed, endogenous cells. We examined the influence of novel three-dimensional bioartificial microenvironments on function and maturation of hepatocyte-like cells differentiated from iPSCs and grown within an acellular, liver-derived extracellular matrix (ECM) scaffold. In parallel, we also compared a bioplotted poly-L -lactic acid (PLLA) scaffold that allows for cell growth in three dimensions and formation of cell-cell contacts but is infused with type I collagen (PLLA-collagen scaffold) alone as a "deconstructed" control scaffold with narrowed biological diversity. iPSC-derived hepatocytes cultured within both scaffolds remained viable, became polarized, and formed bile canaliculi-like structures; however, cells grown within ECM scaffolds had significantly higher P450 (CYP2C9, CYP3A4, CYP1A2) mRNA levels and metabolic enzyme activity compared with iPSC hepatocytes grown in either bioplotted PLLA collagen or Matrigel sandwich control culture. Additionally, the rate of albumin synthesis approached the level of primary cryopreserved hepatocytes with lower transcription of fetal-specific genes, alpha-fetoprotein and CYP3A7, compared with either PLLA-collagen scaffolds or sandwich culture. These studies show that two acellular, three-dimensional culture systems increase the function of iPSC-derived hepatocytes. However, scaffolds derived from ECM alone induced further hepatocyte maturation compared with bioplotted PLLA-collagen scaffolds. This effect is likely mediated by the complex composition of ECM scaffolds in contrast to bioplotted scaffolds, suggesting their utility for in vitro hepatocyte assays or drug discovery. SIGNIFICANCE Through the use of novel technology to develop three-dimensional (3D) scaffolds, the present study demonstrated that hepatocyte-like cells derived via induced pluripotent stem cell (iPSC) technology mature on 3D extracellular matrix scaffolds as a result of 3D matrix structure and scaffold biology. The result is an improved hepatic phenotype with increased synthetic and catalytic potency, an improvement on the blunted phenotype of iPSC-derived hepatocytes, a critical limitation of iPSC technology. These findings provide insight into the influence of 3D microenvironments on the viability, proliferation, and function of iPSC hepatocytes to yield a more mature population of cells for cell toxicity studies and disease modeling

    Novel CNGA3 and CNGB3 mutations in two Pakistani families with achromatopsia

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    PURPOSE: To identify the genetic defect in two Pakistani families with autosomal recessive achromatopsia. METHODS: Two families (RP26 and RP44) were originally diagnosed with retinal dystrophy based upon their medical history. To localize the causative genes in these families, homozygosity mapping was performed using Affymetrix 10K single nucleotide polymorphism (SNP) arrays. Sequence analysis was used to find the mutations in candidate genes cyclic nucleotide-gated channel alpha-3 (CNGA3; family RP26) and cyclic nucleotide-gated channel beta-3 (CNGB3; family RP44). Control individuals were analyzed by allele-specific PCR for the CNGA3 mutation and BstXI restriction analysis for the CNGB3 mutation. After genetic analysis, clinical diagnosis was re-evaluated by electroretinography and color vision testing. During the course of this study, selected affected members of family RP26 were given pink glasses as supportive therapy. RESULTS: Sequence analysis of the positional candidate genes identified a novel missense mutation in CNGA3 (c.822G>T; p.R274S) in family RP26, and a novel CNGB3 frameshift mutation (c.1825delG; p.V609WfsX9) in family RP44. Clinical re-evaluation after genetic analysis revealed that both families have segregating autosomal recessive achromatopsia. CONCLUSIONS: Genetic analysis of two Pakistani families with retinal disease enabled the establishment of the correct diagnosis of achromatopsia. Two novel mutations were identified in CNGA3 and CNGB3 that are both specifically expressed in cone photoreceptors. Re-evaluation of the clinical status revealed that both families had achromatopsia. The use of pink glasses in patients was helpful in reducing photophobia and enabled rod-mediated vision

    A novel mutation in GRK1 causes Oguchi disease in a consanguineous Pakistani family

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    Contains fulltext : 76070.pdf (publisher's version ) (Open Access)PURPOSE: The purpose of this study was to identify the underlying molecular genetic defect in a large consanguineous Pakistani family with Oguchi disease who had been given a diagnosis of autosomal recessive retinitis pigmentosa. METHODS: The family was genotyped with the Affymetrix 10K single nucleotide polymorphism array. Fine-mapping of a common homozygous region on chromosome 13q was performed using fluorescent microsatellite markers. Mutation analysis was done by direct sequencing of the candidate gene GRK1 located in the region. The segregation of a novel mutation in the family and the frequency of the identified mutation in the Pakistani population were determined by StuI RFLP analysis. RESULTS: Genetic mapping supported the diagnosis of typical Oguchi disease in a Pakistani family and also resulted in the identification of a novel nonsense mutation (c.614C>A; p.S205X) in exon 1 of GRK1. This mutation is predicted to result in premature termination of the protein product, thereby affecting the phototransduction cascade. A clinical reappraisal of the family revealed that all patients homozygous for this variant had Oguchi disease. CONCLUSIONS: This is the first report to describe a mutation causing typical Oguchi disease in a large consanguineous Pakistani family. This mutation segregated in eight affected members.6 p

    Pharmacokinetic and exposure-response analysis of pertuzumab in patients with HER2-positive metastatic gastric or gastroesophageal junction cancer

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    Purpose: To characterize the pharmacokinetics (PK) of pertuzumab and trastuzumab in patients with HER2-positive metastatic gastric or gastroesophageal junction cancer in the randomized, double-blind, phase III JACOB study (NCT01774786), and to evaluate the appropriateness of the pertuzumab regimen in these patients. Methods: Patients received 840 mg intravenous pertuzumab or placebo plus trastuzumab q3w and chemotherapy. Pertuzumab and trastuzumab were administered until disease progression or unacceptable toxicity. Chemotherapy was administered for up to six cycles or disease progression or unacceptable toxicity. Serum concentrations of pertuzumab and trastuzumab were measured. Pertuzumab PK was characterized across treatment cycles. The impact of anti-drug antibodies (ADAs) on pertuzumab PK and the impact of pertuzumab on trastuzumab PK were assessed. An exploratory exposure-efficacy analysis was also conducted. Results: In total, 374 patients in the pertuzumab arm had evaluable PK data. The mean observed pertuzumab steady-state serum trough (minimum) concentration (C) ± standard deviation was 114 ± 51.8 μg/mL. The target pertuzumab C of ≥ 20 μg/mL was reached in 99.3% of patients at Cycle 5 (steady state) and beyond. Greater than 90% of patients were above the PK target right after the first pertuzumab dose. There was no apparent impact of ADAs on pertuzumab PK nor of pertuzumab on trastuzumab PK. There were no differences in overall survival across Cycle 1 pertuzumab (C) or Cycle 5 pertuzumab (C) exposure quartiles. Conclusions: Pertuzumab exposure in JACOB was consistent with prior studies in advanced gastric cancer and breast cancer. The 840 mg q3w dose allowed the majority of patients in JACOB to achieve target pertuzumab concentrations and appears to be an appropriate dose selectio

    The Glasgow Benefit Inventory: a systematic review of the use and value of an otorhinolaryngological generic patient-recorded outcome measure

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    The Glasgow Benefit Inventory (GBI) is a validated, generic patient-recorded outcome measure widely used in otolaryngology to report change in quality of life post-intervention.To date, no systematic review has made (i) a quality assessment of reporting of Glasgow Benefit Inventory outcomes; (ii) a comparison between Glasgow Benefit Inventory outcomes for different interventions and objectives; (iii) an evaluation of subscales in describing the area of benefit; (iv) commented on its value in clinical practice and research.Systematic review.'Glasgow Benefit Inventory' and 'GBI' were used as keywords to search for published, unpublished and ongoing trials in PubMed, EMBASE, CINAHL and Google in addition to an ISI citation search for the original validating Glasgow Benefit Inventory paper between 1996 and January 2015.Papers were assessed for study type and quality graded by a predesigned scale, by two authors independently. Papers with sufficient quality Glasgow Benefit Inventory data were identified for statistical comparisons. Papers with 50% and gave sufficient Glasgow Benefit Inventory total and subscales for meta-analysis. For five of the 11 operation categories (vestibular schwannoma, tonsillectomy, cochlear implant, middle ear implant and stapes surgery) that were most likely to have a single clear clinical objective, score data had low-to-moderate heterogeneity. The value in the Glasgow Benefit Inventory having both positive and negative scores was shown by an overall negative score for the management of vestibular schwannoma. The other six operations gave considerable heterogeneity with rhinoplasty and septoplasty giving the greatest percentages (98% and 99%) most likely because of the considerable variations in patient selection. The data from these operations should not be used for comparative purposes. Five papers also reported the number of patients that had no or negative benefit, a potentially a more clinically useful outcome to report. Glasgow Benefit Inventory subscores for tonsillectomy were significantly different from ear surgery suggesting different areas of benefitThe Glasgow Benefit Inventory has been shown to differentiate the benefit between surgical and medical otolaryngology interventions as well as 'reassurance'. Reporting benefit as percentages with negative, no and positive benefit would enable better comparisons between different interventions with varying objectives and pathology. This could also allow easier evaluation of factors that predict benefit. Meta-analysis data are now available for comparison purposes for vestibular schwannoma, tonsillectomy, cochlear implant, middle ear implant and stapes surgery. Fuller report of the Glasgow Benefit Inventory outcomes for non-surgical otolaryngology interventions is encouraged

    Cigarette smoking among adults -- United States, 1988

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    Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30010/1/0000378.pd

    Early Laser for Burn Scars (ELABS): protocol for a multi-centre randomised, controlled trial of both the effectiveness and cost-effectiveness of the treatment of hypertrophic burn scars with Pulsed Dye Laser and standard care compared to standard care alone [version 1; peer review: 2 approved].

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    This paper outlines the protocol for a study that is being carried out at multiple centres across the UK in the next three years. It is a Research for Patient Benefit (RfPB) study funded by the National Institute for Healthcare Research (NIHR). The aim is to assess the effectiveness of treating hypertrophic burns scars with pulsed dye laser (PDL) at an early stage of scar formation. The objective is to improve Quality of Life for the patient by improving both the appearance and quality of burn scarring, as well as reducing its psychological impact. This is a parallel-arm randomised, controlled trial to compare PDL and standard care against standard care alone. The difference is measured between baseline and six-month follow-up. Recruits are within three months of healing from a burn injury; with wounds showing a defined potential for hypertrophic scarring. A total of 120 patients are recruited in a multi-centre study; with randomisation in a 1:1 allocation to each arm. The treatment arm receives 3 PDL treatments at six-week intervals in addition to standard care, whereas the control arm receives standard care alone. The primary outcome is the patient-rated part of the Patient and Observer Scar Scale (POSAS). Psychological and psycho-social impact is evaluated using the CARe burn scale (UWE, Bristol) and Quality Adjusted Life Years (QALY) is determined using the Short-Form Health Survey (SF-12). The study evaluates both the cost-effectiveness through an economic analysis and the patient-reported experience of the treatment by phone interviews

    An IRAK1-PIN1 signalling axis drives intrinsic tumour resistance to radiation therapy

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    Drug-based strategies to overcome tumour resistance to radiotherapy (R-RT) remain limited by the single-agent toxicity of traditional radiosensitizers (for example, platinums) and a lack of targeted alternatives. In a screen for compounds that restore radiosensitivity in p53 mutant zebrafish while tolerated in non-irradiated wild-type animals, we identified the benzimidazole anthelmintic oxfendazole. Surprisingly, oxfendazole acts via the inhibition of IRAK1, a kinase thus far implicated in interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) immune responses. IRAK1 drives R-RT in a pathway involving IRAK4 and TRAF6 but not the IL-1R/TLR-IRAK adaptor MyD88. Rather than stimulating nuclear factor-κB, radiation-activated IRAK1 prevented apoptosis mediated by the PIDDosome complex (comprising PIDD, RAIDD and caspase-2). Countering this pathway with IRAK1 inhibitors suppressed R-RT in tumour models derived from cancers in which TP53 mutations predict R-RT. Moreover, IRAK1 inhibitors synergized with inhibitors of PIN1, a prolyl isomerase essential for IRAK1 activation in response to pathogens and, as shown here, in response to ionizing radiation. These data identify an IRAK1 radiation-response pathway as a rational chemoradiation therapy target

    Tomato: a crop species amenable to improvement by cellular and molecular methods

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    Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.
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