Construction of a Gammaretrovirus with a Novel Tropism and Wild-Type Replication Kinetics Capable of Using Human APJ as Entry Receptor

We have constructed a replication-competent gammaretrovirus (SL3-AP) capable of using the human G-protein-coupled receptor hAPJ as its entry receptor. The envelope protein of the virus was made by insertion of the 13-amino-acid peptide ligand for hAPJ, flanked by linker sequences, into one of the variable loops of the receptor binding domain of SL3-2, a murine leukemia virus (MLV) that uses the xenotropic-polytropic virus receptor Xpr1 and which has a host range limited to murine cells. This envelope protein can utilize hAPJ as well as murine Xpr1 for entry into host cells with equal efficiencies. In addition, the SL3-AP virus replicates in cells expressing either of its receptors, hAPJ and murine Xpr1, and causes resistance to superinfection and downregulation of hAPJ in infected cells. Thus, SL3-AP is the first example of a retargeted replication-competent retrovirus, with replication characteristics and receptor interference properties similar to those of natural isolates.     

Assessing telomeric DNA content in pediatric cancers using whole-genome sequencing data

Background

Telomeres are the protective arrays of tandem TTAGGG sequence and associated proteins at the termini of chromosomes. Telomeres shorten at each cell division due to the end-replication problem and are maintained above a critical threshold in malignant cancer cells to prevent cellular senescence or apoptosis. With the recent advances in massive parallel sequencing, assessing telomere content in the context of other cancer genomic aberrations becomes an attractive possibility. We present the first comprehensive analysis of telomeric DNA content change in tumors using whole-genome sequencing data from 235 pediatric cancers.

Results

To measure telomeric DNA content, we counted telomeric reads containing TTAGGGx4 or CCCTAAx4 and normalized to the average genomic coverage. Changes in telomeric DNA content in tumor genomes were clustered using a Bayesian Information Criterion to determine loss, no change, or gain. Using this approach, we found that the pattern of telomeric DNA alteration varies dramatically across the landscape of pediatric malignancies: telomere gain was found in 32% of solid tumors, 4% of brain tumors and 0% of hematopoietic malignancies. The results were validated by three independent experimental approaches and reveal significant association of telomere gain with the frequency of somatic sequence mutations and structural variations.

Conclusions

Telomere DNA content measurement using whole-genome sequencing data is a reliable approach that can generate useful insights into the landscape of the cancer genome. Measuring the change in telomeric DNA during malignant progression is likely to be a useful metric when considering telomeres in the context of the whole genome.     

High-performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum: Application to a bioequivalence study

A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of anti platelet agent, clopidogrel, in human serum. The analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. A mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 mL/L; pH 5.7) and acetonitrile (56:44 v/v) was used and chromatographic separation was achieved using C18 analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05-10 microg/mL of CCA in human serum. The total run time of analysis was 5.5 min and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 microg/mL, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.     

Determination of oseltamivir carboxylic acid in human serum by solid phase extraction and high performance liquid chromatography with UV detection

A new straightforward method based on cloud-point extraction (CPE) has been developed, optimized and validated for the determination of venlafaxine in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection. The non-ionic surfactant Triton X-114 (polyethylene glycol tert-octylphenyl ether) was chosen as the extract solvent. Separation was obtained using a reversed-phase Diamonsil column (C(18), 250mmx4.6mm I.D., 5mum) and a mobile phase composed of acetonitrile-phosphate buffer solution (pH 3.0)-triethylamine (33.5:66.5:0.4). Fluorescence detection was used (lambda(ex) 276nm, lambda(em) 598nm). Maprotiline was used as the internal standard. Under the optimum conditions, the linear range of venlafaxine in human plasma was 10-800ngmL(-1) (r(2)=0.9995). The limit of detection (LOD) was less than 2ngmL(-1) (S/N=3) and the limit of quantification (LOQ) was less than 10ngmL(-1) (S/N=10). The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine capsules in nine healthy volunteers.     

Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine.     

Using paracrine effects of Ad-MSCs on keratinocyte cultivation and fabrication of epidermal sheets for improving clinical applications

Recent advances in wound healing have made cell therapy a potential approach for the treatment of various types of skin defects such as trauma, burns, scars and diabetic leg ulcers. Cultured keratinocytes have been applied to burn patients since 1981. Patients with acute and chronic wounds can be treated with autologous/allograft cultured keratinocytes. There are various methods for cultivation of epidermal keratinocytes used in cell therapy. One of the important properties of an efficient cell therapy is the preservation of epidermal stem cells. Mesenchymal Stem Cells (MSCs) are major regulatory cells involved in the acceleration of wound healing via induction of cell proliferation, angiogenesis and stimulating the release of paracrine signaling molecules. Considering the beneficial effects of MSCs on wound healing, the main aim of the present study is investigating paracrine effects of Adipose-derived Mesenchymal Stem Cell (Ad-MSCs) on cultivation of keratinocytes with focusing on preservation of stem cells and their differentiation process. We further introduced a new approach for culturing isolated keratinocytes in vitro in order to generate epidermal keratinocyte sheets without using a feeder layer. To do so, Ad-MSC conditioned medium was applied as an alternative to commercial media for keratinocyte cultivation. In this study, the expression of several stem/progenitor cell (P63, K19 and K14) and differentition (K10, IVL and FLG) markers was examined using real time PCR on days 7, 14 and 21 of culture in keratinocytes in Ad-MSC conditioned medium. P63 and α6 integrin expression was also evaluated via flow cytometry. The results were compared with control group including keratinocytes cultured in EpiLife medium and our data indicated that this Ad-MSC conditioned medium is a good alternative for keratinocyte cultivation and producing epidermal sheets for therapeutic and clinical purposes. The reasons are the expression of stem cell and differentiation markers and overcoming the requirement for feeder layer which leads to a xenograft-free transplantation. Besides, this approach has low cost and is easier to perform. However, more in vitro and in vivo experiments as well as safety evaluation required before clinical applications.     

The Therapeutic Potential of PI3K/Akt/mTOR Inhibitors in Breast Cancer: Rational and Progress

Breast cancer (BC) is the most commonly diagnosed cancer in women. The PI3K/AKT/mTOR pathway is among the most frequently dysregulated pathways in patients with BC. The activation of this pathway is associated with increased cell growth and clinical outcome, and its overexpression is associated with a poor prognosis. It has been proposed that it may be of importance as a potential therapeutic target in the treatment of BC. The aim of current review is to provide an overview of the potential utility of PI3K/Akt/mTOR inhibitors in patients with BC, with particular emphasis on recent preclinical and clinical studies. J. Cell. Biochem. 119: 213–222, 2018. © 2017 Wiley Periodicals, Inc.