Aptamer-Based Sandwich Assay for Measurement of Thymidine Kinase 1 in Serum of Cancerous Patients.

Thymidine kinase 1 (TK1) is traditionally a serum biomarker that is elevated in the early stages of malignancies. The diagnostic and prognostic role of TK1 for screening and monitoring human malignancies has recently been investigated. Anti-human TK1 aptamers were selected through 12 iterative rounds of systematic evolution of ligands by exponential enrichment from a DNA library. The aptamer pool of round 12 was amplified, and the polymerase chain reaction product was cloned on the TA vector. Of the 85 colonies obtained, 52 were identified as positive clones. These aptamers were screened for TK1 with surface plasmon resonance, where apta37 and apta69 showed the highest affinity for TK1. The TK1_apta37 and TK1_apta69 aptamers were used in a sandwich assay platform and successfully detected TK1 in the concentration range of 54-3500 pg mL-1. Clinical samples from 60 cancerous patients were also tested with this assay system and compared using the conventional antibody-based enzyme-linked immunosorbent assay kit. The aptamer sandwich assay demonstrated a dynamic range for TK1 at clinically relevant serum levels, covering subpicogram per milliliter concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. The results of this study demonstrate the screening capability of the aptamer sandwich assay platform and its potential applicability to the point-of-care testing system.

The enhancement of differentiating adipose derived mesenchymal stem cells toward hepatocyte like cells using gelatin cryogel scaffold.

Liver tissue engineering creates a promising methodology for developing functional tissue to restore or improve the function of lost or damaged liver by using appropriate cells and biologically compatible scaffolds. The present paper aims to study the hepatogenic potential of human adipose derived mesenchymal stem cells (hADSCs) on a 3D gelatin scaffold in vitro. For this purpose, mesenchymal stem cells were isolated from human adipose tissue and characterized by flowcytometry analysis and mesodermal lineage differentiation capacity. Then, porous cryogel scaffolds were fabricated by cryogelating the gelatin using glutaraldehyde as the crosslinking agent. The structure of the scaffolds as well as the adhesion and proliferation of the cells were then determined by Scanning Electron Microscopy (SEM) analysis and MTT assay, respectively. The efficiency of hepatic differentiation of hADSCs on 2D and 3D culture systems has been assessed by means of morphological, cytological, molecular and biochemical approaches. Based on the results of flowcytometry, the isolated cells were positive for hMSC specific markers and negative for hematopoietic markers. Further, the multipotency of these cells was confirmed by adipogenic and osteogenic differentiation and the highly porous structure of scaffolds was characterized by SEM images. Biocompatibility was observed in the fabricated gelatin scaffolds and the adhesion and proliferation of hADSCs were promoted without any cytotoxicity effects. In addition, compared to 2D TCPS, the fabricated scaffolds provided more appropriate microenvironment resulting in promoting the differentiation of hADSCs toward hepatocyte-like cells with higher expression of hepatocyte-specific markers and appropriate functional characteristics such as increased levels of urea biosynthesis and glycogen storage. Finally, the created 3D gelatin scaffold could provide an appropriate matrix for hepatogenic differentiation of hADSCs, which could be considered for liver tissue engineering applications.

Human Bone Marrow Mesenchymal Stem Cell Behaviors on PCL/Gelatin Nanofibrous Scaffolds Modified with A Collagen IV-Derived RGD-Containing Peptide.

OBJECTIVE: We introduce an RGD (Arg-Gly-Asp)-containing peptide of collagen IV origin that possesses potent cell adhesion and proliferation properties. MATERIALS AND METHODS: In this experimental study, the peptide was immobilized on an electrospun nanofibrous polycaprolactone/gelatin (PCL/Gel) hybrid scaffold by a chemical bonding approach to improve cell adhesion properties of the scaffold. An iodine-modified phenylalanine was introduced in the peptide to track the immobilization process. Native and modified scaffolds were characterized with scanning electronmicroscopy (SEM) and fourier transform infrared spectroscopy (FTIR). We studied the osteogenic and adipogenic differentiation potential of human bone marrow-derived mesenchymal stem cells (hBMSCs). In addition, cell adhesion and proliferation behaviors of hBMSCs on native and peptide modified scaffolds were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4',6-diamidino-2-phenylindole (DAPI) staining, and the results compared with tissue culture plate, as the control. RESULTS: FTIR results showed that the peptide successfully immobilized on the scaffold.MTT assay and DAPI staining results indicated that peptide immobilization had a dramatic effect on cell adhesion and proliferation. CONCLUSION: This peptide modified nanofibrous scaffold can be a promising biomaterial for tissue engineering and regenerative medicine with the use of hBMSCs.

Alpha 1-antitrypsin activity is markedly decreased in Wegener's granulomatosis.

Alpha 1-antitrypsin (A1AT) is the most abundant proteinase inhibitor in plasma and the main inhibitor of Proteinase 3, the target antigen of antineutrophil cytoplasmic antibodies (ANCAs) that predominant in Wegeners' granulomatosis. Α1AT deficiency correlated with ANCA-associated vasculitis. This study explores the trypsin inhibitory capacity (TIC), specific activity, and phenotypic deficiency of Α1AT in Wegener's granulomatosis. Twenty-seven WG patients were studied. ANCA was tested by IIF and ELISA. Serum a1-anti-trypsin levels were quantified in WG patients and healthy controls by immunoturbidimetric assay. Serum TIC was assessed by the enzymatic colorimetric assay. Phenotypes of A1AT were detected by Isoelectric Focusing. A1AT concentration was equivalent in patients and controls; however, serum TIC (P = 0.001) and specific activity of A1AT (P = 0.001) were dramatically lower in WG patients. Five patients had deficient phenotypes of A1AT: MZ (n = 3), MS (n = 1) and SS (n = 1). This was correlated with an increase in the prevalence of deficient phenotypes of A1AT in WG (P = 0.01). Trypsin inhibitory capacity and specific activity of A1AT were decreased in WG patients and may be involve in disease pathogenesis and can worsen the clinical manifestations. This A1AT deficiency probably resulted from oxidative inactivation and/or enzymatic degradation of A1AT. This could result in localized deficiency of A1AT in vessel wall interfaces and lead to severe disease.