Identification of Candidate miRNAs and Predication of Their Role in Keratoconus

Keratoconus (KTCN, OMIM 148300) is known as an eye degenerative disease leading to stromal thinning and conical shape of the cornea. These structural changes can be accompanied by loss of visual function in advanced cases. To date, in spite of recent advances in the investigation of molecular mechanisms which result in Keratoconus, there’s still a lack of information about the role of miRNAs in this disorder. Accordingly, this study aims to find miRNA’s aberrantly expression in KTCN suffering cases and to predict their role by investigating their possible interactions with significantly KTCN correlated genes. The data were comprised of 25 normal and 25 KTCN cases. Weighted gene co-expression network analysis approach was used to construct a protein-coding gene co-expression network and investigate the significant modules. Gene with the higher module membership (MM) and gene significance (GS) in the selected modules were supposed to be more KTCN relevant genes.  Totally 2492 protein-coding genes (PCGs) and 99 miRNAs were up-regulated and 213 PCGs and 31 miRNAs were down-regulated. Significant correlation with the KTCN was observed in three modules, including brown, green-yellow, and salmon from the total of 15 modules. Genes in significant modules have been enriched to gene expression regulation related biological processes such as negative regulation of protein secretion, intra-Golgi vesicle-mediated transport, regulation of mRNA 3’-end processing, and cytoskeleton related gene ontologies such as modulation of the mitochondrial cytoskeleton. Up-regulated miRNAs that interact with down-regulated mRNAs within significant modules include miR-1305, miR-544a, miR-1245a, miR-4635, miR-4266

Mechanisms of simvastatin myotoxicity: The role of autophagy flux inhibition.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin\u27s myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts

Development of advanced operators for enhanced on-chip biosensing in digital microfluidic platforms

Over the past two decades, digital microfluidics (DMF) has grown significantly as a powerful tool for lab-on-a-chip (LOC) applications. Since the introduction of DMF with its primary capabilities in sample manipulation (transport, splitting and mixing) in the droplet format, many advances have been made towards the development of platforms capable of performing entire processes of biochemical assays in an automated fashion. These advances include the progress made towards the fabrication methods, and the development of techniques for sample manipulation and integration of biosensors. Despite the general success in such developments, DMF still lacks capabilities in sample manipulation (including the droplets and their contents, e.g. cells and microbeads), specifically for biosensing, in which sample preparation and post-sensing removal of the sample are required. Therefore, in majority of the applications, DMF has been used for processing parts of the entire assay, and after biosensing, the recovery of the chip was hindered due to the contamination of the biosensor for its hydrophilic behavior. This thesis aims at the development of advanced operators for accurate pre-sensing sample preparation, biosensing and post sensing sample removal. For this purpose, an unequal droplet splitting method is developed based on the geometrical modification of one actuating electrode, which enables dispensing/splitting droplets with a wide range of volumes and with an accuracy of over 99%. An electrohydrodynamic technique is developed for rapid mixing inside the stationary droplets, enhancing the mixing time and eliminating the need for frequent and cyclic transport of the droplet on the chip. A dielectrophoretic-gravity driven technique is developed for concentrating and focusing the particles and cells inside the sample droplet. Also, a systematic study has been performed on the surface properties and geometry of the biosensors to optimize their geometry and configuration on DMF devices for complete sample removal after biosensing. Finally, the application of the developed techniques for enhanced on-chip biosensing is shown for detection of Cryptosporidium as a proof of concept.Applied Science, Faculty ofEngineering, School of (Okanagan)Graduat

Investigating Programmed Cell Death and Tumor Invasion in a Three-Dimensional (3D) Microfluidic Model of Glioblastoma

Glioblastoma multiforme (GBM) is a rapidly progressive and deadly form of brain tumor with a median survival rate of ~15 months. GBMs are hard to treat and significantly affect the patient’s physical and cognitive abilities and quality of life. Temozolomide (TMZ)—an alkylating agent that causes DNA damage—is the only chemotherapy choice for the treatment of GBM. However, TMZ also induces autophagy and causes tumor cell resistance and thus fails to improve the survival rate among patients. Here, we studied the drug-induced programmed cell death and invasion inhibition capacity of TMZ and a mevalonate cascade inhibitor, simvastatin (Simva), in a three-dimensional (3D) microfluidic model of GBM. We elucidate the role of autophagy in apoptotic cell death by comparing apoptosis in autophagy knockdown cells (Atg7 KD) against their scrambled counterparts. Our results show that the cells were significantly less sensitive to drugs in the 3D model as compared to monolayer culture systems. An immunofluorescence analysis confirmed that apoptosis is the mechanism of cell death in TMZ- and Simva-treated glioma cells. However, the induction of apoptosis in the 3D model is significantly lower than in monolayer cultures. We have also shown that autophagy inhibition (Atg7 KD) did not change TMZ and Simva-induced apoptosis in the 3D microfluidic model. Overall, for the first time in this study we have established the simultaneous detection of drug induced apoptosis and autophagy in a 3D microfluidic model of GBM. Our study presents a potential ex vivo platform for developing novel therapeutic strategies tailored toward disrupting key molecular pathways involved in programmed cell death and tumor invasion in glioblastoma

Label-Free Capacitive Biosensor for Detection of Cryptosporidium

Cryptosporidium, an intestinal protozoan pathogen, is one of the leading causes of diarrhea in healthy adults and death in children. Detection of Cryptosporidium oocysts has become a high priority to prevent potential outbreaks. In this paper, a label-free interdigitated-based capacitive biosensor has been introduced for the detection of Cryptosporidium oocysts in water samples. Specific anti-Cryptosporidium monoclonal antibodies (IgG3) were covalently immobilized onto interdigitated gold electrodes as the capture probes, and bovine serum albumin was used to avoid non-specific adsorption. The immobilization of the antibodies was confirmed by measuring the change in the contact angle. The detection was achieved by measuring the relative change in the capacitive/dielectric properties due to the formation of Cryptosporidium-antibody complex. The biosensor has been tested for different concentrations of Cryptosporidium. The results show that the biosensor developed can accurately distinguish different numbers of captured cells and densities on the surface of the biosensor. The number of Cryptosporidium oocysts captured on the electrode surface was confirmed using a fluorescein isothiocyanate (FITC) immunofluorescence assay. The response from the developed biosensor has been mainly dependent on the concentration of Cryptosporidium under optimized conditions. The biosensor showed a linear detection range between 15 and 153 cells/mm² and a detection limit of 40 cells/mm². The label-free capacitive biosensor developed has a great potential for detecting Cryptosporidium in environmental water samples. Furthermore, under optimized conditions, this label-free biosensor can be extended for detection of other biomarkers for biomedical and environmental analyses.Applied Science, Faculty ofNon UBCEngineering, School of (Okanagan)ReviewedFacult

A bioengineering method for modeling alveolar Rhabdomyosarcoma and assessing chemotherapy responses

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Treatment of RMS usually includes primary tumor resection along with systemic chemotherapy. Two-dimensional (2D) cell culture systems and animal models have been extensively used for investigating the potential efficacy of new RMS treatments. However, RMS cells behave differently in 2D culture than in vivo, which has recently inspired the adoption of three-dimensional (3D) culture environments. In the current paper, we will describe the detailed methodology we have developed for fabricating a 3D engineered model to study alveolar RMS (ARMS) in vitro. This model consists of a thermally cross-linked collagen disk laden with RMS cells that mimics the structural and bio-chemical aspects of the tumor extracellular matrix (ECM). This process is highly reproducible and produces a 3D engineered model that can be used to analyze the cytotoxicity and autophagy induction of drugs on ARMS cells. The most improtant bullet points are as following: • We fabricated 3D model of ARMS. • The current ARMS 3D model can be used for screening of chemotherapy drugs. • We developed methods to detect apoptosis and autophagy in ARMS 3D model to detect the mechansims of chemotherapy agents

Fat harvesting site is an important determinant of proliferation and pluripotency of adipose-derived stem cells

To define the optimal fat harvest site and detect any potential differences in adipose-derived stem cells (ASCs) proliferation properties in camels, aspirates from the abdomen and hump sites were compared. Obtained results revealed that ASCs from both abdomen and hump exhibited spindle-shaped and fibroblast-like morphology with hump-derived ASCs being smaller in size and narrower in overall appearance than abdominal ASCs. Abdominal ASCs required a greater time for proliferation than the hump-derived cells. These results were further confirmed with a tetrazolium-based colorimetric assay (MTT) which showed a greater cell proliferation rate for hump ASCs than for the abdomen. Under inductive conditions, ASCs from both abdominal and hump fat deposits maintained their lineage differentiation potential into adipogenic, chondrogenic, and osteogenic lineages during subsequent passages without any qualitative difference. However, expression of alkaline phosphatase was higher in osteogenic differentiated cells from the hump compared with those of the abdomen. Moreover, the increase in calcium content in hump-derived stem cells was higher than that in abdominal-derived stem cells. In conclusion, our findings revealed that ASCs can be obtained from different anatomical locations, although ASCs from the hump fat region may be the ideal stem cell sources for use in cell-based therapies. (C) 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved