Development Of EZ DNA Diagnostic Kit For The Detection Of Homozygous Deletion Of SMN1 Gene In Spinal Muscular Atrophy (SMA) Patients

Spinal Muscular Atrophy (SMA) is the second most frequent fatal autosomal recessive disorder of childhood. The incidence of this disease is approximately 1 in 10000 live births. SMA is characterized by progressive muscle weakness resulting from degeneration and loss of motor neurons in the anterior horn of spinal cord. The responsible genes for SMA are Survival of Motor Neuron (SMN). SMN1 and SMN2 genes share over 99.8% sequence homology and they can be distinguished by base changes in both exons 7 and 8. SMN1 gene is not detectable in majority of SMA cases due to either deletion or conversion. Conventionally, the homozygous deletion of SMN1 gene is detected using Polymerase Chain Reaction-Restriction Enzyme (PCR-RE) method. This method is time consuming, expensive and requires restriction enzyme digestion and a considerably high amount of amplified DNA to be visible after digestion. This may lead to false-negative results. To avoid these problems, we have developed an alternative method using an allele-specific primer for the molecular diagnosis of SMA which is more time-saving and cost-effective. The freeze dry platform was applied to the multiplex AS-PCR for the development of a thermostabilize diagnostic kit. A total of one hundred and forty three samples of clinically suspected SMA were included in this study. Conventional molecular diagnosis using PCR-RE was done to detect the presence or absent of SMN1 deletion in these samples

Jurnal Arkeologi Siddhayatra Vol.14 No.2 Tahun 2009

Dalam terbitan kali ini berisi lima artikel, Artikel Nurhadi Rangkuti menyoroti hasil penelitian arkeologi di Pulau Bangka sejak tahun 1990-2008. Artikel kedua ditulis oleh Irfanuddin Wahud Marzuki mengungkapkan hasil survei tinggalan perang dunia II di Halmahera Utara khususnya Kecamatan Kao dan di Kecamatan Malifut. Artikel Retno Purwanyi menyoroti Museum Sriwijaya Palembang belum berfungsi sebagai pusat informasi sejarah dan arkeologi Kerajaan Sriwijaya. Kristantina Indriastuti tertarik dengan pemanfaatan atau revitalisasi sumber daya budaya.Penulis menilai dalam pengelolaan yang profesional maka tinggalan budaya prasejarah Sumsel dapat dimanfaatkan secara optimal untuk pembangunan kebudayaan nasional. Pemanfaatan sumber daya arkeologi menjadi sorotan Sondang M Siregar yang mencoba penerapannya pada sumber daya arkeologi di kawasan Danau Ranau Kab Ogan Komering Ulu Selatan Propinsi Sumatera Selatan

Molecular analysis of promoter region of the SMN2 gene in the patients of spinal musculatr atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by the absence of the full length SMN protein (FL-SMN) as a result of mutation or deletion of SMN1 gene. The isoform to this gene, SMN2 gene, with mutation in 1 base pair, encodes for 10% of FL-SMN protein and is reported to decrease the severity of the disease when there is an increase gene dosage. There are 3 clinical types of SMA; type I, type II and type III. Type I SMA is the most severe type and only a small amount of FL-SMN protein is present in these individuals. We postulated that the difference in the promoter region of SMN2 gene produces the different level of FL-SMN protein. To verify this hypothesis, the DNA samples of 69 SMA patients who were referred to the Human Genome Center, USM were extracted from their blood. The SMN1 deletion analysis was performed, followed by the SMN2 copy no. analysis and NAIP deletion analysis to remove any clinical bias as NAIP gene deletion and SMN2 copy number have been reported to be associated with SMA disease severity. Only 10 SMA patients from different clinical types (type I=2, type II=3, type III=5) with homozygous deletion of the SMN1 and 2 copies of the SMN2 and deletion in NAIP were finally recruited. Primers were designed for the amplification of the SMN2 promoter region. Bioinformatics analysis was performed to identify the crucial transcription factor binding sites within the reported ~4.6 kb promoter region. As the core promoter region is still unknown (unreported), we analyzed 15 ORFs and 24 nested ORFs with 15 TATA boxes reflecting the diverse functional integrity of this region. The promoter prediction and core promoter prediction was also performed. Based on the bioinformatics analysis and the designed primers, PCR amplification was done for different regions in the promoter and the PCR products were subjected to direct DNA sequencing. The results were analyzed by Vector NTI suite 9, ClustalX and Gene Doc softwares. The molecular analysis confirmed the absence of any mutation in the promoter region of the SMN2 gene between normal healthy individuals (total 2) and SMA patients. In 4 patients and 1 normal healthy individual the CA repeats were found to be increased which we think cause no effect in disease progression and severity. In conclusion, there was no mutation found in the promoter region of the SMN2 gene among the SMA patients of different clinical types and normal controls. Further analysis involving the cloning of the promoter regions with highest probability of involvement in expression of the SMN2 gene using luciferase assay is ongoing. The results will be useful for the subsequent phase of the study involving the transcription initiation of the SMN2 gene

Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

Natural killer (NK) cells are innate immune cells that can remove viral-infected tumour cells without antigen priming. This characteristic offers NK cells an edge over other immune cells as a potential therapy for nasopharyngeal carcinoma (NPC). In this study, we report how cytotoxicity was evaluated in target NPC cell lines and patient-derived xenograft (PDX) cells with effector NK-92, a commercially available NK cell line, by using xCELLigence RTCA system (a real-time, label-free impedance-based monitoring platform). Cell viability, proliferation and cytotoxicity were examined by RTCA. Cell morphology, growth and cytotoxicity were also monitored by microscopy. RTCA and microscopy showed that both target and effector cells were able to proliferate normally and to maintain original morphology in co-culture medium as they were in their own respective culture medium. As target and effector (T:E) cell ratios increased, cell viability as measured by arbitrary cell index (CI) values in RTCA decreased in all cell lines and PDX cells. NPC PDX cells were more sensitive to the cytotoxicity effect of NK-92 cells, than the NPC cell lines. These data were substantiated by GFP-based microscopy. We have shown how the RTCA system can be used for a high throughput screening of the effects of NK cells in cancer studies to obtain data such as cell viability, proliferation and cytotoxicity

Exposure of MDA-MB-231 Cells to Dielectrophoretic Fields for Electroporation and Cancer Diagnostics

This paper presents the experimental analysis of exposing breast cancer cells (MDA-MB-231) to dielectrophoretic (DEP) fields on a curved microelectrode platform. The platform was composed of arrays of curved microelectrodes which were patterned onto a glass slide and samples containing MDA-MB-231 cells were pipetted onto the surface of the platform. The MDA-MB-231 cells were suspended in DMEM media to assess the cells behaviour and effects when exposed to DEP fields in different concentrations of DMEM media. Finite element method was utilised to characterise the electric field gradient and DEP field and numerical simulations were used to determine DEP response of the cells. We performed experimental work to observe DEP effects and confirmed the DEP responses obtained from the simulations

Dynamic tracking of human umbilical cord mesenchymal stem cells (hUC-MSCs) following intravenous administration in mice model

Introduction: In the past decades, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have sparked interest in cellular therapy due to their immunomodulatory properties. Nevertheless, the fate of hUC-MSCs in the body remains poorly understood. This study aimed to investigate the biodistribution, homing and clearance of systemically administered hUC-MSCs in healthy BALB/c mice model. Methods: hUC-MSCs were labelled with GFP-Luc2 protein, followed by characterisation with flow cytometry. Upon intravenous infusion of transduced hUC-MSCs into the healthy BALB/c mice, the cells were dynamically monitored through the bioluminescent imaging (BLI) approach. Results: Transduction of hUC-MSCs with GFP-Luc2 not only preserved the characteristics of MSCs, but also allowed live monitoring of transduced cells in the mice model. Upon systemic administration, BLI showed that transduced hUC-MSCs first localised predominantly in the lungs of healthy BALB/c mice and mainly remained in the lungs for up to 3 days before eventually cleared from the body. At terminal sacrifice, plasma chemistry biomarkers remained unchanged except for C-peptide levels, which were significantly reduced in the hUC-MSCs group. Histopathological findings further revealed that hUC-MSCs infusion did not cause any adverse effects and toxicity to lung, liver and heart tissues. Conclusions: Collectively, systemically administrated hUC-MSCs was safe and demonstrated dynamic homing capacity before eventually disappearing from the body

Dielectrophoretic deformation of breast cancer cells for lab on a chip applications

This paper presents the development and experimental analysis of a curved microelectrode platform for the DEP deformation of breast cancer cells (MDA-MB-231). The platform is composed of arrays of curved DEP microelectrodes which are patterned onto a glass slide and samples containing MDA-MB-231 cells are pipetted onto the platform's surface. Finite element method is utilised to characterise the electric field gradient and DEP field. The performance of the system is assessed with MDA-MB-231 cells in a low conductivity 1% DMEM suspending medium. We applied sinusoidal wave AC potential at peak to peak voltages of 2, 5, and 10 Vpp at both 10 kHz and 50 MHz. We observed cell blebbing and cell shrinkage and analyzed the percentage of shrinkage of the cells. The experiments demonstrated higher percentage of cell shrinkage when cells are exposed to higher frequency and peak to peak voltage electric field.</p

Dielectrophoretic deformation of breast cancer cells for lab on a chip applications

This paper presents the development and experimental analysis of a curved microelectrode platform for the DEP deformation of breast cancer cells (MDA-MB-231). The platform is composed of arrays of curved DEP microelectrodes which are patterned onto a glass slide and samples containing MDA-MB-231 cells are pipetted onto the platform's surface. Finite element method is utilised to characterise the electric field gradient and DEP field. The performance of the system is assessed with MDA-MB-231 cells in a low conductivity 1% DMEM suspending medium. We applied sinusoidal wave AC potential at peak to peak voltages of 2, 5, and 10 Vpp at both 10 kHz and 50 MHz. We observed cell blebbing and cell shrinkage and analyzed the percentage of shrinkage of the cells. The experiments demonstrated higher percentage of cell shrinkage when cells are exposed to higher frequency and peak to peak voltage electric field

Dielectrophoresis-based microfluidic platforms for cancer diagnostics

The recent advancement of dielectrophoresis (DEP)-enabled microfluidic platforms is opening new opportunities for potential use in cancer disease diagnostics. DEP is advantageous because of its specificity, low cost, small sample volume requirement, and tuneable property for microfluidic platforms. These intrinsic advantages have made it especially suitable for developing microfluidic cancer diagnostic platforms. This review focuses on a comprehensive analysis of the recent developments of DEP enabled microfluidic platforms sorted according to the target cancer cell. Each study is critically analyzed, and the features of each platform, the performance, added functionality for clinical use, and the types of samples, used are discussed. We address the novelty of the techniques, strategies, and design configuration used in improving on existing technologies or previous studies. A summary of comparing the developmental extent of each study is made, and we conclude with a treatment of future trends and a brief summary