Methods: Combination of throat and nasal swab specimens was subjected to viral RNA extraction. For screening, the extracted RNA was subjected to real-time RT-PCR targeting upstream of E gene, open reading frame 1b and open reading frame 1a. For confirmation, the RNA was subjected to RT-PCR targeting partial part of the RNA-dependent RNA polymerase and nucleocapsid, followed by amplification of complete N gene region. Nucleotide sequencing of the first Malaysian case of MERS-CoV was performed following the confirmation with real-time RT-PCR detection.
Results: Initial analysis of partial RNA-dependent RNA polymerase and N gene revealed that the nucleotides had high similarity to Jeddah_1_2013 strain. Analysis of complete N gene region (1 242 nucleotides) from the case showed high similarity and yet distinct to the nucleotide sequences of camel-derived MERS-CoV.
Conclusions: From the finding, there are possibilities that the patient acquired the infection from zoonotic transmission from dromedary camels.
METHODOLOGY: The expression of stemness markers for DPSC and SHED was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR). Alkaline phosphatase assay was used to compare the osteoblastic differentiation of these cells (2D culture). Then, cells were seeded on the scaffold and incubated for 21 days. Morphology assessment using field emission scanning electron microscopy (FESEM) was done while osteogenic differentiation was detected using ALP assay (3D culture).
RESULTS: The morphology of cells was mononucleated, fibroblast-like shaped cells with extended cytoplasmic projection. In RT-PCR study, DPSC and SHED expressed GAPDH, CD73, CD105, and CD146 while negatively expressed CD11b, CD34 and CD45. FESEM results showed that by day 21, dental stem cells have a round like morphology which is the morphology of osteoblast as compared to day 7. The osteogenic potential using ALP assay was significantly increased (p
OBJECTIVE: A new variant of the equine fsh (efsh) gene was cloned, sequenced, and expressed in Pichia pastoris (P. pastoris) GS115 yeast expression system.
MATERIALS AND METHODS: The full-length cDNAs of the efshα and efshβ chains were amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified efsh chains were cloned into the pPIC9 vector and transferred into P. pastoris. The secretion of recombined eFSH using P. pastoris expression system was confirmed by Western blotting and immunoprecipitation (IP) methods.
RESULTS: The DNA sequence of the efshβ chain accession number JX861871, predicted two putative differential nucleotide arrays, both of which are located in the 3'UTR. Western blotting showed a molecular mass of 13 and 18 kDa for eFSHα and eFSHβ subunits, respectively. The expression of desired protein was confirmed by protein G immunoprecipitation kit.
CONCLUSIONS: eFSH successfully expressed in P. pastoris. These findings lay a foundation to improve ovulation and embryo recovery rates as well as the efficiency of total embryo-transfer process in mares.
Objectives: In this study, we aimed to report the complete nucleotide sequence of Malaysian isolate of Rice tungro spherical virus Seberang Perai (RTSV-SP) for the first time. RTSV-SP was characterized and its evolutionary relationship with previously reported Indian and Philippines isolates were elucidated.
Materials and Methods: RTSV-SP isolate was isolated from a recent outbreak in a paddy field in Seberang Perai zone of Malaysia. Its complete genome was amplified by RT-PCR, cloned and sequenced.
Results: Sequence analysis indicated that the genome of RTSV-SP consisted of 12,173 nucleotides (nt). Comparative analysis of 6 complete genome sequences using Clustal Omega showed that Seberang Perai isolate shared the highest nucleotide identity (96.04%) with Philippine-A isolate, except that the sORF-2 of RTSV-SP is shorter than RTSV Philippine-A by 27 amino acid residues. RTSV-SP found to cluster in Southeast Asia (SEA) group based on the whole genome sequence phylogenetic analysis using MEGA X software.
Conclusions: Phylogenetic classification of RTSV isolates based on the complete nucleotide sequences showed more distinctive clustering pattern with the addition of RTSV-SP whole genome to the available isolates. Present study described the isolation and molecular characterization of RTSV-SP.
OBJECTIVES: The current study aimed to identify copy number alterations (CNAs) in OSCC using array comparative genomic hybridization (array CGH) and to correlate the CNAs with clinico-pathologic parameters and clinical outcomes.
MATERIALS AND METHODS: Using array CGH, genome-wide profiling was performed on 75 OSCCs. Selected genes that were harboured in the frequently amplified and deleted regions were validated using quantitative polymerase chain reaction (qPCR). Thereafter, pathway and network functional analysis were carried out using Ingenuity Pathway Analysis (IPA) software.
RESULTS: Multiple chromosomal regions including 3q, 5p, 7p, 8q, 9p, 10p, 11q were frequently amplified, while 3p and 8p chromosomal regions were frequently deleted. These findings were in confirmation with our previous study using ultra-dense array CGH. In addition, amplification of 8q, 11q, 7p and 9p and deletion of 8p chromosomal regions showed a significant correlation with clinico-pathologic parameters such as the size of the tumour, metastatic lymph nodes and pathological staging. Co-amplification of 7p, 8q, 9p and 11q regions that harbored amplified genes namely CCND1, EGFR, TPM2 and LRP12 respectively, when combined, continues to be an independent prognostic factor in OSCC.
CONCLUSION: Amplification of 3q, 5p, 7p, 8q, 9p, 10p, 11q and deletion of 3p and 8p chromosomal regions were recurrent among OSCC patients. Co-alteration of 7p, 8q, 9p and 11q was found to be associated with clinico-pathologic parameters and poor survival. These regions contain genes that play critical roles in tumourigenesis pathways.
MAIN METHODS: Human bone marrow derived MSCs were isolated, expanded in vitro and transfected with adiponectin gene containing plasmid vector. Total RNA was extracted and cDNA was prepared by reverse transcription polymerase chain reaction (RT-PCR). The expression of adiponectin gene and protein in GM-MSCs was analyzed by PCR and Western blotting respectively. The secretion of adiponectin protein from GM-MSCs was analyzed by enzyme-linked immunosorbent assay.
KEY FINDINGS: The expression of adiponectin gene and plasmid DNA was detected in GM-MSCs but not in control group of MSCs. Adiponectin gene expression was detected in GM-MSCs at 2, 7, 14, 21 and 28days after transfection. Western blotting analysis revealed the expression of adiponectin protein only in GM-MSCs. The GM-MSCs stably secreted adiponectin protein into culture media at least for 4weeks.
SIGNIFICANCE: GM-MSCs express and secret adiponectin protein. Therefore, these adiponectin secreting GM-MSCs could be instrumental for the supplementation of adiponectin in the treatment of adiponectin deficiency related diseases.