Development And Isolation Of Dna Microsatellite Markers For The Characterisation And Identification Of Mystus Nemurus (C & V)

Mystus nemurus or better known as 'ikan Baung' is an indigenous species In Malaysia. The popularity of this freshwater fish among the locals had made it an economically important aquaculture candidate. Of the so many DNA marker systems available, microsatellites are particularly useful for population studies and have been proven to be the most efficient due to their codominant modes of inheritance. Two methods were used to isolate microsatellites in M. nemurus, namely Random Amplified Hybridisation Microsatellites (RAHMs) and Random Amplified Microsatellites (RAMs). A total of 88 micro satellite sequences and 18 cryptic simple regions were produced. The majority (90%) of these microsatellites were detected by the RAMs method while the RAHMs method required further optimisation and modification. The isolation of microsatellites resulted in 33 primer pairs being designed

Rapid and non-radioactive detection method of microsatellites in Mystus nemurus: a refined technique

A simple and rapid method of DNA microsatellite isolation based on the Random Amplified Microsatellites (RAMs) PCR technique was used in this study. The work presented here is part of a continuous effort in refining and perfecting the technique for more rapid, effective and optimum productivity in single locus microsatellite marker development for the River catfish, Mystus nemurus. The current refined protocol for microsatellite isolation was able to detect a total of 135 microsatellite regions resulting in 42 unique genomic sequences being submitted to GenBank. This refined technique is able to reduce the total time required from peR cloning till sequencing specific microsatellite regions to less than three and a half months

Newly developed microsatellite markers of Mystus nemurus tested for cross-species amplification in two distantly related aquacultured catfish species

The work reported here is an attempt to explore the possibility of DNA microsatellite loci transfer (cross-species amplification) to other economically important aquacultured catfish species other than its source species. A total of 25 new microsatellite loci developed for riverine catfish, Mystus nemurus were successfully cross-amplified in two distantly related catfish species within the suborder Siluroidei. Five out of the 19 loci that successfully cross-amplified in Pangasius micronemus were polymorphic, while for Clarias batrachus, cross-amplification was successful using 17 polymorphic loci. The observed heterozygosities were high for all the three catfishes. The results indicated that microsatellite loci could be as polymorphic in non-source species as in the source species

The cytotoxcity activity of in vitro isolated and expanded cytotoxic T-lymphocytes and natural killer cells in bladder cancer

The expanding roles of the immune system in tumourigenesis have established immunotherapy as a potential mainstream cancer therapeutic modality. Ex vivo expanded and activated cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells have been found to be efficacious in the treatment of various types of cancers. One of the biggest limitations is the ability to generate and store cytotoxic immune cells in larger numbers without losing its cytotoxicity. Consequently, we evaluated the in vitro cytotoxic activity of freshly cultured and cryopreserved CTLs and NK cells that were expanded in vitro. We also compared the synergistic cytotoxic activity of CTLs and NK cells in combination. The cytotoxic activity was measured in bladder cancer cell lines, EJ28 (invasive) and RT112 (minimally-invasive). All experiments were run in three replicates. The cellular phenotype of the isolated and expanded effector cells was characterised using flow cytometry. MTT assay was performed to assess the dose- and time-dependent cell-mediated cytotoxic activity in the bladder cancer cells. An effector to target ratio of 1:1, 2:1, 5:1, 10:1 and 20:1 was tested after 4 h, 12 h and 24 h incubation. The fresh in vitro expanded effector cells had a high percentage of cell viability and expressed cytotoxic markers CD8+ and CD56+ in the CTL and NK cell cultures, respectively. Although the expansion capacity of the cryopreserved cells was limited, the expression of the functional markers and cytotoxic activity of these effector cells were maintained. All the effector cells exhibited significant cytotoxic activity at the effector to target ratio of 5:1 at 4 hours of co-culture. This was confirmed through the real-time observation of the morphological changes of the cells using an inverted phase contrast and time-lapse confocal microscope. The ex vivo generated CTLs and NK cells appear to retain their functionality, especially in recognizing their allogeneic target and thus, serve as a foundation to build on for future therapeutic applications

CYP2D6 genetic polymorphisms and phenotypes in different ethnicities of Malaysian breast cancer patients

The cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) is an enzyme that is predominantly involved in the metabolism of tamoxifen. Genetic polymorphisms of the CYP2D6 gene may contribute to inter-individual variability in tamoxifen metabolism, which leads to the differences in clinical response to tamoxifen among breast cancer patients. In Malaysia, the knowledge on CYP2D6 genetic polymorphisms as well as metabolizer status in Malaysian breast cancer patients remains unknown. Hence, this study aimed to comprehensively identify CYP2D6 genetic polymorphisms among 80 Malaysian breast cancer patients. The genetic polymorphisms of all the 9 exons of CYP2D6 gene were identified using high-resolution melting analysis and confirmed by DNA sequencing. Seven CYP2D6 alleles consisting of CYP2D6*1, CYP2D6*2, CYP2D6*4, CYP2D6*10, CYP2D6*39, CYP2D6*49, and CYP2D6*75 were identified in this study. Among these alleles, CYP2D6*10 is the most common allele in both Malaysian Malay (54.8%) and Chinese (71.4%) breast cancer patients, whereas CYP2D6*4 in Malaysian Indian (28.6%) breast cancer patients. In relation to CYP2D6 genotype, CYP2D6*10/*10 is more frequently observed in both Malaysian Malay (28.9%) and Chinese (57.1%) breast cancer patients, whereas CYP2D6*4/*10 is more frequently observed in Malaysian Indian (42.8%) breast cancer patients. In terms of CYP2D6 phenotype, 61.5% of Malaysian Malay breast cancer patients are predicted as extensive metabolizers in which they are most likely to respond well to tamoxifen therapy. However, 57.1% of Chinese as well as Indian breast cancer patients are predicted as intermediate metabolizers and they are less likely to gain optimal benefit from the tamoxifen therapy. This is the first report of CYP2D6 genetic polymorphisms and phenotypes in Malaysian breast cancer patients for different ethnicities. These data may aid clinicians in selecting an optimal drug therapy for Malaysian breast cancer patients, hence improve the clinical outcome of the patients

Human mesenchymal stromal cells modulate T-cell immune response via transcriptomic regulation

Background aims: Mesenchymal stromal cells (MSCs) have been identified as pan-immunosuppressant in various in vitro and in vivo inflammatory models. Although the immunosuppressive activity of MSCs has been explored in various contexts, the precise molecular signaling pathways that govern inhibitory functions remain poorly elucidated. Methods: By using a microarray-based global gene expression profiling system, this study aimed to decipher the underlying molecular pathways that may mediate the immunosuppressive activity of umbilical cord–derived MSCs (UC-MSCs) on activated T cells. Results: In the presence of UC-MSCs, the proliferation of activated T cells was suppressed in a dose-depended manner by cell-to-cell contact mode via an active cell-cycle arrest at the G0/G1 phase of the cell cycle. The microarray analysis revealed that particularly, IFNG, CXCL9, IL2, IL2RA and CCND3 genes were down-regulated, whereas IL11, VSIG4, GFA1, TIMP3 and BBC3 genes were up-regulated by UC-MSCs. The dysregulated gene clusters associated with immune-response-related ontologies, namely, lymphocyte proliferation or activation, apoptosis and cell cycle, were further analyzed. Conclusions: Among the nine canonical pathways identified, three pathways (namely T-helper cell differentiation, cyclins and cell cycle regulation, and gap/tight junction signalling pathways) were highly enriched with these dysregulated genes. The pathways represent putative molecular pathways through which UC-MSCs elicit immunosuppressive activity toward activated T cells. This study provides a global snapshot of gene networks and pathways that contribute to the ability of UC-MSCs to suppress activated T cells

Immunohistochemical expression of NANOG in urothelial carcinoma of the bladder

Urothelial carcinoma is a common malignant neoplasm that has a poor prognosis and a high frequency of recurrence and metastasis. Constant disease surveillance with periodic and long term cystoscopy examination is necessary for management of the disease. However, the monitoring and therapy regimen is expensive, incurring a massive burden to patients and the government. Therefore, the development of specific biomarkers for urothelial carcinoma at an early stage and recurrence detection becomes a priority. Homeobox genes are a family of genes that are involved in tumourigenesis. They might be potential prognostic markers for urothelial carcinoma. The study investigated the expression pattern of NANOG which is one of a homeobox gene in different stages and grades of urothelial carcinoma. NANOG expressions were also correlated with patient demographic factors and clinicopathological parameters. The expression of NANOG in 100 formalin-fixed paraffin-embedded urothelial carcinoma tissues was determined by immunohistochemistry. Immunohistochemistry showed positive expression of NANOG in all specimens with detection in the cytoplasm, nuclei and the nuclear membrane of the cancer cells. The immunohistochemical expression of NANOG increased across stages and grades of the tumour. The expression of NANOG was not significantly associated with demographic factors; gender (p = 0.376), race (p = 0.718) and age (p = 0.058) as well as with most of the clinicopathological parameters; pathological stage (p = 0.144), grade (p = 0.625), lymph node involvement (p = 0.174) and distant metastasis (p = 0.228). However, NANOG expression showed significant correlation with tumour invasion (p = 0.019). We concluded that NANOG might be a potential biomarker for early diagnosis of urothelial carcinoma of the bladder

miR-137-mediated loss of KDM5B expression leads to suppression of the malignant phenotype of bladder cancer cells

The oncogenic role of KDM5B is implicated in the pathogenesis of many cancers including bladder cancer (BC). KDM5B is a histone demethylase enzyme that modifies the chromatin structure to specify cellular transcriptional states. Overexpression of KDM5B in cancer cells is correlated with an increased proliferative capacity. Intriguingly, KDM5B is a cancer/testis antigen; while its expression in tumours is ectopically amplified, KDM5B expression in normal conditions is limited to embryonic stem cells (ESCs) and the testis in adults. These unique characteristics make KDM5B a potential pan-cancer therapeutic target. Thus, this study was aimed at identifying potential regulators of KDM5B. Since KDM5B expression in ESCs is orchestrated by microRNAs (miRNAs) and the expression of many miRNAs are altered in BC, we hypothesized that miRNAs may be the switch that can abate KDM5B expression to mitigate the BC malignant phenotype. Based on IHC- and RT-QPCR analysis, we found that KDM5B protein and transcript levels were differentially expressed in cancer tissues and cell lines, respectively. Amongst several in silico-predicted putative KDM5B-targeting miRNAs, the in vitro basal expression of miR-137 was inversely correlated with KDM5B expression. We demonstrated that the overexpression of miR-137 significantly attenuated KDM5B expression, induced G1 cell-cycle arrest, suppressed cell growth and blocked invasion and migration of BC cells. In contrast, downregulation of miR-137 expression led to the reverse effect. By integrating in silico screens of miR-137 putative target genes and microarray data using the Ingenuity Pathway Analysis (IPA), we revealed that miR-137 possibly exerts control over the cell-cycle through Rb and adenylyl cyclic signalling pathways by targeting key regulators of cyclin A. We also showed that miR-137 gain-of-function increased the expression of tumor suppressor, JDP2. While our results suggest that miR-137 can mitigate the KDM5B-associated BC phenotype, further studies on understanding the effect on aberrant histone methylation patterns are warranted

Oncolytic effects of the recombinant newcastle disease virus, rAF-IL12, against colon cancer cells in vitro and in tumor-challenged NCr-foxn1nu nude mice

Colon cancer remains one of the main cancers causing death in men and women worldwide as certain colon cancer subtypes are resistant to conventional treatments and the development of new cancer therapies remains elusive. Alternative modalities such as the use of viral-based therapeutic cancer vaccine is still limited, with only the herpes simplex virus (HSV) expressing granulocyte-macrophage colony- stimulating factor (GM-CSF) or talimogene laherparepvec (T-Vec) being approved in the USA and Europe so far. Therefore, it is imperative to continue the search for a new treatment modality. This current study evaluates a combinatorial therapy between the oncolytic Newcastle disease virus (NDV) and interleukin-12 (IL-12) cytokine as a potential therapeutic vaccine to the current anti-cancer drugs. Several in vitro analyses such as MTT assay, Annexin V/FITC flow cytometry, and cell cycle assay were performed to evaluate the cytotoxicity effect of recombinant NDV, rAF-IL12. Meanwhile, serum cytokine, serum biochemical, histopathology of organs and TUNEL assay were carried out to assess the anti-tumoral effects of rAF-IL12 in HT29 tumor-challenged nude mice. The apoptosis mechanism underlying the effect of rAF-IL12 treatment was also investigated using NanoString Gene expression analysis. The recombinant NDV, rAF-IL12 replicated in HT29 colon cancer cells as did its parental virus, AF2240-i. The rAF-IL12 treatment had slightly better cytotoxicity effects towards HT29 cancer cells when compared to the AF2240-i as revealed by the MTT, Annexin V FITC and cell cycle assay. Meanwhile, the 28-day treatment with rAF-IL12 had significantly (p < 0.05) perturbed the growth and progression of HT29 tumor in NCr-Foxn1nu nude mice when compared to the untreated and parental wild-type NDV strain AF2240-i. The rAF-IL12 also modulated the immune system in nude mice by significantly (p < 0.05) increased the level of IL-2, IL-12, and IFN-γ cytokines. Treatment with rAF-IL12 had also significantly (p < 0.05) increased the expression level of apoptosis-related genes such as Fas, caspase-8, BID, BAX, Smad3 and granzyme B in vitro and in vivo. Besides, rAF-IL12 intra-tumoral delivery was considered safe and was not hazardous to the host as evidenced in pathophysiology of the normal tissues and organs of the mice as well as from the serum biochemistry profile of liver and kidney. Therefore, this study proves that rAF-IL12 had better cytotoxicity effects than its parental AF2240-i and could potentially be an ideal treatment for colon cancer in the near future

Overexpression of CTAG1B is a potential biomarker in bladder cancer

Urothelial cell carcinoma (UCC) is the most common form of bladder cancer and is associated with the need for life-long surveillance once a patient is diagnosed with a non-invasive disease. Due to the long-term risk of recurrence and the need for life-long routine monitoring and therapy, the cost per UCC patient from diagnosis to death is the highest of all cancers. The development of non-invasive biomarkers of recurrence and progression can increase survival, decrease treatment costs and improve patient quality of life. However, to date, no biomarker(s) have been endorsed for the use in the clinical management of UCC, especially in predicting risk of progression and recurrence. CTAG1B was previously found to be highly expressed in high-stage and grade bladder cancer, albeit in Caucasian cohorts. However, despite its potential as a target for cancer immunotherapy, the effect of expression modulation on cellular phenotypes has never been reported. In this study, we overexpressed CTAG1B in an invasive bladder cancer cell line, EJ28 after we confirmed that this cell line minimally expressed CTAG1B. The cells were transfected with CTAG1B-pcDNA3.1(-) and the level of expression was confirmed by qRT-PCR. Once the expression was confirmed to persist up to 72h post-transfection, the transfected cells were subjected to various phenotypic assays. In addition, the pattern of CTAG1B expression in a cohort of Malaysian bladder cancer paraffin-embedded tissues was also determined using immunohistochemistry. The effect of CTAG1B overexpression on the cell cycle, migratory and proliferative potential was observed. The changes in phenotype were compared with that of untransfected and mock controls. CTAG1B was overexpressed 20 times as compared to the untransfected and mock controls in the transfected cells. CTAG1B overexpression resulted in cells to migrate slower at 24h post-transfection but proliferate significantly faster after 72-96h post-transfection. In addition, CTAG1B was more frequently expressed in advanced bladder cancer stages and grades. The findings from this study contribute to the current knowledge of CTAG1B‟s role in tumourigenesis. Further functional studies will contribute towards realising the potential of CTAG1B as a biomarker for predicting the risk of progression and recurrence of bladder cancer