The protective role of tocotrienol on corticosterone induced oxidative stress during preimplantation embryonic development in mice / Shahidee Zainal Abidin

Excessive amount of glucocorticoid [cortisol in human or corticosterone (CORT) in rodent] induces oxidative stress (OS) in the cell leading to DNA damage and it has been proven by previous studies. Conversely, it is well documented that tocotrienol (TCT), a potent antioxidant was able to protect cells by neutralizing excessive reactive oxygen species (ROS). Hence, this study was designed to determine the effect of TCT supplementation on the quality and development of embryos and DNA damage level in embryos ofCORT-treated mice. Female mice were given TCT orally at three different doses i.e. 30, 60, and 90 mg kg" BW, concurrent with 10 mg kg" BW of CORT intraperitoneally (ip) for 14 days. Mice were superovulated and paired individually overnight with stud male mice. After 48 hours post-coitum, female mice were euthanized to collect 2-cell stage of embryos. The morphological observation and in vitro development of embryos were accessed and monitored under an inverted microscope and the percentage ofDNA damage was analysed via Comet assay. It was found that oral supplementation of 90 mg kg" BW of TCT in CORT-treated mice were able to normalize the number of fragmented embryos and improve the number of embryos that reach the blastocyst stage. No DNA damage was noted in all CORTtreated groups supplemented with TCT. Supplementation of TCT also suppresses the level of 8-hydroxy-2'-deoxyguanosine (8-0HdG) and restored catalase (CAT) activity toward control. The findings of this study indicate that TCT supplementation in CORT-treated mice was able to reverse the effect of CORT-induced fragmentation and oxidative DNA damage in embryos. Thus, the molecular mechanisms by which TCT suppresses oxidative stress and promotes the quality of embryo need to be investigated in detail in future studie

In silico analysis of mRNA:miR-3099 interaction

Introduction: MicroRNAs (miRNA) are small non-coding RNAs and have crucial role in gene expression and protein synthesis regulation, especially in nervous system and brain development. A novel miR-3099 was found highly express throughout embryogenesis especially in the developing central nervous system. Moreover, miR-3099 was also expressed upon neuronal differentiation in in vitro system suggesting that miR-3099 is a potential regulator during neuronal development. Therefore, objective of this study is to predict target genes of miR-3099 via in-silico analysis. These analyses will predict potential downstream targets for miR-3099 and their relationship to signalling pathways with special focus on neuronal function and brain development. Methods: Four different prediction software, miRDB, miRanda, TargetScan and DIANA micro-T, were employed to identify the candidate target genes of miR-3099. The predicted downstream targeted genes were selected based on the database criteria, prior to BioVenn clustering to identify the common targeted genes. The targeted genes that were predicted by at least three different databases were subjected to DAVID bioinformatics analysis to understand the biological process and function of these targeted genes. Results: Based on the analysis, a total of 1676 predicted genes were targeted by miR-3099. Of these, 73 genes were predicted by three software and 22 genes were predicted by all the four software. Majority of the targeted genes were annotated as involved in positive regulation of transcription activity and were identified as related to neuronal and brain development. Hence, the predicted downstream targets of miR-3099 warrant further investigation to validate the in silico analysis

The expression profile of miR-3099 during neural development of Ts1Cje mouse model of down syndrome

MicroRNA-3099 (miR-3099) plays a crucial role in regulating neuronal differentiation and development of the central nervous system (CNS). The miR-3099 is a pro-neuronal miRNA that promotes neural stem/progenitor cell (NSPC) differentiation into neuronal lineage by suppressing astrogliogenesis. Down syndrome (DS) brain exhibited increased astrogliogenesis and reduced neuronal cell density. The involvement of miR-3099 in the neurodevelopment of DS has not been investigated and potentially responsible for the neurogenic-to-gliogenic shift phenomenon observed in DS brain. To investigate the role of miR-3099 during DS brain development, neural/progenitor cell proliferation and differentiation, we profiled miR-3099 expression level in the Ts1Cje, a mouse model for DS. We analysed the Ts1Cje whole brain at embryonic day (E) 10.5, E14.5 and P1.5, proliferating neurospheres and differentiating neurospheres at 3, 9 and 15 days in vitro (DIV). Expression of miR-3099 in both the developing mouse brain and the differentiating neurosphere was not significantly different between Ts1Cje and wild type controls. In contrast, the expression level of miR-3099 was significantly higher (p<0.05) in proliferating NSPC derived from the Ts1Cje compared to wild-type. Further molecular profiling of NPSC and glial cell markers indicated that the expression of Sox2 (p<0.01) and Gfap (p<0.05) were significantly downregulated in Ts1Cje neurospheres as compared to that of wild type, respectively. While there were no significant differences in Tuj1 and Nestin expression levels between the Ts1Cje and wild type neurospheres, their expression levels were ~3-fold upregulated and ~2.6 downregulated Ts1Cje group, respectively. The findings suggest that dysregulation of miR-3099 affects NSPC lineage commitment as indicated by altered postmitotic neuronal cell markers. Further molecular characterisation and gene expression profiling of other neuronal and glial markers will help refine the analysis of gene-gene interactions underlying the neuropathologies of DS

In silico prediction and validation of Gfap as miR-3099 target in the mouse brain

MicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 via in-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset

Construction and validation of a mammalian expression vector for in utero electroporation study of miR-3099 in the mouse neocortex

Introduction: MiR-3099 was reported to play a role in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development. To further explore its potential regulatory effects on embryonic brain development, this study aims to construct and validate an expression vector of miR-3099 for future gain-of-function and loss-of-function studies. Methods: pCAG-eGFP vector was modified to include IRES2 and miR-3099 with 150bp upstream and downstream genomic sequences. The newly constructed vector, pCAG-miR-3099-IRES2-eGFP, consists of CAG promoter. The in vitro expression level of miR-3099 was measured using stem-loop RT-qPCR after it was transfected into 293FT cell. Later, the vector was electroporated into the embryonic brain at E15.5. Three days later, the E18.5 embryonic brain was harvested and cryopreserved. Immunohistochemistry was performed by using antibody against eGFP to validate the in utero expression of the transgene in the neocortex of the brain. Results: Our finding showed that, the expression level of miR-3099 was significantly upregulated (p<0.001) in cells transfected with miR-3099 vector as compared to both negative and empty plasmid control groups. In addition, the expression of eGFP was noted in the brain section indicating that the vectors with or without miR-3099 transgene were successfully transfected into and expressed in the neocortex upon electroporation. Conclusion: The bicistronic expression vector of miR-3099 which was driven by the CAG promoter was successfully constructed, validated and sufficiently delivered to brain cells via the in utero electroporation approach. The regulatory roles of miR-3099 in embryonic brain development can be manipulated using similar approach

miR-3099 promotes neurogenesis and inhibits astrogliogenesis during murine neural development

MicroRNA-3099 is highly expressed during neuronal differentiation and development of the central nervous system. Here we characterised the role of miR-3099 during neural differentiation and embryonic brain development using a stable and regulatable mouse embryonic stem cell culture system for miR-3099 expression and in utero electroporation of miR-3099 expression construct into E15.5 embryonic mouse brains. In the in vitro system, miR-3099 overexpression upregulated gene related to neuronal markers such as Tuj1, NeuN, Gat1, vGluT1 and vGluT2. In contrast, gene related to astrocyte markers (Gfap, S100β and Slc1a3) were suppressed upon overexpression of miR-3099. Furthermore, miR-3099 overexpression between E15.5 and E18.5 mouse embryonic brains led to disorganised neuronal migration potentially due to significantly decreased Gfap+ cells. Collectively, our results indicated that miR-3099 plays a role in modulating and regulating expression of key markers involved in neuronal differentiation. In silico analysis was also performed to identify miR-3099 homologues in the human genome, and candidates were validated by stem-loop RT-qPCR. Analysis of the miR-3099 seed sequence AGGCUA against human transcriptomes revealed that a potential miRNA, mds21 (Chr21:39186698-39186677) (GenBank accession ID: MK521584), was 100% identical to the miR-3099 seed sequence. Mds21 expression was observed and validated in various human cell lines (293FT, human Wharton's jelly and dental pulp mesenchymal stem cells, and MCF-7, MDA-MB-231, C-Sert, SW780, RT112, 5637, EJ28 and SH-SY5Y cells), with the highest levels detected in human mesenchymal stem cell lines. The analysis validated mds21 as a novel miRNA and a novel homologue of miR-3099 in the human genome

Anti-angiogenic effect of polygonum species: a comprehensive review of literature

Angiogenesis is a physiological, tightly regulated process which is characterized by the development of new blood vessels. Compounds with the potential to control angiogenesis would be highly valuable as therapeutics, as an imbalance in angiogenesis may lead to several pathological disorders, including cancer, retinopathy, and arthritis. In this study, the anti-angiogenic effect of Polygonum sp. has been comprehensively reviewed and this plant also has been known to possess other medicinal benefits such as antioxidative, anti-inflammatory, anti-proliferative, and anti-tumor agents. Hence, this study systematically identified the evidence reporting the anti-angiogenic effects of Polygonum sp. Four electronic databases, namely PubMed, Ovid MEDLINE, Scopus and Web of Science were searched for relevant articles. Based on the pre-set eligibility criteria, 50 relevant articles were identified, and ten qualified articles were selected and reviewed. It was demonstrated that four namely P. cuspidatum, P. barbatum, P. hydropiper, and P. perfoliatum showed anti-angiogenic activities mainly through inhibition of the vascular endothelial growth factor-signaling pathways. Therefore, these species Polygonum have the potential to be developed as natural anti-angiogenic agents for prevention and treatment of various diseases related to pathological angiogenesis

Development and validation of high resolution melting assays for high-throughput screening of BDNF rs6265 and DAT1 rs40184

Introduction: One of the commonly used techniques for mutation screening is High Resolution Melting (HRM) analysis. HRM is a post PCR method that relies on the detection of the fluorescent signals acquired due to the release of DNA intercalated dyes upon the melting of dsDNA to ssDNA. The method is simple, inexpensive and does not require post PCR-handling, making it suitable for high throughput screening. Methods: This study aimed to develop and validate HRM technique for the screening of two disease-associated single nucleotide polymorphisms (SNPs) namely BDNF rs6265 and DAT1 rs40184 using a total of 30 gDNA samples. The obtained results were confirmed and validated by sequencing. Results: HRM analysis showed that the predicted genotypes of BDNF rs6265 and DAT1 rs40184 among all the gDNA samples were in 100% concordance with the sequencing results, making it an accurate and sensitive method for the detection of SNPs. Conclusions: The application of HRM can accurately determine the genotype of BDNF rs6265 and DAT1 rs40184 SNPs, making it a promising tool for rapid and high-throughput screening of targeted SNPs in a large population study

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1] and [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1

Characterization of miR-3099-mediated posttranscriptional of target genes regulation during neurogenesis in mice

MicroRNAs (miRNAs) are a family of small non-coding RNAs with potent regulatory roles in metabolism, neurodevelopment, neuroplasticity, apoptosis, and other neurobiological processes. MiRNAs function through partial complementary base-pairing with specific target mRNAs, resulting in the repression of translational processes or the promotion of mRNA deadenylation leading to degradation. In 2011, miR-3099 was found to be expressed as early as in the blastocyst stage, in which the expression was maintained until the developing E11.5 mouse brain. The expression of miR-3099 was further restricted to the cortical plate of the developing mouse brain between E13.5 and E17.5, coinciding with the time that the majority of the cells are committed to neuronal cell lineage. Moreover, the miR-3099 was also found to be highly expressed in differentiating P19 cell (2-fold upregulation) when comparing to the proliferating P19 cell. Therefore, this study aims to understand the role of miR-3099 during neuro-differentiation and corticogenesis in the mouse model. The expression of miR-3099 was found elevated by 2-3 folds in 46C mouse embryonic stem (mES) cell upon neural induction. Then, predicted target gene of miR-3099 was further analysed by using four different prediction algorithms (miRDB, miRanda, TargetScan and DIANA-micro-T-CDS) and DAVID bioinformatics analysis with emphasis on target genes related to brain development and function. Based on the prediction, nearly 70% of the predicted target genes were expressed in the nervous system. Of these predicted target genes, Gfap was chosen as a candidate for downstream validation because it had been implicated in an important pathway in the brain known as the JAK-STAT signalling pathway, which controls the onset of astrocyte formation. By using the luciferase reporter gene system, Gfap was negatively inhibited by miR-3099. Furthermore, overexpression of miR-3099 was performed in vitro and in vivo for better understanding of the role of miR3099 during neuro-differentiation and brain development. In vitro, a transgenic mES cell that carried miR-3099 was overexpressed and differentiated for 17 days. The gene expression profile was carried out by using stem-loop RT-qPCR for different marker analysis such as proliferative, neural progenitor, neuron, astrocyte and oligodendrocyte markers. The analysis revealed that the overexpression of miR-3099 promoted neuronal differentiation and suppressed the astrogliogenesis in the in vitro system. In the in vivo system, the overexpression of miR-3099 caused disorganised neuronal migration potentially due to downregulation of Gfap. Heretofore, the human homologue of miR-3099 has not been found or reported. In silico analysis via seed sequence similarity search in GEO database found that mds21 to be novel miRNA that has 100% identical at seed region and 64% closed to miR-3099 mature sequence. Interestingly, the expression of mds21 was found to be expressed in various human cell line and tissue, including the brain suggesting that mds21 might be a potential miR-3099 homologue in the human genome. Collectively, this study has shown that miR-3099 plays an essential role in modulating and regulating key markers involved in neuronal differentiation and neural cell function. The degree of functional conservation between miR-3099 and mds21 is not clear, and further validations are needed to characterise them further