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Fulltext Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin

Tham HY, Song AA, Yusoff K, Tan GH

Biotechniques, 2020 09;69(3):161-170.
PMID: 32787565 DOI: 10.2144/btn-2020-0034

Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/XhoI, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using NcoI/XhoI resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein.

Matched MeSH terms: Gene Expression Regulation/genetics
Fulltext Molecular Regulation of Lipogenesis, Adipogenesis and Fat Deposition in Chicken

Nematbakhsh S, Pei Pei C, Selamat J, Nordin N, Idris LH, Abdull Razis AF

Genes (Basel), 2021 03 13;12(3).
PMID: 33805667 DOI: 10.3390/genes12030414

In the poultry industry, excessive fat deposition is considered an undesirable factor, affecting feed efficiency, meat production cost, meat quality, and consumer's health. Efforts to reduce fat deposition in economically important animals, such as chicken, can be made through different strategies; including genetic selection, feeding strategies, housing, and environmental strategies, as well as hormone supplementation. Recent investigations at the molecular level have revealed the significant role of the transcriptional and post-transcriptional regulatory networks and their interaction on modulating fat metabolism in chickens. At the transcriptional level, different transcription factors are known to regulate the expression of lipogenic and adipogenic genes through various signaling pathways, affecting chicken fat metabolism. Alternatively, at the post-transcriptional level, the regulatory mechanism of microRNAs (miRNAs) on lipid metabolism and deposition has added a promising dimension to understand the structural and functional regulatory mechanism of lipid metabolism in chicken. Therefore, this review focuses on the progress made in unraveling the molecular function of genes, transcription factors, and more notably significant miRNAs responsible for regulating adipogenesis, lipogenesis, and fat deposition in chicken. Moreover, a better understanding of the molecular regulation of lipid metabolism will give researchers novel insights to use functional molecular markers, such as miRNAs, for selection against excessive fat deposition to improve chicken production efficiency and meat quality.

Matched MeSH terms: Gene Expression Regulation/genetics*
Prospective Advances in Circular RNA Investigation

Sulaiman SA, Abdul Murad NA, Mohamad Hanif EA, Abu N, Jamal R

Adv Exp Med Biol, 2018 9 28;1087:357-370.
PMID: 30259380 DOI: 10.1007/978-981-13-1426-1_28

circRNAs have emerged as one of the key regulators in many cellular mechanisms and pathogenesis of diseases. However, with the limited knowledge and current technologies for circRNA investigations, there are several challenges that need to be addressed for. These include challenges in understanding the regulation of circRNA biogenesis, experimental designs, and sample preparations to characterize the circRNAs in diseases as well as the bioinformatics pipelines and algorithms. In this chapter, we discussed the above challenges and possible strategies to overcome those limitations. We also addressed the differences between the existing applications and technologies to study the circRNAs in diseases. By addressing these challenges, further understanding of circRNAs roles and regulations as well as the discovery of novel circRNAs could be achieved.

Matched MeSH terms: Gene Expression Regulation/genetics
Functional characterization of two variants in the 3'-untranslated region (UTR) of transcription factor 4 gene and their association with schizophrenia in sib-pairs from multiplex families

Mohamed ZI, Tee SF, Chow TJ, Loh SY, Yong HS, Bakar AKA, et al.

Asian J Psychiatr, 2019 Feb;40:76-81.
PMID: 30771755 DOI: 10.1016/j.ajp.2019.02.001

Transcription factor 4 (TCF4) gene plays an important role in nervous system development and it always associated with the risk of schizophrenia. Since miRNAs regulate targetgenes by binding to 3'UTRs of target mRNAs, the functional variants located in 3'UTR of TCF4 are highly suggested to affect the gene expressions in schizophrenia. To test the hypothesis regarding the effects of the variants located in 3'UTR of TCF4, we conducted an in silico analysis to identify the functional variants and their predicted functions. In this study, we sequenced the 3'UTR of TCF4 in 13 multiplex schizophrenia families and 14 control families. We found two functional variants carried by three unrelated patients. We determined that the C allele of rs1272363 and the TC insert of rs373174214 might suppress post- transcriptional expression. Secondly, we cloned the region that flanked these two variants into a dual luciferase reporter system and compared the luciferase activities between the pmirGLO-TCF4 (control), pmirGLO-TCF4-rs373174214 and pmirGLO-TCF4-rs1273263. Both pmirGLO-TCF4-rs373174214 and pmirGLO-TCF4-rs1273263 caused lower reporter gene activities, as compared to the control. However, only the C allele of rs1272363 reduced the luciferase activity significantly (p = 0.0231). Our results suggested that rs1273263 is a potential regulator of TCF4 expression, and might be associated with schizophrenia.

Matched MeSH terms: Gene Expression Regulation/genetics*
Methylation of the filaggrin gene promoter does not affect gene expression and allergy

Tan HT, Ellis JA, Koplin JJ, Martino D, Dang TD, Suaini N, et al.

Pediatr Allergy Immunol, 2014 Oct;25(6):608-10.
PMID: 24912553 DOI: 10.1111/pai.12245
Matched MeSH terms: Gene Expression Regulation/genetics
Fulltext Over-expression of Nicotinamide phosphoribosyltransferase in mouse cells confers protective effect against oxidative and ER stress-induced premature senescence

Nuriliani A, Nakahata Y, Ahmed R, Khaidizar FD, Matsui T, Bessho Y

Genes Cells, 2020 Aug;25(8):593-602.
PMID: 32533606 DOI: 10.1111/gtc.12794

A main feature of aged organisms is the accumulation of senescent cells. Accumulated senescent cells, especially stress-induced premature senescent cells, in aged organisms lead to the decline of the regenerative potential and function of tissues. We recently reported that the over-expression of NAMPT, which is the rate-limiting enzyme in mammalian NAD+ salvage pathway, delays replicative senescence in vitro. However, whether Nampt-overexpressing cells are tolerant of stress-induced premature senescence remains unknown. Here, we show that primary mouse embryonic fibroblasts derived from Nampt-overexpressing transgenic mice (Nampt Tg-MEF cells) possess resistance against stress-induced premature senescence in vitro. We found that higher oxidative or endoplasmic reticulum (ER) stress is required to induce premature senescence in Nampt Tg-MEF cells compared to wild-type cells. Moreover, we found that Nampt Tg-MEF cells show acute expression of unfolded protein response (UPR)-related genes, which in turn would have helped to restore proteostasis and avoid cellular senescence. Our results demonstrate that NAMPT/NAD+ axis functions to protect cells not only from replicative senescence, but also from stress-induced premature senescence in vitro. We anticipate that in vivo activation of NAMPT activity or increment of NAD+ would protect tissues from the accumulation of premature senescent cells, thereby maintaining healthy aging.

Matched MeSH terms: Gene Expression Regulation/genetics
Fulltext In Silico Prediction and Validation of Gfap as an miR-3099 Target in Mouse Brain

Abidin SZ, Leong JW, Mahmoudi M, Nordin N, Abdullah S, Cheah PS, et al.

Neurosci Bull, 2017 Aug;33(4):373-382.
PMID: 28597341 DOI: 10.1007/s12264-017-0143-0

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.

Matched MeSH terms: Gene Expression Regulation/genetics*
An Overview of Circular RNAs

Awasthi R, Singh AK, Mishra G, Maurya A, Chellappan DK, Gupta G, et al.

Adv Exp Med Biol, 2018 9 28;1087:3-14.
PMID: 30259353 DOI: 10.1007/978-981-13-1426-1_1

Circular RNAs (cirRNAs) are long, noncoding endogenous RNA molecules and covalently closed continuous loop without 5'-3' polarity and polyadenylated tail which are largely concentrated in the nucleus. CirRNA regulates gene expression by modulating microRNAs and functions as potential biomarker. CirRNAs can translate in vivo to link between their expression and disease. They are resistant to RNA exonuclease and can convert to the linear RNA by microRNA which can then act as competitor to endogenous RNA. This chapter summarizes the evolutionary conservation and expression of cirRNAs, their identification, highlighting various computational approaches on cirRNA, and translation with a focus on the breakthroughs and the challenges in this new field.

Matched MeSH terms: Gene Expression Regulation/genetics
Functional Analysis of Circular RNAs

Shanmugapriya, Huda HA, Vijayarathna S, Oon CE, Chen Y, Kanwar JR, et al.

Adv Exp Med Biol, 2018 9 28;1087:95-105.
PMID: 30259360 DOI: 10.1007/978-981-13-1426-1_8

Circular RNAs characterize a class of widespread and diverse endogenous RNAs which are non-coding RNAs that are made by back-splicing events and have covalently closed loops with no polyadenylated tails. Various indications specify that circular RNAs (circRNAs) are plentiful in the human transcriptome. However, their participation in biological processes remains mostly undescribed. To date thousands of circRNAs have been revealed in organisms ranging from Drosophila melanogaster to Homo sapiens. Functional studies specify that these transcripts control expression of protein-coding linear transcripts and thus encompass a key component of gene expression regulation. This chapter provide a comprehensive overview on functional validation of circRNAs. Furthermore, we discuss the recent modern methodologies for the functional validation of circRNAs such as RNA interference (RNAi) gene silencing assay, luciferase reporter assays, circRNA gain-of-function investigation via overexpression of circular transcript assay, RT-q-PCR quantification, and other latest applicable assays. The methods described in this chapter are demonstrated on the cellular model.

Matched MeSH terms: Gene Expression Regulation/genetics*
Fulltext Effect of dietary lead on intestinal nutrient transporters mRNA expression in broiler chickens

Ebrahimi R, Faseleh Jahromi M, Liang JB, Soleimani Farjam A, Shokryazdan P, Idrus Z

Biomed Res Int, 2015;2015:149745.
PMID: 25695048 DOI: 10.1155/2015/149745

Lead- (Pb-) induced oxidative stress is known to suppress growth performance and feed efficiency in broiler chickens. In an attempt to describe the specific underlying mechanisms of such phenomenon we carried out the current study. Ninety-six one-day-old broiler chicks were randomly assigned to 2 dietary treatment groups of 6 pen replicates, namely, (i) basal diet containing no lead supplement (control) and (ii) basal diet containing 200 mg lead acetate/kg of diet. Following 3 weeks of experimental period, jejunum samples were collected to examine the changes in gene expression of several nutrient transporters, antioxidant enzymes, and heat shock protein 70 (Hsp70) using quantitative real-time PCR. The results showed that addition of lead significantly decreased feed intake, body weight gain, and feed efficiency. Moreover, with the exception of GLUT5, the expression of all sugar, peptide, and amino acid transporters was significantly downregulated in the birds under Pb induced oxidative stress. Exposure to Pb also upregulated the antioxidant enzymes gene expression together with the downregulation of glutathione S-transferase and Hsp70. In conclusion, it appears that Pb-induced oxidative stress adversely suppresses feed efficiency and growth performance in chicken and the possible underlying mechanism for such phenomenon is downregulation of major nutrient transporter genes in small intestine.

Matched MeSH terms: Gene Expression Regulation/genetics*
Fulltext Differential expression of basal microRNAs' patterns in human dental pulp stem cells

Vasanthan P, Govindasamy V, Gnanasegaran N, Kunasekaran W, Musa S, Abu Kasim NH

J Cell Mol Med, 2015 Mar;19(3):566-80.
PMID: 25475098 DOI: 10.1111/jcmm.12381

MicroRNAs (miRNAs) are small non-coding RNAs that regulate translation of mRNA into protein and play a crucial role for almost all biological activities. However, the identification of miRNAs from mesenchymal stem cells (MSCs), especially from dental pulp, is poorly understood. In this study, dental pulp stem cells (DPSCs) were characterized in terms of their proliferation and differentiation capacity. Furthermore, 104 known mature miRNAs were profiled by using real-time PCR. Notably, we observed 19 up-regulated miRNAs and 29 significantly down-regulated miRNAs in DPSCs in comparison with bone marrow MSCs (BM-MSCs). The 19 up-regulated miRNAs were subjected to ingenuity analysis, which were composed into 25 functional networks. We have chosen top 2 functional networks, which comprised 10 miRNA (hsa-miR-516a-3p, hsa-miR-125b-1-3p, hsa-miR-221-5p, hsa-miR-7, hsa-miR-584-5p, hsa-miR-190a, hsa-miR-106a-5p, hsa-mir-376a-5p, hsa-mir-377-5p and hsa-let-7f-2-3p). Prediction of target mRNAs and associated biological pathways regulated by each of this miRNA was carried out. We paid special attention to hsa-miR-516a-3p and hsa-miR-7-5p as these miRNAs were highly expressed upon validation with qRT-PCR analysis. We further proceeded with loss-of-function analysis with these miRNAs and we observed that hsa-miR-516a-3p knockdown induced a significant increase in the expression of WNT5A. Likewise, the knockdown of hsa-miR-7-5p increased the expression of EGFR. Nevertheless, further validation revealed the role of WNT5A as an indirect target of hsa-miR-516a-3p. These results provide new insights into the dynamic role of miRNA expression in DPSCs. In conclusion, using miRNA signatures in human as a prediction tool will enable us to elucidate the biological processes occurring in DPSCs.

Matched MeSH terms: Gene Expression Regulation/genetics*
Microarray profiling of secretory-phase endometrium from patients with recurrent miscarriage

Othman R, Omar MH, Shan LP, Shafiee MN, Jamal R, Mokhtar NM

Reprod Biol, 2012 Jul;12(2):183-99.
PMID: 22850470

The aim of the present study was to identify differentially expressed genes and their related biological pathways in the secretory phase endometrium from patients with recurrent miscarriage (RM) and fertile subjects. Endometrial samples from RM and fertile patients were analyzed using the Affymetrix GeneChip® ST Array. The bioinformatic analysis using the Partek Genomic Suite revealed 346 genes (175 up-regulated and 171 down-regulated) that were differentially expressed in the endometrium of RM patients compared to the fertile subjects (fold change ≥1.5, p<0.005). Validation step using quantitative real-time polymerase chain reaction (qPCR) confirmed a similar expression pattern of four exemplary genes: one up-regulated gene (fibroblast growth factor 9, FGF9) and three down-regulated genes: integrin β3 (ITGB3), colony stimulating factor 1 (CSF1) and matrix-metalloproteinases 19 (MMP19). The Gene Set Enrichment Analysis (GSEA) and the Pathway Studio software have found 101 signaling pathways (p<0.05) associated with the affected genes including the FGFR3 /signal transducer and activator of transcription (STAT) pathway and the CSF1R/STAT pathway. Cell adhesion, cell differentiation and angiogenesis were among biological processes indicated by this system. In conclusion, microarray technique is a useful tool to study gene expression in the secretory phase-endometrium of RM patients. The differences in endometrial gene expressions between healthy and RM subjects contribute to an increase in our knowledge on molecular mechanisms of RM development and may improve the outcome of pregnancies in high-risk women with RM.

Matched MeSH terms: Gene Expression Regulation/genetics
Effects of epidermal growth factor on the proliferation and cell cycle regulation of cultured human amnion epithelial cells

Fatimah SS, Tan GC, Chua KH, Tan AE, Hayati AR

J Biosci Bioeng, 2012 Aug;114(2):220-7.
PMID: 22578596 DOI: 10.1016/j.jbiosc.2012.03.021

Human amnion epithelial cells (HAECs) hold great promise in tissue engineering for regenerative medicine. Large numbers of HAECs are required for this purpose. Hence, exogenous growth factor is added to the culture medium to improve epithelial cells proliferation. The aim of the present study was to determine the effects of epidermal growth factor (EGF) on the proliferation and cell cycle regulation of cultured HAECs. HAECs at P1 were cultured for 7 days in medium containing an equal volume mix of HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of EGF (0, 5, 10, 20, 30 and 50 ng/ml EGF) in reduced serum. Morphology, growth kinetics and cell cycle analysis using flow cytometry were assessed. Quantitative gene expression for cell cycle control genes, pluripotent transcription factors, epithelial genes and neuronal genes were also determined. EGF enhanced HAECs proliferation with optimal concentration at 10 ng/ml EGF. EGF significantly increased the proportion of HAECs at S- and G2/M-phase of the cell cycle compared to the control. At the end of culture, HAECs remained as diploid cells under cell cycle analysis. EGF significantly decreased the mRNA expression of p21, pRb, p53 and GADD45 in cultured HAECs. EGF also significantly decreased the pluripotent genes expression: Oct-3/4, Sox2 and Nanog; epithelial genes expression: CK14, p63, CK1 and Involucrin; and neuronal gene expression: NSE, NF-M and MAP 2. The results suggested that EGF is a strong mitogen that promotes the proliferation of HAECs through cell cycle regulation. EGF did not promote HAECs differentiation or pluripotent genes expression.

Matched MeSH terms: Gene Expression Regulation/genetics
Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells

Alhaji SY, Nordin N, Ngai SC, Al Abbar A, Mei L, Abdullah S

Gene, 2020 Oct 20;758:144958.
PMID: 32683073 DOI: 10.1016/j.gene.2020.144958

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

Matched MeSH terms: Gene Expression Regulation/genetics
Fulltext Confirmation of five novel susceptibility loci for systemic lupus erythematosus (SLE) and integrated network analysis of 82 SLE susceptibility loci

Molineros JE, Yang W, Zhou XJ, Sun C, Okada Y, Zhang H, et al.

Hum Mol Genet, 2017 03 15;26(6):1205-1216.
PMID: 28108556 DOI: 10.1093/hmg/ddx026

We recently identified ten novel SLE susceptibility loci in Asians and uncovered several additional suggestive loci requiring further validation. This study aimed to replicate five of these suggestive loci in a Han Chinese cohort from Hong Kong, followed by meta-analysis (11,656 cases and 23,968 controls) on previously reported Asian and European populations, and to perform bioinformatic analyses on all 82 reported SLE loci to identify shared regulatory signatures. We performed a battery of analyses for these five loci, as well as joint analyses on all 82 SLE loci. All five loci passed genome-wide significance: MYNN (rs10936599, Pmeta = 1.92 × 10-13, OR = 1.14), ATG16L2 (rs11235604, Pmeta = 8.87 × 10 -12, OR = 0.78), CCL22 (rs223881, Pmeta = 5.87 × 10-16, OR = 0.87), ANKS1A (rs2762340, Pmeta = 4.93 × 10-15, OR = 0.87) and RNASEH2C (rs1308020, Pmeta = 2.96 × 10-19, OR = 0.84) and co-located with annotated gene regulatory elements. The novel loci share genetic signatures with other reported SLE loci, including effects on gene expression, transcription factor binding, and epigenetic characteristics. Most (56%) of the correlated (r2 > 0.8) SNPs from the 82 SLE loci were implicated in differential expression (9.81 × 10-198 genes. Transcription factor binding sites for p53, MEF2A and E2F1 were significantly (P gene ontology, protein domains, and cell type-specific expression. In summary, we provide evidence of five novel SLE susceptibility loci. Integrated bioinformatics using all 82 loci revealed that SLE susceptibility loci share many gene regulatory features, suggestive of conserved mechanisms of SLE etiopathogenesis.

Matched MeSH terms: Gene Expression Regulation/genetics
Relative quantification of human β‑defensins gene expression in pterygium and normal conjunctiva samples

Abubakar SA, Isa MM, Omar N, Tan SW

Mol Med Rep, 2020 Dec;22(6):4931-4937.
PMID: 33174018 DOI: 10.3892/mmr.2020.11560

The human ocular surface produces highly conserved cationic peptides. Human β‑defensins (HBDs) serve an important role in innate and adaptive immunity. They are primarily expressed in epithelial cells in response to infection and provide the first line of defence against invading microbes. Defensin β1 (DEFB1) is constitutively expressed and regulated by inflammatory mediators including interferon‑γ, lipopolysaccharide and peptidoglycans. DEFB4A is locally induced in response to microbial infection while DEFB109 is induced via Toll‑like receptor 2. The present study examined the expression of the HBD DEFB1, DEFB4A and DEFB109 genes in pterygium. The pterygium tissues and normal conjunctiva samples were obtained from 18 patients undergoing pterygium surgery. The reverse transcription‑quantitative polymerase chain reaction method was employed to determine the expression of DEFB1, DEFB4A and DEFB109 genes. The results revealed that the expression of DEFB1 and DEFB4A was significantly higher and upregulated in pterygium samples when compared with normal conjunctiva samples from each patient (P<0.05), while the expression of DEFB109 was observed to be lower in pterygium samples when compared with normal samples from the same patient. Previous studies have revealed that DEFB1 and DEFB4A genes are present in low concentrations inside the human eye, and they are upregulated during the maturation of keratinocytes, suggesting a possible role in cell differentiation. The DEFB109 gene is present in higher concentrations inside the human eye, though it is newly discovered. It has also been reported that DEFB1 may be involved in carcinogenesis epithelial tumours. Collectively, the current data suggests that HBDs may serve a crucial role in the pathogenesis and development of pterygia, and thus may be considered as novel molecular targets in understanding pterygia development.

Matched MeSH terms: Gene Expression Regulation/genetics
Fulltext Pilot study and bioinformatics analysis of differentially expressed genes in adipose tissues of rats with excess dietary intake

Yuan JC, Yogarajah T, Lim SK, Yvonne Tee GB, Khoo BY

Mol Med Rep, 2020 05;21(5):2063-2072.
PMID: 32323762 DOI: 10.3892/mmr.2020.11012

Excessive adipose tissue accumulation is an increasing health problem worldwide. The present study aimed to determine differentially expressed genes (DEGs) that are associated with the excessive accumulation of adipose tissues by PCR arrays in an excess dietary intake animal model. For this purpose, male Sprague Dawley rats were randomly assigned to 2 groups: Control (given an ordinary diet) and experimental (given twice the amount of the ordinary diet). After 2 months of feeding, the abdominal cavities of the rats from each group were opened, then subcutaneous and visceral adipose tissues were removed. The adipose tissues collected were then used for total RNA extraction and then reverse transcribed to cDNA, which was then used as a template to identify the DEGs of 84 transcripts for rat obesity by RT2 Profiler PCR Arrays. The results showed significant downregulation of bombesin‑like receptor 3 (BRS3) and uncoupling protein 1 (UCP1) in visceral adipose tissues of experimental rats compared with those of the control rats, and differential gene expression analysis showed an association with fat cell differentiation and regulation of triglyceride sequestration, as well as fatty acid binding. The gene expression patterns observed in the present study, which may be associated with peroxisome proliferator‑activated receptor‑γ (PPARG) on excessive visceral adipose tissue accumulation, may be useful in identifying a group of surrogate biomarkers for the early diet‑induced accumulation of visceral adipose tissue detection in humans. The biomarkers can also be the specific targets for drug development to reduce excessive visceral adipose tissue accumulation in the body and its associated diseases.

Matched MeSH terms: Gene Expression Regulation/genetics*
Fulltext Reconstructing directed gene regulatory network by only gene expression data

Zhang L, Feng XK, Ng YK, Li SC

BMC Genomics, 2016 Aug 18;17 Suppl 4:430.
PMID: 27556418 DOI: 10.1186/s12864-016-2791-2

BACKGROUND: Accurately identifying gene regulatory network is an important task in understanding in vivo biological activities. The inference of such networks is often accomplished through the use of gene expression data. Many methods have been developed to evaluate gene expression dependencies between transcription factor and its target genes, and some methods also eliminate transitive interactions. The regulatory (or edge) direction is undetermined if the target gene is also a transcription factor. Some methods predict the regulatory directions in the gene regulatory networks by locating the eQTL single nucleotide polymorphism, or by observing the gene expression changes when knocking out/down the candidate transcript factors; regrettably, these additional data are usually unavailable, especially for the samples deriving from human tissues.
RESULTS: In this study, we propose the Context Based Dependency Network (CBDN), a method that is able to infer gene regulatory networks with the regulatory directions from gene expression data only. To determine the regulatory direction, CBDN computes the influence of source to target by evaluating the magnitude changes of expression dependencies between the target gene and the others with conditioning on the source gene. CBDN extends the data processing inequality by involving the dependency direction to distinguish between direct and transitive relationship between genes. We also define two types of important regulators which can influence a majority of the genes in the network directly or indirectly. CBDN can detect both of these two types of important regulators by averaging the influence functions of candidate regulator to the other genes. In our experiments with simulated and real data, even with the regulatory direction taken into account, CBDN outperforms the state-of-the-art approaches for inferring gene regulatory network. CBDN identifies the important regulators in the predicted network: 1. TYROBP influences a batch of genes that are related to Alzheimer's disease; 2. ZNF329 and RB1 significantly regulate those 'mesenchymal' gene expression signature genes for brain tumors.
CONCLUSION: By merely leveraging gene expression data, CBDN can efficiently infer the existence of gene-gene interactions as well as their regulatory directions. The constructed networks are helpful in the identification of important regulators for complex diseases.

Matched MeSH terms: Gene Expression Regulation/genetics
Fulltext Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

Gholami K, Loh SY, Salleh N, Lam SK, Hoe SZ

PLoS One, 2017;12(6):e0176368.
PMID: 28591185 DOI: 10.1371/journal.pone.0176368

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Matched MeSH terms: Gene Expression Regulation/genetics
Involvement of NF-κB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A

Mohan S, Abdelwahab SI, Kamalidehghan B, Syam S, May KS, Harmal NS, et al.

Phytomedicine, 2012 Aug 15;19(11):1007-15.
PMID: 22739412 DOI: 10.1016/j.phymed.2012.05.012

The plant Artocarpus obtusus is a tropical plant that belongs to the family Moraceae. In the present study a xanthone compound Pyranocycloartobiloxanthone A (PA) was isolated from this plant and the apoptosis mechanism was investigated. PA induced cytotoxicity was observed using MTT assay. High content screening (HCS) was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Reactive oxygen species formation was investigated on treated cells by using fluorescent analysis. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition mRNA levels of Bax and Bcl2 were also checked using RT-PCR. Caspase 3/7, 8 and 9 were measured for their induction while treatment. The involvement of NF-κB was analyzed using HCS assay. The results showed that PA possesses the characteristics of selectively inducing cell death of tumor cells as no inhibition was observed in non-tumorigenic cells even at 30 μg/ml. Treatment of MCF7 cells with PA induced apoptosis with cell death-transducing signals, that regulate the MMP by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol. The release of cytochrome c triggered the activation of caspases-9, then activates downstream executioner caspase-3/7 and consequently cleaved specific substrates leading to apoptotic changes. This form of apoptosis was found closely associated with the extrinsic pathway caspase (caspase-8) and inhibition of translocation of NF-κB from cytoplasm to nucleus. The results demonstrated that PA induced apoptosis of MCF7 cells through NF-κB and Bcl2/Bax signaling pathways with the involvement of caspases.

Matched MeSH terms: Gene Expression Regulation/genetics

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