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Deciphering the potential of baicalin as an antiviral agent for Chikungunya virus infection

Oo A, Rausalu K, Merits A, Higgs S, Vanlandingham D, Bakar SA, et al.

Antiviral Res, 2018 02;150:101-111.
PMID: 29269135 DOI: 10.1016/j.antiviral.2017.12.012

The past decade has seen the re-emergence of Chikungunya virus (CHIKV) as a major global health threat, affecting millions around the world. Although fatal infections are rare among infected patients, the occurrence of long-lasting polyarthralgia has a significant impact on patients' quality of lives and ability to work. These issues were the stimuli for this study to determine the potential of baicalin, a bioflavonoid, as the novel antiviral compound against CHIKV. It was found that baicalin was well tolerated by Vero, BHK-21 and HEK 293T cells with maximal nontoxic doses >600 μM, ≈ 350 μM and ≈110 μM, respectively. Antiviral assays indicated that baicalin was the most effective inhibitor when tested for its direct virucidal activity with EC50 ≈ 7 μM, followed by inhibition of virus entry into the host cell, attachment of virus particle to cellular receptors and finally intracellular replication of viral RNA genome. In silico analysis using molecular docking demonstrated close interactions between baicalin and CHIKV envelope protein with considerably strong binding affinity of -9.7 kcal/mol. qRT-PCR analysis revealed that baicalin had the greatest effect on the synthesis of viral negative stand RNA with EC50 ≈ 0.4 μM followed by the inhibition of synthesis of positive-strand genomic (EC50 ≈ 13 μM) and subgenomic RNAs (EC50 ≈ 14 μM). These readings indicate that the compound efficiently inhibits replicase complexes formation but is a less potent inhibitor of existing replicase complexes. Coherent with this hypothesis, the use of recombinant CHIKV replicons harboring Renilla luciferase marker showed that replication of corresponding replicon RNAs was only slightly downregulated at higher doses of baicalin, with EC50 > 100 μM. Immunofluorescence and western blotting experiments demonstrated dose-dependent inhibition of expression of different viral proteins. It was also observed that levels of important protein markers for cellular autophagy (LC3) and apoptosis (Bax) were reduced in baicalin treatment groups as compared with untreated virus infected controls. In summary, given its low toxicity and high efficacy against CHIKV, baicalin has great potential to be developed as the novel antiviral compound for CHIKV. In vivo studies to evaluate its activity in a more complexed system represent a necessary step for future analysis.

Matched MeSH terms: Genes, Reporter
Fulltext The matrix (M) protein of Newcastle disease virus binds to human bax through its BH3 domain

Molouki A, Hsu YT, Jahanshiri F, Abdullah S, Rosli R, Yusoff K

Virol J, 2011;8:385.
PMID: 21810274 DOI: 10.1186/1743-422X-8-385

The underlying mechanisms by which Newcastle disease virus (NDV) kills cancer cells are still unclear. Recent discoveries have shown that many viruses contain Bcl-2 homology-like domains which enabled their interaction with Bcl-2 family members, and thereby accounting for their virulence and pathogenicity. Alignment of the protein sequences of Malaysian strain of NDV, known as AF2240, with those from members of the human Bcl-2 family showed many similar regions; most notably we found that its matrix (AF2240-M) protein, large (AF2240-L) protein and fusion (AF2240-F) protein all contain BH3-like regions. In addition, there are BH1-like domains in these proteins, where AF2240-F and Mcl-1 share 55% identity within this region. To further investigate our hypothesis that the presence of the BH3-like domains in these proteins may convey cytotoxicity, AF2240-M and AF2240-F genes were cloned into pFLAG and pEGFP.N2 vectors and transfected into HeLa cells. The expression of these constructs promoted cell death. As shown by flow cytometry, AF2240-M protein with deleted BH3-like region showed five-fold decrease in apoptosis. Moreover, the construct containing the N-terminal of AF2240-M showed nearly the same cell death rate as to that of the full-length protein, strongly suggesting that the BH3-like domain within this protein participates in promoting cell death. Moreover, AF2240-M transfection promoted Bax redistribution to mitochondria. Therefore, to determine whether there is any direct interaction between NDV viral proteins with some members of the Bcl-2 family, various constructs were co-transfected into HeLa cells. Co-immunoprecipitation trials showed that the AF2240-M indeed directly interacted with Bax protein via its BH3-domain, as the mutant proteins failed to interact with Bax. AF2240-F failed to interact with any of the tested proteins, although Bcl-XL slowed down the rate of cell death caused by this construct by nearly five-fold. In a parallel experiment, the level of expression of endogenous Bax and Bcl-2 after infection of HeLa cells with NDV was assessed by qRT-PCR, but no statistically significant change was observed. Consequently, the Bax/Bcl-2 ratio at the mRNA level did not alter. Overall, our study has shed additional light into the mechanisms by which NDV induces apoptosis.

Matched MeSH terms: Genes, Reporter
Fulltext Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1)

Ahmad MK, Tabana YM, Ahmed MA, Sandai DA, Mohamed R, Ismail IS, et al.

Malays J Med Sci, 2017 Dec;24(6):29-38.
PMID: 29379384 DOI: 10.21315/mjms2017.24.6.4

Background: A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication.
Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones.
Results: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing.
Conclusion: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.

Matched MeSH terms: Genes, Reporter
Design and synthesis of mono and bicyclic tetrapeptides thioester as potent inhibitor of histone deacetylases

Hoque MA, Islam MS, Islam MN, Kato T, Nishino N, Ito A, et al.

Amino Acids, 2014 Oct;46(10):2435-44.
PMID: 25048030 DOI: 10.1007/s00726-014-1800-5

Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that have an effect on gene regulation. The naturally occurring cyclic depsipeptide FK228 containing disulfide and Largazole possessing thioester functionalities act as pro-drugs and share the same HDAC inhibition mechanism in cell. Inspired from these facts, we have reported bicyclic tetrapeptide disulfide HDAC inhibitors resembling FK228 with potent activity and enhanced selectivity. In the present study, we report the design and synthesis of several mono and bicyclic tetrapeptide thioester HDAC inhibitors that share the inhibition mechanism similar to Largazole. Most of the compounds showed HDAC1 and HDAC4 inhibition and p21 promoting activity in nanomolar ranges. Among these the monocyclic peptides 1, 2 and bicyclic peptide, 4 are notable demanding more advanced research to be promising anticancer drug candidates.

Matched MeSH terms: Genes, Reporter/drug effects
Fulltext MicroRNAs regulating cluster of differentiation 46 (CD46) in cardioembolic and non-cardioembolic stroke

Tan JR, Tan KS, Yong FL, Armugam A, Wang CW, Jeyaseelan K, et al.

PLoS One, 2017;12(2):e0172131.
PMID: 28199366 DOI: 10.1371/journal.pone.0172131

Ischemic stroke is a major cause of mortality and morbidity globally. Among the ischemic stroke subtypes, cardioembolic stroke is with poor functional outcome (Modified Rankin score ≥ 2). Early diagnosis of cardioembolic stroke will prove beneficial. This study examined the microRNAs targeting cluster of differentiation 46 (CD46), a potential biomarker for cardioembolic stroke. CD46 mRNA level was shown to be differentially expressed (p < 0.001) between cardioembolic stroke (median = 1.32) and non-cardioembolic stroke subtypes (large artery stroke median = 5.05; small vessel stroke median = 6.45). Bioinformatic search showed that miR-19a, -20a, -185 and -374b were found to target CD46 mRNA and further verified by luciferase reporter assay. The levels of miRNAs targeting CD46 were significantly reduced (p < 0.05) in non-cardioembolic stroke patients (large artery stroke median: miR-19a = 0.63, miR-20a = 0.42, miR-185 = 0.32, miR-374b = 0.27; small artery stroke median: miR-19a = 0.07, miR-20a = 0.06, miR-185 = 0.07, miR-374b = 0.05) as compared to cardioembolic stroke patients (median: miR-19a = 2.69, miR-20a = 1.36, miR-185 = 1.05, miR-374b = 1.23). ROC curve showed that the miRNAs could distinguish cardioembolic stroke from non-cardioembolic stroke with better AUC value as compared to CD46. Endogenous expression of CD46 in Human Umbilical Vein Endothelial Cells (HUVECs) were found to be regulated by miR-19a and miR-20a. Thus implicating that miR-19a and -20a may play a role in pathogenesis of cardioembolic stroke, possibly via the endothelial cells.

Matched MeSH terms: Genes, Reporter
Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein

Govender N, Wong MY

Phytopathology, 2017 04;107(4):483-490.
PMID: 27918241 DOI: 10.1094/PHYTO-02-16-0062-R

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

Matched MeSH terms: Genes, Reporter
Fulltext Characterisation of Drosophila Ubx CPTI000601 and hth CPTI000378 protein trap lines

Choo SW, Beh CY, Russell S, White R

ScientificWorldJournal, 2014;2014:191535.
PMID: 25389534 DOI: 10.1155/2014/191535

In Drosophila, protein trap strategies provide powerful approaches for the generation of tagged proteins expressed under endogenous control. Here, we describe expression and functional analysis to evaluate new Ubx and hth protein trap lines generated by the Cambridge Protein Trap project. Both protein traps exhibit spatial and temporal expression patterns consistent with the reported endogenous pattern in the embryo. In imaginal discs, Ubx-YFP is expressed throughout the haltere and 3rd leg imaginal discs, while Hth-YFP is expressed in the proximal regions of haltere and wing discs but not in the pouch region. The Ubx (CPTI000601) line is semilethal as a homozygote. No T3/A1 to T2 transformations were observed in the embryonic cuticle or the developing midgut. The homozygous survivors, however, exhibit a weak haltere phenotype with a few wing-like marginal bristles on the haltere capitellum. Although hth (CPTI000378) is completely lethal as a homozygote, the hth (CPTI000378) /hth (C1) genotype is viable. Using a hth deletion (Df(3R)BSC479) we show that hth (CPTI000378) /Df(3R)BSC479 adults are phenotypically normal. No transformations were observed in hth (CPTI000378), hth (CPTI000378) /hth (C1), or hth (CPTI000378) /Df(3R)BSC479 embryonic cuticles. We have successfully characterised the Ubx-YFP and Hth-YFP protein trap lines demonstrating that the tagged proteins show appropriate expression patterns and produce at least partially functional proteins.

Matched MeSH terms: Genes, Reporter
Fulltext Endogenous HIF2A reporter systems for high-throughput functional screening

Zaini MN, Patel SA, Syafruddin SE, Rodrigues P, Vanharanta S

Sci Rep, 2018 08 13;8(1):12063.
PMID: 30104738 DOI: 10.1038/s41598-018-30499-2

Tissue-specific transcriptional programs control most biological phenotypes, including disease states such as cancer. However, the molecular details underlying transcriptional specificity is largely unknown, hindering the development of therapeutic approaches. Here, we describe novel experimental reporter systems that allow interrogation of the endogenous expression of HIF2A, a critical driver of renal oncogenesis. Using a focused CRISPR-Cas9 library targeting chromatin regulators, we provide evidence that these reporter systems are compatible with high-throughput screening. Our data also suggests redundancy in the control of cancer type-specific transcriptional traits. Reporter systems such as those described here could facilitate large-scale mechanistic dissection of transcriptional programmes underlying cancer phenotypes, thus paving the way for novel therapeutic approaches.

Matched MeSH terms: Genes, Reporter/genetics*
Isolation and characterization of an oil palm constitutive promoter derived from a translationally control tumor protein (TCTP) gene

Masura SS, Parveez GK, Ti LL

Plant Physiol Biochem, 2011 Jul;49(7):701-8.
PMID: 21549610 DOI: 10.1016/j.plaphy.2011.04.003

We have characterized an oil palm (Elaeis guineensis Jacq.) constitutive promoter that is derived from a translationally control tumor protein (TCTP) gene. The TCTP promoter was fused transcriptionally with the gusA reporter gene and transferred to monocot and dicot systems in order to study its regulatory role in a transient expression study. It was found that the 5' region of TCTP was capable of driving the gusA expression in all the oil palm tissues tested, including immature embryo, embryogenic callus, embryoid, young leaflet from mature palm, green leaf, mesocarp and stem. It could also be used in dicot systems as it was also capable of driving gusA expression in tobacco leaves. The results indicate that the TCTP promoter could be used for the production of recombinant proteins that require constitutive expression in the plant system.

Matched MeSH terms: Genes, Reporter
Recombinant hepatitis B virus core particles: association, dissociation and encapsidation of green fluorescent protein

Lee KW, Tan WS

J Virol Methods, 2008 Aug;151(2):172-180.
PMID: 18584885 DOI: 10.1016/j.jviromet.2008.05.025

The recombinant hepatitis B virus (HBV) core antigen (HBcAg) expressed in Escherichia coli self-assembles into icosahedral capsids of about 35 nm which can be exploited as gene or drug delivery vehicles. The association and dissociation properties of the C-terminally truncated HBcAg with urea and guanidine hydrochloride (GdnHCl) were studied. Transmission electron microscopy (TEM) revealed that the dissociated HBcAg was able to re-associate into particles when the applied denaturing agents were physically removed. In order to evaluate the potential of the particles in capturing molecules, purified green fluorescent protein (GFP) was applied to the dissociated HBcAg for encapsidation. The HBcAg particles harbouring the GFP molecules were purified using sucrose density gradient ultracentrifugation and analysed using native agarose gel electrophoresis and TEM. A method for the encapsidation of GFP in HBcAg particles which has the potential to capture drugs or nucleic acids was established.

Matched MeSH terms: Genes, Reporter
PNMA2 mediates heterodimeric interactions and antagonizes chemo-sensitizing activities mediated by members of PNMA family

Lee YH, Pang SW, Tan KO

Biochem Biophys Res Commun, 2016 Apr 22;473(1):224-229.
PMID: 27003254 DOI: 10.1016/j.bbrc.2016.03.083

PNMA2, a member of the Paraneoplastic Ma Family (PNMA), was identified through expression cloning by using anti-sera from patients with paraneoplastic disorder. Tissue expression studies showed that PNMA2 was predominantly expressed in normal human brain; however, the protein was shown to exhibit abnormal expression profile as it was found to be expressed in a number of tumour tissues obtained from paraneopalstic patients. The abnormal expression profile of PNMA2 suggests that it might play an important role in tumorigenesis; however, apart from protein expression and immunological studies, the physiological role of PNMA2 remains unclear. In order to determine potential role of PNMA2 in tumorigenesis, and its functional relationship with PNMA family members, MOAP-1 (PNMA4) and PNMA1, expression constructs encoding the respective proteins were generated for both in vitro and in vivo studies. Our investigations showed that over-expressed MOAP-1 and PNMA1 promoted apoptosis and chemo-sensitization in MCF-7 cells as evidenced by condensed nuclei and Annexin-V positive MCF-7 cells; however, the effects mediated by these proteins were significantly inhibited or abolished when co-expressed with PNMA2 in MCF-7 cells. Furthermore, co-immunoprecipitation study showed that PNMA1 and MOAP-1 failed to associate with each other but readily formed respective heterodimer with PNMA2, suggesting that PNMA2 functions as antagonist of MOAP-1 and PNMA1 through heterodimeric interaction.

Matched MeSH terms: Genes, Reporter
Tricistronic expression of MOAP-1, Bax and RASSF1A in cancer cells enhances chemo-sensitization that requires BH3L domain of MOAP-1

Lee YH, Pang SW, Revai Lechtich E, Shah K, Simon SE, Ponnusamy S, et al.

J Cancer Res Clin Oncol, 2020 Jul;146(7):1751-1764.
PMID: 32377840 DOI: 10.1007/s00432-020-03231-9

PURPOSE: Although important for apoptosis, the signaling pathway involving MOAP-1(Modulator of Apoptosis 1), RASSF1A (RAS association domain family 1A), and Bax (Bcl-2 associated X protein) is likely to be dysfunctional in many types of human cancers due to mechanisms associated with gene mutation and DNA hyper-methylation. The purpose of the present study was to assess the potential impact of generating physiologically relevant signaling pathway mediated by MOAP-1, Bax, and RASSF1A (MBR) in cancer cells and chemo-drug resistant cancer cells.
METHODS: The tricistronic expression construct that encodes MOAP-1, Bax, and RASSF1A (MBR) or its mutant, MOAP-1∆BH3L, Bax and RASSF1A (MBRX) was expressed from an IRES (Internal Ribosome Entry Site)-based tricistronic expression vector in human breast cancer cells, including MCF-7, MCF-7-CR (cisplatin resistant) and triple negative breast cancer cells, BMET05, for functional characterization through in vitro and in vivo models.
RESULTS: Transient expression of MBR potently promoted dose-dependent apoptotic signaling and chemo-sensitization in the cancer cells, as evidenced by loss of cell viability, nuclei condensation and Annexin-V positive staining while stable expression of MBR in MCF-7 cells significantly reduced the number of MBR stable clone by 86% and the stable clone exhibited robust chemo-drug sensitivity. In contrast, MBRX stable clone exhibited chemo-drug resistance while transiently over-expressed MOAP-1ΔBH3L inhibited the apoptotic activity of MBR. Moreover, the spheroids derived from the MBR stable clone displayed enhanced chemo-sensitivity and apoptotic activity. In mouse xenograft model, the tumors derived from MBR stable clone showed relatively high level of tumor growth retardation associated with the increase in apoptotic activity, leading to the decreases in both tumor weight and volume.
CONCLUSIONS: Expression of MBR in cancer cells induces apoptotic cell death with enhanced chemo-sensitization requiring the BH3L domain of MOAP-1. In animal model, the expression of MBR significantly reduces the growth of tumors, suggesting that MBR is a potent apoptotic sensitizer with potential therapeutic benefits for cancer treatment.

Matched MeSH terms: Genes, Reporter
Fulltext Production of Lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer

Siew, Ching Ngai, Ramasamy, Rajesh, Syahril Abdullah

Pertanika Journal of Science & Technology, 2012;20(2):269-281.
MyJurnal

Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike nonviralmethods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome,which can lead to long-term transgene expression. Lentiviruses have advantages over other types ofviruses due to their capacity to transduce non-dividing cells. An optimized generation of lentivirusescarrying green fluorescent protein (GFP) reporter gene driven by either UbC (LV/UbC/GFP) orCMV (LV/CMV/GFP) promoter is described in this paper. The lentiviruses were produced by cotransfectinglentiviral expression constructs and packaging mix into 293FT lentivirus producer cell lines.Lipofectamine was highly efficient in transfecting the cells compared to Transfast and Polyethyleneimine(PEI). Following cell transfection, syncytia were clearly visible at day 2. Lentiviruses were harvestedat days 1, 2 and 3 post-transfection. The highest transduction efficiency was read from LV/CMV/GFPharvested at day 2 post-transfection and LV/UbC/GFP harvested at day 3 post-transfection. Finally,the GFP expression in COS-7 cells was determined at day 2 and day 14 post-transduction for transientand stable GFP expression. It was found that the GFP expression declined overtime. However, thetransduction efficiency and duration of the transgene expression in COS-7 cells transduced with LV/CMV/GFP were higher compared to LV/UbC/GFP. In conclusion, we have successfully produced lentivirusescarrying GFP with different promoters and shown that the viruses were able to infect COS-7 cells atdifferent efficiencies. Meanwhile, the generation of the active lentiviruses will allow us to proceed to the subsequent analysis of the effect of regulatory elements in future study.

Matched MeSH terms: Genes, Reporter
Fulltext Evaluation of luciferase and GFP-expressing Nipah viruses for rapid quantitative antiviral screening

Lo MK, Nichol ST, Spiropoulou CF

Antiviral Res, 2014 Jun;106:53-60.
PMID: 24680955 DOI: 10.1016/j.antiviral.2014.03.011

Nipah virus (NiV) outbreaks have occurred in Malaysia, India, and Bangladesh, and the virus continues to cause annual outbreaks of fatal human encephalitis in Bangladesh due to spillover from its bat host reservoir. Due to its high pathogenicity, its potential use for bio/agro-terrorism, and to the current lack of approved therapeutics, NiV is designated as an overlap select agent requiring biosafety level-4 containment. Although the development of therapeutic monoclonal antibodies and soluble protein subunit vaccines have shown great promise, the paucity of effective antiviral drugs against NiV merits further exploration of compound libraries using rapid quantitative antiviral assays. As a proof-of-concept study, we evaluated the use of fluorescent and luminescent reporter NiVs for antiviral screening. We constructed and rescued NiVs expressing either Renilla luciferase or green fluorescent protein, and characterized their reporter signal kinetics in different cell types as well as in the presence of several inhibitors. The 50% effective concentrations (EC50s) derived for inhibitors against both reporter viruses are within range of EC50s derived from virus yield-based dose-response assays against wild-type NiV (within 1Log10), thus demonstrating that both reporter NiVs can serve as robust antiviral screening tools. Utilizing these live NiV-based reporter assays requires modest instrumentation, and circumvents the time and labor-intensive steps associated with cytopathic effect or viral antigen-based assays. These reporter NiVs will not only facilitate antiviral screening, but also the study of host cell components that influence the virus life cycle.

Matched MeSH terms: Genes, Reporter*
Development and characterization of a cell line TTCF from endangered mahseer Tor tor (Ham.)

Yadav K, Lakra WS, Sharma J, Goswami M, Singh A

Fish Physiol Biochem, 2012 Aug;38(4):1035-1045.
PMID: 22203177 DOI: 10.1007/s10695-011-9588-7

Tor tor is an important game and food fish of India with a distribution throughout Asia from the trans-Himalayan region to the Mekong River basin to Malaysia, Pakistan, Bangladesh and Indonesia. A new cell line named TTCF was developed from the caudal fin of T. tor for the first time. The cell line was optimally maintained at 28°C in Leibovitz-15 (L-15) medium supplemented with 20% fetal bovine serum (FBS). The propagation of TTCF cells showed a high plating efficiency of 63.00%. The cytogenetic analysis revealed a diploid count of 100 chromosomes at passage 15, 30, 45 and 60 passages. The viability of the TTCF cell line was found to be 72% after 6 months of cryopreservation in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 578- and 655-bp sequences of 16S rRNA and cytochrome oxidase subunit I (COI) genes of mitochondrial DNA (mtDNA) respectively. TTCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids. Further, immunocytochemistry studies confirm its fibroblastic morphology of cells. Genotoxicity assessment of H₂O₂ in TTCF cell line revealed the utility of TTCF cell line as in vitro model for aquatic toxicological studies.

Matched MeSH terms: Genes, Reporter
Escherichia coli bactofection using Lipofectamine

Narayanan K, Lee CW, Radu A, Sim EU

Anal Biochem, 2013 Aug 15;439(2):142-4.
PMID: 23608053 DOI: 10.1016/j.ab.2013.04.010

Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.

Matched MeSH terms: Genes, Reporter
Fulltext Development of Transgenic Papaya through Agrobacterium-Mediated Transformation

Azad MA, Rabbani MG, Amin L, Sidik NM

Int J Genomics, 2013;2013:235487.
PMID: 24066284 DOI: 10.1155/2013/235487

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi.

Matched MeSH terms: Genes, Reporter
Fulltext Transcriptional activation of zebrafish fads2 promoter and its transient transgene expression in yolk syncytial layer of zebrafish embryos

Tay SS, Kuah MK, Shu-Chien AC

Sci Rep, 2018 03 01;8(1):3874.
PMID: 29497119 DOI: 10.1038/s41598-018-22157-4

The front-end desaturases (Fads) are rate-limiting enzymes responsible for production of long-chain polyunsaturated fatty acids (LC-PUFA). The full spectrum of the transcriptional regulation of fads is still incomplete, as cloning of fads promoter is limited to a few species. Here, we described the cloning and characterisation of the zebrafish fads2 promoter. Using 5'-deletion and mutation analysis on this promoter, we identified a specific region containing the sterol regulatory element (SRE) which is responsible for the activation of the fads2 promoter. In tandem, two conserved CCAAT boxes were also present adjacent to the SRE and mutation of either of these binding sites attenuates the transcriptional activation of the fads2 promoter. An in vivo analysis employing GFP reporter gene in transiently transfected zebrafish embryos showed that this 1754 bp upstream region of the fads2 gene specifically directs GFP expression in the yolk syncytial layer (YSL) region. This indicates a role for LC-PUFA in the transport of yolk lipids through this tissue layer. In conclusion, besides identifying novel core elements for transcriptional activation in zebrafish fads2 promoter, we also reveal a potential role for fads2 or LC-PUFA in YSL during development.

Matched MeSH terms: Genes, Reporter
Fulltext Optogenetic control of iPS cell-derived neurons in 2D and 3D culture systems using channelrhodopsin-2 expression driven by the synapsin-1 and calcium-calmodulin kinase II promoters

Lee SY, George JH, Nagel DA, Ye H, Kueberuwa G, Seymour LW

J Tissue Eng Regen Med, 2019 Mar;13(3):369-384.
PMID: 30550638 DOI: 10.1002/term.2786

Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.

Matched MeSH terms: Genes, Reporter
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: Genes, Reporter

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