Displaying publications 21 - 40 of 79 in total

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  1. Determination of CYP3A4 Inducing Properties of Compounds Using a Laboratory-Developed Cell-Based Assay
    Seah TC, Tay YL, Tan HK, Muhammad TS, Wahab HA, Tan ML
    Int J Toxicol, 2015 08 12;34(5):454-68.
    PMID: 26268769 DOI: 10.1177/1091581815599335
    A cell-based assay to measure cytochrome P450 3A4 (CYP3A4) induction was developed to screen for potential CYP3A4 inducers. This 96-well format assay utilizes HepG2 cells transfected with a gene construct of CYP3A4 proximal promoter linked to green fluorescence protein (GFP) gene, and the expression of the GFP is then measured quantitatively. Bergamottin at 5 to 25 µmol/L produced low induction relative to the positive control. Both curcumin and lycopene were not found to affect the expression of GFP, suggesting no induction properties toward CYP3A4. Interestingly, resveratrol produced significant induction from 25 µmol/L onward, which was similar to omeprazole and may warrant further studies. In conclusion, the present study demonstrated that this cell-based assay can be used as a tool to evaluate the potential CYP3A4 induction properties of compounds. However, molecular docking data have not provided satisfactory pointers to differentiate between CYP3A4 inducers from noninducers or from inhibitors, more comprehensive molecular screening may be indicated.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  2. Fulltext A functional henipavirus envelope glycoprotein pseudotyped lentivirus assay system
    Khetawat D, Broder CC
    Virol J, 2010 Nov 12;7:312.
    PMID: 21073718 DOI: 10.1186/1743-422X-7-312
    BACKGROUND: Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.

    RESULTS: Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.

    CONCLUSIONS: Together, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.

    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  3. 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: Green Fluorescent Proteins/genetics*; Green Fluorescent Proteins/isolation & purification
  4. Statistical optimization of green fluorescent protein production from Escherichia coli BL21(DE3)
    Chew FN, Tan WS, Boo HC, Tey BT
    Prep Biochem Biotechnol, 2012;42(6):535-50.
    PMID: 23030465 DOI: 10.1080/10826068.2012.660903
    An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.
    Matched MeSH terms: Green Fluorescent Proteins/isolation & purification*; Green Fluorescent Proteins/standards*; Green Fluorescent Proteins/chemistry
  5. Fulltext Chemotaxis movement and attachment of Agrobacterium tumefaciens to Phalaenopsis violacea orchid tissues: an assessment of early factors influencing the efficiency of gene transfer
    Subramaniam, Sreeramanan, Balasubramaniam, Vinod, Poobathy, Ranjetta, Sreenivasan, Sasidharan, Rathinam, Xavier
    Trop Life Sci Res, 2009;20(1):-.
    MyJurnal
    An early step in the Agrobacterium-mediated transformation of Phalaenopsis violacea orchid was investigated to elucidate the plant-bacterium interaction. Directed movement in response to chemical attractants is of crucial importance to Agrobacterium tumefaciens strains. Chemotaxis of A. tumefaciens strains (EHA 101 and 105) towards wounded orchid tissues has been studied by using swarm agar plates. The results obtained indicate a minor role for chemotaxis in determining host specificity and suggest that it could not be responsible for the absence of tumourigenesis in P. violacea orchid under natural conditions. The spectrometric GUS and green fluorescent protein (GFP) assays provided information on the amount of inoculated A. tumefaciens that effectively bound to various orchid tissues. It can be concluded that, at least during the two early steps of interaction, A. tumefaciens appears to be compatible with P. violacea, indicating a potential basis for genetic transformation.
    Matched MeSH terms: Green Fluorescent Proteins
  6. Inadvertent expression of dengue 2 virus NS3-EGFP fusion protein in Escherlchla coll using the pEGFP-N 1 mammalian expression vector
    Norazizah S, AbuBakar S
    JUMMEC, 1999;4:41-46.
    Dengue 2 New Guinea C (NGC) virus NS3 protein, a potentially important virulence factor was cloned to the N-terminus of the Aeqirorea victoria enhanced green fluorescent protein (EGFP) using the pEGFP-N1 mammalian expression vector. During amplification of the recombinant plasmid in E. coli, transformants expressing the EGFP were detected in vivo when viewed using fluorescence microscopy. This inadvertent expression of the recombinant fusion protein was confirmed further by detection of the T7.Tag peptide cloned to the aluino terminal of the fusion protein using T 7.Tag specific monoclonal antibody. These findings represent perhaps the first reported expression of the T7.Tag-NS3-EGFP fusion protein using the pEGFP-N1 mammalian expression vector in E. coli. KEYWORDS: Dengue, NS3, pEGFP-N1, fusion protein.
    Matched MeSH terms: Green Fluorescent Proteins
  7. Nucleotide sequencing, cloning, and expression of Capra hircus Heme Oxygenase-1 in caprine islets to promote insulin secretion in vitro
    Vakhshiteh F, Allaudin ZN, Lila MA, Abbasiliasi S, Ajdari Z
    Mol Biotechnol, 2015 Jan;57(1):75-83.
    PMID: 25218408 DOI: 10.1007/s12033-014-9803-8
    Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  8. Fulltext Limited expression of non-integrating CpG-free plasmid is associated with increased nucleosome enrichment
    Habib O, Mohd Sakri R, Ghazalli N, Chau DM, Ling KH, Abdullah S
    PLoS One, 2020;15(12):e0244386.
    PMID: 33347482 DOI: 10.1371/journal.pone.0244386
    CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.
    Matched MeSH terms: Green Fluorescent Proteins/genetics
  9. In vitro evidence consistent with an interaction between wild-type and mutant SOD1 protein associated with canine degenerative myelopathy
    Qi Y, Montague P, Loney C, Campbell C, Shafie INF, Anderson TJ, et al.
    Eur J Neurosci, 2019 12;50(12):3896-3905.
    PMID: 31336405 DOI: 10.1111/ejn.14526
    Canine degenerative myelopathy (DM) is a progressive neurological disorder that may be considered to be a large animal model for specific forms of the fatal human disease, familial amyotrophic lateral sclerosis (fALS). DM is associated with a c118G>A mutation of the superoxide dismutase 1 (Sod1) gene, and a significant proportion of cases are inherited in an autosomal recessive manner in contrast to the largely, but not exclusively, dominant mode of inheritance in fALS. The consensus view is that these Sod1/SOD1 mutations result in a toxic gain of function but the mechanisms remain unclear. Here we used an in vitro neuroblastoma cell line transfection system to monitor wild-type and mutant forms of SOD1 fusion proteins containing either a Cherry or an enhanced green fluorescent protein (EGFP) tag. These fusion proteins retained SOD1 enzymatic activity on a native gel assay system. We demonstrate that SOD1 aggregate density is significantly higher in DM transfectants compared to wild-type. In addition, we show by co-immunoprecipitation and confocal microscopy, evidence for a potential interaction between wild-type and mutant forms of SOD1 in co-transfected cells. While in vitro studies have shown SOD1 heterodimer formation in fALS models, this is the first report for DM SOD1. Therefore, despite for the majority of cases there is a difference in the mode of inheritance between fALS and DM, a similar interaction between wild-type and mutant SOD1 forms can occur. Clarifying the role of SOD1 in DM may also be of benefit to understanding the role of SOD1 in fALS.
    Matched MeSH terms: Green Fluorescent Proteins/genetics
  10. Direct recovery of enhanced green fluorescent protein from unclarified Escherichia coli homogenate using ion exchange chromatography in stirred fluidized bed
    Song CP, Ooi CW, Tey BT, Lu CX, Liu BL, Chang YK
    Int J Biol Macromol, 2020 Dec 01;164:4455-4465.
    PMID: 32937154 DOI: 10.1016/j.ijbiomac.2020.09.051
    A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10-3 mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed.
    Matched MeSH terms: Green Fluorescent Proteins/isolation & purification*
  11. Fulltext Single-step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGE
    Chew FN, Tan WS, Ling TC, Tey BT
    Electrophoresis, 2009 Sep;30(17):3017-3023.
    PMID: 19685471 DOI: 10.1002/elps.200900246
    Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.
    Matched MeSH terms: Green Fluorescent Proteins/isolation & purification*
  12. A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma
    Weihs F, Peh A, Dacres H
    Anal Chim Acta, 2020 Mar 15;1102:99-108.
    PMID: 32044001 DOI: 10.1016/j.aca.2019.12.044
    Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples.
    Matched MeSH terms: Green Fluorescent Proteins/chemistry
  13. Localization and characterization of val-opsin isoform-expressing cells in the brain of adult zebrafish
    Hang CY, Kitahashi T, Parhar IS
    J. Comp. Neurol., 2014 Dec 1;522(17):3847-60.
    PMID: 25043553 DOI: 10.1002/cne.23645
    In addition to vision, light information is used to regulate a range of animal physiology. Such nonimage-forming functions of light are mediated by nonvisual photoreceptors expressed in distinct neurons in the retina and the brain in most vertebrates. A nonvisual photoreceptor vertebrate ancient long opsin (VAL-opsin) possesses two functional isoforms in the zebrafish, encoded by valopa and valopb, which has received little attention. To delineate the neurochemical identities of valop cells and to test for colocalization of the valop isoforms, we used in situ hybridization to characterize the expression of the valop genes along with that of neurotransmitters and a neuropeptide known to be present at the sites of valop expression. Double labeling showed that the thalamic valop population coexpresses valopa and valopb. All the thalamic valop cells overlapped with a GABAergic cell mass that continues from the anterior nucleus to the intercalated thalamic nucleus. A novel valopa cell population found in the superior raphe was serotonergic in nature. A valopb cell population in the Edinger-Westphal nucleus was identified as containing thyrotropin-releasing hormone. Valopb cells localized in the hindbrain intermediate reticular formation were noncholinergic in nature (nonmotorneurons). Thus, the presence of valop cell populations in different brain regions with coexpression of neurotransmitters and neuropeptides and the colocalization of valop isoforms in the thalamic cell population indicate regulatory and functional complexity of VAL-opsin in the brain of the zebrafish.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  14. A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter
    Omidvar V, Siti Nor Akmar A, Marziah M, Maheran AA
    Plant Cell Rep, 2008 Sep;27(9):1451-9.
    PMID: 18563415 DOI: 10.1007/s00299-008-0565-2
    The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  15. Evaluation of in vitro cytochrome P450 induction and inhibition activity of deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber L
    Koe XF, Lim EL, Seah TC, Amanah A, Wahab HA, Adenan MI, et al.
    Food Chem Toxicol, 2013 Oct;60:98-108.
    PMID: 23876819 DOI: 10.1016/j.fct.2013.07.030
    Drug metabolism involving cytochrome P450 (CYP) enzymes is a key determinant of significant drug interactions. Deoxyelephantopin was evaluated for its effects on the expression of mRNAs encoding CYP1A2, CYP2D6 and CYP3A4, and protein expression and resultant enzymatic activity. The mRNA and protein expression of cytochrome isoforms were carried out using an optimized multiplex qRT-PCR assay and Western blot analysis, respectively. Human CYP3A4 protein expression was determined using an optimized hCYP3A4-HepG2 cell-based assay and the enzymatic activity was evaluated using P450-Glo™ CYP3A4 assay. The molecular interaction and possible inhibition of deoxyelephantopin of the CYP3A4 enzyme was determined in silico and further validated using substrate-specific CYP3A4 inhibition assays. Deoxyelephantopin produced no significant effect on the CYP1A2 and CYP2D6 mRNA and protein expression. However, it has a weak induction effect on CYP3A4 at the transcriptional level. In silico docking simulation showed that deoxyelephantopin has a weak interaction with CYP3A4 enzyme and it minimally affects the metabolism of CYP3A4 substrates. Deoxyelephantopin is not an in vitro CYP1A2 and CYP2D6 inducer. It is both a weak in vitro CYP3A4 inducer and inhibitor and is unlikely to elicit a clinically significant effect in human.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  16. Combined use of in ovo electroporation and cultured neurons for gene function analysis of embryogenesis in the chicken optic tectum
    Yang C, Li X, Li Q, Zhang B, Li H, Lin J
    Neuroreport, 2017 Dec 06;28(17):1180-1185.
    PMID: 28953094 DOI: 10.1097/WNR.0000000000000903
    Chicken embryos are used widely in the fields of developmental biology and neurobiology. The chicken embryo also serves as a model to analyze gene expression and function using in ovo electroporation. Plasmids may be injected into the spinal cord or tectum of the chicken central nervous system by microinjection for electroporation. Here, we developed a novel method that combines in ovo electroporation and neuronal culturing to study gene function in the chicken tectum during embryo development. Our method can be used to study in-vivo and in-vitro exogenous genes' function. In addition, live cell imaging microscopy, immunostaining, and transfection can be used with our method to study neuronal growth, development, neurite growth and retraction, and axonal pathfinding. Our result showed that axons were present in isolated neurons after culturing for 24 h, and cell debris was low after replacing the media at 48 h. Many GFP-expressing neurons were observed in the cultured cells after 48 h. We successfully cultured the neurons for 3 weeks. Together, this method combines in ovo electroporation and neuronal culturing advantages and is more convenient for the gene function analysis.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  17. De novo hotspot variants in CYFIP2 cause early-onset epileptic encephalopathy
    Nakashima M, Kato M, Aoto K, Shiina M, Belal H, Mukaida S, et al.
    Ann Neurol, 2018 04;83(4):794-806.
    PMID: 29534297 DOI: 10.1002/ana.25208
    OBJECTIVE: The cytoplasmic fragile X mental retardation 1 interacting proteins 2 (CYFIP2) is a component of the WASP-family verprolin-homologous protein (WAVE) regulatory complex, which is involved in actin dynamics. An obvious association of CYFIP2 variants with human neurological disorders has never been reported. Here, we identified de novo hotspot CYFIP2 variants in neurodevelopmental disorders and explore the possible involvement of the CYFIP2 mutants in the WAVE signaling pathway.

    METHODS: We performed trio-based whole-exome sequencing (WES) in 210 families and case-only WES in 489 individuals with epileptic encephalopathies. The functional effect of CYFIP2 variants on WAVE signaling was evaluated by computational structural analysis and in vitro transfection experiments.

    RESULTS: We identified three de novo CYFIP2 variants at the Arg87 residue in 4 unrelated individuals with early-onset epileptic encephalopathy. Structural analysis indicated that the Arg87 residue is buried at an interface between CYFIP2 and WAVE1, and the Arg87 variant may disrupt hydrogen bonding, leading to structural instability and aberrant activation of the WAVE regulatory complex. All mutant CYFIP2 showed comparatively weaker interactions to the VCA domain than wild-type CYFIP2. Immunofluorescence revealed that ectopic speckled accumulation of actin and CYFIP2 was significantly increased in cells transfected with mutant CYFIP2.

    INTERPRETATION: Our findings suggest that de novo Arg87 variants in CYFIP2 have gain-of-function effects on the WAVE signaling pathway and are associated with severe neurological disorders. Ann Neurol 2018;83:794-806.

    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  18. Fulltext Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery
    Volak A, LeRoy SG, Natasan JS, Park DJ, Cheah PS, Maus A, et al.
    J Neurooncol, 2018 Sep;139(2):293-305.
    PMID: 29767307 DOI: 10.1007/s11060-018-2889-2
    The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  19. Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent
    Jafari S, Hosseini MS, Hajian M, Forouzanfar M, Jafarpour F, Abedi P, et al.
    Mol. Reprod. Dev., 2011 Aug;78(8):576-84.
    PMID: 21721066 DOI: 10.1002/mrd.21344
    In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism; Green Fluorescent Proteins/chemistry
  20. Fulltext Gene expression profiles in primary duodenal chick cells following transfection with avian influenza virus H5 DNA plasmid encapsulated in silver nanoparticles
    Jazayeri SD, Ideris A, Shameli K, Moeini H, Omar AR
    Int J Nanomedicine, 2013;8:781-90.
    PMID: 23459681 DOI: 10.2147/IJN.S39074
    In order to develop a systemically administered safe and effective nonviral gene delivery system against avian influenza virus (AIV) that induced cytokine expression, the hemagglutinin (H5) gene of AIV, A/Ck/Malaysia/5858/04 (H5N1) and green fluorescent protein were cloned into a coexpression vector pIRES (pIREGFP-H5) and formulated using green synthesis of silver nanoparticles (AgNPs) with poly(ethylene glycol) and transfected into primary duodenal cells taken from 18-day-old specific-pathogen-free chick embryos. The AgNPs were prepared using moderated temperature and characterized for particle size, surface charge, ultraviolet-visible spectra, DNA loading, and stability. AgNPs and AgNP-pIREGFP-H5 were prepared in the size range of 13.9 nm and 25 nm with a positive charge of +78 ± 0.6 mV and +40 ± 6.2 mV, respectively. AgNPs with a positive surface charge could encapsulate pIREGFP-H5 efficiently. The ultraviolet-visible spectra for AgNP-pIREGFP-H5 treated with DNase I showed that the AgNPs were able to encapsulate pIREGFP-H5 efficiently. Polymerase chain reaction showed that AgNP-pIREGFP-H5 entered into primary duodenal cells rapidly, as early as one hour after transfection. Green fluorescent protein expression was observed after 36 hours, peaked at 48 hours, and remained stable for up to 60 hours. In addition, green fluorescent protein expression generally increased with increasing DNA concentration and time. Cells were transfected using Lipocurax in vitro transfection reagent as a positive control. A multiplex quantitative mRNA gene expression assay in the transfected primary duodenal cells via the transfection reagent and AgNPs with pIREGFP-H5 revealed expression of interleukin (IL)-18, IL-15, and IL-12β.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism; Green Fluorescent Proteins/chemistry
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