Displaying publications 21 - 40 of 79 in total

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  1. Dengue virus type 2 NS3 protease and NS2B-NS3 protease precursor induce apoptosis
    Shafee N, AbuBakar S
    J Gen Virol, 2003 Aug;84(Pt 8):2191-2195.
    PMID: 12867651 DOI: 10.1099/vir.0.19022-0
    Apoptosis was detected in Vero cell cultures expressing transfected dengue virus type 2 (DENV-2) genes. Approximately 17.5 and 51.5 % of cells expressing NS3 serine protease and NS2B-NS3(185) serine protease precursor protein [NS2B-NS3(185)(pro)] genes, respectively, were apoptotic. The percentage of apoptotic cells was significantly higher in cell cultures expressing NS2B-NS3(185)(pro). NS2B-NS3(185)(pro) was detected as NS2B-NS3(185)(pro)-EGFP fusion protein in cytoplasmic vesicular structures in the apoptotic cells. Site-directed mutagenesis which replaced His(51) with Ala within the protease catalytic triad significantly reduced the ability of the expressed NS3 and NS2B-NS3(185)(pro) to induce apoptosis. Results from the present study showed that DENV-2-encoded NS3 serine protease induces apoptosis, which is enhanced in cells expressing its precursor, NS2B-NS3(185)(pro). These findings suggest the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.
    Matched MeSH terms: Green Fluorescent Proteins
  2. 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
  3. Fulltext Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars
    Sahebi M, Hanafi MM, Rafii MY, Azizi P, Abiri R, Kalhori N, et al.
    Biomed Res Int, 2017;2017:9064129.
    PMID: 28191468 DOI: 10.1155/2017/9064129
    Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  4. Fulltext Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-COV-2 Main Protease (3CLpro)
    Rothan HA, Teoh TC
    Mol Biotechnol, 2021 Mar;63(3):240-248.
    PMID: 33464543 DOI: 10.1007/s12033-021-00299-7
    The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism; Green Fluorescent Proteins/chemistry
  5. Fulltext Mucosal genetic immunization through microsphere-based oral carriers
    Rosli R, Nograles N, Hanafi A, Nor Shamsudin M, Abdullah S
    Hum Vaccin Immunother, 2013 Oct;9(10):2222-7.
    PMID: 24051430 DOI: 10.4161/hv.25325
    Polymeric carriers in the form of cellulose acetate phthalate (CAP) and alginate (ALG) microspheres were used for encapsulation of plasmid DNA for oral mucosal immunization. Access into the intestinal mucosa by pVAX1 eukaryotic expression plasmid vectors carrying gene-coding sequences, either for the cholera enterotoxin B subunit (ctxB) immunostimulatory antigen or the green fluorescent protein (GFP), delivered from both types of microsphere carriers were examined in orally immunized BALB/c mice. Demonstration of transgene protein expression and IgA antibody responses at local mucosal sites suggest immunological response to a potential oral DNA vaccine formulated within the microsphere carriers.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/immunology
  6. Efficacy of Human Cell-Seeded Muscle-Stuffed Vein Conduit in Rat Sciatic Nerve Repair
    Ramli K, Gasim AI, Ahmad AA, Htwe O, Mohamed Haflah NH, Law ZK, et al.
    Tissue Eng Part A, 2019 10;25(19-20):1438-1455.
    PMID: 30848172 DOI: 10.1089/ten.TEA.2018.0279
    We investigated the efficacy of a muscle-stuffed vein (MSV) seeded with neural-transdifferentiated human mesenchymal stem cells as an alternative nerve conduit to repair a 15-mm sciatic nerve defect in athymic rats. Other rats received MSV conduit alone, commercial polyglycolic acid conduit (Neurotube®), reverse autograft, or were left untreated. Motor and sensory functions as well as nerve conductivity were evaluated for 12 weeks, after which the grafts were harvested for histological analyses. All rats in the treatment groups demonstrated a progressive increase in the mean Sciatic Functional Index (motor function) and nerve conduction amplitude (electrophysiological function) and showed positive withdrawal reflex (sensory function) by the 10th week of postimplantation. Autotomy, which is associated with neuropathic pain, was severe in rats treated with conduit without cells; there was mild or no autotomy in the rats of other groups. Histologically, harvested grafts from all except the untreated groups exhibited axonal regeneration with the presence of mature myelinated axons. In conclusion, treatment with MSV conduit is comparable to that of other treatment groups in supporting functional recovery following sciatic nerve injury; and the addition of cells in the conduit alleviates neuropathic pain. Impact Statement It is shown that pretreated muscle-stuffed vein conduit is comparable to that of commercial nerve conduit and autograft in supporting functional recovery following peripheral nerve injury. The addition of neural-differentiated mesenchymal stem cells in the conduit is shown to alleviate neuropathic pain.
    Matched MeSH terms: Green Fluorescent Proteins/metabolism
  7. 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
  8. Transgenic oil palm: production and projection
    Parveez GK, Masri MM, Zainal A, Majid NA, Yunus AM, Fadilah HH, et al.
    Biochem Soc Trans, 2000 Dec;28(6):969-72.
    PMID: 11171275
    Oil palm is an important economic crop for Malaysia. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date. Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation. This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering. Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil. Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020.
    Matched MeSH terms: Green Fluorescent Proteins
  9. Fulltext Burkholderia pseudomallei kills Caenorhabditis elegans through virulence mechanisms distinct from intestinal lumen colonization
    Ooi SK, Lim TY, Lee SH, Nathan S
    Virulence, 2012 Oct 01;3(6):485-96.
    PMID: 23076282 DOI: 10.4161/viru.21808
    The nematode Caenorhabditis elegans is hypersusceptible to Burkholderia pseudomallei infection. However, the virulence mechanisms underlying rapid lethality of C. elegans upon B. pseudomallei infection remain poorly defined. To probe the host-pathogen interaction, we constructed GFP-tagged B. pseudomallei and followed bacterial accumulation within the C. elegans intestinal lumen. Contrary to slow-killing by most bacterial pathogens, B. pseudomallei caused fairly limited intestinal lumen colonization throughout the period of observation. Using grinder-defective mutant worms that allow the entry of intact bacteria also did not result in full intestinal lumen colonization. In addition, we observed a significant decline in C. elegans defecation and pharyngeal pumping rates upon B. pseudomallei infection. The decline in defecation rates ruled out the contribution of defecation to the limited B. pseudomallei colonization. We also demonstrated that the limited intestinal lumen colonization was not attributed to slowed host feeding as bacterial loads did not change significantly when feeding was stimulated by exogenous serotonin. Both these observations confirm that B. pseudomallei is a poor colonizer of the C. elegans intestine. To explore the possibility of toxin-mediated killing, we examined the transcription of the C. elegans ABC transporter gene, pgp-5, upon B. pseudomallei infection of the ppgp-5::gfp reporter strain. Expression of pgp-5 was highly induced, notably in the pharynx and intestine, compared with Escherichia coli-fed worms, suggesting that the host actively thwarted the pathogenic assaults during infection. Collectively, our findings propose that B. pseudomallei specifically and continuously secretes toxins to overcome C. elegans immune responses.
    Matched MeSH terms: Green Fluorescent Proteins/genetics
  10. 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
  11. Fulltext Lactococcus lactis M4, a potential host for the expression of heterologous proteins
    Noreen N, Hooi WY, Baradaran A, Rosfarizan M, Sieo CC, Rosli MI, et al.
    Microb Cell Fact, 2011;10:28.
    PMID: 21518457 DOI: 10.1186/1475-2859-10-28
    Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.
    Matched MeSH terms: Green Fluorescent Proteins/biosynthesis; Green Fluorescent Proteins/genetics
  12. Transient Expression of Green Fluorescent Protein in Integrase-Defective Lentiviral Vector-Transduced 293T Cell Line
    Nordin F, Hamid ZA, Chan L, Farzaneh F, Hamid MK
    Methods Mol Biol, 2016;1448:159-73.
    PMID: 27317180 DOI: 10.1007/978-1-4939-3753-0_12
    Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. They retain high transduction efficiency for efficient gene transfer in various cell types both in vitro and in vivo. IDLV is produced via a combined mutations introduced on the HIV-based lentiviral to disable their integration potency. Therefore, IDLV is considered safer than the wild-type integrase-proficient lentiviral vector as they could avoid the potential insertional mutagenesis associated with the nonspecific integration of transgene into target cell genome afforded by the wild-type vectors.Here we describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. The efficiency of the IDLV in expressing the enhanced green fluorescent protein (GFP) reporter gene in transduced human monocyte (U937) cell lines was investigated. Expression of the transgene was driven by the spleen focus-forming virus (SFFV) LTRs. Transduction efficiency was studied using both the IDLV (ID-SFFV-GFP) and their wild-type counterparts (integrase-proficient SFFV-GFP). GFP expression was analyzed by fluorescence microscope and FACS analysis.Based on the results, the number of the GFP-positive cells in ID-SFFV-GFP-transduced U937 cells decreased rapidly over time. The percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. In wild-type SFFV-GFP-transduced cells, GFP expression is remained consistently at about 100 %. These data confirmed that the transgene expression in the ID-SFFV-GFP-transduced cells is transient in dividing cells. The lack of an origin of replication due to mutation of integrase enzymes in the ID-SFFV-GFP virus vector has caused the progressive loss of the GFP expression in dividing cells.Integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.
    Matched MeSH terms: Green Fluorescent Proteins/genetics*
  13. 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
  14. Formation and characterization of pDNA-loaded alginate microspheres for oral administration in mice
    Nograles N, Abdullah S, Shamsudin MN, Billa N, Rosli R
    J Biosci Bioeng, 2012 Feb;113(2):133-40.
    PMID: 22093752 DOI: 10.1016/j.jbiosc.2011.10.003
    Alginate, a natural polysaccharide, was explored in this study as an oral delivery vehicle of a mammalian expression vector into the murine intestinal mucosa. Alginate microspheres were produced through water-in-oil (W/O) emulsification method. Average diameter sizes of microspheres were 46.88 μm±3.07 μm with significant size reduction upon utilization of 1.0% Span80. Plasmid DNA (pDNA) carrying green fluorescent protein reporter gene (GFP), pVAX-GFP, was encapsulated within microspheres at efficiencies of 72.9 to 74.4%, carrying maximum load of 6 μg pDNA. Alginate microspheres demonstrated shrinkage in pH 1.2 and swelling in pH 9.0 with pDNA release about twice the amount released in acidic environment. Oral delivery of pVAX-GFP loaded-microspheres, at 50 μg, 100 μg and 150 μg dose, was performed on BALB/c mice. Tissue biodistribution, investigated through flow cytometric analysis, demonstrated GFP positive intestinal cells (<1.0%) with 1.3-fold higher levels for the 100 μg dose; therefore suggesting feasibility of the approach for oral gene delivery and vaccination.
    Matched MeSH terms: Green Fluorescent Proteins/analysis; Green Fluorescent Proteins/genetics
  15. Lentivirus vector driven by polybiquitin C promoter without woodchuck posttranscriptional regulatory element and central polypurine tract generates low level and short-lived reporter gene expression
    Ngai SC, Rosli R, Nordin N, Veerakumarasivam A, Abdullah S
    Gene, 2012 May 1;498(2):231-6.
    PMID: 22366305 DOI: 10.1016/j.gene.2012.01.071
    Lentivirus (LV) encoding woodchuck posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) driven by CMV promoter have been proven to act synergistically to increase both transduction efficiency and gene expression. However, the inclusion of WPRE and cPPT in a lentiviral construct may pose safety risks when administered to human. A simple lentiviral construct driven by an alternative promoter with proven extended duration of gene expression without the two regulatory elements would be free from the risks. In a non-viral gene delivery context, gene expression driven by human polybiquitin C (UbC) promoter resulted in higher and more persistent expression in mouse as compared to cytomegalovirus (CMV) promoter. In this study, we measured the efficiency and persistency of green fluorescent protein (GFP) reporter gene expression in cells transduced with LV driven by UbC (LV/UbC/GFP) devoid of the WPRE and cPPT in comparison to the established LV construct encoding WPRE and cPPT, driven by CMV promoter (LV/CMV/GFP). However, we found that LV/UbC/GFP was inferior to LV/CMV/GFP in many aspects: (i) the titer of virus produced; (ii) the levels of reporter gene expression when MOI value was standardized; and (iii) the transduction efficiency in different cell types. The duration of reporter gene expression in selected cell lines was also determined. While the GFP expression in cells transduced with LV/CMV/GFP persisted throughout the experimental period of 14 days, expression in cells transduced with LV/UbC/GFP declined by day 2 post-transduction. In summary, the LV driven by the UbC promoter without the WPRE and cPPT does not exhibit enhanced or durable transgene expression.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  16. Fulltext DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells
    Ngai SC, Rosli R, Al Abbar A, Abdullah S
    Biomed Res Int, 2015;2015:346134.
    PMID: 25961011 DOI: 10.1155/2015/346134
    Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.
    Matched MeSH terms: Green Fluorescent Proteins/genetics
  17. Fulltext Neuronal connectivity between habenular glutamate-kisspeptin1 co-expressing neurons and the raphe 5-HT system
    Nathan FM, Ogawa S, Parhar IS
    J Neurochem, 2015 Nov;135(4):814-29.
    PMID: 26250886 DOI: 10.1111/jnc.13273
    The habenula, located on the dorsal thalamic surface, is an emotional and reward processing center. As in the mammalian brain, the zebrafish habenula is divided into dorsal (dHb) and ventral (vHb) subdivisions that project to the interpeduncular nucleus and median raphe (MR) respectively. Previously, we have shown that kisspeptin 1 (Kiss1) expressing in the vHb, regulates the serotonin (5-HT) system in the MR. However, the connectivity between the Kiss1 neurons and the 5-HT system remains unknown. To resolve this issue, we generated a specific antibody against zebrafish Kiss1 receptor (Kiss-R1); using this primary antibody we found intense immunohistochemical labeling in the ventro-anterior corner of the MR (vaMR) but not in 5-HT neurons, suggesting the potential involvement of interneurons in 5-HT modulation by Kiss1. Double-fluorescence labeling showed that the majority of habenular Kiss1 neurons are glutamatergic. In the MR region, Kiss1 fibers were mainly seen in close association with glutamatergic neurons and only scarcely within GABAergic and 5-HT neurons. Our findings indicate that the habenular Kiss1 neurons potentially modulate the 5-HT system primarily through glutamatergic neurotransmission via as yet uncharacterized interneurons. The neuropeptide kisspeptin (Kiss1) play a key role in vertebrate reproduction. We have previously shown modulatory role of habenular Kiss1 in the raphe serotonin (5-HT) systems. This study proposed that the habenular Kiss1 neurons modulate the 5-HT system primarily through glutamatergic neurotransmission, which provides an important insight for understanding of the modulation of 5-HT system by the habenula-raphe pathway.
    Matched MeSH terms: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism
  18. 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: Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism*; Green Fluorescent Proteins/chemistry
  19. 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
  20. Overcoming the challenge of transduction of human T-cells with chimeric antigen receptor (CAR) specific for ERBB2 antigen
    Munisvaradass R, Ding SSL, Ee AHK, Syahril Abdullah, Mok PL, Kumar S, et al.
    Sains Malaysiana, 2017;46:1831-1838.
    Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the
    overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor
    (CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is
    specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are
    able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering
    it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human
    T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction
    protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene.
    CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted
    into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These
    lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabeadconjugated
    CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected
    and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models
    to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells.
    Matched MeSH terms: Green Fluorescent Proteins
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