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/metabolism
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
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
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/metabolism
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/metabolism
The migration of gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode to the preoptic area (POA) from embryonic day 13 is important for successful reproduction during adulthood. Whether maternal glucocorticoid exposure alters GnRH neuronal morphology and number in the offspring is unknown. This study determines the effect of maternal dexamethasone (DEX) exposure on enhanced green fluorescent protein (EGFP) driven by GnRH promoter neurons (TG-GnRH) in transgenic rats dual-labelled with GnRH immunofluorescence (IF-GnRH). The TG-GnRH neurons were examined in intact male and female rats at different postnatal ages, as a marker for GnRH promoter activity. Pregnant females were subcutaneously injected with DEX (0.1 mg/kg) or vehicle daily during gestation days 13-20 to examine the number of GnRH neurons in P0 male offspring. The total number of TG-GnRH neurons and TG-GnRH/IF-GnRH neuronal ratio increased from P0 and P5 stages to P47-52 stages, suggesting temporal regulation of GnRH promoter activity during postnatal development in intact rats. In DEX-treated P0 males, the number of IF-GnRH neurons decreased within the medial septum, organum vasculosom of the lamina terminalis (OVLT) and anterior hypothalamus. The percentage of TG-GnRH neurons with branched dendritic structures decreased in the OVLT of DEX-P0 males. These results suggest that maternal DEX exposure affects the number and dendritic development of early postnatal GnRH neurons in the OVLT/POA, which may lead to altered reproductive functions in adults.
Matched MeSH terms: Green Fluorescent Proteins/metabolism
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/metabolism
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/metabolism
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/metabolism
Bacterial toxin-antitoxin systems usually comprise of a pair of genes encoding a stable toxin and its cognate labile antitoxin and are located in the chromosome or in plasmids of several bacterial species. Chromosomally-encoded toxin-antitoxin systems are involved in bacterial stress responses and activation of the toxins usually leads to cell death or dormancy. Overexpression of the chromosomally-encoded YoeB toxin from the yefM-yoeB toxin-antitoxin locus of the Gram-positive bacterium Streptococcus pneumoniae has been shown to cause cell death in S. pneumoniae as well as E. coli.
Matched MeSH terms: Green Fluorescent Proteins/metabolism
Gonadotropin-inhibitory hormone (GnIH) neurons project to GnRH neurons to negatively regulate reproductive function. To fully explore the projections of the GnIH neurons, we created transgenic rats carrying an enhanced green fluorescent protein (EGFP) tagged to the GnIH promoter. With these animals, we show that EGFP-GnIH neurons are localized mainly in the dorsomedial hypothalamic nucleus (DMN) and project to the hypothalamus, telencephalon, and diencephalic thalamus, which parallels and confirms immunocytochemical and gene expression studies. We observed an age-related reduction in c-Fos-positive GnIH cell numbers in female rats. Furthermore, GnIH fiber appositions to GnRH neurons in the preoptic area were lessened in middle-aged females (70 weeks old) compared with their younger counterparts (9-12 weeks old). The fiber density in other brain areas was also reduced in middle-aged female rats. The expression of estrogen and progesterone receptors mRNA in subsets of EGFP-GnIH neurons was shown in laser-dissected single EGFP-GnIH neurons. We then examined estradiol-17β and progesterone regulation of GnIH neurons, using c-Fos presence as a marker. Estradiol-17β treatment reduced c-Fos labeling in EGFP-GnIH neurons in the DMN of young ovariectomized adult females but had no effect in middle-aged females. Progesterone had no effect on the number of GnIH cells positive for c-Fos. We conclude that there is an age-related decline in GnIH neuron number and GnIH inputs to GnRH neurons. We also conclude that the response of GnIH neurons to estrogen diminishes with reproductive aging.
Matched MeSH terms: Green Fluorescent Proteins/metabolism
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: Green Fluorescent Proteins/metabolism
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/metabolism