O-GlcNAc transferase promotes glioblastoma by modulating genes responsible for cell survival, invasion, and inflammation.

Metabolic reprogramming has emerged as one of the key hallmarks of cancer cells. Various metabolic pathways are dysregulated in cancers, including the hexosamine biosynthesis pathway. Protein O-GlcNAcylation is catalyzed by the enzyme O-GlcNAc transferase (OGT), an effector of hexosamine biosynthesis pathway that is found to be upregulated in most cancers. Posttranslational O-GlcNAcylation of various signaling and transcriptional regulators could promote cancer cell maintenance and progression by regulating gene expression, as gene-specific transcription factors and chromatin regulators are among the most highly O-GlcNAcylated proteins. Here, we investigated the role of OGT in glioblastoma. We demonstrate that OGT knockdown and chemical inhibition led to reduced glioblastoma cell proliferation and downregulation of many genes known to play key roles in glioblastoma cell proliferation, migration, and invasion. We show that genes downregulated due to OGT reduction are also known to be transcriptionally regulated by transcriptional initiation/elongation cofactor BRD4. We found BRD4 to be O-GlcNAcylated in glioblastoma cells; however, OGT knockdown/inhibition neither changed its expression nor its chromatin association on promoters. Intriguingly, we observed OGT knockdown led to reduced Pol II-Ser2P chromatin association on target genes without affecting other transcription initiation/elongation factors. Finally, we found that chemical inhibition of BRD4 potentiated the effects of OGT inhibition in reducing glioblastoma cell proliferation, invasion, and migration. We propose BRD4 and OGT act independently in the transcriptional regulation of a common set of genes and that combined inhibition of OGT and BRD4 could be utilized therapeutically for more efficient glioblastoma cell targeting than targeting of either protein alone.

Developmental modeling of hepatogenesis using obese iPSCs-hepatocyte differentiation uncovers pathological features.

Obesity is a multigene disorder. However, in addition to genetic factors, environmental determinants also participate in developing obesity and related pathologies. Thus, obesity could be best described as a combination of genetic and environmental perturbations often having its origin during the early developmental period. Environmental factors such as energy-dense food and sedentary lifestyle are known to be associated with obesogenicity. However, the combinatorial effects of gene-environment interactions are not well understood. Understanding the role of multiple genetic variations leading to subtle gene expression changes is not practically possible in monogenic or high-fat-fed animal models of obesity. In contrast, human induced pluripotent stem cells (hiPSCs) from individuals with familial obesity or an obesogenic genotype could serve as a good model system. Herein, we have used hiPSCs generated from normal and genetically obese subjects and differentiated them into hepatocytes in cell culture. We show that hepatocytes from obese iPSCs store more lipids and show increased cell death than normal iPSCs. Whole transcriptome analyses in both normal and obese iPSCs treated with palmitate compared to control revealed LXR-RXR and hepatic fibrosis pathways were enriched among other pathways in obese iPSCs compared to normal iPSCs. Among other genes, increased CD36 and CAV1 expression and decreased expression of CES1 in obese iPSCs could have been responsible for excess lipid accumulation, resulting in differential expression of genes associated with hepatic fibrosis, a key feature of non-alcoholic fatty liver disease (NAFLD). Our results demonstrate that iPSCs derived from genetically obese subjects could serve as an excellent model to understand the effects of this multigene disorder on organ development and may uncover pathologies of NAFLD, which is highly associated with obesity.

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI).

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) as well as neural progenitor cells (NPCs) derived from hESCs at high efficiency. Lentiviral particles were generated by co-transfecting HEK293T cells using entry vectors (carrying shRNAs) along with packaging plasmids (pAX and pMD2.G) using the low-cost cationic polymer polyethylenimine (PEI). Viral particles were concentrated using ultracentrifugation, which resulted in average titers above 5 x 107. Both hESCs and NPCs could be infected at high efficiencies using these lentiviral particles, as shown by puromycin selection and stable expression in hESCs, as well as transient GFP expression in NPCs. Furthermore, western blot analysis showed a significant reduction in the expression of genes targeted by shRNAs. In addition, the cells retained their pluripotency as well as differentiation potential, as evidenced by their subsequent differentiation into different lineages of CNS. The current protocol deals with the delivery of shRNAs; however, the same approach could be used for the ectopic expression of cDNAs for overexpression studies.

Stem cell fate determination through protein O-GlcNAcylation.

Embryonic and adult stem cells possess the capability of self-renewal and lineage-specific differentiation. The intricate balance between self-renewal and differentiation is governed by developmental signals and cell-type-specific gene regulatory mechanisms. A perturbed intra/extracellular environment during lineage specification could affect stem cell fate decisions resulting in pathology. Growing evidence demonstrates that metabolic pathways govern epigenetic regulation of gene expression during stem cell fate commitment through the utilization of metabolic intermediates or end products of metabolic pathways as substrates for enzymatic histone/DNA modifications. UDP-GlcNAc is one such metabolite that acts as a substrate for enzymatic mono-glycosylation of various nuclear, cytosolic, and mitochondrial proteins on serine/threonine amino acid residues, a process termed protein O-GlcNAcylation. The levels of GlcNAc inside the cells depend on the nutrient availability, especially glucose. Thus, this metabolic sensor could modulate gene expression through O-GlcNAc modification of histones or other proteins in response to metabolic fluctuations. Herein, we review evidence demonstrating how stem cells couple metabolic inputs to gene regulatory pathways through O-GlcNAc-mediated epigenetic/transcriptional regulatory mechanisms to govern self-renewal and lineage-specific differentiation programs. This review will serve as a primer for researchers seeking to better understand how O-GlcNAc influences stemness and may catalyze the discovery of new stem-cell-based therapeutic approaches.

Polylysine-modified polyethylenimine polymer can generate genetically engineered mesenchymal stem cells for combinational suicidal gene therapy in glioblastoma.

Glioblastoma remains the most resistant malignant brain tumor owing to the lack of an efficient delivery system for therapeutic genes or drugs, especially in outgrowing tumor islands. Cell-based delivery systems such as mesenchymal stem cells (MSCs) are a potential candidate in this regard. Conventionally, MSCs have been genetically modified for cancer therapy by using viral vectors that can illicit oncogenicity and limit their use in clinical trials. In this study, we have used nonviral agents such as the polylysine-modified polyethylenimine (PEI-PLL) copolymer to generate genetically engineered MSCs with suicidal genes, namely, HSV-TK and TRAIL. Our results demonstrated that an intratumoral injection of polymer-double-transfected MSCs along with prodrug ganciclovir injections can induce a significant synergistic therapeutic response both in vitro and in vivo compared to single plasmid transfections or untransfected MSCs. The proliferation marker Ki67 and the angiogenesis marker VEGF were also significantly reduced in treatment groups, whereas the TUNEL assay demonstrated that apoptosis is significantly increased after treatment. Our findings suggest that the PEI-PLL copolymer can successfully modify MSCs with therapeutic genes and can produce a pronounced impact during glioblastoma therapy. This study proposes a potential nonviral approach to develop a cell-based therapy for the treatment of glioma. STATEMENT OF SIGNIFICANCE: In this study, we have used a polylysine-modified polyethylenimine polymer (PEI-PLL) copolymer, a non viral transfection agent, for gene delivery in mesenchymal stem cells. These PEI-PLL-transfected mesenchymal stem cells with HSV-TK and TRAIL genes have the potential to treat glioma both in vitro and in vivo. This combinational therapy through PEI-PLL-transfected mesenchymal stem cells can provide cost-effective, low immunogenic, and tumor-targeted delivery of suicideal genes (HSV-TK and TRAIL) for promising glioblastoma treatment.

RA-Induced Transcriptional Silencing of Checkpoint Kinase-2 through Promoter Methylation by Dnmt3b Is Required for Neuronal Differentiation of P19 Cells.

In a previous study, we identified several novel targets of Dnmt3b using a chromatin library from retinoic acid (RA)-treated P19 cells. The present study describes the regulation of expression and function of checkpoint kinase (Chk2), which was one of the target genes of Dnmt3b. Chromatin immunoprecipitation followed by quantitative PCR analysis showed that recruitment of Dnmt3b on Chk2 promoter is induced following RA treatment of P19 cells. Both bisulfite genomic sequence and COBRA analyses showed that the methylation level of Chk2 promoter is progressively increased during RA-induced neuronal differentiation of P19 cells. Concomitantly, both mRNA and protein expression of Chk2 are reduced as determined by real-time PCR and Western blot analysis, respectively. Suppression of Dnmt3b expression by lentiviral-mediated shRNA resulted in increased expression and reduced methylation of Chk2, which clearly showed that Dnmt3b is responsible for transcriptional silencing of Chk2 gene in RA-treated P19 cells. Neuronal differentiation of P19 cells was inhibited upon enforced Chk2 expression in P19 cells, which showed that the decrease in endogenous expression of Chk2 is essential for normal differentiation. Ectopic Chk2 expression also negatively regulated cell cycle arrest and apoptosis following RA treatment, which could also contribute to impaired neuronal differentiation. Together, this study described the regulation of Chk2 expression through promoter methylation and also presented a novel role of Chk2 during neuronal differentiation, which is independent of its previously known function in DNA damage response.

Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinson's Disease (PD).

Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by motor deficits which result from the progressive loss of dopaminergic neurons. Gene therapy using growth factors such as VEGF seems to be a viable approach for potential therapeutic treatment of PD. In this study, we utilized a novel non-viral gene carrier designated as PEI-PLL synthesized by our laboratory to deliver VEGF gene to study its effect by using both cell culture as well as animal models of PD. For cell culture experiments, we utilized 6-hydroxydopamine (6-OHDA) mediated cell death model of MN9D cells following transfection with either a control plasmid or VEGF expressing plasmid. As compared to control transfected cells, PEI-PLL mediated VEGF gene delivery to MN9D cells resulted in increased cell viability, increase in the number of Tyrosine hydroxylase (TH) positive cells and decreased apoptosis following 6-OHDA insult. Next, we studied the therapeutic potential of PEI-PLL mediated VEGF gene delivery in SNPc by using unilateral 6-OHDA Medial forebrain bundle (MFB) lesion model of PD in rats. VEGF administration prevented the loss of motor functions induced by 6-OHDA as determined by behavior analysis. Similarly, VEGF inhibited the 6-OHDA mediated loss of DA neurons in Substantia Nigra Pars Compacta (SNPc) as well as DA nerve fibers in striatum as determined by TH immunostaining. In addition, PEI-PLL mediated VEGF gene delivery also prevented apoptosis and microglial activation in PD rat models. Together, these results clearly demonstrated the beneficial effects of PEI-PLL mediated VEGF gene delivery on dopaminergic system in both cell culture and animal models of PD. STATEMENT OF SIGNIFICANCE: In this report, we exploited the potential of PEI-PLL to deliver VEGF gene for the potential therapeutic treatment of PD by using both cell culture and animal models of PD. To the best of our knowledge, this is the first report describing the use of novel polymeric gene carriers for the delivery of VEGF gene to DA neurons with improved transfection efficiency. Finally, the study will lead to a significant advancement in the field of non-viral PD gene therapy treatment.

Epigenetic regulation of Dpp6 expression by Dnmt3b and its novel role in the inhibition of RA induced neuronal differentiation of P19 cells.

DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells. Dnmt3b enriched chromatin library from RA treated P19 cells identified dipeptidyl peptidase 6 (Dpp6) gene as a novel target of Dnmt3b. Further, quantitative ChIP analysis showed that the amount of Dnmt3b recruited on Dpp6 promoter was equal in both RA treated as well as untreated p19 cells. Bisulfite genomic sequencing, COBRA, and methylation specific PCR analysis revealed that Dpp6 promoter was heavily methylated in both RA treated and untreated P19 cells. Dnmt3b was responsible for transcriptional silencing of Dpp6 gene as depletion of Dnmt3b resulted in increased mRNA and protein expression of Dpp6. Consequently, the average methylation of Dpp6 gene promoter was reduced to half in Dnmt3b knockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6 gene promoter and regulated its expression and methylation in P19 cells. RA induced neuronal differentiation was inhibited upon ectopic expression of Dpp6 in P19 cells. Taken together, the present study described epigenetic silencing of Dpp6 expression by DNA methylation and established that its ectopic expression can act as negative signal during RA induced neuronal differentiation of P19 cells.

Doxycycline can stimulate cytoprotection in neural stem cells with oxygen-glucose deprivation-reoxygenation injury: a potential approach to enhance effectiveness of cell transplantation therapy.

A substantial loss of transplanted neural stem cells is a major limitation to cell transplantation therapy of stroke. In this study, we provided in vitro evidence that doxycycline preconditioning of neural stem cells have resulted in decreased cell death and increased cell viability after oxygen-glucose deprivation-reoxygenation conditions that best mimics cerebral ischemia-reperfusion injury. Resistance to oxidative stress is one of the mechanisms of doxycycline-induced cytoprotection in neural stem cells as it significantly reduced the superoxide anion production. Moreover, doxycycline preconditioning also induced the expression of Nrf2 which is a basic transcription factor for a series of antioxidative and cytoprotective genes. Collectively, we suggested that doxycycline preconditioning of neural stem cells is a potential strategy to improve effectiveness of cell transplantation therapy.

PtdIns (3,4,5) P3 recruitment of Myo10 is essential for axon development.

Myosin X (Myo10) with pleckstrin homology (PH) domains is a motor protein acting in filopodium initiation and extension. However, its potential role has not been fully understood, especially in neuronal development. In the present study the preferential accumulation of Myo10 in axon tips has been revealed in primary culture of hippocampal neurons with the aid of immunofluorescence from anti-Myo10 antibody in combination with anti-Tuj1 antibody as specific marker. Knocking down Myo10 gene transcription impaired outgrowth of axon with loss of Tau-1-positive phenotype. Interestingly, inhibition of actin polymerization by cytochalasin D rescued the defect of axon outgrowth. Furthermore, ectopic expression of Myo10 with enhanced green fluorescence protein (EGFP) labeled Myo10 mutants induced multiple axon-like neurites in a motor-independent way. Mechanism studies demonstrated that the recruitment of Myo10 through its PH domain to phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) was essential for axon formation. In addition, in vivo studies confirmed that Myo10 was required for neuronal morphological transition during radial neuronal migration in the developmental neocortex.

Production of surfactin from Bacillus subtilis MZ-7 grown on pharmamedia commercial medium.

BACKGROUND: Commercial medium (Pharmamedia) was investigated for the production of surfactin by Bacillus subtilis MZ-7. Different media (defined, semi-defined, and complex media) were compared for the production of surfactin after fixing the least influential variables in standardized fermentation conditions. Carbohydrate and nitrogen supplements were also tried to improve production in Pharmamedia. RESULTS: Surfactin production was confirmed using PCR along with other analytical techniques and monitored by RP-HPLC and MALDI-TOF-MS. We found that optimized and brain heart infusion media were best for production of surfactin (280 mg/L) and a relatively comparable production with Pharmamedia (220 mg/L), however, supplementing Pharmamedia with Fe+ (4.0 mM) and sucrose (2 g/L) leads to a maximum production of about (300 mg/L). CONCLUSION: Cottonseed-derived medium proved to be a suitable substrate for the production of bioactive substances including surfactin, a useful compound in both medical and biotechnological fields. The medium provided not only higher product accumulations but at considerably lower cost with potential for large scale industrial applications.