Effectiveness of play therapy on level of anxiety among children undergoing nebulization in selected hospitals, Salem

A study was done to evaluate the effectiveness of play therapy on level of anxiety among children undergoing nebulization in selected hospitals, Salem. Quasi experimental post test only control group design was adopted where purposive sampling technique was used to select the 30 children between 1-5 years of age from Sri Gokulam hospital (experimental group) and SKS hospital (control group), Salem. Play therapy was provided to the experimental group before and during the nebulization and in the control group no distraction was implemented. Post test level of anxiety was assessed in both groups with the help of Modified Spence Children Anxiety Scale. Data was collected from 29-07-2013 to 27-08-2013. Data was analyzed by using descriptive and inferential statistics. During post test on experimental group, 11(36.7%) children had no anxiety and 19(63.3%) had mild anxiety whereas in control group 25(83.3%) children had moderate anxiety and 5(16.7%) had severe anxiety. In experimental group, the post test mean percentage was 27.5% and in control group the mean percentage was 58.4% revealing a difference of 30.9%. Significant difference was found in the post test mean values of level of anxiety (t=16.5) at p<0.001 in experimental group. In experimental group there was no association of anxiety with the demographic variables whereas in control group there was an association of anxiety with education of care giver and previous exposure to hospital at p < 0.05 level. This study implies that play therapy was an effective intervention in reducing the level of anxiety among children undergoing nebulization therapy

Molecular Dynamics Simulation Studies of GLUT4: Substrate-Free and Substrate-Induced Dynamics and ATP-Mediated Glucose Transport Inhibition

BACKGROUND: Glucose transporter 4 (GLUT4) is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states). Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport. CONCLUSIONS/SIGNIFICANCE: In the absence of a crystal structure for any glucose transporter, this study provides mechanistic details of the conformational changes in GLUT4 induced by substrate and its regulator

O-GlcNAc transferase maintains metabolic homeostasis in response to CDK9 inhibition

Co-targeting of O-GlcNAc transferase (OGT) and the transcriptional kinase cyclin-dependent kinase 9 (CDK9) is toxic to prostate cancer cells. As OGT is an essential glycosyltransferase, identifying an alternative target showing similar effects is of great interest. Here, we used a multiomics approach (transcriptomics, metabolomics, and proteomics) to better understand the mechanistic basis of the combinatorial lethality between OGT and CDK9 inhibition. CDK9 inhibition preferentially affected transcription. In contrast, depletion of OGT activity predominantly remodeled the metabolome. Using an unbiased systems biology approach (weighted gene correlation network analysis), we discovered that CDK9 inhibition alters mitochondrial activity/flux, and high OGT activity is essential to maintain mitochondrial respiration when CDK9 activity is depleted. Our metabolite profiling data revealed that pantothenic acid (vitamin B5) is the metabolite that is most robustly induced by both OGT and OGT+CDK9 inhibitor treatments but not by CDK9 inhibition alone. Finally, supplementing prostate cancer cell lines with vitamin B5 in the presence of CDK9 inhibitor mimics the effects of co-targeting OGT and CDK9.Peer reviewe

CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation

Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymerase II, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism

Inhibition of CDK9 activity compromises global splicing in prostate cancer cells

Cyclin-dependent kinase 9 (CDK9) phosphorylates RNA polymerase II to promote productive transcription elongation. Here we show that short-term CDK9 inhibition affects the splicing of thousands of mRNAs. CDK9 inhibition impairs global splicing and there is no evidence for a coordinated response between the alternative splicing and the overall transcriptome. Alternative splicing is a feature of aggressive prostate cancer (CRPC) and enables the generation of the anti-androgen resistant version of the ligand-independent androgen receptor, AR-v7. We show that CDK9 inhibition results in the loss of AR and AR-v7 expression due to the defects in splicing, which sensitizes CRPC cells to androgen deprivation. Finally, we demonstrate that CDK9 expression increases as PC cells develop CRPC-phenotype both in vitro and also in patient samples. To conclude, here we show that CDK9 inhibition compromises splicing in PC cells, which can be capitalized on by targeting the PC-specific addiction androgen receptor.Peer reviewe

The impact of transcription on metabolism in prostate and breast cancers

Metabolic dysregulation is regarded as an important driver in cancer development and progression. The impact of transcriptional changes on metabolism has been intensively studied in hormone-dependent cancers, and in particular, in prostate and breast cancer. These cancers have strong similarities in the function of important transcriptional drivers, such as the oestrogen and androgen receptors, at the level of dietary risk and epidemiology, genetics and therapeutically. In this review, we will focus on the function of these nuclear hormone receptors and their downstream impact on metabolism, with a particular focus on lipid metabolism. We go on to discuss how lipid metabolism remains dysregulated as the cancers progress. We conclude by discussing the opportunities that this presents for drug repurposing, imaging and the development and testing of new therapeutics and treatment combinations

VPRBP functions downstream of the androgen receptor and OGT to restrict p53 activation in prostate cancer

Androgen receptor (AR) is a major driver of prostate cancer initiation and progression. O-GlcNAc transferase (OGT), the enzyme that catalyzes the covalent addition of UDP-N-acetylglucosamine (UDP-GlcNAc) to serine and threonine residues of proteins, is often highly expressed in prostate cancer with its expression correlated with high Gleason score. In this study, we have identified an AR and OGT coregulated factor, Vpr (HIV-1) binding protein (VPRBP) also known as DDB1 and CUL4 Associated Factor 1 (DCAF1). We show that VPRBP is regulated by the AR at the transcript level, and stabilized by OGT at the protein level. VPRBP knockdown in prostate cancer cells led to a significant decrease in cell proliferation, p53 stabilization, nucleolar fragmentation, and increased p53 recruitment to the chromatin. In human prostate tumor samples, VPRBP protein overexpression correlated with AR amplification, OGT overexpression, a shorter time to postoperative biochemical progression and poor clinical outcome. In clinical transcriptomic data, VPRBP expression was positively correlated with the AR and also with AR activity gene signatures. IMPLICATIONS: In conclusion, we have shown that VPRBP/DCAF1 promotes prostate cancer cell proliferation by restraining p53 activation under the influence of the AR and OGT

Inhibition of O-GlcNAc transferase renders prostate cancer cells dependent on CDK9

O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed a specific and potent inhibitor targeting this enzyme, and here, we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from patients with metastatic prostate cancer, but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells, but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer-specific vulnerabilities