scholarly journals AUY922 induces retinal toxicity through attenuating TRPM1

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Che-Hung Shen ◽  
Chi-Che Hsieh ◽  
Kuan-Ying Jiang ◽  
Chih-Yu Lin ◽  
Nai-Jung Chiang ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Enrichment Analysis ◽  
Retinal Toxicity ◽  
Ros Production ◽  
Proteomic Profiling ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Hsp90 Chaperone ◽  
Chaperone Complex ◽  
Regulated Gene Expression

Abstract Background Ocular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed. Methods The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)‑based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches. Results Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922. Conclusions Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

scholarly journals Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Nathan L Absalom ◽  
Vivian W Y Liao ◽  
Kavitha Kothur ◽  
Dinesh C Indurthi ◽  
Bruce Bennetts ◽  
...  
Keyword(s):  
De Novo ◽  
Drug Effects ◽  
Receptor Expression ◽  
Aminobutyric Acid ◽  
Loss Of Function ◽  
Molecular Phenotype ◽  
Gain Of Function ◽  
Gene Encoding ◽  
Epileptic Encephalopathies ◽  
Receptor Phenotype

Abstract Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

scholarly journals LncRNA PCAT1 Interacts with DKC1 to Regulate Proliferation, Invasion and Apoptosis in NSCLC Cells via the VEGF/AKT/Bcl2/Caspase9 Pathway

2021 ◽  
Vol 30 ◽  
pp. 096368972098607
Author(s):  
Shi-Yuan Liu ◽  
Zhi-Yu Zhao ◽  
Zhe Qiao ◽  
Shao-Min Li ◽  
Wei-Ning Zhang
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Growth Factor ◽  
Cell Apoptosis ◽  
Rna Binding ◽  
A549 Cells ◽  
Nonsmall Cell Lung Cancer ◽  
Nsclc Cell ◽  
Loss Of Function ◽  
Proliferation And Invasion

Long noncoding RNAs (lncRNAs) are increasingly recognized as indispensable components of the regulatory network in the progression of various cancers, including nonsmall cell lung cancer (NSCLC). The lncRNA prostate cancer associated transcript 1 (PCAT1) has been involved in tumorigenesis of multiple malignant solid tumors, but it is largely unknown that what is the role of lncRNA-PCAT1 and how it functions in the progression of lung cancer. Herein, we observed that lncRNA PCAT1 expression was upregulated in both human NSCLC tissues and cell lines, which was determined by qualitative polymerase chain reaction analysis. Then, gain-and loss-of-function manipulations were performed in A549 cells by transfection with a specific short interfering RNA against PCAT1 or a pcDNA-PCAT1 expression vector. The results showed that PCAT1 not only promoted NSCLC cell proliferation and invasion but also inhibited cell apoptosis. Bioinformatics and expression correlation analyses revealed that there was a potential interaction between PCAT1 and the dyskerin pseudouridine synthase 1 (DKC1) protein, an RNA-binding protein. Then, RNA pull-down assays with biotinylated probes and transcripts both confirmed that PCAT1 directly bounds with DKC1 that could also promote NSCLC cell proliferation and invasion and inhibit cell apoptosis. Moreover, the effects of PCAT1 and DKC1 on NSCLC functions are synergistic. Furthermore, PCAT1 and DKC1 activated the vascular endothelial growth factor (VEGF)/protein kinase B (AKT)/Bcl-2/caspase9 pathway in NSCLC cells, and inhibition of epidermal growth factor receptor, AKT, or Bcl-2 could eliminate the effect of PCAT1/DKC1 co-overexpression on NSCLC cell behaviors. In conclusion, lncRNA PCAT1 interacts with DKC1 to regulate proliferation, invasion, and apoptosis in NSCLC cells via the VEGF/AKT/Bcl-2/caspase9 pathway.

scholarly journals Emerging Role of ODC1 in Neurodevelopmental Disorders and Brain Development

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 470
Author(s):  
Jeremy W. Prokop ◽  
Caleb P. Bupp ◽  
Austin Frisch ◽  
Stephanie M. Bilinovich ◽  
Daniel B. Campbell ◽  
...  
Keyword(s):  
Brain Development ◽  
Neural Progenitor Cells ◽  
Neurodevelopmental Disorder ◽  
Fetal Brain ◽  
Missense Variant ◽  
Neural Progenitor ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Systematic Analysis ◽  
Function Expression

Ornithine decarboxylase 1 (ODC1 gene) has been linked through gain-of-function variants to a rare disease featuring developmental delay, alopecia, macrocephaly, and structural brain anomalies. ODC1 has been linked to additional diseases like cancer, with growing evidence for neurological contributions to schizophrenia, mood disorders, anxiety, epilepsy, learning, and suicidal behavior. The evidence of ODC1 connection to neural disorders highlights the need for a systematic analysis of ODC1 genotype-to-phenotype associations. An analysis of variants from ClinVar, Geno2MP, TOPMed, gnomAD, and COSMIC revealed an intellectual disability and seizure connected loss-of-function variant, ODC G84R (rs138359527, NC_000002.12:g.10444500C > T). The missense variant is found in ~1% of South Asian individuals and results in 2.5-fold decrease in enzyme function. Expression quantitative trait loci (eQTLs) reveal multiple functionally annotated, non-coding variants regulating ODC1 that associate with psychiatric/neurological phenotypes. Further dissection of RNA-Seq during fetal brain development and within cerebral organoids showed an association of ODC1 expression with cell proliferation of neural progenitor cells, suggesting gain-of-function variants with neural over-proliferation and loss-of-function variants with neural depletion. The linkage from the expression data of ODC1 in early neural progenitor proliferation to phenotypes of neurodevelopmental delay and to the connection of polyamine metabolites in brain function establish ODC1 as a bona fide neurodevelopmental disorder gene.

scholarly journals Somatic production of reactive oxygen species does not predict its production in sperm cells across Drosophila melanogaster lines

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Biz R. Turnell ◽  
Luisa Kumpitsch ◽  
Anne-Cécile Ribou ◽  
Klaus Reinhardt
Keyword(s):  
Reactive Oxygen Species ◽  
Drosophila Melanogaster ◽  
Reactive Oxygen ◽  
Future Research ◽  
Ros Production ◽  
Oxygen Species ◽  
Loss Of Function ◽  
Wild Type ◽  
Ros Scavenging ◽  
Transport Chain

Abstract Objective Sperm ageing has major evolutionary implications but has received comparatively little attention. Ageing in sperm and other cells is driven largely by oxidative damage from reactive oxygen species (ROS) generated by the mitochondria. Rates of organismal ageing differ across species and are theorized to be linked to somatic ROS levels. However, it is unknown whether sperm ageing rates are correlated with organismal ageing rates. Here, we investigate this question by comparing sperm ROS production in four lines of Drosophila melanogaster that have previously been shown to differ in somatic mitochondrial ROS production, including two commonly used wild-type lines and two lines with genetic modifications standardly used in ageing research. Results Somatic ROS production was previously shown to be lower in wild-type Oregon-R than in wild-type Dahomey flies; decreased by the expression of alternative oxidase (AOX), a protein that shortens the electron transport chain; and increased by a loss-of-function mutation in dj-1β, a gene involved in ROS scavenging. Contrary to predictions, we found no differences among these four lines in the rate of sperm ROS production. We discuss the implications of our results, the limitations of our study, and possible directions for future research.

scholarly journals Identification and Characterization of Genes That Interact With lin-12 in Caenorhabditis elegans

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1675-1695 ◽  
Author(s):  
Frans E Tax ◽  
James H Thomas ◽  
Edwin L Ferguson ◽  
H Robert Horvitzt
Keyword(s):  
Cell Fate ◽  
Low Frequency ◽  
Signal Transduction Pathway ◽  
Temperature Shift ◽  
Egg Laying ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Suppressor Mutations

Abstract We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-l7, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup1 7 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup1 7and lag-2, suggest that both genes act at approximately the same time as lin-12in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.

scholarly journals Rare Gain-of-Function KCND3 Variant Associated with Cerebellar Ataxia, Parkinsonism, Cognitive Dysfunction, and Brain Iron Accumulation

2021 ◽  
Vol 22 (15) ◽  
pp. 8247
Author(s):  
Cheng-Tsung Hsiao ◽  
Thomas F. Tropea ◽  
Ssu-Ju Fu ◽  
Tanya M. Bardakjian ◽  
Pedro Gonzalez-Alegre ◽  
...  
Keyword(s):  
Cerebellar Ataxia ◽  
Iron Accumulation ◽  
Molecular Spectra ◽  
Loss Of Function ◽  
Brain Iron ◽  
Gain Of Function ◽  
Progressive Cerebellar Ataxia ◽  
Voltage Dependent ◽  
Window Current

Loss-of-function mutations in the KV4.3 channel-encoding KCND3 gene are linked to neurodegenerative cerebellar ataxia. Patients suffering from neurodegeneration associated with iron deposition may also present with cerebellar ataxia. The mechanism underlying brain iron accumulation remains unclear. Here, we aim to ascertain the potential pathogenic role of KCND3 variant in iron accumulation-related cerebellar ataxia. We presented a patient with slowly progressive cerebellar ataxia, parkinsonism, cognitive impairment, and iron accumulation in the basal ganglia and the cerebellum. Whole exome sequencing analyses identified in the patient a heterozygous KCND3 c.1256G>A (p.R419H) variant predicted to be disease-causing by multiple bioinformatic analyses. In vitro biochemical and immunofluorescence examinations revealed that, compared to the human KV4.3 wild-type channel, the p.R419H variant exhibited normal protein abundance and subcellular localization pattern. Electrophysiological investigation, however, demonstrated that the KV4.3 p.R419H variant was associated with a dominant increase in potassium current amplitudes, as well as notable changes in voltage-dependent gating properties leading to enhanced potassium window current. These observations indicate that, in direct contrast with the loss-of-function KCND3 mutations previously reported in cerebellar ataxia patients, we identified a rare gain-of-function KCND3 variant that may expand the clinical and molecular spectra of neurodegenerative cerebellar disorders associated with brain iron accumulation.

scholarly journals Propofol attenuates lung ischemia/reperfusion injury though the involvement of the MALAT1/microRNA-144/GSK3β axis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Jian-Ping Zhang ◽  
Wei-Jing Zhang ◽  
Miao Yang ◽  
Hua Fang
Keyword(s):  
Cell Apoptosis ◽  
Ischemia Reperfusion Injury ◽  
Glycogen Synthase ◽  
Ischemia Reperfusion ◽  
Inflammatory Factors ◽  
Therapeutic Effects ◽  
Protective Effects ◽  
Loss Of Function

Abstract Background Propofol, an intravenous anesthetic, was proven to protect against lung ischemia/reperfusion (I/R) injury. However, the detailed mechanism of Propofol in lung I/R injury is still elusive. This study was designed to explore the therapeutic effects of Propofol, both in vivo and in vitro, on lung I/R injury and the underlying mechanisms related to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-144 (miR-144)/glycogen synthase kinase-3β (GSK3β). Methods C57BL/6 mice were used to establish a lung I/R injury model while pulmonary microvascular endothelial cells (PMVECs) were constructed as hypoxia/reperfusion (H/R) cellular model, both of which were performed with Propofol treatment. Gain- or loss-of-function approaches were subsequently employed, followed by observation of cell apoptosis in lung tissues and evaluation of proliferative and apoptotic capabilities in H/R cells. Meanwhile, the inflammatory factors, autophagosomes, and autophagy-related proteins were measured. Results Our experimental data revealed that Propofol treatment could decrease the elevated expression of MALAT1 following I/R injury or H/R induction, indicating its protection against lung I/R injury. Additionally, overexpressing MALAT1 or GSK3β promoted the activation of autophagosomes, proinflammatory factor release, and cell apoptosis, suggesting that overexpressing MALAT1 or GSK3β may reverse the protective effects of Propofol against lung I/R injury. MALAT1 was identified to negatively regulate miR-144 to upregulate the GSK3β expression. Conclusion Overall, our study demonstrated that Propofol played a protective role in lung I/R injury by suppressing autophagy and decreasing release of inflammatory factors, with the possible involvement of the MALAT1/miR-144/GSK3β axis.

Implementation and clinical outcomes of pharmacogenetic CYP2C19 testing for clopidogrel therapy in real-world clinical practice.

2021 ◽  
Vol 28 (Supplement_1) ◽  
Author(s):  
POD O"drisceoil ◽  
TK Kiernan ◽  
SA Arnous
Keyword(s):  
Real World ◽  
Point Of Care ◽  
Coronary Intervention ◽  
Buccal Swab ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Funding Sources ◽  
Patients At Risk ◽  
Clopidogrel Therapy ◽  
Stable Cad

Abstract Funding Acknowledgements Type of funding sources: None. BACKGROUNDCYP2C19 loss-of-function (LOF) polymorphisms are associated with adverse ischaemic events after PCI. The use of a point-of-care assay (POC) to routinely genotype patients immediately post PCI could rapidly identify patients at risk of adverse cardiac outcomes. PURPOSE To investigate the incidence of CYP2C19 polymorphisms (*2, *17) and 30-day MACE in patients presenting to catheter laboratory for PCI (See table 1).METHODS We performed a single centre prospective analysis of patients presenting to a cardiac catheterisation laboratory for percutaneous coronary intervention. Participants underwent prospective rapid point-of-care genotyping of CYP2C19 major alleles (2*,17*), using the SpartanRx PCR device via buccal swab sample. All patients provided written consent. RESULTS:A total of 120 tests were performed, 51 patients were normal allele carriers (*1), 31 patients were carriers of LOF alleles (*2) and 38 patients were carriers of gain of function alleles (*17). All tests results returned in one hour. Rate of dyslipidaemia was significantly different between three groups (55% vs. 63% vs. 36%; p = 0.050). A numerically higher proportion of LOF allele carriers received clopidogrel prior to undergoing pharmacogenetic testing but this was not statistically significant (52% vs. 35% vs. 34%; p = 0.09). Two cases of MACE at 30 day follow up occurred in the loss-of-function group. Both cases received clopidogrel.CONCLUSIONSWe have demonstrated that a rapid POC of CYP2C19 testing can take place in a real-world setting. Our incidence rate of LOF carriers is concordant with international published literature. We found 52% of LOF carriers were commenced on clopidogrel therapy prior to genetic analysis. Comparison of CPY2C19 Metabolisers genotype Loss of function normal Gain of function p values baseline characteristics age in years, median (range) 65 (43-82) 64 (43-85) 65 (42-89) 0.717 Male, N (%) 21 (68%) 43 (64%) 27 (71%) 0.198 Hypertensive, N (%) 16 (52%) 29 (57%) 24 (50%) 0.623 Dyslipidaemia. N (%) 17 (55%) 32 (63%) 14 (36%) 0.050 Indication, N (%) St-Elevation MI 12 (39%) 18 (35%) 11 (29%) 0.558 NSTEMI 5 (16%) 15 (29%) 14 (37%) 0.142 Unstable Angina 5 (16%) 7 (14%) 3 (8%) 0.518 Stable CAD 9 (29%) 11 (22%) 10 (26%) 0.731 Antiplatelet, N (%) Ticagrelor 15 (48%) 33 (65%) 25 (66%) 0.09 Clopidogrel 16 (52%) 18 (35%) 13 (34%) Complication, N (%) 30-day MACE 2 (6.5%) 0 0 0.01

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