Multiomic analysis of oral keratinocytes chronically exposed to shisha

Background: Tobacco is smoked in different form including cigarettes and water pipes. One popular form of water pipe smoking especially in Middle Eastern countries is shisha smoking. Shisha has been associated with various diseases including oral cancer. However, genomic alterations and gene expression changes associated with chronic shisha exposure have not been previously investigated. Objectives: Whole‐exome sequencing and gene expression profiling of immortalized human oral keratinocytes (OKF6/TERT1) cells chronically treated with 0.5% shisha extract for a period of 8 months was undertaken to characterize molecular alterations associated with shisha exposure. Methods: Genomic DNA and RNA were extracted and preprocessed as per manufacturer's instruction and subjected to whole‐exome and transcriptome sequencing using Illumina HiSeq2500 platform. Exome was analyzed using GATK pipeline whereas RNA‐Seq data was analyzed using HiSat2 and HTSeq along with DESeq to elucidate differentially expressed genes. Results: Whole‐exome sequence analysis led to identification of 521 somatic missense variants corresponding to 389 genes RNA‐Seq data revealed 247 differentially expressed genes (≥2‐fold, P‐value<0.01) in shisha treated cells compared to parental cells. Pathway analysis of differentially expressed genes revealed that interferon‐signaling pathway was significantly affected. We predict activation of MAPK1 pathway which is known to play a key role in oral cancer. We also observed allele specific expression of mutant LIMA1 based on RNA‐Seq dataset. Conclusion: Our findings provide insights into genomic alterations and gene expression pattern associated with oral keratinocytes chronically exposed to shisha

A biochemical and ultrastructural evaluation of the type 2 Gaucher mouse

Gaucher mice, created by targeted disruption of the glucocerebrosidase gene, are totally deficient in glucocerebrosidase and have a rapidly deteriorating clinical course analogous to the most severely affected type 2 human patients. An ultrastructural study of tissues from these mice revealed glucocerebroside accumulation in bone marrow, liver, spleen, and brain. This glycolipid had a characteristic elongated tubular structure and was contained in lysosomes, as demonstrated by colocalization with both ingested carbon particles and cathepsin D. In the central nervous system (CNS), glucocerebroside was diffusely stored in microglia cells and in brainstem and spinal cord neurons, but not in neurons of the cerebellum or cerebral cortex. This rostralcaudal pattern of neuronal lipid storage in these Gaucher mice replicates the pattern seen in type 2 human Gaucher patients and clearly demonstrates that glycosphingolipid catabolism and/or accumulation varies within different brain regions. Surprisingly, the cellular pathology of tissue from these Gaucher mice was relatively mild, and suggests that the early and rapid demise of both Gaucher mice and severely affected type 2 human neonates may be the result of both a neurotoxic metabolite, such as glucosylsphingosine, and other factors, such as skin water barrier dysfunction secondary to the absence of glucocerebrosidase activity

Endoplasmic reticulum and lysosomal Ca2+ stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts.

Mutations in β-glucocerebrosidase (encoded by GBA1) cause Gaucher disease (GD), a lysosomal storage disorder, and increase the risk of developing Parkinson disease (PD). The pathogenetic relationship between the two disorders is unclear. Here, we characterised Ca2+ release in fibroblasts from type I GD and PD patients together with age-matched, asymptomatic carriers, all with the common N370S mutation in β-glucocerebrosidase. We show that endoplasmic reticulum (ER) Ca2+ release was potentiated in GD and PD patient fibroblasts but not in cells from asymptomatic carriers. ER Ca2+ signalling was also potentiated in fibroblasts from aged healthy subjects relative to younger individuals but not further increased in aged PD patient cells. Chemical or molecular inhibition of β-glucocerebrosidase in fibroblasts and a neuronal cell line did not affect ER Ca2+ signalling suggesting defects are independent of enzymatic activity loss. Conversely, lysosomal Ca2+ store content was reduced in PD fibroblasts and associated with age-dependent alterations in lysosomal morphology. Accelerated remodelling of Ca2+ stores by pathogenic GBA1 mutations may therefore feature in PD

Dietary soy and meat proteins induce distinct physiological and gene expression changes in rats

This study reports on a comprehensive comparison of the effects of soy and meat proteins given at the recommended level on physiological markers of metabolic syndrome and the hepatic transcriptome. Male rats were fed semi-synthetic diets for 1 wk that differed only regarding protein source, with casein serving as reference. Body weight gain and adipose tissue mass were significantly reduced by soy but not meat proteins. The insulin resistance index was improved by soy, and to a lesser extent by meat proteins. Liver triacylglycerol contents were reduced by both protein sources, which coincided with increased plasma triacylglycerol concentrations. Both soy and meat proteins changed plasma amino acid patterns. The expression of 1571 and 1369 genes were altered by soy and meat proteins respectively. Functional classification revealed that lipid, energy and amino acid metabolic pathways, as well as insulin signaling pathways were regulated differently by soy and meat proteins. Several transcriptional regulators, including NFE2L2, ATF4, Srebf1 and Rictor were identified as potential key upstream regulators. These results suggest that soy and meat proteins induce distinct physiological and gene expression responses in rats and provide novel evidence and suggestions for the health effects of different protein sources in human diets

High Throughput Screening for Small Molecule Therapy for Gaucher Disease Using Patient Tissue as the Source of Mutant Glucocerebrosidase

Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development

Neuronopathic Gaucher disease in the mouse: viable combined selective saposin C deficiency and mutant glucocerebrosidase (V394L) mice with glucosylsphingosine and glucosylceramide accumulation and progressive neurological deficits

Gaucher disease is caused by defective acid β-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C−/−) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities ∼30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred ∼48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C−/− alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease

Visual short-term memory deficits associated with GBA mutation and Parkinson's disease.

Individuals with mutation in the lysosomal enzyme glucocerebrosidase (GBA) gene are at significantly high risk of developing Parkinson's disease with cognitive deficit. We examined whether visual short-term memory impairments, long associated with patients with Parkinson's disease, are also present in GBA-positive individuals-both with and without Parkinson's disease. Precision of visual working memory was measured using a serial order task in which participants observed four bars, each of a different colour and orientation, presented sequentially at screen centre. Afterwards, they were asked to adjust a coloured probe bar's orientation to match the orientation of the bar of the same colour in the sequence. An additional attentional 'filtering' condition tested patients' ability to selectively encode one of the four bars while ignoring the others. A sensorimotor task using the same stimuli controlled for perceptual and motor factors. There was a significant deficit in memory precision in GBA-positive individuals-with or without Parkinson's disease-as well as GBA-negative patients with Parkinson's disease, compared to healthy controls. Worst recall was observed in GBA-positive cases with Parkinson's disease. Although all groups were impaired in visual short-term memory, there was a double dissociation between sources of error associated with GBA mutation and Parkinson's disease. The deficit observed in GBA-positive individuals, regardless of whether they had Parkinson's disease, was explained by a systematic increase in interference from features of other items in memory: misbinding errors. In contrast, impairments in patients with Parkinson's disease, regardless of GBA status, was explained by increased random responses. Individuals who were GBA-positive and also had Parkinson's disease suffered from both types of error, demonstrating the worst performance. These findings provide evidence for dissociable signature deficits within the domain of visual short-term memory associated with GBA mutation and with Parkinson's disease. Identification of the specific pattern of cognitive impairment in GBA mutation versus Parkinson's disease is potentially important as it might help to identify individuals at risk of developing Parkinson's disease

Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype

<p>Abstract</p> <p>Background</p> <p>A finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level.</p> <p>Results</p> <p>We propose a new finite state machine able to detect and correct aberrant molecular phenotype given by mutated genetic transcripts. The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced. We experimentally demostrated in <it>in vitro </it>translation system that a viable protein can be autonomously assembled.</p> <p>Conclusion</p> <p>Our work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.</p