Publications by authors named "Harsha Gowda"

146 Publications

Rapid exome sequencing: revolutionises the management of acutely unwell neonates.

Eur J Pediatr 2021 Jun 18. Epub 2021 Jun 18.

Birmingham Heartlands Hospital, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK.

Diagnosing acutely unwell infants with a potential genetic diagnosis can be challenging for healthcare professionals. Evidence suggests that up to 13% of critically unwell infants on the neonatal intensive care unit (NICU) have an underlying molecular diagnosis and when identified directly affects treatment decisions in 83%. On 1st October 2019, the National Health Service England (NHSE) launched a nationally commissioned service so that rapid whole-exome sequencing can be offered to critically unwell babies and children with a likely monogenic disorder who are admitted to NICU and paediatric intensive care unit (PICU). We present 7 cases from two neonatal units in the West Midlands (UK), where rapid exome sequencing has revealed a genetic diagnosis. Early genetic diagnosis in this cohort has influenced management in all (100%) cases, and in 57% (4 in 7 cases), it has helped in the decision to reorientate care. In some cases, early diagnosis has reduced the need for invasive and unnecessary investigations and avoided the need for post-mortem investigations. The genetic diagnosis has helped in counselling the families regarding the recurrence risk for future pregnancies. In some cases, this has provided parents with the reassurance of a low recurrence. In others, it has resulted in the offer of prenatal diagnosis or assisted conception technologies. What is Known: • Rapid whole-exome sequencing was commissioned in the UK in October 2019. • It is available for critically unwell babies with a likely monogenic aetiology. What is New: • It helps management planning for rare genetic disorders and future pregnancies counselling. • It can reduce the need for invasive investigations and overall intensive care costs.
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http://dx.doi.org/10.1007/s00431-021-04115-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8212268PMC
June 2021

Whole-Exome Sequencing Analysis of Oral Squamous Cell Carcinoma Delineated by Tobacco Usage Habits.

Front Oncol 2021 31;11:660696. Epub 2021 May 31.

Institute of Bioinformatics, International Technology Park, Bangalore, India.

Oral squamous cell carcinoma (OSCC) is a common cancer of the oral cavity in India. Cigarette smoking and chewing tobacco are known risk factors associated with OSCC. However, genomic alterations in OSCC with varied tobacco consumption history are not well-characterized. In this study, we carried out whole-exome sequencing to characterize the mutational landscape of OSCC tumors from subjects with different tobacco consumption habits. We identified several frequently mutated genes, including , , , , , , and . and exhibited mutually exclusive mutation patterns. We identified recurrent amplifications in the 1q31, 7q35, 14q11, 22q11, and 22q13 regions and observed amplification of in 25% of samples with tobacco consumption history. We observed genomic alterations in several genes associated with PTK6 signaling. We observed alterations in clinically actionable targets including , , , , , and . We observed enrichment of signature 29 in 40% of OSCC samples from tobacco chewers. Signature 15 associated with defective DNA mismatch repair was enriched in 80% of OSCC samples. was mutated in 36% of samples and harbored truncating as well as missense variants. We observed copy number alterations in 67% of OSCC samples. Several genes associated with non-receptor tyrosine kinase signaling were affected in OSCC. These molecules can serve as potential candidates for therapeutic targeting in OSCC.
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http://dx.doi.org/10.3389/fonc.2021.660696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8200776PMC
May 2021

Molecular alterations in oral cancer between tobacco chewers and smokers using serum proteomics.

Cancer Biomark 2021 May 14. Epub 2021 May 14.

Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Karnataka, India.

Background: Tobacco exposure (through smoking or chewing) is one of the predominant risk factors associated with the development of oral squamous cell carcinoma (OSCC). Despite the growing number of patients diagnosed with OSCC, there are few circulating biomarkers for identifying individuals at a higher risk of developing the disease. Successful identification of candidate molecular markers for risk assessment could aid in the early detection of oral lesions and potentially used for community screening of high-risk populations.

Objective: Identification of differentially expressed proteins in the serum of oral cancer patients which can serve as biomarkers for the diagnosis of the onset of oral cancer among tobacco users.

Methods: We employed a tandem mass tag (TMT)-based quantitative proteomics approach to study alterations in the serum proteomes of OSCC patients based on their tobacco exposure habits (chewing and smoking) compared to healthy individuals with no history of using any form of tobacco or any symptoms of the disease.
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http://dx.doi.org/10.3233/CBM-203077DOI Listing
May 2021

Temporal Quantitative Proteomics Reveals Proteomic and Phosphoproteomic Alterations Associated with Adaptive Response to Hypoxia in Melanoma Cells.

Cancers (Basel) 2021 Apr 30;13(9). Epub 2021 Apr 30.

Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, QLD-4006, Australia.

Hypoxia is a common feature in various solid tumours, including melanoma. Cancer cells in hypoxic environments are resistant to both chemotherapy and radiation. Hypoxia is also associated with immune suppression. Identification of proteins and pathways that regulate cancer cell survival in hypoxic environments can reveal potential vulnerabilities that can be exploited to improve the efficacy of anticancer therapies. We carried out temporal proteomic and phosphoproteomic profiling in melanoma cell lines to identify hypoxia-induced protein expression and phosphorylation changes. By employing a TMT-based quantitative proteomics strategy, we report the identification and quantitation of >7000 proteins and >10,000 phosphosites in melanoma cell lines grown in hypoxia. Proteomics data show metabolic reprogramming as one of the prominent adaptive responses in hypoxia. We identify several novel hypoxia-mediated phosphorylation changes that have not been reported before. They reveal kinase signalling pathways that are potentially involved in modulating cellular response to hypoxia. In addition to known protein expression changes, we identify several novel proteomic alterations associated with adaptive response to hypoxia. We show that cancer cells require the ubiquitin-proteasome system to survive in both normoxia and hypoxia. Inhibition of proteasome activity affects cell survival and may provide a novel therapeutic avenue to target cancer cells in hypoxia. Our study can serve as a valuable resource to pursue novel candidates to target hypoxia in cancers and improve the efficacy of anticancer therapies.
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http://dx.doi.org/10.3390/cancers13092175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124723PMC
April 2021

Proteomic and phosphoproteomic profiling of shammah induced signaling in oral keratinocytes.

Sci Rep 2021 Apr 30;11(1):9397. Epub 2021 Apr 30.

Institute of Bioinformatics, International Technology Park, Bangalore, India.

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.
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http://dx.doi.org/10.1038/s41598-021-88345-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087671PMC
April 2021

Molecular Profiling Associated with Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2)-Mediated Carcinogenesis in Gastric Cancer.

J Proteome Res 2021 05 12;20(5):2687-2703. Epub 2021 Apr 12.

Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore 575018, India.

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death worldwide. We showed previously that calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2), a serine-threonine kinase, is highly expressed in gastric cancer and leads to progression. In the present study, we identified the molecular networks involved in CAMKK2-mediated progression of gastric adenocarcinoma. Treatment of gastric cancer cell lines with a CAMKK2 inhibitor, STO-609, resulted in decreased cell migration, invasion, and colony-forming ability and a G1/S-phase arrest. In addition, tandem mass tag (TMT)-based quantitative proteomic analysis resulted in the identification of 7609 proteins, of which 219 proteins were found to be overexpressed and 718 downregulated (1.5-fold). Our data identified several key downregulated proteins involved in cell division and cell proliferation, which included DNA replication licensing factors, replication factor C, origin recognition complex, replication protein A and GINS, and mesenchymal markers, upon CAMKK2 inhibition. Immunoblotting and immunofluorescence results showed concordance with our mass spectroscopy data. Taken together, our study supports CAMKK2 as a novel therapeutic target in gastric cancer.
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http://dx.doi.org/10.1021/acs.jproteome.1c00008DOI Listing
May 2021

Implementing less invasive surfactant administration on a neonatal unit.

Arch Dis Child Educ Pract Ed 2021 Apr 8. Epub 2021 Apr 8.

Neonatal Unit, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK.

There is increasing evidence reflected in both UK 2019 NICE and European guidelines suggesting that less invasive surfactant administration (LISA) reduces the need for mechanical ventilation and reduces the combined outcome of death or bronchopulmonary dysplasia, and is now the optimal method for surfactant delivery in spontaneously breathing babies. Despite this, uptake in England has been slow compared with Europe. This quality improvement project outlines the process of implementing LISA in a neonatal intensive care unit over a 2-year period, the barriers and challenges which were encountered, and how they were overcome.
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http://dx.doi.org/10.1136/archdischild-2020-320574DOI Listing
April 2021

Proteomic Alterations Associated with Oral Cancer Patients with Tobacco Using Habits.

OMICS 2021 Apr 31;25(4):255-268. Epub 2021 Mar 31.

Institute of Bioinformatics, International Technology Park, Bangalore, India.

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits.
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http://dx.doi.org/10.1089/omi.2021.0001DOI Listing
April 2021

Protocol for purification and identification of MHC class I immunopeptidome from cancer cell lines.

STAR Protoc 2021 Mar 18;2(1):100385. Epub 2021 Mar 18.

Cancer Precision Medicine Group, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.

Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides.
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http://dx.doi.org/10.1016/j.xpro.2021.100385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982746PMC
March 2021

An integrated approach for identification of a panel of candidate genes arbitrated for invasion and metastasis in oral squamous cell carcinoma.

Sci Rep 2021 Mar 18;11(1):6208. Epub 2021 Mar 18.

Institute of Life Sciences, Bhubaneswar, India.

Oral squamous cell carcinoma (OSCC) is known for its aggressiveness associated with poor prognosis. The molecular mechanisms underlying the invasion and metastasis are still poorly understood. An improved understanding of these mechanisms shall precede the development of new diagnostic tools and targeted therapies. We report an integrated approach using bioinformatics to predict candidate genes, coupled with proteomics and immunohistochemistry for validating their presence and involvement in OSCC pathways heralding invasion and metastasis. Four genes POSTN, TNC, CAV1 and FSCN1 were identified. A protein-protein interaction network analysis teamed with pathway analysis led us to propose the role of the identified genes in invasion and metastasis in OSCC. Further analyses of archived FFPE blocks of various grades of oral cancer was carried out using TMT-based mass spectrometry and immunohistochemistry. Results of this study expressed a strong communiqué and interrelationship between these candidate genes. This study emphasizes the significance of a molecular biomarker panel as a diagnostic tool and its correlation with the invasion and metastatic pathway of OSCC. An insight into the probable association of CAF's and these biomarkers in the evolution and malignant transformation of OSCC further magnifies the molecular-biological spectrum of OSCC tumour microenvironment.
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http://dx.doi.org/10.1038/s41598-021-85729-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973753PMC
March 2021

Molecular alterations in oral cancer using high-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue.

J Cell Commun Signal 2021 Sep 8;15(3):447-459. Epub 2021 Mar 8.

Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed To Be University), Mangalore, Karnataka, 575018, India.

Loss of cell differentiation is a hallmark for the progression of oral squamous cell carcinoma (OSCC). Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues constitute a valuable resource for studying the differentiation of OSCC and can offer valuable insights into the process of tumor progression. In the current study, we performed LC-MS/MS-based quantitative proteomics of FFPE specimens from pathologically-confirmed well-differentiated, moderately-differentiated, and poorly-differentiated OSCC cases. The data were analyzed in four technical replicates, resulting in the identification of 2376 proteins. Of these, 141 and 109 were differentially expressed in moderately-differentiated and poorly differentiated OSCC cases, respectively, compared to well-differentiated OSCC. The data revealed significant metabolic reprogramming with respect to lipid metabolism and glycolysis with proteins belonging to both these processes downregulated in moderately-differentiated OSCC when compared to well-differentiated OSCC. Signaling pathway analysis indicated the alteration of extracellular matrix organization, muscle contraction, and glucose metabolism pathways across tumor grades. The extracellular matrix organization pathway was upregulated in moderately-differentiated OSCC and downregulated in poorly differentiated OSCC, compared to well-differentiated OSCC. PADI4, an epigenetic enzyme transcriptional regulator, and its transcriptional target HIST1H1B were both found to be upregulated in moderately differentiated and poorly differentiated OSCC, indicating epigenetic events underlying tumor differentiation. In conclusion, the findings support the advantage of using high-resolution mass spectrometry-based FFPE archival blocks for clinical and translational research. The candidate signaling pathways identified in the study could be used to develop potential therapeutic targets for OSCC.
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http://dx.doi.org/10.1007/s12079-021-00609-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8222490PMC
September 2021

Proteomics-based approach for differentiation of age-related macular degeneration sub-types.

Indian J Ophthalmol 2021 03;69(3):647-654

L&T Opthalmic Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.

Purpose: Age-related macular degeneration (AMD) is one of the leading causes of irreversible central vision loss in the elderly population. The current study aims to find non-invasive prognostic biomarkers in the urine specimens of the AMD patients.

Methods: Peripheral blood and urine samples were collected from 23 controls and 61 AMD patients. Genomic DNA was extracted from the buffy coat of peripheral blood. Allele specific PCR was used to assay SNPs in complement factor H (CFH), complement component 3 (C3). Comparative proteomic analysis of urine samples from early AMD, choroidal neovascular membrane (CNVM), geographic atrophy (GA), and healthy controls was performed using isobaric labelling followed by mass spectrometry. Validation was performed using enzyme-linked immunosorbent assay (ELISA).

Results: Comparative proteomic analysis of urine samples identified 751 proteins, of which 383 proteins were found to be differentially expressed in various groups of AMD patients. Gene ontology classification of differentially expressed proteins revealed the majority of them were involved in catalytic functions and binding activities. Pathway analysis showed cell adhesion molecule pathways (CAMs), Complement and coagulation cascades, to be significantly deregulated in AMD. Upon validation by ELISA, SERPINA-1 (Alpha1 antitrypsin), TIMP-1 (Tissue inhibitor of matrix metaloprotease-1), APOA-1 (Apolipoprotein A-1) were significantly over-expressed in AMD (n = 61) patients compared to controls (n = 23). A logistic model of APOA-1 in combination with CFH and C3 polymorphisms predicted the risk of developing AMD with 82% accuracy.

Conclusion: This study gives us a preliminary data on non-invasive predictive biomarkers for AMD, which can be further validated in a large cohort and translated for diagnostic use.
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http://dx.doi.org/10.4103/ijo.IJO_470_20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7942106PMC
March 2021

Identification of novel dysregulated circular RNAs in early-stage breast cancer.

J Cell Mol Med 2021 Apr 5;25(8):3912-3921. Epub 2021 Feb 5.

Department of Molecular Oncology, Cancer Institute (WIA), Chennai, India.

Breast cancer is a major cause of cancer-related death in women worldwide. Non-coding RNAs are a potential resource to be used as an early diagnostic biomarker for breast cancer. Circular RNAs are a recently identified group of non-coding RNA with a significant role in disease development with potential utility in diagnosis/prognosis in cancer. In this study, we identified 26 differentially expressed circular RNAs associated with early-stage breast cancer. RNA sequencing and two circRNA detection tools (find_circ and DCC) were used to understand the circRNA expression signature in breast cancer. We identified hsa_circ_0006743 (circJMJD1C) and hsa_circ_0002496 (circAPPBP1) to be significantly up-regulated in early-stage breast cancer tissues. Co-expression analysis identified four pairs of circRNA-miRNA (hsa_circ_0023990 : hsa-miR-548b-3p, hsa_circ_0016601 : hsa_miR-1246, hsa_circ_0001946 : hsa-miR-1299 and hsa_circ_0000117:hsa-miR-502-5p) having potential interaction. The miRNA target prediction and network analysis revealed mRNA possibly regulated by circRNAs. We have thus identified circRNAs of diagnostic implications in breast cancer and also observed circRNA-miRNA interaction which could be involved in breast cancer development.
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http://dx.doi.org/10.1111/jcmm.16324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051735PMC
April 2021

Signaling alterations in oral keratinocytes in response to shisha and crude tobacco extract.

J Oral Pathol Med 2021 May 22;50(5):459-469. Epub 2021 Jan 22.

Institute of Bioinformatics, International Technology Park, Bangalore, India.

Background: Tobacco consumption in smoking and non-smoking forms has been consequential in the rise of oral cancer cases. Among different forms, epidemiological studies from Middle Eastern countries and rural parts of northern India have reported increasing association of oral cancer with waterpipe (hookah) smoking. However, molecular mechanisms and role played by waterpipe smoking in the onset of oral carcinogenesis remains unexplored.

Methods: In this study, immortalized normal human oral keratinocytes were chronically treated with extracts of two varieties of waterpipe tobacco-crude tobacco and processed shisha. Phenotypic changes and molecular aberrations were examined using cell culture-based assays and mass spectrometry-based quantitative proteomic analysis, respectively. Bioinformatics analysis was utilized to analyze proteomics data and identify dysregulated pathways.

Results: Our data indicate that chronic treatment with waterpipe tobacco extracts increased proliferation, invasion, migration, and significant dysregulation of protein expression in oral keratinocytes. Altered expression of proteins involved in interferon signaling pathway were observed with both varieties of tobacco. Overexpression of cholesterol metabolism and vesicle-mediated transport proteins were identified exclusively in cells treated with crude tobacco extract. Bioinformatics analyses revealed different oncogenic response in oral cells based on the type of waterpipe tobacco used.

Conclusions: This study may serve as a useful resource in understanding the early onset of oral cancer attributed to waterpipe smoking.
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http://dx.doi.org/10.1111/jop.13154DOI Listing
May 2021

CusVarDB: A tool for building customized sample-specific variant protein database from next-generation sequencing datasets.

F1000Res 2020 11;9:344. Epub 2020 May 11.

Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, 575018, India.

Cancer genome sequencing studies have revealed a number of variants in coding regions of several genes. Some of these coding variants play an important role in activating specific pathways that drive proliferation. Coding variants present on cancer cell surfaces by the major histocompatibility complex serve as neo-antigens and result in immune activation. The success of immune therapy in patients is attributed to neo-antigen load on cancer cell surfaces. However, which coding variants are expressed at the protein level can't be predicted based on genomic data. Complementing genomic data with proteomic data can potentially reveal coding variants that are expressed at the protein level. However, identification of variant peptides using mass spectrometry data is still a challenging task due to the lack of an appropriate tool that integrates genomic and proteomic data analysis pipelines. To overcome this problem, and for the ease of the biologists, we have developed a graphical user interface (GUI)-based tool called CusVarDB. We integrated variant calling pipeline to generate sample-specific variant protein database from next-generation sequencing datasets. We validated the tool with triple negative breast cancer cell line datasets and identified 423, 408, 386 and 361 variant peptides from BT474, MDMAB157, MFM223 and HCC38 datasets, respectively.
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http://dx.doi.org/10.12688/f1000research.23214.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684676PMC
March 2021

Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells.

Front Oncol 2020 5;10:1666. Epub 2020 Nov 5.

Institute of Bioinformatics, International Technology Park, Bangalore, India.

Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.
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http://dx.doi.org/10.3389/fonc.2020.01666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675040PMC
November 2020

Inhaled nitric oxide in managing preterm infants with suspected pulmonary hypoplasia.

Acta Paediatr 2021 03 24;110(3):1066-1067. Epub 2020 Nov 24.

University Hospitals of Birmingham: Heartlands, Birmingham, UK.

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http://dx.doi.org/10.1111/apa.15656DOI Listing
March 2021

High-quality nuclear genome for Sarcoptes scabiei-A critical resource for a neglected parasite.

PLoS Negl Trop Dis 2020 10 1;14(10):e0008720. Epub 2020 Oct 1.

Cell and Molecular Biology Department, Infectious Diseases Program, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

The parasitic mite Sarcoptes scabiei is an economically highly significant parasite of the skin of humans and animals worldwide. In humans, this mite causes a neglected tropical disease (NTD), called scabies. This disease results in major morbidity, disability, stigma and poverty globally and is often associated with secondary bacterial infections. Currently, anti-scabies treatments are not sufficiently effective, resistance to them is emerging and no vaccine is available. Here, we report the first high-quality genome and transcriptomic data for S. scabiei. The genome is 56.6 Mb in size, has a a repeat content of 10.6% and codes for 9,174 proteins. We explored key molecules involved in development, reproduction, host-parasite interactions, immunity and disease. The enhanced 'omic data sets for S. scabiei represent comprehensive and critical resources for genetic, functional genomic, metabolomic, phylogenetic, ecological and/or epidemiological investigations, and will underpin the design and development of new treatments, vaccines and/or diagnostic tests.
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http://dx.doi.org/10.1371/journal.pntd.0008720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591027PMC
October 2020

Mutational Landscape of Esophageal Squamous Cell Carcinoma in an Indian Cohort.

Front Oncol 2020 20;10:1457. Epub 2020 Aug 20.

Institute of Bioinformatics, International Technology Park, Bangalore, India.

Esophageal squamous cell carcinoma (ESCC) is the most common histological subtype of esophageal cancer in India. Cigarette smoking and chewing tobacco are known risk factors associated with ESCC. However, genomic alterations associated with ESCC in India are not well-characterized. In this study, we carried out exome sequencing to characterize the mutational landscape of ESCC tumors from subjects with a varied history of tobacco usage. Whole exome sequence analysis of ESCC from an Indian cohort revealed several genes that were mutated or had copy number changes. ESCC from tobacco chewers had a higher frequency of C:G > A:T transversions and 2-fold enrichment for mutation signature 4 compared to smokers and non-users of tobacco. Genes, such as , and were found to be frequently mutated in Indian cohort. Mutually exclusive mutation patterns were observed in ---, and - gene pairs. Recurrent amplifications were observed in 3q22-3q29, 11q13.3-q13.4, 7q22.1-q31.1, and 8q24 regions. Approximately 53% of tumors had genomic alterations in making this pathway a promising candidate for targeted therapy. In conclusion, we observe enrichment of mutation signature 4 in ESCC tumors from patients with a history of tobacco chewing. This is likely due to direct exposure of esophagus to tobacco carcinogens when it is chewed and swallowed. Genomic alterations were frequently observed in PIK3CA-AKT pathway members independent of the history of tobacco usage. PIK3CA pathway can be potentially targeted in ESCC which currently has no effective targeted therapeutic options.
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http://dx.doi.org/10.3389/fonc.2020.01457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469928PMC
August 2020

Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.

Nat Commun 2020 08 24;11(1):4225. Epub 2020 Aug 24.

Department of Surgery, Division of Hepatobiliary and Pancreatic Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, 08826, South Korea.

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.
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http://dx.doi.org/10.1038/s41467-020-17880-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445288PMC
August 2020

Quantitative Proteomics of Urinary Bladder Cancer Cell Lines Identify UAP1 as a Potential Therapeutic Target.

Genes (Basel) 2020 07 8;11(7). Epub 2020 Jul 8.

Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.

Bladder carcinoma (BC) incidence and mortality rates are increasing worldwide. The development of novel therapeutic strategies is required to improve clinical management of this cancer. Aberrant protein expression may lead to cancer initiation and progression. Therefore, the identification of these potential protein targets and limiting their expression levels would provide alternative treatment options. In this study, we utilized a liquid-chromatography tandem mass spectrometry-based global proteomics approach to identify differentially expressed proteins in bladder cancer cell lines. A total of 3913 proteins were identified in this study, of which 479 proteins were overexpressed and 141 proteins were downregulated in 4 out of 6 BC cell lines when compared with normal human urothelial cell line (TERT-NHUC). We evaluated the role of UDP-N-acetylhexosamine pyrophosphorylase (UAP1) in bladder cancer pathogenesis. The silencing of UAP1 led to reduction in proliferation, invasion, colony formation and migration capability of bladder cancer cell lines. Thus, our study reveals UAP1 as a promising therapeutic target for bladder cancer.
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http://dx.doi.org/10.3390/genes11070763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397020PMC
July 2020

Proteomic profiling of medulloblastoma reveals novel proteins differentially expressed within each molecular subgroup.

Clin Neurol Neurosurg 2020 09 17;196:106028. Epub 2020 Jun 17.

Department of Neuropathology, National Institute of Mental Health and Neurosciences [NIMHANS], Bangalore, India. Electronic address:

Objectives: The objective of the study was to identify novel medulloblastoma (MB) biomarkers through proteomic profiling, correlate it with the molecular subgroups of MB and assess the clinical significance.

Methods: Archived paraffin embedded tumor tissue blocks from 118 MB patients, operated at our institute were retrieved. Clinical information was documented from the hospital database. Tumours were stratified into molecular subgroups using the IHC markers- β Catenin, GAB-1, YAP-1 and p53. Six fresh MB tumour tissues and two control cerebellar tissues were subjected to proteomic profiling to study differential protein expression in molecular subgroups using high resolution mass spectrometry. Prominent signalling pathways activated in each subgroup were identified using the Panther pathway software.

Results: Non WNT/SHH group was the most common (61.1 %), followed by SHH and WNT. p53 immunopositivity did not correlate with prognosis in any subgroup. Proteomic profiling revealed several novel proteins differentially expressed between MB molecular subgroups. Signalling pathways exclusively enriched in each molecular subgroup were also identified. The top upregulated proteins were PMEL and FBN2 in the WNT subgroup, SYNGR2 in the SHH subgroup and GFAP, IMPG2 and MAGEA10 in the Non WNT/Non SHH group. We validated GFAP by immunohistochemistry on the archived samples (n = 118) and noted two types of staining pattern in MBs - reactive (stellate) astrocytes and tumour cell staining. GFAP immunopositivity in tumor cells of SHH subgroup correlated with a better prognosis.

Conclusions: Proteomic profile identified several novel proteins differentially regulated within the molecular subgroups that could serve as potential diagnostic /prognostic biomarkers. Notably, GFAP, which was derived from proteomics data, when validated by IHC, revealed a variable staining pattern in MB tumours. The prognostic significance of GFAP in SHH tumor patients further points at the heterogeneity of this subgroup. The study also throws light on the signaling pathways activated in MB and in turn its plausible role in the tumorigenesis.
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http://dx.doi.org/10.1016/j.clineuro.2020.106028DOI Listing
September 2020

Altered mitochondrial proteome and functional dynamics in patients with rheumatoid arthritis.

Mitochondrion 2020 09 13;54:8-14. Epub 2020 Jun 13.

Disease Biology Laboratory, School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Deemed to be University, Bhubaneswar, Odisha, India. Electronic address:

The autoimmune inflammatory disease, Rheumatoid arthritis (RA), has known imbalances in energy metabolism and superoxide levels thus may have an etiology associated with mitochondrial dysfunction. We thus evaluated the presence of a differential mitochondrial proteome as well as other characteristics including mitochondrial mass, membrane potential (Ψm), total cellular ATP and superoxide levels. Eighteen mitochondrial proteins were down-regulated while four were up-regulated in RA patients in comparison to the healthy controls (HC). A significant decrease in mitochondrial Ψm, superoxides and cellular ATP levels was observed in RA with constant mitochondrial mass suggesting mitochondrial dysfunction responsible for functional disparity in RA.
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http://dx.doi.org/10.1016/j.mito.2020.06.005DOI Listing
September 2020

VapBC22 toxin-antitoxin system from is required for pathogenesis and modulation of host immune response.

Sci Adv 2020 Jun 3;6(23):eaba6944. Epub 2020 Jun 3.

Tuberculosis Research Laboratory, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Harya na-121001, India.

Virulence-associated protein B and C toxin-antitoxin (TA) systems are widespread in prokaryotes, but their precise role in physiology is poorly understood. We have functionally characterized the VapBC22 TA system from . Transcriptome analysis revealed that overexpression of VapC22 toxin in results in reduced levels of metabolic enzymes and increased levels of ribosomal proteins. Proteomics studies showed reduced expression of virulence-associated proteins and increased levels of cognate antitoxin, VapB22 in the Δ mutant strain. Furthermore, both the Δ mutant and VapB22 overexpression strains of were susceptible to killing upon exposure to oxidative stress and showed attenuated growth in guinea pigs and mice. Host transcriptome analysis suggests upregulation of the transcripts involved in innate immune responses and tissue remodeling in mice infected with the Δ mutant strain. Together, we demonstrate that the VapBC22 TA system belongs to a key regulatory network and is essential for pathogenesis.
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http://dx.doi.org/10.1126/sciadv.aba6944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269643PMC
June 2020

Intracranial Aneurysm Biomarker Candidates Identified by a Proteome-Wide Study.

OMICS 2020 08 11;24(8):483-492. Epub 2020 Jun 11.

Division of Neuroanesthesia, Department of Anesthesia and Intensive Care, Post Graduate Institute of Medical Education and Research, Chandigarh, India.

The scientific basis of intracranial aneurysm (IA) formation, its rupture and further development of cerebral vasospasm is incompletely understood. Aberrant protein expression may drive structural alterations of vasculature found in IA. Deciphering the molecular mechanisms underlying these events will lead to identification of early detection biomarkers and in turn, improved treatment outcomes. To unravel differential protein expression in three clinical subgroups of IA patients: (1) unruptured aneurysm, (2) ruptured aneurysm without vasospasm, (3) ruptured aneurysm who developed vasospasm, we performed untargeted quantitative proteomic analysis of aneurysm tissue and serum samples from three subgroups of IA patients and control subjects. Candidate molecules were then validated in a larger cohort of patients using enzyme-linked immunosorbent assay. A total of 937 and 294 proteins were identified from aneurysm tissue and serum samples, respectively. Several proteins that are known to maintain structural integrity of vasculature were found to be dysregulated in the context of aneurysm. , a glycoprotein, was significantly upregulated in both tissue and serum samples of unruptured aneurysm patients. We employed a larger cohort of subjects ( = 26) and validated as a potential biomarker for screening of unruptured aneurysms. Samples from ruptured aneurysms with vasospasm showed significant upregulation of , a protease, compared with ruptured aneurysms without vasospasm. We validated as a potential biomarker for vasospasm in a larger cohort ( = 52). This study reports the first global proteomic analysis of the entire clinical spectrum of IA. Furthermore, this study suggests and as potential biomarkers for unruptured aneurysm and cerebral vasospasm, respectively.
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http://dx.doi.org/10.1089/omi.2020.0057DOI Listing
August 2020

Marizomib suppresses triple-negative breast cancer via proteasome and oxidative phosphorylation inhibition.

Theranostics 2020 6;10(12):5259-5275. Epub 2020 Apr 6.

QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Brisbane QLD 4006, Australia.

: Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required. : We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, using breast cancer lines and using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model . : We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity. : Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients.
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http://dx.doi.org/10.7150/thno.42705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196287PMC
April 2020

Phosphoproteomic analysis identifies CLK1 as a novel therapeutic target in gastric cancer.

Gastric Cancer 2020 09 24;23(5):796-810. Epub 2020 Apr 24.

Institute of Bioinformatics, International Technology Park, Bangalore, Bangalore, 560066, India.

Background: Phosphorylation is an important regulatory mechanism of protein activity in cells. Studies in various cancers have reported perturbations in kinases resulting in aberrant phosphorylation of oncoproteins and tumor suppressor proteins.

Methods: In this study, we carried out quantitative phosphoproteomic analysis of gastric cancer tissues and corresponding xenograft samples. Using these data, we employed bioinformatics analysis to identify aberrant signaling pathways. We further performed molecular inhibition and silencing of the upstream regulatory kinase in gastric cancer cell lines and validated its effect on cellular phenotype. Through an ex vivo technology utilizing patient tumor and blood sample, we sought to understand the therapeutic potential of the kinase by recreating the tumor microenvironment.

Results: Using mass spectrometry-based high-throughput analysis, we identified 1,344 phosphosites and 848 phosphoproteins, including differential phosphorylation of 177 proteins (fold change cut-off ≥ 1.5). Our data showed that a subset of differentially phosphorylated proteins belonged to splicing machinery. Pathway analysis highlighted Cdc2-like kinase (CLK1) as upstream kinase. Inhibition of CLK1 using TG003 and CLK1 siRNA resulted in a decreased cell viability, proliferation, invasion and migration as well as modulation in the phosphorylation of SRSF2. Ex vivo experiments which utilizes patient's own tumor and blood to recreate the tumor microenvironment validated the use of CLK1 as a potential target for gastric cancer treatment.

Conclusions: Our data indicates that CLK1 plays a crucial role in the regulation of splicing process in gastric cancer and that CLK1 can act as a novel therapeutic target in gastric cancer.
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http://dx.doi.org/10.1007/s10120-020-01062-8DOI Listing
September 2020

MAP2K1 is a potential therapeutic target in erlotinib resistant head and neck squamous cell carcinoma.

Sci Rep 2019 12 11;9(1):18793. Epub 2019 Dec 11.

Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India.

Epidermal growth factor receptor (EGFR) targeted therapies have shown limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients despite its overexpression. Identifying molecular mechanisms associated with acquired resistance to EGFR-TKIs such as erlotinib remains an unmet need and a therapeutic challenge. In this study, we employed an integrated multi-omics approach to delineate mechanisms associated with acquired resistance to erlotinib by carrying out whole exome sequencing, quantitative proteomic and phosphoproteomic profiling. We observed amplification of several genes including AXL kinase and transcription factor YAP1 resulting in protein overexpression. We also observed expression of constitutively active mutant MAP2K1 (p.K57E) in erlotinib resistant SCC-R cells. An integrated analysis of genomic, proteomic and phosphoproteomic data revealed alterations in MAPK pathway and its downstream targets in SCC-R cells. We demonstrate that erlotinib-resistant cells are sensitive to MAPK pathway inhibition. This study revealed multiple genetic, proteomic and phosphoproteomic alterations associated with erlotinib resistant SCC-R cells. Our data indicates that therapeutic targeting of MAPK pathway is an effective strategy for treating erlotinib-resistant HNSCC tumors.
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http://dx.doi.org/10.1038/s41598-019-55208-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906491PMC
December 2019

Dickkopf Homolog 3 Acts as a Potential Tumor Suppressor in Gallbladder Cancer.

Front Oncol 2019 29;9:1121. Epub 2019 Oct 29.

Institute of Bioinformatics, International Tech Park, Bangalore, India.

Gallbladder cancer (GBC) is a common malignancy of biliary tract cancers and its incidence has been rising rapidly worldwide. The prognosis for this disease is dismal as most of the symptoms are non-specific leading to a definitive diagnosis only at a late stage. Loss of gene is associated with a possible tumor suppressor role in human cancers. The role and regulation of DKK3 in GBC have not been studied. We found that DKK3 expression levels were low in GBC patients and cell lines. Treatment of GBC cell lines with demethylating agent 5-Aza- 2'-deoxycytidine enhances its expression, establishing impact of methylation on DKK3 expression. We observed low expression of DKK3 in gallbladder adenocarcinoma tumors and highly invasive GBC cell lines. We showed that overexpression of DKK3 can decrease cell invasion, proliferation, and colony forming ability of GBC cells. Our data thus demonstrated the DKK3 gene is a potential tumor suppressor gene in GBC and aberrant promoter methylation could be involved in its downregulation, which may play a role in the tumorigenesis and aggressiveness of GBC.
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http://dx.doi.org/10.3389/fonc.2019.01121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828847PMC
October 2019

Proteome-wide changes in primary skin keratinocytes exposed to diesel particulate extract-A role for antioxidants in skin health.

J Dermatol Sci 2019 11 16;96(2):114-124. Epub 2019 Oct 16.

L'Oréal Research and Innovation, Aulnay sous bois, 93600, France; L'Oréal India Pvt. Ltd., Beary's Global Research Triangle, Bangalore, 560067, India. Electronic address:

Background: Skin acts as a protective barrier against direct contact with pollutants but inhalation and systemic exposure have indirect effect on keratinocytes. Exposure to diesel exhaust has been linked to increased oxidative stress.

Objective: To investigate global proteomic alterations in diesel particulate extract (DPE)/ its vapor exposed skin keratinocytes.

Methods: We employed Tandem Mass Tag (TMT)-based proteomics to study effect of DPE/ DPE vapor on primary skin keratinocytes.

Results: We observed an increased expression of oxidative stress response protein NRF2, upon chronic exposure of primary keratinocytes to DPE/ its vapor which includes volatile components such as polycyclic aromatic hydrocarbons (PAHs). Mass spectrometry-based quantitative proteomics led to identification 4490 proteins of which 201 and 374 proteins were significantly dysregulated (≥1.5 fold, p ≤ 0.05) in each condition, respectively. Proteins involved in cellular processes such as cornification (cornifin A), wound healing (antileukoproteinase) and differentiation (suprabasin) were significantly downregulated in primary keratinocytes exposed to DPE/ DPE vapor. These results were corroborated in 3D skin models chronically exposed to DPE/ DPE vapor. Bioinformatics analyses indicate that DPE and its vapor affect distinct molecular processes in skin keratinocytes. Components of mitochondrial oxidative phosphorylation machinery were seen to be exclusively overexpressed upon chronic DPE vapor exposure. In addition, treatment with an antioxidant like vitamin E partially restores expression of proteins altered upon exposure to DPE/ DPE vapor.

Conclusions: Our study highlights distinct adverse effects of chronic exposure to DPE/ DPE vapor on skin keratinocytes and the potential role of vitamin E in alleviating adverse effects of environmental pollution.
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http://dx.doi.org/10.1016/j.jdermsci.2019.08.009DOI Listing
November 2019