Publications by authors named "Ruchi Shukla"

52 Publications

NMR based CSF metabolomics in tuberculous meningitis: correlation with clinical and MRI findings.

Metab Brain Dis 2022 Jan 14. Epub 2022 Jan 14.

Department of Neurology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, 226014, India.

We report the potential role of H Nuclear Magnetic Resonance (NMR) based metabolomics in tuberculous meningitis (TBM). We also correlate the significant metabolites with clinical-radiological parameters. Forty-three patients with TBM were included, and their severity of meningitis was graded as stages I to III, and patients with positive Mycobacterium tuberculosis or its nucleic acid was considered as definite TBM. H NMR-based metabolomic study was performed on (CSF) samples, and the significant metabolites compared to healthy controls were identified. Outcome at three months was defined as death, poor and good based on the modified Rankin Scale. These metabolites were compared between definite and probable groups of TBM, and also correlated with MRI findings. About 11 metabolites were found to be significant for distinguishing TBM from the controls. In TBM, lactate, glutamate, alanine, arginine, 2-hydroxyisobutyrate, formate, and cis-aconitate were upregulated, and glucose, fructose, glutamine, and myo-inositol were downregulated compared to the controls. For differentiating TBM from the controls, the AUC of the ROC curve generated using these significant metabolites was 0.99, with a 95% confidence interval from 0.96 to 1, demonstrating that these metabolites were able to classify cases with good sensitivity and specificity. Lactate concentration in CSF correlated with hemoglobin, CSF glucose, and infarction. The outcome did not correlate with metabolomics parameters. NMR-based CSF metabolomics have a potential role in differentiating TBM from the controls.
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http://dx.doi.org/10.1007/s11011-021-00860-yDOI Listing
January 2022

Predictors of fever response in tuberculous meningitis: A clinical, MRI and biomarker study.

Eur J Clin Invest 2022 Feb 2;52(2):e13701. Epub 2021 Nov 2.

Department of Anesthesiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India.

Background: Central nervous system (CNS) has a different immune surveillance system; therefore, fever at admission and timeline of fever response after antitubercular treatment (ATT) may follow a different course in CNS infection. We report the predictors of fever response in tuberculous meningitis (TBM) including the effect of tumour necrosis factor-α (TNF-α) in cerebrospinal fluid (CSF) and its gene expression at mRNA of peripheral blood mononuclear cells (PBMCs).

Methods: Fifty-seven patients with TBM were prospectively evaluated. Their clinical findings and severity of meningitis were recorded. The expression of TNF-α gene in PBMCs was quantified by real-time polymerase chain reaction and TNF-α concentration in CSF by cytokine bead array both in the patients and 14 matched controls.

Results: All the patients had history of fever for a median duration of 75 days. The admission temperature ranged between 37.2°C and 40°C and correlated with CSF cell counts (p < 0.05). Cranial MRI was abnormal in 54 (94.7%) and revealed exudates in 33(57.9%), hydrocephalus in 27(47.4%), infarction in 27(47.4%) and tuberculoma in 33(57.9%) patients. Fever subsided after a median duration of 18 (2 60) days of treatment. Twelve (21.8%) patients only became afebrile within 10 days. The expression of TNF-α gene correlated with CSF concentration of TNF-α (p = 0.02) and independently predicted duration of defervescence [adjusted hazard ratio 1.02 (95% CI 1.00-1.04; p = 0.01).

Conclusion: In the patients with TBM, defervescence takes longer time, and TNF-α gene expression predicts the duration of defervescence. Future studies are needed to evaluate the role of TNF-α-modifying drugs in TBM.
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http://dx.doi.org/10.1111/eci.13701DOI Listing
February 2022

HCV Activates Somatic L1 Retrotransposition-A Potential Hepatocarcinogenesis Pathway.

Cancers (Basel) 2021 Oct 11;13(20). Epub 2021 Oct 11.

Newcastle University Centre for Cancer, Biosciences Institute, Faculty of Medical Sciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

Hepatitis C virus (HCV) is a common cause of hepatocellular carcinoma (HCC). The activation and mutagenic consequences of L1 retrotransposons in virus-associated-HCC have been documented. However, the direct influence of HCV upon L1 elements is unclear, and is the focus of the present study. L1 transcript expression was evaluated in a publicly available liver tissue RNA-seq dataset from patients with chronic HCV hepatitis (CHC), as well as healthy controls. L1 transcript expression was significantly higher in CHC than in controls. L1orf1p (a L1 encoded protein) expression was observed in six out of 11 CHC livers by immunohistochemistry. To evaluate the influence of HCV on retrotransposition efficiency, in vitro engineered-L1 retrotransposition assays were employed in Huh7 cells in the presence and absence of an HCV replicon. An increased retrotransposition rate was observed in the presence of replicating HCV RNA, and persisted in cells after viral clearance due to sofosbuvir (PSI7977) treatment. Increased retrotransposition could be due to dysregulation of the DNA-damage repair response, including homologous recombination, due to HCV infection. Altogether these data suggest that L1 expression can be activated before oncogenic transformation in CHC patients, with HCV-upregulated retrotransposition potentially contributing to HCC genomic instability and a risk of transformation that persists post-viral clearance.
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http://dx.doi.org/10.3390/cancers13205079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8533982PMC
October 2021

Key features of the environment promoting liver cancer in the absence of cirrhosis.

Sci Rep 2021 08 18;11(1):16727. Epub 2021 Aug 18.

Faculty of Medical Sciences, Newcastle University Translational and Clinical Research Institute, Newcastle-upon-Tyne, NE2 4HH, UK.

The prevalence of obesity and non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) is rising, even in the absence of cirrhosis. We aimed to develop a murine model that would facilitate further understanding of NAFLD-HCC pathogenesis. A total of 144 C3H/He mice were fed either control or American lifestyle (ALIOS) diet, with or without interventions, for up to 48 weeks of age. Gross, liver histology, immunohistochemistry (IHC) and RNA-sequencing data were interpreted alongside human datasets. The ALIOS diet promoted obesity, elevated liver weight, impaired glucose tolerance, non-alcoholic fatty liver disease (NAFLD) and spontaneous HCC. Liver weight, fasting blood glucose, steatosis, lobular inflammation and lipogranulomas were associated with development of HCC, as were markers of hepatocyte proliferation and DNA damage. An antioxidant diminished cellular injury, fibrosis and DNA damage, but not lobular inflammation, lipogranulomas, proliferation and HCC development. An acquired CD44 phenotype in macrophages was associated with type 2 diabetes and NAFLD-HCC. In this diet induced NASH and HCC (DINAH) model, key features of obesity associated NAFLD-HCC have been reproduced, highlighting roles for hepatic steatosis and proliferation, with the acquisition of lobular inflammation and CD44 positive macrophages in the development of HCC-even in the absence of progressive injury and fibrosis.
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http://dx.doi.org/10.1038/s41598-021-96076-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373870PMC
August 2021

Feasibility and usefulness of tele-follow-up in the patients with tuberculous meningitis.

Trans R Soc Trop Med Hyg 2021 10;115(10):1153-1159

Department of Neurology, Vivekananda Polyclinic and Institute of Medical Sciences, Lucknow, Uttar Pradesh 226007, India.

Background: Tuberculous meningitis (TBM) is the most severe form of tuberculosis and these patients need close follow-up because of a high frequency of complications. The coronavirus disease 2019 pandemic and lockdown resulted in an interruption in physical follow-up. In this situation, tele-follow-up may be helpful. We report the feasibility and usefulness of a telephonic follow-up in patients with TBM.

Methods: Patients with TBM managed by us from January 2017 to March 2020 were included from the TBM registry. Their presenting symptoms, and clinical and investigation findings were noted. We contacted these patients telephonically and their clinical status was obtained using a questionnaire. Based on the telephonic information, outcomes were categorized as death, poor or good. Patients with the new medical problems were advised as to relevant investigations and the reports were obtained through WhatsApp for prescribing treatment.

Results: The telephone numbers of 103 of 144 (71.5%) patients were viable. Twenty-seven (26.2%) patients died, 15 (19.7%) had a poor outcome and 61 (80.2%) had a good outcome. Twenty-five (32.9%) patients had new medical problems: 18 TBM related and 7 TBM unrelated. The medical problems of 23 patients could be managed telephonically and only 3 (4%) patients needed a physical visit. Sixty-five (85.5%) patients happily answered the questionnaire and willing responders needed a treatment modification more frequently than the reluctant responders (p=0.008). Patients on active antitubercular treatment needed treatment modification more frequently (80% vs 21.3%).

Conclusions: Tele-follow-up is feasible in 96% of TBM patients and is beneficial, cost effective and overcomes the barrier of distance.
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http://dx.doi.org/10.1093/trstmh/trab069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8194613PMC
October 2021

A Role in the Genetic Predisposition to NAFLD-HCC?

Cancers (Basel) 2021 Mar 19;13(6). Epub 2021 Mar 19.

Translational and Clinical Research Institute, Faculty of Medical Sciences, The Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.

Obesity and non-alcoholic fatty liver disease (NAFLD) are contributing to the global rise in deaths from hepatocellular carcinoma (HCC). The pathogenesis of NAFLD-HCC is not well understood. The severity of hepatic steatosis, steatohepatitis and fibrosis are key pathogenic mechanisms, but animal studies suggest altered immune responses are also involved. Genetic studies have so far highlighted a major role of gene variants promoting fat deposition in the liver ( rs738409; rs58542926). Here, we have considered single-nucleotide polymorphisms (SNPs) in candidate immunoregulatory genes ( rs2596542; rs187115; rs7421861 and rs10204525), in 594 patients with NAFLD and 391 with NAFLD-HCC, from three European centres. Associations between age, body mass index, diabetes, cirrhosis and SNPs with HCC development were explored. and SNPs were associated with both progression to cirrhosis and NAFLD-HCC development, while SNPs were specifically associated with NAFLD-HCC risk, regardless of cirrhosis. rs7421861 was independently associated with NAFLD-HCC development, while rs10204525 acquired significance after adjusting for other risks, being most notable in the smaller numbers of women with NAFLD-HCC. The study highlights the potential impact of inter individual variation in immune tolerance induction in patients with NAFLD, both in the presence and absence of cirrhosis.
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http://dx.doi.org/10.3390/cancers13061412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003582PMC
March 2021

Activation of transcription factor circuity in 2i-induced ground state pluripotency is independent of repressive global epigenetic landscapes.

Nucleic Acids Res 2020 08;48(14):7748-7766

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, WGH, University of Edinburgh, Edinburgh EH4 2XU, UK.

Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3β (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes.
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http://dx.doi.org/10.1093/nar/gkaa529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641322PMC
August 2020

DNA Methylation Directs Polycomb-Dependent 3D Genome Re-organization in Naive Pluripotency.

Cell Rep 2019 11;29(7):1974-1985.e6

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XU, UK. Electronic address:

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs.
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http://dx.doi.org/10.1016/j.celrep.2019.10.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856714PMC
November 2019

Author Correction: Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

Nat Commun 2018 12 19;9(1):5398. Epub 2018 Dec 19.

Division of Medical Biotechnology, Paul-Ehrlich-Institute, D-63225, Langen, Germany.

This Article contains an error in the author affiliations. The correct affiliation for author Ruchi Shukla is 'MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK', and is not 'Mater Research Institute - University of Queensland, TRI Building, Woolloongabba QLD 4102, Australia'.
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http://dx.doi.org/10.1038/s41467-018-07917-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300592PMC
December 2018

An In Silico Investigation of Potential EGFR Inhibitors for the Clinical Treatment of Colorectal Cancer.

Curr Top Med Chem 2018 ;18(27):2355-2366

Center of Excellence in Biotechnology Research, College of Science, King Saud University, Riyadh, Saudi Arabia.

Colorectal cancer possesses the third highest diagnostic rate and is the second leading cause of cancer death in the USA as reported by NIH. Epidermal Growth Factor Receptor (EGFR), a transmembrane protein, participates in PLC gamma-1, RAS-RAF-MEK-MAPKs, phosphatidylinositol-3 kinase, Akt pathways and plays a key role in normal functioning of cell division, cell differentiation, apoptosis and migration. This protein is found to be overexpressed in more than 60% of the colorectal cancers. Overexpressed EGFR advances the tumorigenic properties through cell cycle dysregulation and activates signaling pathways linked to cancer such as WNT/β-catenin, transforming growth factor β (TGF-β) and phosphoinositide-3-kinase (PI3K). Inhibiting the overexpressed EGFR protein has been proposed for the treatment and many inhibitors have been reported suppressing the activity of EGFR. However, patients in malignant state of cancer show resistance to those inhibitors, which open a wide space to research for the discovery of novel inhibitors. The present study employed Molecular Docking and Virtual Screening to find novel inhibitors with high affinity against EGFR. Molecular docking of existing inhibitors resulted in the compound titled as BGB-283 (PubChem CID-89670174) having the highest score, which was subjected to similarity search to retrieve the drugs with similar structure. The virtual screening concluded a compound SCHEMBL18435602 (PubChem CID-126517400) which revealed a better affinity with the target protein. A comparative study of both the compounds showed equivalent pharmacokinetic properties. These identified drugs have a high potential to act as EGFR inhibitors and can show promising results in the research of colorectal cancer.
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http://dx.doi.org/10.2174/1568026619666181129144107DOI Listing
April 2019

L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis.

Genome Res 2018 05 11;28(5):639-653. Epub 2018 Apr 11.

Mater Research Institute-University of Queensland, Woolloongabba, QLD 4102, Australia.

The retrotransposon Long Interspersed Element 1 (LINE-1 or L1) is a continuing source of germline and somatic mutagenesis in mammals. Deregulated L1 activity is a hallmark of cancer, and L1 mutagenesis has been described in numerous human malignancies. We previously employed retrotransposon capture sequencing (RC-seq) to analyze hepatocellular carcinoma (HCC) samples from patients infected with hepatitis B or hepatitis C virus and identified L1 variants responsible for activating oncogenic pathways. Here, we have applied RC-seq and whole-genome sequencing (WGS) to an mouse model of hepatic carcinogenesis and demonstrated for the first time that L1 mobilization occurs in murine tumors. In 12 HCC nodules obtained from 10 animals, we validated four somatic L1 insertions by PCR and capillary sequencing, including T subfamily elements, and one G subfamily example. One of the T insertions carried a 3' transduction, allowing us to identify its donor L1 and to demonstrate that this full-length T element retained retrotransposition capacity in cultured cancer cells. Using RC-seq, we also identified eight tumor-specific L1 insertions from 25 HCC patients with a history of alcohol abuse. Finally, we used RC-seq and WGS to identify three tumor-specific L1 insertions among 10 intra-hepatic cholangiocarcinoma (ICC) patients, including one insertion traced to a donor L1 on Chromosome 22 known to be highly active in other cancers. This study reveals L1 mobilization as a common feature of hepatocarcinogenesis in mammals, demonstrating that the phenomenon is not restricted to human viral HCC etiologies and is encountered in murine liver tumors.
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http://dx.doi.org/10.1101/gr.226993.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932605PMC
May 2018

Structural basis for the in vitro known acyl-depsipeptide 2 (ADEP2) inhibition to Clp 2 protease from Mycobacterium tuberculosis.

Bioinformation 2016 21;12(3):92-97. Epub 2016 Jun 21.

Bioinformatics Research Laboratory, Eminent Biosciences, Vijaynagar, Indore - 452010, India.

Inhibition of Mycobacterium tuberculosis Clp 2 protease has emerged as an attractive therapeutic option for treatment. Acyldepsipeptides (ADEPs) is known as an inhibitor for Clp 2 protease. Therefore, it is of interest to document its affinity, enzyme activity and ADME profiles. We report the predicted binding affinity of all known Clp 2 inhibitors like IDR-10001 and IDR-10011 against Clp2 protease using MolDock algorithm aided molecular docking. The predicted activity (using Molinspiration server) and ADMET properties (AdmetSAR server) were estimated for these compounds. This data suggest ADEP2 having improved binding features with Mtb Clp 2 having acceptable ADMET properties. This is in agreement with known in vitro data for ADEP2 inhibition with Mtb Clp 2 protease.
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http://dx.doi.org/10.6026/97320630012092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267950PMC
June 2016

Retrotransposons and genetic instability in hepatocellular carcinoma.

Authors:
Ruchi Shukla

Hepat Oncol 2017 Jan 18;4(1):5-8. Epub 2017 Jul 18.

Northern Institute for Cancer Research, Newcastle University, NE2 4HH, UK.

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http://dx.doi.org/10.2217/hep-2017-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095407PMC
January 2017

Dihedral-Angle-Controlled Crossover from Static Hole Delocalization to Dynamic Hopping in Biaryl Cation Radicals.

Angew Chem Int Ed Engl 2017 01 29;56(1):266-269. Epub 2016 Nov 29.

Department of Chemistry, Marquette University, Milwaukee, WI, 53201-1881, USA.

In cases of coherent charge-transfer mechanism in biaryl compounds the rates follow a squared cosine trend with varying dihedral angle. Herein we demonstrate using a series of biaryl cation radicals with varying dihedral angles that the hole stabilization shows two different regimes where the mechanism of the hole stabilization switches over from (static) delocalization over both aryl rings to (dynamic) hopping. The experimental data and DFT calculations of biaryls with different dihedral angles unequivocally support that a crossover from delocalization to hopping occurs at a unique dihedral angle where the electronic coupling (H ) is one half of reorganization (λ), that is, H =λ/2. The implication of this finding in non-coherent charge-transfer rates is being investigated.
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http://dx.doi.org/10.1002/anie.201609695DOI Listing
January 2017

Mutual epithelium-macrophage dependency in liver carcinogenesis mediated by ST18.

Hepatology 2017 05 30;65(5):1708-1719. Epub 2016 Dec 30.

Center for Genomic Science of [email protected], Fondazione Istituto Italiano di Tecnologia, Milan, Italy.

The ST18 gene has been proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis has been lacking thus far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 messenger RNA (mRNA) and protein were detectable neither in normal liver nor in cultured hepatoblasts, but were readily expressed after subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as coculture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre-established tumors caused rapid tumor involution associated with pervasive morphological changes, proliferative arrest, and apoptosis in tumor cells, as well as depletion of tumor-associated macrophages, vascular ectasia, and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in hepatoblasts. Finally, RNA sequencing of ST18-depleted tumors before involution revealed down-regulation of inflammatory response genes, pointing to the suppression of nuclear factor kappa B-dependent transcription.

Conclusion: ST18 expression in epithelial cells is induced by tumor-associated macrophages, contributing to the reciprocal feed-forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18-dependent epithelium-macrophage interactions in a therapeutic setting. (Hepatology 2017;65:1708-1719).
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http://dx.doi.org/10.1002/hep.28942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412898PMC
May 2017

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

Nat Commun 2016 Jan 8;7:10286. Epub 2016 Jan 8.

Division of Medical Biotechnology, Paul-Ehrlich-Institute, D-63225 Langen, Germany.

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.
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http://dx.doi.org/10.1038/ncomms10286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729875PMC
January 2016

Mechanism of Adsorptive Removal of Methylene Blue Using Dried Biomass of Rhizopus oryzae.

Appl Biochem Biotechnol 2015 Sep 4;177(2):541-55. Epub 2015 Aug 4.

Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, 784028, India.

Adsorption is an efficient way to remove synthetic dyes from industrial effluent. Here, we show mechanism of adsorptive removal of cationic dye methylene blue (MB) from its aqueous solution using dried biomass of Rhizopus oryzae as a biosorbent. The optimum pH and temperature for adsorption was found to be 7.0 and 28 °C, respectively. Scanning electron microscopy (SEM) of the biomass suggested distinct changes in surface topology post-MB adsorption, while Fourier transform infrared (FTIR) study indicated chemical interaction between the surface of the biomass and MB. Chemical modification of -OH and -C=O groups of biomass reduced the MB adsorption and corroborated with the FTIR analyses. Kinetics study revealed that the adsorption rate was fast initially and reached equilibrium at 4 h following a pseudo-second-order-kinetics. The adsorption isotherm followed Freundlich isotherm model with n value of 1.1615.The dried biomass of R. oryzae can be used as a potent biosorbent for the removal of MB.
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http://dx.doi.org/10.1007/s12010-015-1761-5DOI Listing
September 2015

DNA immunoprecipitation semiconductor sequencing (DIP-SC-seq) as a rapid method to generate genome wide epigenetic signatures.

Sci Rep 2015 May 14;5:9778. Epub 2015 May 14.

MRC Human Genetics Unit at the Institute of Genetics and Molecular Medicine at the University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.

Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; "DIP-SC-seq") on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets.
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http://dx.doi.org/10.1038/srep09778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435000PMC
May 2015

Evidence of rapid recovery from perceptual odor adaptation using a new stimulus paradigm.

Atten Percept Psychophys 2014 May;76(4):1093-105

Department of Psychology, University of Florida, 114 Psychology Building, P. O. Box 112250, Gainesville, Florida, 32611, USA.

By attenuating neural and perceptual responses to sustained stimulation, adaptation enhances the detection of new, transient stimuli. Disadaptation serves a similarly important role as a temporal filter for chemoreceptor cells, producing rapid recovery of sensitivity upon termination of the adapting odorant. Previous research from our laboratory indicated that a rapid form of odor adaptation can be measured using a novel, simultaneous-odorant paradigm. In the present study, we extended the earlier method by measuring recovery from adaptation. Perceptual odor adaptation was measured by estimating psychophysical detection thresholds in a group of college-aged student volunteers (N = 20; 12 females, eight males) for a self-adapting odorant, vanilla extract. To induce adaptation, the time between the onset of the adapting odorant and the onset of the target odorant was systematically varied. By first quantifying adaptation, recovery of sensitivity could therefore be investigated by using different time points following the termination of the adapting odorant. Consistent with our previous work, thresholds estimated in the presence of the simultaneous adapting odorant were significantly increased, reflecting a decrease in sensitivity due to adaptation. Conversely, approximately 100 ms following termination of the adapting stimulus (the briefest delay tested), sensitivity began to rapidly recover. Nevertheless, some residual adaptation was evident at the longest offset delay of 500 ms. These findings suggest that the recovery from adaptation proceeds at least as rapidly as the onset of adaptation, a finding that is consistent with physiological evidence from olfactory receptors. These data also suggest the effectiveness of this new odorant paradigm in characterizing the temporal characteristics underlying these critical olfactory mechanisms.
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http://dx.doi.org/10.3758/s13414-013-0620-0DOI Listing
May 2014

Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma.

Cell 2013 Mar;153(1):101-11

Division of Genetics and Genomics, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush EH25 9RG, UK.

LINE-1 (L1) retrotransposons are mobile genetic elements comprising ~17% of the human genome. New L1 insertions can profoundly alter gene function and cause disease, though their significance in cancer remains unclear. Here, we applied enhanced retrotransposon capture sequencing (RC-seq) to 19 hepatocellular carcinoma (HCC) genomes and elucidated two archetypal L1-mediated mechanisms enabling tumorigenesis. In the first example, 4/19 (21.1%) donors presented germline retrotransposition events in the tumor suppressor mutated in colorectal cancers (MCC). MCC expression was ablated in each case, enabling oncogenic β-catenin/Wnt signaling. In the second example, suppression of tumorigenicity 18 (ST18) was activated by a tumor-specific L1 insertion. Experimental assays confirmed that the L1 interrupted a negative feedback loop by blocking ST18 repression of its enhancer. ST18 was also frequently amplified in HCC nodules from Mdr2(-/-) mice, supporting its assignment as a candidate liver oncogene. These proof-of-principle results substantiate L1-mediated retrotransposition as an important etiological factor in HCC.
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http://dx.doi.org/10.1016/j.cell.2013.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898742PMC
March 2013

Transcriptional regulation of the human tumor suppressor DOK1 by E2F1.

Mol Cell Biol 2012 Dec 1;32(23):4877-90. Epub 2012 Oct 1.

International Agency for Research on Cancer, Lyon, France.

The expression of the tumor suppressor DOK1 is repressed in a variety of human tumors as a result of hypermethylation of its promoter region. However, the molecular mechanisms by which DOK1 expression is regulated have been poorly investigated. Here, we show that the expression of DOK1 is regulated mainly by the transcription factor E2F1. We identified three putative E2F1 response elements (EREs) in the DOK1 promoter region. E2F1 had a relatively higher binding affinity for the ERE located between bp -498 and -486 compared with the other two EREs. E2F1 gene silencing strongly inhibited DOK1 expression. E2F1-driven DOK1 transcription occurred in the presence of cellular stresses, such as accumulation of DNA damage induced by etoposide. DOK1 silencing promoted cell proliferation and protected against etoposide-induced apoptosis, indicating that DOK1 acts as a key mediator of cellular stress-induced cell death. Most importantly, we observed that DNA methylation of the DOK1 core promoter region found in head and neck cancer cell lines hampered the recruitment of E2F1 to the DOK1 promoter and compromised DOK1 expression. In summary, our data show that E2F1 is a key factor in DOK1 expression and provide novel insights into the regulation of these events in cancer cells.
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http://dx.doi.org/10.1128/MCB.01050-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497605PMC
December 2012

Extensive somatic L1 retrotransposition in colorectal tumors.

Genome Res 2012 Dec 11;22(12):2328-38. Epub 2012 Sep 11.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

L1 retrotransposons comprise 17% of the human genome and are its only autonomous mobile elements. Although L1-induced insertional mutagenesis causes Mendelian disease, their mutagenic load in cancer has been elusive. Using L1-targeted resequencing of 16 colorectal tumor and matched normal DNAs, we found that certain cancers were excessively mutagenized by human-specific L1s, while no verifiable insertions were present in normal tissues. We confirmed de novo L1 insertions in malignancy by both validating and sequencing 69/107 tumor-specific insertions and retrieving both 5' and 3' junctions for 35. In contrast to germline polymorphic L1s, all insertions were severely 5' truncated. Validated insertion numbers varied from up to 17 in some tumors to none in three others, and correlated with the age of the patients. Numerous genes with a role in tumorigenesis were targeted, including ODZ3, ROBO2, PTPRM, PCM1, and CDH11. Thus, somatic retrotransposition may play an etiologic role in colorectal cancer.
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http://dx.doi.org/10.1101/gr.145235.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514663PMC
December 2012

Charge delocalization in self-assembled mixed-valence aromatic cation radicals.

Langmuir 2012 Jan 29;28(1):71-83. Epub 2011 Sep 29.

Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53201-1881, United States.

The spontaneous assembly of aromatic cation radicals (D(+•)) with their neutral counterpart (D) affords dimer cation radicals (D(2)(+•)). The intermolecular dimeric cation radicals are readily characterized by the appearance of an intervalence charge-resonance transition in the NIR region of their electronic spectra and by ESR spectroscopy. The X-ray crystal structure analysis and DFT calculations of a representative dimer cation radical (i.e., the octamethylbiphenylene dimer cation radical) have established that a hole (or single positive charge) is completely delocalized over both aromatic moieties. The energetics and the geometrical considerations for the formation of dimer cation radicals is deliberated with the aid of a series of cyclophane-like bichromophoric donors with drastically varied interplanar angles between the cofacially arranged aryl moieties. X-ray crystallography of a number of mixed-valence cation radicals derived from monochromophoric benzenoid donors established that they generally assemble in 1D stacks in the solid state. However, the use of polychromophoric intervalence cation radicals, where a single charge is effectively delocalized among all of the chromophores, can lead to higher-order assemblies with potential applications in long-range charge transport. As a proof of concept, we show that a single charge in the cation radical of a triptycene derivative is evenly distributed on all three benzenoid rings and this triptycene cation radical forms a 2D electronically coupled assembly, as established by X-ray crystallography.
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http://dx.doi.org/10.1021/la202611wDOI Listing
January 2012

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers.

Int J Cancer 2012 Jun 22;130(11):2484-94. Epub 2011 Sep 22.

International Agency for Research on Cancer (IARC), Lyon, France.

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.
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http://dx.doi.org/10.1002/ijc.26299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422753PMC
June 2012

NF-kappaB protects human papillomavirus type 38 E6/E7-immortalized human keratinocytes against tumor necrosis factor alpha and UV-mediated apoptosis.

J Virol 2011 Sep 29;85(17):9013-22. Epub 2011 Jun 29.

International Agency for Research on Cancer, 150 cours Albert-Thomas, 69372 Lyon Cedex 08, France.

Constitutive activation of NF-κB signaling is a key event in virus- and non-virus-induced carcinogenesis. We have previously reported that cutaneous human papillomavirus type 38 (HPV38) displays transforming properties in in vitro and in vivo experimental models. However, the involvement of NF-κB signaling in HPV38-induced cell growth transformation remains to be determined. In this study, we showed that HPV38 E6 and E7 activate NF-κB and that inhibition of the pathway with the IκBα superrepressor sensitizes HPV38E6E7-immortalized human keratinocytes to tumor necrosis factor alpha (TNF-α)- and UVB radiation-mediated apoptosis. Accordingly, inhibition of NF-κB signaling resulted in the downregulation of NF-κB-regulated antiapoptotic genes, including cIAP1, cIAP2, and xIAP genes. These findings demonstrate a critical role of NF-κB activity in the survival of HPV38E6E7-immortalized human keratinocytes exposed to cytokine or UV radiation. Our data provide additional evidence for cooperation between beta HPV infection and UV irradiation in skin carcinogenesis.
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http://dx.doi.org/10.1128/JVI.00002-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165856PMC
September 2011

Cutaneous human papillomavirus type 38 E7 regulates actin cytoskeleton structure for increasing cell proliferation through CK2 and the eukaryotic elongation factor 1A.

J Virol 2011 Sep 22;85(17):8477-94. Epub 2011 Jun 22.

International Agency for Research on Cancer, Lyon, France.

We previously reported that the oncoproteins E6 and E7 from cutaneous human papillomavirus type 38 (HPV38) can immortalize primary human keratinocytes in vitro and sensitize transgenic mice to develop skin cancer in vivo. Immunofluorescence staining revealed that human keratinocytes immortalized by HPV38 E6 and E7 display fewer actin stress fibers than do control primary keratinocyte cells, raising the possibility of a role of the viral oncoproteins in the remodeling of the actin cytoskeleton. In this study, we show that HPV38 E7 induces actin stress fiber disruption and that this phenomenon correlates with its ability to downregulate Rho activity. The downregulation of Rho activity by HPV38 E7 is mediated through the activation of the CK2-MEK-extracellular signal-regulated kinase (ERK) pathway. In addition, HPV38 E7 is able to induce actin fiber disruption by binding directly to eukaryotic elongation factor 1A (eEF1A) and abolishing its effects on actin fiber formation. Finally, we found that the downregulation of Rho activity by HPV38 E7 through the CK2-MEK-ERK pathway facilitates cell growth proliferation. Taken together, our data support the conclusion that HPV38 E7 promotes keratinocyte proliferation in part by negatively regulating actin cytoskeleton fiber formation through the CK2-MEK-ERK-Rho pathway and by binding to eEF1A and inhibiting its effects on actin cytoskeleton remodeling.
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http://dx.doi.org/10.1128/JVI.02561-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165781PMC
September 2011

Proinflammatory cytokine TNF-α increases the stability of hepatitis B virus X protein through NF-κB signaling.

Carcinogenesis 2011 Jul 31;32(7):978-85. Epub 2011 Mar 31.

Infections and Cancer Biology Group, International Agency for Research on Cancer, 150 cours Albert-Thomas, 69372 Lyon Cedex 08, France.

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV-induced hepatocellular carcinoma (HCC). HBx interacts with several cell signaling molecules, leading to activation of various transcription factors including nuclear factor-kappaB (NF-κB). Activated NF-κB signaling is implicated in many human cancers including HCC. Here, we present evidence that the NF-κB signaling activator, tumor necrosis factor (TNF)-α, induces the accumulation of HBx in cells by increasing protein stability due to reduced proteasomal degradation. The effects of TNF-α on HBx protein stability are mediated via activated NF-κB effector kinases IKKα and IKKβ and p65. The non-IKK-phosphorylable p65-S534A mutant did not induce HBx protein stability; hence, phosphorylation of p65 by IKK is a key step in TNF-α-induced stabilization of HBx. Phospho-p65 showed higher affinity to HBx compared with the non-phosphorylable p65 mutant, suggesting that the interaction of phospho-p65 with HBx might be important for HBx stabilization. We also show that the increased level of HBx in cells cooperates with TNF-α toward activation of NF-κB and expression of NF-κB-regulated genes, indicating a positive feedback loop between HBx and NF-κB signaling. Overall, our study provides evidence for interplay between HBx and NF-κB signaling, which may account for HBV-mediated liver carcinogenesis.
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http://dx.doi.org/10.1093/carcin/bgr057DOI Listing
July 2011

Probing the arenium-ion (protontransfer) versus the cation-radical (electron transfer) mechanism of Scholl reaction using DDQ as oxidant.

J Org Chem 2010 Jul;75(14):4748-60

Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53201-1881, USA.

DDQ/H(+) system readily oxidizes a variety of electron donors with oxidation potential as high as approximately 1.7 V to the corresponding cation radicals. A re-examination of the controversial arenium-ion versus cation-radical mechanisms for Scholl reaction using DDQ/H(+) together with commonly utilized FeCl(3) as oxidants led us to demonstrate that the reaction proceeds largely via a cation-radical mechanism. The critical experimental evidence in support of a cation-radical pathway for the Scholl reaction includes the following: (i) There is no reaction in Scholl precursors in a mixture of dichloromethane and various acids (10% v/v). (ii) The necessity to use powerful oxidants such as ferric chloride (FeCl(3)) or DDQ/H(+) for Scholl reactions is inconsistent with the arenium-ion mechanism in light of the fact that aromatization of the dihydro intermediates (formed via arenium-ion mechanism) can be easily accomplished with rather weak oxidants such as iodine or air. (iii) Various Scholl precursors with oxidation potentials 1.7 V vs SCE do not react. (iv) Finally, the feasibility of the dicationic intermediate, formed by loss of two electrons, has been demonstrated by its generation from a tetraphenylene derivative using DDQ/H(+) as an oxidant.
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http://dx.doi.org/10.1021/jo100611kDOI Listing
July 2010

Crossover from single-step tunneling to multistep hopping for molecular triplet energy transfer.

Science 2010 Jun;328(5985):1547-50

Department of Chemistry and Argonne-Northwestern Solar Energy Research (ANSER) Center, Northwestern University, Evanston, IL 60208, USA.

Triplet energy transfer (TT), a key process in molecular and organic electronics, generally occurs by either strongly distance-dependent single-step tunneling or weakly distance-dependent multistep hopping. We have synthesized a series of pi-stacked molecules consisting of a benzophenone donor, one to three fluorene bridges, and a naphthalene acceptor, and studied the rate of TT from benzophenone to naphthalene across the fluorene bridge using femtosecond transient absorption spectroscopy. We show that the dominant TT mechanism switches from tunneling to wire-like hopping between bridge lengths 1 and 2. The crossover observed for TT can be determined by direct observation of the bridge-occupied state.
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http://dx.doi.org/10.1126/science.1189354DOI Listing
June 2010

Direct observation of electron-transfer-induced conformational transformation (molecular actuation) in a bichromophoric electron donor.

J Phys Chem B 2010 Nov 21;114(45):14592-5. Epub 2010 May 21.

Marquette University, Department of Chemistry, P.O. Box 1881, Milwaukee, Wisconsin 53201-1881, USA.

With the aid of laser-flash photolysis, the one-electron oxidation of conformationally mobile tetramethoxydibenzobicyclo[4.4.1]undecane (1), using photoexcited chloranil as an oxidant, allows us to show that extended 1(+•) undergoes a conformational transformation to π-stacked folded 1(+•) on a microsecond time scale (τ ≈ 1 μs), which is at least six times longer than that found for the conformationally locked model compound.
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http://dx.doi.org/10.1021/jp102357wDOI Listing
November 2010
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