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Article: Good Laboratory and Research practices-Lecture series and workshop 20-09-14-IISER Mohali.

Thakur, Keshav

2015 Volume 22, Issue 2, Page(s) 119–120

Language	English
Publishing date	2015-06-12
Publishing country	United States
Document type	Journal Article
ZDB-ID	2576191-2
ISSN	0976-3260 ; 0972-7531
ISSN (online)	0976-3260
ISSN	0972-7531
DOI	10.5214/ans.0972.7531.221211
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Article ; Online: Detection of endocrine and metabolism disrupting xenobiotics in milk-derived fat samples by fluorescent protein-tagged nuclear receptors and live cell imaging.

Thakur, Keshav / Goud, Emmagouni Sharath Kumar / Jawa, Yashika / Keswani, Chetan / Onteru, Suneel / Singh, Dheer / Singh, Surya P / Roy, Partha / Tyagi, Rakesh K

Toxicology mechanisms and methods

2022 Volume 33, Issue 4, Page(s) 293–306

Abstract: Nuclear receptors (NRs) are ligand-modulated transcription factors that regulate multiple physiological functions in our body. Many NRs in their unliganded state are localized in the cytoplasm. The ligand-inducible nuclear translocation of NRs provides a ...

Abstract	Nuclear receptors (NRs) are ligand-modulated transcription factors that regulate multiple physiological functions in our body. Many NRs in their unliganded state are localized in the cytoplasm. The ligand-inducible nuclear translocation of NRs provides a valuable tool for studying the NR-ligand interactions and their downstream effects. The translocation response of NRs can be studied irrespective of the nature of the interacting ligand (agonist, antagonist, or a small molecule modulator). These nuclear translocation studies offer an advantage over promoter-reporter-based transcription assays where transcription response is observed only with the activating hormones or agonistic ligands. Globally, milk serves as a major dietary source. However, suspected presence of endocrine/metabolism-disrupting chemicals like bisphenols, parabens, organochlorine pesticides, carbamates, non-steroidal anti-inflammatory drugs, chloramphenicol, brominated flame retardants, etc. has been reported. Considering that these chemicals may impart serious developmental and metabolism-related health concerns, it is essential to develop assays suitable for the detection of xenobiotics present at differing levels in milk. Since milk samples cannot be used directly on cultured cells or for microscopy, a combination of screening strategies has been developed herein based on the revelation that i) lipophilic NR ligands can be successfully retrieved in milk-fat; ii) milk-fat treatment of cells is compatible with live-cell imaging studies; and finally, iii) treatment of cells with xenobiotics-spiked and normal milk derived fat provides a visual and quantifiable response of NR translocation in living cells. Utilizing a milk-fat extraction method and Green Fluorescent Protein (GFP) tagged NRs expressed in cultured mammalian cells, followed by an assessment of NR response proved to be an effective approach for screening xenobiotics present in milk samples.HighlightsDiverse endocrine and metabolism-disrupting chemicals are suspected to contaminate milk.Nuclear receptors serve as 'xenosensors' for assessing the presence of xenobiotics in milk.Nuclear import of steroid receptors with (ant)agonist can be examined in live cells.Lipophilic xenobiotics are extracted and observed enriched in milk-fat fraction.A comprehensive cell-based protocol aids in the detection of xenobiotics in milk.
MeSH term(s)	Animals ; Milk/chemistry ; Milk/metabolism ; Xenobiotics/toxicity ; Ligands ; Receptors, Cytoplasmic and Nuclear ; Receptors, Steroid/metabolism ; Endocrine Disruptors/toxicity ; Endocrine Disruptors/analysis ; Mammals/metabolism
Chemical Substances	Xenobiotics ; Ligands ; Receptors, Cytoplasmic and Nuclear ; Receptors, Steroid ; Endocrine Disruptors
Language	English
Publishing date	2022-10-09
Publishing country	England
Document type	Journal Article
ZDB-ID	2081252-8
ISSN	1537-6524 ; 1537-6516 ; 1051-7235
ISSN (online)	1537-6524
ISSN	1537-6516 ; 1051-7235
DOI	10.1080/15376516.2022.2128704
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Article: Milk metabolites and neurodegeneration: Is there crosstalk?

Thakur, Keshav / Anand, Akshay

Annals of neurosciences

2015 Volume 22, Issue 4, Page(s) 239–243

Abstract: Milk has been considered as a natural source of nutrition for decades. Milk is known to be nutrient-rich which aids the growth and development of the human body. Milk contains both macro- and micronutrients. Breast milk is widely regarded as the optimal ... ...

Abstract	Milk has been considered as a natural source of nutrition for decades. Milk is known to be nutrient-rich which aids the growth and development of the human body. Milk contains both macro- and micronutrients. Breast milk is widely regarded as the optimal source of neonatal nutrition due to its composition of carbohydrates, proteins, minerals and antibodies. However, despite the wide use of milk products, investigations into the role of milk in degenerative diseases have been limited. This review will examine the relationship between the β-casein gene found in bovine milk and disease states by using age-related macular degeneration as an example.
Language	English
Publishing date	2015-10-31
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	2576191-2
ISSN	0976-3260 ; 0972-7531
ISSN (online)	0976-3260
ISSN	0972-7531
DOI	10.5214/ans.0972.7531.220410
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Article: Angiogenesis-Centered Molecular Cross-Talk in Amyotrophic Lateral Sclerosis Survival: Mechanistic Insights.

Thakur, Keshav / Tiwari, Abha / Sharma, Kaushal / Modgil, Shweta / Khosla, Radhika / Anand, Akshay

Critical reviews in eukaryotic gene expression

2020 Volume 30, Issue 2, Page(s) 137–151

Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized with progressive muscle atrophy. We have attempted to establish the link between angiogenesis and cellular survival in the pathogenesis of ALS by compiling evidence ... ...

Abstract	Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized with progressive muscle atrophy. We have attempted to establish the link between angiogenesis and cellular survival in the pathogenesis of ALS by compiling evidence described in various scientific reports. The phenotypes of human ALS have earlier been captured in the mutant SOD1 mice as well as by targeted deletion of the hypoxia response element (HRE) from the promoter of the mouse gene for vascular endothelial growth factor (VEGF). Indirect evidence shows that angiogenesis can help prevent oxidative stress, and hence, enhance cell survival. VEGF and angiogenin chiefly regulate the process of angiogenesis. Transactive response DNA-binding protein 43 (TDP-43) is usually found inside the nucleus, but in large number of cases of ALS, it accumulates in the cytoplasm (TDP-43 proteinopathy). Interestingly, TDP-43 proteinopathy is found to be aggravated in the presence of the OPTN mutation, which is the genetic factor that is responsible for such accumulation. Interaction of TDP-43 with progranulin can further affect the angiogenesis in ALS patients by regulating activity of VEGF receptors, but conclusive evidence is needed to establish its role in pathogenesis of ALS. Certain mutations in UBQLN2 and UBQLN4 indicate that ubiquitination has a role in ALS pathobiology, but its link to angiogenesis has not been adequately studied. Recent studies have shown that several mutations in RNA-binding proteins (RBPs) can also cause ALS. Conclusively, in this review, we have attempted to argue the role of angiogenesis in enhanced ALS survival rate is probably regulated with the activation of NF-κβ. Additionally, interaction between OPTN and TDP-43 can also impact the transcription of various angiogenic molecules. Whether targeting angiogenic substances or TDP-43 can provide clues about extending ALS survival rate, in combination with current treatments, can only be evaluated after additional studies.
Language	English
Publishing date	2020-06-17
Publishing country	United States
Document type	Journal Article
ZDB-ID	1071345-1
ISSN	1045-4403
ISSN	1045-4403
DOI	10.1615/CritRevEukaryotGeneExpr.2020031020
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Article ; Online: Evaluation of (Anti)androgenic Activities of Environmental Xenobiotics in Milk Using a Human Liver Cell Line and Androgen Receptor-Based Promoter-Reporter Assay.

Agrawal, Himanshu / Thakur, Keshav / Mitra, Shreyasi / Mitra, Debarghya / Keswani, Chetan / Sircar, Debabrata / Onteru, Suneel / Singh, Dheer / Singh, Surya P / Tyagi, Rakesh K / Roy, Partha

ACS omega

2022 Volume 7, Issue 45, Page(s) 41531–41547

Abstract: The recent reports on milk consumption and its associated risk with hormone related disorders necessitates the evaluation of dairy products for the presence of endocrine disrupting chemicals (EDCs) and ensure the safety of consumers. In view of this, we ... ...

Abstract	The recent reports on milk consumption and its associated risk with hormone related disorders necessitates the evaluation of dairy products for the presence of endocrine disrupting chemicals (EDCs) and ensure the safety of consumers. In view of this, we investigated the possible presence of (anti)androgenic contaminants in raw and commercialized milk samples. For this purpose, a novel HepARE-Luc cell line that stably expresses human androgen receptor (AR) and the androgen responsive luciferase reporter gene was generated and used in the present study. Treatment of this cell line with androgens and corresponding antiandrogen (flutamide) stimulated or inhibited expression of reporter luciferase, respectively. Real time polymerase chain reaction and immunostaining results exhibited transcription response and translocation of AR from the cytoplasm to the nucleus in response to androgen. Observations implied that a cell-based xenobiotic screening assay
Language	English
Publishing date	2022-11-04
Publishing country	United States
Document type	Journal Article
ISSN	2470-1343
ISSN (online)	2470-1343
DOI	10.1021/acsomega.2c05344
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Article: Can Cheiromancy Predict Mean Survival or Fatality of a Patient with Amyotrophic Lateral Sclerosis?

Anand, Akshay / Prabhakar, Sudesh / Thakur, Keshav / Tyagi, Rahul / Shastri, Vinod / Tripathi, Devi Prasad / Sharma, Suresh

Journal of neurosciences in rural practice

2020 Volume 11, Issue 2, Page(s) 256–260

Abstract: ... ...

Abstract	Background
Language	English
Publishing date	2020-05-02
Publishing country	United States
Document type	Journal Article
ZDB-ID	2601242-X
ISSN	0976-3155 ; 0976-3147
ISSN (online)	0976-3155
ISSN	0976-3147
DOI	10.1055/s-0040-1703969
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Article ; Online: Identifying putative cerebrospinal fluid biomarkers of amyotrophic lateral sclerosis in a north Indian population.

Khosla, Radhika / Rain, Manjari / Chawathey, Shreyas / Modgil, Shweta / Tyagi, Rahul / Thakur, Keshav / Pannu, Viraaj / Sharma, Suresh Kumar / Anand, Akshay

Muscle & nerve

2020 Volume 62, Issue 4, Page(s) 528–533

Abstract: Introduction: Evidence-based information about cerebrospinal fluid (CSF) levels of biomarkers in patients with amyotrophic lateral sclerosis (ALS) is limited.: Methods: Vascular endothelial growth factor (VEGF) and its receptor vascular endothelial ... ...

Abstract	Introduction: Evidence-based information about cerebrospinal fluid (CSF) levels of biomarkers in patients with amyotrophic lateral sclerosis (ALS) is limited. Methods: Vascular endothelial growth factor (VEGF) and its receptor vascular endothelial growth factor receptor 2 (VEGFR2), optineurin (OPTN), monocyte chemoattractant protein-1 (MCP-1), angiogenin (ANG), and TAR DNA-binding protein (TDP-43) were quantified by enzyme-linked immunoassay in the CSF of 54 patients with sporadic ALS and 32 controls in a case-control study design. Results: CSF levels of VEGF (P = .014) and ANG (P = .009) were decreased, whereas VEGFR2 was higher (P = .002) in patients with ALS than in controls. TDP-43 positively correlated with MCP-1 (P = .003), VEGF (P < .001), and VEGFR2 (P < .001) in patients with ALS. Discussion: Our findings suggest possible utility of VEGF, VEGFR2, and ANG as biomarkers for use in ALS treatment trials.
MeSH term(s)	Adult ; Amyotrophic Lateral Sclerosis/cerebrospinal fluid ; Amyotrophic Lateral Sclerosis/diagnosis ; Biomarkers/cerebrospinal fluid ; Case-Control Studies ; Cell Cycle Proteins/cerebrospinal fluid ; Chemokine CCL2/cerebrospinal fluid ; DNA-Binding Proteins/cerebrospinal fluid ; Female ; Humans ; India ; Male ; Membrane Transport Proteins/cerebrospinal fluid ; Middle Aged ; Ribonuclease, Pancreatic/cerebrospinal fluid ; Vascular Endothelial Growth Factor A/cerebrospinal fluid ; Vascular Endothelial Growth Factor Receptor-2/cerebrospinal fluid
Chemical Substances	Biomarkers ; CCL2 protein, human ; Cell Cycle Proteins ; Chemokine CCL2 ; DNA-Binding Proteins ; Membrane Transport Proteins ; OPTN protein, human ; TARDBP protein, human ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factor Receptor-2 (EC 2.7.10.1) ; angiogenin (EC 3.1.27.-) ; Ribonuclease, Pancreatic (EC 3.1.27.5)
Language	English
Publishing date	2020-08-13
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	438353-9
ISSN	1097-4598 ; 0148-639X
ISSN (online)	1097-4598
ISSN	0148-639X
DOI	10.1002/mus.27026
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Article: Revisiting the dilution factor as vital parameter for sensitivity of ELISA assay in CSF and Plasma.

Thakur, Keshav / Sharma, Suresh / Prabhakar, Sudesh / Gupta, Pawan / Anand, Akshay

Annals of neurosciences

2015 Volume 22, Issue 1, Page(s) 37–42

Abstract: Background: Enzyme Linked Immunosorbent Assay (ELISA) is very sensitive assay which provides quantitative data about expression of antigens. However, its utility is based on certain parameters which vary in the experimental situations.: Purpose: We ... ...

Abstract	Background: Enzyme Linked Immunosorbent Assay (ELISA) is very sensitive assay which provides quantitative data about expression of antigens. However, its utility is based on certain parameters which vary in the experimental situations. Purpose: We aimed to analyse the dilution factor as an important parameter for determining the sensitivity of ELISA in human samples. Methods: Total of n = 57 ALS patients and n = 48 normal controls were selected for the study. All the patients were recruited from, Department for Neurology and Anaesthesia, PGIMER. Blood and CSF sample was collected and ELISA run was performed in both plasma and blood sample. ELISA of OPTN and TDP-43 was employed to check the respective protein concentration in CSF and Plasma. Results: There was no significant difference which was reported for Plasma as well as CSF values of TDP-43 and OPTN. Dilution test prior to actual experiment made a significant impact in deciding the actual concentration of sample and led to overshootingbeyond range of reference protein. Conclusion: Negative results from our study highlights the significance of determining the dilution factor as an important parameter for conduct of ELISA.
Language	English
Publishing date	2015-06-29
Publishing country	United States
Document type	Journal Article
ZDB-ID	2576191-2
ISSN	0976-3260 ; 0972-7531
ISSN (online)	0976-3260
ISSN	0972-7531
DOI	10.5214/ans.0972.7531.220108
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Article ; Online: ALS and oxidative stress: the neurovascular scenario.

Anand, Akshay / Thakur, Keshav / Gupta, Pawan Kumar

Oxidative medicine and cellular longevity

2013 Volume 2013, Page(s) 635831

Abstract: Oxidative stress and angiogenic factors have been placed as the prime focus of scientific investigations after an establishment of link between vascular endothelial growth factor promoter (VEGF), hypoxia, and amyotrophic lateral sclerosis (ALS) ... ...

Abstract	Oxidative stress and angiogenic factors have been placed as the prime focus of scientific investigations after an establishment of link between vascular endothelial growth factor promoter (VEGF), hypoxia, and amyotrophic lateral sclerosis (ALS) pathogenesis. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter and mutant superoxide dismutase 1 (SOD1) which are characterised by atrophy and muscle weakness resulted in phenotype resembling human ALS in mice. This results in lower motor neurodegeneration thus establishing an important link between motor neuron degeneration, vasculature, and angiogenic molecules. In this review, we have presented human, animal, and in vitro studies which suggest that molecules like VEGF have a therapeutic, diagnostic, and prognostic potential in ALS. Involvement of vascular growth factors and hypoxia response elements also highlights the converging role of oxidative stress and neurovascular network for understanding and treatment of various neurodegenerative disorders like ALS.
MeSH term(s)	Amyotrophic Lateral Sclerosis/pathology ; Animals ; Blood-Brain Barrier/metabolism ; Blood-Brain Barrier/pathology ; Humans ; Motor Neurons/metabolism ; Motor Neurons/pathology ; Nervous System/blood supply ; Nervous System/pathology ; Oxidative Stress ; Vascular Endothelial Growth Factor A/metabolism
Chemical Substances	Vascular Endothelial Growth Factor A
Language	English
Publishing date	2013-12-03
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	2455981-7
ISSN	1942-0994 ; 1942-0994
ISSN (online)	1942-0994
ISSN	1942-0994
DOI	10.1155/2013/635831
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Article: Vascular endothelial growth factor and monocyte chemoattractant protein-1 levels unaltered in symptomatic atherosclerotic carotid plaque patients from north India.

Khurana, Dheeraj / Mathur, Deepali / Prabhakar, Sudesh / Thakur, Keshav / Anand, Akshay

Frontiers in neurology

2013 Volume 4, Page(s) 27

Abstract: We aimed to identify the role of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein (MCP-1) as a serum biomarker of symptomatic carotid atherosclerotic plaque in North Indian population. Individuals with symptomatic carotid ... ...

Abstract	We aimed to identify the role of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein (MCP-1) as a serum biomarker of symptomatic carotid atherosclerotic plaque in North Indian population. Individuals with symptomatic carotid atherosclerotic plaque have high risk of ischemic stroke. Previous studies from western countries have shown an association between VEGF and MCP-1 levels and the incidence of ischemic stroke. In this study, venous blood from 110 human subjects was collected, 57 blood samples of which were obtained from patients with carotid plaques, 38 neurological controls without carotid plaques, and another 15 healthy controls who had no history of serious illness. Serum VEGF and MCP-1 levels were measured using commercially available enzyme-linked immunosorbent assay. We also correlated the data clinically and carried out risk factor analysis based on the detailed questionnaire obtained from each patient. For risk factor analysis, a total of 70 symptomatic carotid plaque cases and equal number of age and sex matched healthy controls were analyzed. We found that serum VEGF levels in carotid plaque patients did not show any significant change when compared to either of the controls. Similarly, there was no significant upregulation of MCP-1 in the serum of these patients. The risk factor analysis revealed that hypertension, diabetes, and physical inactivity were the main correlates of carotid atherosclerosis (p < 0.05). Prevalence of patients was higher residing in urban areas as compared to rural region. We also found that patients coming from mountain region were relatively less vulnerable to cerebral atherosclerosis as compared to the ones residing at non mountain region. On the contrary, smoking, obesity, dyslipidemia, alcohol consumption, and tobacco chewing were not observed as the determinants of carotid atherosclerosis risk in North India (p > 0.05). We conclude that the pathogenesis of carotid plaques may progress independent of these inflammatory molecules. In parallel, risk factor analysis indicates hypertension, diabetes, and sedentary lifestyle as the most significant risk factors of ischemic stroke identified in North India. This could be helpful in early identification of subjects at risk for stroke and devising health care strategies.
Language	English
Publishing date	2013-04-02
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2564214-5
ISSN	1664-2295
ISSN	1664-2295
DOI	10.3389/fneur.2013.00027
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