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Article ; Online: CRISPR-Cas12a assisted recombinase based strand invading isothermal amplification platform designed for targeted detection of Bacillus anthracis Sterne.

Patnaik, Abhinandan / Rai, Sharad Kumar / Dhaked, Ram Kumar

International journal of biological macromolecules

2024 Volume 263, Issue Pt 1, Page(s) 130216

Abstract: Detection of a pathogen is crucial prior to all prophylaxis and post exposure treatment, as it can prevent further disease manifestation. In this study, we have developed a nucleic acid pre-amplification based CRISPR diagnostic for detection and ... ...

Abstract	Detection of a pathogen is crucial prior to all prophylaxis and post exposure treatment, as it can prevent further disease manifestation. In this study, we have developed a nucleic acid pre-amplification based CRISPR diagnostic for detection and surveillance of Bacillus anthracis Sterne. Strand Invasion Based isothermal Amplification (SIBA) platform and Cas12a (CRISPR endo-nuclease) was used to develop CRISPR-SIBA, a multifaceted diagnostic platform. SIBA was employed as the isothermal pre-amplification platform. CRISPR-Cas12a based collateral trans-cleavage reaction was used to ensure and enhance the specificity of the system. Efficiency of the detection system was evaluated by detecting Bacillus anthracis Sterne in complex wastewater sample backgrounds. Previously reported, Prophage 3, Cya and Pag genes of Bacillus anthracis were used as targets for this assay. The amplification system provided reliable and specific detection readout, with a sensitivity limit of 100 colony forming units in 40 min. The endpoint fluorescence from CRISPR collateral cleavage reactions gave a detection limit of 10
MeSH term(s)	Recombinases/genetics ; Bacillus anthracis/genetics ; CRISPR-Cas Systems/genetics ; Biological Assay ; Deoxyadenosines ; Thionucleosides
Chemical Substances	Recombinases ; 5'-deoxy-5'-S-isobutylthioadenosine (35899-54-8) ; Deoxyadenosines ; Thionucleosides
Language	English
Publishing date	2024-02-18
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	282732-3
ISSN	1879-0003 ; 0141-8130
ISSN (online)	1879-0003
ISSN	0141-8130
DOI	10.1016/j.ijbiomac.2024.130216
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Article: Recent Advancements and Novel Approaches Contributing to the Present Arsenal of Prophylaxis and Treatment Strategies Against Category A Bacterial Biothreat Agents.

Patnaik, Abhinandan / Rai, Sharad Kumar / Dhaked, Ram Kumar

Indian journal of microbiology

2023 Volume 63, Issue 2, Page(s) 161–172

Abstract: Bacterial pathogens have always been a part of the ecosystem in which we thrive. Some pathogens have caused deadly outbreaks in the past and have been exploited as an agent of threat. Natural hotspots for these biological pathogens are widely distributed ...

Abstract	Bacterial pathogens have always been a part of the ecosystem in which we thrive. Some pathogens have caused deadly outbreaks in the past and have been exploited as an agent of threat. Natural hotspots for these biological pathogens are widely distributed throughout the world and hence they remain clinically important. Technological advancement and change in general lifestyle has driven the evolution of these pathogens into more virulent and resistant variants. There has been a growing concern over the development of multidrug-resistant bacterial strains that could be used as bioweapons. This rapid change in pathogens also propels the field of science to develop and innovate new strategies and methodologies which are superior and safer to the existing ones. Some bacterial agents like-
Language	English
Publishing date	2023-05-27
Publishing country	India
Document type	Journal Article ; Review
ZDB-ID	413422-9
ISSN	0973-7715 ; 0046-8991
ISSN (online)	0973-7715
ISSN	0046-8991
DOI	10.1007/s12088-023-01075-8
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Article ; Online: Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis.

Dixit, Shivani / Parashar, Jagrati / Dhaked, Ram Kumar / Kumar, Abdhesh / Saxena, Nandita

International immunopharmacology

2024 Volume 132, Page(s) 111986

Abstract: Background: Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the ... ...

Abstract	Background: Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. Methods: Ricin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin-biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. Results: The LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90-62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. Conclusion: The developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.
MeSH term(s)	Ricin/immunology ; Ricin/analysis ; Enzyme-Linked Immunosorbent Assay/methods ; Animals ; Humans ; Rabbits ; Streptavidin ; Biotin ; Limit of Detection ; Antibodies, Monoclonal/immunology ; Cross Reactions ; Ricinus communis/immunology ; Mice ; Reproducibility of Results ; Seeds/immunology ; Seeds/chemistry
Chemical Substances	Ricin (9009-86-3) ; Streptavidin (9013-20-1) ; Biotin (6SO6U10H04) ; Antibodies, Monoclonal
Language	English
Publishing date	2024-04-03
Publishing country	Netherlands
Document type	Journal Article ; Validation Study
ZDB-ID	2043785-7
ISSN	1878-1705 ; 1567-5769
ISSN (online)	1878-1705
ISSN	1567-5769
DOI	10.1016/j.intimp.2024.111986
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Article ; Online: Silibinin ameliorates abrin induced hepatotoxicity by attenuating oxidative stress, inflammation and inhibiting Fas pathway.

Saxena, Nandita / Dhaked, Ram Kumar / Nagar, D P

Environmental toxicology and pharmacology

2022 Volume 93, Page(s) 103868

Abstract: Abrin is a toxin from the seeds of Abrus precatorius. Abrin is considerably more toxic than ricin and a potent bio-warfare agent. The mechanism of abrin induced hepatotoxicity remains unclear. Silibinin has antioxidant, anti-inflammatory and ... ...

Abstract	Abrin is a toxin from the seeds of Abrus precatorius. Abrin is considerably more toxic than ricin and a potent bio-warfare agent. The mechanism of abrin induced hepatotoxicity remains unclear. Silibinin has antioxidant, anti-inflammatory and hepatoprotective activities. But, its therapeutic potential in abrin toxicity is unknown. In view of these facts, the purpose of this study was to delineate the mechanisms and ameliorative role of silibinin against abrin induced hepatotoxicity. Parameters related to liver functions, oxidative stress, inflammation, Fas pathway and histopathology were evaluated in the liver of BALB/c mice after abrin exposure. Abrin intoxication resulted in hepatotoxicity, oxidative stress, inflammation, altered histopathology and increased Fas pathway signaling. Silibinin improves survival of abrin-exposed mice by decreasing serum liver enzymes and reinstating the antioxidant capacity. Silibinin also inhibits abrin-induced inflammation and Fas pathway. Present study for the first time demonstrates the hepatoprotective potential of silibinin against abrin toxicity.
MeSH term(s)	Abrin/toxicity ; Animals ; Antioxidants/pharmacology ; Chemical and Drug Induced Liver Injury/drug therapy ; Chemical and Drug Induced Liver Injury/metabolism ; Chemical and Drug Induced Liver Injury/prevention & control ; Drug Interactions ; Inflammation/chemically induced ; Inflammation/drug therapy ; Mice ; Oxidative Stress/drug effects ; Signal Transduction/drug effects ; Silybin/pharmacology ; fas Receptor/antagonists & inhibitors ; fas Receptor/metabolism
Chemical Substances	Antioxidants ; Fas protein, mouse ; fas Receptor ; Abrin (1393-62-0) ; Silybin (4RKY41TBTF)
Language	English
Publishing date	2022-04-30
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1318302-3
ISSN	1872-7077 ; 1382-6689
ISSN (online)	1872-7077
ISSN	1382-6689
DOI	10.1016/j.etap.2022.103868
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Article: Immunological detection assays for recombinant Shiga toxin &

Gupta, Pallavi / Dhaked, Ram Kumar

The Indian journal of medical research

2019 Volume 149, Issue 3, Page(s) 412–417

Abstract: Background & objectives: : Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin ... ...

Abstract	Background & objectives: : Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs to be rapid, accurate, reliable and must be extensively evaluated under field conditions. The aim of this study was to develop rapid, sensitive and specific detection method for Stx. Methods: : Mice and rabbits were immunized with purified recombinant Shiga toxin B (rStxB). Using these antibodies dot ELISA, sandwich ELISA and flow through assay were developed. Results: : The high-titre antibodies specifically reacted with purified rStxB. Dot-ELISA, sandwich ELISA and flow-through assay were developed and standardized that could detect StxB with limit of detection (LOD) of 9.75, 9.7 ng/ml and 0.46 μg/cassette, respectively. Interpretation & conclusions: : The rStxB was used to produce antibodies to avoid handling of pathogen. The Flow through assay 'developed was specific, rapid and field amenable.
MeSH term(s)	Animals ; Antibodies, Bacterial/genetics ; Antibodies, Bacterial/immunology ; Arthritis, Reactive/diagnosis ; Arthritis, Reactive/genetics ; Arthritis, Reactive/microbiology ; Dysentery, Bacillary/diagnosis ; Dysentery, Bacillary/genetics ; Dysentery, Bacillary/microbiology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli/genetics ; Hemolytic-Uremic Syndrome/diagnosis ; Hemolytic-Uremic Syndrome/genetics ; Hemolytic-Uremic Syndrome/microbiology ; Humans ; Mice ; Shiga Toxin/genetics ; Shiga Toxin/isolation & purification ; Shigella dysenteriae/genetics ; Shigella dysenteriae/pathogenicity
Chemical Substances	Antibodies, Bacterial ; Shiga Toxin (75757-64-1)
Language	English
Publishing date	2019-06-27
Publishing country	India
Document type	Journal Article
ZDB-ID	390883-5
ISSN	0971-5916 ; 0019-5340
ISSN	0971-5916 ; 0019-5340
DOI	10.4103/ijmr.IJMR_308_17
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Article ; Online: Immunological detection assays for recombinant Shiga toxin & Shigella dysenteriae

Pallavi Gupta / Ram Kumar Dhaked

Indian Journal of Medical Research, Vol 149, Iss 3, Pp 412-

2019 Volume 417

Abstract: Background & objectives: Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs ... ...

Abstract	Background & objectives: Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs to be rapid, accurate, reliable and must be extensively evaluated under field conditions. The aim of this study was to develop rapid, sensitive and specific detection method for Stx. Methods: Mice and rabbits were immunized with purified recombinant Shiga toxin B (rStxB). Using these antibodies dot ELISA, sandwich ELISA and flow through assay were developed. Results: The high-titre antibodies specifically reacted with purified rStxB. Dot-ELISA, sandwich ELISA and flow-through assay were developed and standardized that could detect StxB with limit of detection (LOD) of 9.75, 9.7 ng/ml and 0.46 μg/cassette, respectively. Interpretation & conclusions: The rStxB was used to produce antibodies to avoid handling of pathogen. The Flow through assay 'developed was specific, rapid and field amenable.
Keywords	Anti-Shiga toxin B serum - dot-enzyme-linked immunosorbent assay - flow-through assay - immunological assays - Shiga toxin - Shigella dysenteriae ; Medicine ; R
Language	English
Publishing date	2019-01-01T00:00:00Z
Publisher	Wolters Kluwer Medknow Publications
Document type	Article ; Online
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Article ; Online: Designing, synthesis and evaluation of derived analogues of selected small molecule non-peptidic inhibitors against serotype BoNT/ F.

Chauhan, Ritika / Chauhan Kushwah, Vinita / Agnihotri, Surabhi / Vimal, Manorama / Saxena, Nandita / Dhaked, Ram Kumar

Toxicon : official journal of the International Society on Toxinology

2022 Volume 222, Page(s) 106981

Abstract: Botulinum neurotoxins are lethal Biowarfare categorized in group A of selected agents, by CDC USA. The unavailability of counter-measures against these neurotoxins has been a matter of extensive research. The 8-hydroxyquinoline (8-HQ) scaffold is ... ...

Abstract	Botulinum neurotoxins are lethal Biowarfare categorized in group A of selected agents, by CDC USA. The unavailability of counter-measures against these neurotoxins has been a matter of extensive research. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. We have reported 8-HQ compounds NSC1014 and NSC1011 as potential small molecule inhibitors against BoNT/F. In the present study, analogues of NSC84087 and NSC1014 were designed, synthesized and studied for their inhibitory role against BoNT/F intoxication through in silico study, in vitro and in-vivo assays. ∼25 in-house synthesized small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay and then further assessed through endopeptidase assay. The binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in-vivo efficacy of these compounds was evaluated in mice model. Analogues C87.9, C87.10 and C87.12 of compound NSC84087 and C14.10, C14.11 and C14.13 of NSC1014 showed promising results through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed KD values in sub-micromolar range signifying high affinity interaction. The IC
Language	English
Publishing date	2022-11-26
Publishing country	England
Document type	Journal Article
ZDB-ID	204479-1
ISSN	1879-3150 ; 0041-0101
ISSN (online)	1879-3150
ISSN	0041-0101
DOI	10.1016/j.toxicon.2022.106981
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Article ; Online: E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide targets abrin and ricin toxicity: Hitting two toxins with one stone.

Phatak, Pooja / Chauhan, Vinita / Dhaked, Ram Kumar / Pathak, Uma / Saxena, Nandita

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

2021 Volume 143, Page(s) 112134

Abstract: The efficacy of small molecule inhibitors (SMIs) against the enzymatic activity of Shiga toxin prompted the evaluation of their efficacy on related toxins viz. ricin and abrin. Ricin, like Shiga toxin, is listed as a category B bioweapon and belongs to ... ...

Abstract	The efficacy of small molecule inhibitors (SMIs) against the enzymatic activity of Shiga toxin prompted the evaluation of their efficacy on related toxins viz. ricin and abrin. Ricin, like Shiga toxin, is listed as a category B bioweapon and belongs to the type II family of ribosome inactivating proteins (RIPs). Abrin though structurally and functionally similar to ricin, is considerably more toxic. In the present study, 35 compounds were evaluated in A549 cells in in vitro assays, of which 5 offered protection against abrin and 2 against ricin, with IC
MeSH term(s)	A549 Cells ; Abrin/antagonists & inhibitors ; Abrin/toxicity ; Acrylamides/chemical synthesis ; Acrylamides/pharmacology ; Animals ; Antidotes/chemical synthesis ; Antidotes/pharmacology ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Humans ; Inhibitory Concentration 50 ; Lethal Dose 50 ; Lung/drug effects ; Lung/metabolism ; Lung/pathology ; Male ; Mice, Inbred BALB C ; Poisoning/etiology ; Poisoning/prevention & control ; Protein Biosynthesis/drug effects ; Ricin/antagonists & inhibitors ; Ricin/toxicity ; Mice
Chemical Substances	Acrylamides ; Antidotes ; E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide ; Abrin (1393-62-0) ; Ricin (9009-86-3)
Language	English
Publishing date	2021-08-31
Publishing country	France
Document type	Comparative Study ; Journal Article
ZDB-ID	392415-4
ISSN	1950-6007 ; 0753-3322 ; 0300-0893
ISSN (online)	1950-6007
ISSN	0753-3322 ; 0300-0893
DOI	10.1016/j.biopha.2021.112134
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Article ; Online: Identification of Inhibitors against Botulinum Neurotoxins: 8-Hydroxyquinolines Hold Promise.

Chauhan, Ritika / Chauhan, Vinita / Sonkar, Priyanka / Dhaked, Ram Kumar

Mini reviews in medicinal chemistry

2019 Volume 19, Issue 20, Page(s) 1694–1706

Abstract: Botulinum neurotoxins (BoNTs) are the most toxic category A biological warfare agents. There is no therapeutics available for BoNT intoxication yet, necessitating the development of a medical countermeasure against these neurotoxins. The discovery of ... ...

Abstract	Botulinum neurotoxins (BoNTs) are the most toxic category A biological warfare agents. There is no therapeutics available for BoNT intoxication yet, necessitating the development of a medical countermeasure against these neurotoxins. The discovery of small molecule-based drugs has revolutionized in the last two decades resulting in the identification of several small molecule inhibitors of BoNTs. However, none progressed to clinical trials. 8-Hydroxyquinolines scaffold-based molecules are important 'privileged structures' that can be exploited as inhibitors of a diverse range of targets. In this review, our study of recent reports suggests the development of 8-hydroxyquinoline derived molecules as a potential drug may be on the horizon.
MeSH term(s)	Animals ; Clostridium botulinum/chemistry ; Clostridium botulinum/drug effects ; Humans ; Molecular Structure ; Neurotoxins/antagonists & inhibitors ; Oxyquinoline/chemistry ; Oxyquinoline/pharmacology ; Small Molecule Libraries/chemistry ; Small Molecule Libraries/pharmacology
Chemical Substances	Neurotoxins ; Small Molecule Libraries ; Oxyquinoline (5UTX5635HP)
Language	English
Publishing date	2019-09-19
Publishing country	Netherlands
Document type	Journal Article ; Review
ZDB-ID	2104081-3
ISSN	1875-5607 ; 1389-5575
ISSN (online)	1875-5607
ISSN	1389-5575
DOI	10.2174/1389557519666190906120228
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Article: Squamous Cell Carcinoma Penis in a Case of Urethral Stricture Due to Lichen Sclerosus Balanitis Xerotica Obliterans: A Case Report and Review of Literature.

Taneja, Yogesh / Ram, Priyatama / Dhaked, Santosh Kumar / Sen, Tridib Kumar

Journal of clinical and diagnostic research : JCDR

2017 Volume 11, Issue 7, Page(s) PD17–PD18

Abstract: Penile carcinoma is considered a delayed sequel of lichen sclerosus. It is important to recognize this not so uncommon complication in time as survival of patients with Squamous Cell Carcinoma (SCC) depends on early diagnosis and treatment. We describe a ...

Abstract	Penile carcinoma is considered a delayed sequel of lichen sclerosus. It is important to recognize this not so uncommon complication in time as survival of patients with Squamous Cell Carcinoma (SCC) depends on early diagnosis and treatment. We describe a case of a 49-year-old male presenting with urethral stricture due to lichen sclerosus. He was treated for stricture disease and later on developed SCC penis after ten years of presentation.
Language	English
Publishing date	2017-07-01
Publishing country	India
Document type	Case Reports
ZDB-ID	2775283-5
ISSN	0973-709X ; 2249-782X
ISSN (online)	0973-709X
ISSN	2249-782X
DOI	10.7860/JCDR/2017/26330.10254
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