Dr. Nidhi Puranik | Yeungnam University (original) (raw)

Papers by Dr. Nidhi Puranik

Current Pharmaceutical Design

: Multiple sclerosis (MS) is a central nervous system (CNS) immune-mediated disease that mainly s... more : Multiple sclerosis (MS) is a central nervous system (CNS) immune-mediated disease that mainly strikes young adults and leaves them disabled. MS is an autoimmune illness that causes the immune system to attack the brain and spinal cord. The myelin sheaths, which insulate the nerve fibers, are harmed by our own immune cells, and this interferes with brain signal transmission. Numbness, tingling, mood swings, memory problems, exhaustion, agony, vision problems, and/or paralysis are just a few of the symptoms. Despite technological advancements and significant research efforts in recent years, diagnosing MS can still be difficult. Each patient's MS is distinct due to a heterogeneous and complex pathophysiology with diverse types of disease courses. There is a pressing need to identify markers that will allow for more rapid and accurate diagnosis and prognosis assessments to choose the best course of treatment for each MS patient. The cerebrospinal fluid (CSF) is an excellent source of particular indicators associated with MS pathology. CSF contains molecules that represent pathological processes such as inflammation, cellular damage, and loss of blood-brain barrier integrity. Oligoclonal bands, neurofilaments, MS-specific miRNA, lncRNA, IgG-index, and anti-aquaporin 4 antibodies are all clinically utilised indicators for CSF in MS diagnosis. In recent years, a slew of new possible biomarkers have been presented. In this review, we look at what we know about CSF molecular markers and how they can aid in the diagnosis and differentiation of different MS forms and treatment options, and monitoring and predicting disease progression, therapy response, and consequences during such opportunistic infections.

IOP Publishing eBooks, May 1, 2023

IOP Publishing eBooks, May 1, 2023

IOP Publishing eBooks, May 1, 2023

Paper-Based Diagnostic Devices for Infectious Diseases

For improved cost-effectiveness and temperaturestability, a ready to use lateral flow assay (LFA)... more For improved cost-effectiveness and temperaturestability, a ready to use lateral flow assay (LFA) is developed in this work for detecting inflammation/infection biomarker C-reactive protein (CRP) in human patient samples on the basis of aptamers. In prescreening investigations, an aptamer with CRP affinity was immobilized on microarray chips in forward and sandwich formats to optimize assay conditions. We suggest these microarray techniques as a resource-sparing and fast-screening instrument for evaluation of various conditions. The capability of the aptamer to detect CRP was shown. Optimized assay conditions were consequently transferred to the LFA-platform. Here we could demonstrate for the first time an aptamer-based LFA for the detection of CRP in human patient samples in pathologically relevant concentrations. The cutoff for CRP detection is set at 10 mg/L, providing a distinctive "yes" (≥10 mg/L CRP) or "no" (<10 mg/L CRP) answer for the patient. The resulting aptamer-based LFA is promising with regard to its application as point-of-care testing (POCT) for efficient monitoring, especially of patients affected by frequent infections or inflammations.

International Journal of Nanomedicine

Although various treatments are currently being developed, lung cancer still has a very high mort... more Although various treatments are currently being developed, lung cancer still has a very high mortality rate. Moreover, while various strategies for the diagnosis and treatment of lung cancer are being used in clinical settings, in many cases, lung cancer does not respond to treatment and presents reducing survival rates. Cancer nanotechnology, also known as nanotechnology in cancer, is a relatively new topic of study that brings together scientists from a variety of fields, including chemistry, biology, engineering, and medicine. The use of lipid-based nanocarriers to aid drug distribution has already had a significant impact in several scientific fields. Lipid-based nanocarriers have been demonstrated to help stabilize therapeutic compounds, overcome barriers to cellular and tissue absorption, and improve in vivo drug delivery to specific target areas. For this reason, lipid-based nanocarriers are being actively researched and used for lung cancer treatment and vaccine development. This review discusses the improvements in drug delivery achieved with lipid-based nanocarriers, the obstacles that still exist with in vivo applications, and the current clinical and experimental applications of lipid-based nanocarriers in lung cancer treatment and management.

Endocrine, Metabolic & Immune Disorders - Drug Targets

: Diabetes mellitus (DM) is the most common metabolic disorder that occurs due to the loss, or im... more : Diabetes mellitus (DM) is the most common metabolic disorder that occurs due to the loss, or impaired function of insulin‐secreting pancreatic beta cells, which are of two types - type 1 (T1D) and type 2 (T2D). To cure DM, the replacement of the destroyed pancreatic beta cells of islet of Langerhans is the most widely practiced treatment. For this, isolating neuronal stem cells and cultivating them as a source of renewable beta cells is a significant breakthrough in medicine. The functions, growth, and gene expression of insulin-producing pancreatic beta cells and neurons are very similar in many ways. A diabetic patient's neural stem cells (obtained from the hippocampus and olfactory bulb) can be used as a replacement source of beta cells for regenerative therapy to treat diabetes. The same protocol used to create functional neurons from progenitor cells can be used to create beta cells. Recent research suggests that replacing lost pancreatic beta cells with autologous transplantation of insulin-producing neural progenitor cells may be a perfect therapeutic strategy for diabetes, allowing for a safe and normal restoration of function and a reduction in potential risks and a long-term cure.

archives of razi institute, Sep 26, 2021

Wound infections are among public health problems worldwide. However, progress has been made in i... more Wound infections are among public health problems worldwide. However, progress has been made in improving surgical techniques and antibiotic treatments. Misuse/overuse of antibiotics to prevent and treat bacterial infections eventually leads to increased bacterial resistance with rising incidences of multi-drug resistant (MDR) bacterial strains. The wider dissemination of antibiotics may ultimately result in ineffectiveness to antibiotic therapy, thereby complicating/graving the outcome of a patient. In the present study, a 60-year-old male patient having wound infection with MDR bacterium that ultimately required surgical amputation of the toe was investigated. For the confirmation of MDR bacterium, two culture media viz., MacConkeyAgar and Mueller Hinton Agar media were used. The sensitivity of the isolated strain for various antibiotics was tested using the disc diffusion method. The wound sample was found positive for Gram-positive bacterium that was identified as Clostridium perfringens. The bacterium was screened for 40 antibiotics, and among all the antibiotics, it was found sensitive for only Piperacillin/Tazobactam antibiotic combination. C. perfringens bacterium caused the gas gangrene in the infected wound part of the patient. Amputation of the gangreneaffected foot part was performed by surgery, and with good medical care, the person recovered fast. To the best of our knowledge, this is the first-ever report of MDRC. perfringens single isolate harboring resistance against at least 40 antibiotics tested. More research is needed to develop really new and effective medicines that do not cross-react with antibiotics now in use and have robust activity against MDR organisms.

International Journal of Infectious Diseases, 2016

antigen (PGA) with nanoparticlessandwich ELISA, compared to the traditional sandwich ELISA Result... more antigen (PGA) with nanoparticlessandwich ELISA, compared to the traditional sandwich ELISA Results: Sandwich ELISA achieved sensitivity of 93%, specificity of 92.5%, positive predictive value (PPV) of 95.7% and negativepredictive value (NPV) of 88%, while nano-sandwich ELISA achieved sensitivity, specificity, PPV and NPV of 95.8%, 95%, 97.2% and 92.6%, respectively. Conclusion: In conclusion, our research study provides that nano-sandwich ELISA isa well-established reference test for diagnosis of giardiasis.

International Journal of Infectious Diseases, 2016

Background: Anthrax, a disease of bioterrorism and public health importance is caused by the Gram... more Background: Anthrax, a disease of bioterrorism and public health importance is caused by the Gram positive, spore-forming bacterium, Bacillus anthracis. Anthrax toxin, a tripartite toxin is composed of protective antigen (PA), lethal factor (LF) or edema factor (EF). PA is the major protein which facilitates the entry of toxin component either of lethal factor or edema factor. Recombinant PA has been a suitable target for anthrax vaccine worldwide. However, instead of full PA, its domains are reported to provide protection. LF also contributes to immuno-protection against anthrax. Therefore, in this study, a chimeric protein consisting of both, PA and LF was developed as candidate vaccine for anthrax. Methods & Materials: A chimera was made by fusion of immunodominant portion of PA (Domains 2-4) and LF (Domain 1) genes.. The construct was cloned in pET32a+ vector and expressed in E. coli host. The recombinant chimeric protein was purified by immobilized metal affinity chromatography. The 4-6 week old Balb/c mice were injected intraperitonealy with three doses of chimeric protein (20 g each mouse) at two week interval. The first dose was given with Freund's complete adjuvant and the subesequent doses were given with incomplete Freund's adjuvant. The serum IgG and its subtypes were determined by plate ELISA. Results: The chimeric protein (PA-LF) was purified up to homogeneity and the production yield was 15 mg/l of the shake flask culture. The chimera elicited good immune response against both the toxins i.e.

IOP Publishing eBooks, May 1, 2023

Journal of Experimental Agriculture International, 2022

Chickpea is the second most significant legume grain, growing in 14.80 mhs regions all over the w... more Chickpea is the second most significant legume grain, growing in 14.80 mhs regions all over the world. It contributes roughly 14.24 million people to the global food basket each year. Desi and Kabuli chickpeas are widely grown in India, and both cultivars have distinct physiochemical characteristics such as seed size and shape, hydration capacity, coat thickness, and protein content. The genetic basis of chickpea cultivars has been limited as a result of agricultural techniques. Molecular markers, on the other hand, have proven to be useful tools for accurate quantification and characterisation within plant species. Markers can be an excellent way to improve chickpeas quantitatively because they are a high source of protein and constitute 80% of total dry weight. Markers such as RAPD (Random amplified polymeric DNA), SSR (Single sequence repeat), AFLP (amplified fragment length polymorphism) and ISSR (Inter simple sequence repeat) have aided in improving breeding efficiency and thus...

Journal of Bioterrorism and Biodefense, 2020

Anthrax, a disease of public health as well as bio-warfare potential is caused by Gram positive, ... more Anthrax, a disease of public health as well as bio-warfare potential is caused by Gram positive, non-motile and spore forming Bacillus Anthracis. Bacteraemia and toxaemia of B. anthracis is responsible to cause lethality in anthrax-infected host. In this study, GroEL (HSP60) gene was cloned in pET28a+ vector and expressed in heterologous system E. coli. Over expressed recombinant GroEL was purified by IMAC chromatography and analysed on 10% SDS-PAGE. In western blot analysis, the rGroEL reacted with mouse anti-his antibody and anti-B. anthracis Sterne mouse serum. The immunogenicity of GroEL with aluminium hydroxide was evaluated in BALB/c mouse model. The endpoint titer of anti-rGroEL circulating antibodies in mouse serum was found high by indirect ELISA after first and second booster doses. IgG1 and IgG2b isotypes were found dominating in comparison to other isotypes. Humoral immune response generated by rGroEL indicated that it is immuno-dominant antigen of B. anthracis, which co...

Defence Life Science Journal, 2020

Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as p... more Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as public health point of view. The virulence factors of B. anthracis are encoded by the two plasmids, pXO1 and pXO2. Protective antigen (PA), an 83 kDa protein encoded by pXO1 along with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa), makes the anthrax toxin responsible for causing the disease. Current detection and diagnostic systems for anthrax are mostly based on PA, a potential biomarker of B. anthracis. The objective of the present study was to produce and purify the PA for development of a sandwich ELISA for its detection. In this study, pYS5 plasmid containing the full PA gene was transformed into an 8 proteases deficient Bacillus anthracis host BH480. The PA was produced under shake flask conditions and purified using the gel filtration chromatography. The reactivity of PA with rabbit and mouse anti-PA antibodies was confirmed by Western blotting. The antibodies were purifi...

Defence Life Science Journal, 2020

Detection of spores of Bacillus anthracis, the causative agent of anthrax in human and animals in... more Detection of spores of Bacillus anthracis, the causative agent of anthrax in human and animals in environment is cumbersome due to the presence of spores of other closely related Bacillus species. The Extractable Antigen 1 (EA1), an extracellular protein is considered as a biomarker for detection of B. anthracis spores. In the present work, we have cloned and expressed the recombinant EA1 protein in soluble form in Escherichia coli. Optimisation of culture conditions and cultivation media was carried out to achieve enhanced soluble expression of recombinant EA1 protein. Further, the batch fermentation process was also developed using optimised conditions for scale up production of recombinant EA1 protein. The final yield of protein purified employing affinity chromatography was 42.64 mg/l of culture during batch fermentation process. The polyclonal antibodies were raised against recombinant EA in rabbit and mice and used to develop an ELISA for detection of B. anthracis spores. The ...

Journal of Applied Microbiology, 2020

Category A classified Bacillus anthracis is highly fatal pathogen that causes anthrax and creates... more Category A classified Bacillus anthracis is highly fatal pathogen that causes anthrax and creates challenges for global security and public health. In this study, development of a safe and ideal next‐generation subunit anthrax vaccine has been evaluated in mouse model.

Defence Life Science Journal, 2019

Protective antigen (PA) produced by Bacillus anthracis is a highly immunogenic protein. Therefore... more Protective antigen (PA) produced by Bacillus anthracis is a highly immunogenic protein. Therefore, it has significant importance in serodiagnosis as well as a vaccine candidate for anthrax. In the present study, codons for PA gene were optimised and synthesised for its expression in Escherichia coli. Various expression conditions were optimised for scaled up production of rPA. The final yield of affinity chromatography purified protein was 40.8 mg/l during batch fermentation. For further purification, affinity purified protein was diafiltered and subjected to anion exchange chromatography. SDS-PAGE and Western blot was used to characterise the purified rPA protein. The diagnostic potential of purified rPA was evaluated in Western blot using standards reference serum AVR 801 and cutaneous anthrax clinical sera. The results of the present study established the optimum production of rPA in E. coli after codon optimisation for its subsequent use in diagnosis of anthrax infection.

Defence Science Journal, 2019

Anthrax, caused by Bacillus anthracis is an important disease of biowarfare and public health imp... more Anthrax, caused by Bacillus anthracis is an important disease of biowarfare and public health importance. It is imperative to develop a simple system which can detect and differentiate B. anthracis from other closely related species. The surface array protein (Sap), which is secreted during the early growth phase of bacteria can be an important biomarker for detection of B. anthracis. In the present study, we have developed a rapid flow through membrane ELISA for detection of B. anthracis. Polyclonal antibodies were used to develop a sandwich plate ELISA, which could detect 3.9 ng/ml of recombinant Sap. B. anthracis bacteria grown in culture broth could be detected after 5 h of growth. Finally, a rapid flow through membrane ELISA was developed which can be accomplished just within 2 minutes, instead of 3-4 h as required in sandwich plate ELISA. The results established that the developed flow through membrane ELISA may be used for detection of B. anthracis. The proposed method is rap...

Biologicals, 2019

In this study, an ELISA was developed for simultaneous detection of antibodies against both the i... more In this study, an ELISA was developed for simultaneous detection of antibodies against both the important toxins of B. anthracis i.e. protective antigen (PA) and lethal factor (LF). A chimera of PA and LF was made by fusion and cloned and expressed in E. coli. The purified recombinant protein was used in plate ELISA for serodiagnosis of anthrax. The chimera could detect antibodies against both the toxins of Bacillus anthracis. The human serum samples (n = 98) collected from anthrax endemic and non-endemic areas were tested employing ELISA. The ELISA gave sensitivity of 100% (95% Confidence Interval [CI], 92.13 to 100) and specificity of 97.78% (95% Confidence Interval [CI], 88.23 to 99.94) with a J index of 0.97. The efficiency of ELISA was found to be 98.9% with the positive predictive value (PPV) and negative predictive value (NPV) of 97.8% and 100%, respectively. The chimera of PA and LF could be a better diagnostic antigen for serodiagnosis as the assay detects antibodies against both the toxins in early as well delayed infection cases of anthrax. Therefore, it can be a very useful tool for the surveillance as well as for confirmation of cutaneous anthrax cases.

Current Pharmaceutical Design

: Multiple sclerosis (MS) is a central nervous system (CNS) immune-mediated disease that mainly s... more : Multiple sclerosis (MS) is a central nervous system (CNS) immune-mediated disease that mainly strikes young adults and leaves them disabled. MS is an autoimmune illness that causes the immune system to attack the brain and spinal cord. The myelin sheaths, which insulate the nerve fibers, are harmed by our own immune cells, and this interferes with brain signal transmission. Numbness, tingling, mood swings, memory problems, exhaustion, agony, vision problems, and/or paralysis are just a few of the symptoms. Despite technological advancements and significant research efforts in recent years, diagnosing MS can still be difficult. Each patient's MS is distinct due to a heterogeneous and complex pathophysiology with diverse types of disease courses. There is a pressing need to identify markers that will allow for more rapid and accurate diagnosis and prognosis assessments to choose the best course of treatment for each MS patient. The cerebrospinal fluid (CSF) is an excellent source of particular indicators associated with MS pathology. CSF contains molecules that represent pathological processes such as inflammation, cellular damage, and loss of blood-brain barrier integrity. Oligoclonal bands, neurofilaments, MS-specific miRNA, lncRNA, IgG-index, and anti-aquaporin 4 antibodies are all clinically utilised indicators for CSF in MS diagnosis. In recent years, a slew of new possible biomarkers have been presented. In this review, we look at what we know about CSF molecular markers and how they can aid in the diagnosis and differentiation of different MS forms and treatment options, and monitoring and predicting disease progression, therapy response, and consequences during such opportunistic infections.

IOP Publishing eBooks, May 1, 2023

IOP Publishing eBooks, May 1, 2023

IOP Publishing eBooks, May 1, 2023

Paper-Based Diagnostic Devices for Infectious Diseases

For improved cost-effectiveness and temperaturestability, a ready to use lateral flow assay (LFA)... more For improved cost-effectiveness and temperaturestability, a ready to use lateral flow assay (LFA) is developed in this work for detecting inflammation/infection biomarker C-reactive protein (CRP) in human patient samples on the basis of aptamers. In prescreening investigations, an aptamer with CRP affinity was immobilized on microarray chips in forward and sandwich formats to optimize assay conditions. We suggest these microarray techniques as a resource-sparing and fast-screening instrument for evaluation of various conditions. The capability of the aptamer to detect CRP was shown. Optimized assay conditions were consequently transferred to the LFA-platform. Here we could demonstrate for the first time an aptamer-based LFA for the detection of CRP in human patient samples in pathologically relevant concentrations. The cutoff for CRP detection is set at 10 mg/L, providing a distinctive "yes" (≥10 mg/L CRP) or "no" (<10 mg/L CRP) answer for the patient. The resulting aptamer-based LFA is promising with regard to its application as point-of-care testing (POCT) for efficient monitoring, especially of patients affected by frequent infections or inflammations.

International Journal of Nanomedicine

Although various treatments are currently being developed, lung cancer still has a very high mort... more Although various treatments are currently being developed, lung cancer still has a very high mortality rate. Moreover, while various strategies for the diagnosis and treatment of lung cancer are being used in clinical settings, in many cases, lung cancer does not respond to treatment and presents reducing survival rates. Cancer nanotechnology, also known as nanotechnology in cancer, is a relatively new topic of study that brings together scientists from a variety of fields, including chemistry, biology, engineering, and medicine. The use of lipid-based nanocarriers to aid drug distribution has already had a significant impact in several scientific fields. Lipid-based nanocarriers have been demonstrated to help stabilize therapeutic compounds, overcome barriers to cellular and tissue absorption, and improve in vivo drug delivery to specific target areas. For this reason, lipid-based nanocarriers are being actively researched and used for lung cancer treatment and vaccine development. This review discusses the improvements in drug delivery achieved with lipid-based nanocarriers, the obstacles that still exist with in vivo applications, and the current clinical and experimental applications of lipid-based nanocarriers in lung cancer treatment and management.

Endocrine, Metabolic & Immune Disorders - Drug Targets

: Diabetes mellitus (DM) is the most common metabolic disorder that occurs due to the loss, or im... more : Diabetes mellitus (DM) is the most common metabolic disorder that occurs due to the loss, or impaired function of insulin‐secreting pancreatic beta cells, which are of two types - type 1 (T1D) and type 2 (T2D). To cure DM, the replacement of the destroyed pancreatic beta cells of islet of Langerhans is the most widely practiced treatment. For this, isolating neuronal stem cells and cultivating them as a source of renewable beta cells is a significant breakthrough in medicine. The functions, growth, and gene expression of insulin-producing pancreatic beta cells and neurons are very similar in many ways. A diabetic patient's neural stem cells (obtained from the hippocampus and olfactory bulb) can be used as a replacement source of beta cells for regenerative therapy to treat diabetes. The same protocol used to create functional neurons from progenitor cells can be used to create beta cells. Recent research suggests that replacing lost pancreatic beta cells with autologous transplantation of insulin-producing neural progenitor cells may be a perfect therapeutic strategy for diabetes, allowing for a safe and normal restoration of function and a reduction in potential risks and a long-term cure.

archives of razi institute, Sep 26, 2021

Wound infections are among public health problems worldwide. However, progress has been made in i... more Wound infections are among public health problems worldwide. However, progress has been made in improving surgical techniques and antibiotic treatments. Misuse/overuse of antibiotics to prevent and treat bacterial infections eventually leads to increased bacterial resistance with rising incidences of multi-drug resistant (MDR) bacterial strains. The wider dissemination of antibiotics may ultimately result in ineffectiveness to antibiotic therapy, thereby complicating/graving the outcome of a patient. In the present study, a 60-year-old male patient having wound infection with MDR bacterium that ultimately required surgical amputation of the toe was investigated. For the confirmation of MDR bacterium, two culture media viz., MacConkeyAgar and Mueller Hinton Agar media were used. The sensitivity of the isolated strain for various antibiotics was tested using the disc diffusion method. The wound sample was found positive for Gram-positive bacterium that was identified as Clostridium perfringens. The bacterium was screened for 40 antibiotics, and among all the antibiotics, it was found sensitive for only Piperacillin/Tazobactam antibiotic combination. C. perfringens bacterium caused the gas gangrene in the infected wound part of the patient. Amputation of the gangreneaffected foot part was performed by surgery, and with good medical care, the person recovered fast. To the best of our knowledge, this is the first-ever report of MDRC. perfringens single isolate harboring resistance against at least 40 antibiotics tested. More research is needed to develop really new and effective medicines that do not cross-react with antibiotics now in use and have robust activity against MDR organisms.

International Journal of Infectious Diseases, 2016

antigen (PGA) with nanoparticlessandwich ELISA, compared to the traditional sandwich ELISA Result... more antigen (PGA) with nanoparticlessandwich ELISA, compared to the traditional sandwich ELISA Results: Sandwich ELISA achieved sensitivity of 93%, specificity of 92.5%, positive predictive value (PPV) of 95.7% and negativepredictive value (NPV) of 88%, while nano-sandwich ELISA achieved sensitivity, specificity, PPV and NPV of 95.8%, 95%, 97.2% and 92.6%, respectively. Conclusion: In conclusion, our research study provides that nano-sandwich ELISA isa well-established reference test for diagnosis of giardiasis.

International Journal of Infectious Diseases, 2016

Background: Anthrax, a disease of bioterrorism and public health importance is caused by the Gram... more Background: Anthrax, a disease of bioterrorism and public health importance is caused by the Gram positive, spore-forming bacterium, Bacillus anthracis. Anthrax toxin, a tripartite toxin is composed of protective antigen (PA), lethal factor (LF) or edema factor (EF). PA is the major protein which facilitates the entry of toxin component either of lethal factor or edema factor. Recombinant PA has been a suitable target for anthrax vaccine worldwide. However, instead of full PA, its domains are reported to provide protection. LF also contributes to immuno-protection against anthrax. Therefore, in this study, a chimeric protein consisting of both, PA and LF was developed as candidate vaccine for anthrax. Methods & Materials: A chimera was made by fusion of immunodominant portion of PA (Domains 2-4) and LF (Domain 1) genes.. The construct was cloned in pET32a+ vector and expressed in E. coli host. The recombinant chimeric protein was purified by immobilized metal affinity chromatography. The 4-6 week old Balb/c mice were injected intraperitonealy with three doses of chimeric protein (20 g each mouse) at two week interval. The first dose was given with Freund's complete adjuvant and the subesequent doses were given with incomplete Freund's adjuvant. The serum IgG and its subtypes were determined by plate ELISA. Results: The chimeric protein (PA-LF) was purified up to homogeneity and the production yield was 15 mg/l of the shake flask culture. The chimera elicited good immune response against both the toxins i.e.

IOP Publishing eBooks, May 1, 2023

Journal of Experimental Agriculture International, 2022

Chickpea is the second most significant legume grain, growing in 14.80 mhs regions all over the w... more Chickpea is the second most significant legume grain, growing in 14.80 mhs regions all over the world. It contributes roughly 14.24 million people to the global food basket each year. Desi and Kabuli chickpeas are widely grown in India, and both cultivars have distinct physiochemical characteristics such as seed size and shape, hydration capacity, coat thickness, and protein content. The genetic basis of chickpea cultivars has been limited as a result of agricultural techniques. Molecular markers, on the other hand, have proven to be useful tools for accurate quantification and characterisation within plant species. Markers can be an excellent way to improve chickpeas quantitatively because they are a high source of protein and constitute 80% of total dry weight. Markers such as RAPD (Random amplified polymeric DNA), SSR (Single sequence repeat), AFLP (amplified fragment length polymorphism) and ISSR (Inter simple sequence repeat) have aided in improving breeding efficiency and thus...

Journal of Bioterrorism and Biodefense, 2020

Anthrax, a disease of public health as well as bio-warfare potential is caused by Gram positive, ... more Anthrax, a disease of public health as well as bio-warfare potential is caused by Gram positive, non-motile and spore forming Bacillus Anthracis. Bacteraemia and toxaemia of B. anthracis is responsible to cause lethality in anthrax-infected host. In this study, GroEL (HSP60) gene was cloned in pET28a+ vector and expressed in heterologous system E. coli. Over expressed recombinant GroEL was purified by IMAC chromatography and analysed on 10% SDS-PAGE. In western blot analysis, the rGroEL reacted with mouse anti-his antibody and anti-B. anthracis Sterne mouse serum. The immunogenicity of GroEL with aluminium hydroxide was evaluated in BALB/c mouse model. The endpoint titer of anti-rGroEL circulating antibodies in mouse serum was found high by indirect ELISA after first and second booster doses. IgG1 and IgG2b isotypes were found dominating in comparison to other isotypes. Humoral immune response generated by rGroEL indicated that it is immuno-dominant antigen of B. anthracis, which co...

Defence Life Science Journal, 2020

Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as p... more Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as public health point of view. The virulence factors of B. anthracis are encoded by the two plasmids, pXO1 and pXO2. Protective antigen (PA), an 83 kDa protein encoded by pXO1 along with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa), makes the anthrax toxin responsible for causing the disease. Current detection and diagnostic systems for anthrax are mostly based on PA, a potential biomarker of B. anthracis. The objective of the present study was to produce and purify the PA for development of a sandwich ELISA for its detection. In this study, pYS5 plasmid containing the full PA gene was transformed into an 8 proteases deficient Bacillus anthracis host BH480. The PA was produced under shake flask conditions and purified using the gel filtration chromatography. The reactivity of PA with rabbit and mouse anti-PA antibodies was confirmed by Western blotting. The antibodies were purifi...

Defence Life Science Journal, 2020

Detection of spores of Bacillus anthracis, the causative agent of anthrax in human and animals in... more Detection of spores of Bacillus anthracis, the causative agent of anthrax in human and animals in environment is cumbersome due to the presence of spores of other closely related Bacillus species. The Extractable Antigen 1 (EA1), an extracellular protein is considered as a biomarker for detection of B. anthracis spores. In the present work, we have cloned and expressed the recombinant EA1 protein in soluble form in Escherichia coli. Optimisation of culture conditions and cultivation media was carried out to achieve enhanced soluble expression of recombinant EA1 protein. Further, the batch fermentation process was also developed using optimised conditions for scale up production of recombinant EA1 protein. The final yield of protein purified employing affinity chromatography was 42.64 mg/l of culture during batch fermentation process. The polyclonal antibodies were raised against recombinant EA in rabbit and mice and used to develop an ELISA for detection of B. anthracis spores. The ...

Journal of Applied Microbiology, 2020

Category A classified Bacillus anthracis is highly fatal pathogen that causes anthrax and creates... more Category A classified Bacillus anthracis is highly fatal pathogen that causes anthrax and creates challenges for global security and public health. In this study, development of a safe and ideal next‐generation subunit anthrax vaccine has been evaluated in mouse model.

Defence Life Science Journal, 2019

Protective antigen (PA) produced by Bacillus anthracis is a highly immunogenic protein. Therefore... more Protective antigen (PA) produced by Bacillus anthracis is a highly immunogenic protein. Therefore, it has significant importance in serodiagnosis as well as a vaccine candidate for anthrax. In the present study, codons for PA gene were optimised and synthesised for its expression in Escherichia coli. Various expression conditions were optimised for scaled up production of rPA. The final yield of affinity chromatography purified protein was 40.8 mg/l during batch fermentation. For further purification, affinity purified protein was diafiltered and subjected to anion exchange chromatography. SDS-PAGE and Western blot was used to characterise the purified rPA protein. The diagnostic potential of purified rPA was evaluated in Western blot using standards reference serum AVR 801 and cutaneous anthrax clinical sera. The results of the present study established the optimum production of rPA in E. coli after codon optimisation for its subsequent use in diagnosis of anthrax infection.

Defence Science Journal, 2019

Anthrax, caused by Bacillus anthracis is an important disease of biowarfare and public health imp... more Anthrax, caused by Bacillus anthracis is an important disease of biowarfare and public health importance. It is imperative to develop a simple system which can detect and differentiate B. anthracis from other closely related species. The surface array protein (Sap), which is secreted during the early growth phase of bacteria can be an important biomarker for detection of B. anthracis. In the present study, we have developed a rapid flow through membrane ELISA for detection of B. anthracis. Polyclonal antibodies were used to develop a sandwich plate ELISA, which could detect 3.9 ng/ml of recombinant Sap. B. anthracis bacteria grown in culture broth could be detected after 5 h of growth. Finally, a rapid flow through membrane ELISA was developed which can be accomplished just within 2 minutes, instead of 3-4 h as required in sandwich plate ELISA. The results established that the developed flow through membrane ELISA may be used for detection of B. anthracis. The proposed method is rap...

Biologicals, 2019

In this study, an ELISA was developed for simultaneous detection of antibodies against both the i... more In this study, an ELISA was developed for simultaneous detection of antibodies against both the important toxins of B. anthracis i.e. protective antigen (PA) and lethal factor (LF). A chimera of PA and LF was made by fusion and cloned and expressed in E. coli. The purified recombinant protein was used in plate ELISA for serodiagnosis of anthrax. The chimera could detect antibodies against both the toxins of Bacillus anthracis. The human serum samples (n = 98) collected from anthrax endemic and non-endemic areas were tested employing ELISA. The ELISA gave sensitivity of 100% (95% Confidence Interval [CI], 92.13 to 100) and specificity of 97.78% (95% Confidence Interval [CI], 88.23 to 99.94) with a J index of 0.97. The efficiency of ELISA was found to be 98.9% with the positive predictive value (PPV) and negative predictive value (NPV) of 97.8% and 100%, respectively. The chimera of PA and LF could be a better diagnostic antigen for serodiagnosis as the assay detects antibodies against both the toxins in early as well delayed infection cases of anthrax. Therefore, it can be a very useful tool for the surveillance as well as for confirmation of cutaneous anthrax cases.