Rajnikant Dixit - Academia.edu (original) (raw)

Papers by Rajnikant Dixit

Background: Periodic ingestion of a protein-rich blood meal by adult female mosquitoes causes a d... more Background: Periodic ingestion of a protein-rich blood meal by adult female mosquitoes causes a drastic metabolic change in their innate physiological status, which is referred to as ‘metabolic switch. Although the down-regulation of olfactory factors is key to restrain host-attraction, how the gut ‘metabolic switch’ modulates brain functions, and resilience physiological homeostasis remains unexplored. Methods: To uncover a possible correlation of gut metabolic switching and brain function, we carried out a comparative RNAseq analysis of naïve and blood-fed mosquito’s brain. Spatio-temporal expression of neuro-signaling and neuro-modulatory genes was monitored through Real-Time PCR. To establish a proof-of-concept, we followed LC/MS-based absolute quantification of different neurotransmitters (NT) and compared their levels in the brain as well as in the gut of the mosquitoes. To correlate how microbiome influences gut-brain-axis communication, we performed a comparative gut metagen...

BMC Pulmonary Medicine

Background The increasing trend of Chronic Obstructive Pulmonary Disease (COPD) in becoming the t... more Background The increasing trend of Chronic Obstructive Pulmonary Disease (COPD) in becoming the third leading cause of deaths by 2020 is of great concern, globally as well as in India. Dysregulation of protease/anti-protease balance in COPD has been reported to cause tissue destruction, inflammation and airway remodelling; which are peculiar characteristics of COPD. Therefore, it is imperative to explore various serum proteases involved in COPD pathogenesis, as candidate biomarkers. COPD and Asthma often have overlapping symptoms and therefore involvement of certain proteases in their pathogenesis would render accurate diagnosis of COPD to be difficult. Methods Serum samples from controls, COPD and Asthma patients were collected after requisite institutional ethics committee approvals. The preliminary analysis qualitatively and quantitatively analyzed various serum proteases by ELISA and mass spectrometry techniques. In order to identify a distinct biomarker of COPD, serum neutrophi...

In humans, dysregulation of the antioxidant defense system has a detrimental impact on male ferti... more In humans, dysregulation of the antioxidant defense system has a detrimental impact on male fertility and reproductive physiology. Establishing such a correlation in disease vectors may pave a new way to manipulate male’s reproductive physiology, but remains the least attended. Since long - term storage of healthy and viable sperm earmarks male’s reproductive competency, we tested whether the anti-oxidative system protein also influences male fertility in the mosquito An. stephensi. We showed that a testis-specific HPX12 is critical for maintaining the physiological homeostasis of male mosquito’s reproductive organs. Disruption of this antioxidant enzyme by dsRNA silencing in the male mosquito severely impairs the reproductive potential of mated blood-fed female mosquitoes,resulting in a loss of 60% eggs. Our data demonstrate that increased ROS in the HPX12 mRNA depleted mosquitoes is an ultimate cause of sperm disabilities both qualitatively as well as quantitatively.

In the adult female mosquito, successful blood meal acquisition is accomplished by salivary gland... more In the adult female mosquito, successful blood meal acquisition is accomplished by salivary glands, which releases a cocktail of proteins to counteract vertebrate host’s immune-homeostasis. However, the biological relevance of many salivary proteins remains unknown. Here, we characterize a salivary specific Heme peroxidase family member HPX12, originally identified from Plasmodium vivax infected salivary RNAseq data of the mosquito Anopheles stephensi. We demonstrate that dsRNA silencing mediated mRNA depletion of salivary AsHPX12 (80-90%), causes enhanced host attraction but reduced blood-meal acquisition abilities, by increasing probing propensity (31%), as well as probing time (100–200s, P<0.0001) as compared to control (35-90s) mosquitoes group. Altered expression of the salivary secretory and antennal proteins may account for an unusual fast release of salivary cocktail proteins, but the slowing acquisition of blood meal, possibly due to salivary homeostasis disruption of As...

Journal of Vector Borne Diseases

Immunology & Cell Biology

Parasitology and Microbiology Research [Working Title]

Malaria is one of the most deadly diseases infecting humans. Advances in elimination and vector c... more Malaria is one of the most deadly diseases infecting humans. Advances in elimination and vector control have reduced the global malaria burden in the past decade; however, the emerging threat of drug resistance and suboptimal vaccine efficacies threaten global eradication efforts. Unlocking novel drug and vaccine targets while simultaneously mitigating spread of resistant strains seems to be the need of the hour. Protein-protein interactions (PPIs), an integral part of hostpathogen cross-talk and parasite survival, have only recently emerged as promising drug targets. Large PPI networks (interactome) are being developed to better our understanding of various parasite biochemical pathways. In this chapter, we throw light on several newly characterized protein-protein interactions between the host (humans) and parasite (plasmodium) in key processes such as hemoglobin degradation, enzyme regulation, protein export, egress, invasion, and drug resistance and further discuss their viability for development as novel chemotherapeutic targets.

In our preceding study (Sharma et al., 2019; BioRxiv) we showed that in the gut lumen Plasmodium ... more In our preceding study (Sharma et al., 2019; BioRxiv) we showed that in the gut lumen Plasmodium vivax follows a unique strategy of immuno-suppression by disabling gut flora proliferation. Here, we further demonstrate that post gut invasion, a shrewd molecular relationship with individual tissues such as midgut, hemocyte, salivary glands, and strategic changes in the genetic makeup of P. vivax favors its survival in the mosquito host. A transient suppression of metabolic machinery by early oocysts, and increased immunity against late oocysts suggested a unique mechanism of gut homeostasis restoration and Plasmodium population regulation. Though a hyper immune response of hemocyte was a key to remove free circulating sporozoites, but a strong suppression of salivary metabolic activities, may favor successful survival of invaded sporozoites. Finally, genetic alteration of P. vivax ensures evasion of mosquito responses. Conclusively, our system-wide RNAseq analysis provides first genet...

Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in... more Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in the adult female mosquito may provide an opportunity to design new molecular strategy to disrupt human-mosquito interactions. However, despite the great progress in the field of mosquito olfaction and chemo-detection, little is known that how the sex-specific specialization of the olfactory system enables adult female mosquitoes to derive and manage complex blood feeding associated behavioral responses. A comprehensive RNAseq analysis of prior and post blood meal olfactory system of An. culicifacies mosquito revealed that a minor but unique change in the nature and regulation of key olfactory genes play a pivotal role in managing diverse behavioral responses. Age dependent transcriptional profiling demonstrated that adult female mosquito’s chemosensory system gradually learned and matured to drive the host-seeking and blood feeding behavior at the age of 5-6 days. A zeitgeber time scale...

Experimental Parasitology

Experimental parasitology, 2018

Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and fie... more Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and field deployable technique for nucleic acid amplification under isothermal conditions. In this study, we have developed and optimized four different visualization methods of loop-mediated isothermal amplification (LAMP) assay to detect Pfcrt K76T mutants of P. falciparum and compared their important features for one-pot in-field applications. Even though all the four tested LAMP methods could successfully detect K76T mutants of P. falciparum, however considering the time, safety, sensitivity, cost and simplicity, the malachite green and HNB based methods were found more efficient. Among four different visual dyes uses to detect LAMP products accurately, hydroxynaphthol blue and malachite green could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk. Our results indicated that the LAMP offers an interesting novel and convenient...

Frontiers in physiology, 2018

Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in... more Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in the adult female mosquitoes may provide an opportunity to design new molecular strategy to disrupt human-mosquito interactions. Although there is a great progress in the field of mosquito olfaction and chemo-detection, little is known about the sex-specific evolution of the specialized olfactory system of adult female mosquitoes that enables them to drive and manage the complex blood-feeding associated behavioral responses. A comprehensive RNA-Seq analysis of prior and post blood meal olfactory system of mosquito revealed a minor but unique change in the nature and regulation of key olfactory genes that may play a pivotal role in managing diverse behavioral responses. Based on age-dependent transcriptional profiling, we further demonstrated that adult female mosquito's chemosensory system gradually learned and matured to drive the host-seeking and blood feeding behavior at the age o...

Frontiers in immunology, 2018

Mosquitoes that transmit many deadly infectious diseases also need to keep fighting against many ... more Mosquitoes that transmit many deadly infectious diseases also need to keep fighting against many microbial infections. Constitutive expression of multiple antimicrobial peptides (AMPs) in almost all body tissues is believed to facilitate the effective management of these local infections. When any infection breaches the local barrier, AMPs are induced rapidly in non-target tissues such as hemocytes (HCs) and establish their co-ordination with systemic immune effectors to clear off the body infection. But how interorgan immune communication is managed during local and systemic infections remain largely unknown. To understand this interorgan molecular relationship, we identified, extensively profiled and compared the expression of AMPs in three important mosquito tissues. midgut, fat body (FB), and HCs.-mediated AMPs silencing suggests that mosquito tissues are able to manage an optimal expression of AMPs at the physiological level. We also examined the possible contribution of two im...

Molecular and biochemical parasitology, Mar 6, 2018

Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- ac... more Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- activity, which are known as "metacaspases". Although this family of cysteine proteases is structurally similar to caspases with Cys-His dyad but their evolutionary significance and functional relevance remains largely unknown. These proteases are considered to be an important target against malaria due to their absence in humans. In this report, we have biochemically characterized metacaspase-2 (PfMCA-2) of P.falciparum. Enzymatic assay showed that PfMCA-2 efficiently cleaved arginine/lysine specific peptide, but not caspase-specific substrate. Consistently, PfMCA-2 activity was sensitive to effector caspases inhibitor, Z-FA-FMK, and mildly inhibited by aprotinin and E-64. However, general caspase inhibitors such as Z-VAD-FMK and Z-DEVD-FMK had no effect on PfMCA-2 activity. Z-FA-FMK inhibits parasite growth with an ICvalue of 2.7 μM along with the notable morphological changes....

Scientific Reports, 2017

Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloro... more Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In-house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/μl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria. P. falciparum malaria is still one of the most threatening diseases and fast emergence of resistance to anti-malarial drugs remains one of the challenges to control and eliminate malaria, leading to the renaissance of malaria incidences and death 1. Currently available molecular tools for rapid detection and tracking of anti-malarial drug resistance are primarily based on the parasite gene markers. Chloroquine (CQ) resistance, which is mainly caused by the point mutation at codon K76T of the P. falciparum chloroquine resistance transporter (Pfcrt) gene; is widely used as a molecular marker for the detection of CQ resistance. This molecular marker is highly correlated with increased clinical CQ tolerance and treatment failure 2-4. However other markers i.e. P. falciparum multidrug resistance-1 (Pfmdr1) especially codons 86, 184, 1034 and 1042 are known to associated with CQ resistance 5,6. Although other molecular methods are valuable for anti-malarial drug resistance detection but are, costlier, time-consuming and require highly sophisticated laboratory facilities with trained personnel including DNA sequencing 7. Since these methods cannot cope with the current demand of field based assays; there is an urgent need to evaluate a new simple, easy to use, rapid, less time consuming and cost effective field based method for detecting anti-malarial drug resistance. In recent years various isothermal amplification based techniques have been developed with distinct features which are widely used for the detection of various pathogenic species 8-10. Loop mediated isothermal amplification assay (LAMP) is a fast emerging innovative isothermal amplification tool eliminating the use of highly sophisticated and costly thermo cycler based techniques because there is no requirement of denaturation of DNA template, completing whole reaction in a single step 8. In comparison to other available isothermal amplification methods, LAMP is easy to use as one step method with high sensitivity and specificity 11. This method employs strand displacement synthesis primed by specially designed five set of primers, two outer (F3, B3) and two inner

Biochemistry, 2016

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper resid... more Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Background: Periodic ingestion of a protein-rich blood meal by adult female mosquitoes causes a d... more Background: Periodic ingestion of a protein-rich blood meal by adult female mosquitoes causes a drastic metabolic change in their innate physiological status, which is referred to as ‘metabolic switch. Although the down-regulation of olfactory factors is key to restrain host-attraction, how the gut ‘metabolic switch’ modulates brain functions, and resilience physiological homeostasis remains unexplored. Methods: To uncover a possible correlation of gut metabolic switching and brain function, we carried out a comparative RNAseq analysis of naïve and blood-fed mosquito’s brain. Spatio-temporal expression of neuro-signaling and neuro-modulatory genes was monitored through Real-Time PCR. To establish a proof-of-concept, we followed LC/MS-based absolute quantification of different neurotransmitters (NT) and compared their levels in the brain as well as in the gut of the mosquitoes. To correlate how microbiome influences gut-brain-axis communication, we performed a comparative gut metagen...

BMC Pulmonary Medicine

Background The increasing trend of Chronic Obstructive Pulmonary Disease (COPD) in becoming the t... more Background The increasing trend of Chronic Obstructive Pulmonary Disease (COPD) in becoming the third leading cause of deaths by 2020 is of great concern, globally as well as in India. Dysregulation of protease/anti-protease balance in COPD has been reported to cause tissue destruction, inflammation and airway remodelling; which are peculiar characteristics of COPD. Therefore, it is imperative to explore various serum proteases involved in COPD pathogenesis, as candidate biomarkers. COPD and Asthma often have overlapping symptoms and therefore involvement of certain proteases in their pathogenesis would render accurate diagnosis of COPD to be difficult. Methods Serum samples from controls, COPD and Asthma patients were collected after requisite institutional ethics committee approvals. The preliminary analysis qualitatively and quantitatively analyzed various serum proteases by ELISA and mass spectrometry techniques. In order to identify a distinct biomarker of COPD, serum neutrophi...

In humans, dysregulation of the antioxidant defense system has a detrimental impact on male ferti... more In humans, dysregulation of the antioxidant defense system has a detrimental impact on male fertility and reproductive physiology. Establishing such a correlation in disease vectors may pave a new way to manipulate male’s reproductive physiology, but remains the least attended. Since long - term storage of healthy and viable sperm earmarks male’s reproductive competency, we tested whether the anti-oxidative system protein also influences male fertility in the mosquito An. stephensi. We showed that a testis-specific HPX12 is critical for maintaining the physiological homeostasis of male mosquito’s reproductive organs. Disruption of this antioxidant enzyme by dsRNA silencing in the male mosquito severely impairs the reproductive potential of mated blood-fed female mosquitoes,resulting in a loss of 60% eggs. Our data demonstrate that increased ROS in the HPX12 mRNA depleted mosquitoes is an ultimate cause of sperm disabilities both qualitatively as well as quantitatively.

In the adult female mosquito, successful blood meal acquisition is accomplished by salivary gland... more In the adult female mosquito, successful blood meal acquisition is accomplished by salivary glands, which releases a cocktail of proteins to counteract vertebrate host’s immune-homeostasis. However, the biological relevance of many salivary proteins remains unknown. Here, we characterize a salivary specific Heme peroxidase family member HPX12, originally identified from Plasmodium vivax infected salivary RNAseq data of the mosquito Anopheles stephensi. We demonstrate that dsRNA silencing mediated mRNA depletion of salivary AsHPX12 (80-90%), causes enhanced host attraction but reduced blood-meal acquisition abilities, by increasing probing propensity (31%), as well as probing time (100–200s, P<0.0001) as compared to control (35-90s) mosquitoes group. Altered expression of the salivary secretory and antennal proteins may account for an unusual fast release of salivary cocktail proteins, but the slowing acquisition of blood meal, possibly due to salivary homeostasis disruption of As...

Journal of Vector Borne Diseases

Immunology & Cell Biology

Parasitology and Microbiology Research [Working Title]

Malaria is one of the most deadly diseases infecting humans. Advances in elimination and vector c... more Malaria is one of the most deadly diseases infecting humans. Advances in elimination and vector control have reduced the global malaria burden in the past decade; however, the emerging threat of drug resistance and suboptimal vaccine efficacies threaten global eradication efforts. Unlocking novel drug and vaccine targets while simultaneously mitigating spread of resistant strains seems to be the need of the hour. Protein-protein interactions (PPIs), an integral part of hostpathogen cross-talk and parasite survival, have only recently emerged as promising drug targets. Large PPI networks (interactome) are being developed to better our understanding of various parasite biochemical pathways. In this chapter, we throw light on several newly characterized protein-protein interactions between the host (humans) and parasite (plasmodium) in key processes such as hemoglobin degradation, enzyme regulation, protein export, egress, invasion, and drug resistance and further discuss their viability for development as novel chemotherapeutic targets.

In our preceding study (Sharma et al., 2019; BioRxiv) we showed that in the gut lumen Plasmodium ... more In our preceding study (Sharma et al., 2019; BioRxiv) we showed that in the gut lumen Plasmodium vivax follows a unique strategy of immuno-suppression by disabling gut flora proliferation. Here, we further demonstrate that post gut invasion, a shrewd molecular relationship with individual tissues such as midgut, hemocyte, salivary glands, and strategic changes in the genetic makeup of P. vivax favors its survival in the mosquito host. A transient suppression of metabolic machinery by early oocysts, and increased immunity against late oocysts suggested a unique mechanism of gut homeostasis restoration and Plasmodium population regulation. Though a hyper immune response of hemocyte was a key to remove free circulating sporozoites, but a strong suppression of salivary metabolic activities, may favor successful survival of invaded sporozoites. Finally, genetic alteration of P. vivax ensures evasion of mosquito responses. Conclusively, our system-wide RNAseq analysis provides first genet...

Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in... more Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in the adult female mosquito may provide an opportunity to design new molecular strategy to disrupt human-mosquito interactions. However, despite the great progress in the field of mosquito olfaction and chemo-detection, little is known that how the sex-specific specialization of the olfactory system enables adult female mosquitoes to derive and manage complex blood feeding associated behavioral responses. A comprehensive RNAseq analysis of prior and post blood meal olfactory system of An. culicifacies mosquito revealed that a minor but unique change in the nature and regulation of key olfactory genes play a pivotal role in managing diverse behavioral responses. Age dependent transcriptional profiling demonstrated that adult female mosquito’s chemosensory system gradually learned and matured to drive the host-seeking and blood feeding behavior at the age of 5-6 days. A zeitgeber time scale...

Experimental Parasitology

Experimental parasitology, 2018

Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and fie... more Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and field deployable technique for nucleic acid amplification under isothermal conditions. In this study, we have developed and optimized four different visualization methods of loop-mediated isothermal amplification (LAMP) assay to detect Pfcrt K76T mutants of P. falciparum and compared their important features for one-pot in-field applications. Even though all the four tested LAMP methods could successfully detect K76T mutants of P. falciparum, however considering the time, safety, sensitivity, cost and simplicity, the malachite green and HNB based methods were found more efficient. Among four different visual dyes uses to detect LAMP products accurately, hydroxynaphthol blue and malachite green could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk. Our results indicated that the LAMP offers an interesting novel and convenient...

Frontiers in physiology, 2018

Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in... more Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in the adult female mosquitoes may provide an opportunity to design new molecular strategy to disrupt human-mosquito interactions. Although there is a great progress in the field of mosquito olfaction and chemo-detection, little is known about the sex-specific evolution of the specialized olfactory system of adult female mosquitoes that enables them to drive and manage the complex blood-feeding associated behavioral responses. A comprehensive RNA-Seq analysis of prior and post blood meal olfactory system of mosquito revealed a minor but unique change in the nature and regulation of key olfactory genes that may play a pivotal role in managing diverse behavioral responses. Based on age-dependent transcriptional profiling, we further demonstrated that adult female mosquito's chemosensory system gradually learned and matured to drive the host-seeking and blood feeding behavior at the age o...

Frontiers in immunology, 2018

Mosquitoes that transmit many deadly infectious diseases also need to keep fighting against many ... more Mosquitoes that transmit many deadly infectious diseases also need to keep fighting against many microbial infections. Constitutive expression of multiple antimicrobial peptides (AMPs) in almost all body tissues is believed to facilitate the effective management of these local infections. When any infection breaches the local barrier, AMPs are induced rapidly in non-target tissues such as hemocytes (HCs) and establish their co-ordination with systemic immune effectors to clear off the body infection. But how interorgan immune communication is managed during local and systemic infections remain largely unknown. To understand this interorgan molecular relationship, we identified, extensively profiled and compared the expression of AMPs in three important mosquito tissues. midgut, fat body (FB), and HCs.-mediated AMPs silencing suggests that mosquito tissues are able to manage an optimal expression of AMPs at the physiological level. We also examined the possible contribution of two im...

Molecular and biochemical parasitology, Mar 6, 2018

Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- ac... more Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- activity, which are known as "metacaspases". Although this family of cysteine proteases is structurally similar to caspases with Cys-His dyad but their evolutionary significance and functional relevance remains largely unknown. These proteases are considered to be an important target against malaria due to their absence in humans. In this report, we have biochemically characterized metacaspase-2 (PfMCA-2) of P.falciparum. Enzymatic assay showed that PfMCA-2 efficiently cleaved arginine/lysine specific peptide, but not caspase-specific substrate. Consistently, PfMCA-2 activity was sensitive to effector caspases inhibitor, Z-FA-FMK, and mildly inhibited by aprotinin and E-64. However, general caspase inhibitors such as Z-VAD-FMK and Z-DEVD-FMK had no effect on PfMCA-2 activity. Z-FA-FMK inhibits parasite growth with an ICvalue of 2.7 μM along with the notable morphological changes....

Scientific Reports, 2017

Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloro... more Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In-house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/μl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria. P. falciparum malaria is still one of the most threatening diseases and fast emergence of resistance to anti-malarial drugs remains one of the challenges to control and eliminate malaria, leading to the renaissance of malaria incidences and death 1. Currently available molecular tools for rapid detection and tracking of anti-malarial drug resistance are primarily based on the parasite gene markers. Chloroquine (CQ) resistance, which is mainly caused by the point mutation at codon K76T of the P. falciparum chloroquine resistance transporter (Pfcrt) gene; is widely used as a molecular marker for the detection of CQ resistance. This molecular marker is highly correlated with increased clinical CQ tolerance and treatment failure 2-4. However other markers i.e. P. falciparum multidrug resistance-1 (Pfmdr1) especially codons 86, 184, 1034 and 1042 are known to associated with CQ resistance 5,6. Although other molecular methods are valuable for anti-malarial drug resistance detection but are, costlier, time-consuming and require highly sophisticated laboratory facilities with trained personnel including DNA sequencing 7. Since these methods cannot cope with the current demand of field based assays; there is an urgent need to evaluate a new simple, easy to use, rapid, less time consuming and cost effective field based method for detecting anti-malarial drug resistance. In recent years various isothermal amplification based techniques have been developed with distinct features which are widely used for the detection of various pathogenic species 8-10. Loop mediated isothermal amplification assay (LAMP) is a fast emerging innovative isothermal amplification tool eliminating the use of highly sophisticated and costly thermo cycler based techniques because there is no requirement of denaturation of DNA template, completing whole reaction in a single step 8. In comparison to other available isothermal amplification methods, LAMP is easy to use as one step method with high sensitivity and specificity 11. This method employs strand displacement synthesis primed by specially designed five set of primers, two outer (F3, B3) and two inner

Biochemistry, 2016

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper resid... more Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.