Bal R Singh | Institute of Advanced Sciences (original) (raw)

Papers by Bal R Singh

Research paper thumbnail of Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions

Research paper thumbnail of Rapid biotoxin immunoassays and activity assays in a multiplexing chip

OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information), 2011

Research paper thumbnail of Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin

Applied and Environmental Microbiology, Dec 15, 2018

Research paper thumbnail of The identification and biochemical characterization of drug-like compounds that inhibit botulinum neurotoxin serotype A endopeptidase activity

Research paper thumbnail of Rapid, Sensitive Detection and Quantification of Toxins from Complex Biological Matrices

Research paper thumbnail of Expeditious Molecular Weight Determination of Native Proteins by Transverse Pore Gradient Polyacrylamide Gel Electrophoresis Using the PhastGel System

Research paper thumbnail of A Phenotypic Switch of Differentiated Glial Cells to Dedifferentiated Cells Is Regulated by Folate Receptor α

Stem Cells, 2019

In a previous study, we showed that folate receptor-α (FRα) translocates to the nucleus where it ... more In a previous study, we showed that folate receptor-α (FRα) translocates to the nucleus where it acts as a transcription factor and upregulates Hes1, Oct4, Sox2, and Klf4 genes responsible for pluripotency. Here, we show that acetylation and phosphorylation of FRα favor its nuclear translocation in the presence of folate and can cause a phenotypic switch from differentiated glial cells to dedifferentiated cells. shRNA-FRα mediated knockdown of FRα was used to confirm the role of FRα in dedifferentiation. Ocimum sanctum hydrophilic fraction-1 treatment not only blocks the folate mediated dedifferentiation of glial cells but also promotes redifferentiation of dedifferentiated glial cells, possibly by reducing the nuclear translocation of ~38 kDa FRα and subsequent interaction with chromatin assembly factor-1. Stem Cells 2019;37:1441–1454

Research paper thumbnail of A Targeted Therapeutic Rescues Botulinum Toxin-A Poisoned Neurons

Nature Precedings, 2008

Botulinum neurotoxin (BoNT), a Category A biothreat agent, is the most potent poison known to man... more Botulinum neurotoxin (BoNT), a Category A biothreat agent, is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. BoNT acts specifically by blocking neurotransmission primarily at peripheral nerve-muscle junctions causing severe flaccid muscle paralysis, which is fatal if proper medical care is not provided. The neurotoxin acts by specifically entering the presynaptic nerve endings where it interferes with the biochemical machinery involved in the process of neurotransmitter release, i.e., neuroexocytosis. Most serotypes of BoNT are known to remain active for weeks to months after entering the nerves, but BoNT/A is the most potent and long lasting in causing muscle paralysis. An effective medical countermeasure strategy requires developing a drug that could rescue poisoned neuromuscular synapses, and would include its efficient delivery specifically to presynaptic nerve terminals. Here we report rescuing of botulinum poisoned nerv...

Research paper thumbnail of Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin

Applied and Environmental Microbiology, 2018

The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerv... more The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P ) value and other favorable characteristics (P. Leeson, Nature 481:455–456, 2012, https://doi.org/10.1038/481455a ). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602–2607, 2007, https://doi.org/10.1073/pnas.0611...

Research paper thumbnail of Role of critical elements in botulinum neurotoxin complex in toxin routing across intestinal and bronchial barriers

PloS one, 2018

The highly potent botulinum neurotoxin serotype A (BoNT/A) inhibits neurotransmitter release at n... more The highly potent botulinum neurotoxin serotype A (BoNT/A) inhibits neurotransmitter release at neuromuscular junctions resulting in flaccid muscle paralysis, respiratory arrest and death. In order to reach their neuronal cell targets, BoNT/A must cross epithelial cell barriers lining the intestines and airways. The toxin is produced as a large protein complex comprised of the neurotoxin and non-toxic neurotoxin-associated proteins (NAPs). Although NAPs are known to protect the toxin from harsh environments, their role in the movement of BoNT/A across epithelial barriers has not been fully characterized. In the current study, movement of the toxin across epithelial cells was examined macroscopically using a sensitive near infrared fluorescence transcytosis assay and microscopically using fluorescently labeled toxin and confocal microscopy. The studies show that the BoNT/A complex internalizes more rapidly than the pure toxin. The studies also show that one NAP protein, hemaglutinin ...

Research paper thumbnail of A novel role of C-terminus in introducing a functionally flexible structure critical for the biological activity of botulinum neurotoxin

Scientific reports, Jan 11, 2018

Botulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flacci... more Botulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flaccid muscle paralysis and potentially death. The light chain is responsible for its intracellular toxicity through its endopeptidase activity. Available crystal structures of BoNT/A light chains (LCA) are based on various truncated versions (tLCA) of the full-length LCA (fLCA) and do not necessarily reflect the true structure of LCA in solution. The understanding of the mechanism of action, longevity of intoxication, and an improved development of endopeptidase inhibitors are dependent on first having a better insight into the structure of LCA in solution. Using an array of biophysical techniques, we report that the fLCA structure is significantly more flexible than tLCA in solution, which may be responsible for its dramatically higher enzymatic activity. This seems to be achieved by a much stronger, more rapid binding to substrate (SNAP-25) of the fLCA compared to tLCA. These results sugge...

Research paper thumbnail of Strategies to Design Inhibitors of Clostridium Botulinum Neurotoxins

Infectious Disorders - Drug Targets, 2007

Research paper thumbnail of Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach

Applied biochemistry and biotechnology, Jan 16, 2016

Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The... more Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we ob...

Research paper thumbnail of Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

The Protein Journal, 2014

Research paper thumbnail of The identification and biochemical characterization of drug-like compounds that inhibit botulinum neurotoxin serotype A endopeptidase activity

Research paper thumbnail of RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin

Research paper thumbnail of In vitro selection of RNA aptamers that inhibit the activity of type A botulinum neurotoxin

Biochemical and Biophysical Research Communications, 2010

Research paper thumbnail of Endopeptidase Activities of Botulinum Neurotoxin Type B Complex, Holotoxin, and Light Chain

Applied and Environmental Microbiology, 2010

Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadl... more Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum , and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities o...

Research paper thumbnail of Parallel Botulinum Neurotoxin/A Immuno- and Enzyme Activity Assays Using the Versatile RapiDx Platform

Research paper thumbnail of Additional file 1 of Dramatic neurological and biological effects by botulinum neurotoxin type A on SH-SY5Y neuroblastoma cells, beyond the blockade of neurotransmitter release

Additional file 1: Table S1. BoNT/A effects on the neurological system related functional groups ... more Additional file 1: Table S1. BoNT/A effects on the neurological system related functional groups with gene list and fold changes from the DAVID Analysis. Table S2. BoNT related cellular biological functions. Table S3. Cell structure related functional groups and gene fold changes. Table S4. Cell fate determination functional groups and gene fold changes.

Research paper thumbnail of Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions

Research paper thumbnail of Rapid biotoxin immunoassays and activity assays in a multiplexing chip

OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information), 2011

Research paper thumbnail of Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin

Applied and Environmental Microbiology, Dec 15, 2018

Research paper thumbnail of The identification and biochemical characterization of drug-like compounds that inhibit botulinum neurotoxin serotype A endopeptidase activity

Research paper thumbnail of Rapid, Sensitive Detection and Quantification of Toxins from Complex Biological Matrices

Research paper thumbnail of Expeditious Molecular Weight Determination of Native Proteins by Transverse Pore Gradient Polyacrylamide Gel Electrophoresis Using the PhastGel System

Research paper thumbnail of A Phenotypic Switch of Differentiated Glial Cells to Dedifferentiated Cells Is Regulated by Folate Receptor α

Stem Cells, 2019

In a previous study, we showed that folate receptor-α (FRα) translocates to the nucleus where it ... more In a previous study, we showed that folate receptor-α (FRα) translocates to the nucleus where it acts as a transcription factor and upregulates Hes1, Oct4, Sox2, and Klf4 genes responsible for pluripotency. Here, we show that acetylation and phosphorylation of FRα favor its nuclear translocation in the presence of folate and can cause a phenotypic switch from differentiated glial cells to dedifferentiated cells. shRNA-FRα mediated knockdown of FRα was used to confirm the role of FRα in dedifferentiation. Ocimum sanctum hydrophilic fraction-1 treatment not only blocks the folate mediated dedifferentiation of glial cells but also promotes redifferentiation of dedifferentiated glial cells, possibly by reducing the nuclear translocation of ~38 kDa FRα and subsequent interaction with chromatin assembly factor-1. Stem Cells 2019;37:1441–1454

Research paper thumbnail of A Targeted Therapeutic Rescues Botulinum Toxin-A Poisoned Neurons

Nature Precedings, 2008

Botulinum neurotoxin (BoNT), a Category A biothreat agent, is the most potent poison known to man... more Botulinum neurotoxin (BoNT), a Category A biothreat agent, is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. BoNT acts specifically by blocking neurotransmission primarily at peripheral nerve-muscle junctions causing severe flaccid muscle paralysis, which is fatal if proper medical care is not provided. The neurotoxin acts by specifically entering the presynaptic nerve endings where it interferes with the biochemical machinery involved in the process of neurotransmitter release, i.e., neuroexocytosis. Most serotypes of BoNT are known to remain active for weeks to months after entering the nerves, but BoNT/A is the most potent and long lasting in causing muscle paralysis. An effective medical countermeasure strategy requires developing a drug that could rescue poisoned neuromuscular synapses, and would include its efficient delivery specifically to presynaptic nerve terminals. Here we report rescuing of botulinum poisoned nerv...

Research paper thumbnail of Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin

Applied and Environmental Microbiology, 2018

The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerv... more The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P ) value and other favorable characteristics (P. Leeson, Nature 481:455–456, 2012, https://doi.org/10.1038/481455a ). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602–2607, 2007, https://doi.org/10.1073/pnas.0611...

Research paper thumbnail of Role of critical elements in botulinum neurotoxin complex in toxin routing across intestinal and bronchial barriers

PloS one, 2018

The highly potent botulinum neurotoxin serotype A (BoNT/A) inhibits neurotransmitter release at n... more The highly potent botulinum neurotoxin serotype A (BoNT/A) inhibits neurotransmitter release at neuromuscular junctions resulting in flaccid muscle paralysis, respiratory arrest and death. In order to reach their neuronal cell targets, BoNT/A must cross epithelial cell barriers lining the intestines and airways. The toxin is produced as a large protein complex comprised of the neurotoxin and non-toxic neurotoxin-associated proteins (NAPs). Although NAPs are known to protect the toxin from harsh environments, their role in the movement of BoNT/A across epithelial barriers has not been fully characterized. In the current study, movement of the toxin across epithelial cells was examined macroscopically using a sensitive near infrared fluorescence transcytosis assay and microscopically using fluorescently labeled toxin and confocal microscopy. The studies show that the BoNT/A complex internalizes more rapidly than the pure toxin. The studies also show that one NAP protein, hemaglutinin ...

Research paper thumbnail of A novel role of C-terminus in introducing a functionally flexible structure critical for the biological activity of botulinum neurotoxin

Scientific reports, Jan 11, 2018

Botulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flacci... more Botulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flaccid muscle paralysis and potentially death. The light chain is responsible for its intracellular toxicity through its endopeptidase activity. Available crystal structures of BoNT/A light chains (LCA) are based on various truncated versions (tLCA) of the full-length LCA (fLCA) and do not necessarily reflect the true structure of LCA in solution. The understanding of the mechanism of action, longevity of intoxication, and an improved development of endopeptidase inhibitors are dependent on first having a better insight into the structure of LCA in solution. Using an array of biophysical techniques, we report that the fLCA structure is significantly more flexible than tLCA in solution, which may be responsible for its dramatically higher enzymatic activity. This seems to be achieved by a much stronger, more rapid binding to substrate (SNAP-25) of the fLCA compared to tLCA. These results sugge...

Research paper thumbnail of Strategies to Design Inhibitors of Clostridium Botulinum Neurotoxins

Infectious Disorders - Drug Targets, 2007

Research paper thumbnail of Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach

Applied biochemistry and biotechnology, Jan 16, 2016

Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The... more Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we ob...

Research paper thumbnail of Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

The Protein Journal, 2014

Research paper thumbnail of The identification and biochemical characterization of drug-like compounds that inhibit botulinum neurotoxin serotype A endopeptidase activity

Research paper thumbnail of RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin

Research paper thumbnail of In vitro selection of RNA aptamers that inhibit the activity of type A botulinum neurotoxin

Biochemical and Biophysical Research Communications, 2010

Research paper thumbnail of Endopeptidase Activities of Botulinum Neurotoxin Type B Complex, Holotoxin, and Light Chain

Applied and Environmental Microbiology, 2010

Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadl... more Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum , and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities o...

Research paper thumbnail of Parallel Botulinum Neurotoxin/A Immuno- and Enzyme Activity Assays Using the Versatile RapiDx Platform

Research paper thumbnail of Additional file 1 of Dramatic neurological and biological effects by botulinum neurotoxin type A on SH-SY5Y neuroblastoma cells, beyond the blockade of neurotransmitter release

Additional file 1: Table S1. BoNT/A effects on the neurological system related functional groups ... more Additional file 1: Table S1. BoNT/A effects on the neurological system related functional groups with gene list and fold changes from the DAVID Analysis. Table S2. BoNT related cellular biological functions. Table S3. Cell structure related functional groups and gene fold changes. Table S4. Cell fate determination functional groups and gene fold changes.