Journal articles
Steg LC, Shireby GL, Imm J, Davies JP, Franklin A, Flynn R, Namboori SC, Bhinge A, Jeffries AR, Burrage J, et al (In Press). Novel epigenetic clock for fetal brain development predicts prenatal age for cellular stem cell models and derived neurons.
Abstract:
Novel epigenetic clock for fetal brain development predicts prenatal age for cellular stem cell models and derived neurons
AbstractInduced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and iPSC-derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.
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Hawkins S, Namboori SC, Tariq A, Blaker C, Flaxman C, Dey NS, Henley P, Randall A, Rosa A, Stanton LW, et al (2022). Upregulation of β-catenin due to loss of miR-139 contributes to motor neuron death in amyotrophic lateral sclerosis.
Stem Cell Reports,
17(7), 1650-1665.
Abstract:
Upregulation of β-catenin due to loss of miR-139 contributes to motor neuron death in amyotrophic lateral sclerosis.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons (MNs). There are no effective treatments and patients usually die within 2-5 years of diagnosis. Emerging commonalities between familial and sporadic cases of this complex multifactorial disorder include disruption to RNA processing and cytoplasmic inclusion bodies containing TDP-43 and/or FUS protein aggregates. Both TDP-43 and FUS have been implicated in RNA processing functions, including microRNA biogenesis, transcription, and splicing. In this study, we explore the misexpression of microRNAs in an iPSC-based disease model of FUS ALS. We identify the downregulation of miR-139, an MN-enriched microRNA, in FUS and sporadic ALS MN. We discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator β-catenin is a major driver of MN degeneration in ALS. Our results highlight the importance of homeostatic RNA networks in ALS.
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Steg LC, Shireby GL, Imm J, Davies JP, Franklin A, Flynn R, Namboori SC, Bhinge A, Jeffries AR, Burrage J, et al (2021). Novel epigenetic clock for fetal brain development predicts prenatal age for cellular stem cell models and derived neurons.
Mol Brain,
14(1).
Abstract:
Novel epigenetic clock for fetal brain development predicts prenatal age for cellular stem cell models and derived neurons.
Induced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and embryonic stem cells and their derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.
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Namboori SC, Thomas P, Ames R, Hawkins S, Garrett LO, Willis CRG, Rosa A, Stanton LW, Bhinge A (2021). Single cell transcriptomics identifies master regulators of neurodegeneration in SOD1 ALS motor neurons. bioRxiv 593129; doi: https://doi.org/10.1101/593129
Namboori SC, Thomas P, Ames R, Hawkins S, Garrett LO, Willis CRG, Rosa A, Stanton LW, Bhinge A (2021). Single-cell transcriptomics identifies master regulators of neurodegeneration in SOD1 ALS iPSC-derived motor neurons.
Stem Cell Reports,
16(12), 3020-3035.
Abstract:
Single-cell transcriptomics identifies master regulators of neurodegeneration in SOD1 ALS iPSC-derived motor neurons.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by the loss of motor neurons. We utilized single-cell transcriptomics to uncover dysfunctional pathways in degenerating motor neurons differentiated from SOD1 E100G ALS patient-derived induced pluripotent stem cells (iPSCs) and respective isogenic controls. Differential gene expression and network analysis identified activation of developmental pathways and core transcriptional factors driving the ALS motor neuron gene dysregulation. Specifically, we identified activation of SMAD2, a downstream mediator of the transforming growth factor β (TGF-β) signaling pathway as a key driver of SOD1 iPSC-derived motor neuron degeneration. Importantly, our analysis indicates that activation of TGFβ signaling may be a common mechanism shared between SOD1, FUS, C9ORF72, VCP, and sporadic ALS motor neurons. Our results demonstrate the utility of single-cell transcriptomics in mapping disease-relevant gene regulatory networks driving neurodegeneration in ALS motor neurons. We find that ALS-associated mutant SOD1 targets transcriptional networks that perturb motor neuron homeostasis.
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Lim S, Bhinge A, Bragado Alonso S, Aksoy I, Aprea J, Cheok CF, Calegari F, Stanton LW, Kaldis P (2017). Cyclin-Dependent Kinase-Dependent Phosphorylation of Sox2 at Serine 39 Regulates Neurogenesis.
Mol Cell Biol,
37(16).
Abstract:
Cyclin-Dependent Kinase-Dependent Phosphorylation of Sox2 at Serine 39 Regulates Neurogenesis.
Sox2 is known to be important for neuron formation, but the precise mechanism through which it activates a neurogenic program and how this differs from its well-established function in self-renewal of stem cells remain elusive. In this study, we identified a highly conserved cyclin-dependent kinase (Cdk) phosphorylation site on serine 39 (S39) in Sox2. In neural stem cells (NSCs), phosphorylation of S39 enhances the ability of Sox2 to negatively regulate neuronal differentiation, while loss of phosphorylation is necessary for chromatin retention of a truncated form of Sox2 generated during neurogenesis. We further demonstrated that nonphosphorylated cleaved Sox2 specifically induces the expression of proneural genes and promotes neurogenic commitment in vivo Our present study sheds light on how the level of Cdk kinase activity directly regulates Sox2 to tip the balance between self-renewal and differentiation in NSCs.
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Bhinge A, Namboori SC, Zhang X, VanDongen AMJ, Stanton LW (2017). Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis.
Stem Cell Reports,
8(4), 856-869.
Abstract:
Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis.
Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS), it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN)-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential.
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Wang J, Jenjaroenpun P, Bhinge A, Angarica VE, Sol AD, Nookaew I, Kuznetsov VA, Stanton LW (2017). Single-cell gene expression analysis reveals regulators of distinct cell subpopulations among developing human neurons.
Genome Research,
27(11), 1783-1794.
Abstract:
Single-cell gene expression analysis reveals regulators of distinct cell subpopulations among developing human neurons
© 2017 Wang et al. The stochastic dynamics and regulatory mechanisms that govern differentiation of individual human neural precursor cells (NPC) into mature neurons are currently not fully understood. Here, we used single-cell RNA-sequencing (scRNA-seq) of developing neurons to dissect/identify NPC subtypes and critical developmental stages of alternative lineage specifications. This study comprises an unsupervised, high-resolution strategy for identifying cell developmental bifurcations, tracking the stochastic transcript kinetics of the subpopulations, elucidating regulatory networks, and finding key regulators. Our data revealed the bifurcation and developmental tracks of the two NPC subpopulations, and we captured an early (24 h) transition phase that leads to alternative neuronal specifications. The consequent up-regulation and down-regulation of stage- and subpopulation-specific gene groups during the course of maturation revealed biological insights with regard to key regulatory transcription factors and lincRNAs that control cellular programs in the identified neuronal subpopulations.
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Bhinge A, Namboori SC, Bithell A, Soldati C, Buckley NJ, Stanton LW (2016). MiR-375 is Essential for Human Spinal Motor Neuron Development and May be Involved in Motor Neuron Degeneration.
Stem Cells,
34(1), 124-134.
Abstract:
MiR-375 is Essential for Human Spinal Motor Neuron Development and May be Involved in Motor Neuron Degeneration.
The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons.
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Er JC, Leong C, Teoh CL, Yuan Q, Merchant P, Dunn M, Sulzer D, Sames D, Bhinge A, Kim D, et al (2015). NeuO: a fluorescent chemical probe for live neuron labeling.
Angew Chem Int Ed Engl,
54(8), 2442-2446.
Abstract:
NeuO: a fluorescent chemical probe for live neuron labeling.
To address existing limitations in live neuron imaging, we have developed NeuO, a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation inâ€
vivo and inâ€
vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications.
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Bhinge A, Poschmann J, Namboori SC, Tian X, Jia Hui Loh S, Traczyk A, Prabhakar S, Stanton LW (2014). MiR-135b is a direct PAX6 target and specifies human neuroectoderm by inhibiting TGF-β/BMP signaling.
EMBO J,
33(11), 1271-1283.
Abstract:
MiR-135b is a direct PAX6 target and specifies human neuroectoderm by inhibiting TGF-β/BMP signaling.
Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development, and recently, the TF PAX6 was shown to be critical for human NE specification. However, microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach, we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs, including hsa-miR-135b. MiR-135b was activated during NE development, and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways, thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage.
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Polioudakis D, Bhinge AA, Killion PJ, Lee B-K, Abell NS, Iyer VR (2013). A Myc–microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes. Nucleic Acids Research, 41(4), 2239-2254.
Johnson R, Richter N, Bogu GK, Bhinge A, Teng SW, Choo SH, Andrieux LO, de Benedictis C, Jauch R, Stanton LW, et al (2012). A genome-wide screen for genetic variants that modify the recruitment of REST to its target genes.
PLoS Genet,
8(4).
Abstract:
A genome-wide screen for genetic variants that modify the recruitment of REST to its target genes.
Increasing numbers of human diseases are being linked to genetic variants, but our understanding of the mechanistic links leading from DNA sequence to disease phenotype is limited. The majority of disease-causing nucleotide variants fall within the non-protein-coding portion of the genome, making it likely that they act by altering gene regulatory sequences. We hypothesised that SNPs within the binding sites of the transcriptional repressor REST alter the degree of repression of target genes. Given that changes in the effective concentration of REST contribute to several pathologies-various cancers, Huntington's disease, cardiac hypertrophy, vascular smooth muscle proliferation-these SNPs should alter disease-susceptibility in carriers. We devised a strategy to identify SNPs that affect the recruitment of REST to target genes through the alteration of its DNA recognition element, the RE1. A multi-step screen combining genetic, genomic, and experimental filters yielded 56 polymorphic RE1 sequences with robust and statistically significant differences of affinity between alleles. These SNPs have a considerable effect on the the functional recruitment of REST to DNA in a range of in vitro, reporter gene, and in vivo analyses. Furthermore, we observe allele-specific biases in deeply sequenced chromatin immunoprecipitation data, consistent with predicted differenes in RE1 affinity. Amongst the targets of polymorphic RE1 elements are important disease genes including NPPA, PTPRT, and CDH4. Thus, considerable genetic variation exists in the DNA motifs that connect gene regulatory networks. Recently available ChIP-seq data allow the annotation of human genetic polymorphisms with regulatory information to generate prior hypotheses about their disease-causing mechanism.
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(2012). An integrated encyclopedia of DNA elements in the human genome. Nature, 489(7414), 57-74.
(2011). A User's Guide to the Encyclopedia of DNA Elements (ENCODE). PLoS Biology, 9(4), e1001046-e1001046.
Lee B-K, Bhinge AA, Battenhouse A, McDaniell RM, Liu Z, Song L, Ni Y, Birney E, Lieb JD, Furey TS, et al (2011). Cell-type specific and combinatorial usage of diverse transcription factors revealed by genome-wide binding studies in multiple human cells.
Genome Research,
22(1), 9-24.
Abstract:
Cell-type specific and combinatorial usage of diverse transcription factors revealed by genome-wide binding studies in multiple human cells
Cell-type diversity is governed in part by differential gene expression programs mediated by transcription factor (TF) binding. However, there are few systematic studies of the genomic binding of different types of TFs across a wide range of human cell types, especially in relation to gene expression. In the ENCODE Project, we have identified the genomic binding locations across 11 different human cell types of CTCF, RNA Pol II (RNAPII), and MYC, three TFs with diverse roles. Our data and analysis revealed how these factors bind in relation to genomic features and shape gene expression and cell-type specificity. CTCF bound predominantly in intergenic regions while RNAPII and MYC preferentially bound to core promoter regions. CTCF sites were relatively invariant across diverse cell types, while MYC showed the greatest cell-type specificity. MYC and RNAPII co-localized at many of their binding sites and putative target genes. Cell-type specific binding sites, in particular for MYC and RNAPII, were associated with cell-type specific functions. Patterns of binding in relation to gene features were generally conserved across different cell types. RNAPII occupancy was higher over exons than adjacent introns, likely reflecting a link between transcriptional elongation and splicing. TF binding was positively correlated with the expression levels of their putative target genes, but combinatorial binding, in particular of MYC and RNAPII, was even more strongly associated with higher gene expression. These data illuminate how combinatorial binding of transcription factors in diverse cell types is associated with gene expression and cell-type specific biology.
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Lee B-K, Bhinge AA, Iyer VR (2011). Wide-ranging functions of E2F4 in transcriptional activation and repression revealed by genome-wide analysis. Nucleic Acids Research, 39(9), 3558-3573.
Smith AE, Chronis C, Christodoulakis M, Orr SJ, Lea NC, Twine NA, Bhinge A, Mufti GJ, Thomas NSB (2009). Epigenetics of human T cells during the G0-->G1 transition.
Genome Res,
19(8), 1325-1337.
Abstract:
Epigenetics of human T cells during the G0-->G1 transition.
We investigated functional epigenetic changes that occur in primary human T lymphocytes during entry into the cell cycle and mapped these at the single-nucleosome level by ChIP-chip on tiling arrays for chromosomes 1 and 6. We show that nucleosome loss and flanking active histone marks define active transcriptional start sites (TSSs). Moreover, these signatures are already set at many inducible genes in quiescent cells prior to cell stimulation. In contrast, there is a dearth of the inactive histone mark H3K9me3 at the TSS, and under-representation of H3K9me2 and H3K9me3 defines the body of active genes. At the DNA level, cytosine methylation (meC) is enriched for nucleosomes that remain at the TSS, whereas in general there is a dearth of meC at TSSs. Furthermore, a drop in meC also marks 3' transcription termination, and a peak of meC occurs at stop codons. This mimics the 3' nucleosomal distribution in yeast, which we show does not occur in human T cells.
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Shivaswamy S, Bhinge A, Zhao Y, Jones S, Hirst M, Iyer VR (2008). Dynamic remodeling of individual nucleosomes across a eukaryotic genome in response to transcriptional perturbation.
PLoS Biol,
6(3).
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Dynamic remodeling of individual nucleosomes across a eukaryotic genome in response to transcriptional perturbation.
The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high-throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.
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Birney E, Stamatoyannopoulos JA, Dutta A, GuigĂł R, Gingeras TR, Margulies EH, Weng Z, Snyder M, Dermitzakis ET, Thurman RE, et al (2007). Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.
Nature,
447(7146), 799-816.
Abstract:
Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project
We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function. ©2007 Nature Publishing Group.
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Bhinge AA, Kim J, Euskirchen GM, Snyder M, Iyer VR (2007). Mapping the chromosomal targets of STAT1 by Sequence Tag Analysis of Genomic Enrichment (STAGE).
Genome Research,
17(6), 910-916.
Abstract:
Mapping the chromosomal targets of STAT1 by Sequence Tag Analysis of Genomic Enrichment (STAGE)
Identifying the genome-wide binding sites of transcription factors is important in deciphering transcriptional regulatory networks. ChIP-chip (Chromatin immunoprecipitation combined with microarrays) has been widely used to map transcription factor binding sites in the human genome. However, whole genome ChIP-chip analysis is still technically challenging in vertebrates. We recently developed STAGE as an unbiased method for identifying transcription factor binding sites in the genome. STAGE is conceptually based on SAGE, except that the input is ChIP-enriched DNA. In this study, we implemented an improved sequencing strategy and analysis methods and applied STAGE to map the genomic binding profile of the transcription factor STAT1 after interferon treatment. STAT1 is mainly responsible for mediating the cellular responses to interferons, such as cell proliferation, apoptosis, immune surveillance, and immune responses. We present novel algorithms for STAGE tag analysis to identify enriched loci with high specificity, as verified by quantitative ChIP. STAGE identified several previously unknown STAT1 target genes, many of which are involved in mediating the response to interferon-γ signaling. STAGE is thus a viable method for identifying the chromosomal targets of transcription factors and generating meaningful biological hypotheses that further our understanding of transcriptional regulatory networks.
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Bhinge A, Chakrabarti P, Uthanumallian K, Bajaj K, Chakraborty K, Varadarajan R (2004). Accurate detection of protein:ligand binding sites using molecular dynamics simulations.
Structure,
12(11), 1989-1999.
Abstract:
Accurate detection of protein:ligand binding sites using molecular dynamics simulations.
Accurate prediction of location of cavities and surface grooves in proteins is important, as these are potential sites for ligand binding. Several currently available programs for cavity detection are unable to detect cavities near the surface or surface grooves. In the present study, an optimized molecular dynamics based procedure is described for detection and quantification of interior cavities as well as surface pockets. This is based on the observation that the mobility of water in such pockets is significantly lower than that of bulk water. The algorithm efficiently detects surface grooves that are sites of protein-ligand and protein-protein interaction. The algorithm was also used to substantially improve the performance of an automated docking procedure for docking monomers of nonobligate protein-protein complexes. In addition, it was applied to predict key residues involved in the binding of the E. coli toxin CcdB with its inhibitor. Predictions were subsequently validated by mutagenesis experiments.
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Kim J, Bhinge AA, Morgan XC, Iyer VR (2004). Mapping DNA-protein interactions in large genomes by sequence tag analysis of genomic enrichment. Nature Methods, 2(1), 47-53.
Dasgupta S, Chandran V, Bhinge A, Sewlikar S, Nimbkar A, Datta D (2004). Role of L-lysine HCl in adoptive immune therapy towards development of suitable tuberculosis vaccination.
Indian J Exp Biol,
42(8), 758-765.
Abstract:
Role of L-lysine HCl in adoptive immune therapy towards development of suitable tuberculosis vaccination.
L-Lysine HCI is being proposed to be a possible biocompatible adjuvant to enhance immune response by virtue of its probable non-specific bridging action and cellular proliferation properties. This proposal has been tried to be substantiated by designing an in vitro culture protocol, varying the concentration of L-lysine HCI and its further in vivo application. Splenic lymphocyte population has been extracted from mice and co-cultured with extracted mice macrophage population in presence of either Bacille Calmette Guerrin (BCG) or Hepatitis B surface antigen (HbsAg) and added L-lysine hydrochloride in culture media. Post incubation of these cultures, "taught" cell population has been adoptively transferred in naïve mice. These mice were then challenged by respective antigen dose, Change in Immune response with this challenge was noted. Antibody titre was followed in all the experiments as a measure of immune response. In adoptive immune transfer experiment of with HbsAg (AIT-HbsAg), similar to that with BCG (AIT-BCG), after the incubation period, antibody titre was higher in added lysine containing cultures in comparison with the control ones. Post transfer followed by antigen challenge, in AIT-BCG the expected augmentation in immune response was hardly visible. But in AIT-HbsAg, with the help of lysine booster, the animals responded better as far as the antibody titre is concerned.
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Dasgupta S, Bhinge A, Chandran V, Sewlikar S, Nimbkar A, Datta D (2003). Role of L-lysine HCl in immunopotentiation towards development of suitable tuberculosis vaccination.
Vaccine,
21(32), 4722-4727.
Abstract:
Role of L-lysine HCl in immunopotentiation towards development of suitable tuberculosis vaccination.
L-Lysine HCl is being proposed to be a possible biocompatible adjuvant to enhance immune response by virtue of its probable non-specific bridging action and cellular proliferation properties. This proposal has been tried to be substantiated by carrying out experimentation where L-lysine HCl has been used as an adjuvant (various groups based on mode of application and frequency of booster dose were designed) in tuberculosis vaccination experiments with heat killed Mycobacterium tuberculosis (MTB) and Bacille Calmette Guerin (BCG). Antibody titre has been followed in all the experiments as a measure of immune response. Amongst the various groups designed, group 1A (L-lysine HCl was given at a separate site as that of the antigen; lysine booster was given to this group intermittently, i.e. lysine given on 0th, 7th, 14th, 21st days of immunization) came out as the stand-alone leader. This mode and frequency of application was then compared with a group which received a standard adjuvant, viz. alhydrogel. Results were obtained which showed the following order in terms of decreasing antibody titre: alhydrogel group > lysine group > control group. Considering the biocompatible nature of lysine in comparison with the reportedly hazardous nature of alum adjuvants, we propose L-lysine HCl as a probable adjuvant in vaccination.
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Chakravarty S, Bhinge A, Varadarajan R (2002). A procedure for detection and quantitation of cavity volumes proteins. Application to measure the strength of the hydrophobic driving force in protein folding.
J Biol Chem,
277(35), 31345-31353.
Abstract:
A procedure for detection and quantitation of cavity volumes proteins. Application to measure the strength of the hydrophobic driving force in protein folding.
Accurate identification of cavities is important in the study of protein structure, stability, design, and ligand binding. Identification and quantitation of cavities is a nontrivial problem because most cavities are connected to the protein exterior. We describe a computational procedure for quantitating cavity volumes and apply this to derive an estimate of the hydrophobic driving force in protein folding. A grid-based Monte Carlo procedure is used to position water molecules on the surface of a protein. A Voronoi procedure is used to identify and quantitate empty space within the solvated protein. Additional cavities not detected by other existing procedures can be identified. Most of these are close to surface concavities. Residue volumes for both the interior and the surface residues as well as cavity volumes are in good agreement with volumes calculated from fully hydrated protein structures obtained from molecular dynamic simulations. We show that the loss of stability because of cavity-creating mutations correlates better with cavity volumes determined by this procedure than with cavity volumes determined by other methods. Available structural and thermodynamic data for a number of cavity-containing mutants were analyzed to obtain estimates of 26.1 cal x mol(-1) x A(-3) and 18.5 cal x mol(-1) x A(-2) for the relative contributions of cavity formation and the hydrophobic effect to the observed stability changes. The present estimate for the hydrophobic driving force is at the lower end of estimates derived from model compound studies and considerably lower than previous estimates of approximately 50 cal x mol(-1) x A(-2) derived from protein mutational data. In the absence of structural rearrangement, on average, deletion of a single methylene group is expected to result in losses in stability of 0.41 and 0.70 kcal x mol(-1) resulting from decrease in hydrophobicity and packing, respectively.
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Datta D, Bhinge A, Chandran V (2001). Lysine: is it worth more?.
Cytotechnology,
36(1-3), 3-32.
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Lysine: is it worth more?
Lysine, an essential cationic amino acid, has a positively charged R group. The structure of lysine is given as (H(3)N(+)-)CH(-COO(-))-CH(2)-CH(2)-CH(2)-CH(2)-N(+)H(3).While the anabolic role(s) of the molecule has been in focus for quite a few decades now, its biological properties, e.g. role in cellular proliferation in vitro (both anchorage dependent and anchorage independent) and in vivo, its ability to induce strong inflammatory and immune responses - both humoral and cell mediated, its role in augmented healing of all types of wounds in animal models as well as in human subjects (both acute and chronic), as well as its role in inducing extensive angiogenic responses, have never received reasonable attention so far. In the current brief and indicative review (rather than exhaustive reviews of each area), we intend to bring these biological properties of the molecule to focus while discussing a few other interesting aspects - lysine as a food preservative as well as its possible role(s) in immune therapy. While the areas look extremely divergent, we propose a common denominator in the form of a possible molecular mechanism of action of the molecule in all these diverse situations.
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Datta D, Kundu PK, Biswas S, Dasgupta S, Bhinge A, Chandran V (2000). Effect of cationic amino acid, L-lysine and its polymers on the growth and secretion of hybridoma cell line OKT-3.
Hybridoma,
19(4), 339-346.
Abstract:
Effect of cationic amino acid, L-lysine and its polymers on the growth and secretion of hybridoma cell line OKT-3.
Apart from their pivotal roles in anabolic protein synthesis, cationic amino acids, particularly, L-lysine HCl and its oligomers, up to molecular weight 1000, showed a remarkable property of cellular growth stimulation both in vitro and in vivo. L- and D-Lysine HCl, at a maximal stimulatory concentration of 7 microg/mL of added load of the amino acid, supported a characteristic time-scaled cellular expansion in vitro, and L-lysine-mediated cell expansion in batch cultures always showed a stimulation index (S.I.) ranging up to approximately 35, compared with the matched control populations. Variable S.I. was possibly due to factors such as seeding density, type of media additives, number of passages the cells have undergone before being stimulated, etc. Beyond and before maximal stimulatory concentration of the amino acid, there is a sharp decline in the cellular growth-promoting activity of monomeric L-lysine HCl in vitro, thereby showing a clear concentration window for maximum cellular growth promotion. While the essential amino acid does not have any dedicated cell surface receptor, the monomeric and oligomeric amino acid molecule(s) possibly mediates the serum-derived growth factor-receptor binding on the cell membrane by having two cationic charge centres at two ends of the molecule. Beyond a cutoff molecular weight of 1000, oligomeric lysines did not show any positive effects on either cell division and secretion.
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