Publications

23. Maiguel D, Faridi MH, Wei C, Kuwano Y, Balla KM, Hernandez D, Barth CJ, Lugo G, Donnelly M, Nayer A, Moita LF, Schürer S, Traver D, Ruiz P, Vazquez-Padron RI, Ley K, Reiser J, Gupta V., Small molecule-mediated activation of the integrin CD11b/CD18 reduces inflammatory disease, Science Signaling, 2011 Sep 6;4(189):ra57. PMID: 21900205

This work was featured in a Highlight in Nature Reviews in Immunology, 11, 638 (October 2011) and in a Highlight in Acta Pharmacologica Sinica (2011) 32: 1309–1310.
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22. Wei C, El Hindi S, Li J, Fornoni A, Goes N, Sageshima J, Maiguel D, Karumanchi SA, Yap HK, Saleem M, Zhang Q, Nikolic B, Chaudhuri A, Daftarian P, Salido E, Torres A, Salifu M, Sarwal MM, Schaefer F, Morath C, Schwenger V, Zeier M, Gupta V, Roth D, Rastaldi MP, Burke G, Ruiz P, Reiser J., Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis. Nature Medicine, 2011 Jul 31;17(8):952-60. doi: 10.1038/nm.2411. PMID: 21804539

This work was featured as a Cover article and in a Highlight in the same journal.
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21. Cheng, Q., Ogihara, M., and Gupta, V., Learning condition-dependent dynamical PPI networks from conflict-sensitive phosphorylation dynamics, In Proceedings of the IEEE Conference on Biomedicine and Computational Biology (IEEE-BIBM), 2011. B459.
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20. Cheng, Q., Gupta, V., and Ogihara, M., Inferring conflicting phosphorylation dynamics, In Proceedings of the ACM Conference on Computational Biology and Biomedicine (ACM-BCB), 2011.
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19. Faridi MH, Maiguel D, Brown BT, Suyama E, Barth CJ, Hedrick M, Vasile S, Sergienko E, Schürer S, Gupta V., High-throughput screening based identification of small molecule antagonists of integrin CD11b/CD18 ligand binding, Biochem Biophys Res Commun, 2010, Mar 26;394(1):194-9. Epub 2010 Feb 25. PMID: 20188705.
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18. Reiser J, Gupta V, Kistler AD. Toward the development of podocyte-specific drugs, Kidney Int. 2010 Apr;77(8):662-8. Epub 2010 Feb 3. PMID: 20130528.
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17. Faridi MH, Maiguel D, Barth CJ, Stoub D, Day R, Schürer S, Gupta V. Identification of novel agonists of the integrin CD11b/CD18, Bioorg Med Chem Lett, 2009 Dec 15;19(24):6902. NIHMS154324.
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16. Qureshi AH, Chaoji V, Maiguel D, Faridi MH, Barth CJ, Salem SM, Singhal M, Stoub D, Krastins B, Ogihara M, Zaki MJ, Gupta V., Proteomic and phospho-proteomic profile of human platelets in basal, resting state: insights into integrin signaling, PLoS One, 2009 Oct 27;4(1):e7627.
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15. Banerjee A., Sergienko E., Vasile S., Gupta V., Vuori K. and Wipf P., Triple Hybrids of Steroids, Spiroketals, and Oligopeptides as New Biomolecular Chimeras, Organic Letters, 2009 Jan 1;11(1):65.
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14. Gupta, V., Alonso J.-L., Sugimori T., Issafi M., Xiong J.-P. and Arnaout M. A., Role of the beta-subunit arginine/lysine finger in integrin heterodimer formation and function, Journal of Immunology, 2008 Mar 1;180(5):3613.
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13. Wei C., Möller C.C., Altintas M.M., Li J., Schwarz K., Zacchigna S., Xie L., Henger A., Schmid H., Rastaldi M.P., Cowan P., Kretzler M., Parrilla R., Bendayan M., Gupta V., Nikolic B., Kalluri R., Carmeliet P., Mundel P. and Reiser J., Modification of kidney barrier function by the urokinase receptor, Nature Medicine, 2008 Jan;14(1):55-63. Epub 2007 Dec 16.
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12. Park, J.Y., Arnaout, M.A. and Gupta V., A simple, no-wash cell-adhesion based high throughput assay for the discovery of small molecule regulators of the integrin CD11b/CD18, Journal of Biomolecular Screening, 2007 Apr;12(3):406-17.
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11. Gupta V., Gylling A., Alonso, J.-L., Sugimori T., Ianakiev, P., Xiong J.-P. and Arnaout M. A.,The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18, Blood, 2007 Apr 15;109(8):3513-20. Epub 2006 Dec 14 (DOI 10.1182/blood-2005-11-056689).

This work was featured in a Highlight in the same journal.
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10. Gupta# V., Cherkassky A., Chatis P., Joseph R., Johnson A. J., Erickson T. and Dimeo J., Directly labeled mRNA produces highly precise and unbiased differential gene expression data, Nucleic Acids Research, 2003, 31, e13.
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9. Werner MH, Nagata T, Gupta V, Goger M, Chait BT, Sâli A, Kim WY, Shigesada K, Ito Y. Molecular insights into the origin of the acute human leukemias revealed from the three-dimensional structure and sub-cellular localization of AML1, Clinical Cancer Res, 1999, 5, 3780S.

8. Werner M. H., Gupta V., Lambert L.J. and Nagata T., Uniform 13C/15N-labeling of DNA by tandem repeat amplification, Methods in Enzymology, 2001, 338, 283-304.
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7. Nagata* T., Gupta* V., Sorce D., Kim W-Y., Sâli A., Chait B. T., Shigesada K., Ito Y. and Werner M. H., Immunoglobulin-motif DNA recognition and heterodimerization for the PEBP2/CBF Runt-domain, Nature Structural Biology, 1999, 615-619.
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6. Goger* M., Gupta* V., Kim W-Y., Shigesada K., Ito Y. and Werner M. H. ,Molecular insights into PEBP2/CBF-SMMHC associated acute leukemias revealed from the three-dimensional structure of PEBP2/CBF beta, Nature Structural Biology, 1999, 620-623.
File link.

5. Gupta V., Parthasarathsy S. and Zaki M. J., Arithmetic and Logic Operations with DNA, Proceedings of the 3rd DIMACS Workshop on DNA Based Computers 1997, 212-220.
File link

4. Gupta V. and Kool E. T. A Novel Self-Cleaving DNA Nucleoside, Chemical Communications, 1997, 1425-26.
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3. Gupta V. Studies Aimed at Controlled Chemical Cleavage of DNA. Thesis, University Of Rochester, 1997.

2. Jayaram B., Aneja N., Rajsekaran E., Arora V., Das A., Ranganathan V. and Gupta V., Modeling DNA in Aqueous Solution, Journal of Scientific and Industrial Research, 1994, 53 (2), 88-105.

1. Gupta V. Computer Modeling and Theoretical Studies of Drug-DNA Interactions. Thesis, Indian institute of Technology, Delhi; 1992.

Details on older papers

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Faridi MH, Maiguel D, Brown BT, Suyama E, Barth CJ, Hedrick M, Vasile S, Sergienko E, Schürer S, Gupta V., High-throughput screening based identification of small molecule antagonists of integrin CD11b/CD18 ligand binding, Biochem Biophys Res Commun, 2010 Article in press Download file

Abstract

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.

bbrc

Figure Legend: Novel compounds inhibit CD11b/CD18 dependent adhesion of mouse neutrophils. (A) Chemical structures of identified hit compounds. (B) A histogram showing adhesion of mouse neutrophils to immobilized Fg in the control physiologic buffer containing 1 mM Ca2+ and Mg2+ (Con), with agonists (Mn2+ and PMA), and with agonists in the presence of one of the newly identified antagonists.

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Faridi MH, Maiguel D, Barth CJ, Stoub D, Day R, Schürer S, Gupta V., Identification of novel agonists of the integrin CD11b/CD18, Bioorg Med Chem Lett, 2009 Dec 15;19(24):6902. Download file

Abstract

We report the identification of novel small molecule agonists of integrin CD11b/CD18, which increased, in a dose-dependent manner, the adhesion of the integrin CD11b/CD18 expressing cells to two physiologically relevant ligands: Fibrinogen and iC3b. Compound 6 showed an ex vivo EC(50) of 10.5 microM and in vitro selectivity for binding to the recombinant alphaA-domain of CD11b/CD18. In silico docking experiments suggest that the compounds recognized a hydrophobic cleft in the ligand-binding alphaA-domain, implying an allosteric mechanism of modulation of integrin affinity by this novel compound.

bmcl

Figure Legend: Structural Model. Two views of a structural model showing proposed binding mode of the novel agonist in the zoomed-in activation-sensitive ?7 helix (?-helix on the right in A) region of the CD11b A-domain (blue ribbon). B shows a 90° rotated view of the same pocket as shown in A. Compound 6 is shown as orange stick model. Interacting residues from the activation-sensitive hydrophobic region are also shown as blue sticks and are labeled. ?7 helix, ?1 helix and the F-strand are also labeled. Dashed lines highlight potential key interactions between agonist and ?A.

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Qureshi AH, Chaoji V, Maiguel D, Faridi MH, Barth CJ, Salem SM, Singhal M, Stoub D, Krastins B, Ogihara M, Zaki MJ, Gupta V., Proteomic and phospho-proteomic profile of human platelets in basal, resting state: insights into integrin signaling, PLoS One, 2009 Oct 27;4(1):e7627. Download file

Abstract

During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin alphaIIbbeta3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin alphaIIbbeta3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin alphaIIbbeta3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future.

plos

Figure Legend: Schematic workflow of LC-MS/MS based platelet proteomic profiling. A. Workflow used in the proteomic analysis of human platelets. Isolated platelets were lysed and the extracted proteins were size-fractionated using 1D-SDS PAGE. The coomassie-stained gel lanes were cut in 14–16 equally sized sections, and in-gel digested with trypsin. Subsequently, extracted peptide mixture from each gel slice was independently analyzed using LC-MS/MS to obtain a list of unique platelet proteins. B. Workflow used in the phospho-proteomic analysis of platelets. Isolated platelets were lysed and the extracted total lysate was digested in-solution with trypsin. The trypsinized samples were enriched for phospho-peptides using an IMAC column and the enriched peptide mixtures were analyzed using LC-MS/MS to obtain a list of unique platelet phospho-proteins.

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Banerjee A., Sergienko E., Vasile S., Gupta V., Vuori K. and Wipf P., Triple Hybrids of Steroids, Spiroketals, and Oligopeptides as New Biomolecular Chimeras, Organic Letters, 2009 Jan 1;11(1):65. Download file

Abstract

An oxidative enol ether rearrangement was the key methodology in the construction of steroid-spiroketal-RGD peptides. Biological studies demonstrated potent integrin CD11b/CD18 antagonistic effects.

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Gupta*, V., Alonso* J.-L., Sugimori T., Issafi M., Xiong J.-P. and Arnaout M. A., Role of the beta-subunit arginine/lysine finger in integrin heterodimer formation and function, Journal of Immunology, 2008 Mar 1;180(5):3613. Download file

Abstract

Formation of the integrin alphabeta heterodimer is essential for cell surface expression and function. At the core of the alphabeta interface is a conserved Arg/Lys "finger" from the beta-subunit that inserts into a cup-like "cage" formed of two layers of aromatic residues in the alpha-subunit. We evaluated the role of this residue in heterodimer formation in an alphaA-lacking and an alphaA-containing integrin alphaVbeta3 and alphaMbeta2 (CD11b/CD18), respectively. Arg261 of beta3 was mutated to Ala or Glu; the corresponding Lys252 of beta2 was mutated to Ala, Arg, Glu, Asp, or Phe; and the effects on heterodimer formation in each integrin examined by ELISA and immunoprecipitation in HEK 293 cells cotransfected with plasmids encoding the alpha- and beta-subunits. The Arg261Glu (but not Arg261Ala) substitution significantly impaired cell surface expression and heterodimer formation of alphaVbeta3. Although Lys252Arg, and to a lesser extent Lys252Ala, were well tolerated, each of the remaining substitutions markedly reduced cell surface expression and heterodimer formation of CD11b/CD18. Lys252Arg and Lys252Ala integrin heterodimers displayed a significant increase in binding to the physiologic ligand iC3b. These data demonstrate an important role of the Arg/Lys finger in formation of a stable integrin heterodimer, and suggest that subtle changes at this residue affect the activation state of the integrin.

arg

Figure Legend: Structural Model. A, Three-dimensional crystal structure of unliganded integrin {alpha}V?3 head segment (as a ribbon diagram with the {alpha}V propeller domain in blue and the ?A domain in red) showing the position of the arginine finger (Arg261, carbon atoms in green, amides in blue) projecting from the 310 helix. Loop 3 (long arrow) and the ADMIDAS cation (magenta sphere, short arrow) are indicated. The four metal ions at the base of the propeller are indicated as orange spheres. B, Higher magnification of the Arg/Lys coordination pocket, with a portion of the aromatic side chains forming the upper ring of the cup-like "cage" structure of {alpha}V propeller. Orientation is the same as in A. C, Structure alignment of all eight mammalian ?-subunits around the Arg/Lys finger (boxed). The amino acid sequence of strands ?D and ?D', loop 3 and the 310 helix are indicated in single letter code. The ?4-subunit structure model was generated using Modeler with the {alpha}V?3 structure (PDB code: 1jv2) as template.

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Wei C., Möller C.C., Altintas M.M., Li J., Schwarz K., Zacchigna S., Xie L., Henger A., Schmid H., Rastaldi M.P., Cowan P., Kretzler M., Parrilla R., Bendayan M., Gupta V., Nikolic B., Kalluri R., Carmeliet P., Mundel P. and Reiser J., Modification of kidney barrier function by the urokinase receptor, Nature Medicine, 2008 Jan;14(1):55-63. Epub 2007 Dec 16. Download file

Abstract

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin. Mice lacking uPAR (Plaur(-/-)) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active beta3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate alphavbeta3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of alphavbeta3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

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Park, J.Y., Arnaout, M.A. and Gupta# V., A simple, no-wash cell-adhesion based high throughput assay for the discovery of small molecule regulators of the integrin CD11b/CD18, Journal of Biomolecular Screening, 2007 Apr;12(3):406-17. Download file.
# Senior Corresponding author.

Abstract

The leukocyte specific integrin CD11b/CD18 plays a key role in the biological function of these cells and represents a validated therapeutic target for inflammatory diseases. Currently, the low affinity interaction between CD11b/CD18 integrin and its respective ligand poses a challenge in the development of cell-based adhesion assays for the high throughput screening (HTS) environment. Here we describe a simple cell-based adhesion assay that can be readily used for high-throughput screening for the discovery of functional regulators of CD11b/CD18. The assay consistently produces acceptable Z’-values (>0.5) for HTS. After testing the assay using two established blocking antibodies as reference biologicals, we performed a proof-of-concept primary screen using a library of 6,612 compounds and identified both agonist and antagonist hits.

hts

Figure Legend: HTS Assay.Example photomicrographs from a 384-well plate assay showing cells remaining adherent upon completion of the no-wash cell adhesion assay. Cells were transferred to wells coated with an increasing amount of Fg and incubated in buffer containing either Ca2+ and Mg2+ (1 mM of each) or 1 mM Mn2+ for 20 min at 37 °C. Nonadherent cells were allowed to detach by gentle inversion of the plates, and the adherent cells were fixed with 1.1% formaldehyde. After washing the plate with the automated plate washer and staining cells with DAPI, cells were imaged using an automated microscope. The amount of Fg used to coat the wells is indicated. Cell adhesion to the uncoated well surface is shown (no block).

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Featured in  

Gupta V., Gylling A., Alonso, J.-L., Sugimori T., Ianakiev, P., Xiong J.-P. and Arnaout M. A. ,The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18, Blood, 2007 Apr 15;109(8):3513-20. Epub 2006 Dec 14 (DOI 10.1182/blood-2005-11-056689). Download file.

Abstract

Crystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligand-bound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal b-tail domain (bTD) and the ligand-binding bA domain of the bent conformation, in regulating interaction of integrin CD11b/CD18 (aMb2) with its physiologic ligand iC3b. We replaced the bTD CD loop residues D658GMD of the CD18 (b2) subunit with the equivalent D672SSG of the b3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca2+ plus Mg2+ (at 1mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b, compared to wild-type receptors. K562 expressing the bTD-modified integrins bound in Ca2+/Mg2+ to the bA-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal bTD as an allosteric modulator of integrin activation.

btd7

Figure Legend: A mechanistic model for the conformational changes during integrin activation and signaling. (A) Schematic of the domain structure of low affinity leg-bent CD11b/CD18 (CD11b and CD18 in dark and light gray, respectively). bTD contacts the bA and Hybrid domains and the two legs are in close proximity. The aA is occupied by a metal ion in the low affinity MIDAS (marked with asterisk); the bottom of a7 helix of inactive aA (shown with a thick black arrow) is not engaged by bA. (B) Inside-out activation disrupts the bTD contact with bA, which unlocks the bA F/a7 loop and allows the inward shift of the a1 helix leading to the formation of a stable aA-bound bA MIDAS (marked with asterisk) which in turn stabilizes aA in the open (high affinity state) (open semicircle). (C, D) Ligand (L) (black circle on a stick) bound at aA MIDAS stabilizes the occupancy of bA MIDAS by endogenous aA, opening up the bA/hybrid hinge to different degrees (double headed arrows), with larger hinge opening forcing genuextension and leg separation (D), resulting in outside-in signaling.

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Gupta# V., Cherkassky A., Chatis P., Joseph R., Johnson A. J., Erickson T. and Dimeo J., Directly labeled mRNA produces highly precise and unbiased differential gene expression data, Nucleic Acids Research, 2003, 31, e13. Download file.
# Senior Corresponding author.

Abstract

Microarray based gene expression studies allow simultaneous analysis of relative amounts of messenger RNA (mRNA) for thousands of genes using fluorescently labeled nucleic acid targets. Most common methods use enzymatic techniques, such as oligo-dT primed reverse transcription to produce labeled cDNA. These labeling methods have a number of shortcomings, including enzyme-introduced labeling and sequence bias, laborious protocols, high experiment-to-experiment variability and an inability to detect small changes in expression levels. Here, we describe a novel labeling methodology that uses platinum-linked cyanine dyes to directly chemically label mRNA from as little as 2mg of total RNA. We show that the gene expression data produced using the labeled mRNA method has very high precision, low error, no labeling bias and a dynamic range over several orders of magnitude. This allows a greater accuracy in the identification of differentially expressed genes and cuts down on the need for running too many replicate assays. Small changes in gene expression can now be detected in large-scale gene expression profiling assays using this simple, easy and quick procedure.

btd7

Figure Legend: Precision of the data obtained using novel RNA labeling technology. We performed five assays with Cyanine labeled cDNA target and seven assays with Cyanine labeled mRNA targets. The combined scatter plots from each set are shown. Since the probes were spotted in duplicate on these microarrays, each data point in the scatter plot has the benefit of 10 separate measurements for the cDNA target assays and 14 separate measurements for the mRNA target assays. Error bars representing the standard deviations for each gene have been added to the scatter plots for the respective experiments to demonstrate the amount of variability in each of the labeling assays. (i) Scatter plot for microarray experiments using standard labeled cDNA target (five experiments). (ii) Scatter plot for microarray experiments using labeled mRNA target (seven experiments). Note that the differential expression axes are set at 1.753 in the labeled mRNA assays as compared with the traditional 23 in labeled cDNA assays. An implication from these experiments is that fewer microarray experiments are necessary to determine with a high degree of statistical rigor that a given gene is truly up, down or non-differentially expressed.

Werner M. H., Gupta V., Lambert L.J. and Nagata T., Uniform 13C/15N-labeling of DNA by tandem repeat amplification, Methods in Enzymology, 2001, 338, 283-304. Download file.

Abstract

An optimized procedure has been described for the large-scale production of stable isotopeenriched duplex oligonucleotides of designed sequence. Large-scale production of labeled nucleotide triphosphates can be produced in this procedure simultaneously with labeled proteins, thereby providing synthetic dNMP precursors at no additional cost. The procedure is robust, with a minimum product:template yield of 800:1 overall, and produces > 99% single-length product. Tandem repeat PCR amplification is a general approach to large scale synthesis of duplex oligonucleotides and may have applications to both NMR and X-ray methods, particularly for product lengths in excess of 25 base pairs where failed sequences from solid-phase synthesis can be difficult to remove chromatographically. A drawback of the present approach is that the product is a duplex of two equal-length strands, making single-stranded products more difficult to prepare. For this reason, it could be preferable to produce single-stranded products by the [figure: see text] method of Zimmer and Crothers. Although a single base type can be selectively enriched in this approach, chemical synthesis will provide greater flexibility for labeled DNAs requiring site-specific labels at only one or a small number of nucleotide positions in the sequence. Therefore, maximum flexibility in labeling patterns can be realized by judicious choice of labeling method appropriate to the type of DNA product and extent of isotopic enrichment desired.

btd7

Figure Legend: PCR Amplification of Tandemly-repeated Template Sequences. The course of synthesis is followed by 0.7% agarose gel electrophoresis (right). a) A tandem repeat of the desired sequence is added to the reaction mixture as a blunt-ended duplex. b) Thermal cycling converts the blunt-ended duplex into a self-priming repeat, creating a pool of different length DNAs 2-20 kilobasepairs in length (Pool A, lane 1). c) Pool A is diluted 10-fold in Step 2 into a series of reactions which create long tandem repeats. At the beginning of Step 2, additional primer of monomer duplex DNA containing a single repeat of the desired sequence can be added to increase the overall yield. It is imagined that both linear and branched DNAs might be formed during amplification. Extensive thermal cycling (d, lane 2) followed by restriction with EcoRV (e, lane 3) results in milligram quantities of single-length DNA product of the desired sequence. The enhanced product yield resulting from the addition of single repeat DNA at point c) can be appreciated by comparison of lanes 3 and 4.

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Nagata* T., Gupta* V., Sorce D., Kim W-Y., Sâli A., Chait B. T., Shigesada K., Ito Y. and Werner M. H., Immunoglobulin-motif DNA recognition and heterodimerization for the PEBP2/CBF Runt-domain, Nature Structural Biology, 1999, 615-619. Download file.
* Joint first authors.

Abstract

The polyomavirus enhancer binding protein 2 (PEBP2) or core binding factor (CBF) is a heterodimeric enhancer binding protein that is associated with genetic regulation of hematopoiesis and osteogenesis. Aberrant forms of PEBP2/CBF are implicated in the cause of the acute human leukemias and in a disorder of bone development known as cleidocranial dysplasia. The common denominator in the natural and mutant forms of this protein is a highly conserved domain of PEBP2/CBF alpha, termed the Runt domain (RD), which is responsible for both DNA binding and heterodimerization with the beta subunit of PEBP2/CBF. The three-dimensional structure of the RD bound to DNA has been determined to be an S-type immunoglobulin fold, establishing a structural relationship between the RD and the core DNA binding domains of NF-kappaB, NFAT1, p53 and the STAT proteins. NMR spectroscopy of a 43.6 kD RD-beta-DNA ternary complex identified the surface of the RD in contact with the beta subunit, suggesting a mechanism for the enhancement of RD DNA binding by beta. Analysis of leukemogenic mutants within the RD provides molecular insights into the role of this factor in leukemogenesis and cleidocranial dysplasia.

aml1 aml1tube

Figure Legend: Three-dimensional structure of the Runt domain and its interaction with the beta-subunit. Left, Stereosuperposition of backbone heavy atoms of the 47 Runt domain three-dimensional structures, residues 51–178. The conformation of the N-terminal residues 51–62 and C-terminal residues 169–178 were poorly defined by the NOE data. Residues 179–188 are unstructured, display negative heteronuclear NOEs and are not shown. The C-terminal segment (residues 169–178) displayed heteronuclear NOEs less than or equal to 0.6, suggestive of some dynamic motion in this portion of the molecule. Heteronuclear NOEs were near zero for the N-terminal residues 51–61. Right, A tube model of the Runt domain three-dimensional structure, with its DNA-binding surface shown in blue and the heterodimerization surface shown in red.

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Goger* M., Gupta* V., Kim W-Y., Shigesada K., Ito Y. and Werner M. H. ,Molecular insights into PEBP2/CBF-SMMHC associated acute leukemias revealed from the three-dimensional structure of PEBP2/CBF beta, Nature Structural Biology, 1999, 620-623. Download file.
* Joint first authors

Abstract

PEBP2/CBF is a heterodimeric transcription factor essential for genetic regulation of hematopoiesis and osteogenesis. DNA binding by PEBP2/CBF alpha is accomplished by a highly conserved DNA binding domain, the Runt domain (RD), whose structure adopts an S-type immunoglobulin fold when bound to DNA. The supplementary subunit beta enhances DNA binding by the RD in vitro, but its role in the control of gene expression has remained largely unknown in vivo. Chromosome 16 inversion creates a chimeric gene product fusing PEBP2/CBF beta to a portion of the smooth muscle myosin heavy chain (PEBP2/CBF beta-SMMHC) that is causally associated with the onset of acute myeloid leukemia in humans. The three-dimensional structure of PEBP2/CBF beta has been determined in solution and is shown to adopt a fold related to the beta-barrel oligomer binding motif. Direct analysis of a 43.6 kD ternary RD-beta-DNA complex identifies the likely surface of beta in contact with the RD. The structure of PEBP2/CBF beta enables a molecular understanding of the capacity of PEBP2/CBF beta-SMMHC to sequester PEBP2/CBF alpha in the cytoplasm and therefore provides a molecular basis for understanding leukemogenic transformation.

cbfb cbfbtube

Figure Legend: Three-dimensional structure of PEBP2/CBFbeta. Left, A ribbon model of the PEBP2/CBFb three-dimensional structure summarizing the a helices (cyan), 310-helix (blue) and anti-parallel beta sheets (green) generated from a stereosuperposition of the final 25 simulated annealing structures using the backbone of the protein for residues 4–141. Right, A tube model of the PEBP2/CBFbeta three-dimensional structure, with its heterodimerization surface shown in red.

Gupta V., Parthasarathsy S. and Zaki M. J., Arithmetic and Logic Operations with DNA, Proceedings of the 3rd DIMACS Workshop on DNA Based Computers 1997, 212-220. Download file.

Abstract

The use of DNA molecules to solve hard computational problems has been demonstrated in recent studies. However, the question of suitability of DNA for solving simple computer operations, such as boolean or arithmetic operations, has largely been unaddressed. Incorporation of these operations in DNA computing is essential for solving a wide range of applications. We present a bit encoding scheme, modeling the input/output mechanisms of an electronic computer, and show how a sequence of such operations can be executed in a single test tube producing a unique result.

btd7

Figure Legend: Bit Operations with DNA: Example of a "series" operation. A. The first operation : (1001 NAND 0101) B. The whole series : (((1001 NAND 0101) XOR 0001) ADD 0001) C. A cartoon of the structure generated.

Gupta V. and Kool E. T. A Novel Self-Cleaving DNA Nucleoside, Chemical Communications, 1997, 1425-26. Download file.

Abstract

An allyl-modified nucleoside has been shown to self-alkylate and depurinate on treatment with iodine and heat; placement in DNA strands yields site-directed cleavage of the DNA.

selfcleaving

Figure Legend: Reaction Scheme for the Self-Cleavage of the Novel DNA Nucleoside.

Jayaram B., Aneja N., Rajsekaran E., Arora V., Das A., Ranganathan V. and Gupta V. ,Modeling DNA in Aqueous Solution, Journal of Scientific and Industrial Research 1994, 53 (2), 88-105.

Thesis

Gupta V. Computer Modeling and Theoretical Studies of Drug-DNA Interactions. Indian institute of Technology, Delhi, India, 1992.

Gupta V. Studies Aimed at Controlled Chemical Cleavage of DNA. University Of Rochester, Rochester, NY, 1997. Download file.

Journal Cover Design

Designed cover for the Journal "Chemistry and Biology" for the article: Kevin Ryan and Eric T. Kool, Triplex-directed self-assembly of an artificial sliding clamp on duplex DNA, Chemistry & Biology 1998, 5, 59-67. Journal link

catenane

Figure Legend: A CPK model of DNA catenane with double-stranded DNA shown in white and blue and the sisngle-stranded DNA circle shown in red.

Patents and Patent Applications

1. Gupta V. and Werner M. H., Methods for Preparing Polynucleotide Sequences and Uses Thereof, Patent number US6194179, 2001. Download file.

2. Gupta V. and Werner M. H., Methods for Preparing Quantities of Preselected Polynucleotide Sequences and Uses Thereof, WO/2001/006017. Download file.

3. Jacobs A. A., Gupta V. and Nikolic B., Clinically Intelligent Diagnostic Devices and Methods, Patent Pending US2002/0095073. Download file.

4. Jacobs A. A., Gupta V. and Nikolic B., Clinically Intelligent Diagnostic Devices and Methods, WO/2002/042775. Download file.

5. Luehrsen K. R., Gupta V. et al., P450 Single Nucleotide Polymorphism Biochip Analysis. WO/2002/083839. Download file.

6. Brush, C.K. and Gupta V., Post-Synthetic Labeling of Nucleic Acids, Assays Using Nucleic Acids that are Labeled Post-Synthetically, Single Nucleotide Polymorphism Detection, and Associated Compounds and Microarrays, Patent Pending US2003/0119005. Download file.

7. Jacobs A. A., Gupta V. and Nikolic B., Methods and Devices for Detecting Hydrocarbons and petroleum products, WO/2003/012390. Download file.

8. Brush, C.K. and Gupta V., Post-Synthetic Labeling of Nucleic Acids and uses thereof, WO/2003/052115. Download file.

9. Xia, J. and Gupta V. et al., Compositions and Methods for Rolling Circle Amplification, Patent Pending US2004/0014078. Download file.

10. Chui B., Elghanian R. E., Gupta V. et al., P450 Single Nucleotide Polymorphism Biochip Analysis, Patent Pending US2004/0229222. Download file.

11. Jacobs A. A., Gupta V. and Nikolic B., Clinically Intelligent Diagnostic Devices and Methods, Patent number US6905816. Download file.

12. Jacobs A. A., Gupta V. and Nikolic B., Clinically Intelligent Diagnostic Devices and Methods, Patent Pending US2005/0191694. Download file.

13. Gupta V., Nikolic B. and Gupta V., Water-based personal moisturizers and lubricants, in particular vaginal lubricants, and uses thereof, Patent Pending US2006/0204557. Download file.

14. Chui B., Elghanian R. E., Gupta V. et al., P450 Single Nucleotide Polymorphism Biochip Analysis, Patent Number US6986992. Download file.

15. Jacobs A. A., Gupta V. and Nikolic B., Clinically Intelligent Diagnostic Devices and Methods, Patent Pending US2006/0240453. Download file.