Vanderbilt University School of Medicine

Stein, Roland W. , Ph.D.
Professor of Molecular Physiology and Biophysics
Mark Collie Chair in Diabetes Resesarch
Professor of Cell and Developmental Biology

Lab Url:

Phone Number: 615-322-7026


Stein, Roland's picture

Office Address   Mailing Address

723 Light Hall

702 Light Hall 0615

Research Keywords
Acetylation, Chromatin, Developmental biology, Endocrinology, Gene regulation, Kinase, Knockout, Phosphorylation, Post-transcriptional modification, Signal transduction, Transcription, Transcription factor, phosphorylation,Cancer,Chromatin,Developmental biology,Diabetes,Gene regulation,Genomics,Knockout,Mass spectroscopy,Phosphorylation,Post-transcriptional modification,Protein Structure,Proteomics,Transcription,Transcription factor

Research Specialty
Focused on defining the transcription factors involved in controlling the expression of Pdx-1 and MafA. In addition, we are examining how transcriptional factors influence beta cell formation and function.

Research Description
Until recently, little detailed information was known about the factors controlling pancreas development and islet beta cell function. However, our understanding has increased greatly with the identification and molecular characterization of the islet-enriched MafA, MafB, and PDX-1 transcription factors. Gene knockouts performed on these and other pancreas-enriched factors are helping to elucidate the events influencing pancreatic morphogenesis. Because of their unique expression pattern and fundamental significance to beta cells, work here is focused on defining the transcription factors involved in controlling the expression of Pdx-1 and MafA. In addition, we are examining how transcriptional factors influence beta cell formation and function. Our recent results indicate that MafA and MafB strongly impact whether a cell becomes a producer in the islet of the insulin (beta) or glucagon (alpha) hormone. Both animal and cell culture models are used in these studies, with comprehensive and diverse methods from Cre/loxP conditional gene inactivation to mass spectrometry involved in addressing our experimental questions.

Understanding the mechanisms involved in controlling of pancreatic islet beta-cell specific transcription will likely lead to the development of therapeutic approaches that will prevent, correct, or at least delay the decline in beta cell function observed in diabetics. In fact, considerable efforts are focused on trying to develop an unlimited source of insulin-producing beta-like cells from adult and embryonic stem cells, as a consequence of success in reversing type 1 diabetes by islet transplantation. We believe that long-term success in this endeavor will require a fundamental understanding of the regulatory factors that are required for controlling the specialized genetic programs associated with beta cells. Our hope is that successful completion of our proposed studies will provide information important for generating acceptable islet-like cells for therapeutic treatment.

The beta cell biology community here is also very interactive and supportive, with eight groups meeting weekly to discuss their most recent findings. This gives students, post-docs, and faculty a routine opportunity to obtain input from a group of experts.

Artner, I and Stein, R. Insulin Gene Transcription: The Factors involved in Cell-type-Specific and Glucose-Regulated Expression in Islet beta Cells are also Essential during Pancreatic Development . , (Review Article- in press), , 2007.

Artner, I., Blanchi, B., Raum, J.C., Guo, M., Kaneko, T., Cordes, S., Sieweke, M., and Stein, R.. MafB is required for islet beta cell maturation. Proc Natl Acad Sci U S A, 104(10):3853-8, , 2007.

Oetjen E., Blume R., Cierny I., Schlag C., Kutschenko A., Kratzner R., Stein R., Knepel W.. nhibition of MafA transcriptional activity and human insulin gene transcription by interleukin-1beta and mitogen-activated protein kinase kinase kinase in pancreatic islet beta cells.. Diabetologia, in press, , 2007.

Wiebe PO, Kormish JD, Roper VT, Fujitani Y, Alston NI, Zaret KS, Wright CV, Stein RW, Gannon M.. Ptf1a binds to and activates area III, a highly conserved region of the Pdx1 promoter that mediates early pancreas-wide Pdx1 expression.. Mol. Cell. Biol. , 27:4093-104., , 2007.

Wiebe, PO, Kormish, JD, Roper, VT, Fujitani, Y, Alston, NI, Zaret, KS, Wright, CV, Stein, RW, Gannon, M. Ptf1a binds to and activates area III, a highly conserved region of the Pdx1 promoter that mediates early pancreas-wide Pdx1 expression. Mol Cell Biol, 27(11), 4093-104, 2007. PMCID:1900007

Artner, I., Lelay, J., Hang, Y., Elgazhi, L., Schisler, JC., Henderson, E., Sosa-Pineda, B., and Stein, R. . MafB: A Transcription Factor of the Glucagon Gene in Islet alpha Cells is also Expressed in Alpha Cell Progenitors. Diabetes , 55:297-304, , 2006.

Boyer DF, Fujitani Y, Gannon M, Powers AC, Stein RW, Wright CV.. Complementation rescue of Pdx1 null phenotype demonstrates distinct roles of proximal and distal cis-regulatory sequences in pancreatic and duodenal expression.. Dev. Biol. , 298:616-631, , 2006.

Boyer, DF, Fujitani, Y, Gannon, M, Powers, AC, Stein, RW, Wright, CV. Complementation rescue of Pdx1 null phenotype demonstrates distinct roles of proximal and distal cis-regulatory sequences in pancreatic and duodenal expression. Dev Biol, 298(2), 616-31, 2006.

Fujitani, Y, Fujitani, S, Boyer, DF, Gannon, M, Kawaguchi, Y, Ray, M, Shiota, M, Stein, RW, Magnuson, MA, Wright, CV. Targeted deletion of a cis-regulatory region reveals differential gene dosage requirements for Pdx1 in foregut organ differentiation and pancreas formation. Genes Dev, 20(2), 253-66, 2006. PMCID:1356115

Le Lay, J. and and Stein, R. . Involvement of PDX-1 in Activation of Human Insulin Gene Transcription. J. of Endocrinology, 188(2):287-94, , 2006.

Poitout, V., Hagman. D., Stein, R., Artner, I., Robertson, R.P., and Harmon, J.S. . Regulation of the insulin gene by glucose and fatty acids . J. Nutr. , 136:873-876, (Review Article), 2006.

Raum, J. Gerrish, K., Henderson, E., Guo, M., and Stein, R.. . Islet Beta-Cell Specific Expression of the MafA Transcription Factor is Regulated by FoxA2, Nkx2.2 and PDX-1 Binding to Conserved -8118 to -7748 Sequences. Mol. Cell. Biol., 26(15):5735-43, , 2006.

Tweedie, E., Artner, I, Crawford, L, Poffenberger, G, Thorens, G, Stein, R, Powers, A, and Gannon, M . Maintenance of Hnf6 in postnatal islets impairs terminal differentiation and function of beta cells. Diabetes , 55:3264-3270, , 2006.

Ueki, K., Okada, T., Dahlren, G.M., Peters, J.L., Shacman, J.G., Zhang, M., Artner, I., Satin, L.S., Stein, R., Kennedy, R.T., Kahn, C.R., and Kulkarni, R.N. . Total Insulin/IGF-1 Resistance in Pancreatic Beta-Cells causes Overt Diabetes. Nature Med. , 38:583-588, , 2006.

Harmon, J.S., Stein, R. and Robertson, R.P.. Oxidative stress-mediated, post-translational loss of MafA protein as a contributing mechanism to loss of insulin gene expression in glucotoxic beta cells. J. Biol. Chem., 280:11107-11113, , 2005.

Kitamura, Y., Kitamura, T., Kruse, J. -P., Raum, J., Stein, R., Wei, G, and Accili, D. . Dual regulation of FoxO1 activity protects against pancreatic beta cell failure through NeuroD and MafA induction. Cell Metabolism, 2:153-163, , 2005.

Van Velkinburgh, J.C., Samaras, S., Gerrish, K., Artner, I., and Stein, R.. Interactions between Areas I and II direct pdx-1 expression specifically to islet cell types of the mature and developing pancreas. . J. Biol. Chem. , 280:38438-44, , 2005.

Zhao, Li, Guo, Min, Matsuoka, Taka-Aki, Hagman, Derek K, Parazzoli, Susan D, Poitout, Vincent, Stein, Roland. The islet beta cell-enriched MafA activator is a key regulator of Insulin gene transcription. J. Biol. Chem., 280, 11887-11894, 2005.

Cockburn, B.N., Bermano, G., Boodram, L.-L., Telllucksingh, S., Tsuchiya, T., Mahabir, D., Allan, A.B., Stein, R., Docherty, K., and Bell, G.I.. Identification and functional characterization of a novel diabetes-associated mutation (E224K) in the IPF-1 (MODY4) gene in an Indo-Trinidadian family. J. Clin. Endo. and Metab., 89, 971-978, 2004.

Gerrish, Kevin, Van Velkinburgh, Jennifer C, Stein, Roland. Conserved transcriptional regulatory domains of the pdx-1 gene. Mol Endocrinol, 18(3), 533-48, 2004.

Le Lay, John, Matsuoka, Taka-Aki, Henderson, Eva, Stein, Roland. Identification of a novel PDX-1 binding site in the human insulin gene enhancer. J Biol Chem, 279(21), 22228-35, 2004.

Matsuoka, Taka-aki, Artner, Isabella, Henderson, Eva, Means, Anna, Sander, Maike, Stein, Roland. The MafA transcription factor appears to be responsible for tissue-specific expression of insulin. Proc Natl Acad Sci U S A, 101(9), 2930-3, 2004. PMCID:365722

Qiu, Yi, Guo, Min, Huang, Suming, Stein, Roland. Acetylation of the BETA2 transcription factor by p300-associated factor is important in insulin gene expression. J Biol Chem, 279(11), 9796-802, 2004.

Review: Poitout, V., Stein, R., and Rhodes, C.J. . Insulin gene expression and biosynthesis. . International Textbook of Diabetes Mellitus. , Volume III, E. Ferrannini, P. Zimmet, R. DeFronzo and H. Keen. Eds. (NYC, New York; John Wiley & Sons), pp. 97-124, 2004.

Cissell, Michelle A, Zhao, Li, Sussel, Lori, Henderson, Eva, Stein, Roland. Transcription factor occupancy of the insulin gene in vivo. Evidence for direct regulation by Nkx2.2. J Biol Chem, 278(2), 751-6, 2003.

Griffin, David, Penberthy, W Todd, Lum, Helen, Stein, Roland W, Taylor, William L. Isolation of the B3 transcription factor of the Xenopus TFIIIA gene. Gene, 313, 179-88, 2003.

Martin, Cyrus C, Svitek, Christina A, Oeser, James K, Henderson, Eva, Stein, Roland, O''Brien, Richard M. Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression. Biochem J, 371(Pt 3), 675-86, 2003. PMCID:1223330

Matsuoka, Taka-aki, Zhao, Li, Artner, Isabella, Jarrett, Harry W, Friedman, David, Means, Anna, Stein, Roland. Members of the large Maf transcription family regulate insulin gene transcription in islet beta cells. Mol Cell Biol, 23(17), 6049-62, 2003. PMCID:180917

Moates, J.M., Nanda, S., Cissell, M., and Stein, R. . The BETA2 transcription factor binds to and activates expression of the glucokinase gene in beta cells. Diabetes, 52, 403-408, 2003.

Samaras, Susan E, Zhao, Li, Means, Anna, Henderson, Eva, Matsuoka, Taka-Aki, Stein, Roland. The islet beta cell-enriched RIPE3b1/Maf transcription factor regulates pdx-1 expression. J Biol Chem, 278(14), 12263-70, 2003.

Petersen, H.V., Jensen, J.N., Stein, R., and Serup, P. Glucose induced MAPK signaling influences NeuroD1-mediated activation and nuclear localization. FEBS Letters, 528, 1-3, 2002.

Qiu, Yi, Guo, Min, Huang, Suming, Stein, Roland. Insulin gene transcription is mediated by interactions between the p300 coactivator and PDX-1, BETA2, and E47. Mol Cell Biol, 22(2), 412-20, 2002. PMCID:139753

Samaras, Susan E, Cissell, Michelle A, Gerrish, Kevin, Wright, Christopher V E, Gannon, Maureen, Stein, Roland. Conserved sequences in a tissue-specific regulatory region of the pdx-1 gene mediate transcription in Pancreatic beta cells: role for hepatocyte nuclear factor 3 beta and Pax6. Mol Cell Biol, 22(13), 4702-13, 2002. PMCID:133887

Shih, D.Q., Heimesaat, M.E., Kuwajima, S., Stein, R., Wright, C.V.E., Stoffel, M. Defects in Pancreatic Beta-Cell function in Mice with Combined Heterozygous Mutations in Pdx-1, Hnf-1 alpha and Hnf-3 beta. Proc. Natl. Acad. Sci., 99, 3818-3823, 2002.

Gerrish, K, Cissell, M A, Stein, R. The role of hepatic nuclear factor 1 alpha and PDX-1 in transcriptional regulation of the pdx-1 gene. J Biol Chem, 276(51), 47775-84, 2001.

Matsuoka , T, Zhao, L, Stein, R. The DNA binding activity of the RIPE3b1 transcription factor of insulin appears to be influenced by tyrosine phosphorylation. J Biol Chem, 276(25), 22071-6, 2001.

Review: Stein, R. Insulin gene transcription: the factors involved in cell-type-specific and glucose-regulated expression in islet beta cells are also essential during pancreatic development. Handbook of Physiology. Section 7: The Endocrine System, II(A. Cherrington and J. Jefferson. Eds. (Washington, D.C. American Physiology Society), 25-78, 2001.

Gerrish, K, Gannon, M, Shih, D, Henderson, E, Stoffel, M, Wright, C V, Stein, R. Pancreatic beta cell-specific transcription of the pdx-1 gene. The role of conserved upstream control regions and their hepatic nuclear factor 3beta sites. J Biol Chem, 275(5), 3485-92, 2000.

Peshavaria, M, Cissell, M A, Henderson, E, Petersen, H V, Stein, R. The PDX-1 activation domain provides specific functions necessary for transcriptional stimulation in pancreatic beta-cells. Mol Endocrinol, 14(12), 1907-17, 2000.

Review: Gannon, M, Gerrish, K., Stein, R., and Wright, C.V.E. Role of pdx-1 in pancreas development. Review for Frontiers in Diabetes: Molecular pathogenesis of MODYs. Editors: F.M. Matschinsky and M.A. Magnuson, S. Karger Publishers., , 166-180, 2000.

Review: Gerrish, K., Samaras, S., Cissell, M.A., Wright, C.V.E., and Stein, R. Regulation of pdx-1 gene expression. Review for Molecular Basis of Pancreatic Development and Function. Editors: M.A. Hussein, C.P. Miller, and J.F. Habener Kluwer Academic Publishers, Nowell, MA., , 251-263, 2000.

Zhao, L, Cissell, M A, Henderson, E, Colbran, R, Stein, R. The RIPE3b1 activator of the insulin gene is composed of a protein(s) of approximately 43 kDa, whose DNA binding activity is inhibited by protein phosphatase treatment. J Biol Chem, 275(14), 10532-7, 2000.

Huang, S, Qiu, Y, Stein, R W, Brandt, S J. p300 functions as a transcriptional coactivator for the TAL1/SCL oncoprotein. Oncogene, 18(35), 4958-67, 1999.

Sharma, A, Moore, M, Marcora, E, Lee, J E, Qiu, Y, Samaras, S, Stein, R. The NeuroD1/BETA2 sequences essential for insulin gene transcription colocalize with those necessary for neurogenesis and p300/CREB binding protein binding. Mol Cell Biol, 19(1), 704-13, 1999. PMCID:83927

Petersen, H.V., Peshavaria, M., Pedersen, A.A., Philippe, J., Stein, R., Madsen, O., and Serup, P. "Glucose Stimulates the Activation Domain Potential of the PDX-1 Homeodomain Transcription Factor.". FEBS Lett., 431, 362-366, 1998.

Qiu, Y, Sharma, A, Stein, R. p300 mediates transcriptional stimulation by the basic helix-loop-helix activators of the insulin gene. Mol Cell Biol, 18(5), 2957-64, 1998. PMCID:110675

Carty, M.D., Lillquist, J.S., Peshavaria, M., Stein, R., and Soeller, W.C. "Identification of Cis- and Trans-active Factors Regulating Islet Amyloid Polypeptide Expression in Pancreatic b cells.". J. Biol. Chem., 272, 11986-11993, 1997.

Fernandes, A., King, L.C., Guz, Y., Stein, R., Wright, C.V.E., and Teitelman, G. "Differentiation of New Insulin Producing Cells is Induced by Injury in Adult Pancreatic Islets.". Endocrinology, 138, 1750-1762, 1997.

Peshavaria, M, Henderson, E, Sharma, A, Wright, C V, Stein, R. Functional characterization of the transactivation properties of the PDX-1 homeodomain protein. Mol Cell Biol, 17(7), 3987-96, 1997. PMCID:232251

Peshavaria, M. and Stein, R. "PDX-1: An Activator of Genes Involved in Pancreatic Development and Islet Gene Expression.". Review for Pancreas Growth and Regeneration, Editor: Nora Sarvetnick, Molecular Biology Intelligence Unit, Publishers: Karger Landes Systems, , 96-103., 1997.

Sharma, A, Henderson, E, Gamer, L, Zhuang, Y, Stein, R. Analysis of the role of E2A-encoded proteins in insulin gene transcription. Mol Endocrinol, 11(11), 1608-17, 1997.

Wu, K L, Gannon, M, Peshavaria, M, Offield, M F, Henderson, E, Ray, M, Marks, A, Gamer, L W, Wright, C V, Stein, R. Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene. Mol Cell Biol, 17(10), 6002-13, 1997. PMCID:232449

Offield, M F, Jetton, T L, Labosky, P A, Ray, M, Stein, R W, Magnuson, M A, Hogan, B L, Wright, C V. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development, 122(3), 983-95, 1996.

Guz, Y., Montminy, M.R., Stein, R., Gamer, L.W., Wright, C.V.E., and Teitelman, G. "Expression of the STF-1 Homeobox Protein Identifies Common Precursors for Endocrine and Exocrine Pancreatic Cells.". Development, 121, 11-18, 1995.

Olson, L.K., Sharma, A., Wright, C.V.E., Towle, H.C., Robertson, R.P., and Stein, R. "Reduction of Insulin Gene Transcription in HIT-T15 b cells Chronically Exposed to a Supraphysiological Glucose Concentration is Associated with Loss of STF-1 Transcription Factor Expression.". Proc. Natl. Acad. Sci., 92, 9127-9131, 1995.

Robinson, G L, Henderson, E, Massari, M E, Murre, C, Stein, R. c-jun inhibits insulin control element-mediated transcription by affecting the transactivation potential of the E2A gene products. Mol Cell Biol, 15(3), 1398-404, 1995. PMCID:230364

Sharma, A, Fusco-DeMane, D, Henderson, E, Efrat, S, Stein, R. The role of the insulin control element and RIPE3b1 activators in glucose-stimulated transcription of the insulin gene. Mol Endocrinol, 9(11), 1468-76, 1995.

Sharma, A, Olson, L K, Robertson, R P, Stein, R. The reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to high glucose concentration is associated with the loss of RIPE3b1 and STF-1 transcription factor expression. Mol Endocrinol, 9(9), 1127-34, 1995.

Stoffel, M., Stein, R., Wright, C.V.E., Espinosa, R. III, Le Beau, M.M., and Bell, G.I. "Localization of Human Homeodomain Transcription Factor Insulin Promoter Factor 1 (IPF1) to Chromosome Band 13q12.1". Genomics, 28, 125-126, 1995.

Henderson, E, Stein, R. c-jun inhibits transcriptional activation by the insulin enhancer, and the insulin control element is the target of control. Mol Cell Biol, 14(1), 655-62, 1994. PMCID:358415

Peshavaria, M, Gamer, L, Henderson, E, Teitelman, G, Wright, C V, Stein, R. XIHbox 8, an endoderm-specific Xenopus homeodomain protein, is closely related to a mammalian insulin gene transcription factor. Mol Endocrinol, 8(6), 806-16, 1994.

Robinson, G L, Cordle, S R, Henderson, E, Weil, P A, Teitelman, G, Stein, R. Isolation and characterization of a novel transcription factor that binds to and activates insulin control element-mediated expression. Mol Cell Biol, 14(10), 6704-14, 1994. PMCID:359201

Robinson, G L, Peshavaria, M, Henderson, E, Shieh, S Y, Tsai, M J, Teitelman, G, Stein, R. Expression of the trans-active factors that stimulate insulin control element-mediated activity appear to precede insulin gene transcription. J Biol Chem, 269(4), 2452-60, 1994.

Sharma, A, Stein, R. Glucose-induced transcription of the insulin gene is mediated by factors required for beta-cell-type-specific expression. Mol Cell Biol, 14(2), 871-9, 1994. PMCID:358441

Stein, R, Henderson, E, Cordle, S R. Analysis of an insulin gene transcription control element. Positive and negative regulation appears to be mediated by different element sequences. FEBS Lett, 338(2), 187-90, 1994.

Cordle, S R, Henderson, E, Masuoka, H, Weil, P A, Stein, R. Pancreatic beta-cell-type-specific transcription of the insulin gene is mediated by basic helix-loop-helix DNA-binding proteins. Mol Cell Biol, 11(3), 1734-8, 1991. PMCID:369485

Cordle, S R, Whelan, J, Henderson, E, Masuoka, H, Weil, P A, Stein, R. Insulin gene expression in nonexpressing cells appears to be regulated by multiple distinct negative-acting control elements. Mol Cell Biol, 11(5), 2881-6, 1991. PMCID:360077

Hicks, B A, Stein, R, Efrat, S, Grant, S, Hanahan, D, Demetriou, A A. Transplantation of beta cells from transgenic mice into nude athymic diabetic rats restores glucose regulation. Diabetes Res Clin Pract, 14(3), 157-64, 1991.

Pietenpol, J A, M??nger, K, Howley, P M, Stein, R W, Moses, H L. Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor beta 1 and by the retinoblastoma gene product. Proc Natl Acad Sci U S A, 88(22), 10227-31, 1991. PMCID:52901

Pietenpol, J A, Holt, J T, Stein, R W, Moses, H L. Transforming growth factor beta 1 suppression of c-myc gene transcription: role in inhibition of keratinocyte proliferation. Proc Natl Acad Sci U S A, 87(10), 3758-62, 1990. PMCID:53982

Pietenpol, J A, Stein, R W, Moran, E, Yaciuk, P, Schlegel, R, Lyons, R M, Pittelkow, M R, M??nger, K, Howley, P M, Moses, H L. TGF-beta 1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains. Cell, 61(5), 777-85, 1990.

Stein, R W, Corrigan, M, Yaciuk, P, Whelan, J, Moran, E. Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity. J Virol, 64(9), 4421-7, 1990. PMCID:247911

Whelan, J, Cordle, S R, Henderson, E, Weil, P A, Stein, R. Identification of a pancreatic beta-cell insulin gene transcription factor that binds to and appears to activate cell-type-specific expression: its possible relationship to other cellular factors that bind to a common insulin gene sequence. Mol Cell Biol, 10(4), 1564-72, 1990. PMCID:362261

Stein, R W, Whelan, J. Insulin gene enhancer activity is inhibited by adenovirus 5 E1a gene products. Mol Cell Biol, 9(10), 4531-4, 1989. PMCID:362538

Whelan, J, Poon, D, Weil, P A, Stein, R. Pancreatic beta-cell-type-specific expression of the rat insulin II gene is controlled by positive and negative cellular transcriptional elements. Mol Cell Biol, 9(8), 3253-9, 1989. PMCID:362369

Postdoctoral Position Available

Postdoctoral Position Details
Postdoctoral Research Fellow Position Available Immediately

Term: at least 2 years

Salary and Benefits: Aprox.$38,000 plus benefits

Project(s): see research description

Please send CV to:
Roland Stein, Ph.D.

Molecular Physiology & Biophysics

702 Light Hall

Nashville, TN 37232-0615


Updated Date

Vanderbilt University is committed to principles of equal opportunity and affirmative action.
Copyright 2008 Vanderbilt University School of Medicine
The office of Biomedical Research Education & Training All rights reserved.
For questions or problems concerning this page, please submit a help ticket.