Cathepsin D is a lysosomal hydrolase involved in intra- and extracellular proteolysis. This enzyme is aberrantly produced and processed in malignancy, and most notably is over-secreted into the tumor cell microenvironment. This hyper-secretion may lead to excessive degradation of the extracellular matrix, and contribute to tumor progression and metastases. These phenomena have been established in vitro, and there is evidence that Cathepsin D is similarly dysregulated in human breast cancer patients. Because breast cancer lacks an effective screening or surveillance biomarker, here we address the hypothesis that serum Cathepsin D activity may be useful to assess the presence or progression of breast cancer in females. While representative histologic sections from various disease-specific cohorts confirm previous findings that increased Cathepsin D production and secretion correlate with tumor progression, we report no difference in serum Cathepsin D activity between patients who are disease free, patients with pre-invasive or limited invasive disease, and patients with metastatic disease. Furthermore, in patients with known metastatic disease, there were no clinical variables associated with significantly different serum Cathepsin D activity. However, the immunohistochemical localization of Cathepsin D expression in histopathologic sections from breast cancer patients correlates with disease progression. Based on the serum results, and in contradistinction to Cathepsin D localization in breast cancer tissues, our findings support using Cathepsin D as a reliable histopathology biomarker for disease progression, but not for serum screening. Abbott, D. E. Margaryan, N. V. Jeruss, J. S. Khan, S. Kaklamani, V. Winchester, D. Hansen, N. Rademaker, A. Khalkhali-Ellis, Z. Hendrix, M. J. 0 2009-11-21T07:19:50Z 2010-01-15 2009-11-20 %0 Journal Article %A Abbott, D. E. %A Margaryan, N. V. %A Jeruss, J. S. %A Khan, S. %A Kaklamani, V. %A Winchester, D. %A Hansen, N. %A Rademaker, A. %A Khalkhali-Ellis, Z. %A Hendrix, M. J. %D 2010 %T Reevaluating cathepsin D as a biomarker for breast cancer: Serum activity levels versus histopathology. %J Cancer Biol Ther %V 9 %N 1 %M 19923884 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19923884 %X Cathepsin D is a lysosomal hydrolase involved in intra- and extracellular proteolysis. This enzyme is aberrantly produced and processed in malignancy, and most notably is over-secreted into the tumor cell microenvironment. This hyper-secretion may lead to excessive degradation of the extracellular matrix, and contribute to tumor progression and metastases. These phenomena have been established in vitro, and there is evidence that Cathepsin D is similarly dysregulated in human breast cancer patients. Because breast cancer lacks an effective screening or surveillance biomarker, here we address the hypothesis that serum Cathepsin D activity may be useful to assess the presence or progression of breast cancer in females. While representative histologic sections from various disease-specific cohorts confirm previous findings that increased Cathepsin D production and secretion correlate with tumor progression, we report no difference in serum Cathepsin D activity between patients who are disease free, patients with pre-invasive or limited invasive disease, and patients with metastatic disease. Furthermore, in patients with known metastatic disease, there were no clinical variables associated with significantly different serum Cathepsin D activity. However, the immunohistochemical localization of Cathepsin D expression in histopathologic sections from breast cancer patients correlates with disease progression. Based on the serum results, and in contradistinction to Cathepsin D localization in breast cancer tissues, our findings support using Cathepsin D as a reliable histopathology biomarker for disease progression, but not for serum screening. %+ Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. 8611 false false 1 Cancer biology & therapy Cancer Biol Ther 2010-01-15 aheadofprint JOURNAL ARTICLE 19923884 Publisher 2009-11-21T07:19:50Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19923884 9 2010 Gene silencing approaches afford investigators the ability to gain important insight into the normal functional requirements of specific epidermal proteins and promise to yield a powerful therapeutic means to dampen the level of proteins that are mutated or frequently overexpressed in skin disease. The efficient and tractable delivery of siRNAs into epidermal keratinocytes is seminal to this process. Here, we describe techniques for transient and long-term silencing of a representative gene product, namely desmoglein 1, in primary human epidermal keratinocytes maintained as submerged cultures or three-dimensional organotypic raft cultures. As a complement to epidermal-specific gene targeting strategies in mice, these technical approaches permit relatively rapid loss-of-function studies purely in keratinocytes without some of the potential influences present in situ, such as an immune system or vasculature. Simpson, C. L. Kojima, S. Getsios, S. 0 2009-11-14T07:17:28Z 2009-11-13 %0 Journal Article %A Simpson, C. L. %A Kojima, S. %A Getsios, S. %D 2010 %T RNA interference in keratinocytes and an organotypic model of human epidermis. %J Methods Mol Biol %V 585 %P 127-146 %M 19908001 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19908001 %X Gene silencing approaches afford investigators the ability to gain important insight into the normal functional requirements of specific epidermal proteins and promise to yield a powerful therapeutic means to dampen the level of proteins that are mutated or frequently overexpressed in skin disease. The efficient and tractable delivery of siRNAs into epidermal keratinocytes is seminal to this process. Here, we describe techniques for transient and long-term silencing of a representative gene product, namely desmoglein 1, in primary human epidermal keratinocytes maintained as submerged cultures or three-dimensional organotypic raft cultures. As a complement to epidermal-specific gene targeting strategies in mice, these technical approaches permit relatively rapid loss-of-function studies purely in keratinocytes without some of the potential influences present in situ, such as an immune system or vasculature. %+ Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. Simpson, Cory L Kojima, Shin-Ichiro Getsios, Spiro 8593 false false Methods in molecular biology (Clifton, N.J.) Methods Mol Biol 127-146 2010-01-01 ppublish Journal Article 19908001 In-Process 2009-11-17T07:21:51Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19908001 585 2010 The significance of intracellular beta-amyloid (Abeta(42)) accumulation is increasingly recognized in Alzheimer's disease (AD) pathogenesis. Abeta removal mechanisms that have attracted attention include IDE/neprilysin degradation and antibody-mediated uptake by immune cells. However, the role of the ubiquitin-proteasome system (UPS) in the disposal of cellular Abeta has not been fully explored. The E3 ubiquitin ligase Parkin targets several proteins for UPS degradation, and Parkin mutations are the major cause of autosomal recessive Parkinson's disease. We tested whether Parkin has cross-function to target misfolded proteins in AD for proteasome-dependent clearance in SH-SY5Y and primary neuronal cells. Wild-type Parkin greatly decreased steady-state levels of intracellular Abeta(42), an action abrogated by proteasome inhibitors. Intracellular Abeta(42) accumulation decreased cell viability and proteasome activity. Accordingly, Parkin reversed both effects. Changes in mitochondrial ATP production from Abeta or Parkin did not account for their effects on the proteasome. Parkin knock-down led to accumulation of Abeta. In AD brain, Parkin was found to interact with Abeta and its levels were reduced. Thus, Parkin is cytoprotective, partially by increasing the removal of cellular Abeta through a proteasome-dependent pathway. (c) 2009 Wiley-Liss, Inc. Rosen, K. M. Moussa, C. E. Lee, H. K. Kumar, P. Kitada, T. Qin, G. Fu, Q. Querfurth, H. W. 0 2009-09-06T12:00:08Z 2009-07-18 %0 Journal Article %A Rosen, K. M. %A Moussa, C. E. %A Lee, H. K. %A Kumar, P. %A Kitada, T. %A Qin, G. %A Fu, Q. %A Querfurth, H. W. %D 2010 %T Parkin reverses intracellular beta-amyloid accumulation and its negative effects on proteasome function. %J J Neurosci Res %V 88 %N 1 %P 167-178 %M 19610108 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19610108 %X The significance of intracellular beta-amyloid (Abeta(42)) accumulation is increasingly recognized in Alzheimer's disease (AD) pathogenesis. Abeta removal mechanisms that have attracted attention include IDE/neprilysin degradation and antibody-mediated uptake by immune cells. However, the role of the ubiquitin-proteasome system (UPS) in the disposal of cellular Abeta has not been fully explored. The E3 ubiquitin ligase Parkin targets several proteins for UPS degradation, and Parkin mutations are the major cause of autosomal recessive Parkinson's disease. We tested whether Parkin has cross-function to target misfolded proteins in AD for proteasome-dependent clearance in SH-SY5Y and primary neuronal cells. Wild-type Parkin greatly decreased steady-state levels of intracellular Abeta(42), an action abrogated by proteasome inhibitors. Intracellular Abeta(42) accumulation decreased cell viability and proteasome activity. Accordingly, Parkin reversed both effects. Changes in mitochondrial ATP production from Abeta or Parkin did not account for their effects on the proteasome. Parkin knock-down led to accumulation of Abeta. In AD brain, Parkin was found to interact with Abeta and its levels were reduced. Thus, Parkin is cytoprotective, partially by increasing the removal of cellular Abeta through a proteasome-dependent pathway. %+ Department of Neurology, Tufts University School of Medicine, Boston, Massachusetts, USA. 7280 false false 1 Journal of neuroscience research J Neurosci Res 167-178 2010-01-01 ppublish JOURNAL ARTICLE 19610108 In-Process 2009-11-13T07:20:37Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19610108 88 2010 The tissue-specific regulation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) is coordinated by intronic and extragenic cis-acting elements that influence its transcriptional activity. The promoter apparently lacks sequences to drive cell-type specific expression. We previously identified a number of intronic elements that were associated with DNase I hypersensitive sites (DHS) and bound the hepatocyte nuclear factor 1 (HNF1) transcription factor. Moreover, we demonstrated the likely involvement of HNF1 in the regulation of CFTR expression in vivo. Here we investigate DHS in introns 16 and 17a of the CFTR gene, which are evident in intestinal and pancreatic cell lines, and determine the transcription factors that interact with these sites. Of particular interest were factors known to interact with HNF1 in co-ordinated expression of genes in the gastrointestinal tract. We demonstrate that though sequences within these DHS bind HNF1, CDX2 and PBX1 in vitro, only PBX1 show a robust in vivo interaction. These data contribute to our understanding of the complexity of cell-type-specific CFTR regulatory mechanisms. McCarthy, V. A. Ott, C. J. Phylactides, M. Harris, A. 0 2009-09-30T06:15:37Z 2009-09-24 2009-09-29 %0 Journal Article %A McCarthy, V. A. %A Ott, C. J. %A Phylactides, M. %A Harris, A. %D 2009 %T Interaction of intestinal and pancreatic transcription factors in the regulation of CFTR gene expression. %J Biochim Biophys Acta %V 1789 %N 11-12 %P 709-718 %M 19782160 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19782160 %X The tissue-specific regulation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) is coordinated by intronic and extragenic cis-acting elements that influence its transcriptional activity. The promoter apparently lacks sequences to drive cell type-specific expression. We previously identified a number of intronic elements that were associated with DNase I hypersensitive sites (DHS) and bound the hepatocyte nuclear factor 1 (HNF1) transcription factor. Moreover, we demonstrated the likely involvement of HNF1 in the regulation of CFTR expression in vivo. Here we investigate DHS in introns 16 and 17a of the CFTR gene, which are evident in intestinal and pancreatic cell lines, and determine the transcription factors that interact with these sites. Of particular interest were factors known to interact with HNF1 in coordinated expression of genes in the gastrointestinal tract. We demonstrate that though sequences within these DHS bind HNF1, CDX2, and PBX1 in vitro, only PBX1 show a robust in vivo interaction. These data contribute to our understanding of the complexity of cell-type-specific CFTR regulatory mechanisms. %+ Paediatric Molecular Genetics, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK. 8415 false false 11-12 Biochimica et biophysica acta Biochim Biophys Acta 709-718 ppublish JOURNAL ARTICLE 19782160 In-Data-Review 2009-11-17T07:21:58Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19782160 1789 2009 The mechanism of hypertrophic scar reduction using silicone gel sheeting remains elusive. We hypothesize that the decrease in scar formation is due to occlusion and homeostasis of the barrier layer. Using an established model of hypertrophic scarring, rabbits were divided into four groups and scars were tape-stripped or occluded with Kelocote, Cavilon, or Indermil, with each rabbit serving as its own internal control. All wounds were harvested on day 28 and examined histologically to measure the scar elevation index (SEI), epithelial thickness, and cellularity. Immunohistochemistry fluorescence was used to quantify inflammation in the dermis. Transepidermal water loss (TEWL) was measured for each occlusive agent and tape stripping. Ultrastructural analysis was performed by electron microscopy. Kelocote, Cavilon, and Indermil all significantly decreased SEI when compared with controls. Each of the occlusive treatments was shown to decrease TEWL while tape stripping increased TEWL. Tape stripping significantly increased the SEI, epithelial thickness, and cellularity. Immunostaining for macrophages showed increased density of inflammatory cells in the tape-stripped scars. Under electron microscopy, the tape-stripped wounds displayed extensive inflammation and keratinocyte damage. Both unwounded skin and occlusion-treated scars did not display these characteristics. In conclusion, hypertrophic scarring was reduced regardless of occlusive method used. Furthermore, repeated disruption of the permeability barrier by tape stripping led to an increase in scarring. Ultrastructural analysis suggests that occluded wounds may be in an advanced state of wound repair. Occlusion may mediate its effects through establishing homeostasis of the epidermal barrier layer. O'Shaughnessy, K. D. De La Garza, M. Roy, N. K. Mustoe, T. A. 0 2009-09-24T06:15:33Z 2009-09-23 %0 Journal Article %A O'Shaughnessy, K. D. %A De La Garza, M. %A Roy, N. K. %A Mustoe, T. A. %D 2009 %T Homeostasis of the epidermal barrier layer: a theory of how occlusion reduces hypertrophic scarring. %J Wound Repair Regen %V 17 %N 5 %P 700-708 %M 19769722 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19769722 %X The mechanism of hypertrophic scar reduction using silicone gel sheeting remains elusive. We hypothesize that the decrease in scar formation is due to occlusion and homeostasis of the barrier layer. Using an established model of hypertrophic scarring, rabbits were divided into four groups and scars were tape-stripped or occluded with Kelocote, Cavilon, or Indermil, with each rabbit serving as its own internal control. All wounds were harvested on day 28 and examined histologically to measure the scar elevation index (SEI), epithelial thickness, and cellularity. Immunohistochemistry fluorescence was used to quantify inflammation in the dermis. Transepidermal water loss (TEWL) was measured for each occlusive agent and tape stripping. Ultrastructural analysis was performed by electron microscopy. Kelocote, Cavilon, and Indermil all significantly decreased SEI when compared with controls. Each of the occlusive treatments was shown to decrease TEWL while tape stripping increased TEWL. Tape stripping significantly increased the SEI, epithelial thickness, and cellularity. Immunostaining for macrophages showed increased density of inflammatory cells in the tape-stripped scars. Under electron microscopy, the tape-stripped wounds displayed extensive inflammation and keratinocyte damage. Both unwounded skin and occlusion-treated scars did not display these characteristics. In conclusion, hypertrophic scarring was reduced regardless of occlusive method used. Furthermore, repeated disruption of the permeability barrier by tape stripping led to an increase in scarring. Ultrastructural analysis suggests that occluded wounds may be in an advanced state of wound repair. Occlusion may mediate its effects through establishing homeostasis of the epidermal barrier layer. %+ Wound Healing Research Laboratory, Department of Surgery, Division of Plastic Surgery, Feinberg School of Medicine, Northwestern University, 675 N. St. Clair St., Chicago, IL 60611, USA. O'Shaughnessy, Kristina D De La Garza, Mauricio Roy, Nakshatra K Mustoe, Thomas A 8390 false false 5 Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society Wound Repair Regen 700-708 ppublish Journal Article 19769722 In-Process 2009-09-24T06:15:33Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19769722 17 2009 We test the hypothesis that the fibrinogen-thrombin formulation of fibrin sealant combined with fibroblasts and PDGF-BB enhance cutaneous wound healing. Four formulations varying in fibrinogen and thrombin concentration were applied to full-thickness biopsy wounds in the rabbit ear cutaneous wound healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 x 10(5) cells/wound) embedded in the fibrinogen component. At post-wounding day 7, there was no difference in the diluted vs. non-diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/mL fibrinogen, 167 U/mL thrombin; n=10 wounds) that was 167% (p<0.05) of the nondiluted FS containing cells (50 mg/mL fibrinogen, 250 U/mL thrombin; n=10 wounds). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone (p<0.05) for each diluted formulation although no differences in this parameter were observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF-BB (3 mg; n=4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over two-fold (p<0.01) over FS alone. Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in fibrin sealant. Mogford, J. E. Tawil, B. Jia, S. Mustoe, T. A. 0 2009-09-06T11:56:51Z 2009-08-08 %0 Journal Article %A Mogford, J. E. %A Tawil, B. %A Jia, S. %A Mustoe, T. A. %D 2009 %T Fibrin sealant combined with fibroblasts and platelet-derived growth factor enhance wound healing in excisional wounds. %J Wound Repair Regen %V 17 %N 3 %P 405-410 %M 19660049 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19660049 %X We test the hypothesis that the fibrinogen-thrombin formulation of fibrin sealant combined with fibroblasts and PDGF-BB enhance cutaneous wound healing. Four formulations varying in fibrinogen and thrombin concentration were applied to full-thickness biopsy wounds in the rabbit ear cutaneous wound healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 x 10(5) cells/wound) embedded in the fibrinogen component. At post-wounding day 7, there was no difference in the diluted vs. non-diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/mL fibrinogen, 167 U/mL thrombin; n=10 wounds) that was 167% (p<0.05) of the nondiluted FS containing cells (50 mg/mL fibrinogen, 250 U/mL thrombin; n=10 wounds). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone (p<0.05) for each diluted formulation although no differences in this parameter were observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF-BB (3 mg; n=4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over two-fold (p<0.01) over FS alone. Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in fibrin sealant. %K Administration, Topical %K Animals %K Cells, Cultured %K Disease Models, Animal %K Fibrin Tissue Adhesive/*administration & dosage %K Fibroblasts/*cytology %K Platelet-Derived Growth Factor/*administration & dosage/therapeutic use %K Rabbits %K Skin/*injuries/pathology %K Tissue Adhesives/*administration & dosage %K Treatment Outcome %K Wound Healing/*drug effects/physiology %K Wounds and Injuries/*drug therapy/pathology %+ Wound Healing Research Laboratory, Division of Plastic and Reconstructive Surgery, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. Mogford, Jon E Tawil, Bill Jia, Shengxian Mustoe, Thomas A 4223 false false 3 Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society Wound Repair Regen Administration, Topical; Animals; Cells, Cultured; Disease Models, Animal; Fibrin Tissue Adhesive/*administration & dosage; Fibroblasts/*cytology; Platelet-Derived Growth Factor/*administration & dosage/therapeutic use; Rabbits; Skin/*injuries/pathology; Tissue Adhesives/*administration & dosage; Treatment Outcome; Wound Healing/*drug effects/physiology; Wounds and Injuries/*drug therapy/pathology 405-410 ppublish Journal Article 19660049 MEDLINE 2009-11-18T07:16:32Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19660049 17 2009 Bacterial biofilms have gained increasing visibility in recent years as a ubiquitous form of survival for microorganisms in myriad environments. A number of in vivo models exist for the study of biofilms in the setting of medically relevant implanted foreign bodies. Growing evidence has demonstrated the presence of bacterial biofilms in the setting of a number of chronic wound states including pressure sores, diabetic foot ulcers, and venous stasis ulcers. Here we present a novel murine cutaneous wound system that directly demonstrates delayed reepithelialization caused by the presence of a bacterial biofilm. We established biofilms using either Staphylococcus aureus or Staphylococcus epidermidis in splinted cutaneous punch wounds created on the backs of normal C57Bl6/J mice. Wound reepithelialization was significantly delayed by bacterial biofilms. This effect was specifically dependent on the ability of the bacteria to form biofilms as demonstrated by exogenous administration of biofilm inhibiting peptides and the use of mutant Staphylococcus spp. deficient in biofilm formation. This represents the first direct evidence for the effect of bacterial biofilms on cutaneous wound healing. Schierle, C. F. De la Garza, M. Mustoe, T. A. Galiano, R. D. 0 2009-09-06T11:56:52Z 2009-08-08 %0 Journal Article %A Schierle, C. F. %A De la Garza, M. %A Mustoe, T. A. %A Galiano, R. D. %D 2009 %T Staphylococcal biofilms impair wound healing by delaying reepithelialization in a murine cutaneous wound model. %J Wound Repair Regen %V 17 %N 3 %P 354-359 %M 19660043 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19660043 %X Bacterial biofilms have gained increasing visibility in recent years as a ubiquitous form of survival for microorganisms in myriad environments. A number of in vivo models exist for the study of biofilms in the setting of medically relevant implanted foreign bodies. Growing evidence has demonstrated the presence of bacterial biofilms in the setting of a number of chronic wound states including pressure sores, diabetic foot ulcers, and venous stasis ulcers. Here we present a novel murine cutaneous wound system that directly demonstrates delayed reepithelialization caused by the presence of a bacterial biofilm. We established biofilms using either Staphylococcus aureus or Staphylococcus epidermidis in splinted cutaneous punch wounds created on the backs of normal C57Bl6/J mice. Wound reepithelialization was significantly delayed by bacterial biofilms. This effect was specifically dependent on the ability of the bacteria to form biofilms as demonstrated by exogenous administration of biofilm inhibiting peptides and the use of mutant Staphylococcus spp. deficient in biofilm formation. This represents the first direct evidence for the effect of bacterial biofilms on cutaneous wound healing. %K Animals %K *Biofilms %K Disease Models, Animal %K Male %K Mice %K Mice, Inbred C57BL %K Skin/injuries/*pathology %K Staphylococcal Infections/microbiology/pathology %K Staphylococcus aureus/*physiology %K Staphylococcus epidermidis/*physiology %K Wound Healing/*physiology %K Wound Infection/*microbiology/pathology %K Wounds and Injuries/*pathology %+ Laboratory for Repair and Regenerative Medicine, Division of Plastic & Reconstructive Surgery, Northwestern University, Chicago, Illinois, USA. Schierle, Clark F De la Garza, Mauricio Mustoe, Thomas A Galiano, Robert D 4224 false false 3 Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society Wound Repair Regen Animals; *Biofilms; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Skin/injuries/*pathology; Staphylococcal Infections/microbiology/pathology; Staphylococcus aureus/*physiology; Staphylococcus epidermidis/*physiology; Wound Healing/*physiology; Wound Infection/*microbiology/pathology; Wounds and Injuries/*pathology 354-359 ppublish Journal Article 19660043 MEDLINE 2009-11-18T07:16:32Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19660043 17 2009 Ischemia is a common underlying factor in a number of pathologic conditions ranging from cardiac dysfunction to delayed wound healing. Previous efforts have shown the resulting hypoxia activates the hypoxia inducible factor, a transcription factor with signaling effects through an intranuclear hypoxia response element (HRE). We hypothesized that ischemic conditions should activate these hypoxic signaling pathways in a measurable manner. We tested our hypothesis using variations of an established rabbit ear ischemic wound model and an HRE-luciferase-reporter gene construct. This plasmid construct was transfected into the ears of young, female New Zealand White rabbits, harvested at day 7 and processed to yield a reactive solution. Luminometry was used to quantify luciferase expression in each solution as a marker for HRE activation in each wound. Quantitative readings of hypoxic signaling as measured by luminescence yielded profound and statistically significant differences between the various ischemic models. Our results suggest that the biologic systems for hypoxic signaling can be used to detect local ischemia. HRE-luciferase transfection is an effective tool for quantifying the degree of tissue hypoxia. The caudal ischemic rabbit ear model showed significantly higher levels of hypoxia. Use of a validated model that produces sufficient tissue levels of hypoxia is recommended for meaningful study of ischemic wound healing. Said, H. K. Roy, N. K. Gurjala, A. N. Mustoe, T. A. 0 2009-09-06T11:56:52Z 2009-07-21 %0 Journal Article %A Said, H. K. %A Roy, N. K. %A Gurjala, A. N. %A Mustoe, T. A. %D 2009 %T Quantifying tissue level ischemia: hypoxia response element-luciferase transfection in a rabbit ear model. %J Wound Repair Regen %V 17 %N 4 %P 473-479 %M 19614911 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19614911 %X Ischemia is a common underlying factor in a number of pathologic conditions ranging from cardiac dysfunction to delayed wound healing. Previous efforts have shown the resulting hypoxia activates the hypoxia inducible factor, a transcription factor with signaling effects through an intranuclear hypoxia response element (HRE). We hypothesized that ischemic conditions should activate these hypoxic signaling pathways in a measurable manner. We tested our hypothesis using variations of an established rabbit ear ischemic wound model and an HRE-luciferase-reporter gene construct. This plasmid construct was transfected into the ears of young, female New Zealand White rabbits, harvested at day 7 and processed to yield a reactive solution. Luminometry was used to quantify luciferase expression in each solution as a marker for HRE activation in each wound. Quantitative readings of hypoxic signaling as measured by luminescence yielded profound and statistically significant differences between the various ischemic models. Our results suggest that the biologic systems for hypoxic signaling can be used to detect local ischemia. HRE-luciferase transfection is an effective tool for quantifying the degree of tissue hypoxia. The caudal ischemic rabbit ear model showed significantly higher levels of hypoxia. Use of a validated model that produces sufficient tissue levels of hypoxia is recommended for meaningful study of ischemic wound healing. %K Animals %K Cell Hypoxia/*genetics/physiology %K Ear/blood supply/injuries %K Female %K Ischemia/*genetics/physiopathology %K Luciferases/*genetics %K *Models, Animal %K Rabbits %K Response Elements/*genetics %K Severity of Illness Index %K Skin/blood supply/injuries %K Transfection %K Wound Healing/physiology %+ Division of Plastic Surgery, University of Washington, Seattle, Washington, USA. Said, Hakim K Roy, Nakshatra K Gurjala, Anandev N Mustoe, Thomas A 4225 false false 4 Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society Wound Repair Regen Animals; Cell Hypoxia/*genetics/physiology; Ear/blood supply/injuries; Female; Ischemia/*genetics/physiopathology; Luciferases/*genetics; *Models, Animal; Rabbits; Response Elements/*genetics; Severity of Illness Index; Skin/blood supply/injuries; Transfection; Wound Healing/physiology 473-479 ppublish Journal Article 19614911 MEDLINE 2009-09-25T06:16:09Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19614911 17 2009 Self-expanding stent design systems for carotid artery stenting (CAS) have morphed from nontapered (NTS) to tapered (TS); however, the impact of this change is unknown. We reviewed the outcomes of CAS with these two broad categories of stents in a single-center retrospective review of 308 CAS procedures from May 2001 to July 2007. Nitinol self-expanding TS or NTS coupled with cerebral embolic protection devices were used to treat extracranial carotid occlusive disease. Data analysis included demographics, procedural records, duplex exams, and conventional arteriography. Mean follow-up was 18 months (range 1-69). Restenosis was defined as >or=80% in-stent carotid artery stenosis by angiography. The mean age of the entire cohort was 71.3 years (75% men, 25% women). Of the 308 cases, 233 were de novo lesions and 75 had a prior ipsilateral carotid endarterectomy (n = 44) or external beam radiation exposure (n = 31). Preprocedure neurological symptoms were present in 30% of patients. TS were used in 156 procedures and NTS in 152 procedures. The 30-day ipsilateral stroke and death rates were 1.3% and 0.3%, respectively. An additional three (1.0%) posterior circulation strokes occurred. There was no statistically significant difference in the 30-day total stroke rates between TS (3.2%, n = 5) and NTS (1.3%, n = 2) (p = 0.5). At midterm follow-up, restenosis or asymptomatic occlusion was detected in eight cases (2.6%). All occurred in arteries treated with NTS, and this was statistically different when compared to arteries treated with TS (p = 0.03). Furthermore, a post-hoc subgroup analysis revealed significant correlation (chi(2) = 0.02) for restenosis in "hostile necks" when separated by TS vs. NTS. Early CAS outcomes between TS and NTS are comparable. In contrast, self-expanding nitinol TS may have a lower incidence of significant restenosis or asymptomatic occlusion when compared to NTS. Brown, K. E. Usman, A. Kibbe, M. R. Morasch, M. D. Matsumura, J. S. Pearce, W. H. Amaranto, D. J. Eskandari, M. K. 0 2009-09-06T11:55:55Z 2009-01-06 2009-01-09 %0 Journal Article %A Brown, K. E. %A Usman, A. %A Kibbe, M. R. %A Morasch, M. D. %A Matsumura, J. S. %A Pearce, W. H. %A Amaranto, D. J. %A Eskandari, M. K. %D 2009 %T Carotid stenting using tapered and nontapered stents: associated neurological complications and restenosis rates. %J Ann Vasc Surg %V 23 %N 4 %P 439-445 %M 19128933 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19128933 %X Self-expanding stent design systems for carotid artery stenting (CAS) have morphed from nontapered (NTS) to tapered (TS); however, the impact of this change is unknown. We reviewed the outcomes of CAS with these two broad categories of stents in a single-center retrospective review of 308 CAS procedures from May 2001 to July 2007. Nitinol self-expanding TS or NTS coupled with cerebral embolic protection devices were used to treat extracranial carotid occlusive disease. Data analysis included demographics, procedural records, duplex exams, and conventional arteriography. Mean follow-up was 18 months (range 1-69). Restenosis was defined as >or=80% in-stent carotid artery stenosis by angiography. The mean age of the entire cohort was 71.3 years (75% men, 25% women). Of the 308 cases, 233 were de novo lesions and 75 had a prior ipsilateral carotid endarterectomy (n = 44) or external beam radiation exposure (n = 31). Preprocedure neurological symptoms were present in 30% of patients. TS were used in 156 procedures and NTS in 152 procedures. The 30-day ipsilateral stroke and death rates were 1.3% and 0.3%, respectively. An additional three (1.0%) posterior circulation strokes occurred. There was no statistically significant difference in the 30-day total stroke rates between TS (3.2%, n = 5) and NTS (1.3%, n = 2) (p = 0.5). At midterm follow-up, restenosis or asymptomatic occlusion was detected in eight cases (2.6%). All occurred in arteries treated with NTS, and this was statistically different when compared to arteries treated with TS (p = 0.03). Furthermore, a post-hoc subgroup analysis revealed significant correlation (chi(2) = 0.02) for restenosis in "hostile necks" when separated by TS vs. NTS. Early CAS outcomes between TS and NTS are comparable. In contrast, self-expanding nitinol TS may have a lower incidence of significant restenosis or asymptomatic occlusion when compared to NTS. %K Aged %K Aged, 80 and over %K Alloys %K Angioplasty, Balloon/*adverse effects/*instrumentation/mortality %K Carotid Stenosis/diagnosis/mortality/*therapy %K Female %K Humans %K Ischemic Attack, Transient/*etiology/mortality %K Male %K Middle Aged %K Proportional Hazards Models %K Prosthesis Design %K Recurrence %K Retrospective Studies %K Risk Assessment %K Risk Factors %K Severity of Illness Index %K *Stents %K Stroke/*etiology/mortality %K Time Factors %K Treatment Outcome %+ Division of Vascular Surgery, Northwestern University, Chicago, IL, USA. Brown, Katherine E Usman, Asad Kibbe, Melina R Morasch, Mark D Matsumura, Jon S Pearce, William H Amaranto, Daniel J Eskandari, Mark K 2785 false false 4 Annals of vascular surgery Ann Vasc Surg Aged; Aged, 80 and over; Alloys; Angioplasty, Balloon/*adverse effects/*instrumentation/mortality; Carotid Stenosis/diagnosis/mortality/*therapy; Female; Humans; Ischemic Attack, Transient/*etiology/mortality; Male; Middle Aged; Proportional Hazards Models; Prosthesis Design; Recurrence; Retrospective Studies; Risk Assessment; Risk Factors; Severity of Illness Index; *Stents; Stroke/*etiology/mortality; Time Factors; Treatment Outcome 439-445 ppublish Comparative Study 19128933 MEDLINE 2009-09-06T11:55:55Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19128933 23 2009 Prosthetic devices that come into contact with blood ultimately fail secondary to thrombus formation. This limits the utility of a variety of materials used to surgically treat cardiovascular disease, including vascular grafts and stents, as well as sensors and catheters placed within the circulatory system. Moreover, systemic anticoagulation that is used to prevent malfunction of these devices has potential for serious complications. It is known that nitric oxide (NO) produced via the endothelium imparts thromboresistant properties to native blood vessels. Thus, if NO were delivered locally to the site of the prosthetic material, it has the potential to halt thrombus formation while limiting life-threatening side effects. This review serves to examine the variety of NO-releasing materials that have been created with the two different classes of NO donors, the diazeniumdiolates and S-nitrosothiols, and the clinical applications of these prosthetics for potential future use. Varu, V. N. Tsihlis, N. D. Kibbe, M. R. 0 2009-09-06T11:55:55Z 2008-09-17 2008-09-19 %0 Journal Article %A Varu, V. N. %A Tsihlis, N. D. %A Kibbe, M. R. %D 2009 %T Basic science review: nitric oxide--releasing prosthetic materials. %J Vasc Endovascular Surg %V 43 %N 2 %P 121-131 %M 18799500 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18799500 %X Prosthetic devices that come into contact with blood ultimately fail secondary to thrombus formation. This limits the utility of a variety of materials used to surgically treat cardiovascular disease, including vascular grafts and stents, as well as sensors and catheters placed within the circulatory system. Moreover, systemic anticoagulation that is used to prevent malfunction of these devices has potential for serious complications. It is known that nitric oxide (NO) produced via the endothelium imparts thromboresistant properties to native blood vessels. Thus, if NO were delivered locally to the site of the prosthetic material, it has the potential to halt thrombus formation while limiting life-threatening side effects. This review serves to examine the variety of NO-releasing materials that have been created with the two different classes of NO donors, the diazeniumdiolates and S-nitrosothiols, and the clinical applications of these prosthetics for potential future use. %K Animals %K Anticoagulants/therapeutic use %K Azo Compounds/therapeutic use %K Blood Coagulation/*drug effects %K *Blood Vessel Prosthesis %K Blood Vessel Prosthesis Implantation/adverse effects/*instrumentation %K *Coated Materials, Biocompatible %K Drug Therapy, Combination %K *Drug-Eluting Stents %K Humans %K Nitric Oxide Donors/administration & dosage/*therapeutic use %K Prosthesis Design %K Prosthesis Failure %K S-Nitrosothiols/therapeutic use %K Thrombosis/blood/etiology/*prevention & control %+ Division of Vascular Surgery, Northwestern University, Chicago, IL 60611, USA. Varu, Vinit N Tsihlis, Nick D Kibbe, Melina R 2789 false false 2 Vascular and endovascular surgery Vasc Endovascular Surg Animals; Anticoagulants/therapeutic use; Azo Compounds/therapeutic use; Blood Coagulation/*drug effects; *Blood Vessel Prosthesis; Blood Vessel Prosthesis Implantation/adverse effects/*instrumentation; *Coated Materials, Biocompatible; Drug Therapy, Combination; *Drug-Eluting Stents; Humans; Nitric Oxide Donors/administration & dosage/*therapeutic use; Prosthesis Design; Prosthesis Failure; S-Nitrosothiols/therapeutic use; Thrombosis/blood/etiology/*prevention & control 121-131 ppublish Journal Article 18799500 MEDLINE 2009-09-06T11:55:55Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18799500 43 2009 Graft choice remains an area of contention in anterior cruciate ligament reconstruction. Poorer cosmetic results and anterior knee pain remain a problem in the use of autologous patellar tendon grafts despite excellent clinical results when compared with autologous hamstring tendon grafts. Using a 2-incision technique to harvest the patellar tendon grafts has been shown to decrease the risk of anterior knee pain to a level comparable to hamstring tendon grafts. Proper graft tunnel placement and orientation also remain controversial with several recent researchers arguing the ability to perform an anatomic reconstruction using a conventional endoscopic transtibial technique. We will describe a relatively simple and cosmetically acceptable 2-incision technique for harvesting a bone-tendon-bone graft. In addition, we will describe the bony landmarks that should be used to ensure proper anatomic graft placement and the appropriate angles that need to be used for the tibial tunnel to drill the femoral tunnel in an anatomic position and carry out a successful endoscopic transtibial tunnel anterior cruciate ligament reconstruction. Purnell, M. L. Larson, A. I. 0 2009-11-18T07:17:28Z 2009-11-17 %0 Journal Article %A Purnell, M. L. %A Larson, A. I. %D 2009 %T Mini-incision patellar tendon harvest and anterior cruciate ligament reconstruction using critical bony landmarks. %J Sports Med Arthrosc %V 17 %N 4 %P 234-241 %M 19910781 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19910781 %X Graft choice remains an area of contention in anterior cruciate ligament reconstruction. Poorer cosmetic results and anterior knee pain remain a problem in the use of autologous patellar tendon grafts despite excellent clinical results when compared with autologous hamstring tendon grafts. Using a 2-incision technique to harvest the patellar tendon grafts has been shown to decrease the risk of anterior knee pain to a level comparable to hamstring tendon grafts. Proper graft tunnel placement and orientation also remain controversial with several recent researchers arguing the ability to perform an anatomic reconstruction using a conventional endoscopic transtibial technique. We will describe a relatively simple and cosmetically acceptable 2-incision technique for harvesting a bone-tendon-bone graft. In addition, we will describe the bony landmarks that should be used to ensure proper anatomic graft placement and the appropriate angles that need to be used for the tibial tunnel to drill the femoral tunnel in an anatomic position and carry out a successful endoscopic transtibial tunnel anterior cruciate ligament reconstruction. %+ Orthopaedic Associates of Aspen and Glenwood, 100 E. Main St., Aspen, CO 81611, USA. mark@purnell.com Purnell, Mark L Larson, Andrew I 8605 false false 4 Sports medicine and arthroscopy review Sports Med Arthrosc 234-241 2009-12-01 ppublish Journal Article 19910781 In-Process 2009-11-18T07:17:28Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19910781 17 2009 PURPOSE OF REVIEW: Food allergy, a growing clinical and public health problem in the United States and worldwide, is likely determined by multiple environmental and genetic factors. The purpose of this review is to summarize recent advances in food allergy genetic research. RECENT FINDINGS: There is compelling evidence that genetic factors may play a role in food allergy. However, the specific genetic loci that may modulate individual risk of food allergy remain to be identified. To date, only a limited number of candidate gene association studies of food allergy have been reported. Polymorphism(s) in nine genes have been associated with the incidence of food allergy or food allergy severity in at least one study. But most of these findings remain to be replicated in independent populations. In contrast, there are considerable advances in genetics of other allergic diseases such as asthma and atopic dermatitis. Although asthma and atopic dermatitis often coexist with food allergy, the relevance of their candidate genes to food allergy remains to be evaluated. SUMMARY: Genetics in food allergy is a promising research area but is still in its infancy. More studies are needed to dissect susceptible genes of food allergy. A genome-wide association approach may serve as a powerful tool to identify novel genes related to food allergy. Furthermore, the role of gene-environment interaction, gene-gene interaction, and epigenetics in food allergy remains largely unexplored. Given the complex nature of food allergy, future studies need to integrate environment, genomics, and epigenomics in order to better understand the multifaceted etiology and biological mechanisms of food allergy. Hong, X. Tsai, H. J. Wang, X. 0 2009-10-25T06:20:54Z 2009-10-24 %0 Journal Article %A Hong, X. %A Tsai, H. J. %A Wang, X. %D 2009 %T Genetics of food allergy. %J Curr Opin Pediatr %V 21 %N 6 %P 770-776 %M 19851108 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19851108 %X PURPOSE OF REVIEW: Food allergy, a growing clinical and public health problem in the United States and worldwide, is likely determined by multiple environmental and genetic factors. The purpose of this review is to summarize recent advances in food allergy genetic research. RECENT FINDINGS: There is compelling evidence that genetic factors may play a role in food allergy. However, the specific genetic loci that may modulate individual risk of food allergy remain to be identified. To date, only a limited number of candidate gene association studies of food allergy have been reported. Polymorphism(s) in nine genes have been associated with the incidence of food allergy or food allergy severity in at least one study. But most of these findings remain to be replicated in independent populations. In contrast, there are considerable advances in genetics of other allergic diseases such as asthma and atopic dermatitis. Although asthma and atopic dermatitis often coexist with food allergy, the relevance of their candidate genes to food allergy remains to be evaluated. SUMMARY: Genetics in food allergy is a promising research area but is still in its infancy. More studies are needed to dissect susceptible genes of food allergy. A genome-wide association approach may serve as a powerful tool to identify novel genes related to food allergy. Furthermore, the role of gene-environment interaction, gene-gene interaction, and epigenetics in food allergy remains largely unexplored. Given the complex nature of food allergy, future studies need to integrate environment, genomics, and epigenomics in order to better understand the multifaceted etiology and biological mechanisms of food allergy. %+ Mary Ann and J. Milburn Smith Child Health Research Program, Department of Pediatrics, Northwestern University Feinberg School of Medicine and Children's Memorial Hospital and Children's Memorial Research Center, Chicago, Illinois, USA. 8526 false false 6 Current opinion in pediatrics Curr Opin Pediatr 770-776 2009-12-01 ppublish JOURNAL ARTICLE 19851108 Publisher 2009-11-19T07:23:14Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19851108 21 2009 Historically, it has been assumed that oxidative stress contributes to tumor initiation and progression solely by inducing genomic instability. Recent studies indicate that reactive oxygen species are upregulated in tumors and can lead to aberrant induction of signaling networks that cause tumorigenesis and metastasis. Here we review the role of redox-dependent signaling pathways and transcription factors that regulate tumorigenesis. Weinberg, F. Chandel, N. S. 0 2009-09-06T11:55:02Z 2009-07-24 2009-07-25 %0 Journal Article %A Weinberg, F. %A Chandel, N. S. %D 2009 %T Reactive oxygen species-dependent signaling regulates cancer. %J Cell Mol Life Sci %V 66 %N 23 %P 3663-3673 %M 19629388 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19629388 %X Historically, it has been assumed that oxidative stress contributes to tumor initiation and progression solely by inducing genomic instability. Recent studies indicate that reactive oxygen species are upregulated in tumors and can lead to aberrant induction of signaling networks that cause tumorigenesis and metastasis. Here we review the role of redox-dependent signaling pathways and transcription factors that regulate tumorigenesis. %+ Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University Medical School, Chicago, IL 60611, USA. 1039 false false 23 Cellular and molecular life sciences : CMLS Cell Mol Life Sci 3663-3673 2009-12-01 ppublish JOURNAL ARTICLE 19629388 In-Process 2009-11-17T07:20:41Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19629388 66 2009 Numerous mutations in the human A-type lamin gene (LMNA) cause the premature aging disease, progeria. Some of these are located in the alpha-helical central rod domain required for the polymerization of the nuclear lamins into higher order structures. Patient cells with a mutation in this domain, 433G>A (E145K) show severely lobulated nuclei, a separation of the A- and B-type lamins, alterations in pericentric heterochromatin, abnormally clustered centromeres, and mislocalized telomeres. The induction of lobulations and the clustering of centromeres originate during postmitotic nuclear assembly in daughter cells and this early G1 configuration of chromosomes is retained throughout interphase. In vitro analyses of E145K-lamin A show severe defects in the assembly of protofilaments into higher order lamin structures. The results show that this central rod domain mutation affects nuclear architecture in a fashion distinctly different from the changes found in the most common form of progeria caused by the expression of LADelta50/progerin. The study also emphasizes the importance of lamins in nuclear assembly and chromatin organization. Taimen, P. Pfleghaar, K. Shimi, T. Moller, D. Ben-Harush, K. Erdos, M. R. Adam, S. A. Herrmann, H. Medalia, O. Collins, F. S. Goldman, A. E. Goldman, R. D. 0 2009-11-22T07:24:56Z 2009-11-19 2009-11-21 %0 Journal Article %A Taimen, P. %A Pfleghaar, K. %A Shimi, T. %A Moller, D. %A Ben-Harush, K. %A Erdos, M. R. %A Adam, S. A. %A Herrmann, H. %A Medalia, O. %A Collins, F. S. %A Goldman, A. E. %A Goldman, R. D. %D 2009 %T A progeria mutation reveals functions for lamin A in nuclear assembly, architecture, and chromosome organization. %J Proc Natl Acad Sci U S A %M 19926845 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19926845 %X Numerous mutations in the human A-type lamin gene (LMNA) cause the premature aging disease, progeria. Some of these are located in the alpha-helical central rod domain required for the polymerization of the nuclear lamins into higher order structures. Patient cells with a mutation in this domain, 433G>A (E145K) show severely lobulated nuclei, a separation of the A- and B-type lamins, alterations in pericentric heterochromatin, abnormally clustered centromeres, and mislocalized telomeres. The induction of lobulations and the clustering of centromeres originate during postmitotic nuclear assembly in daughter cells and this early G1 configuration of chromosomes is retained throughout interphase. In vitro analyses of E145K-lamin A show severe defects in the assembly of protofilaments into higher order lamin structures. The results show that this central rod domain mutation affects nuclear architecture in a fashion distinctly different from the changes found in the most common form of progeria caused by the expression of LADelta50/progerin. The study also emphasizes the importance of lamins in nuclear assembly and chromatin organization. %+ Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611. 8615 false false Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A 2009-11-19 aheadofprint JOURNAL ARTICLE 19926845 Publisher 2009-11-22T07:24:56Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19926845 2009 We have recently discovered that de-N-acetyl GM3 [NeuNH(2)LacCer, d-GM3], a derivative of ganglioside GM3, is specifically expressed in metastatic tumor cells and that its expression correlates with an enhanced metastatic phenotype. Although the classic N-acetylated form of GM3 (NeuAcLacCer, c-GM3) is found in both normal and tumor cells, metastatic tumor cells (but not other cells) predominantly express d-GM3 (82-95% of total GM3). d-GM3 expression is mainly found in metastatic melanomas, but not in benign nevi or the majority of primary melanomas. Using metastatic (d-GM3-positive) and poorly invasive (d-GM3-negative) human melanoma cell lines, we found that d-GM3 stimulates cell migration and invasion by increasing the expression and activation of urokinase-like plasminogen activator (uPA). Further studies showed that d-GM3 activates matrix metalloproteinase-2 (MMP-2), but not MMP-9, when uPA receptor signaling is activated. These results implicate d-GM3 as a specific marker for metastatic melanoma and a novel therapeutic target for neoplastic diseases. [Cancer Res 2009;69(22):8662-9]. Liu, J. W. Sun, P. Yan, Q. Paller, A. S. Gerami, P. Ho, N. Vashi, N. Le Poole, I. C. Wang, X. Q. 0 2009-11-13T07:19:52Z 2009-11-10 2009-11-12 %0 Journal Article %A Liu, J. W. %A Sun, P. %A Yan, Q. %A Paller, A. S. %A Gerami, P. %A Ho, N. %A Vashi, N. %A Le Poole, I. C. %A Wang, X. Q. %D 2009 %T De-N-acetyl GM3 promotes melanoma cell migration and invasion through urokinase plasminogen activator receptor signaling-dependent MMP-2 activation. %J Cancer Res %V 69 %N 22 %P 8662-8669 %M 19903858 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19903858 %X We have recently discovered that de-N-acetyl GM3 [NeuNH(2)LacCer, d-GM3], a derivative of ganglioside GM3, is specifically expressed in metastatic tumor cells and that its expression correlates with an enhanced metastatic phenotype. Although the classic N-acetylated form of GM3 (NeuAcLacCer, c-GM3) is found in both normal and tumor cells, metastatic tumor cells (but not other cells) predominantly express d-GM3 (82-95% of total GM3). d-GM3 expression is mainly found in metastatic melanomas, but not in benign nevi or the majority of primary melanomas. Using metastatic (d-GM3-positive) and poorly invasive (d-GM3-negative) human melanoma cell lines, we found that d-GM3 stimulates cell migration and invasion by increasing the expression and activation of urokinase-like plasminogen activator (uPA). Further studies showed that d-GM3 activates matrix metalloproteinase-2 (MMP-2), but not MMP-9, when uPA receptor signaling is activated. These results implicate d-GM3 as a specific marker for metastatic melanoma and a novel therapeutic target for neoplastic diseases. %+ Department of Oncology, the First Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China. 8588 false false 22 Cancer research Cancer Res 8662-8669 2009-11-15 ppublish JOURNAL ARTICLE 19903858 In-Process 2009-11-17T07:21:11Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19903858 69 2009 Leukocyte migration across endothelial cell borders (paracellular) and through endothelial cells (transcellular) appear to be distinct processes. During paracellular migration, membrane from a parajunctional reticulum of interconnected vesicles, the endothelial lateral border recycling compartment (LBRC), moves to surround the leukocyte in a kinesin-mediated, microtubule-dependent manner. We show that transcellular migration likewise requires targeted trafficking of LBRC membrane. We show that in addition to platelet/endothelial cell adhesion molecule (PECAM; CD31), CD99 and junctional adhesion molecule A (JAM-A), but apparently not vascular endothelial cell-specific cadherin (cadherin 5, CD144), are components of the LBRC. During transcellular migration, LBRC membrane invests the transmigrating leukocyte. Intracellular adhesion molecule 1 (ICAM-1) on the apical endothelial surface is enriched around adherent leukocytes. Depolymerization of microtubules has no effect on ICAM-1 enrichment but blocks targeted trafficking of LBRC membrane and transcellular migration by >90%. Similar to their effects on paracellular transmigration, antibodies against PECAM or CD99, but not JAM-A, block transcellular migration. We conclude that similar molecular mechanisms promote both para- and transcellular migration. Mamdouh, Z. Mikhailov, A. Muller, W. A. 0 2009-11-07T07:18:19Z 2009-11-11 2009-11-06 %0 Journal Article %A Mamdouh, Z. %A Mikhailov, A. %A Muller, W. A. %D 2009 %T Transcellular migration of leukocytes is mediated by the endothelial lateral border recycling compartment. %J J Exp Med %M 19887395 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19887395 %X Leukocyte migration across endothelial cell borders (paracellular) and through endothelial cells (transcellular) appear to be distinct processes. During paracellular migration, membrane from a parajunctional reticulum of interconnected vesicles, the endothelial lateral border recycling compartment (LBRC), moves to surround the leukocyte in a kinesin-mediated, microtubule-dependent manner. We show that transcellular migration likewise requires targeted trafficking of LBRC membrane. We show that in addition to platelet/endothelial cell adhesion molecule (PECAM; CD31), CD99 and junctional adhesion molecule A (JAM-A), but apparently not vascular endothelial cell-specific cadherin (cadherin 5, CD144), are components of the LBRC. During transcellular migration, LBRC membrane invests the transmigrating leukocyte. Intracellular adhesion molecule 1 (ICAM-1) on the apical endothelial surface is enriched around adherent leukocytes. Depolymerization of microtubules has no effect on ICAM-1 enrichment but blocks targeted trafficking of LBRC membrane and transcellular migration by >90%. Similar to their effects on paracellular transmigration, antibodies against PECAM or CD99, but not JAM-A, block transcellular migration. We conclude that similar molecular mechanisms promote both para- and transcellular migration. %+ Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611. 8573 false false The Journal of experimental medicine J Exp Med 2009-11-11 aheadofprint JOURNAL ARTICLE 19887395 Publisher 2009-11-13T07:20:34Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19887395 2009 Hypoxia can cause stress and structural changes to the epithelial cytoskeleton. The intermediate filament (IF) network is known to reorganize in response to stress. We examined whether rats exposed to hypoxia had altered keratin IF expression in their alveolar epithelial type II (ATII) cells. There was a significant decrease in keratin protein levels in hypoxic ATII cells compared with those in ATII cells isolated from normoxic rats. To define the mechanisms regulating this process we studied changes to the keratin IF network in A549 cells (an alveolar epithelial cell line) exposed to 1.5% oxygen. We observed a time-dependent disassembly-degradation of keratin 8 and 18 proteins, which was associated with an increase in reactive oxygen species (ROS). Hypoxia-treated A549 cells deficient in mitochondrial DNA or A549 cells treated with a small interfering RNA against the Rieske iron-sulfur protein of mitochondrial complex III did not have increased levels of ROS nor was the keratin IF network disassembled and degraded. The superoxide dismutase (SOD)/catalase mimetic (EUK-134) prevented the hypoxia-mediated keratin IF degradation as did the overexpression of SOD1 but not of SOD2. Accordingly, we provide evidence that hypoxia promotes the disassembly and degradation of the keratin IF network via mitochondrial complex III-generated reactive oxygen species.-Na, N., Chandel, N. S., Litvan, J., Ridge, K. M. Mitochondrial reactive oxygen species are required for hypoxia-induced degradation of keratin intermediate filaments. Na, N. Chandel, N. S. Litvan, J. Ridge, K. M. 0 2009-11-11T07:21:44Z 2009-11-06 2009-11-10 %0 Journal Article %A Na, N. %A Chandel, N. S. %A Litvan, J. %A Ridge, K. M. %D 2009 %T Mitochondrial reactive oxygen species are required for hypoxia-induced degradation of keratin intermediate filaments. %J FASEB J %M 19897662 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19897662 %X Hypoxia can cause stress and structural changes to the epithelial cytoskeleton. The intermediate filament (IF) network is known to reorganize in response to stress. We examined whether rats exposed to hypoxia had altered keratin IF expression in their alveolar epithelial type II (ATII) cells. There was a significant decrease in keratin protein levels in hypoxic ATII cells compared with those in ATII cells isolated from normoxic rats. To define the mechanisms regulating this process we studied changes to the keratin IF network in A549 cells (an alveolar epithelial cell line) exposed to 1.5% oxygen. We observed a time-dependent disassembly-degradation of keratin 8 and 18 proteins, which was associated with an increase in reactive oxygen species (ROS). Hypoxia-treated A549 cells deficient in mitochondrial DNA or A549 cells treated with a small interfering RNA against the Rieske iron-sulfur protein of mitochondrial complex III did not have increased levels of ROS nor was the keratin IF network disassembled and degraded. The superoxide dismutase (SOD)/catalase mimetic (EUK-134) prevented the hypoxia-mediated keratin IF degradation as did the overexpression of SOD1 but not of SOD2. Accordingly, we provide evidence that hypoxia promotes the disassembly and degradation of the keratin IF network via mitochondrial complex III-generated reactive oxygen species.-Na, N., Chandel, N. S., Litvan, J., Ridge, K. M. Mitochondrial reactive oxygen species are required for hypoxia-induced degradation of keratin intermediate filaments. %+ *Division of Pulmonary and Critical Care Medicine andDepartment of Cell Molecular Biology, Northwestern University Medical School, Chicago, Illinois, USA. 8579 false false The FASEB journal : official publication of the Federation of American Societies for Experimental Biology FASEB J 2009-11-06 aheadofprint JOURNAL ARTICLE 19897662 Publisher 2009-11-11T07:21:44Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19897662 2009 The regulated expression of large human genes can depend on long-range interactions to establish appropriate three-dimensional structures across the locus. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encompasses 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation. The flanking loci, ASZ1 and CTTNBP2, show very different tissue-specific expression. The mechanisms governing control of CFTR expression remain poorly understood, although they are known to involve intronic regulatory elements. Here, we show a complex looped structure of the CFTR locus in cells that express the gene, which is absent from cells in which the gene is inactive. By using chromatin conformation capture (3C) with a bait probe at the CFTR promoter, we demonstrate close interaction of this region with sequences in the middle of the gene about 100 kb from the promoter and with regions 3' to the locus that are about 200 kb away. We show that these interacting regions correspond to prominent DNase I hypersensitive sites within the locus. Moreover, these sequences act cooperatively in reporter gene constructs and recruit proteins that modify chromatin structure. The model for CFTR gene expression that is revealed by our data provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions. We anticipate that these observations will enable original approaches to designing regulated transgenes for tissue-specific gene therapy protocols. Ott, C. J. Blackledge, N. P. Kerschner, J. L. Leir, S. H. Crawford, G. E. Cotton, C. U. Harris, A. 0 2009-11-11T07:23:01Z 2009-11-06 2009-11-10 %0 Journal Article %A Ott, C. J. %A Blackledge, N. P. %A Kerschner, J. L. %A Leir, S. H. %A Crawford, G. E. %A Cotton, C. U. %A Harris, A. %D 2009 %T Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus. %J Proc Natl Acad Sci U S A %M 19897727 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19897727 %X The regulated expression of large human genes can depend on long-range interactions to establish appropriate three-dimensional structures across the locus. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encompasses 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation. The flanking loci, ASZ1 and CTTNBP2, show very different tissue-specific expression. The mechanisms governing control of CFTR expression remain poorly understood, although they are known to involve intronic regulatory elements. Here, we show a complex looped structure of the CFTR locus in cells that express the gene, which is absent from cells in which the gene is inactive. By using chromatin conformation capture (3C) with a bait probe at the CFTR promoter, we demonstrate close interaction of this region with sequences in the middle of the gene about 100 kb from the promoter and with regions 3' to the locus that are about 200 kb away. We show that these interacting regions correspond to prominent DNase I hypersensitive sites within the locus. Moreover, these sequences act cooperatively in reporter gene constructs and recruit proteins that modify chromatin structure. The model for CFTR gene expression that is revealed by our data provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions. We anticipate that these observations will enable original approaches to designing regulated transgenes for tissue-specific gene therapy protocols. %+ Human Molecular Genetics Program, Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, 2300 Children's Plaza #211, Chicago, IL 60614. 8582 false false Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A 2009-11-06 aheadofprint JOURNAL ARTICLE 19897727 Publisher 2009-11-11T07:23:01Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19897727 2009 Tissue engineering scaffolds capable of gene delivery can provide a structure that supports tissue formation while also inducing the expression of inductive factors. Sustained release strategies are hypothesized to maintain elevated plasmid concentrations locally that can enhance gene transfer. In this report, we investigate the relationship between plasmid release kinetics and the extent and duration of transgene expression. Scaffolds were fabricated from polymer microspheres modified with cationic polymers (polyethylenimine, poly(l-lysine), poly(allylamine hydrochloride), polydiallyldimethylammonium) or polydopamine (PD), with PD enhancing incorporation and slowing release. In vivo implantation of scaffolds into the peritoneal fat pad had no significant changes in the level and duration of transgene expression between PD and unmodified scaffolds. Control studies with plasmid dried onto scaffolds, which exhibited a rapid release, and scaffolds with extended leaching to reduce initial quantities released had similar levels and duration of expression. Changing the plasmid design, from a cytomegalovirus (CMV) to an ubiquitin C (UbC) promoter substantially altered the duration of expression. These studies suggest that the initial dose released and vector design affect the extent and duration of transgene expression, which may be sustained over several weeks, potentially leading to numerous applications in cell transplantation and regenerative medicine. Aviles, M. O. Lin, C. H. Zelivyanskaya, M. Graham, J. G. Boehler, R. M. Messersmith, P. B. Shea, L. D. 0 2009-11-08T07:21:35Z 2009-11-03 2009-11-07 %0 Journal Article %A Aviles, M. O. %A Lin, C. H. %A Zelivyanskaya, M. %A Graham, J. G. %A Boehler, R. M. %A Messersmith, P. B. %A Shea, L. D. %D 2009 %T The contribution of plasmid design and release to in vivo gene expression following delivery from cationic polymer modified scaffolds. %J Biomaterials %M 19892398 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19892398 %X Tissue engineering scaffolds capable of gene delivery can provide a structure that supports tissue formation while also inducing the expression of inductive factors. Sustained release strategies are hypothesized to maintain elevated plasmid concentrations locally that can enhance gene transfer. In this report, we investigate the relationship between plasmid release kinetics and the extent and duration of transgene expression. Scaffolds were fabricated from polymer microspheres modified with cationic polymers (polyethylenimine, poly(l-lysine), poly(allylamine hydrochloride), polydiallyldimethylammonium) or polydopamine (PD), with PD enhancing incorporation and slowing release. In vivo implantation of scaffolds into the peritoneal fat pad had no significant changes in the level and duration of transgene expression between PD and unmodified scaffolds. Control studies with plasmid dried onto scaffolds, which exhibited a rapid release, and scaffolds with extended leaching to reduce initial quantities released had similar levels and duration of expression. Changing the plasmid design, from a cytomegalovirus (CMV) to an ubiquitin C (UbC) promoter substantially altered the duration of expression. These studies suggest that the initial dose released and vector design affect the extent and duration of transgene expression, which may be sustained over several weeks, potentially leading to numerous applications in cell transplantation and regenerative medicine. %+ Departments of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd, Evanston, IL 60208, USA. 8576 false false Biomaterials Biomaterials 2009-11-03 aheadofprint JOURNAL ARTICLE 19892398 Publisher 2009-11-08T07:21:35Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19892398 2009 Elevated CO(2) levels (hypercapnia) frequently occur in patients with obstructive pulmonary diseases and are associated with increased mortality. However, the effects of hypercapnia on non-neuronal tissues and the mechanisms that mediate these effects are largely unknown. Here, we develop Drosophila as a genetically tractable model for defining non-neuronal CO(2) responses and response pathways. We show that hypercapnia significantly impairs embryonic morphogenesis, egg laying, and egg hatching even in mutants lacking the Gr63a neuronal CO(2) sensor. Consistent with previous reports that hypercapnic acidosis can suppress mammalian NF-kappaB-regulated innate immune genes, we find that in adult flies and the phagocytic immune-responsive S2* cell line, hypercapnia suppresses induction of specific antimicrobial peptides that are regulated by Relish, a conserved Rel/NF-kappaB family member. Correspondingly, modest hypercapnia (7-13%) increases mortality of flies inoculated with E. faecalis, A. tumefaciens, or S. aureus. During E. faecalis and A. tumefaciens infection, increased bacterial loads were observed, indicating that hypercapnia can decrease host resistance. Hypercapnic immune suppression is not mediated by acidosis, the olfactory CO(2) receptor Gr63a, or by nitric oxide signaling. Further, hypercapnia does not induce responses characteristic of hypoxia, oxidative stress, or heat shock. Finally, proteolysis of the Relish IkappaB-like domain is unaffected by hypercapnia, indicating that immunosuppression acts downstream of, or in parallel to, Relish proteolytic activation. Our results suggest that hypercapnic immune suppression is mediated by a conserved response pathway, and illustrate a mechanism by which hypercapnia could contribute to worse outcomes of patients with advanced lung disease, who frequently suffer from both hypercapnia and respiratory infections. Helenius, I. T. Krupinski, T. Turnbull, D. W. Gruenbaum, Y. Silverman, N. Johnson, E. A. Sporn, P. H. Sznajder, J. I. Beitel, G. J. 0 2009-10-24T06:20:02Z 2009-10-21 2009-10-23 %0 Journal Article %A Helenius, I. T. %A Krupinski, T. %A Turnbull, D. W. %A Gruenbaum, Y. %A Silverman, N. %A Johnson, E. A. %A Sporn, P. H. %A Sznajder, J. I. %A Beitel, G. J. %D 2009 %T Elevated CO2 suppresses specific Drosophila innate immune responses and resistance to bacterial infection. %J Proc Natl Acad Sci U S A %V 106 %N 44 %P 18710-18715 %M 19846771 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19846771 %X Elevated CO(2) levels (hypercapnia) frequently occur in patients with obstructive pulmonary diseases and are associated with increased mortality. However, the effects of hypercapnia on non-neuronal tissues and the mechanisms that mediate these effects are largely unknown. Here, we develop Drosophila as a genetically tractable model for defining non-neuronal CO(2) responses and response pathways. We show that hypercapnia significantly impairs embryonic morphogenesis, egg laying, and egg hatching even in mutants lacking the Gr63a neuronal CO(2) sensor. Consistent with previous reports that hypercapnic acidosis can suppress mammalian NF-kappaB-regulated innate immune genes, we find that in adult flies and the phagocytic immune-responsive S2* cell line, hypercapnia suppresses induction of specific antimicrobial peptides that are regulated by Relish, a conserved Rel/NF-kappaB family member. Correspondingly, modest hypercapnia (7-13%) increases mortality of flies inoculated with E. faecalis, A. tumefaciens, or S. aureus. During E. faecalis and A. tumefaciens infection, increased bacterial loads were observed, indicating that hypercapnia can decrease host resistance. Hypercapnic immune suppression is not mediated by acidosis, the olfactory CO(2) receptor Gr63a, or by nitric oxide signaling. Further, hypercapnia does not induce responses characteristic of hypoxia, oxidative stress, or heat shock. Finally, proteolysis of the Relish IkappaB-like domain is unaffected by hypercapnia, indicating that immunosuppression acts downstream of, or in parallel to, Relish proteolytic activation. Our results suggest that hypercapnic immune suppression is mediated by a conserved response pathway, and illustrate a mechanism by which hypercapnia could contribute to worse outcomes of patients with advanced lung disease, who frequently suffer from both hypercapnia and respiratory infections. %+ Department of Biochemistry, Northwestern University, Evanston, IL 60208, USA. 8518 false false 44 Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A 18710-18715 2009-11-03 ppublish JOURNAL ARTICLE 19846771 In-Process 2009-11-06T07:15:06Z http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19846771 106 2009