Abnormal development such as in DD and cancer can influence activities within the surrounding tis... more Abnormal development such as in DD and cancer can influence activities within the surrounding tissues. Hence an association of these diseases with normal tissue repair processes should be expected, compromising the distinction between the genes causing the development of such a disease and the genes involved in the homeostatic response. Response to perturbations may be regulated at different levels [Daran-Lapujade et al., 2007 and processes are linked extensively,
Non-alcoholic fatty liver disease (NAFLD) is a consequence of sedentary life style and high fat d... more Non-alcoholic fatty liver disease (NAFLD) is a consequence of sedentary life style and high fat diets with an estimated prevalence of about 30% in western countries. It is associated with insulin resistance, obesity, glucose intolerance and drug toxicity. Additionally, polymorphisms within, e.g., APOC3, PNPLA3, NCAN, TM6SF2 and PPP1R3B, correlate with NAFLD. Several studies have already investigated later stages of the disease. This study explores the early steatosis stage of NAFLD with the aim of identifying molecular mechanisms underlying the etiology of NAFLD. We analyzed liver biopsies and serum samples from patients with high- and low-grade steatosis (also pre-disease states) employing transcriptomics, ELISA-based serum protein analyses and metabolomics. Here, we provide a detailed description of the various related datasets produced in the course of this study. These datasets may help other researchers find new clues for the etiology of NAFLD and the mechanisms underlying its ...
In the second half of 2014 the Manchester Institute of Biotechnology, based in Manchester (UK), h... more In the second half of 2014 the Manchester Institute of Biotechnology, based in Manchester (UK), hosted the first SupraBiology congress, an event attended by representatives of different academic institutions and industry based in both the UK and China. The congress was aimed to serve as a platform to discuss and promote potential collaborations between the UK and China on the subject of Systems Biology and High Performance Computing. The event, sponsored by the "BBSRC China Partnering Awards" and ISBE, was organised as a sequence of talks addressing the different aspects of Systems Biology that can benefit from High Performance Computing. A general discussion session followed where the scientific, technical, and logistic aspects of the prospected UK-China collaborations were examined.
ABSTRACT Dupuytren’s disease (DD) is a benign fibroproliferative tumour of unknown aetiopathogene... more ABSTRACT Dupuytren’s disease (DD) is a benign fibroproliferative tumour of unknown aetiopathogenesis affecting the palm of the hand, which often causes progressive, permanent contracture of the digits. Previous studies provide compelling evidence that genetic dysregulation plays an important role. Macroscopically affected areas demonstrate phenotypic differences between two structurally distinct fibrotic elements in DD (i.e. the nodule and the cord). In addition, the fat adjacent to and the skin overlying the nodule is thought to be of pathological relevance in relation to development of the disease. Abnormal Dupuytren fibroblasts are considered to be responsible for causing the disease. Whether any significant changes seen in these fibroblasts are due to genetic alterations alone or a consequence of a metabolic dysregulation has not to our knowledge been shown yet. In this study, we have used a metabolomic analysis of Dupuytren fibroblast cultures derived from different DD tissue phenotypes and compare early (primary) cultures to late passages in order to identify the most representative passage for the disease. Using Fourier transform infrared spectroscopy and Raman microspectroscopy we obtained metabolic profiles of endogenous (fingerprint) and secreted (footprint) metabolites from (1) DD cords and nodules from the palm against the unaffected transverse palmar fascia (internal control), (2) DD cords and nodules with the cushioning fat surrounding the nodule, and with the skin overlying the nodule in one DD patient. We then compared metabolic profiles of those in (1) and (2) between different DD patients and identified biologically important metabolites. Metabolic differences were identified between fibroblast cell samples in DD fibroblasts and in control fibroblasts through variable passages from the different sites cultured in O2 conditions in same culture media. We believe that metabolomic strategies offer a novel approach to investigate biomarkers in DD.
. The hierarchical subdivision of cellular biochemistry. From top to bottom, the levels are those... more . The hierarchical subdivision of cellular biochemistry. From top to bottom, the levels are those of DNA, mRNA, proteins, and metabolism. From www.siliconcell.net, with permission.
Non-alcoholic fatty liver disease comprises a broad spectrum of disease states ranging from simpl... more Non-alcoholic fatty liver disease comprises a broad spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis. As a result of increases in the prevalences of obesity, insulin resistance, and hyperlipidemia, the number of people with hepatic steatosis continues to increase. Differences in susceptibility to steatohepatitis and its progression to cirrhosis have been attributed to a complex interplay of genetic and external factors all addressing the intracellular network. Increase in sugar or refined carbohydrate consumption results in an increase of insulin and insulin resistance that can lead to the accumulation of fat in the liver. Here we demonstrate how a multidisciplinary approach encompassing cellular reprogramming, transcriptomics, proteomics, metabolomics, modeling, network reconstruction, and data management can be employed to unveil the mechanisms underlying the progression of steatosis. Proteomics revealed reduced AKT/mTOR signaling in fibroblasts derived from steatosis patients and further establishes that the insulin-resistant phenotype is present not only in insulin-metabolizing central organs, e.g., the liver, but is also manifested in skin fibroblasts. Transcriptome data enabled the generation of a regulatory network based on the transcription factor SREBF1, linked to a metabolic network of glycerolipid, and fatty acid biosynthesis including the downstream transcriptional targets of SREBF1 which include LIPIN1 (LPIN) and low density lipoprotein receptor. Glutathione metabolism was among the pathways enriched in steatosis patients in comparison to healthy controls. By using a model of the glutathione pathway we predict a significant increase in the flux through glutathione synthesis as both gamma-glutamylcysteine synthetase and glutathione synthetase have an increased flux. We anticipate that a larger cohort of patients and matched controls will confirm our preliminary findings presented here.
Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease resulting from an aberrant... more Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease resulting from an aberrant BCR.ABL gene and protein. To predict BCR.ABL protein abundance and phosphorylation in individual cells in a population of CML cells, we modelled BCR.ABL protein regulation through associated miRNAs using a systems approach. The model rationalizes the level of BCR.ABL protein heterogeneity in CML cells in correlation with the heterogeneous BCR.ABL mRNA levels. We also measured BCR.ABL mRNA and BCR.ABLp phosphorylation in individual cells. The experimental data were consistent with the modelling results, thereby partly validating the model. Provided it is tested further, the model may be used to support effective therapeutic strategies including the combined application of a tyrosine kinase inhibitor and miRNAs targeting BCR.ABL. It appears able to predict different effects of the two types of drug on cells with different expression levels and consequently different effects on the generation of resistance.
Background: Proteomics is increasingly becoming an important tool for the study of many different... more Background: Proteomics is increasingly becoming an important tool for the study of many different aspects of plant functions, such as investigating the molecular processes underlying in plant physiology, development, differentiation and their interaction with the environments. To investigate the cassava (Manihot esculenta Crantz) proteome, we extracted proteins from somatic embryos, plantlets and tuberous roots of cultivar SC8 and separated them by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Analysis by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) yielded a total of 383 proteins including isoforms, classified into 14 functional groups. The majority of these were carbohydrate and energy metabolism associated proteins (27.2%), followed by those involved in protein biosynthesis (14.4%). Subsequent analysis has revealed that 54, 59, 74 and 102 identified proteins are unique to the somatic embryos, shoots, adventitious roots and tuberous roots, respectively. Some of these proteins may serve as signatures for the physiological and developmental stages of somatic embryos, shoots, adventitious roots and tuberous root. Western blotting results have shown high expression levels of Rubisco in shoots and its absence in the somatic embryos. In addition, high-level expression of α-tubulin was found in tuberous roots, and a low-level one in somatic embryos. This extensive study effectively provides a huge data set of dynamic protein-related information to better understand the molecular basis underlying cassava growth, development, and physiological functions.
The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules... more The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.
Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder affecting the palm of the ... more Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder affecting the palm of the hand, resulting in progressive and irreversible digital contracture. In view of the abnormal gene dysregulation found in DD, and its potential effect on metabolites at a functional level, we chose to examine the metabolic profile involved in DD. Using Fourier transform infrared (FT-IR) spectroscopy to generate metabolic fingerprints of cultured cells, we compared the profiles of DD cords and nodules (1) against the unaffected transverse palmar fascia (internal control), (2) against carpal ligamentous fascia (external control), and (3) against fibroblasts from fat surrounding the nodule and skin overlying the nodule (environmental control). We also determined the effects of serial passaging of the cells on DD fingerprints. Subsequently, gas chromatographymass spectrometry (GC-MS) was employed for metabolic profiling in order to identify metabolites characteristic of the DD tissue phenotypes. We developed a robust metabolomic analysis procedure of DD using cultured fibroblasts derived from DD tissues. Our carefully controlled culture conditions, combined with assessment of metabolic phenotypes by FT-IR and GC-MS, enabled us to demonstrate metabolic differences between DD and unaffected transverse palmar fascia and between DD and healthy control tissue. In early passage (0-3) the metabolic differences were clear, but cells from subsequent passages (4-6) started to lose this distinction between diseased and non-diseased origin. The dysregulated metabolites we identified were leucine, phenylalanine, lysine, cysteine, aspartic acid, glycerol-3-phosphate and the vitamin precursor to coenzyme A. Early passage DD cells exhibit a clear metabolic profile, in which central metabolic pathways appear to be involved. Experimental conditions have been identified in which these DD data are reproducible. The experimental reproducibility will be useful in DD diagnostics and for DD systems biology.
Purpose Dupuytren's disease (DD) is a fibroproliferative disorder of unknown etiopathogenesis, wh... more Purpose Dupuytren's disease (DD) is a fibroproliferative disorder of unknown etiopathogenesis, which may cause progressive, permanent contracture of digits. Previous studies provide compelling evidence that genetic alterations play an important role. Macroscopically affected areas demonstrate phenotypic differences between the two structurally distinct fibrotic elements in DD (ie, the nodule and the cord). In this study, we set out to (1) compare gene expression profiles between DD and transverse carpal fascia of control subjects (external control); (2) profile DD cords and nodules from the palm against the unaffected transverse palmar fascia (internal control); and (3) identify biologically important candidate genes from the transcriptome profiles. Methods RNA samples from DD nodules (n ϭ 4), cords (n ϭ 4), and internal control (n ϭ 4) as well as external control (n ϭ 4) from unaffected individuals were subjected to differential gene expression profile analysis. Changes of more than 2-fold in DD groups and controls were recorded. Quantitative reverse transcriptase-polymerase chain reactions were performed to validate 16 implicated genes, which included developmental control genes, matrix metalloproteinases, and apoptotic genes. Results Several genes associated with DD formation were common across all 6 pairwise analyses. Genes markedly upregulated shared common expression levels across all pairwise analysis studies. Pairs involving the DD nodule arrays were notably distinguishable from all other permutations. The majority of genes dysregulated in the DD cords demonstrated an increase in fold change when compared with the DD nodule tissues. Key collagens, collagenases, metalloproteinases, and inhibitors were identified. Genes involved in cytoskeleton development and lipid metabolism were markedly dysregulated. Confirmations of these alterations were obtained in quantitative reverse transcriptase-polymerase chain reaction. Conclusions These data demonstrate a gradation in expression of certain genes in DD tissue phenotypes compared with control fascia. Transcriptome profiling is predictive not only of disease but also of disease phenotype. These results indicate a number of important candidate genes associated with DD formation, which may provide clues for molecular mechanisms involved in DD pathogenesis. ( J Hand Surg 2008;33A:359 -372.
Th e quasi-neoplastic nodular palmar fi bromatosis [1] called Dupuytren's disease (DD) often caus... more Th e quasi-neoplastic nodular palmar fi bromatosis [1] called Dupuytren's disease (DD) often causes permanent fl exion contracture of the metacarpophalangeal and proximal interphalangeal joints of the digits [2,3] ), leading to loss of function, deformity of the hand, and permanent contracture of the involved digits . Although DD does not metastasize , it may invade locally within the palmar aponeurosis of the hand (sparingly supplied with blood vessels) and it is progressive with a high rate of recurrence after surgical excision [6], often requiring amputation of the aff ected digit . Th e three stages of DD growth (proliferative, involutional, and residual) appear to involve dysdiff er en tiation into myofi broblasts . DD is associated with abundance of collagen, fi bronectin, integrins, cytokines and many other growth factors , as well as altered expression of several genes , but unlike the involve ment of known oncogenes and suppressor genes in cancer development , our knowledge of the exact aetiopathogenesis of DD remains poor despite signifi cant understanding of its biology.
Abnormal development such as in DD and cancer can influence activities within the surrounding tis... more Abnormal development such as in DD and cancer can influence activities within the surrounding tissues. Hence an association of these diseases with normal tissue repair processes should be expected, compromising the distinction between the genes causing the development of such a disease and the genes involved in the homeostatic response. Response to perturbations may be regulated at different levels [Daran-Lapujade et al., 2007 and processes are linked extensively,
Non-alcoholic fatty liver disease (NAFLD) is a consequence of sedentary life style and high fat d... more Non-alcoholic fatty liver disease (NAFLD) is a consequence of sedentary life style and high fat diets with an estimated prevalence of about 30% in western countries. It is associated with insulin resistance, obesity, glucose intolerance and drug toxicity. Additionally, polymorphisms within, e.g., APOC3, PNPLA3, NCAN, TM6SF2 and PPP1R3B, correlate with NAFLD. Several studies have already investigated later stages of the disease. This study explores the early steatosis stage of NAFLD with the aim of identifying molecular mechanisms underlying the etiology of NAFLD. We analyzed liver biopsies and serum samples from patients with high- and low-grade steatosis (also pre-disease states) employing transcriptomics, ELISA-based serum protein analyses and metabolomics. Here, we provide a detailed description of the various related datasets produced in the course of this study. These datasets may help other researchers find new clues for the etiology of NAFLD and the mechanisms underlying its ...
In the second half of 2014 the Manchester Institute of Biotechnology, based in Manchester (UK), h... more In the second half of 2014 the Manchester Institute of Biotechnology, based in Manchester (UK), hosted the first SupraBiology congress, an event attended by representatives of different academic institutions and industry based in both the UK and China. The congress was aimed to serve as a platform to discuss and promote potential collaborations between the UK and China on the subject of Systems Biology and High Performance Computing. The event, sponsored by the "BBSRC China Partnering Awards" and ISBE, was organised as a sequence of talks addressing the different aspects of Systems Biology that can benefit from High Performance Computing. A general discussion session followed where the scientific, technical, and logistic aspects of the prospected UK-China collaborations were examined.
ABSTRACT Dupuytren’s disease (DD) is a benign fibroproliferative tumour of unknown aetiopathogene... more ABSTRACT Dupuytren’s disease (DD) is a benign fibroproliferative tumour of unknown aetiopathogenesis affecting the palm of the hand, which often causes progressive, permanent contracture of the digits. Previous studies provide compelling evidence that genetic dysregulation plays an important role. Macroscopically affected areas demonstrate phenotypic differences between two structurally distinct fibrotic elements in DD (i.e. the nodule and the cord). In addition, the fat adjacent to and the skin overlying the nodule is thought to be of pathological relevance in relation to development of the disease. Abnormal Dupuytren fibroblasts are considered to be responsible for causing the disease. Whether any significant changes seen in these fibroblasts are due to genetic alterations alone or a consequence of a metabolic dysregulation has not to our knowledge been shown yet. In this study, we have used a metabolomic analysis of Dupuytren fibroblast cultures derived from different DD tissue phenotypes and compare early (primary) cultures to late passages in order to identify the most representative passage for the disease. Using Fourier transform infrared spectroscopy and Raman microspectroscopy we obtained metabolic profiles of endogenous (fingerprint) and secreted (footprint) metabolites from (1) DD cords and nodules from the palm against the unaffected transverse palmar fascia (internal control), (2) DD cords and nodules with the cushioning fat surrounding the nodule, and with the skin overlying the nodule in one DD patient. We then compared metabolic profiles of those in (1) and (2) between different DD patients and identified biologically important metabolites. Metabolic differences were identified between fibroblast cell samples in DD fibroblasts and in control fibroblasts through variable passages from the different sites cultured in O2 conditions in same culture media. We believe that metabolomic strategies offer a novel approach to investigate biomarkers in DD.
. The hierarchical subdivision of cellular biochemistry. From top to bottom, the levels are those... more . The hierarchical subdivision of cellular biochemistry. From top to bottom, the levels are those of DNA, mRNA, proteins, and metabolism. From www.siliconcell.net, with permission.
Non-alcoholic fatty liver disease comprises a broad spectrum of disease states ranging from simpl... more Non-alcoholic fatty liver disease comprises a broad spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis. As a result of increases in the prevalences of obesity, insulin resistance, and hyperlipidemia, the number of people with hepatic steatosis continues to increase. Differences in susceptibility to steatohepatitis and its progression to cirrhosis have been attributed to a complex interplay of genetic and external factors all addressing the intracellular network. Increase in sugar or refined carbohydrate consumption results in an increase of insulin and insulin resistance that can lead to the accumulation of fat in the liver. Here we demonstrate how a multidisciplinary approach encompassing cellular reprogramming, transcriptomics, proteomics, metabolomics, modeling, network reconstruction, and data management can be employed to unveil the mechanisms underlying the progression of steatosis. Proteomics revealed reduced AKT/mTOR signaling in fibroblasts derived from steatosis patients and further establishes that the insulin-resistant phenotype is present not only in insulin-metabolizing central organs, e.g., the liver, but is also manifested in skin fibroblasts. Transcriptome data enabled the generation of a regulatory network based on the transcription factor SREBF1, linked to a metabolic network of glycerolipid, and fatty acid biosynthesis including the downstream transcriptional targets of SREBF1 which include LIPIN1 (LPIN) and low density lipoprotein receptor. Glutathione metabolism was among the pathways enriched in steatosis patients in comparison to healthy controls. By using a model of the glutathione pathway we predict a significant increase in the flux through glutathione synthesis as both gamma-glutamylcysteine synthetase and glutathione synthetase have an increased flux. We anticipate that a larger cohort of patients and matched controls will confirm our preliminary findings presented here.
Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease resulting from an aberrant... more Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease resulting from an aberrant BCR.ABL gene and protein. To predict BCR.ABL protein abundance and phosphorylation in individual cells in a population of CML cells, we modelled BCR.ABL protein regulation through associated miRNAs using a systems approach. The model rationalizes the level of BCR.ABL protein heterogeneity in CML cells in correlation with the heterogeneous BCR.ABL mRNA levels. We also measured BCR.ABL mRNA and BCR.ABLp phosphorylation in individual cells. The experimental data were consistent with the modelling results, thereby partly validating the model. Provided it is tested further, the model may be used to support effective therapeutic strategies including the combined application of a tyrosine kinase inhibitor and miRNAs targeting BCR.ABL. It appears able to predict different effects of the two types of drug on cells with different expression levels and consequently different effects on the generation of resistance.
Background: Proteomics is increasingly becoming an important tool for the study of many different... more Background: Proteomics is increasingly becoming an important tool for the study of many different aspects of plant functions, such as investigating the molecular processes underlying in plant physiology, development, differentiation and their interaction with the environments. To investigate the cassava (Manihot esculenta Crantz) proteome, we extracted proteins from somatic embryos, plantlets and tuberous roots of cultivar SC8 and separated them by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Analysis by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) yielded a total of 383 proteins including isoforms, classified into 14 functional groups. The majority of these were carbohydrate and energy metabolism associated proteins (27.2%), followed by those involved in protein biosynthesis (14.4%). Subsequent analysis has revealed that 54, 59, 74 and 102 identified proteins are unique to the somatic embryos, shoots, adventitious roots and tuberous roots, respectively. Some of these proteins may serve as signatures for the physiological and developmental stages of somatic embryos, shoots, adventitious roots and tuberous root. Western blotting results have shown high expression levels of Rubisco in shoots and its absence in the somatic embryos. In addition, high-level expression of α-tubulin was found in tuberous roots, and a low-level one in somatic embryos. This extensive study effectively provides a huge data set of dynamic protein-related information to better understand the molecular basis underlying cassava growth, development, and physiological functions.
The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules... more The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.
Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder affecting the palm of the ... more Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder affecting the palm of the hand, resulting in progressive and irreversible digital contracture. In view of the abnormal gene dysregulation found in DD, and its potential effect on metabolites at a functional level, we chose to examine the metabolic profile involved in DD. Using Fourier transform infrared (FT-IR) spectroscopy to generate metabolic fingerprints of cultured cells, we compared the profiles of DD cords and nodules (1) against the unaffected transverse palmar fascia (internal control), (2) against carpal ligamentous fascia (external control), and (3) against fibroblasts from fat surrounding the nodule and skin overlying the nodule (environmental control). We also determined the effects of serial passaging of the cells on DD fingerprints. Subsequently, gas chromatographymass spectrometry (GC-MS) was employed for metabolic profiling in order to identify metabolites characteristic of the DD tissue phenotypes. We developed a robust metabolomic analysis procedure of DD using cultured fibroblasts derived from DD tissues. Our carefully controlled culture conditions, combined with assessment of metabolic phenotypes by FT-IR and GC-MS, enabled us to demonstrate metabolic differences between DD and unaffected transverse palmar fascia and between DD and healthy control tissue. In early passage (0-3) the metabolic differences were clear, but cells from subsequent passages (4-6) started to lose this distinction between diseased and non-diseased origin. The dysregulated metabolites we identified were leucine, phenylalanine, lysine, cysteine, aspartic acid, glycerol-3-phosphate and the vitamin precursor to coenzyme A. Early passage DD cells exhibit a clear metabolic profile, in which central metabolic pathways appear to be involved. Experimental conditions have been identified in which these DD data are reproducible. The experimental reproducibility will be useful in DD diagnostics and for DD systems biology.
Purpose Dupuytren's disease (DD) is a fibroproliferative disorder of unknown etiopathogenesis, wh... more Purpose Dupuytren's disease (DD) is a fibroproliferative disorder of unknown etiopathogenesis, which may cause progressive, permanent contracture of digits. Previous studies provide compelling evidence that genetic alterations play an important role. Macroscopically affected areas demonstrate phenotypic differences between the two structurally distinct fibrotic elements in DD (ie, the nodule and the cord). In this study, we set out to (1) compare gene expression profiles between DD and transverse carpal fascia of control subjects (external control); (2) profile DD cords and nodules from the palm against the unaffected transverse palmar fascia (internal control); and (3) identify biologically important candidate genes from the transcriptome profiles. Methods RNA samples from DD nodules (n ϭ 4), cords (n ϭ 4), and internal control (n ϭ 4) as well as external control (n ϭ 4) from unaffected individuals were subjected to differential gene expression profile analysis. Changes of more than 2-fold in DD groups and controls were recorded. Quantitative reverse transcriptase-polymerase chain reactions were performed to validate 16 implicated genes, which included developmental control genes, matrix metalloproteinases, and apoptotic genes. Results Several genes associated with DD formation were common across all 6 pairwise analyses. Genes markedly upregulated shared common expression levels across all pairwise analysis studies. Pairs involving the DD nodule arrays were notably distinguishable from all other permutations. The majority of genes dysregulated in the DD cords demonstrated an increase in fold change when compared with the DD nodule tissues. Key collagens, collagenases, metalloproteinases, and inhibitors were identified. Genes involved in cytoskeleton development and lipid metabolism were markedly dysregulated. Confirmations of these alterations were obtained in quantitative reverse transcriptase-polymerase chain reaction. Conclusions These data demonstrate a gradation in expression of certain genes in DD tissue phenotypes compared with control fascia. Transcriptome profiling is predictive not only of disease but also of disease phenotype. These results indicate a number of important candidate genes associated with DD formation, which may provide clues for molecular mechanisms involved in DD pathogenesis. ( J Hand Surg 2008;33A:359 -372.
Th e quasi-neoplastic nodular palmar fi bromatosis [1] called Dupuytren's disease (DD) often caus... more Th e quasi-neoplastic nodular palmar fi bromatosis [1] called Dupuytren's disease (DD) often causes permanent fl exion contracture of the metacarpophalangeal and proximal interphalangeal joints of the digits [2,3] ), leading to loss of function, deformity of the hand, and permanent contracture of the involved digits . Although DD does not metastasize , it may invade locally within the palmar aponeurosis of the hand (sparingly supplied with blood vessels) and it is progressive with a high rate of recurrence after surgical excision [6], often requiring amputation of the aff ected digit . Th e three stages of DD growth (proliferative, involutional, and residual) appear to involve dysdiff er en tiation into myofi broblasts . DD is associated with abundance of collagen, fi bronectin, integrins, cytokines and many other growth factors , as well as altered expression of several genes , but unlike the involve ment of known oncogenes and suppressor genes in cancer development , our knowledge of the exact aetiopathogenesis of DD remains poor despite signifi cant understanding of its biology.
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