Davor Jurišić

An integrated proteomics approach for studying the molecular pathogenesis of Dupuytren's disease

Dupuytrens disease (DD) is a fibromatosis characterized by non‐malignant transformation of palmar fascia leading to permanent contraction of one or more fingers. Despite the extensive knowledge of its clinical pathogenesis, the aetiology of this disease remains obscure. In the present paper, we report for the first time on the proteomic profiling of diseased versus unaffected patient‐matched palmar fasciae tissues from DD patients using two‐dimensional gel electrophoresis coupled with mass spectrometry analysis. The herein identified proteins were then used to create the protein–protein interaction network (interactome). Such an integrated approach revealed the involvement of several different molecular processes related to DD progression, including extra‐ and intra‐cellular signalling, oxidative stress, cytoskeletal changes, and alterations in cellular metabolism. In particular, autocrine regulation through ERBB‐2 and IGF‐1R receptors and the Akt signalling pathway have emerged as novel components of pro‐survival signalling in Dupuytrens fibroblasts and thus might provide a basis for a new therapeutic strategy in Dupuytrens disease. Copyright

Running for time: circadian rhythms and melanoma

Circadian timing system includes an input pathway transmitting environmental signals to a core oscillator that generates circadian signals responsible for the peripheral physiological or behavioural events. Circadian 24-h rhythms regulate diverse physiologic processes. Deregulation of these rhythms is associated with a number of pathogenic conditions including depression, diabetes, metabolic syndrome and cancer. Melanoma is a less common type of skin cancer yet more aggressive often with a lethal ending. However, little is known about circadian control in melanoma and exact functional associations between core clock genes and development of melanoma skin cancer. This paper, therefore, comprehensively analyses current literature data on the involvement of circadian clock components in melanoma development. In particular, the role of circadian rhythm deregulation is discussed in the context of DNA repair mechanisms and influence of UV radiation and artificial light exposure on cancer development. The role of arylalkylamine N-acetyltransferase (AANAT) enzyme and impact of melatonin, as a major output factor of circadian rhythm, and its protective role in melanoma are discussed in details. We hypothesise that further understanding of clock genes’ involvement and circadian regulation might foster discoveries in the field of melanoma diagnostics and treatment.

Screening of potential prodrugs on cells derived from Dupuytren's disease patients

Dupuytrens disease (DD) is a fibroproliferative disorder, the cure for which is still limited to surgical excision of the affected fascia, often leading to high recurrence rates. Due to this fact, non-surgical treatments are being investigated, among them those targeting molecular processes of proliferation and differentiation in Dupuytrens cell cultures. Drugs with antiproliferative action may be valuable in DD treatment. Through characterization of changes on DD-specific cells, we, therefore, decided to test the therapeutic potential of new cytostatic drugs for DD treatment and/or for reduction of post-operative recurrence rates. The N-sulfonylpyrimidine derivative, amidino-substituted benzimidazo[1,2-a]quinoline, and amidino dihydrothienothienyl[2,3-c]quinolone hydrochloride, known to affect proliferation processes, were tested for their antiproliferative activity on primary fibroblasts/myofibroblasts cell cultures derived from the palmar fascia of patients with DD. Only amidino dihydrothienothienyl[2,3-c]quinolone hydrochloride acted in a highly specific manner on cells derived from diseased fascia of DD patients and exhibited a low cytotoxic effect. This result might be a consequence of its specific activity on cytoskeleton changes occurring in differentiating cells. A similar short-term differential antiproliferative effect was observed by the N-sulfonylpyrimidine derivative that was, however, completely lost after 6- and 14-day treatments. The amidino-substituted benzimidazo[1,2-a]quinoline exerted a strong non-specific, dose-related antiproliferative activity on cell types.

Functional genomics in identification of drug targets in Dupuytren's contracture

Although functional genomics methods offer new viewpoint on molecular processes involved in particular pathological state, these methods, in particular proteomics, are still under-represented in Dupuytrens contracture research. However, several recent papers based on functional genomics technologies represent a breakthrough in studying Dupuytrens contracture as they revealed new molecular players that had been impossible to detect by traditional molecular biology methods. Using computational tools to provide biological context for such broad arrays of data accelerates the process of homing in on the potential molecular markers and drug targets. Interactomes, maps of protein-protein interactions characteristic for the disease and as such putative models of its molecular pathology, are especially useful for this purpose, facilitating the transition from global screening methods to specific experiments aimed at therapy development. The combination of these approaches in Dupuytrens contracture research might therefore facilitate the discovery of novel therapeutic targets and diagnostic markers indicative of disease progression.

Inflammatory Gene Expression Upon TGF-β1-Induced p38 Activation in Primary Dupuytren's Disease Fibroblasts

Objectives: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytrens disease (DD) patients. Methods: Primary non-Dupuytrens disease cells (ND) were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-β1 and/or inhibitor of p38 phosphorylation. Results: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11, and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro. Conclusions: TGF-β1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed toward inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence.

A polymorphic GGC repeat in the NPAS2 gene and its association with melanoma

Circadian clock regulation in mammals is controlled by feedback loops of a set of circadian genes. One of these circadian genes, NPAS2, encodes for a member of the bHLH-PAS class of transcription factors and is expressed in the forebrain and in some peripheral organs such as liver and skin. Other biological processes are also regulated by circadian genes. For example, NPAS2 is involved in cell proliferation, DNA damage repair and malignant transformation. Aberrant expression of clock genes has been previously observed in melanoma which led to our effort to sequence the NPAS2 promoter region in this cancer type. The NPAS2 putative promoter and 5′ untranslated region of ninety-three melanoma patients and ninety-six control subjects were sequenced and several variants were identified. Among these is a novel microsatellite comprising a GGC repeat with different alleles ranging from 7 to 13 repeats located in the 5′ untranslated exon. Homozygosity of an allele with nine repeats (9/9) was more prevalent in melanoma than in control subjects (22.6% and 13.5%, respectively, P: 0.0206) suggesting that some NPAS2 variants might contribute to melanoma susceptibility. Impact statement This report describes a variable microsatellite repeat sequence located in the 5′ untranslated exon of NSPAS2, a gene encoding a clock transcription factor. Significantly, this study is the first to show that a variant copy number GGC repeat sequence in the NPAS2 clock gene associates with melanoma risk and which may be useful in the assessment of melanoma predisposition.

Simultaneous dermal graft harvesting and de-fatting

Even after careful excision of full-thickness skin graft, some fat usually remains on the undersurface of the graft and has to be removed by additional trimming using sharp, curved scissors. The de-fatting of the fat on the dermal side of the full-thickness skin graft is usually a time-consuming procedure. We would like to propose an extremely simple modification of excision and simultaneous de-fatting of dermal skin graft. The ellipse shape of the dermal grafting is elevated with a # 15 blade rolling the epidermal surface of the graft over a syringe with one hand, exposing the dermal side to be simultaneously de-fatted with the blade in the other hand (Figure 1). Better exposition of the dermal side of the skin graft can be additionally facilitated by grasping the top of