The Effect of Endozym β-split, a Commercial Enzyme Preparation Used for Aroma Release, on Tannat Wine Glycosides

Commercial preparations with glycosidase activities are used to enhance wine aroma, but they arenot extensively characterized. The aim of this work was to study the effect of three enzymes on aromaimprovement in Tannat red wine. After the selection of the most appropriate enzyme for further testing,its effectiveness on the hydrolysis of the total glycosides present in Tannat wine was measured. The threepreparations showed high β-glucosidase and α-arabinosidase activities (in the range of 10 to 174 U/mL orU/g), but very low levels of α-rhamnosidase (less than 1 U/mL). The β-glucosidases studied remained activein the presence of Tannat wine. The selected enzyme, Endozym β-split, supplemented with α-rhamnosidase,resulted in almost 30% hydrolysis of the glycosides in Tannat wine, when added at a concentration higherthan that recommended by the manufacturers. A sensory evaluation showed that the enzyme-treated wineswere significantly different from the controls, suggesting that at least a part of the hydrolyzed glycosidesin the Tannat wine were aroma precursors. However, it cannot be assumed that all commercial enzymeswould be effective in hydrolyzing aroma precursors just because they show glycosidase activities. Higherconcentrations of Endozym β-split than that recommended by the manufacturer are necessary to reachan appreciable level of glycoside hydrolysis. Supplementation of Endozym β-split with α-rhamnosidase isrecommended in this enzyme – wine system for the greater release of the aroma from the glycosidicallyboundprecursors

Constrained metal-on-metal hip arthroplasty: ever heard of a 50-year survival story?

Background The history of total hip arthroplasty dates back to the first half of the twentieth century. Data on hip endoprostheses implanted during the 1960s and 1970s suggest widely varying survival rates of the prosthesis. Case A case of a patient who underwent total hip arthroplasty in 1972 using a Sivash prosthesis, developed in 1956 in the former Soviet Union, is presented in this article. The prosthesis has remained unrevised in the patient’s body for 50 years and he continues to be widely free of implant-related symptoms. Despite the constrained metal-on-metal design of the implant, which can lead to adverse reactions to metal debris, no elevated systemic metal ion levels were detected. Conclusion The likelihood of encountering patients with prosthesis survival beyond 50 years is still rare. Nevertheless, changing demographics and the steadily improving designs and materials of hip endoprostheses may likely result in such cases

A new submodelling technique for multi-scale finite element computation of electromagnetic fields: application in bioelectromagnetism

Complex multi-scale Finite Element (FE) analyses always involve high number of elements and therefore require very long time of computations. This is caused by the fact, that considered effects on smaller scales have greater influences on the whole model and larger scales. Thus, mesh density should be as high as required by the smallest scale factor. New submodelling routine has been developed to sufficiently decrease the time of computation without loss of accuracy for the whole solution. The presented approach allows manipulation of different mesh sizes on different scales and, therefore total optimization of mesh density on each scale and transfer results automatically between the meshes corresponding to respective scales of the whole model. Unlike classical submodelling routine, the new technique operates with not only transfer of boundary conditions but also with volume results and transfer of forces (current density load in case of electromagnetism), which allows the solution of full Maxwell's equations in FE space. The approach was successfully implemented for electromagnetic solution in the forward problem of Magnetic Field Tomography (MFT) based on Magnetoencephalography (MEG), where the scale of one neuron was considered as the smallest and the scale of whole-brain model as the largest. The time of computation was reduced about 100 times, with the initial requirements of direct computations without submodelling routine of 10 million elements

Depletion of Macrophages Improves Therapeutic Response to Gemcitabine in Murine Pancreas Cancer.

BACKGROUND: The tumor microenvironment (TME) is composed of fibro-inflammatory cells and extracellular matrix (ECM) components. However, the exact contribution of the various TME compartments towards therapeutic response is unknown. Here, we aim to dissect the specific contribution of tumor-associated macrophages (TAMs) towards drug delivery and response in pancreatic ductal adenocarcinoma (PDAC). METHODS: The effect of gemcitabine was assessed in human and murine macrophages, human pancreatic stellate cells (hPSCs), and tumor cells (L3.6pl, BxPC3 and KPC) in vitro. The drug metabolism of gemcitabine was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Preclinical studies were conducted using KrasG12D;p48-Cre and KrasG12D;p53172H;Pdx-Cre mice to investigate gemcitabine delivery at different stages of tumor progression and upon pharmacological TAM depletion. RESULTS: Gemcitabine accumulation was significantly increased in murine PDAC tissue compared to pancreatic intraepithelial neoplasia (PanIN) lesions and healthy control pancreas tissue. In vitro, macrophages accumulated and rapidly metabolized gemcitabine resulting in a significant drug scavenging effect for gemcitabine. Finally, pharmacological TAM depletion enhanced therapeutic response to gemcitabine in tumor-bearing KPC mice. CONCLUSION: Macrophages rapidly metabolize gemcitabine in vitro, and pharmacological depletion improves the therapeutic response to gemcitabine in vivo. Our study supports the notion that TAMs might be a promising therapeutic target in PDAC

Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFβ signalling and EMT

Ubiquitin-dependent mechanisms have emerged as essential regulatory elements controlling cellular levels of Smads and TGFß-dependent biological outputs such as epithelial–mesenchymal transition (EMT). In this study, we identify a HECT E3 ubiquitin ligase known as WWP2 (Full-length WWP2-FL), together with two WWP2 isoforms (N-terminal, WWP2-N; C-terminal WWP2-C), as novel Smad-binding partners. We show that WWP2-FL interacts exclusively with Smad2, Smad3 and Smad7 in the TGFß pathway. Interestingly, the WWP2-N isoform interacts with Smad2 and Smad3, whereas WWP2-C interacts only with Smad7. In addition, WWP2-FL and WWP2-C have a preference for Smad7 based on protein turnover and ubiquitination studies. Unexpectedly, we also find that WWP2-N, which lacks the HECT ubiquitin ligase domain, can also interact with WWP2-FL in a TGFß-regulated manner and activate endogenous WWP2 ubiquitin ligase activity causing degradation of unstimulated Smad2 and Smad3. Consistent with our protein interaction data, overexpression and knockdown approaches reveal that WWP2 isoforms differentially modulate TGFß-dependent transcription and EMT. Finally, we show that selective disruption of WWP2 interactions with inhibitory Smad7 can stabilise Smad7 protein levels and prevent TGFß-induced EMT. Collectively, our data suggest that WWP2-N can stimulate WWP2-FL leading to increased activity against unstimulated Smad2 and Smad3, and that Smad7 is a preferred substrate for WWP2-FL and WWP2-C following prolonged TGFß stimulation. Significantly, this is the first report of an interdependent biological role for distinct HECT E3 ubiquitin ligase isoforms, and highlights an entirely novel regulatory paradigm that selectively limits the level of inhibitory and activating Smads

Cytosolic 5'-nucleotidase 1A is overexpressed in pancreatic cancer and mediates gemcitabine resistance by reducing intracellular gemcitabine metabolites.

BACKGROUND: Cytosolic 5'-nucleotidase 1A (NT5C1A) dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. Here, we investigate NT5C1A expression in pancreatic ductal adenocarcinoma (PDAC) and its impact on gemcitabine metabolism and therapeutic efficacy. METHODS: NT5C1A expression was determined by semiquantitative immunohistochemistry using tissue microarrays. Gemcitabine metabolites and response were assessed in several human and murine PDAC cell lines using crystal violet assays, Western blot, viability assays, and liquid chromatography tandem mass-spectrometry (LC-MS/MS). FINDINGS: NT5C1A was strongly expressed in tumor cells of a large subgroup of resected PDAC patients in two independent patient cohorts (44-56% score 2 and 8-26% score 3, n = 414). In contrast, NT5C1A was expressed at very low levels in the tumor stroma, and neither stromal nor tumoral expression was a prognostic marker for postoperative survival. In vitro, NT5C1A overexpression increased gemcitabine resistance by reducing apoptosis levels and significantly decreased intracellular amounts of cytotoxic dFdCTP in +NT5C1A tumor cells. Co-culture experiments with conditioned media from +NT5C1A PSCs improved gemcitabine efficacy in tumor cells. In vivo, therapeutic efficacy of gemcitabine was significantly decreased and serum levels of the inactive gemcitabine metabolite dFdU significantly increased in mice bearing NT5C1A overexpressing tumors. INTERPRETATION: NT5C1A is robustly expressed in tumor cells of resected PDAC patients. Moreover, NT5C1A mediates gemcitabine resistance by decreasing the amount of intracellular dFdCTP, leading to reduced tumor cell apoptosis and larger pancreatic tumors in mice. Further studies should clarify the role of NT5C1A as novel predictor for gemcitabine treatment response in patients with PDAC

Nuclear Factor of Activated T Cells-dependent Down-regulation of the Transcription Factor Glioma-associated Protein 1 (GLI1) Underlies the Growth Inhibitory Properties of Arachidonic Acid

Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis, and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1 and 4-1BB, in AA-treated cells. We demonstrated that downregulation of the transcription factor GLI1 in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1 and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules, and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and downregulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating NFATc1 nuclear translocation, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants reduced the AA-induced apoptosis, downregulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for the future PUFA-based therapeutic approaches

SPARC dependent collagen deposition and gemcitabine delivery in a genetically engineered mouse model of pancreas cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterised by extensive matrix deposition that has been implicated in impaired drug delivery and therapeutic resistance. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates collagen deposition and is highly upregulated in the activated stroma subtype with poor prognosis in PDAC patients. METHODS: KrasG12D;p48-Cre;SPARC-/- (KC-SPARC-/-) and KrasG12D;p48-Cre;SPARCWT (KC-SPARCWT) were generated and analysed at different stages of carcinogenesis by histological grading, immunohistochemistry for epithelial and stromal markers, survival and preclinical analysis. Pharmacokinetic and pharmacodynamic studies were conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunohistochemistry following gemcitabine treatment (100 mg/kg) in vivo. FINDINGS: Global genetic ablation of SPARC in a KrasG12D driven mouse model resulted in significantly reduced overall and mature collagen deposition around early and advanced pancreatic intraepithelial neoplasia (PanIN) lesions and in invasive PDAC (p < .001). However, detailed pathological scoring and molecular analysis showed no effects on PanIN to PDAC progression, vessel density (CD31), tumour incidence, grading or metastatic frequency. Despite comparable tumour kinetics, ablation of SPARC resulted in a significantly shortened survival in KC-SPARC-/- mice (280 days versus 485 days, p < .03, log-rank-test). Using LC-MS/MS, we show that SPARC dependent collagen deposition does not affect intratumoural gemcitabine accumulation or immediate therapeutic response in tumour bearing KC-SPARCWT and KC-SPARC-/-mice. INTERPRETATION: Global SPARC ablation reduces the collagen-rich microenvironment in murine PDAC. Moreover, global SPARC depletion did not affect tumour growth kinetics, grading or metastatic frequency. Notably, the dense-collagen matrix did not restrict access of gemcitabine to the tumour. These findings may have direct translational implications in clinical trial design

Histone deacetylase 1 and 2 differentially regulate apoptosis by opposing effects on extracellular signal-regulated kinase 1/2

Histone deacetylases (HDACs) are epigenetic regulators that are important for the control of various pathophysiological events. We found that HDAC inhibitors completely abolished transforming growth factor-β1 (TGF-β1)-induced apoptosis in AML-12 and primary mouse hepatocytes. Expression of a dominant-negative mutant of HDAC1 or downregulation of HDAC1 by RNAi both suppressed TGF-β1-induced apoptosis. In addition, overexpression of HDAC1 enhanced TGF-β1-induced apoptosis, and the rescue of HDAC1 expression in HDAC1 RNAi cells restored the apoptotic response of cells to TGF-β1. These data indicate that HDAC1 functions as a proapoptotic factor in TGF-β1-induced apoptosis. In contrast, downregulation of HDAC2 by RNAi increased spontaneous apoptosis and markedly enhanced TGF-β1-induced apoptosis, suggesting that HDAC2 has a reciprocal role in controlling cell survival. Furthermore, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) by MEK1 inhibitor PD98059 or expression of a kinase-dead mutant of MEK1 restored the apoptotic response to TGF-β1 in HDAC1 RNAi cells. Strikingly, HDAC2 RNAi caused an inhibition of ERK1/2, and the spontaneous apoptosis can be abolished by reactivation of ERK1/2. Taken together, our data demonstrate that HDAC1 and 2 reciprocally affect cell viability by differential regulation of ERK1/2; these observations provide insight into the roles and potential mechanisms of HDAC1 and 2 in apoptosis