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To satisfy requirements of high operating current densities, compact layouts, and the possibility to reach magnetic field higher than 20$\,\mathrm{T}$, at temperatures from 4.2$\,\mathrm{K}$ to 20$\,\mathrm{K}$, e.g., necessary for the development of next generation of colliders, the CORC® cable concept is of particular interest. Based on ReBCO high temperature superconducting tapes wound around a copper former, it exploits round cross-section guaranteeing electrical and mechanical isotropy. However, these cables can degrade as a result of the winding process and operating conditions, as ReBCO tapes are strain-sensitive. This paper presents a 3D multi-physics numerical model for characterizing the critical current ($I_{c}$) of bended ReBCO CORC® cables, based on a T–A formulation implemented in COMSOL Multiphysics® coupled with a thermal model of a straight cable, already validated against experimental results. The tape is approximated as a thin shell, taking advantage of its high aspect ratio, and the $I_{c}$ scaling for the tape accounts for the local strain. A pure geometrical strain evaluation on the tape surface allow to properly account for the punctual degradation of $I_{c}$, as an input for the multi-physics model. The assessment of the voltage-current curve for the bent conductor has been compared to experimental results.

High-temperature superconducting-slotted cables, including the VIPER cable, are being investigated as possible candidates for winding the toroidal field coils of compact nuclear fusion machines, such as ARC. The higher performance of HTS materials in terms of magnetic fields and currents allows them to reduce their size compared to machines based on Nb alloys and compounds. However, compactness also means a closer interaction between the plasma products and the coils, creating new technological challenges to be faced. Among the others, the thermal effects induced on the superconducting materials by direct particles (i.e., neutrons), secondary particles and gamma-ray heating, will be stronger than for low-field larger machines and must be quantified. In this paper, a detailed thermo-magnetic model is built up using the software COMSOL Multiphysics to simulate the consequences of the D-T plasma operation products on the first turn of a toroidal field coil of the ARC preliminary machine design. The irradiation-induced heat load on the (RE)Ba2Cu3O7-δ tapes, computed via Monte Carlo simulations, is used as input for the thermal analysis of the VIPER cable. At the same time, a homogenized T-A formulation is developed for checking the performance reduction in the current/field operating conditions.

The study of superconducting tapes requires specialized formulations to account for the high aspect ratio of the tapes and the extremely high conductivity of the thin superconducting layer. This article presents a new formulation coupling the line integral of the magnetic vector potential $a$ defined in the whole domain to the stream function $\psi $ defined on the nodes of the superconducting layer. Results on two benchmarks show that the proposed $a-\psi $ formulation is effective in solving problems where the currents are limited to thin layers also in case of a strong material nonlinearity.

Macrocycles, including macrocyclic peptides, have shown promise for targeting challenging protein–protein interactions (PPIs). One PPI of high interest is between Kelch-like ECH-Associated Protein-1 (KEAP1) and Nuclear Factor (Erythroid-derived 2)-like 2 (Nrf2). Guided by X-ray crystallography, NMR, modeling, and machine learning, we show that the full 20 nM binding affinity of Nrf2 for KEAP1 can be recapitulated in a cyclic 7-mer peptide, c[(D)-β-homoAla-DPETGE]. This compound was identified from the Nrf2-derived linear peptide GDEETGE (KD= 4.3 μM) solely by optimizing the conformation of the cyclic compound, without changing any KEAP1 interacting residue. X-ray crystal structures were determined for each linear and cyclic peptide variant bound to KEAP1. Despite large variations in affinity, no obvious differences in the conformation of the peptide binding residues or in the interactions they made with KEAP1 were observed. However, analysis of the X-ray structures by machine learning showed that locations of strain in the bound ligand could be identified through patterns of subangstrom distortions from the geometry observed for unstrained linear peptides. We show that optimizing the cyclic peptide affinity was driven partly through conformational preorganization associated with a proline substitution at position 78 and with the geometry of the noninteracting residue Asp77 and partly by decreasing strain in the ETGE motif itself. This approach may have utility in dissecting the trade-off between conformational preorganization and strain in other ligand–receptor systems. We also identify a pair of conserved hydrophobic residues flanking the core DxETGE motif which play a conformational role in facilitating the high-affinity binding of Nrf2 to KEAP1.

Dictyostelium discoideumamoebae were exposed to Hg 2 μM corresponding to a sublethal concentration and Hg 10 μM with the first effects on mortality and replication rate. A total of 900 spots were visualized by 2-DE electrophoresis. Two-hundred fifty single proteins were identified by mass spectrometry. Low Hg concentration (2 μM) treatment induced up-regulation of 13 spots, mainly involved in oxidative stress response/detoxification, oxidoreductase activity, and metabolic processes. High Hg concentration (10 μM) treatment showed a different PES with 12 proteins downregulated and only two up-regulated, mainly involved in cellular metabolic processes, metal ion binding, and transferase activity. The analyses for the carbonylation show no changes after 2 μM Hg2+treatment and 13 differentially carbonylated proteins after 10 μM Hg2+involved in a broad range of cellular processes. Our findings provide insight into the mechanisms of physiological adaptation and toxicity to a low and an high mercury concentration, respectively, of Dictyostelium amoebae.

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (β+) or absent (β0) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life1. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease2. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß0or severe ß+mutations in a phase 1/2 trial (NCT02453477) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6–76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10–1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.

The paracrine properties of human amniotic membrane‐derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC‐CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC‐CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC‐CM by cytokine array. Our results strongly support the concept that the administration of hAMC‐CM favors the repair process after acute myocardial infarction. The goal of this study was to elucidate whether human amniotic membrane‐derived mesenchymal stromal cells (hAMCs) can exert beneficial paracrine effects on infarcted rat hearts. In particular, the administration of hAMC‐conditioned medium repaired ischemic damage through cardioprotection and angiogenesis. Finally, several putative active paracrine mediators that might account for the effects observed were identified by gene and protein arrays.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over‐expressed in P19 cells individually or in different combinations. The over‐expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real‐time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation‐contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over‐expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. StemCells2015;33:1187–1199

Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, type IV collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vivo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin‐treated cultures was significantly reduced. Stimulation with type IV collagen resulted in a 1.4‐fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derived from fetal liver progenitor cells. In vivo, megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5‐fluorouracil induced myelosuppression, while only type IV collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM‐producing cells for the establishment of cell niches during bone marrow regeneration. StemCells2014;32:926–937

SynopsisThe research problemThis paper aims to test whether an acquirer’s past earnings quality (proxied by the level of earnings management) serves as an indicator of its trustworthiness in view of the inclusion of an earnout agreement in an acquisition contract.MotivationPrior studies highlighted the benefits of earnout agreements, showing that they help reduce adverse selection problems and valuation risk faced by the acquirers. However, not much attention has been paid to the risks that these contracts entail. The majority of earnouts are based on accounting performance measures, which may be subject to earnings management by the acquirer to reduce, or even avoid, contingent payments. Therefore, the decision to include an earnout in an acquisition may depend on the sellers’ perceived probability that the acquirer will use opportunistic tactics to avoid the contingent payment, as indicated by the acquirer’s past behavior.The test hypothesesH1:The likelihood of inclusion of earnouts in acquisition deals is negatively associated with the level of earnings management in the acquirer’s past financial statements.H2:The relation between earnings management and the use of earnouts is stronger when the bidder has no acquisition track record.Target populationM&A advisors, financial analysts, stakeholders of companies engaging in M&A deals.Adopted methodologyLogit regressions, propensity score matching analyses for robustness.AnalysesUsing a sample of 8,968 acquisition deals completed by US listed companies between 2002 and 2014, we examine the association between the use of earnouts and bidder’s earnings quality (proxied by the level of earnings management) controlling for the determinants of the use of earnouts described by previous literature. We collected acquisitions data from Thomson ONE Banker and accounting information from Compustat. Using logit models, we regress the choice to include an earnout in an M&A deal on several earnings management proxies and on the interaction between the latter variables and bidder’s acquisition track record.FindingsWe find robust evidence that the level of the acquirer’s past earnings management is negatively associated to the probability of inclusion of an earnout in acquisition contracts. This association is stronger if the bidder has no prior acquisition history and is robust to various measures of earnings management and to controls for other external monitoring mechanisms.

The frequency of A3669G single nucleotide polymorphism (SNP) of human glucocorticoid receptor has been reported increased in polycythemia vera. We investigated the frequency of A3669G SNP and its impact on disease phenotype and progression in 499 patients with primary myelofibrosis (PMF). The distribution of the A3669G allele differed between PMF patients and 2 healthy control populations (odds ratio, 1.6 and 1.8). The variant allele at the homozygous state (G/G) was associated with higher white blood cell count, larger spleen index, and higher frequency of circulating CD34+ cells at diagnosis. The latter association remained significant after correction for the JAK2V617F genotype. In patients JAK2V617F mutated, the G/G genotype was associated with shorter overall survival (77.6 months vs 298 months, P = .049) and blast transformation (BT)–free survival (76.7 months vs 261 months; P = .018). The latter association remained significant after correction for the known BT risk factors, such as age, sex, white blood cell count, percentage of blasts, IPSS prognostic score, and homozygosity for JAK2V617F (hazard ratio = 3.3; P = .006). In conclusion, the glucocorticoid receptor A3669G is a susceptibility allele for PMF: it contributes to confer the phenotype of excess myeloproliferation, and it cooperates with the JAK2V617F mutation in determining BT.

Abstract: Implementation of extended lymph node dissection for gastric cancer in western non-specialized centers through tailoring its extent upon disease stage and patient comorbidities was suggested as a wise policy to reduce morbidity and mortality rates, albeit with a potential for undertreatment in elderly and/or comorbid patients. Current definition of R0 resection for gastric cancer lacks consideration of treatment-related variables such as extended lymph node dissection. Few studies to date have tried to fill this gap in such a clinical context. A retrospective evaluation of factors influencing long-term results after R0 surgery was done in a prospective series of a non-specialized western surgical unit during the implementation of D2 lymphadenectomy. Univariate and multivariate analysis of 22 variables were performed on a prospective database of 233 consecutive R0 resections performed by ten different surgeons in 10 years. Endpoint was disease-free survival calculated at 5 and at 10 years. Disease-free survival rates were independently influenced by age, American Society of Anesthesiologists (ASA) status and lymph node ratio. Subset analysis of the status at censor stratified for age and ASA status failed to identify any significant difference in disease recurrence rates. Lymph node ratio was the only treatment-related independent prognostic factor for long-term results after R0 surgery for gastric cancer in the setting of a non-specialized western unit, where the extent of lymph node dissection needs to be tailored on the presence of comorbidities (ASA status).

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit–endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit–endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.

Macrocyclic compounds (MCs) can have complex conformational properties that affect pharmacologically important behaviors such as membrane permeability. We measured the passive permeability of 3600 diverse nonpeptidic MCs and used machine learning to analyze the results. Incorporating selected properties based on the three-dimensional (3D) conformation gave models that predicted permeability with Q2= 0.81. A biased spatial distribution of polar versus nonpolar regions was particularly important for good permeability, consistent with a mechanism in which the initial insertion of nonpolar portions of a MC helps facilitate the subsequent membrane entry of more polar parts. We also examined effects on permeability of 800 substructural elements by comparing matched molecular pairs. Some substitutions were invariably beneficial or invariably deleterious to permeability, while the influence of others was highly contextual. Overall, the work provides insights into how the permeability of MCs is influenced by their 3D conformational properties and suggests design hypotheses for achieving macrocycles with high membrane permeability.

Background: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized. Objective: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors. Methods: Human megakaryocytes were obtained by differentiation of cord blood-derived CD34+cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: We found that human megakaryocytes extended proplatelets in a time-dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin αIIbβ3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb-IX-V, or upon cleavage of GPIbα by the metalloproteinase mocarhagin. Membrane-associated VWF was detected exclusively on proplatelet-forming megakaryocytes, but not on round mature cells that do not extend proplatelets. Conclusions: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio-temporal regulation orchestrated by adhesive proteins, GPIb-IX-V and myosin IIA.

Few investigators have evaluated the usefulness of the JAK2 V617F mutation for explaining the phenotypic variations and for predicting the risk of major clinical events in primary myelofibrosis (PMF). In a transversal survey we assayed by allele-specific polymerase chain reaction (PCR) the JAK2 V617F mutational status in 304 patients with PMF. Multiple DNA samples were collected prospectively from 64 patients, and a highly sensitive quantitative PCR was used as a confirmatory test. In a longitudinal prospective study we determined the progression rate to clinically relevant outcomes in 174 patients who had JAK2 mutation determined at diagnosis. JAK2 V617F was identified in 63.4% of patients. None of the V617F-negative patients who were sequentially genotyped progressed to become V617F positive, whereas progression rate from heterozygous to homozygous mutation was 10 per 100 patient-years. JAK2 V617F mutation contributed to hemoglobin, aquagenic pruritus, and platelet count variability, whereas homozygous mutation was independently associated with higher white blood cell count, larger spleen size, and greater need for cytoreductive therapies. Adjusting for conventional risk factors, V617F mutation independently predicted the evolution toward large splenomegaly, need of splenectomy, and leukemic transformation. We conclude that JAK2 V617F genotype should be considered in any future risk stratification of patients with PMF.

Few investigators have evaluated the usefulness of the JAK2V617F mutation for explaining the phenotypic variations and for predicting the risk of major clinical events in primary myelofibrosis (PMF). In a transversal survey we assayed by allele-specific polymerase chain reaction (PCR) the JAK2V617F mutational status in 304 patients with PMF. Multiple DNA samples were collected prospectively from 64 patients, and a highly sensitive quantitative PCR was used as a confirmatory test. In a longitudinal prospective study we determined the progression rate to clinically relevant outcomes in 174 patients who had JAK2mutation determined at diagnosis. JAK2V617F was identified in 63.4% of patients. None of the V617F-negative patients who were sequentially genotyped progressed to become V617F positive, whereas progression rate from heterozygous to homozygous mutation was 10 per 100 patient-years. JAK2V617F mutation contributed to hemoglobin, aquagenic pruritus, and platelet count variability, whereas homozygous mutation was independently associated with higher white blood cell count, larger spleen size, and greater need for cytoreductive therapies. Adjusting for conventional risk factors, V617F mutation independently predicted the evolution toward large splenomegaly, need of splenectomy, and leukemic transformation. We conclude that JAK2V617F genotype should be considered in any future risk stratification of patients with PMF.

In mussel Mytilus galloprovincialis tissues, metallothionein belongs to two different gene classes, mt10 and mt20, showing differential expression at both basal conditions and under heavy metal challenge. In this study, a new more highly sensitive technique, expression analysis of mt10 and mt20 mRNA levels by quantitative reverse transcription polymerase chain reaction, was used to assess the effects of heavy metal contamination in the digestive glands of mussels caged along the Tunisian coast. To validate the new assay, total metallothionein protein, amount of heavy metals (zinc, copper, cadmium), and a biomarker of oxidative stress such as malondialdehyde content, were assessed in the same tissues. At the investigated sites, the molecular assay showed variations of mt20 relative gene expression levels within one or two orders of magnitude, with maximum values at two sites severely polluted with cadmium, Mahres (100-fold) and Menzel Jemile (165-fold). Changes in mt10 expression were recorded at all sites where copper had significantly accumulated, although fold induction levels were less pronounced than those of mt20. In this paper, gene expression data are discussed in relation to the studied biomarkers, demonstrating that the molecular technique based on the differential expression of mt10 and mt20 genes represents (i) a useful and robust tool for studying and monitoring heavy metal pollution under field conditions, and (ii) an improvement in the application of metallothionein as a biomarker of response to exposure to heavy metals in marine mussels.

Biomarkers on sentinel organisms are utilised worldwide in biomonitoring programs. However, the lack of effective interpretational capacity has hampered their uptake for use for assessment of risk in environmental management. The aim of the present study was to develop and test an objective decision-support or expert system capable of integrating biomarker results into a five-level health-status index. The expert system is based on a set of rules derived from available data on responses to natural and contaminant-induced stress of marine mussels. Integration of parameters includes: level of biological organization; biological significance; mutual inter-relationship; and qualitative trends in a stress gradient. The system was tested on a set of biomarker data obtained from the field and subsequently validated with data from previous studies. The results demonstrate that the expert system can effectively quantify the biological effects of different levels of pollution. The system represents a simple tool for risk assessment of the harmful impact of contaminants by providing a clear indication of the degree of stress syndrome induced by pollutants in mussels.

Marine bivalves of the genus Mytilus are intertidal filter-feeders commonly used as biosensors of coastal pollution. Mussels adjust their functions to ordinary environmental changes, e.g. temperature fluctuations and emersion-related hypoxia, and react to various contaminants, accumulated from the surrounding water and defining a potential health risk for sea-food consumers. Despite the increasing use of mussels in environmental monitoring, their genome and gene functions are largely unexplored. Hence, we started the systematic identification of expressed sequence tags and prepared a cDNA microarray of Mytilus galloprovincialis including 1714 mussel probes (76% singletons, ~50% putatively identified transcripts) plus unrelated controls. To assess the potential use of the gene set represented in MytArray 1.0, we tested different tissues and groups of mussels. The resulting data highlighted the transcriptional specificity of the mussel tissues. Further testing of the most responsive digestive gland allowed correct classification of mussels treated with mixtures of heavy metals or organic contaminants (expression changes of specific genes discriminated the two pollutant cocktails). Similar analyses made a distinction possible between mussels living in the Venice lagoon (Italy) at the petrochemical district and mussels close to the open sea. The suggestive presence of gene markers tracing organic contaminants more than heavy metals in mussels from the industrial district is consistent with reported trends of chemical contamination. Further study is necessary in order to understand how much gene expression profiles can disclose the signatures of pollutants in mussel cells and tissues. Nevertheless, the gene expression patterns described in this paper support a wider characterization of the mussel transcriptome and point to the development of novel environmental metrics.

Metallothioneins are rather ubiquitous metal-binding proteins induced by stressing or physiological stimuli. Two major metallothionein isoforms have been identified in mussel: MT10 and MT20. Nevertheless the high sequence homology, the two isoforms exhibit different expression and inducibility in vivo.We cloned and produced in Escherichia colithe MT20 isoform from Mytilus galloprovincilis.cDNA was subcloned into pGEX-6P.1 vector, in frame with a sequence encoding a glutathione-S-transferase (GST) tail. Recombinant protein was purified to electrophoretic homogeneity by affinity chromatography. After enzymatic cleavage of the GST tail the MT moiety was recovered with a final yield of about 5 mg of protein per litre of bacterial culture. The metal-binding ability of MT20 was assessed by absorption spectroscopy upon addition of cadmium equivalents and the metal release was checked as a function of the environment pH. Moreover the protein was analysed for the propensity to polymerization, typical of this class of protein, before and after exposure to reducing and alkylating agents.

Objective: Adult GH deficiency (GHD) syndrome is characterized by increased risk of atherosclerosis and hence of cardio- and cerebrovascular mortality. Oxidative stress appears to play an important role in early atherogenesis. Oxidized LDL represents an important predictor of cardiovascular risk and is mainly responsible for oxidative damage of the endothelium. Its concentrations are increased in GHD, but the association between this abnormality and oxidative stress is still unclear, due to the discordant results yielded by the few available studies. Design and methods: In 13 GHD patients, plasma lipid peroxide concentrations were measured before and after a 4-month treatment with recombinant human GH (rhGH) and compared with those of 13 age-and sex-matched controls. In the same subjects, the so-called “lag-time”, an index of anti-oxidant activity and thus of plasma oxidative balance, was also measured using a fluorescence kinetics method. Results: Before treatment, peroxide levels were significantly higher in patients than in controls (374.0±31.52 vs268.0±8.51 U.C., p<0.01), whereas the lag-time was significantly lower (113.0±10.70 vs168.0±7.80 min, p<0.01). RhGH administration to patients resulted both in a significant decrease in lipid peroxide levels (from 374.0±31.52 to 336.0±33.17 U.C., p<0.01) and a significant prolongation of lag-time (from 113.0±10.70 to 144.0±15.00 min, p<0.01). After treatment, both parameters were no longer significantly different in patients and controls. Lagtime and peroxide levels at baseline did not show any correlation with IGF-I concentrations in GHD patients. After replacement therapy, however, lag-time was positively (r2= 0.62, p<0.01), and peroxide levels negatively (r2=0.41, p<0.05), correlated with IGF-I levels. Conclusions: These data support the view that adult GHD syndrome is characterized by an unbalance between pro- and anti-oxidant factors with marked preponderance of the former. This abnormality, likely contributing to the increased atherogenic risk of GHD patients, is corrected by short-term GH administration at a dose able to increase, although not to fully normalize, IGF-I levels.

Immunomagnetic CD34+ cell selection (ICS) is a widely employed technology in autotransplant and allotransplant settings. When an haploidentical transplant is performed, a high dose of purified CD34+ cells together with efficient T and B cell depletion are required to minimize the risks of graft versus host disease (GVHD) and Epstein–Barr virus (EBV)-related lymphoma. To ameliorate the performances of the CliniMACS (Miltenyi Biotec) ICS device, we compared 73 ICS performed following the manufacturer's recommended platelet depletion versus 48 performed after adjunctive centrifugations to increase platelet depletion of the leukapheresis (LKF) product. A total of 121 ICS (from single or fractioned LKF products) were carried out on 93 LKF collected from 47 related healthy donors. A statistical significance in terms of CD34+ cell recovery (81.8% vs. 71.2%) was found in favor of the modified ICS procedure (p = 0.0049) with a comparable stem cell purity and viability. The modification of the standard manufacturer's technique for increasing platelet depletion can further improve the recovery of stem cells with no influence on T and B cell depletion. These results demonstrate the negative influence exerted on CD34+ cell recovery by LKF platelet contamination.

We have studied the effects of heavy metals (Hg2+, Cu2+, Cd2+) on growth hormone (GH) activation of tyrosine kinase and Ca2+signaling in the trout (Oncorhynchusmykiss) hepatoma cell line RTH-149. Molecular cloning techniques using primer designed on Oncorhynchusspp. growth hormone receptor (GHR) genes allowed to isolate a highly homologous cDNA fragment from RTH-149 mRNA. Thereafter, cells were analysed by Western blotting or, alternatively, with Ca2+imaging using fura-2/AM. Exposure of cells to ovine GH alone produced a stimulation of the JAK2/STAT5 pathway and intracellular free Ca2+variations similar to what has been observed in mammalian models. Cell pre-exposure to Cu2+, Hg2+or Cd2+affected cell response to GH by enhancing (Cu2+) or inhibiting (Cd2+) the phosphorylation of JAK2 and STAT5. Heavy metals induced the activation of the MAP kinase p38, and pre-exposure to Hg2+or Cu2+followed by GH enhanced the effect of metal alone. Image analysis of fura2-loaded cells indicated that pre-treatment with Hg2+prior to GH produced a considerable increase of the [Ca2+]ivariation produced by either element, while using Cu2+or Cd2+the result was similar but much weaker. Data suggest that heavy metals interfere with GH as follows: Hg2+is nearly ineffective on JAK/STAT and strongly synergistic on Ca2+signaling; Cu2+is activatory on JAK/STAT and slightly activatory on Ca2+; Cd2+is strongly inhibitory on JAK/STAT and slightly activatory on Ca2+; heavy metals could partially activate STAT via p38 independently from GH interaction.

Immunomagnetic cell selection (ICS) of CD34+ cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34+ ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34+ cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34+ cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34+ cells (<500 × 106) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

It is generally accepted that metallothioneins (MTs) are devoted to the regulation of the metabolism of essential trace metals and to chelation of toxic metals. Nowadays, there is increasing evidence that MTs also act as free radical scavengers. We employed wild type mouse embryo fibroblast cell line, GKA1, and its MT-null variant, GKA2, in order to correlate the presence of MTs to the sensitivity of cells to reactive oxygen species (ROS), spontaneously generated by the aerobic cellular metabolism, or chemically induced by hydrogen peroxide. The absence of MTs in GKA2 cells was unambiguously correlated to higher sensitivity to ROS attack, as evaluated by detection and quantification of 8-oxo-2′-deoxyguanosine (8-oxo-G), the first product of oxidative attack to DNA, using Fluorescence-Activated Cell Sorter (FACS). When compared to MT-null cell line, the wild type cells (GKA1) were less sensitive to ROS attack. In GKA1 cells, MT biosynthesis is readily induced by Cd2+treatment, and such an induction caused a further decrease in sensitivity to ROS injury. On the contrary, the MT-null cells (GKA2) expressed no detectable metallothioneins either constitutively, or after heavy metal pretreatment. Indeed, in GKA2 cell line, pretreatment with Cd2+did not reduce but even enhanced the oxidative stress.

Metallothioneins (MT) are ubiquitous low-molecular-weight metal-binding intracellular proteins. We used wild type mouse embryo fibroblasts, GKA1, and its MT-null variant, named GKA2, in order to correlate the presence of MT to the response to a number of different antitumor drugs with different mechanisms of action. We studied sensitivity of GKA1 and GKA2 cells to metal-based compounds having alkylating property, or able to generate reactive oxygen species (ROS); as well as to drugs acting with different mechanisms. The absence of MT in GKA2 cells was correlated to higher sensitivity to the metal-based drugs compared to that of GKA1. No marked differences in sensitivity of two cell lines against gemcitabine, taxol, and vinblastine were observed. No significant change in sensitivity of either GKA1 or GKA2 cells to these non-alkylating drugs was seen after heavy metal pretreatments. In GKA1 cells, MT biosynthesis was induced by copper and cadmium but not by zinc treatment under the conditions of these experiments. Induction of MT was directly proportional to decrease in sensitivity of GKA1 cells to the compounds used in this experiment. In contrast to GKA1 cells, the MT-null cells (GKA2) expressed no detectable metallothionein either constitutively or after treatment with zinc, copper, or cadmium. Nonetheless, heavy metal pretreatment of GKA2 cells did not cause any change in their sensitivity.