P < 0.001). At the genus level, consumption of 3 g/d HMW β-glucan increased Bacteroides (P < 0.003), tended to increase Prevotella (P < 0.1) but decreased Dorea (P < 0.1), whereas diets containing 5 g LMW β-glucan and 3 g LMW β-glucan failed to alter the gut microbiota composition. Bacteroides, Prevotella, and Dorea composition correlated (P < 0.05) with shifts of CVD risk factors, including body mass index, waist circumference, blood pressure, as well as triglyceride levels. Our data suggest that consumption of HMW β-glucan favorably alters the composition of gut microbiota and this altered microbiota profile associates with a reduction of CVD risk markers. Together, our study suggests that β-glucan induced shifts in gut microbiota in a MW-dependent manner and that might be one of the underlying mechanisms responsible for the physiological benefits of β-glucan.The physiological cholesterol-lowering benefits of β-glucan have been well documented, however, whether modulation of gut microbiota by β-glucan is associated with these physiological effects remains unknown. The objectives of this study were therefore to determine the impact of β-glucan on the composition of gut microbiota in mildly hypercholesterolemic individuals and to identify if the altered microbiota are associated with bioactivity of β-glucan in improving risk factors of cardiovascular disease (CVD). Using a randomized, controlled crossover study design, individuals received for 5-week either a treatment breakfast containing 3 g high molecular weight (HMW), 3 g low molecular weight (LMW), 5 g LMW barley β-glucan, or wheat and rice. The American Heart Association (AHA) diet served as the background diet for all treatment groups. Phases were separated by 4-week washout periods. Fecal samples were collected at the end of each intervention phase and subjected to Illumina sequencing of 16S rRNA genes. Results revealed that at the phylum level, supplementation of 3 g/d HMW β-glucan increased Bacteroidetes and decreased Firmicutes abundances compared to control ( D-glucan (β-glucan) is a linear polysaccharide composed of D-glucose monomers joined by (1→4)-glycosidic bonds (∼70%) with an occasional (1→3)-glycosidic bonds (1→4)-β-ds ∼30%; . β-glucads ∼30%;  and has ds ∼30%; , reductids ∼30%; , and weids ∼30%; . Previouds ∼30%; .in vitro fermentation , are considered to be critical factors in its physiological properties . The extA randomized, single-blinded, controlled crossover trial was conducted at the Clinical Nutrition Research Unit at the Richardson Centre for Functional Foods and Nutraceuticals (RCFFN), University of Manitoba  in accordance with the principles expressed in the Declaration of Helsinki. All research procedures were approved by the University of Manitoba Biomedical Research Ethics Board (Ethic reference no. B2010:057). The clinical trial was registered with ClinicalTrials.gov (ID NCT01408719). The endpoint data for body weight, BMI, waist circumference, blood glucose and lipid profiles, and blood pressure for the 19 subjects included in this microbiome study extracted from the parallel clinical trial that focused on cholesterol lowering effect of β-glucan  and weren = 30) with a body mass index  between 20 and 40 were selected on the basis of fasting serum total cholesterol (TC) between 5.0 and 8.0 mmol/L and LDL cholesterol (LDL-C) between 2.7 and 5.0 mmol/L. Potential volunteers were excluded using criteria including: intake of blood lipid lowering medications, herbal or nutritional supplements which are known to affect blood lipids and any dietary restrictions which would affect trial completion. No antibiotics intake was reported at the time of screening. A study physician evaluated the health history of volunteers before they were accepted as subjects. The physician also provided medical supervision throughout the study. Written informed consent was obtained from all subjects. Of the 30 subjects in this study, a subset of 19 subjects who completed the entire protocol and provided fecal samples for all four study phases were selected for the microbiota profile analysis.Male and female subjects aged 27–78 years , LDL-C, and triglyceride (TG) levels were measured. Approximately 15 g of stool samples were collected at the end of each phase between days 30 and 35. Subjects were advised to collect stool samples from three locations of one bowel movement and immediately store the collected samples in their household –20°C freezer. Upon transport, samples were transferred to chilled coolers and then stored for future analysis at –80°C.TM , which included a bead-beating step for the mechanical lysis of the microbial cells. DNA was quantified using a NanoDrop 2000 spectrophotometer . DNA samples were normalized to 20 ng/μl, and quality checked by PCR amplification of the 16S rRNA gene using universal primers 27F (5′-GAAGAGTTTGATCATGGCTCAG-3′) and 342R (5′-CTGCTGCCTCCCGTAG-3′) as described by Stool samples from different phases of the projects were DNA extracted and sequenced at once. For DNA extraction, approximately 200 mg of each fecal sample was used for DNA extraction using ZR Fecal DNA MiniPrep® . Reactions consisted of an initial denaturing step at 94°C for 3 min followed by 35 amplification cycles at 94°C for 45 s, 50°C for 60 s, and 72°C for 90 s; finalized by an extension step at 72°C for 10 min in an Eppendorf Mastercycler® pro . PCR products were then purified using a ZR-96 DNA Clean-up KitTM  to remove primers, dNTPs and reaction components. The V4 library was then generated by pooling 200 ng of each sample, quantified by Picogreen dsDNA . This was followed by multiple dilution steps using pre-chilled hybridization buffer  to bring the pooled amplicons to a final concentration of 5 pM, measured by an Qubit® 2.0 Fluorometer . Finally, 15% of PhiX control library was spiked into the amplicon pool to improve the unbalanced and biased base composition, a known characteristic of low diversity 16S rRNA libraries. Customized sequencing primers for read1 (5′-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA-3′), read2 (5′-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT-3′) and index read (5′-ATTAGAWACCCBDGTAGTCCGGCTGACTGACT-3′) were synthesized and purified by polyacrylamide gel electrophoresis  and added to the MiSeq Reagent Kit v2 . The 150 paired-end sequencing reaction was performed on a MiSeq platform  at the Gut Microbiome and Large Animal Biosecurity Laboratories, Department of Animal Science, University of Manitoba, Canada. All sequencing data are uploaded into the Sequence Read Archive (SRA) of NCBI and are available through accession number SRR2558312.Library construction and Illumina sequencing were performed as described by e – 5 were truncated, and those with a read length shorter than 75 bases were removed from the downstream analysis. Chimeric reads were filtered using UCHIME  was calculated using QIIME. The α rarefaction curve was generated using Chao1 estimator of species richness  with 10 P < 0.05 while trends were observed at P < 0.1.Statistical analyses were performed as described by P-values and test for significant differences of β-diversity among treatment groups. The effects of treatment, period, and treatment by period interaction were considered fixed factors and subject as the random factor.Permutational multivariate analysis of variance . The PLS-DA is a particular case of partial least square regression analysis in which Y is a set of variables describing the categories of a categorical variable on X. In this case, X variables were bacterial genera and Y variables were experimental diets. For this analysis, data were scaled using Unit Variance in SIMCA. Cross-validation was then performed to determine the number of significant PLS components and a permutation testing was conducted to validate the model. To avoid over-parameterization of the model, variable influence on projection value (VIP) was estimated for each genus and genera with VIP < 0.50 were removed from the final model  was performed on genus data to identify the effects of treatments . The clinical parameters and bacterial taxa defined as response (Y) variables and a multivariate platform was used to examine how many variables relate to each other. For each correlation, the correlation coefficient (Spearman’s ρ) and The results of blood glucose, blood pressure, body weight, waist circumference, and BMI were consistent with the results of parallel study that included 30 subjects and focused on examining the blood cholesterol lowering effects of β-glucan (Supplementary Table Table 1). In contrast, β-diversity of the fecal microbial community, or the dissimilarity between communities, measured by weighted UniFrac was affected by β-glucan supplementation . In the pairwise comparison, significant differences were observed among microbial communities between 3 g HMW and 3 g LMW β-glucan , 3 g HMW β-glucan and the control , and 5 g LMW β-glucan and the control . The differences between 5 g LMW and 3 g HMW β-glucan , 5 g LMW and 3 g LMW β-glucan , 3 g LMW β-glucan and control  were not significant. The community composition profile of the above-mentioned three paired comparisons, which showed significant differences in their β-diversity, were further compared using PLS-DA as discussed below .Alpha-diversity indices of fecal microbiota, or the diversity within a community, did not change in response to consumption of β-glucan (Table 2). The remaining five phyla including Cyanobacteria, Synergistetes, Lentisphaerae, Deferribacteres, and Elusimicrobia were in low abundance (<0.1% of community). Abundances of p. Firmicutes and Bacteroidetes were significantly impacted in response to 3 g/d HMW β-glucan consumption, where Firmicutes decreased  and Bacteroidetes increased  compared to the control (Table 2). However, Firmicutes and Bacteroidetes abundances did not change in response to 5 g/d LMW and 3 g/d LMW β-glucan consumption. The abundances of p. Actinobacteria, Verrucomicrobia, Proteobacteria, and Tenericute did not change across all treatments.While majority of OTUs were identified at the genus level, some were only classified at the phylum (p.), class (c.), order (o.), or family (f.) levels. Across all samples, 11 bacterial phyla were identified of which two phyla Firmicutes and Bacteroidetes were abundant (above 1% of community), accounting for approximately 84 and 9% of the entire bacterial community, respectively. Another four bacterial phyla, including Actinobacteria, Verrucomicrobia, Proteobacteria, and Tenericute were present at abundances between 0.1 and 1% of the community (Table 3). Of these 44 taxa, 24 were identified at the genus level, 14 at the family level, four at the order level, and two at the class level. Irrespective of treatment group, g. Bacteroides was the most abundant genera in p. Bacteroidetes, whereas p. Firmicutes were dominated by g. Blautia. Consumption of 3 g/d HMW β-glucan increased the abundances of Bacteroides  compared to control. Consumption of 3 g/d HMW β-glucan also increased the relative abundance of o. Bacteroidales compared to the 3 g LMW β-glucan treatment . Moreover, f. Rikenellaceae and g. Prevotella showed increasing trends (P < 0.1) following 3 g/d HMW β-glucan consumption compared to control (Table 3). In contrast, g. Streptococcus (p. Firmicutes) was significantly decreased by 3 g/d HMW β-glucan compared to control . Additionally, g. Dorea showed a decreasing trend (P < 0.1) in response to 3 g/d HMW β-glucan consumption (Table 3).At the lower taxonomical level and across all samples, 169 distinct bacterial taxa were detected of which 44 had a relative abundance above 0.1% of community .The PLS-DA results  supported the SAS output, however, due to the higher sensitivity of PLS-DA methodology, additional significant differences among treatments, especially in low abundance taxa, were identified. For instance, when comparing 3 g HMW β-glucan vs. control, two additional low abundance taxa, g. Table 4). Weak (Spearman’s ρ between 0.20 and 0.39) and moderate correlations (Spearman’s ρ between 0.40 and 0.59) were observed between parameters associated with CVD including BMI, waist circumference, TC levels, LDL-C, HDL-C, TG, glucose, systolic and diastolic blood pressure, and specific members of bacterial community (Table 4). Bacteria, which were affected by 3 g HMW β-glucan and also showed correlation with parameters associated with CVD, were g. Bacteroides which was negatively correlated with waist circumference  and diastolic BP ; g. Prevotella which showed negative correlation with TG ; and g. Dorea which was positively associated with BMI , waist circumference  and diastolic BP .Spearman non-parametric correlation was conducted to test the association between bacterial taxa and risk factors of CVD . Predicted shifts in glycerolipid metabolism and other glycan degradation were increased by 3 g HMW β-glucan compared with control . Same results for glycerolipid metabolism and other glycan degradation were observed when compare 3 g HMW vs. 3 g LMW β-glucan . However, although 5 g LMW β-glucan showed differences in weighted UniFrac compared with control, only three metabolisms  were predicted to be different using PICRUSt , whereas no differences were observed between 3 g LMW β-glucan and control with both weighted UniFrac or PICRUSt (no difference to be graphed).A total of 41 level 2 and 328 level 3 KOs were identified for the fecal microbiota (Supplementary Tables Tables 2 and 3). The shifts in g. Bacteroides, Prevotella, and Dorea were favorably correlated with an improved CVD risk factor profile, such as BMI, waist circumference, TG, blood pressure (Table 4). Consumption of 5 g/d LMW β-glucan only impacted the β-diversity but not the composition of gut microbiota compared to control . Additionally, the consumption of 3 g/d LMW β-glucan failed to alter either the β-diversity or the composition of gut microbiota.The main finding of this controlled feeding study was that gut microbiota were modulated in response to barley β-glucan consumption on a MW-dependent manner, and that the shifts in gut microbiota was correlated with reduced CVD risk factors. Specifically, consumption of 3 g/d HMW β-glucan resulted in increased abundance of p. Bacteroidetes and decreased abundance of p. Firmicutes, as did several of their respective taxa at the lower taxonomical levels  indicating that certain members of the bacterial community, such as members of p. Bacteroidetes, had taken advantage of HMW β-glucan and perhaps other fermentable substrates in the digesta most likely due to increased exposure time for microbial fermentation. Lending support to this hypothesis, a recent study . Genus Prevotella, however, showed a negative correlation with TG. Additionally, g. Dorea that tended to be decreased by 3 g/d HMW β-glucan was positively associated with BMI, waist circumferences and diastolic blood pressure. Consumption of 3 g/d HMW β-glucan also decreased g. Streptococcus compared to the control. But this genus was not correlated with biochemical markers, anthropometric markers or blood pressure that were measured in the clinical trial. Altogether, these results suggested that consumption of 3 g/d HMW β-glucan for 35 days was able to alter the gut microbiota and the altered microbiota were correlated with a favorable shift in parameters associated with CVD. However, since these metabolic parameters were not changed significantly in the sampled subjects (n = 19), studies with greater sample size would be beneficial to further investigate the causality to see whether the altered gut microbiota improves the CVD risk factors.The present intervention study showed that consuming 3 g/d of HMW β-glucan was able to significantly increase g. In addition, predicted metabolic capacity of bacterial community (generated using PICRUSt) exhibited increased abundances of genes associated with glycerolipid metabolism with HMW β-glucan diet compared to control and LMW β-glucan diets, which has been associated with obesity and type 2 diabetes via energy homeostasis, appetite control, and insulin sensitivity . These oGut health in part is associated with proper richness, diversity and stability of gut microbiome. Recent research shows that individuals with low richness of microbiota are more vulnerable to obesity, insulin resistance and dyslipidemia compared to those with high richness of microbiota . In thisOverall, our results suggest that β-glucan with higher MW was able to alter the gut microbiota and the shifts observed were positively associated with an improved CVD risk factor profile. Although studies with large samples size are needed to confirm the relationship between the altered gut microbiota by β-glucan and the improved CVD health benefits, results from current study are reminiscent of the hypothesis that altering gut microbiota is responsible for the physiological benefits of HMW β-glucan consumption.NA and PJ conceived and designed the experiment. NA, PJ, and YW conducted the clinical trial. YW performed lab analyses. YW, HT, and EK developed the bioinformatics and statistical models and analyzed the data. All authors drafted the manuscript. All authors carefully read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gloydius Ussuriensis venom. The RGD (Arg-Gly-Asp) site of disintegrin dominates its interaction with integrins on the surface of the tumor cells. uPA is over-expressed and plays an important role in tumor cell invasiveness and metastatic progression. The DLM (disintegrin-linker-melittin) linker is uPA-cleavable, enabling DLM to release melittin. We compared binding activity of our synthesized disintegrin with native disintegrin and report that DLM had less binding activity than the native form. uPA-cleavage was evaluated in vitro and the uPA-cleavable linker released melittin. Treating tumors expressing uPA with DLM enhanced tumor cell killing as well as reduced toxicity to erythrocytes and other non-cancerous normal cells. The mechanism behind DLM tumor cell killing was tested using a DNA ladder assay, fluorescent microscopy, flow cytometry, and transmission electron microscopy. Data revealed tumor cell necrosis as the mechanism of cell death, and the fused DLM toxin with an uPA-cleavable linker enhanced tumor selectivity and killing ability.Melittin, which acts as a membrane-disrupting lytic peptide, is not only cytotoxic to tumors, but also vital to normal cells. Melittin had low toxicity when coupled with target peptides. Despite significant research development with the fused toxin, a new fused toxin is needed which has a cleavable linker such that the fused toxin can release melittin after protease cleavage on the tumor cell surface. We describe a novel fused toxin, composed of disintegrin, uPA (urokinase-type plasminogen activator)-cleavable linker, and melittin. Disintegrin is a single strand peptide (73 aa) isolated from  Traditional chemotherapy drugs and radiotherapy is based on the rapid proliferation of cancer cells ,2, and ain vitro and in vivo, such as adhesion, migration, invasion, metastasis, and angiogenesis, by binding to integrins αvβ3, αvβ5, and/or α5β1 [Gloydius Ussurinsi’s venom, is a small disulfide-rich peptide  with properties similar to contortrostatin (has 65 amino acids and an RGD motif).Integrins are cell surface receptors needed for cell adhesion and migration, and they are comprised of heterodimeric α- and β-subunits ,14 that /or α5β1 ,20,21. C/or α5β1 ,23,24. A/or α5β1 . UssurinMelittin (26 amino acids) is a major peptide component of bee venom with high cancer-fighting potency ,31,32,33Pichia pastoris. After optimization of growth and induction conditions, the DLM yield was 160 mg/L (95% purity). DLM had less hemolytic bioactivity, allowing for intravenous drug delivery. DLM had low cytotoxicity to uPA non-expressing HEK-293 cells and strong cytotoxicity against uPA-expressing cells A549 [Previously, we constructed a tumor-activated conjugate DLM and expressed it in lls A549 .We compared the biological activity between DLM and native disintegrin to evaluate uPA-cleavage, and we assayed tumor cytotoxicity as well as safety for erythrocyte and human normal cells. Finally, we investigated how DLM kills tumor cells using a DNA ladder assay, fluorescent microscopy, flow cytometry and transmission electron microscopy (TEM).The DLM can be cleaved by uPA specifically to release the disintegrin domain .In order to show that DLM is less cytotoxic than melittin and that the cleaved DLM is toxic to tumor cells, an erythrocyte hemolysis assay was used. After incubating DLM, DLM + uPA and free melittin at 37 °C for 1 h, DLM was observed to cause minimal hemolysis at 10 μmol/L. When erythrocytes were treated with the same concentrations of DLM + uPA and melittin, significant hemolysis occurred . Free me50 of disintegrin attached to DLM = 130 nM, compared to native disintegrin IC50 102 nM; The disintegrin domain of DLM retains platelet aggregation inhibitory activity, although this domain is attached to DLM. Intact DLM can greatly inhibit platelet aggregation . DLM binding to washed platelets was compared with disintegrin under identical conditions . Neither490). Serial dilutions of DLM show a dose-dependent cytotoxicity against BT-549, MDA-MB-231, SMMC-7721, MCF-7, and SKOV-3 cells. DLM killing of HEK293 was low (2.626 μM DLM) and caused cytoplasmic condensation only. Under an inverted microscope, control MCF-7 cells were normal; with 0.164 µM DLM there was significant change. With 0.328 µM DLM cells were swelling, plasma membranes ruptured, and content released into the extracellular area, suggesting necrosis. MCF-7 cells treated with 1.313 µM DLM did not have complete cell structures.Every tumor cell strain was cultured and treated with DLM and incubated for 72 h  have a clear band and no laddering. DLM-treated cells (1.313 and 2.626 μM) and melittin-treated cells appear as smears. The  pattern .Cells are measured with flow cytometry and observed under a fluorescent microscope. Spots mainly appeared in the Q1 section, implying necrosis, which increased with increasing DLM . Under tMCF-7 cells treated with DLM were necrotic . The ER (endoplasmic reticulum) and the nuclear matrix and cytoplasm had vacuoles .Invasion and metastasis are key to tumor malignancy and these events are complex interactions of host and cells and the extracellular matrix. In this process, the combination of normal epithelial cells and the matrix is achieved by integrins, which are present on epithelial cell surfaces. The ligand is in the matrix. Thus, the integrin family of cell adhesion receptors is vital to initiation, progression and metastasis of malignant tumors and attractive targets for anti-cancer therapy. Due to the affinity of integrin for what, we selected the disintegrin, ussurin, as the carrier because it can deliver the toxin domain, melittin, to cancer cells with high efficiency and accuracy.50 130 nM) and its IC50 is higher than native natural disintegrin (IC50 102 nM). Incubation of native disintegrin and DLM, with washed, inactivated human platelets, revealed no inhibition by either molecule but after ADP activation both molecules efficiently inhibited platelets.Melittin is attached in DLM via a linker to confine its hemolytic activity and to increase safety of delivery. Melittin is released when DLM reaches the target cell and is cleaved by uPA, which is mainly expressed on the cancer cell surface. Then, the cancer cell membrane is ruptured by melittin, and cell death ensues. SDS-PAGE revealed that after DLM treatment with uPA, DLM can be cleaved to release the melittin and disintegrin domains. Intact DLM can inhibit platelet aggregation  and DLM-treated cells were smears, indicating that necrosis occurred.Flow cytometry confirmed that DLM was cytotoxic to MCF-7 cells. Under TEM, normal MCF-7 cells are clear with microvilli, a large irregular nucleus and normal cytoplasmic organelles. After treatment with DLM, MCF-7 cell membranes ruptured, the ER had vacuoles, and organelles were degraded, all indicators of necrosis. DLM may be useful for anti-cancer therapy because it can inhibit cancer cell growth via necrosis and reduce melittin-induced hemolysis and other toxic effects on normal cells. Our work provides a foundation for further exploration of DLM for anti-tumor biological functions and applications.(1)SMMC-7721, a human hepatoma cell line,(2)MCF-7, a human breast carcinoma cell line,(3)BT-549, a human breast carcinoma cell line,(4)MDA-MB-231, a human breast carcinoma cell line,(5)SKOV-3, a human ovarian neoplasm cell line,(6)MCF-10, a human normal breast cell,(7)L-02, a human normal liver cell, and(8)HEK293, a human normal embryonic kidney cell.The cells used in our studies were as follows:2.All cell lines were maintained in RPMI 1640 medium  with 20% fetal bovine serum and 10% DMSO at −80 °C. Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C, in 5% COP. Pastoris strain pPIC9K/DLM was cultured in BMGY, and induced with 0.5% methanol in BMMY media. After 96 h of culture, the medium was harvested by centrifugation and the supernatant was concentrated by ultrafiltration.DLM was expressed and purified as previously described . The selThe concentrated solution was loaded onto a SP-Sepharose Fast Flow column, the eluted protein, was loaded onto a Sephadex G 75 column. Purified DLM was a single band on SDS-PAGE and Western blot (95% purity).v/v)). The suspension was incubated for 60 min at 37 °C with gentle mixing. Samples were then centrifuged and supernatant absorbance was measured at 545 nm. Controls for zero hemolysis (blank) and 100% hemolysis consisted of erythrocytes suspended in PBS and 1% Triton X-100, respectively. Data (means ± SD) were from three independent experiments.As described previously , DLM wasin vitro, a reaction mixture containing 5 µg DLM, 0.5 µg uPA, and 50 µL enzyme buffer  was prepared. The reactant was analyzed by SDS-PAGE after incubating at 37 °C in a water bath for 60 min.The fused toxin DLM contained a uPA-cleavable sequence to link carrier and toxin domains. To verify linker cleavage with uPA The DLM contained equal moles of disintegrin and melittin and the biological activity of disintegrin within the DLM is crucial for a tumor-activated fused toxin. Activity was assessed by measuring disintegrin-mediated inhibition of ADP-induced platelet aggregation.g for 10 min) at 25 °C. The supernatant, PRP, was carefully transferred to a new tube. PRP (1 mL) was placed in a microcentrifuge tube and centrifuged at (8000 g for 5 min) 25 °C. The supernatant was removed and retained as platelet-poor plasma (PPP). PPP was then used as a blank to standardize the platelet aggregometer. Inhibition of ADP-induced platelet aggregation was monitored at 37 °C by adding samples (disintegrin and DLM) over a range of disintegrin concentrations (0–200 nM) to PRP 1 min prior to the addition of ADP. Data (means ± SD) were from three independent experiments.Briefly, the inhibitory activity of native disintegrin and DLM toward ADP-induced platelet aggregation was measured with a platelet aggregometer  with human platelet rich plasma (PRP). Whole human blood (50 mL), obtained from volunteers and drawn into 5 mL tubes, with 0.1 mol/L sodium citrate. Donors had not received any aspirin-based medications for at least two weeks prior to donation. Sample blood was centrifuged ) was added  and PGE1 was added to a concentration of 2.5 mM. A small amount (1–2 mL) of PPP was set aside for a zero-platelet control in the binding experiments. After addition of Tyrode’s buffer, platelets were resuspended by gently stirring the buffer with a disposable plastic pipette; platelets were gently drawn into and expelled from the pipette until minuscule particles of the original pellet were no longer visible. Pelleting and pellet resuspension were repeated three times, and a final resuspension was in 6 mL modified Tyrode’s buffer without PGE1. Platelets were counted, and washed platelets were used in binding experiment with different forms of disintegrin.Disintegrin and DLM binding to washed human platelets was evaluated. PRP and PPP were prepared as described above and supernatant (PPP) was decanted, and the inside wall of the centrifuge tube was wiped. Modified Tyrode’s buffer , respectively . FITC solution was added to native disintegrin or DLM solution dropwise while gently and continuously shaking the protein solution. After FITC solution was added at the molar ratio of 1:2 (DLM or native disintegrin:FITC), the reaction was incubated in the dark for 16 h at 4 °C. The conjugate of the FITC-protein was put into a dialysis bag, and this was dialyzed overnight to separate unbound FITC from the conjugate. Unreacted FITC was removed from FITC-disintegrin or FITC-DLM solution by gel filtration using a gel matrix of Sephadex G25 column (40 × 1.0 cm). After elution with PBS, two peaks formed. The first peak was FITC-protein, which can easily be visualized. The ratio of fluorescein to protein in the conjugates was measured using absorbance at 495 nm (F) and 280 nm (P).FITC (fluorescein isothiocyanate), native disintegrin and DLM were dissolved in buffer (7.56 g NaHCO5 platelets/mL in Tyrode’s buffer) and 1 tube with Tyrode’s buffer as the control, FITC-disintegrin, FITC-DLM were added to the 2 group tubes . ADP was added to one group as a platelet activator  and the same volume of Tyrode’s buffer was added to the other group (inactivated platelets). The mixtures were incubated for 30 min at 37 °C. Platelets were pelleted by centrifugation and fluorescence measured by counting fluorescence in both the supernatant and the pellet with a fluorophotometer . Data (means ± SD) were from three independent experiments.Tubes were divided into two groups, each group had 3 tubes with 200 μL washed platelets . There were six individual wells for each dose. The blank control was the dilution medium without the sample. Then cells were incubated in 96-well Xat-bottomed plates at 37 °C under 5% CO2 for 72 h. After incubation, supernatant was removed and cells were washed with PBS (pH 7.4). Cells were incubated with medium containing 20 μL of the MTT solution (5 mg/mL) at 37 °C for 4 h. To dissolve formazan crystals, 100 μL of DMSO was added after removing the supernatant. OD was measured with an ELISA microplate reader at 490 nm. Cytotoxicity was measured with IC50. Data (means ± SD) were from three independent experiments.Cells  were seeded on 96-well Xat-bottomed plates (3000 cells/well), and incubated at 37 °C under 5% CO6 cells). Every tube was washed twice with cold PBS. Medium (1 mL) was added to each tube containing 10 μL Hoechst 33342 (5 µg/mL). This was mixed thoroughly and incubated at 37 °C for 10 min. Cells were centrifuged at 1500 g for 5 min at 4 °C and supernatant was discarded. Cells were resuspended in 1 mL buffer (1 μg/mL PMA in RPMI 1640 medium). Then 5 μL of PI was added to each cell suspension and mixed thoroughly. Cells were incubated at room temperature for 10 min. After incubation, stained cells were analyzed with flow cytometry immediately .After 48 h of treatment with serially diluted DLM  MCF-7 cells were collected (1 × 107) after a 48 h culture with medium and serially diluted DLM as previously described. A necrosis-positive control group received 2.626 μM melittin and an apoptosis-positive group was treated with 10 μM cis-platinum. Cells were washed three times with PBS, and then resuspended in 350 mL of lysis solution  and 3.5 μL (50 μg/mL) of proteinase K. cells were incubated in a water bath (50 °C) for 2 h. Next, equal volumes of phenol-chloroform-isoamyl alcohol (25:24:1) were added to cells and mixed. The mixture was centrifuged at 12,000 g for 10 min at 4 °C. Supernatant was discarded and 1/10 volumes of 5 mol/L NaCl and 2.5 volumes of ethanol was added to the cells which were centrifuged at 12,000 g for 10 min at 4 °C. Precipitates were washed in 75% ethanol and left to dry at room temperature. Precipitates were re-suspended in 100 μL TE buffer  and incubated with 2 μL (10 mg/mL) RNase for 1 h at 65 °C and then separated by 1% gel electrophoresis.MCF-7 cells were collected (1 × 105) after a 48 h culture with medium and serially diluted DLM as described previously. Cells were washed with precooling PBS twice and then fixed in 2.5% glutaraldehyde at 4 °C for 2 h. Cells were post-fixed in 1% osmium tetroxide at 4 °C for 1 h and then washed with PBS for 20 min twice. Cells were stained with 0.5% aqueous uranyl acetate at room temperature overnight. To dehydrate slides, they were immersed in 30%, 50%, 70%, and 90% ethanol for 10 min at each percent and then 100% ethanol for 10 min twice. Samples were embedded in Epon812 and cut into ultrathin sections. Sections were collected onto grids and stained with 1% (w/v) uranyl acetate and lead citrate. Samples were observed under a JEM-1200EX electron microscope .MCF-7 cells were collected (1 × 10


Diabetes is a major cardiovascular risk factor. However, its influence on the rate of occurrence of cardiovascular (CV) events during a clinical trial that included a diabetes subgroup has not yet been quantified.To establish equations relating baseline diabetes prevalence and incident CV events, based on comparator arms data of major lipid-modifying trials.Meta-analysis of primary outcomes (PO) rates of key prospective trials, for which the baseline proportion of diabetics was reported, including studies having specifically reported CV outcomes within their diabetic subgroups.47 studies, representing 330,376 patients , were analyzed as regards the relationship between CV outcomes rates (including CHD) and the number of diabetics enrolled. Altogether, a total of 18,445 and 16,156 events occurred in the comparator and treatment arms, respectively. There were significant linear relationships between diabetes prevalence and both PO and CHD rates (%/year): y = 0.0299*x + 3.12 [PO] (p = 0.0128); and y = 0.0531*x + 1.54 [CHD] (p = 0.0094), baseline diabetes predicting PO rates between 3.12 %/year (no diabetic included) and 6.11 %/year ; and CHD rates between 1.54 %/year (no diabetic) and 6.85 %/year . The slopes of the equations did not differ according to whether they were derived from primary or secondary prevention trials.Absolute and relative CV risk associated with diabetes at inclusion can be readily predicted using linear equations relating diabetes prevalence to primary outcomes or CHD rates. Key prospective trials have demonstrated the effectiveness of long-term control of conventional risk factors (RFs) to prevent cardiovascular (CV) events. Next to decreasing tobacco use and physical inactivity, indisputable gains were achieved by targeting hypertension and hypercholesterolemia. Nevertheless, there remained a high residual risk of incident CV events in control and comparator arms of these trials, even in patients receiving appropriate standard of care –4. This vs. nondiabetic subgroups of individual trials.Based on epidemiology and prospective studies, type 2 diabetes mellitus (T2DM) significantly increases the absolute risk of developing coronary heart disease (CHD), and confers a higher residual risk of large and small vessel damage. In the microcirculation, such risk is directly related to hyperglycaemia, whereas in large vessels, this residual risk is linked to hypertension, low-density lipoproteins (LDL); non-LDL dyslipidemias; and other metabolic comorbidities –10. As aThe aim of this work was to establish equations relating baseline diabetes prevalence and incident CV events, based on comparator arms data of major clinical trials having investigated the potential CV benefit of various pharmacological or dietary interventions targeting, in the vast majority, lipids and lipoproteins. We performed a systematic meta-analysis of CV outcomes rates of those key prospective studies, for which the baseline proportion of diabetics was reported and, where available, studies having reported CV outcomes of diabetic subgroups –90 Tabl.Table 1Oi) the main purpose of the trial was to study the effect on CHD of a pharmacological or dietary intervention targeting lipids or lipoproteins, with CHD rates as sole primary outcome (PO), or with a major adverse CV event (MACE) composite PO comprising CHD; (ii) to focus exclusively on diabetic patients, or (iii) to report data on a sufficient number of diabetic patients from pre-/post-hoc analyses of DM subgroups of the main trial. Among studies conducted non-exclusively in DM patients, eligible trials had to comply with ≥1 of the following criteria: (i) the main trial had a subgroup of patients already diagnosed with DM at baseline, whose proportion was deemed sufficiently representative (>15 %); or (ii) the trial enrolled at least 100 DM patients, regardless of on-study new-onset diabetes.To be selected for inclusion, major clinical trials with CV outcomes had to meet three requirements: ; low-density lipoprotein cholesterol (LDL-C); high-density lipoprotein cholesterol (HDL-C); non-HDL-cholesterol (non-HDL-C); apolipoprotein B100 (apoB); triglycerides (TG); type of pharmacological or dietary intervention; primary trial outcome; CHD outcomes (see Table 2for CV outcomes categories); and CV events number and rates for each trial.For each study, the following items were analyzed: CV risk category at baseline ; number of patients included; number and proportion of patients with DM at baseline; number of patients in the active or comparator arms; duration of follow-up; age at inclusion; number of males; DM type and duration; HbAResults are presented as means (±1 standard deviation (SD)), or as proportions (%), with between-study range [BSR] described when needed. Linear regression was computed using the least-squares method. Results were considered statistically significant or non-significant (NS) for p <0.05 or p ≥0.05, respectively.n = 42,279) enrolled patients in PP at baseline; 17 of 47  included populations whose CV risk was a mix of PP and SP; and 22 of 47  were SP trials. Lipid values at baseline were (mg/dL): 209 (34) [TC]; 126 (32) [LDL-C]; 44 (7) [HDL-C]; 161 (32) [non-HDL-C]; 99 (19) [apoB] and 162 (27) [TG]. In total, these studies have included 124,115 diabetic patients, representing 42.1 % [BSR 2.3 %–100 %] of the population studied. For studies that reported diabetes duration, it averaged 7.5 (4.9) years, whereas metabolic control assessed by HbA1c was 7.49 (0.68) % (Table Forty-seven studies were selected based on the criteria defined above –90. Theycomparator) and 3.0 (1.9)%/year [BSR 0.3–9.1] (treatment), respectively , whereas y = 0.0267* x +3.76  for SP trials.For all 47 studies, a total of 18,445 and 16,156 events occurred in the comparator and treatment arms, respectively. On an annual basis, this was equivalent to an average rate of occurrence for the primary CV outcome of 3.6 (2.4) %/year [BSR 0.5–11.8] . For diabetic patients, the event rate decreased from 5.0 %/year [<2005] to 4.3 %/year [≥2005], ie. absolute and relative reductions of 0.7 % and 14 % (NS).When comparing PO rates from the comparator arms of studies published prior to 2005 predominantly non-diabetes studies ; 129 (36) [LDL-C]; 44 (7) [HDL-C]; 165 (36) [non-HDL-C]; 98 (21) [apoB] and 160 (25) [TG]. In total, these studies have included 63.189 diabetic patients, representing 21.3 % [BSR 2.3 %–44.2 %] of the population studied (Table predominantly non-diabetes studies investigated the following interventions over a mean (1SD) duration of 4.3 (1.5) years [BSR: 1.0–7.5 years]: statins ; fibrates ; n-3 fatty acids ; niacin ; CETP-inhibitor ; ezetimibe ; and Lp-PLA2 inhibitor  , numbering 12,732 patients, published as pre-/post-hoc sub-group analyses of DM patients   of DM patients at baseline , 77, 81,6] Table .studies focusing on diabetes, 4 of 14 (29 %) enrolled patients who were in PP at baseline; 8 of 14 (57 %) included mixed populations whose CV risk was either PP or SP; and 2 of 14 (14 %) were clinical trials in SP only. Lipid values at baseline were (mg/dL): 200 (19) [TC]; 118 (16) [LDL-C]; 46 (6) [HDL-C]; 154 (19) [non-HDL-C]; and 165 (32) [TG] (Table studies focusing on diabetes investigated the following interventions over a mean (1SD) duration of 4.8 (2.7) years [BSR: 1.8–13.3 years]: statins ; fibrates ; n-3 fatty acids and/or traditional Mediterranean diet ; PPAR-γ agonist ; and PPAR-α/γ agonist   %/year [BSR 0.5–11.8] (comparator) and 3.1 (1.8) %/year [BSR 0.3–7.5] (treatment), respectively.Among the 33 comparator) and 4.0 (2.1) %/year [BSR 0.7–7.8] (treatment), respectively.Amongst the 9 DSS, a total of 1,469 and 1,119 events occurred in the comparator and treatment arms, respectively. On an annual basis, this was equivalent to an average rate of occurrence for the primary CV outcome of 6.1 (3.0) %/year [BSR 2.1–10.8]  %/year [BSR 1.1–9.6] (comparator) and 2.9 (2.4) %/year [BSR 0.8–9.1] (treatment), respectively.Among the 14 de facto CHD, we also examined CHD rate as a separate outcome [Table 1left panels]. Rates of CHD were issued for 21 trials and DSS for comparator and treatment arms, and amounted to [%/year]: 11.1 and 7.2 [4S-DSS]; 1.3 and 0.9 [AFCAPS/TexCAPS]; 1.5 and 1.0 [ASCOT-LLA]; 5.1 and 4.9 [AURORA]; 5.8 and 5.4 [BIP]; 12.0 and 9.3 [CARE-DSS]; 4.9 and 4.5 [CDP (clofibrate)]; 4.9 and 4.1 [CDP (niacin)]; 2.4 and 1.7 [HPS - MRC/BHF]; 2.6 and 2.0 [HPS - MRC/BHF-DSS]; 1.4 and 1.3 [HPS2-THRIVE]; 5.0 and 4.2 [IDEAL]; 2.0 and 2.4 [ILLUMINATE]; 0.8 and 0.6 [JELIS]; 3.1 and 2.5 [LEADER]; 0.5 and 0.3 [MEGA]; 1.0 and 0.9 [SHARP]; 4.3 and 4.0 [STABILITY]; 1.7 and 1.4 [TNT]; 2.6 and 2.1 [TNT-DSS]; and 1.9 and 1.7 [VA Cooperative Study] .In addition to PO rates, which include predominantly non-diabetic studies, including where appropriate the rates for the corresponding DSS, ie 259,151 patients. Both for PO and CHD, there was a highly significant linear relationship between the proportion of diabetics enrolled and events rates, both in comparator arms  and active arms . When comparing the slopes of the equations between PO and the proportion of diabetes at baseline in the comparator arm of studies published < 2005 and from 2005 to 2014, they rose from 0.0129 to 0.0162, ie a relative increase of 26 % (not shown). Such relationships were more pronounced as regards CHD events, exhibiting steeper gradients than those of PO rates, with slope coefficients higher by a relative 78 % [comparator arms] and 110 % [treatment arms]. Vis-à-vis the comparator arms, the slopes of the relationships between proportions of diabetics and events rates in the treatment arms of the same studies were attenuated, by a relative 45 % [PO rates] and 34 % [CHD events] .The relationship between proportion of diabetic patients at inclusion and PO or CHD rates was inferred on the basis of the comparator and treatment arms data from the 33 comparator arms showed that the proportion of diabetics at inclusion predicted PO rates ranging from 3.12 %/year (no diabetic included) to 6.11 %/year . Predicted CHD rates depending on baseline diabetes prevalence ranged from 1.54 %/year (no diabetic included) to 6.85 %/year . This implies that a cohort exclusively composed of diabetic patients would present a PO rate already increased by an absolute 3 %/year due to the mere fact of being diabetic at baseline. Such an out-of-hand absolute increase in events rate due to the diabetic state would further increase to 5.3 %/year when it comes to the risk of incident CHD .Computing occurrence rates of PO and CHD in the treatment arms, it appears that the proportion of diabetics at inclusion predicts PO rates ranging from 2.65 %/year (no diabetic included) to 4.31 %/year . Predicted CHD rates based on diabetes prevalence ranged from 1.64 %/year (no diabetic included) to 5.13 %/year . It follows that a cohort exclusively composed of diabetic patients would present an on-treatment PO rate increased by an absolute 1.7 %/year solely due to the presence of DM at baseline. Such an absolute increase in events rate due to diabetes would further increase to 3.5 %/year for incident CHD risk .By relating incidence rates of PO and CHD in the upper and lower panels).The comparison of these equations linking the proportion of diabetics and outcome rates in comparator vs. treatment arms allows for determining whether being diabetic (apart from the observation that it increases the absolute rate of occurrence of CV events) is associated with an idiosyncratic on-treatment clinical response. As for PO and CHD, diabetic patients were characterized by a clinical response that was better than that calculated for a non-diabetic population that would have been subject to the same therapeutic interventions. Thus, residual CV risk persisting after treatment was further reduced in case of diabetes, in a relative proportion of 14.4 % [PO] and 31.2 % [CHD], respectively  than the risk that would be determined for diabetics if included, as a subgroup, in a clinical trial not focusing on diabetes. This follows from the fact that studies focusing on diabetes had a lower CV risk at inclusion, as well as lesser PO or CHD events during the study. As a result, the impact of DM on CV events must be qualified according to whether it is evaluated from diabetic subgroups of cohorts followed in cardiology (mostly in a macrovascular setting), or whether it is obtained in patients from clinical trials focusing on nutrition or diabetes . In addition, variation in residual risk related to T2DM in key trials may result from inhomogeneity in inclusion criteria; varying baseline CV risk; individual differences in diabetes duration or severity; and heterogeneous RFs exposure among diabetics.It should be noted that mean PO rate in i) at the onset of diabetes  of the hyperbolic product between insulin secretion and insulin sensitivity), and/or (ii) because they embody cardiometabolic comorbidities that increase the macrovascular risk regardless of glucose levels.As opposed to what occurs in microvessels, and unlike a widely held view about it, residual risk targeting large vessels is related to a limited extent only by hyperglycaemia in (pre)diabetes states. Rather, the accrued macrovascular risk is associated with the common form of T2DM . The common pathogenic factors underlying the observed association between hyperglycemia and CHD are involved either (i) that the presence of diabetes at baseline has less adverse effect on the occurrence of certain constituents of the PO, such as all-cause deaths or coronary revascularization; and (ii) that diabetic patients derive more benefits from the different treatment approaches studied than non-diabetic patients as regards the occurrence of macrovascular events [placebo or standard care. When comparing less recent (published <2005) and more contemporary studies (published ≥2005), a decrease in absolute and relative events rates was observed (-28 % and -1 % respectively), suggestive of a reduction in exposure to CV RFs over time and/or of improved overall CV management. Such changes were however not significant and further, diabetic patients benefited less from this trend, reducing the absolute and relative rates by only -14 % and -0.7 %. It seemed therefore appropriate to include all studies in this analysis regardless of publication year.It should be noted that the slopes of the relationships between CV events and percentage of included diabetics were less marked when it came to comparing PO vs. CHD events rates, both in comparator and treatment arms, on one hand, or when it came to comparing PO or CHD events rates in treated arms vs. comparator arms, on the other hand. These observations suggest (r events . In thisIt is noteworthy that the increased risk of CV events due to the presence of a subgroup of diabetics had a pretty similar slope, whatever the CV risk category at baseline. It follows that the excess CV risk associated with the inclusion of people with diabetes in a lipid-modifying trial is relatively independent of study design, expanding the applicability of equations derived from this meta-analysis. There exists a positive relationship between biomarkers and occurrence of CV events ; our metvs. T2DM subgroups, or to newly-diagnosed vs. long-standing T2DM patients [1c values [This study has several limitations. Firstly, the risk estimates attributed to DM were not adjusted for age or other CV RFs comorbid to T2DM and, as in all systematic collection of published data, there is always a potential bias related to publications . Secondlpatients . We werec values . Finallyc values .This study attempted to quantify the impact of diabetes on the occurrence of CV events during a lipid-modifying trial, based on the proportion of known diabetics included. The component of absolute and relative residual CV risk associated with diabetes can be measured from linear equations relating diabetes prevalence to primary outcomes or CHD rates. Such calculations may help clinical study designers when selecting inclusion criteria; cohort size; and planned diabetics’ enrollment, so as to achieve sufficient CV events over time.


Although all these kinases serve as signal transducing proteins by phosphorylating different sites in cardiomyocytes, some of their effects are cardioprotective whereas others are detrimental. Such opposing effects of each signal transduction pathway seem to depend upon the duration and intensity of stimulus as well as the type of kinase isoform for each kinase. In view of the fact that most of these kinases are activated in heart disease and their inhibition has been shown to improve cardiac function, it is suggested that these kinases form excellent targets for drug development for therapy of heart disease.Protein kinases are intimately integrated in different signal transduction pathways for the regulation of cardiac function in both health and disease. Protein kinase A (PKA), Ca Due to their integrative role of phosphorylating specific proteins essential for modifying different biological processes, protein kinases are involved in a number of signal transduction mechanisms ,2. In th2+-calmodulin-dependent protein kinase (CaMK), protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and mitogen-activation protein kinase (MAPK), in cardioprotection against ischemia-reperfusion (I/R) injury as well as in a number of cardiac diseases. It is also planned to discuss the participation of protein kinase-mediated signal transduction mechanisms in the development of cardiac dysfunction due to cardiac hypertrophy, heart failure and cardiomyopathies (This review is intended to focus on the currently known roles of protein kinase A (PKA), Caopathies . Further2C2 complex to dissociate and release the active C monomers [2+ channels and the SR Ca2+ pump regulatory protein PLB. It has shown to be responsive to β-adrenoceptor (β-AR) agonists, catecholamines, for stimulation in cardiomyocytes causing an elevation of intracellular Ca2+ concentration [2-AR transgene increases ventricular function in vivo, and modulates cardiomyocyte gene expression by phosphorylating its cAMP-response element binding protein transcription factor [2+ affinity of the SR Ca2+ pump and calcium current ICa-triggered Ca2+-induced Ca2+-release from the SR Ca2+ stores [2+ pump ATPase in heart failure (HF), PKA-PLB interaction is considered to be a target for drug development for the treatment of HF [2+-handling by PLB, PKA has also been reported to activate RyR2. Although phosphorylation of RyR2 occurs at Ser2030 and Ser2808 [2+ handling and cardiac arrhythmia [2+ leakage is also increased as a consequence of an elevation in PKA-dependent RyR phosphorylation [2+ from SR, which is partially influenced by the PKA-mediated phosphorylation of RyR2 [2+ overload due to excessive Ca2+ release from SR in this setting [PKA is a tetrameric holoenzyme with two catalytic (C) and two regulatory (R) subunits whose activity is dependent on the concentration of cyclic AMP (cAMP) ,8. Thesemonomers . PKA hasmonomers ,12,13,14ntration ,16,17,18n factor ,20. Phos+ stores ,22,23. Snt of HF . In addirhythmia . After βrylation . It shou of RyR2 . This ph setting . However setting  and thus2+ sensitivity of actomyosin ATPase leading to increased cross-bridge cycling and accelerated relaxation [2+ response has been observed to increase HF as a result in the decrease of cTn1 phosphorylation [PKA plays an integral part in phosphorylating myosin and has been shown to change filament orientation and influence contractile mechanics . It has laxation ,37,38,39laxation . The myorylation ,40. It i2+ channel activity, and sensitivity of contractile proteins to Ca2+ [2+ homeostasis [in vivo; this was blunted by the intracoronary administration of PKA inhibitor H89 [The inhibition of PKA has been shown to be beneficial in different studies in which PKA inhibitors, including H89 and KT5720, were observed to decrease infarct size due to myocardial infarction and prevent complications responsible for HF ,42,43. K to Ca2+ ,54,55,56itor H89 . Reducing cell apoptosis may provide a possible therapy for preventing cardiomyocyte death because PKA inhibition decreased the extent of cell death occurring in I/R and HF ,62,63,642+-binding protein. CaMKII is a highly conserved Ser/Thr protein kinase encoded by four genes α, β, γ, and δ [2+-calmodulin for a transient period, the resulting oligomerization of its subunits is associated with a prolonged period of activation [2+ sensitivity [2+ release channels or RyRs, PLB, and SL L-type Ca2+ channels [2+ regulatory proteins subsequently influencing cardiac function [2+ is initially necessary to activate CaMKII, but Ca2+ is not required for prolonged activation which may have a potentially detrimental effect on cardiac function. Prolonged activation of Ca2+ handling proteins by CaMKII due to β-AR stimulation has been reported to induce hypertrophic gene expression and apoptosis [2+concentrations which imply that CaMKII may become activated at very low Ca2+ levels when exposed to ROS [CaMK is another protein kinase which is activated by calcium and calmodulin, a Caγ, and δ ,71. Althtivation ,72,73. Isitivity . CaMKII channels ,76,77 anfunction . Howeverpoptosis ,80,81,82d to ROS . Taken tin vivo [2+-handling [2+ leakage [in vivo, to be involved with pathological cardiac remodeling in a pressure-overload mouse model [et al. [2+ leakage. The enhancement of RyR Ca2+ release could possibly lead to increased chances of ventricular arrhythmia [As with PKA, there have been numerous studies showing both the beneficial and detrimental effects of inhibiting CaMKII in cardiac disease. CaMKII activation may trigger lethal ventricular arrhythmias with increased oxidative stress ,85. Inhiin vivo ,87, as whandling . CaMKII  leakage . It has  leakage , which ise model . It is tse model , implyin [et al.  demonstr [et al. . Overexp [et al. . Acute C [et al. , has alsrhythmia . This derhythmia . These o2+-induced ASK-1 phosphorylation [in vivo [CaMKII δ3 has also been shown to induce cardiomyocyte hypertrophy via the apoptosis signal-regulating kinase (ASK1) in mammalian cells, where KN93 prevented Carylation . These d[in vivo ,98. Over[in vivo . On the [in vivo ,100. Inh[in vivo . Despite[in vivo  suggesti2+-activated, phospholipid-dependent enzyme that consists of 12 distinct isoforms, each containing a highly conserved carboxyl terminal kinase domain that includes an ATP-binding site [2+-dependent active form [2+ and consist of the α, βI, βII, and γ isoforms. nPKCs are activated by DAG independently of Ca2+ and consist of the δ, ε, η and θ isoforms. aPKCs are activated by phosphoinositide-dependent kinase-1, independently of Ca2+, and consist of the ζ and λ isoforms [2+-independent activity, illustrating association with subcellular alterations and cardiac dysfunction in the diabetic heart [PKC is a Caing site . In the ing site ,104, witing site . Inactivive form . There aisoforms ,108,109.isoforms . The PKC1,2 and PKC-ε, during the development of left ventricular hypertrophy induced by pressure overload occurs in membrane and nuclear-cytoskeletal fractions [2+ overload and development of cardiac dysfunction and HF are depicted in Various hormones are known to induce cardiac hypertrophy through the involvement of PKC activation and associated signal transduction mechanisms . Isozymeractions . Anotherractions . PKC-ε hractions ,115,116.ractions . The sig2+-handling in cardiomyocytes [et al. [2+ transients and SR Ca2+ load and were less susceptible to HF, whereas the PKC-β/γ -/- mice displayed severe HF. When treated with ruboxistaurin, there was increased contractility in PKC-β/γ -/- mice, but not PKC-α -/- mice [in vivo. In addition, it was demonstrated that PKC-α-specific inhibitor Ro-318110 augmented cardiac contractility and restored pump function in the lim-/- mouse model of HF [2+ accumulation when administered at the beginning of reperfusion and attenuated the post-I/R cardiac contractile dysfunction [PKC inhibition has been shown to be beneficial in a number of ways, including preventing diabetes-induced abnormal cardiomyocyte mechanics  and enhamyocytes ,121,122.myocytes ,123,124.myocytes . Aldostemyocytes . Ruboxismyocytes . Our labmyocytes . In wildmyocytes . Further [et al.  demonstr-/- mice ; these sel of HF . Gö 6983function ,131. Whefunction . PKC-ζ ifunction . et al. [Not only must one consider the variety of isoforms of PKC when targeting it for inhibition, but a study by Hahn et al.  has showet al. . Althouget al. . Additioet al. . Althouget al. . Inhibitet al. . In the et al. . Thus a c release for preventing cell death [in vitro and in vivo, emphasizing the need for selective PKC inhibitors [By far the most significant effect of PKC activation, particularly PKC-ε, is its involvement with cardioprotection from I/R injury . Activatll death . Fibroblll death . PKC-α all death . Althoughibitors . As discin vivo [PI3K is a heterodimeric complex of p85 and p110 proteins that contain two SH2 domains, thought to function as adaptor motifs linking tyrosine kinases to their substrates ,151,152.in vivo . The catin vivo . All PI3in vivo ,156. Difin vivo ,158. To Inhibition of PI3K was demonstrated to be beneficial in a variety of cardiovascular diseases. It has been shown to reduce ischemia-induced arrhythmias  and inflInhibition of PI3K could also be detrimental to cardiac function. After ischemic reperfusion in isolated rat hearts, upregulation of G protein-coupled estrogen receptor by G-1 was observed to improve the cardiac function recovery and reduce infarct size, where beneficial effects were abolished upon treatment with PI3K inhibitor, wortmannin . Wortman2+ channels in cardiac myocytes localized on the cell surface. Myocytes were found to have contractile defects in vivo when lacking PI3K subunit p110 α [An increase in PI3K activity has also been demonstrated to be effective in reducing atrial fibrosis and improving cardiac conduction in dilated cardiomyopathy transgenic mice. The reduction in PI3K activity, specifically the regulatory subunit β 110 α, was found to increase the susceptibility to atrial fibrillation in patients . It is at p110 α . Increast p110 α . Anothert p110 α  suggestit p110 α . The prot p110 α . Complett p110 α  indicatiThe PI3K/Akt pathway is another protein kinase signal transduction mechanism which is involved in cardioprotection. Heat shock protein 90, shown to be protective against cellular stress prevented apoptosis partially mediated by the PI3K/Akt pathway in cardiomyocytes subjected to hypoxia . Additioet al. [Another study showed that chronic activation of Akt could potentiate myocardial complications, however restoration of PI3K after I/R injury rescued left ventricular function and decreased the extent of injury. This illustrates the possibility that PI3K-dependent and Akt-independent effects are required for more complete cardioprotection . In bothet al.  found thThus, it is evident that the PI3K/Akt pathway is intimately involved in cardioprotection and the use of its inhibitors may produce detrimental effects on cardiac function. It should be noted that the Akt isoforms 1 and 2 are targets of PI3K and demonstrate similar substrate specificities, however Akt1 has been shown to be essential for the physiological growth of the heart whereas Akt2 is essential for maintaining normal glucose metabolism ,190,191.N-terminal kinases (JNK1-3), p38 MAPK, and ERK5. They share an overall 60–70% similarity with variations present between their activation loop sequences and sizes [2+ depletion and Ca2+ repletion has been associated with cardiac dysfunction and apoptosis; these changes were attenuated by pretreatment with taurine [MAPK is a family of proline-direct serine/threonine specific protein kinases. There are four distinct families of kinases: extracellular signal regulated kinases (ERK1 and 2), c-jun nd sizes . Activat taurine . The act taurine . The inh taurine ,201,202. taurine . In spon taurine . Selecti taurine . Treatme taurine . It has  taurine . However taurine . This ma taurine . An incr taurine . In a st taurine . A highl taurine . p38 MAPK has been shown to be involved with the expression of pro-inflammatory cytokines and inflammatory responses responsible for cardiac dysfunction development . Inhibit1/A2A (AMP579) has been shown to reduce myocardial stunning by activation of the p38 MAPK pathway [In view of both detrimental and beneficial effects of different types of MAPK, a great deal of caution should be exercised while using various inhibitors for these targets. Activation of ERK1/2 after pressure overload has been shown to promote cardiomyocyte survival . Since o pathway ,222 wher pathway . When p3 pathway . A const pathway . p38 MAP pathway . Direct  pathway . An inte pathway .in vivo [+ production, increase glycogen synthesis, and improve the recovery of LV work [2+ uptake in cardiomyocytes and reduced Ca2+ release in SR caused by hypoxic preconditioning [et al. [MAPK has been observed to be intimately involved in cardioprotection pathways. In particular, high levels of p38 MAPK activity have been linked with cardioprotective effects during sustained ischemia . Increasin vivo . When inin vivo . Nonethe LV work . Inhibititioning . Anotheritioning . Thus p3itioning . Activatitioning . Selectiitioning . Timing  [et al.  found th [et al. . This obFrom the foregoing discussion, it is evident that various protein kinases are involved in different signal transduction pathways in cardiomyocytes. These enzymes affect cardiac function by phosphorylating different sites engaged in the regulation of cation transport, cellular metabolism, cardiac growth and apoptosis, as well as contractile activity  236,237,239.A wide variety of pharmacologic agents have been used as experimental tools for inhibiting various protein kinases  suggestiCaMKII inhibition is effective in suppressing arrhythmias, as well as reducing hypertrophic response upon inhibition of the CaMKII δC isoform indicating that inhibition of CaMKII, particularly the δC isoform, would reduce cardiac dysfunction ,92,93. H


Biliary tract carcinoma is a rare malignancy with multiple causes, which underlie the different genetic and molecular profiles. Cancer cell lines are affordable models, reflecting the characteristics of the tumor of origin. They represent useful tools to identify molecular targets for treatment. Here, we established and characterized from biological, molecular, and genetic point of view, an Italian intrahepatic cholangiocarcinoma cell line (ICC), the MT-CHC01. MT-CHC01 cells were isolated from a tumor-derived xenograft. Immunophenotypical characterization was evaluated both at early and after stabilization passages. In vitro biological, genetic, and molecular features were also investigated. In vivo tumorigenicity was assessed in NOD/SCID mice. MT-CHC01cells retain epithelial cell markers, EPCAM, CK7, and CK19, and some stemness and pluripotency markers, i.e., SOX2, Nanog, CD49f/integrin-α6, CD24, PDX1, FOXA2, and CD133. They grow as a monolayer, with a population double time of about 40 h; they show a low migration and invasion potential. In low attachment conditions, they are able to form spheres and to growth in anchorage-independent manner. After subcutaneous injection, they retain in vivo tumorigenicity; the expression of biliary markers as CA19-9 and CEA were maintained from primary tumor. The karyotype is highly complex, with a hypotriploid to hypertriploid modal number (3n+/−) (52 to 77 chromosomes); low level of HER2 gene amplification, TP53 deletion, gain of AURKA were identified; K-RAS G12D mutation were maintained from primary tumor to MT-CHC01 cells. We established the first ICC cell line derived from an Italian patient. It will help to study either the biology of this tumor or to test drugs both in vitro and in vivo.The online version of this article (doi:10.1007/s13277-015-4215-3) contains supplementary material, which is available to authorized users. Biliary tract carcinoma (BTC) is a malignant neoplasm derived from cholangiocytes in different tracts of biliary tree. Recent studies suggest that BTC could originate from a combination of cholangiocytes, peribiliary gland around bile duct, and progenitor of hepatocytes or hepatocytes. It can be classified into intrahepatic cholangiocarcinoma (ICC), originating from the bile ducts within the liver, and extrahepatic cholangiocarcinoma (ECC) arising from bile duct outside the liver; ECC can be further classified as perihilar and distal BTC .Incidence and mortality rates from BTC, including ICC (10 % of primary liver cancers in Western countries), are increasing worldwide –5. PatieOpisthorchis viverrini, and Clonorchis sinensis are common in Thailand, Vietnam and Laos [The lack of effective therapies against this tumor prompts to investigate its molecular pathogenesis. Literature data extensively described risk factor promoting BTC; in particular, the etiology is different according to geography and ethnicity: infestation of the liver flukes, and Laos ; hepatoland Laos . Other pand Laos –12. AltoTo better characterize BTC, cancer cell lines represent an important and indispensable tool. By 1980s, a number of BTC cell lines, in particular originating from ECC, has been established and reported in the literature, from Japanese, Korean, Thai, or Chinese patients –32. OnlyIn this work, we describe a human Italian ICC cell line obtained from a patient-derived xenograft (PDX) . This ceThe PDX was obtained from a tumor sample of a 60-year-old Italian woman who underwent surgical resection for ICC. Biological material was obtained from patient who has signed the informed consent, following institutional review board-approved protocols . This institutional study provides molecular genetic analysis, set up of primary cultures and the creation of PDX from tumor biological samples . The tumor was histopathologically classified as pT2b pN0, moderately differentiated (G2) intrahepatic bile duct carcinoma. Tumor sample was also evaluated for the presence of HBV or HCV markers, resulting negative. The primitive tumor was associated with chronic cholecystitis, but not associated with liver cirrhosis or chronic liver disease, primary sclerosing cholangitis, diabetes, obesity, alcohol consumption, and tobacco smoking.3, tumor was explanted and re-implanted in new mice for a second generation. The stabilization was obtained in fourth generation. To obtain the cell line, tumor specimen derived from fourth generation of PDX, was enzymatically digested with collagenase (200 U/ml)  for 3 h at 37 °C. Collagenase was inactivated by two washes in fetal bovine serum (FBS) . Single-cell suspension was obtained by filtering the supernatant through a 70-μm cell strainer . Cells were finally re-suspended in three different culture conditions: complete DMEM, RPMI, and Knockout/DMEM/F-12 media at a cell density of 300,000/mL in six-well tissue culture plates at 37 °C in a humidified atmosphere of 95 % air and 5 % CO2. Media were replaced twice a week. When cells reached 70–80 % of confluence, they were propagated in the optimal culture condition (complete Knockout/DMEM/F-12 medium). The stable cell line was named MT-CHC01.For PDX establishment, non-obese diabetic (NOD)/Shi-severe combined immunodeficient (SCID) female mice (4–6 weeks old) (Charles River Laboratory) were maintained under sterile conditions in micro-isolator cages at the animal facilities of the IRCCS-Candiolo. All animal procedures were approved by the Institutional Ethical Committee for Animal Experimentation (Fondazione Piemontese per la Ricerca sul Cancro) and by the Italian Ministry of Health. Mice were subcutaneously (s.c.) grafted with a fragment of 4 × 4 mm of representative primary tumor. After 4 months, the primary tumor was successfully engrafted in mice at first generation and was named CHC001 PDX; after reaching a volume of about 200 mmThe ICC cell line HuH28  was cultured in RPMI 1640 containing 10 % FBS , 100 U/mL penicillin and 100 μg/mL streptomycin . The extrahepatic cholangiocarcinoma (ECC) WITT cells  were cultured in DMEM (Sigma-Aldrich) 10 % FBS. The authentication of all the cell lines was performed by using Cell_ID system (Promega) comparing their profile with those published on the DMSZ database.The immunophenotype of MT-CHC01 cells was determined by flow cytometric analysis. Cells were washed in 1× PBS containing 0.1 % bovine serum albumin  and 0.01 % sodium-azide. For cell permeabilization, when requested, the Fix and Perm reagent  was used following the manufacturer’s instructions. The following antibodies were used: fluorescein isothiocyanate (FITC)-conjugated mouse anti-CK7 and anti-CK19  and CD44 (BD Bioscience Europe), allophycocyanin (APC)-conjugated mouse mAbs anti-EPCAM (BD), anti-CD34 and anti-CD133 , phycoerytrin-conjugated (PE) anti-CD24, anti-CXCR4, anti-Oct3/4, anti-FOXA1/2, anti-PDX1 , and anti-CD338 , PerCP-Cy 5.5-conjugated anti-SOX2/17, Alexafluor 647-conjugated anti-Nanog and anti-Stro1 (BD), and Alexafluor 488-conjugated anti-PAX6 (BD).The presence of mycoplasma DNA was tested by the PCR kit VenorGeM (Minerva Biolabs) following the manufacturer’s instructions. The primers are specific to the highly conserved 16S rRNA coding region in the mycoplasma genome. Detection requires 1–5 fg of mycoplasma DNA.5 cells were plated in 24-well plates in triplicate in optimal medium. Viable cells were counted at 24, 48, and 72 h after seeding. The average number of cells was calculated in three different experiments.The population doubling time at passage 25 was determined in hemacytometer chamber by staining with trypan-blue dye. Briefly, 1.4 × 10To calculate the population doubling time (DT), we used the following formula: DT = T ln2 / ln(Xe/Xb), in which T is the time duration of culture, Xe is the cell number at the end of the incubation time and Xb is the cell number at the beginning of the incubation time.w/v) soft agar layered over 0.3 % (w/v) base agar in 24-well plates in quadruplicate. Complete medium was added twice a week for the entire period of incubation (3 weeks).Anchorage-independent growth was assessed by colony-formation assay in soft agar culture. Briefly, 100,000 cells were suspended in 0.5 ml 0.3 % . Sphere formation was monitored on days 7, 10, and 14 after seeding.MT-CHC01 cells were seeded onto six-well plates (ultra-low attachment surface) at 1.5 × 10Motility was performed by wound-healing assay and by 8.0-μm transwell chambers . For wound-healing assay, cells were seeded in triplicate in six-well tissue culture plates and allowed to grow until 100 % confluence. The cell layer was gently “wounded.” Cell migration toward the scraped area was observed in nine randomly selected microscopic fields for each time point (up to 72 h). Images were acquired with a Leica DM13000B Inverted Microscope (Leica). The gap distance was analyzed using ImageJ software; 0 % represents the time of wound (T0). Wound closure was calculated in three different experiments. The percentage of closure was calculated as 100 − (T/T0) × 100, where T represents the average wound closures at the different time points. Statistical analysis was performed using one-way ANOVA and multiple comparison test (GraphPad software). The assay was performed in three different experiments.2/well, BD Falcon). The upper and lower cultures were separated by an 8-μm pore size poly-vinyl-pyrrolidone-free polycarbonate filters . The experiments were carried out in triplicates.Motility was also performed by transwell chambers assay  of about 20–25 g/each (Charles River Laboratory) were maintained under sterile conditions in micro-isolator cages at the animal facilities of the IRCCS-Candiolo. All animal procedures were approved by the Institutional Ethical Committee for Animal Experimentation (Fondazione Piemontese per la Ricerca sul Cancro) and by the Italian Ministry of Health.6 MT-CHC01 cells in 50 % growth factor-reduced BD Matrigel . Tumor diameters were measured weekly after cell injection up to tumor engraftment until 35 days after injection.In three independent experiments, ten mice were subcutaneously (s.c.) injected into the right flank under anesthesia (mixture of isoflurane and nitrous oxide) with 3.0 × 102O2 to block endogenous peroxidase activity, followed by washing in PBS at pH 7.4. The sections were incubated with the primary antibodies for AFP, CEA, CA19-9  for 30 min. Rabbit anti-human polyclonal antibody was used for AFP and CEA, and mouse anti-human monoclonal antibody was used for CA19-9. Subsequently, sections were incubated with biotinylated antibody and peroxidase-labeled streptavidin. Staining was completed after incubation with the freshly prepared substrate–chromogen solution of 3,3′-diaminobenzidine (DAB). Finally, sections were counterstained with Meyer’s hematoxylin.The expression of CA19-9, alpha fetoprotein (AFP), and carcinoembryonic antigen (CEA) was evaluated. Tissues derived from primary tumor, PDX, MT-CHC01 cell line, and its xenograft were fixed in 10 % buffered formalin and embedded in paraffin. The sections (4 μm thick) were incubated in 0.01 mol/L citrate solution (pH 6) for 5 min three times at intervals of 20 min; then, the sections were soaked in 3 % HEstablished cell line was subjected to chromosomal analysis using G banding and Multi-Color FISH (M-FISH). Cells were trypsinized using trypsin/EDTA after colcemid treatment (10 μg/ml) for 1 to 3 h, and slides were prepared according to standard methods. Briefly, cells were incubated in 0.075 M KCl hypotonic solution for 20 min and fixed in methanol-glacial acetic acid (3:1). G banding was performed using 2xSSC at 68 °C for 2 min and Wright’s stain for 2 min. Metaphase images were captured using an Olympus BX61 microscope  and analyzed by CytoVision software . An average banding resolution of 300 bands was achieved. Aberrations were described according to the International System for Human Cytogenetic Nomenclature, 2013 .M-FISH was performed with the aim of identifying complex chromosomal rearrangements. The probe cocktail containing 24 differentially labeled chromosome-specific painting probes  was denatured and hybridized to denatured tumor metaphase chromosomes according to the manufacturer’s protocol for the Human Multicolor FISH kit (MetaSystems). Briefly, slides were incubated at 70 °C in saline solution (2xSSC), denatured in NaOH, dehydrated in ethanol series, air-dried, covered with 10 μl of probe cocktail (denatured) and hybridized for 2 days at 37 °C. The slides were then washed with post-hybridization buffers, dehydrated in ethanol series, and counter-stained with 10 μl of DAPI/antifade. The signal detection and analysis of subsequent metaphases used the Metafer system and Metasytems’ ISIS software .Genomic DNA from MT-CHC01 was extracted by Qiamp DNA mini Kit (Qiagen), and high-resolution oligonucleotide comparative genomic hybridization array (CGH) was performed following standard operating procedures from Agilent Technologies. One thousand nanograms of DNA were digested by a double enzymatic digestion (AluIþRsa I), fragmented, amplified, and purified. After the quantification with NanoDrop, 2 μg of genomic DNA of both tumor DNA and control DNA from Promega  were labeled with CY5-dCTPs and CY3-dCTP, respectively, and hybridized on glass array 2 X105 K at 65 °C for 40 h at 20 rpm. Slides were then washed and scanned on an Agilent 4000C dual laser scanner, and images were analyzed with Feature Extraction v10.5 software. Raw txt files were then loaded into Cytogenomics software for data processing and visualization.AURKA, EGFR, HER2, MET, and TP53 gene status was defined evaluating the ratio between the gene copy number (CN) and the relative centromere number (ratio CN/CEN) and the mean copy number of gene (ratio CN/nuclei). A gene amplification was defined when the ratio CN/CEN was ≥2.0 and a gene/centromere gain when the mean copy number was ≥3.FISH analysis was performed using the following probes: ALK (2p23) Break Apart Rearrangement probe , dual color AURKA (20q13)/CEN20 probe , dual color EGFR (7p12)/CEP7 probe (Vysis), dual color HER2 (17q11.2)/CEP17 , dual color MET (7q31.2)/CEP7 probe (Vysis) and dual color TP53 (17p13.1)/CEP17 (Vysis) and Del(5q) Deletion probe . FISH was carried out on interphase cells prepared according to standard techniques. Cells were incubated with the probe for 5 min at 75 °C for co-denaturation and placed in a humidified chamber at 37 °C overnight for the hybridization step. After washing, chromatin was counterstained with DAPI II (Vysis). An average of 100 cells was analyzed using a Olympus BX61 microscope and CytoVision software. ® SV Gel and PCR Clean-Up System , and sense and antisense sequences were obtained using forward and reverse internal primers respectively. Each exon was sequenced using the BigDye Terminator Cycle sequence following the PE Applied Biosystem strategy and Applied Biosystem ABI PRISM3100 DNA Sequencer . Mutations were confirmed performing two independent rounds of PCR amplifications.Genomic DNA was extracted by using QIAamp DNA FFPE Mini kit  following the manufacturer’s instructions. The kinase domain of EGFR coding sequence, from exons 18 to 21, was amplified by using primers and nested polymerase chain reaction (PCR) conditions previously described by Lynch et al. . Exon 2 For the cell isolation, tumor tissue derived from the PDX at the fourth generation was mechanically and enzymatically dissociated. Cells adhered to the dish 24 h after plating, but only 1 week later, they formed scattered colonies. After 1 month in the optimal cell culture conditions, Knockout/DMEM/F-12 medium in the presence of 10 % FBS and P/S, cells were detached for the first time. At the 25th passage, after about 5 months, a stable cell line was obtained and named MT-CHC01. It appears as a homogeneous culture of tumor cells with epithelial morphologic features , CK7 (98.99 %), and 19 (99.78 %) as shown in Fig. The expression of different stemness and pluripotency markers, already known to be typical of human biliary tree  or other5 cells/cm2, the population doubling time was about 40 h  was monitored for other 4 weeks when tumors reached volumes ranging from 1500 to 2100 mm3 , exon 2 of K-RAS, exons 9 and 20 of PI3KCA, and exon 15 of B-RAF was performed in primary tumor, and in MT-CHC01 cell line. As shown in Fig. To further genetically characterize MT-CHC01 cells, we performed the chromosome G-banding analysis. The cytogenetic analysis on ten metaphases of MT-CHC01 cells demonstrated a highly complex karyotype with a hypotriploid to hypertriploid modal number (3n+/−) (52 to 77 chromosomes). Each chromosome harbored either numerical and/or structural aberrations. M-FISH confirmed that the cell line was near-triploid containing multiple chromosomal aberrations and various marker chromosomes. The composite G-banded and M-FISH karyotype Fig. .Fig. 6Gestatus of ALK (2p23), AURKA (20q13), EGFR (7p12), HER2 (17q11.2), MET (7q31.2), and TP53 (17p13.1) genes with relation to the observed chromosomal aberrations. ALK, EGFR, and MET genes showed a trisomic pattern of signals reflecting the near-triploid chromosomal assessment. As shown in Fig. HER2 gene amplification  was observed, consistent with 17q instability and its involvement in various rearrangements with multiple partner chromosomes. Moreover, a loss of TP53 was present if compared to chromosome 17 centromere (TP53 mean copy number 2.2 vs CEN17 mean copy number 3.1) as a consequence of 17p loss detected in the karyotype. We observed a gain of AURKA gene (mean copy number 5.3) and chromosome 20 centromere (mean copy number 4.1) not detectable in the karyotype, as only three copies of chromosome 20 were always present. Finally, to confirm the deletion on chromosome 5 detected by CGH array, we performed a FISH analysis with a dual color probe specific for 5p15.31 and 5q31.2 regions. As expected, a loss of 5q31.2 region was detected if compared to the copy number of 5p15.31 region (5p15.31 mean copy number 5 vs 5q31.2 mean copy number 4)  Fig. .Biliary tract carcinoma is a complex and heterogeneous disease, characterized by different incidence, etiology, genetic, and molecular profiles. In Italy, BTC shows progressive increase in incidence mainly in intrahepatic cholangiocarcinoma .The importance of identifying preclinical models able to closely reflect the characteristics of tumor of origin is crucial. To date, all models available have limits and consist of cell lines derived from oriental patients. None of these models derived from patient living in Europe and in particular in Italy.In this work, we characterized a preclinical model represented by a cell line obtained from an Italian intrahepatic cholangiocarcinoma PDX.The MT-CHC01 cell line was extensively characterized from biological, molecular, and genetic point of view.The MT-CHC01 cells grew as an adherent monolayer with a population doubling time of approximately 40 h. The morphology was clearly epithelial, and this status was confirmed by immunophenotypic analysis, demonstrating that they expressed epithelial cell markers, EPCAM, CK7, and CK19. In soft agar medium, MT-CHC01 cells revealed the ability to grow in anchorage-independent manner as cellular aggregates; in low attachment and serum-free culture conditions in stem cell medium, MT-CHC01 originated spheroid structures. A peculiar characteristic of cancer cells is their ability to migrate; thus, we investigated the in vitro motility of the MT-CHC01 cells. As demonstrated by wound healing and transwell chamber assays, the MT-CHC01 cells showed a low migration potential compared to other BTC cell lines, reflecting the reduced metastatic potential of this tumor.Immunophenotyping characterization demonstrated that MT-CHC01 cells expressed, at high levels, some of stemness and pluripotency markers such as SOX2, Nanog, CD49f, CD24, PDX1, FOXA2, and CD133. Their expression was increased during cell stabilization; at early passage of culture, the other tested markers, with the exception of CD133, CD49f, and Nanog, were scarcely represented. This could suggest that during cell propagation, a clone selection with putative cancer stem cell phenotype occurred, as demonstrated by Rowehl et al. in a colorectal cancer cell line model . The abiIt is known that tumor cells progressively acquired many genetic and molecular alterations. Cytogenetic analysis of MT-CHC01 cells confirmed a highly complex karyotype with a hypotriploid to hypertriploid modal number (52 to 77 chromosomes), multiple chromosomal aberrations, and various marker chromosomes. With the exception of chromosomes 9 and 10, numerical and structural alterations were observed, in particular in chromosomes 1, 4 (loss), 5, 6 (loss), 16 (gain), and 17 (loss). Only few data are available in literature on cytogenetic of BTC, but most of them, derived from BTC cell lines karyotyping, showed chromosomal aberration in chromosomes 2, 4, 5, 7, 8, 9, 13, 17, 18, 19, X, and Y , 45, 46.ALK, EGFR, and MET genes showed a trisomic pattern of signals, low level of HER2 gene amplification, and a gain of AURKA gene.Further, genetic characterization was focused on mutational status of the hotspot of EGFR (from exons 18 to 21) and its principle signal transducers ; only a mutation of KRAS (G12D) was found. Other genomic analyses revealed loss of TP53, In conclusion, we established a human intrahepatic cholangiocarcinoma cell line, derived from an Italian patient, which could represent a useful tool to better characterize this disease in relation to etiology and ethnicity and to develop new therapies for ICC patients.ESM 1(DOCX 2335 kb)Below is the link to the electronic supplementary material.


All the identified proteins were categorized functionally by Gene Ontology (GO) analysis, and gene-pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, protein–protein interaction networks were also built. The proteomics and bioinformatics data presented here provide novel insights into the molecular characteristics and the early modulation of the TEC proteome in the tumor microenvironment.Increasing evidence indicates that tumor-derived endothelial cells (TECs) are more relevant for the study of tumor angiogenesis and for screening antiangiogenic drugs than normal ECs (NECs). In this data article, high-purity (>98%) primary CD105 Specifications TableValue of the data•D-PAGE from tumor tissues.Highly optimized method for primary ECs proteomic analysis by 2•Bioinformatics data can be useful for clarified the heterogeneity of tumor derived ECs.•The differentially expressed proteins indicate the potential function of the TEC in tumor microenvironment.1+ NECs and TECs proteomic analysis by 2D-PAGE and MALDI-MS/MS was presented here. All the identified proteins were categorized by GO, KEGG and protein–protein interaction analysis, to clarify the function of TEC in tumor microenvironment.The data is related to the identification and verification of transgelin-2 as a potential biomarker of tumor-derived lung-cancer endothelial cells by comparative proteomics 2+ NECs and TECs were isolated from a mouse Lewis lung carcinoma model bearing 0.5 cm tumors. Differentially expressed proteins were identified using 2D-PAGE and Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). 2D-PAGE was performed using the GE Ettan™ IPGphor™ 3 and DALTsix system. Proteins were visualized by silver staining, and images were recorded on a GE ImageScanner III system and analyzed with the ImageMaster 2D Platinum software. Mass spectrometry data were obtained in an automated analysis loop using a 4800 Plus MALDI TOF/TOF™ Analyzer , and collected using the 4000 Series Explorer™ software and submitted to database search via GPS Explorer™ (Applied Biosystems). MASCOT Server version 2.2 and NCBI non-redundant database were used for protein identification. A total of 63 spots resulted in the identification of 48 unique proteins (28 up- and 20 down-regulated proteins) were detected by at least 1.5-fold changes in TECs. All the identified proteins were categorized functionally by Gene Ontology (GO) analysis. Gene-pathway annotations were compiled from Kyoto Encyclopedia of Genes and Genomes (KEGG), BioCarta, BioCyc, and Reactome. Protein–protein interaction networks were built using the DIP, MINI, BioGRID, IntAct, and STRING databases, and the data were imported into Cytoscape in order to visualize the graphs.Primary CD1053Primary ECs were purified by combining the enzymatic digestion, differential adherence and magnetic cell-sorting using a CD105 MultiSort Kit, according to the procedure described in the Journal of Proteomics paper 4g for 40 min at 4 °C and then stored at −80 °C. Protein concentration was determined using a Bradford assay kit. A non-linear pH gradient of 3–10 was chosen for isoelectric focusing (IEF). The second-dimension was performed on a 12.5% SDS-PAGE to optimize the separation of proteins from 12 to 97 kDa. Before IEF, a solution containing 50 mM MgCl2, 1 mg/mL DNase 1, and 0.25 mg/mL RNase A was added to the protein samples at ratio of 1:20 (V/V). Aliquots containing 100 μg of protein were resuspended in 250 μL of rehydration solution. Equal amount of sample was loaded in triplicate. After 18 h of rehydration of the IPG strips , IEF was performed using the GE Ettan™ IPGphor™ 3 system at 67,860 V·h. After focusing, the strips were first equilibrated for 15 min in a buffer containing 6 M urea, 20% glycerol, 2%SDS, 2% DTT, and then for 15 min in the same buffer containing 2.5% iodoacetamide instead of DTT. SDS-PAGE was performed on a GE Ettan™ DALTsix system. Finally, the proteins were visualized by silver staining. Briefly, gels were soaked in fix solution  for at least 45 min, rinsed in 30% ethanol and ddw for 3×10 min, respectively. To sensitize, gels were soaked in sensitivity enhancing solution (2 mL of 10% sodium thiosulfate solution per liter) for 2 min (one gel at a time), followed by rinsed in ddw for 2×1 min. For silver reaction, submerged gel in 0.1% silver stain solution [0.1% silver nitrate with 0.08% formalin (37%)] for 20 min, followed by rinsed in ddw for 2×1 min. Developed image in development solution [2% sodium carbonate with 0.04% formalin (37%)] until desired intensity of staining occur, then quickly washed in 5% acetic acid for 10 min, and rinsed in ddw for 5 min to stop the staining. Finally, all gels were rinsed with water  prior to drying or densitometry.NECs and TECs were harvested and suspended in lysis buffer containing 7 M urea, 4% CHAPS, 2 M thiourea, 60 mM DTT, 10 mM Tris, 1 mM EDTA, 0.002% bromophenol blue, and 2% ampholine (pH 3–10) Images were recorded on a GE ImageScanner III system. The gels were analyzed with the ImageMaster 2D Platinum software, and automatic spot matching in conjunction with detailed manual checking of the spot finding, to identify proteins in both the NECs and TECs. The quality of the gels was verified by using the quality control of the software. Spot intensities were expressed as the percentage of the integrated spot density (volume) over the total density of all measured spots. Significantly over-abundant spots were detected at a significance level of 5% and a fold number of >1.5. After statistical analysis, 63 spots were identified in TECs, compared with NECs, and the histograms in p-value < 0.05%) and a fold number of >1.5.Gels were analyzed with the ImageMaster 2D Platinum software. The quality of the gels was verified by using the quality control of the software. Spot intensities were expressed as the percentage of the integrated spot density (volume) over the total density of all measured spots. Significantly over-abundant spots were detected at a significance level of 5% . Digested peptides were desalted using C18 ZipTips® . MS and MS/MS spectra were collected using the 4000 Series Explorer™ software and submitted to database search via GPS Explorer™ (Applied Biosystems). MASCOT Server version 2.2  and the NCBI non-redundant database were used for protein identification. The search parameters were set as follows: taxonomy: Mouse; mass values, monoisotopic; precursor mass tolerance, ±1 Da; fragment mass tolerance, ±0.3 Da; enzyme, trypsin; maximum missed cleavage allowed, 1; modifications, carbamidomethyl Cys (permanent); methionine oxidation (variable); Ser, Thr, and Tyr phosphorylation. Results were scored using the probability-based MASCOT score. Among these identified proteins, many have been identified in previous cancer studies, including Tagln2, Hspd1, Pgam1, Dld, Cct2, Npm1, Arhgdia, Gdi2, Aprt, Park7, Acy1, Capzb, Ctsb, Hnrnpk, Vcp, Enol, Pkm2, Pcna, Pgk1, and Map2k1, which are list in 6® Green I RT-PCR Master Mix kit from Bio-Rad Laboratories, Inc. on a Rotor-Gene 3000 system. The relative mRNA levels of differentially expressed proteins were normalized to that of GAPDH, and NECs were used for calibration. Primers for selected proteins are listed in D-PAGE analysis  and protein–protein interaction analysis results (3 proteins), the mRNA levels of 23 differentially expressed proteins were analyzed by real-time RT-PCR. Total RNA was extracted using TRIzol. RT-PCR analysis was performed by using the SYBRysis see A.7ftp://ftp.ncbi.nih.gov/pub/HomoloGene/build67/homologene.data.” Then, the molecular functions of the all identified proteins were assigned on the basis of a search against the Human Protein Reference Database . Results including biological process, cellular component, and molecular function were shown in To get a precise prediction, multiple bioinformatics methods were performed. First, the mouse genes thus identified were associated with their putative human orthologs using NCBI׳s HomoloGene resource. Homogene annotations were downloaded from “p<0.05), including 58 BP, 23 MF and 18 CC. The top 10 GO terms ranked according to their significance level were listed in Second, all the identified proteins were categorized functionally by GO analysis. GO was downloaded from the GeneOntology website (geneontology.org/ontology/ geneontology_edit.obo). Corresponding mouse GO-gene annotations were downloaded from the NCBI Entrez Gene ftp website (ftp://ftp.ncbi.nih.gov/gene/DATA/gene2go.gz). The GO analysis results, including the biological process (BP), cellular component (CC), and molecular function (MF), were generated. Gene set enrichment analysis revealed that all the differentially expressed proteins were enriched in 99 GO terms (http://www.biocarta.com/), BioCyc, and Reactome. A hypergeometric test was chosen for statistical analysis, and significantly enriched pathways were identified at a corrected p-value of <0.05. Results were listed in Third, gene-pathway annotations were compiled from Kyoto Encyclopedia of Genes and Genomes (KEGG), BioCarta (http://string-db.org/) databases, and the data were imported into Cytoscape in order to visualize the graphs. The graphs was shown in Forth, protein–protein interaction networks were built using the Database of Interacting Proteins (DIP), Molecular Interaction (MINI), Database of Protein and Genetic Interaction (BioGRID), IntAct molecular interaction (IntAct), and STRING (8Lung squamous cell carcinoma specimens from 30 patients (11 lung squamous cell carcinoma and 19 adenocarcinoma) were chosen for IHC analysis. Histopathology reports were also obtained along with the samples, and shown in