The effects of Phycocyanobilin on experimental arthritis involve the reduction in nociception and synovial neutrophil infiltration, inhibition of cytokine production, and modulation of the neuronal proteome.

Introduction: The antinociceptive and pharmacological activities of C-Phycocyanin (C-PC) and Phycocyanobilin (PCB) in the context of inflammatory arthritis remain unexplored so far. In the present study, we aimed to assess the protective actions of these compounds in an experimental mice model that replicates key aspects of human rheumatoid arthritis. Methods: Antigen-induced arthritis (AIA) was established by intradermal injection of methylated bovine serum albumin in C57BL/6 mice, and one hour before the antigen challenge, either C-PC (2, 4, or 8 mg/kg) or PCB (0.1 or 1 mg/kg) were administered intraperitoneally. Proteome profiling was also conducted on glutamate-exposed SH-SY5Y neuronal cells to evaluate the PCB impact on this key signaling pathway associated with nociceptive neuronal sensitization. Results and discussion: C-PC and PCB notably ameliorated hypernociception, synovial neutrophil infiltration, myeloperoxidase activity, and the periarticular cytokine concentration of IFN-γ, TNF-α, IL-17A, and IL-4 dose-dependently in AIA mice. In addition, 1 mg/kg PCB downregulated the gene expression for T-bet, RORγ, and IFN-γ in the popliteal lymph nodes, accompanied by a significant reduction in the pathological arthritic index of AIA mice. Noteworthy, neuronal proteome analysis revealed that PCB modulated biological processes such as pain, inflammation, and glutamatergic transmission, all of which are involved in arthritic pathology. Conclusions: These findings demonstrate the remarkable efficacy of PCB in alleviating the nociception and inflammation in the AIA mice model and shed new light on mechanisms underlying the PCB modulation of the neuronal proteome. This research work opens a new avenue to explore the translational potential of PCB in developing a therapeutic strategy for inflammation and pain in rheumatoid arthritis.

Differences in maternal and neonatal cardiometabolic markers and placenta status by foetal sex. The GESTAFIT project.

AIMS: To explore the differences in some maternal-neonatal metabolic markers and placenta status by foetal sex. METHODS: One hundred thirty-nine Caucasian pregnant women from the GESTAFIT project and their new-borns were included in the present cross-sectional study. Serum cardiometabolic markers (i.e. lipid and glycaemic profile and uric acid) were analysed at late pregnancy and at birth. In placenta, telomeres length, proportion of deleted mitochondrial-DNA and mitochondrial-DNA density, some minerals and interleukin 8, epidermal growth factor, fibroblast growth factor-2 and vascular endothelial growth factor were measured. The study was run between November 2015 and April 2018. RESULTS: Mothers carrying a male showed higher serum triglycerides than mothers carrying a female at late pregnancy (p < .05). Serum total and low-density lipoprotein cholesterol were greater in males' umbilical cord blood artery compared to females' new-borns (both, p < .05). Mothers of males and male new-borns presented higher uric acid than mothers of females and female new-borns at birth (p < .05). Female's placentas presented greater placental-newborn weight ratio, manganese content and fibroblast growth factor-2 (all, p ⩽ .05), and evidence of statistical significance in telomeres length, which were 17% longer (p = .076). CONCLUSION: Our findings show weak differences in some cardiometabolic and placental status markers by foetal sex. Notwithstanding, we observed a slightly more proatherogenic profile in both, mothers carrying males' foetuses and male new-borns. We also found lower serum uric acid and better placenta status in mothers carrying a female. These findings indicate that foetal sex might need to be considered for a more personalized follow-up of pregnancies.

The Influence of Exercise, Lifestyle Behavior Components, and Physical Fitness on Maternal Weight Gain, Postpartum Weight Retention, and Excessive Gestational Weight Gain.

This study examines (a) the influence of exercise, lifestyle behavior components (sedentary time, physical activity, and sleep and dietary patterns), and physical fitness on maternal weight gain, postpartum weight retention, and excessive gestational weight gain and (b) whether exercise protects against the adverse effects of impaired metabolism and nonoptimal body composition related to excessive gestational weight gain. Subjects were assigned to either a supervised concurrent (aerobic + resistance) exercise program followed 3 days/week (n = 47) or a control group (n = 54). Sedentary time, physical activity, sleep and dietary patterns (assessed by accelerometry and questionnaires), muscle strength (handgrip test), and cardiorespiratory fitness (Bruce test) were determined at gestational Weeks 16 and 33 (early-middle and late pregnancy, respectively), and at 6 weeks postpartum. Weight gain and weight retention were calculated using recorded weights at prepregnancy, early-middle, and late pregnancy, and at 6 weeks postpartum. Birth complications, maternal postpartum body composition, cardiometabolic, and inflammatory markers in maternal and umbilical cord arterial and venous blood, and in colostrum, and mature milk were also recorded. The exercise intervention reduced late weight gain (B = -2.7, SE = 0.83, p = .003) and weight retention (B = -2.85, SE = 1.3, p = .03), independent of any lifestyle behavior component or physical fitness, but did not prevent excessive weight gain. Increasing cardiorespiratory fitness, muscle strength, and sleep duration were associated with a smaller mean weight gain and lower excessive weight gain values (p < .05). Among the participants who experienced excessive weight gain, those who were exercisers had a lower body mass index and systemic tumor necrosis factor-alpha concentration, lower umbilical cord venous tumor necrosis factor-alpha and arterial interferon gamma levels, higher cord arterial interleukin-10 levels, and improved placental function compared with controls (p < .05). In summary, exercise may help optimize gestational weight gain and weight retention, and may attenuate the impaired phenotype related to excessive weight gain. Increasing cardiorespiratory fitness, muscle strength, and sleep duration might help to prevent excessive weight gain during pregnancy.

CIGB-300-Regulated Proteome Reveals Common and Tailored Response Patterns of AML Cells to CK2 Inhibition.

Protein kinase CK2 is a highly pleiotropic and ubiquitously expressed Ser/Thr kinase with instrumental roles in normal and pathological states, including neoplastic phenotype in solid tumor and hematological malignancies. In line with previous reports, CK2 has been suggested as an attractive prognostic marker and molecular target in acute myeloid leukemia (AML), a blood malignant disorder that remains as an unmet medical need. Accordingly, this work investigates the complex landscape of molecular and cellular perturbations supporting the antileukemic effect exerted by CK2 inhibition in AML cells. To identify and functionally characterize the proteomic profile differentially modulated by the CK2 peptide-based inhibitor CIGB-300, we carried out LC-MS/MS and bioinformatic analysis in human cell lines representing two differentiation stages and major AML subtypes. Using this approach, 109 and 129 proteins were identified as significantly modulated in HL-60 and OCI-AML3 cells, respectively. In both proteomic profiles, proteins related to apoptotic cell death, cell cycle progression, and transcriptional/translational processes appeared represented, in agreement with previous results showing the impact of CIGB-300 in AML cell proliferation and viability. Of note, a group of proteins involved in intracellular redox homeostasis was specifically identified in HL-60 cell-regulated proteome, and flow cytometric analysis also confirmed a differential effect of CIGB-300 over reactive oxygen species (ROS) production in AML cells. Thus, oxidative stress might play a relevant role on CIGB-300-induced apoptosis in HL-60 but not in OCI-AML3 cells. Importantly, these findings provide first-hand insights concerning the CIGB-300 antileukemic effect and draw attention to the existence of both common and tailored response patterns triggered by CK2 inhibition in different AML backgrounds, a phenomenon of particular relevance with regard to the pharmacologic blockade of CK2 and personalized medicine.

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis.

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.

Interplay between genetics and lifestyle on pain susceptibility in women with fibromyalgia: the al-Ándalus project.

OBJECTIVES: It is widely acknowledged that the experience of pain is promoted by both genetic susceptibility and environmental factors such as engaging in physical activity (PA), and that pain-related cognitions are also important. Thus, the purpose of the present study was to test the association of 64 polymorphisms (34 candidate genes) and the gene-gene, gene-PA and gene-sedentary behaviour interactions with pain and pain-related cognitions in women with FM. METHODS: Saliva samples from 274 women with FM [mean (s.d.) age 51.7 (7.7) years] were collected for extracting DNA. We measured PA and sedentary behaviour by accelerometers for a week, pain with algometry and questionnaires, and pain-related cognitions with questionnaires. To assess the robustness of the results, a meta-analysis was also performed. RESULTS: The rs6311 and rs6313 polymorphisms (5-hydroxytryptamine receptor 2A, HTR2A) were individually related to algometer scores. The interaction of rs4818 (catechol-O-methyltransferase, COMT) and rs1799971 (opioid receptor µ gene, OPRM1) was related to pain catastrophizing. Five gene-behaviour interactions were significant: the interactions of sedentary behaviour with rs1383914 (adrenoceptor alpha 1A, ADRA1A), rs6860 (charged multivesicular body protein 1A, CHMP1A), rs4680 (COMT), rs165599 (COMT) and rs12994338 (SCN9A) on bodily pain subscale of the Short Form 36. Furthermore, the meta-analysis showed an association between rs4680 (COMT) and severity of FM symptoms (codominant model, P-value 0.032). CONCLUSION: The HTR2A gene (individually), COMT and OPRM1 gene-gene interaction, and the interactions of sedentary behaviour with ADRA1A, CHMP1A, COMT and SCN9A genes were associated with pain-related outcomes. Collectively, findings from the present study indicate a modest contribution of genetics and gene-sedentary behaviour interaction to pain and pain catastrophizing in women with FM. Future research should examine whether reducing sedentary behaviour is particularly beneficial for reducing pain in women with genetic susceptibility to pain.

CIGB-300 Anticancer Peptide Differentially Interacts with CK2 Subunits and Regulates Specific Signaling Mediators in a Highly Sensitive Large Cell Lung Carcinoma Cell Model.

Large cell lung carcinoma (LCLC) is one form of NSCLC that spreads more aggressively than some other forms, and it represents an unmet medical need. Here, we investigated for the first time the effect of the anti-CK2 CIGB-300 peptide in NCI-H460 cells as an LCLC model. NCI-H460 cells were highly sensitive toward CIGB-300 cytotoxicity, reaching a peak of apoptosis at 6 h. Moreover, CIGB-300 slightly impaired the cell cycle of NCI-H460 cells. The CIGB-300 interactomics profile revealed in more than 300 proteins that many of them participated in biological processes relevant in cancer. Interrogation of the CK2 subunits targeting by CIGB-300 indicated the higher binding of the peptide to the CK2α' catalytic subunit by in vivo pull-down assays plus immunoblotting analysis and confocal microscopy. The down-regulation of both phosphorylation and protein levels of the ribonuclear protein S6 (RPS6) was observed 48 h post treatment. Altogether, we have found that NCI-H460 cells are the most CIGB-300-sensitive solid tumor cell line described so far, and also, the findings we provide here uncover novel features linked to CK2 targeting by the CIGB-300 anticancer peptide.

Targeting of Protein Kinase CK2 in Acute Myeloid Leukemia Cells Using the Clinical-Grade Synthetic-Peptide CIGB-300.

Protein kinase CK2 has emerged as an attractive therapeutic target in acute myeloid leukemia (AML), an advent that becomes particularly relevant since the treatment of this hematological neoplasia remains challenging. Here we explored for the first time the effect of the clinical-grade peptide-based CK2 inhibitor CIGB-300 on AML cells proliferation and viability. CIGB-300 internalization and subcellular distribution were also studied, and the role of B23/nucleophosmin 1 (NPM1), a major target for the peptide in solid tumors, was addressed by knock-down in model cell lines. Finally, pull-down experiments and phosphoproteomic analysis were performed to study CIGB-interacting proteins and identify the array of CK2 substrates differentially modulated after treatment with the peptide. Importantly, CIGB-300 elicited a potent anti-proliferative and proapoptotic effect in AML cells, with more than 80% of peptide transduced cells within three minutes. Unlike solid tumor cells, NPM1 did not appear to be a major target for CIGB-300 in AML cells. However, in vivo pull-down experiments and phosphoproteomic analysis evidenced that CIGB-300 targeted the CK2α catalytic subunit, different ribosomal proteins, and inhibited the phosphorylation of a common CK2 substrates array among both AML backgrounds. Remarkably, our results not only provide cellular and molecular insights unveiling the complexity of the CIGB-300 anti-leukemic effect in AML cells but also reinforce the rationale behind the pharmacologic blockade of protein kinase CK2 for AML-targeted therapy.

Circulating PTGS2, JAG1, GUCY2C and PGF mRNA in Peripheral Blood and Serum as Potential Biomarkers for Patients with Metastatic Colon Cancer.

Genes involved in the angiogenic process have been proposed for the diagnosis and therapeutic response of metastatic colorectal cancer (CRC). This study aimed to investigate the value of PTGS2, JAG1, GUCY2C and PGF-circulating RNA as biomarkers in metastatic CRC. Blood cells and serum mRNA from 59 patients with metastatic CRC and 47 healthy controls were analyzed by digital PCR. The area under the receiver operating characteristic curve (AUC) was used to estimate the diagnostic value of each mRNA alone or mRNA combinations. A significant upregulation of the JAG1, PTGS2 and GUCY2C genes in blood cells and serum samples from metastatic CRC patients was detected. Circulating mRNA levels in the serum of all genes were significantly more abundant than in blood. The highest discrimination ability between metastatic CRC patients and healthy donors was obtained with PTGS2 (AUC of 0.984) and GUCY2C (AUC of 0.896) in serum samples. Biomarker combinations did not improve the discriminatory capacity of biomarkers separately. Analyzed biomarkers showed no correlation with overall survival or progression-free survival, but GUCY2C and GUCY2C/PTGS2 expression in serum correlated significantly with the response to antiangiogenic agents. These findings demonstrate that assessment of genes involved in the angiogenic process may be a potential non-invasive diagnostic tool for metastatic CRC and its response to antiangiogenic therapy.

Fatigue in Women with Fibromyalgia: A Gene-Physical Activity Interaction Study.

Fatigue is a cardinal symptom in fibromyalgia. Fatigue is assumed to be the result of genetic susceptibility and environmental factors. We aimed at examining the role of genetic susceptibility for fatigue in southern Spanish women with fibromyalgia, by looking at single nucleotide polymorphisms in 34 fibromyalgia candidate-genes, at the interactions between genes, and at the gene-physical activity interactions. We extracted DNA from saliva of 276 fibromyalgia women to analyze gene-polymorphisms. Accelerometers registered physical activity and sedentary behavior. Fatigue was assessed with the Multidimensional Fatigue Inventory. Based on the Bonferroni's and False Discovery Rate values, we found that the genotype of the rs4453709 polymorphism (sodium channel protein type 9 subunit alpha, SCN9A, gene) was related to reduced motivation (AT carriers showed the highest reduced motivation) and reduced activity (AA carriers showed the lowest reduced activity). Carriers of the heterozygous genotype of the rs1801133 (methylene tetrahydrofolate reductase, MTHFR, gene) or rs4597545 (SCN9A gene) polymorphisms who were physically active reported lower scores on fatigue compared to their inactive counterparts. Highly sedentary carriers of the homozygous genotype of the rs7607967 polymorphism (AA/GG genotype; SCN9A gene) presented more reduced activity (a dimension of fatigue) than those with lower levels of sedentary behavior. Collectively, findings from the present study suggest that the contribution of genetics and gene-physical activity interaction to fatigue in fibromyalgia is modest.

Phosphoproteomic Landscape of AML Cells Treated with the ATP-Competitive CK2 Inhibitor CX-4945.

Casein kinase 2 (CK2) regulates a plethora of proteins with pivotal roles in solid and hematological neoplasia. Particularly, in acute myeloid leukemia (AML) CK2 has been pointed as an attractive therapeutic target and prognostic marker. Here, we explored the impact of CK2 inhibition over the phosphoproteome of two cell lines representing major AML subtypes. Quantitative phosphoproteomic analysis was conducted to evaluate changes in phosphorylation levels after incubation with the ATP-competitive CK2 inhibitor CX-4945. Functional enrichment, network analysis, and database mining were performed to identify biological processes, signaling pathways, and CK2 substrates that are responsive to CX-4945. A total of 273 and 1310 phosphopeptides were found differentially modulated in HL-60 and OCI-AML3 cells, respectively. Despite regulated phosphopeptides belong to proteins involved in multiple biological processes and signaling pathways, most of these perturbations can be explain by direct CK2 inhibition rather than off-target effects. Furthermore, CK2 substrates regulated by CX-4945 are mainly related to mRNA processing, translation, DNA repair, and cell cycle. Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy.

Uncovering Tumour Heterogeneity through PKR and nc886 Analysis in Metastatic Colon Cancer Patients Treated with 5-FU-Based Chemotherapy.

Colorectal cancer treatment has advanced over the past decade. The drug 5-fluorouracil is still used with a wide percentage of patients who do not respond. Therefore, a challenge is the identification of predictive biomarkers. The protein kinase R (PKR also called EIF2AK2) and its regulator, the non-coding pre-mir-nc886, have multiple effects on cells in response to numerous types of stress, including chemotherapy. In this work, we performed an ambispective study with 197 metastatic colon cancer patients with unresectable metastases to determine the relative expression levels of both nc886 and PKR by qPCR, as well as the location of PKR by immunohistochemistry in tumour samples and healthy tissues (plasma and colon epithelium). As primary end point, the expression levels were related to the objective response to first-line chemotherapy following the response evaluation criteria in solid tumours (RECIST) and, as the second end point, with survival at 18 and 36 months. Hierarchical agglomerative clustering was performed to accommodate the heterogeneity and complexity of oncological patients' data. High expression levels of nc886 were related to the response to treatment and allowed to identify clusters of patients. Although the PKR mRNA expression was not associated with chemotherapy response, the absence of PKR location in the nucleolus was correlated with first-line chemotherapy response. Moreover, a relationship between survival and the expression of both PKR and nc886 in healthy tissues was found. Therefore, this work evaluated the best way to analyse the potential biomarkers PKR and nc886 in order to establish clusters of patients depending on the cancer outcomes using algorithms for complex and heterogeneous data.

Targeting the hydrophilic regions of recombinant proteins by MS via in-solution buffer-free trypsin digestion.

A desalting step using reversed phase chromatography is a common practice prior to mass spectrometry analysis of proteolytic digests in spite of the detrimental exclusion of the hydrophilic peptides. The detection of such peptides is also important for the complete coverage of protein sequences and the analysis of posttranslational modifications as inquired by regulatory agencies for the commercialization of biotechnological products. The procedure described here, named in-solution buffer-free digestion, simplifies the sample processing and circumvents the above-mentioned limitations by allowing the detection of tryptic hydrophilic peptides via direct ESI-MS analysis. Two DNA recombinant proteins such as HBcAg (hepatitis B core antigen) and fusion VEGF (vascular endothelial growth factor) were analyzed with the proposed in-solution buffer-free digestion allowing the detection of extremely hydrophilic di-, tri- and tetra-peptides, C-terminal His-tail peptide, as well as disulfide-containing peptides. All these molecular species are hardly seen in mass spectrometric analysis using a standard digestion that includes a C18-desalting step. The procedure was also successfully tried on hydrophilic tetra- and hexa-peptides of Ribonuclease B carrying an N-glycosylation site occupied with "high-mannose" N-glycan chains. The in-solution buffer-free digestion constitutes a simple and straightforward approach to analyse the hydrophilic proteolytic peptides which are commonly elusive to the detection by conventional mass spectrometric analysis.

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation.

Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.

GSTM1 gene expression and copy number variation in prostate cancer patients-Effect of chemical exposures and physical activity.

BACKGROUND: Many etiological factors have been related to prostate cancer (CaP) development, progression, and survival, such as age, population origin, geographic area, occupational exposures, and nutrition and lifestyle factors. However, physical activity affords health benefits to cancer patients, including those with CaP. Glutathione S-Transferases enzymes have been linked to CaP because of their role in the detoxification of a wide variety of potential carcinogens, steroid hormones and xenobiotics. Among the different glutathione S-transferases isoforms, null genotype for GSTM1 has been associated with an increased risk of CaP, although data are controversial. As the relationship between copy number variation and gene expression of GSTM1 in CaP remains unexplored, this study analyzed GSTM1 gene expression and/or dosage effect on CaP risk and aggressiveness. The potential protective role of physical activity was also explored. METHODS: Three hundred and seventeen patients (159 non-CaP and 158 CaP) were recruited from the Service of Urology (Hospital Virgen de las Nieves, Granada, Spain) over the period 2012 to 2014 and were followed-up until January 2018 to ensure a correct classification of control and patients. Individuals were classified in each group based on histological analysis of tissue biopsy, along with data on PSA level, Gleason score and T stage in patients with biopsies positive for CaP. Individuals with a negative biopsy were considered as controls. All controls underwent a systematic 20-core ultrasound guided biopsy in order to limit the false negative rate. Genomic DNA was extracted from peripheral blood to determine the exact copy numbers of GSTM1, and RNA was extracted from prostate tissue samples to determine GSTM1 gene expression. Both analyses were performed using the qPCR method. A questionnaire was administered to all patients to assess environmental exposures, lifestyle, and physical activity. The association of GSTM1 copy number variation and expression with the rest of variables was assessed by chi-square test and the Mann-Whitney test. Multiple logistic regression was used to assess which factors were associated with the risk of CaP. RESULTS: The presence of 1 or 2 copies of the GSTM1 gene was not less prevalent in CaP compared to non-CaP patients; however, a significant decreased GSTM1 gene expression was observed in CaP tissue relative to non-CaP tissue (P = 0.003). CaP patients with environmental exposure to dust and smoke, and smoking habit had a significantly decreased GSTM1 gene expression (and near-significantly decreased for living in urban areas) as compared to non-CaP patients with the same exposures. In addition, physical activity was significantly associated with a lower risk of CaP (P = 0.006) and with increased GSTM1 gene expression (P = 0.002). CONCLUSIONS: A reduced GSTM1 gene expression in prostate tissue was observed in CaP patients with some environmental chemical exposures. Intriguingly, physical activity might play a protective role against CaP development, possibly as a result of increasing GSTM1 gene expression in prostate tissue. However, this observation warrants further confirmation.

Identification of candidate genes associated with fibromyalgia susceptibility in southern Spanish women: the al-Ándalus project.

BACKGROUND: Candidate-gene studies on fibromyalgia susceptibility often include a small number of single nucleotide polymorphisms (SNPs), which is a limitation. Moreover, there is a paucity of evidence in Europe. Therefore, we compared genotype frequencies of candidate SNPs in a well-characterised sample of Spanish women with fibromyalgia and healthy non-fibromyalgia women. METHODS: A total of 314 women with a diagnosis of fibromyalgia (cases) and 112 non-fibromyalgia healthy (controls) women participated in this candidate-gene study. Buccal swabs were collected for DNA extraction. Using TaqMan™ OpenArray™, we analysed 61 SNPs of 33 genes related to fibromyalgia susceptibility, symptoms, or potential mechanisms. RESULTS: We observed that the rs841 and rs1799971 GG genotype was more frequently observed in fibromyalgia than in controls (p = 0.04 and p = 0.02, respectively). The rs2097903 AT/TT genotypes were also more often present in the fibromyalgia participants than in their control peers (p = 0.04). There were no differences for the remaining SNPs. CONCLUSIONS: We identified, for the first time, associations of the rs841 (guanosine triphosphate cyclohydrolase 1 gene) and rs2097903 (catechol-O-methyltransferase gene) SNPs with higher risk of fibromyalgia susceptibility. We also confirmed that the rs1799971 SNP (opioid receptor µ1 gene) might confer genetic risk of fibromyalgia. We did not adjust for multiple comparisons, which would be too stringent and yield to non-significant differences in the genotype frequencies between cases and controls. Our findings may be biologically meaningful and informative, and should be further investigated in other populations. Of particular interest is to replicate the present study in a larger independent sample to confirm or refute our findings. On the other hand, by including 61 SNPs of 33 candidate-genes with a strong rationale (they were previously investigated in relation to fibromyalgia susceptibility, symptoms or potential mechanisms), the present research is the most comprehensive candidate-gene study on fibromyalgia susceptibility to date.

Novel interactions of domain III from the envelope glycoprotein of dengue 2 virus with human plasma proteins.

Blood cells and plasma are important media for the four serotypes of dengue virus (DENV1-4) spreading into an infected person. Thus, interactions with human plasma proteins are expected to be decisive in the course of the viral infection. Affinity purification followed by MS analysis (AP/MS) was used to isolate and identify plasma-derived proteins capable to interact with a recombinant protein comprising the domain III of the envelope protein of DENV2 (DIIIE2). The elution of the AP potently inhibits DENV2 infection. Twenty-nine proteins were identified using a label-free approach as specifically captured by DIIIE2. Of these, a direct interaction with C reactive protein, thrombin and Inter-alpha-inhibitor complexes was confirmed by ELISA. Results provide further evidence of a significant representation of proteins from complement and coagulation cascades on DENV2 interactome in human plasma and stand out the domain III of the viral envelope protein as participant on these interactions. A functional clustering analysis highlights the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. BIOLOGICAL SIGNIFICANCE: Early cycles of dengue virus replication take place in human blood cells. Thus, the characterization of the interactome of dengue virus proteins in human plasma can lead to the identification of pivotal interactions for the infection that can eventually constitute the target for the development of methods to control dengue virus-caused disease. In this work we identified 29 proteins from human plasma that potentially interact with the envelope protein of dengue 2 virus either directly or through co-complex formation. C reactive protein, thrombin and Inter-alpha-inhibitor complexes were validated as interactors of the domain III of the envelope protein of dengue 2. Results highlight the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. This finding together with the participation of domain III of the envelope protein on the interactions with human plasma proteins should contribute to a better understanding of dengue virus interactome in human plasma. Such knowledge can contribute to the development of more effective treatments to infected persons.

Influence of the HER2 Ile655Val polymorphism on trastuzumab-induced cardiotoxicity in HER2-positive breast cancer patients: a meta-analysis.

BACKGROUND: The HER2 655 A>G genetic variant has recently been associated with trastuzumab-induced cardiotoxicity in HER2 breast cancer patients. Considering previous results, the aim of our study was to validate the role of this polymorphism as a predictor of the cardiac toxicity of trastuzumab in breast cancer patients. METHODS: Our study population was composed of 78 HER2 breast cancer patients receiving trastuzumab. The HER2 655 A>G (rs1136201) genetic variant was genotyped using TaqMan allelic discrimination technology. Patients were classified on the basis of the occurrence of cardiotoxic events or the absence of cardiotoxic events during 1 year after the first infusion. RESULTS: The HER2 655 A>G polymorphism was significantly associated with cardiotoxicity: AG versus AA [P=0.012, odds ratio (OR)=5.12, 95% confidence interval (CI) 1.43-18.36], AG+GG versus AA (P=0.01, OR=5.72, 95% CI 1.50-21.76), AG versus AA+GG (P=0.005, OR=7.17, 95% CI 1.82-28.29). A meta-analysis combining these data with the results from previous studies confirmed this association. CONCLUSION: Our results support the role of the HER2 655 A>G polymorphism as a genetic marker of trastuzumab-induced cardiotoxicity in HER2-positive breast cancer patients.

HI-bone: a scoring system for identifying phenylisothiocyanate-derivatized peptides based on precursor mass and high intensity fragment ions.

Peptide sequence matching algorithms used for peptide identification by tandem mass spectrometry (MS/MS) enumerate theoretical peptides from the database, predict their fragment ions, and match them to the experimental MS/MS spectra. Here, we present an approach for scoring MS/MS identifications based on the high mass accuracy matching of precursor ions, the identification of a high intensity b1 fragment ion, and partial sequence tags from phenylthiocarbamoyl-derivatized peptides. This derivatization process boosts the b1 fragment ion signal, which turns it into a powerful feature for peptide identification. We demonstrate the effectiveness of our scoring system by implementing it on a computational tool called "HI-bone" and by identifying mass spectra of an Escherichia coli sample acquired on an Orbitrap Velos instrument using Higher-energy C-trap dissociation. Following this strategy, we identified 1614 peptide spectrum matches with a peptide false discovery rate (FDR) below 1%. These results were significantly higher than those from Mascot and SEQUEST using a similar FDR.