Karina Helena Morais Cardozo

Effects of heavy metals and light levels on the biosynthesis of carotenoids and fatty acids in the macroalgae Gracilaria tenuistipitata (var. liui Zhang & Xia)

We present here the effect of heavy metals and of different light intensities on the biosynthesis of fatty acids and pigments in the macroalga Gracilaria tenuistipitata (var. liui Zhang & Xia). In order to verify the fatty acid content, gas chromatography with flame ionization detection (GC-FID) was employed. Pigments (major carotenoids and chlorophyl-a) were monitored by liquid chromatography with diode array detection (HPLC-DAD). Cultures of G. tenuistipitata were exposed to cadmium (Cd2+, 200 ppb) and copper (Cu2+, 200 ppb), as well as to different light conditions (low light: 100 µmol.photons.m-2.s-1, or high light: 1000 µmol.photons.m-2.s-1). Cd2+ and Cu2+ increased the saturated and monounsaturated fatty acid content [14:0, 16:0, 18:0, 18:1 (n-7) and 18:1 (n-9)] and all major pigments (violaxanthin, antheraxanthin, lutein, zeaxanthin, chlorophyll-a and β-carotene). Both heavy metals decreased the levels of polyunsaturated fatty acids (PUFA) [18:2 (n-6), 18:3 (n-6), 18:5 (n-4), 20:4 (n-6), 20:5 (n-3), 22:6 (n-3)]. G. tenuistipitata cultures were exposed to high light intensity for five days and no statistically significant differences were observed in the content of fatty acids. On the other hand, the levels of pigments rose markedly for chlorophyll-a and all of the carotenoids studied.

Young “Healthy” Smokers Have Functional and Inflammatory Changes in the Nasal and the Lower Airways

BACKGROUND Smoking is responsible for most COPD. Although people with COPD often have concomitant nasal disease, there are few studies that report physiologic or inflammatory changes in the upper airways in young asymptomatic smokers. We investigated physiologic and inflammatory changes in the nasal and lower airways of young smokers and if these changes were related to smoking history. METHODS Seventy-two subjects aged between 18 and 35 years (32 healthy nonsmokers and 40 young smokers) participated in this study. We measured nasal mucociliary clearance (MCC), nasal mucus surface contact angle, cell counts, myeloperoxidase and cytokine concentrations in nasal lavage fluid, exhaled breath condensate (EBC) pH, and lung function. RESULTS Smokers had faster MCC, an increased number of cells (macrophages, ciliated cells, and goblet cells), increased lavage myeloperoxidase concentration, and decreased EBC pH compared with nonsmokers. There was a significant inverse relationship between pack-year smoking history and EBC pH. There were no differences in lung function or mucus surface properties comparing smokers to nonsmokers. CONCLUSIONS Young adult smokers have functional and inflammatory changes in the nasal and lower airways and these correlate with smoking history. However, in these young smokers, smoking history was not associated with pulmonary function decline, probably because it is unlikely that spirometry detects early physiologic changes in the airways. TRIAL REGISTRY ClinicalTrials.gov; No.: NCT01877291; URL: www.clinicaltrials.gov.

Variation of mechanical properties and quantitative proteomics of VSMC along the arterial tree

Vascular smooth muscle cells (VSMCs) are thought to assume a quiescent and homogeneous mechanical behavior after arterial tree development phase. However, VSMCs are known to be molecularly heterogeneous in other aspects and their mechanics may play a role in pathological situations. Our aim was to evaluate VSMCs from different arterial beds in terms of mechanics and proteomics, as well as investigate factors that may influence this phenotype. VSMCs obtained from seven arteries were studied using optical magnetic twisting cytometry (both in static state and after stretching) and shotgun proteomics. VSMC mechanical data were correlated with anatomical parameters and ultrastructural images of their vessels of origin. Femoral, renal, abdominal aorta, carotid, mammary, and thoracic aorta exhibited descending order of stiffness (G, P < 0.001). VSMC mechanical data correlated with the vessel percentage of elastin and amount of surrounding extracellular matrix (ECM), which decreased with the distance from the heart. After 48 h of stretching simulating regional blood flow of elastic arteries, VSMCs exhibited a reduction in basal rigidity. VSMCs from the thoracic aorta expressed a significantly higher amount of proteins related to cytoskeleton structure and organization vs. VSMCs from the femoral artery. VSMCs are heterogeneous in terms of mechanical properties and expression/organization of cytoskeleton proteins along the arterial tree. The mechanical phenotype correlates with the composition of ECM and can be modulated by cyclic stretching imposed on VSMCs by blood flow circumferential stress.

Differences in the seminal plasma proteome are associated with oxidative stress levels in men with normal semen parameters

OBJECTIVE To study the seminal plasma proteome in association with semen lipid peroxidation levels in men with normal semen parameters. DESIGN Cross-sectional study. SETTING University andrology and research laboratories. PATIENT(S) A total of 156 normozoospermic men. INTERVENTION(S) Seminal lipid peroxidation levels were assessed in individual samples through thiobarbituric acid reactive substances quantification. Subsequently, lipid peroxidation data were used to divide the samples into the experimental groups: low lipid peroxidation levels (control group, bottom 15%, n = 23) and high lipid peroxidation levels (study group, top 15%, n = 23). Seminal plasma proteins from these groups were pooled (four pools per group, with biological variation between the pools) and used for a shotgun proteomic analysis using a liquid chromatography-tandem mass spectrometry approach. Quantitative data were used for univariate (unpaired Students t test) and multivariate (partial least-squares discriminant analysis, logistic regression, and discriminant analyses) statistical analyses. Significant proteins were also used for functional enrichment analysis. MAIN OUTCOME MEASURE(S) Seminal plasma protein profile and postgenomic pathways of seminal plasma are associated with seminal lipid peroxidation levels. RESULT(S) In total, 629 proteins were quantified in seminal plasma. Of these, 23 proteins were absent or underexpressed and 71 were exclusive or overexpressed in the study group. The main enriched functions in association with seminal lipid peroxidation were unsaturated fatty acids biosynthesis, oxidants and antioxidants activity, cellular response to heat stress, and immune response. Moreover, we suggested mucin-5B as a potential biomarker of semen oxidative stress. CONCLUSION(S) The seminal plasma proteome does reflect semen lipid peroxidation status and, thus, oxidative stress.

Unbiased label-free quantitative proteomic profiling and enriched proteomic pathways in seminal plasma of adult men before and after varicocelectomy

STUDY QUESTION Does the seminal plasma proteomic profile and functional enrichment of gene ontology terms change after microsurgical varicocelectomy? Are there any potential targets for diagnosis or therapeutic intervention in varicocele? SUMMARY ANSWER A shift in state from a responsive-to-stress condition before varicocele correction to a responsive-to-environment condition after varicocelectomy was observed in enriched proteomic pathways. WHAT IS KNOWN ALREADY Varicocele may lead to many adverse effects, including failure of testicular growth and development, and is associated with decreased semen quality and increased semen oxidative stress. Varicocelectomy is the treatment of choice, and is associated with improved semen quality, but little is known regarding the underlying molecular mechanisms and post-genomic pathways following intervention. STUDY DESIGN, SIZE, DURATION A prospective study was carried out including 18 adult men with varicocele. These patients provided one semen sample before they were submitted for bilateral varicocele repair through microsurgical varicocelectomy, and one other semen sample 90 days after the surgery. PARTICIPANTS/MATERIALS, SETTING, METHODS An aliquot of each semen sample was used for unbiased proteomics analysis by a label-free quantitative approach (2D nanoUPLC-ESI-MS(E)). Samples were pooled according to group (normalized to protein content) and run in quadruplicate. These quadruplicate runs provided degrees of freedom in order to compare groups using a non-parametric Mann-Whitney test for quantified proteins. MAIN RESULTS AND THE ROLE OF CHANCE A total of 316 proteins were quantified or identified, of which 91 were exclusively identified or quantified in one of the groups (53 in the pre- and 38 in the post-varicocelectomy group), and 68 were quantified in both groups and submitted to statistical analysis, of which 5 were overrepresented in the pre-varicocelectomy group (P < 0.05). In enriched functional analysis, binding and response to stimulus functions were enriched in a common cluster (present in both groups), nitric oxide metabolism and tetratricopeptide repeat domain-binding functions were enriched in the pre-varicocelectomy group, and response to reactive oxygen species, gluconeogenesis, nicotinamide adenine dinucleotide-binding and protein stabilization were enriched in the post-varicocelectomy. LIMITATIONS, REASONS FOR CAUTION Because a shotgun proteomics analysis was chosen in order to generate a list of putative biomarkers, a targeted follow-up study should be performed to confirm these biomarkers. WIDER IMPLICATIONS OF THE FINDINGS The proteins found in both groups possess functions usually found in human semen. The enriched function analysis demonstrated a shift back to homeostasis after varicocelectomy, suggesting that varicocele correction promotes return of semen to a physiological state. STUDY FUNDING/COMPETING INTEREST(S) The funding for this project was received from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) as a scholarship for Ms Camargo. There was no conflict of interest.

Association between the seminal plasma proteome and sperm functional traits

OBJECTIVE To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. DESIGN Cross-sectional study. SETTING University andrology and research laboratories. PATIENT(S) A total of 156 normozoospermic men. INTERVENTION(S) Sperm mitochondrial activity, acrosome integrity, and DNA fragmentation were evaluated in a semen aliquot. Remaining semen was centrifuged, and seminal plasma was utilized for proteomic analysis (liquid chromatography-tandem mass spectrometry). Patients were divided into percentiles (15%) to form the following groups: substudy 1, high (control, n = 26) and low (study, n = 23) sperm mitochondrial activity; substudy 2, high (control, n = 23) and low (study, n = 22) sperm acrosome integrity; and substudy 3, low (control, n = 22) and high (study, n = 22) sperm DNA fragmentation. Groups were compared using univariate and multivariate analyses. Differentially expressed proteins were used for functional enrichment analysis. MAIN OUTCOME MEASURE(S) Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. RESULT(S) In total, 506, 493, and 464 proteins were observed in substudies 1, 2, and 3, respectively. Enriched functions in substudy 1 were intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (study group). In substudy 2, main enriched functions were phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammation, response to hydrogen peroxide, and lysosomal transport (study group). In substudy 3, enriched functions were prostaglandin biosynthesis and fatty acid binding (study group). We proposed eight, six, and eight seminal biomarkers for substudies 1, 2, and 3, respectively. CONCLUSION(S) Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations.

Determination of testicular function in adolescents with varicocoele – a proteomics approach

The goal of this study was to determine seminal plasma biomarkers of testicular function in adolescents with varicocoele and to verify enriched gene ontology terms associated to these differential proteomes. An observational study was carried out in an academic research environment. A total of 77 adolescent patients were recruited from a local public school, of which 23 were without varicocoele and with normal semen analysis (control group), 37 were with varicocoele and normal semen (VNS) parameters, and 17 were with varicocoele and altered semen (VAS) parameters. Two semen collections were provided with a 1‐week interval, after 2–5 days of ejaculatory abstinence. Seminal plasma proteins were identified and quantified utilizing a label‐free shotgun proteomics approach, generating (i) proteins differentially expressed in each group (control, VNS, and VAS) and putative biomarkers using multivariate statistics followed by discriminant analysis. Confirmatory analysis was performed for two proteins by western blotting. Enriched biological processes and molecular functions were determined using gene ontology analysis. In total, 541 proteins were identified and quantified: 108 exclusive or overexpressed in controls, 26 in the VNS group, and 13 in the VAS group. The suggested biomarkers are Cab45/SDF4 (Q9BRK5), protein lefty‐1 (O75610), DNase I (P24855), PAP2‐alpha (O14494), IBP‐7 (Q16270), HDC (P01860), and CRISP‐3 (P54108). Western blotting results showed that Cab45 was significantly underexpressed in both varicocoele groups, and CRISP‐3 was significantly overexpressed in seminal plasma of adolescents with VAS. In conclusion, specific biomarkers of spermatogenesis and homeostasis are observed in adolescents without varicocoele, and the presence of a palpable varicocoele progressively shifts these adolescents toward initially an immune response, and finally toward a chronic inflammatory profile. This shift is accompanied by decreased semen quality.

Improved serological detection of rheumatoid arthritis: a highly antigenic mimotope of carbonic anhydrase III selected in a murine model by phage display

IntroductionRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1 % of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis.MethodsSera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope.ResultsAfter three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases.ConclusionM12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.

Long-term in vivo polychlorinated biphenyl 126 exposure induces oxidative stress and alters proteomic profile on islets of Langerhans.

It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 μg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early β-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.

Proteome analysis of acute kidney injury – Discovery of new predominantly renal candidates for biomarker of kidney disease ☆

The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are organ and process specific. Here, we have used different tissues from a controlled porcine renal ischemia/reperfusion (I/R) model to identify new, predominantly renal biomarker candidates for kidney disease. Urine and serum samples were analyzed in pre-ischemia, ischemia (60min) and 4, 11 and 16h post-reperfusion, and renal cortex samples after 24h of reperfusion. Peptides were analyzed on the Q-Exactive™. In renal cortex proteome, we observed an increase in the synthesis of proteins in the ischemic kidney compared to the contralateral, highlighted by transcription factors and epithelial adherens junction proteins. Intersecting the set of proteins up- or down-regulated in the ischemic tissue with both serum and urine proteomes, we identified 6 proteins in the serum that may provide a set of targets for kidney injury. Additionally, we identified 49, being 4 predominantly renal, proteins in urine. As prove of concept, we validated one of the identified biomarkers, dipeptidyl peptidase IV, in a set of patients with diabetic nephropathy. In conclusion, we identified 55 systemic proteins, some of them predominantly renal, candidates for biomarkers of renal disease. BIOLOGICAL SIGNIFICANCE The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are predominantly renal. In fact, putative biomarkers for this condition have also been identified in a number of other clinical scenarios, such as cardiovascular diseases, chronic kidney failure or in patients being treated in intensive care units from a number of conditions. Here we propose a comprehensive, sequential screening procedure able to identify and validate potential biomarkers for kidney disease, using kidney ischemia/reperfusion as a paradigm for a kidney pathological event.