Thalidomide measurement in plasma and dried plasma spot by SPE combined with UHPLC-MS/MS for therapeutic drug monitoring.

Aims: To validate an SPE-ultra-HPLC-MS/MS method for thalidomide (THD) measurement in dried plasma spot (DPS). Methods: Extraction included acetonitrile/water clean-up and online SPE. The LOD, LLOQ, linearity, precision, accuracy, recovery, matrix effect, process efficiency, carryover, stability, drug interference and dilution integrity were assessed. Results: The method was linear from 50 to 2000 ng/ml with a LOD of 20 ng/ml and LLOQ of 50 ng/ml. The coefficient of variation for precision was 0.4-7.9% for intra-assay and 1.3-8.9% for interassay, and accuracy was 81.4-97.1%. Adequate matrix effect (100.6-107.0%), recovery (88.7-105.0%) and process efficiency (91.3-109.3%) were registered. DPS was stable for 14 days at room temperature and 45°C ,and for 4 months at -80°C. The method was applied to quantify THD in both wet plasma and DPS from patients with cutaneous lupus receiving THD treatment. The difference between THD wet plasma and DPS concentration was <15%. Conclusion: The method is suitable to quantify THD in DPS.

Mass Spectrometry Multiplexed Detection of SARS-CoV-2.

Testing of large populations for virus infection is now a reality worldwide due to the coronavirus (SARS-CoV-2) pandemic. The demand for SARS-CoV-2 testing using alternatives other than PCR led to the development of mass spectrometry (MS)-based assays. However, MS for SARS-CoV-2 large-scale testing have some downsides, including complex sample preparation and slow data analysis. Here, we describe a high-throughput targeted proteomics method to detect SARS-CoV-2 directly from nasopharyngeal and oropharyngeal swabs. This strategy employs fully automated sample preparation mediated by magnetic particles, followed by detection of SARS-CoV-2 nucleoprotein peptides by turbulent flow chromatography coupled with tandem mass spectrometry.

Proteomic analysis reveals rattlesnake venom modulation of proteins associated with cardiac tissue damage in mouse hearts.

Snake envenomation is a common but neglected disease that affects millions of people around the world annually. Among venomous snake species in Brazil, the tropical rattlesnake (Crotalus durissus terrificus) accounts for the highest number of fatal envenomations and is responsible for the second highest number of bites. Snake venoms are complex secretions which, upon injection, trigger diverse physiological effects that can cause significant injury or death. The components of C. d. terrificus venom exhibit neurotoxic, myotoxic, hemotoxic, nephrotoxic, and cardiotoxic properties which present clinically as alteration of central nervous system function, motor paralysis, seizures, eyelid ptosis, ophthalmoplesia, blurred vision, coagulation disorders, rhabdomyolysis, myoglobinuria, and cardiorespiratory arrest. In this study, we focused on proteomic characterization of the cardiotoxic effects of C. d. terrificus venom in mouse models. We injected venom at half the lethal dose (LD50) into the gastrocnemius muscle. Mouse hearts were removed at set time points after venom injection (1 h, 6 h, 12 h, or 24 h) and subjected to trypsin digestion prior to high-resolution mass spectrometry. We analyzed the proteomic profiles of >1300 proteins and observed that several proteins showed noteworthy changes in their quantitative profiles, likely reflecting the toxic activity of venom components. Among the affected proteins were several associated with cellular deregulation and tissue damage. Changes in heart protein abundance offer insights into how they may work synergistically upon envenomation. SIGNIFICANCE: Venom of the tropical rattlesnake (Crotalus durissus terririficus) is known to be neurotoxic, myotoxic, nephrotoxic and cardiotoxic. Although there are several studies describing the biochemical effects of this venom, no work has yet described its proteomic effects in the cardiac tissue of mice. In this work, we describe the changes in several mouse cardiac proteins upon venom treatment. Our data shed new light on the clinical outcome of the envenomation by C. d. terrificus, as well as candidate proteins that could be investigated in efforts to improve current treatment approaches or in the development of novel therapeutic interventions in order to reduce mortality and morbidity resulting from envenomation.

One-carbon metabolism and global DNA methylation in mothers of individuals with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder, resulting from the failure of normal chromosome 21 segregation. Studies have suggested that impairments within the one-carbon metabolic pathway can be of relevance for the global genome instability observed in mothers of individuals with DS. Based on the association between global DNA hypomethylation, genome instability, and impairments within the one-carbon metabolic pathway, the present study aimed to identify possible predictors, within the one-carbon metabolism, of global DNA methylation, measured by methylation patterns of LINE-1 and Alu repetitive sequences, in mothers of individuals with DS and mothers of individuals without the syndrome. In addition, we investigated one-carbon genetic polymorphisms and metabolites as maternal predisposing factors for the occurrence of trisomy 21 in children. Eighty-three samples of mothers of children with DS with karyotypically confirmed free trisomy 21 (case group) and 84 of mothers who had at least one child without DS or any other aneuploidy were included in the study. Pyrosequencing assays were performed to access global methylation. The results showed that group affiliation (case or control), betaine-homocysteine methyltransferase (BHMT) G742A and transcobalamin 2 (TCN2) C776G polymorphisms, and folate concentration were identified as predictors of global Alu DNA methylation values. In addition, thymidylate synthase (TYMS) 28-bp repeats 2R/3R or 3R/3R genotypes are independent maternal predisposing factors for having a child with DS. This study adds evidence that supports the association of impairments in the one-carbon metabolism, global DNA methylation, and the possibility of having a child with DS.

Improving data quality in liquid chromatography-mass spectrometry metabolomics of human urine.

Signal variation is a common drawback in untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS), mainly due to the complexity of biological matrices and reduced sample preparation, which results in the accumulation of sample components in the column and the ion source. Here we propose a simple, easy to implement approach to improve data quality in untargeted metabolomics by LC-MS. This approach involves the use of a divert valve to direct the column effluent to waste at the beginning of the chromatographic run and during column cleanup and equilibration, in combination with longer column cleanups in between injections. Our approach was tested using urine samples collected from patients after renal transplantation. Analytical responses were contrasted before and after introducing these modifications by analyzing a batch of untargeted metabolomics data. A significant improvement in peak area repeatability was observed for the quality controls, with relative standard deviations (RSDs) for several metabolites decreasing from ∼60% to ∼10% when our approach was introduced. Similarly, RSDs of peak areas for internal standards improved from ∼40% to ∼10%. Furthermore, calibrant solutions were more consistent after introducing these modifications when comparing peak areas of solutions injected at the beginning and the end of each analytical sequence. Therefore, we recommend the use of a divert valve and extended column cleanup as a powerful strategy to improve data quality in untargeted metabolomics, especially for very complex types of samples where minimum sample preparation is required, such as in this untargeted metabolomics study with urine from renal transplanted patients.

Pre-coating decellularized liver with HepG2-conditioned medium improves hepatic recellularization.

Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5 weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.

Establishing a mass spectrometry-based system for rapid detection of SARS-CoV-2 in large clinical sample cohorts.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Here, we develop a high-throughput targeted proteomics assay to detect SARS-CoV-2 nucleoprotein peptides directly from nasopharyngeal and oropharyngeal swabs. A modified magnetic particle-based proteomics approach implemented on a robotic liquid handler enables fully automated preparation of 96 samples within 4 hours. A TFC-MS system allows multiplexed analysis of 4 samples within 10 min, enabling the processing of more than 500 samples per day. We validate this method qualitatively (Tier 3) and quantitatively (Tier 1) using 985 specimens previously analyzed by real-time RT-PCR, and detect up to 84% of the positive cases with up to 97% specificity. The presented strategy has high sample stability and should be considered as an option for SARS-CoV-2 testing in large populations.

The impact of rattlesnake venom on mice cerebellum proteomics points to synaptic inhibition and tissue damage.

Snake envenomation is responsible for more than 130,000 deaths worldwide. In Brazil, the Crotalus rattlesnake is responsible for the second largest number of accidental snake bites in the country. Although there are many descriptions of the clinical and biochemical effects of Crotalus envenoming, there are few works describing the molecular events in the central nervous system of an organism due to envenomation. In this study, we analyzed the proteomic effect of Crotalus durissus terrificus snake venom on mice cerebellums. To monitor the envenomation over time, changes in the protein abundance were evaluated at 1 h, 6 h, 12 h and 24 h after venom injection by mass spectrometry. The analysis of the variation of over 4600 identified proteins over time showed a reduction in components of inhibitory synapse signaling, oxidative stress, and maintenance of neuronal cells, which paralleled increasing tissue damage and apoptosis factors. These analyses revealed the potential protein targets of the C. d. terrificus venom on the murine cerebellum, showing new aspects of the snake envenomation effect. These data may contribute to new therapeutic approaches (i.e., approaches directed at protein targets affected by the envenomation) on the treatment of envenomation by the neurotoxic C. d. terrificus snake venom. SIGNIFICANCE: Snakebites are a neglected global health problem that affects mostly rural and tropical areas of developing countries. It is estimated that over 5.4 million people are bitten by snakes each year, from which 2.7 million people are bitten by venomous snakes, resulting in disabilities such as amputations and in some cases leading to death. The C. d. terrificus snake is the most lethal snake in Brazil. Studying the molecular changes upon envenomation in a specific tissue may lead to a better understanding of the envenomation process by C. d. terrificus snakebites.

Proteome and miRNome profiling of microvesicles derived from medulloblastoma cell lines with stem-like properties reveals biomarkers of poor prognosis.

Primary central nervous system (CNS) tumors are the most common deadly childhood cancer. Several patients with medulloblastoma experience local or metastatic recurrences after standard treatment, a condition associated with very poor prognosis. Current neuroimaging techniques do not accurately detect residual stem-like medulloblastoma cells promoting tumor relapses. In attempt to identify candidate tumor markers that could be circulating in blood or cerebrospinal (CSF) fluid of patients, we evaluated the proteome and miRNome content of extracellular microvesicles (MVs) released by highly-aggressive stem-like medulloblastoma cells overexpressing the pluripotent factor OCT4A. These cells display enhanced tumor initiating capability and resistance to chemotherapeutic agents. A common set of 464 proteins and 10 microRNAs were exclusively detected in MVs of OCT4A-overexpressing cells from four distinct medulloblastoma cell lines, DAOY, CHLA-01-MED, D283-MED, and USP13-MED. The interactome mapping of these exclusive proteins and miRNAs revealed ERK, PI3K/AKT/mTOR, EGF/EGFR, and stem cell self-renewal as the main oncogenic signaling pathways altered in these aggressive medulloblastoma cells. Of these MV cargos, four proteins (UBE2M, HNRNPCL2, HNRNPCL3, HNRNPCL4) and five miRNAs (miR-4449, miR-500b, miR-3648, miR-1291, miR-3607) have not been previously reported in MVs from normal tissues and in CSF. These proteins and miRNAs carried within MVs might serve as biomarkers of aggressive stem-like medulloblastoma cells to improve clinical benefit by helping refining diagnosis, patient stratification, and early detection of relapsed disease.

The impact of rattlesnake venom on mice cerebellum proteomics points to synaptic inhibition and tissue damage

Snake envenomation is responsible for more than 130,000 deaths worldwide. In Brazil, the Crotalus rattlesnake is responsible for the second largest number of accidental snake bites in the country. Although there are many descriptions of the clinical and biochemical effects of Crotalus envenoming, there are few works describing the molecular events in the central nervous system of an organism due to envenomation. In this study, we analyzed the proteomic effect of Crotalus durissus terrificus snake venom on mice cerebellums. To monitor the envenomation over time, changes in the protein abundance were evaluated at 1 h, 6 h, 12 h and 24 h after venom injection by mass spectrometry. The analysis of the variation of over 4600 identified proteins over time showed a reduction in components of inhibitory synapse signaling, oxidative stress, and maintenance of neuronal cells, which paralleled increasing tissue damage and apoptosis factors. These analyses revealed the potential protein targets of the C. d. terrificus venom on the murine cerebellum, showing new aspects of the snake envenomation effect. These data may contribute to new therapeutic approaches (i.e., approaches directed at protein targets affected by the envenomation) on the treatment of envenomation by the neurotoxic C. d. terrificus snake venom. Significance Snakebites are a neglected global health problem that affects mostly rural and tropical areas of developing countries. It is estimated that over 5.4 million people are bitten by snakes each year, from which 2.7 million people are bitten by venomous snakes, resulting in disabilities such as amputations and in some cases leading to death. The C. d. terrificus snake is the most lethal snake in Brazil. Studying the molecular changes upon envenomation in a specific tissue may lead to a better understanding of the envenomation process by C. d. terrificus snakebites.

The impact of rattlesnake venom on mice cerebellum proteomics points to synaptic inhibition and tissue damage

Snake envenomation is responsible for more than 130,000 deaths worldwide. In Brazil, the Crotalus rattlesnake is responsible for the second largest number of accidental snake bites in the country. Although there are many descriptions of the clinical and biochemical effects of Crotalus envenoming, there are few works describing the molecular events in the central nervous system of an organism due to envenomation. In this study, we analyzed the proteomic effect of Crotalus durissus terrificus snake venom on mice cerebellums. To monitor the envenomation over time, changes in the protein abundance were evaluated at 1 h, 6 h, 12 h and 24 h after venom injection by mass spectrometry. The analysis of the variation of over 4600 identified proteins over time showed a reduction in components of inhibitory synapse signaling, oxidative stress, and maintenance of neuronal cells, which paralleled increasing tissue damage and apoptosis factors. These analyses revealed the potential protein targets of the C. d. terrificus venom on the murine cerebellum, showing new aspects of the snake envenomation effect. These data may contribute to new therapeutic approaches (i.e., approaches directed at protein targets affected by the envenomation) on the treatment of envenomation by the neurotoxic C. d. terrificus snake venom. Significance Snakebites are a neglected global health problem that affects mostly rural and tropical areas of developing countries. It is estimated that over 5.4 million people are bitten by snakes each year, from which 2.7 million people are bitten by venomous snakes, resulting in disabilities such as amputations and in some cases leading to death. The C. d. terrificus snake is the most lethal snake in Brazil. Studying the molecular changes upon envenomation in a specific tissue may lead to a better understanding of the envenomation process by C. d. terrificus snakebites.

Integrated proteomics and metabolomics analysis reveals differential lipid metabolism in human umbilical vein endothelial cells under high and low shear stress.

Atherosclerotic plaque development is closely associated with the hemodynamic forces applied to endothelial cells (ECs). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. ECs under low or oscillatory SS (LSS) show upregulation of inflammatory, adhesion, and cellular permeability molecules. On the contrary, cells under high or laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind SS regulation of an atheroprotective phenotype is not completely elucidated. Here we used proteomics and metabolomics to better understand the changes in endothelial cells (human umbilical vein endothelial cells) under in vitro LSS and HSS that promote an atheroprone or atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism, in special cholesterol metabolism, was downregulated in cells under LSS. The low-density lipoprotein receptor (LDLR) showed significant alterations both at the quantitative expression level as well as regarding posttranslational modifications. Under LSS, LDLR was seen at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of a HSS metabolic phenotype in EC under LSS. Altogether, our data suggest that there is significant modulation of lipid metabolism in endothelial cells under different SS intensities and that this could contribute to the atheroprone phenotype of LSS. Statin treatment was able to partially recover the protective profile of these cells.

Daily supplementation with 5 mg of folic acid in Brazilian patients with hereditary spherocytosis.

Patients with hereditary spherocytosis (HS) have increased rates of erythropoiesis and higher folate requirements. In a case-control study of patients with HS, we evaluated the associations between the use of 5 mg folic acid (FA) daily and serum concentrations of folate, unmetabolized folic acid (UMFA), interleukin (IL)-6, IL-8, IL-10, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α); and mRNA expression of dihydrofolate reductase (DHFR), methylene tetrahydrofolate reductase (MTHFR), IL8, IFNG and TNFA genes. Total serum folate and folate forms were measured in 27 patients with HS (21 users [HS-U] and 6 non-users [HS-NU] of supplemental FA) and 54 healthy controls not consuming 5 mg/day supplemental FA. Each patient was matched to two controls based on age, sex and body mass index. The mononuclear leucocyte mRNA expression of relevant genes and their products were determined. Serum folate, UMFA, 5-methyl-tetrahydrofolate (5-methyl-THF) and tetrahydrofolate (THF) concentrations were significantly higher in HS-U compared with matched healthy controls (p<0.001, n=42). HS-NU had lower serum folate concentrations than matched healthy controls (p=0.044, n=12). HS-U and HS-NU presented similar hematological and biochemical markers profiles. No differences were found between HS-U and HS-NU for cytokine serum concentrations and mRNA expression genes. DHFR mRNA expression was higher in HS-U than in HS-NU. The use of high daily doses of FA for treatment of patients with HS may be excessive and is associated with elevated serum UMFA and elevated DHFR mRNA expression. It is not known whether long-term high-dose FA use by patients with HS might have adverse health effects.

Mesenchymal stem cells enhance tumorigenic properties of human glioblastoma through independent cell-cell communication mechanisms.

Mesenchymal stem cells (MSC) display tumor tropism and have been addressed as vehicles for delivery of anti-cancer agents. As cellular components of the tumor microenvironment, MSC also influence tumor progression. However, the contribution of MSC in brain cancer is not well understood since either oncogenic or tumor suppressor effects have been reported for these cells. Here, MSC were found capable of stimulating human Glioblastoma (GBM) cell proliferation through a paracrine effect mediated by TGFB1. Moreover, when in direct cell-cell contact with GBM cells, MSC elicited an increased proliferative and invasive tumor cell behavior under 3D conditions, as well as accelerated tumor development in nude mice, independently of paracrine TGFB1. A secretome profiling of MSC-GBM co-cultures identified 126 differentially expressed proteins and 10 proteins exclusively detected under direct cell-cell contact conditions. Most of these proteins are exosome cargos and are involved in cell motility and tissue development. These results indicate a dynamic interaction between MSC and GBM cells, favoring aggressive tumor cell traits through alternative and independent mechanisms. Overall, these findings indicate that MSC may exert pro-tumorigenic effects when in close contact with tumor cells, which must be carefully considered when employing MSC in targeted cell therapy protocols against cancer.

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.

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 ß-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.

Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers.

OBJECTIVE: To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. PATIENTS AND METHODS: Sperm functional tests were performed in 20 non-smoking men with normal semen quality, according to the World Health Organization (2010) and in 20 smoking patients. These included: evaluation of DNA fragmentation by alkaline Comet assay; analysis of mitochondrial activity using DAB staining; and acrosomal integrity evaluation by PNA binding. The remaining semen was centrifuged and seminal plasma was used for proteomic analysis (liquid chromatography-tandem mass spectrometry). The quantified proteins were used for Venn diagram construction in Cytoscape 3.2.1 software, using the PINA4MS plug-in. Then, differentially expressed proteins were used for functional enrichment analysis of Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes and Reactome, using Cytoscape software and the ClueGO 2.2.0 plug-in. RESULTS: Smokers had a higher percentage of sperm DNA damage (Comet classes III and IV; P < 0.01), partially and fully inactive mitochondria (DAB classes III and IV; P = 0.001 and P = 0.006, respectively) and non-intact acrosomes (P < 0.01) when compared with the control group. With respect to proteomic analysis, 422 proteins were identified and quantified, of which one protein was absent, 27 proteins were under-represented and six proteins were over-represented in smokers. Functional enrichment analysis showed the enrichment of antigen processing and presentation, positive regulation of prostaglandin secretion involved in immune response, protein kinase A signalling and arachidonic acid secretion, complement activation, regulation of the cytokine-mediated signalling pathway and regulation of acute inflammatory response in the study group (smokers). CONCLUSION: In conclusion, cigarette smoking was associated with an inflammatory state in the accessory glands and in the testis, as shown by enriched proteomic pathways. This state causes an alteration in sperm functional quality, which is characterized by decreased acrosome integrity and mitochondrial activity, as well as by increased nuclear DNA fragmentation.

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.

Serum lipidomic profiling as a useful tool for screening potential biomarkers of hepatitis B-related hepatocellular carcinoma by ultraperformance liquid chromatography-mass spectrometry.

BACKGROUND: Chronic hepatitis B (CHB) virus infection is a major cause of hepatocellular carcinoma (HCC), as late diagnosis is the main factor for the poor survival of patients. There is an urgent need for accurate biomarkers for early diagnosis of HCC. The aim of the study was to explore the serum lipidome profiles of hepatitis B-related HCC to identify potential diagnostic biomarkers. METHODS: An ultraperformance liquid chromatography mass spectrometry (UPLC-MS) lipidomic method was used to characterize serum profiles from HCC (n = 32), liver cirrhosis (LC) (n = 30), CHB (n = 25), and healthy subjects (n = 34). Patients were diagnosed by clinical laboratory and imaging evidence and all presented with CHB while healthy controls had normal liver function and no infectious diseases. RESULTS: The UPLC-MS-based serum lipidomic profile provided more accurate diagnosis for LC patients than conventional alpha-fetoprotein (AFP) detection. HCC patients were discriminated from LC with 78 % sensitivity and 64 % specificity. In comparison, AFP showed sensitivity and specificity of 38 % and 93 %, respectively. HCC was differentiated from CHB with 100 % sensitivity and specificity using the UPLC-MS approach. Identified lipids comprised glycerophosphocolines, glycerophosphoserines and glycerophosphoinositols. CONCLUSIONS: UPLC-MS lipid profiling proved to be an efficient and convenient tool for diagnosis and screening of HCC in a high-risk population.