Brevetoxicosis in Florida manatees (Trichechus manatus latirostris): Plasma proteomics for Florida red tide biomarkers.

Acute myocardial infarction (AMI) is a common cause of death for which effective treatments are available provided that diagnosis is rapid. The current diagnostic gold standards are circulating cardiac troponins I and T. However, their slow release delays diagnosis, and their persistence limits their utility in the identification of reinfarction. The aim was to identify candidate biomarkers of AMI. Isolated mouse hearts were perfused with oxygenated protein-free buffer, and coronary effluent was collected after ischemia or during matched normoxic perfusion. Effluents were analyzed using proteomics approaches based on one- or two-dimensional initial separation. Of the 459 proteins identified after ischemia with one-dimensional separation, 320 were not detected in the control coronary effluent. Among these were all classic existing biomarkers of AMI. We also identified the cardiac isoform of myosin-binding protein C in its full-length form and as a 40-kDa degradation product. This protein was not detected in the other murine organs examined, increased markedly with even trivial myocardial infarction, and could be detected in the plasma after myocardial infarction in vivo, a profile compatible with a biomarker of AMI. Two-dimensional fluorescence DIGE of ischemic and control coronary effluents identified more than 200 asymmetric spots verified by swapping dyes. Once again existing biomarkers of injury were confirmed as well as posttranslational modifications of antioxidant proteins such as peroxiredoxins. Perfusing hearts with protein-free buffers provides a platform of graded ischemic injury that allows detailed analysis of protein release and identification of candidate cardiac biomarkers like myosin-binding protein C.

High-fat diet–induced plasma protein and liver changes in obese rats can be attenuated by melatonin supplementation

We investigated the effects of pedal speed on changes in plasma volume, electrolytes and protein during incremental exercise. Ten adult males participated in two, 30 minute incremental cycle ergometer exercise tests at room temperature (22 degrees C, rh = 56%). Exercise load was increased from 20 to 70% of peak VO2. Five minutes were spent at each of six stages which were equally spaced in exercise intensity. Subjects pedaled at 50 (50 RPM) and 90 (90 RPM) rev.min-1. Venous blood samples were drawn prior to exercise and during the last minute of each stage. Relative plasma volume changes showed a progressive hemoconcentration during the exercise. There were no significant differences due to pedal speed as plasma volume loss averaged -7.3% during exercise. [Na+], [Cl-], and [K+] increased significantly during exercise but were not influenced by pedal speed. Changes in plasma protein and albumin concentrations indicated that there was a loss of globulin from the vascular volume in both conditions and an addition of albumin to the plasma in 50 RPM. The difference in plasma albumin dynamics was possibly related to an effect of pedal speed on movement of fluid in the lymphatic vessels of the legs.

The effect of animal age on plasma protein composition was studied in the Mongolian gerbil, Meriones unguiculatus. Values for plasma protein fractions separated by electrophoresis were obtained from bromophenol blue-stained paper strips by elution and by scanning techniques, both of which yielded similar results. Total plasma proteins increased with age from 1 day to 28 months. Maximum albumin concentrations were attained at 3 months and maximum (adult) γ-globulin-fibrinogen concentrations at 28 months. A fast α2-globulin fraction was found in plasma from the youngest age group (1–3 days) but absent in older groups. No sex-related differences in plasma proteins were found for gerbils at 3 and 6 months. Age-related changes in gerbil plasma proteins are similar to changes found during the life span of many other mammalian species.

Because the plasma proteins are so much easier to sample than other body proteins much of the early work on the effects of protein nutrition centred on changes in plasma protein. This accessability was technically an advantage, but it also tended to distort the true picture of the nutritional role of the circulating and fixed tissue proteins respectively. Thus Whipple and his colleagues1 concluded that during protein depletion there was “raiding” of tissue proteins to maintain plasma protein, and that “the blood proteins in these experiments take priority over the organ and tissue proteins”. This conclusion is hardly justified in view of the techniques used to produce protein depletion: the dogs were fed a protein-free diet and plasma proteins were removed by plasma-pheresis. If thereafter plasma protein was made at the expense of tissue protein this is no more than a demonstration of homeostasis. Had it been feasible to remove progressively portions of liver no doubt this organ too would have been regenerated at the expense of other proteins which were not specifically depleted, and it would have been equally logical to conclude that liver protein had priority over other tissues in time of protein shortage. The true pricture is that protein depletion in an intact animal, however, it is produced, affects all the body proteins since they are in dynamic equilibrium2 although the extent and timing of protein loss varies greatly from one organ to another.3 It is convenient, therefore, to consider the effects of nutrition on plasma proteins from three aspects: first, the interrelationship of plasma and tissue protein stores in times of nutritional stress of various kinds; second, the diagnostic value of plasma protein concentrations in assessing nutritional status; and third, the dynamic changes in plasma protein metabolism which occur in response to changes in diet. Only studies in intact mammals especially man, will be considered, since elegant and ingenious studies on isolated perfused organs are described elsewhere.

Trace elements like copper, zinc, iron and selenium have a significant influence on the function of the immune system. We studied plasma levels of trace elements in 53 patients with acute bacterial and viral infections. In bacterial infections (septicaemia, pneumonia, erysipelas and meningitis) the plasma concentrations of selenium, iron and zinc were decreased. Plasma copper was unchanged in patients with erysipelas, but increased in other types of bacterial infections. Although the patients with viral infections showed similar shifts of the trace elements as were observed in patients with bacterial infections, the changes were not as pronounced. A plasma selenium value below 0.8 mumol/l was found in only 6% of the patients with viral infections in contrast to 63% of the patients with septicaemia or 57% of the patients with pneumonia. Furthermore, in viral infections 60% of the zinc values were below the mean level of 12.8 mumol/l observed in healthy controls as compared with 90% of the values in patients with sepsis or 92% of the values in patients with pneumonia. The onset of change in trace elements occurred within a few days and persisted for several weeks. These changes seem to be non-specific and are independent of the agent causing infection. The different types of infections were followed by changes in most of the plasma proteins which are known to be associated with an inflammatory reaction. The changes in plasma proteins were most pronounced in patients with sepsis and pneumonia. Patients with sepsis having a high degree of inflammation did not show a positive correlation between the severity of the disease--as judged by plasma proteins--and the alterations of trace elements.

Many selenoorganic compounds play an important role in biochemical processes and act as antioxidants, enzyme inhibitors or drugs. The effects of a new selenocompound — bis(2-aminophenyl)-diselenide on oxidative/nitrative changes in human plasma proteins induced by peroxynitrite (ONOO−) were studied in vitro and compared with the those of ebselen, a well-known antioxidant. We also studied the role of the tested selenocompounds in peroxynitrite-induced plasma lipid peroxidation. Exposure of the plasma to peroxynitrite (0.1 mM) resulted in an increase in the level of carbonyl groups and nitrotyrosine residues in plasma proteins (estimated using the ELISA method and Western blot analysis). In the presence of different concentrations (0.025–0.1 mM) of the tested selenocompounds, 0.1 mM peroxynitrite caused a distinct decrease in the level of carbonyl group formation and tyrosine nitration in plasma proteins. Moreover, these selenocompounds also inhibited plasma lipid peroxidation induced by ONOO−1 (0.1 mM). The obtained results indicate that in vitro bis(2-aminophenyl)-diselenide and ebselen have very similar protective effects against peroxynitrite-induced oxidative/nitrative damage to human plasma proteins and lipids.

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4-500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430-1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.

We examined plasma cortisol concentration (PCC), cortisol metabolism, and cortisol protein binding (CPB) in chronically cannulated Atlantic salmon (Salmo salar) during seawater (SW) adaptation. PCC rose significantly 2 h after freshwater (FW) to SW changeover but tended to be lower for 6 days thereafter. Plasma chloride concentrations began to stabilize 24-48 h after entry to SW. Cortisol metabolic clearance rate (MCR) was significantly higher 24 h after SW transfer than in FW; CPB and plasma protein concentration were significantly lower, but cortisol production rate was unchanged. The change in plasma chloride after 24 h in SW was negatively correlated with the change in plasma protein (r = -0.915, n = 9, P less than 0.001). The changes in CPB and plasma protein were correlated with the change in MCR (r = -0.643, n = 14, P less than 0.02; r = -0.850, n = 9, P less than 0.005). We hypothesize that these changes in protein concentration reflect an ion-induced expansion of extracellular volume and lead to increased MCR.

The neuroinflammatory response to surgery can be characterized by peripheral acute plasma protein changes in blood, but corresponding, persisting alterations in cerebrospinal fluid (CSF) proteins remain mostly unknown. Using the SOMAscan assay, we define acute and longer-term proteome changes associated with surgery in plasma and CSF. We hypothesized that biological pathways identified by these proteins would be in the categories of neuroinflammation and neuronal function and define neuroinflammatory proteome changes associated with surgery in older patients. SOMAscan analyzed 1305 proteins in blood plasma (n = 14) and CSF (n = 15) samples from older patients enrolled in the Role of Inflammation after Surgery for Elders (RISE) study undergoing elective hip and knee replacement surgery with spinal anesthesia. Systems biology analysis identified biological pathways enriched among the surgery-associated differentially expressed proteins in plasma and CSF. Comparison of postoperative day 1 (POD1) to preoperative (PREOP) plasma protein levels identified 343 proteins with postsurgical changes ( P < .05; absolute value of the fold change [|FC|] > 1.2). Comparing postoperative 1-month (PO1MO) plasma and CSF with PREOP identified 67 proteins in plasma and 79 proteins in CSF with altered levels ( P < .05; |FC| > 1.2). In plasma, 21 proteins, primarily linked to immune response and inflammation, were similarly changed at POD1 and PO1MO. Comparison of plasma to CSF at PO1MO identified 8 shared proteins. Comparison of plasma at POD1 to CSF at PO1MO identified a larger number, 15 proteins in common, most of which are regulated by interleukin-6 (IL-6) or transforming growth factor beta-1 (TGFB1) and linked to the inflammatory response. Of the 79 CSF PO1MO-specific proteins, many are involved in neuronal function and neuroinflammation. SOMAscan can characterize both short- and long-term surgery-induced protein alterations in plasma and CSF. Acute plasma protein changes at POD1 parallel changes in PO1MO CSF and suggest 15 potential biomarkers for longer-term neuroinflammation that warrant further investigation.

More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins-SLC3A2, STMN1, and TAGLN2-in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant overexpression in individual bladder cancer tissues and urine specimens, and thus represents a potential biomarker for noninvasive screening for bladder cancer. Our findings highlight the value of bladder tissue proteome in providing valuable information for future validation studies of potential biomarkers in urothelial carcinoma.

A "red tide" is a harmful algal bloom that occurs when toxic, microscopic algae in seawater proliferate to a higher-than-normal concentration (i.e., bloom), often discoloring the water red, brown, green, or yellow. Red tides can kill fish, birds, and marine mammals and cause illness in humans. Florida red tide is caused by the dinoflagellate Karenia brevis, which produces toxins called brevetoxins and is most commonly found in the Gulf of Mexico; however, K. brevis blooms also can occur along the Atlantic coast. On September 25, 2007, a cluster of respiratory illnesses was reported to the Nassau County Health Department (NCHD) in northeastern Florida. All of the ill persons were employed at a beach restoration worksite by a dredging company operating at Fernandina Beach; they reported symptoms of eye or respiratory irritation (e.g., coughing, sneezing, sniffling, and throat irritation). NCHD and the Florida Department of Health promptly conducted epidemiologic and environmental investigations and determined the illnesses likely were associated with exposure to a red tide along the Atlantic coast. These actions highlight the importance of rapid investigation of health concerns with potential environmental causes to enable timely notification of the public and prevent further illness.

This study aimed to assess mutagenicity biomarkers among Egyptian textile dyeing workers, their alteration with gene polymorphism, and the changes in plasma proteins' expression. Using a detailed questionnaire, a comparative cross-sectional study was conducted on 212 workers (106 textile dyeing-exposed group and 106 control group). CBMN-Cyt assay, ERCC2 gene polymorphism, and plasma protein fractions were analyzed in workers' blood samples. Textile dye workers had significantly higher mutagenicity biomarkers than the control group. Mutant ERCC2 genotypes, dye exposure, exposure period, and formaldehyde levels significantly predicted mutagenicity biomarkers' levels. Dye-exposed workers also showed significant changes in plasma protein fractions. Textile dyeing workers, particularly susceptible genotypes, are at mutagenic risk and have significant changes in plasma protein fractions. Those changes are usually the first detectable response to toxic exposures and can be useful as exposure biomarkers.

The acute phase response of rainbow trout (Oncorhynchus mykiss) plasma proteins to viral, bacterial and fungal inflammatory agents

Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of death worldwide. Efficient non-invasive blood-based biomarkers for CRC early detection and prognosis are urgently needed. To identify novel potential plasma biomarkers, we applied a proximity extension assay (PEA), an antibody-based proteomics strategy to quantify the abundance of plasma proteins in CRC development and cancer-associated inflammation from few μL of plasma sample. Among the 690 quantified proteins, levels of 202 plasma proteins were significantly changed in CRC patients compared to age-and-sex-matched healthy subjects. We identified novel protein changes involved in Th17 activity, oncogenic pathways, and cancer-related inflammation with potential implications in the CRC diagnosis. Moreover, the interferon γ (IFNG), interleukin (IL) 32, and IL17C were identified as associated with the early stages of CRC, whereas lysophosphatidic acid phosphatase type 6 (ACP6), Fms-related tyrosine kinase 4 (FLT4), and MANSC domain-containing protein 1 (MANSC1) were correlated with the late-stages of CRC. Further study to characterize the newly identified plasma protein changes from larger cohorts will facilitate the identification of potential novel diagnostic, prognostic biomarkers for CRC.