The plasma peptides of sepsis.

BACKGROUND: A practical strategy to discover sepsis specific proteins may be to compare the plasma peptides and proteins from patients in the intensive care unit with and without sepsis. The aim was to discover proteins and/or peptides that show greater observation frequency and/or precursor intensity in sepsis. The endogenous tryptic peptides of ICU-Sepsis were compared to ICU Control, ovarian cancer, breast cancer, female normal, sepsis, heart attack, Alzheimer's and multiple sclerosis along with their institution-matched controls, female normals and normal samples collected directly onto ice. METHODS: Endogenous tryptic peptides were extracted from individual sepsis and control EDTA plasma samples in a step gradient of acetonitrile for random and independent sampling by LC-ESI-MS/MS with a set of robust and sensitive linear quadrupole ion traps. The MS/MS spectra were fit to fully tryptic peptides within proteins using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The protein observation frequency of ICU-sepsis versus ICU Control was subsequently tested by Chi square analysis. The average protein or peptide log10 precursor intensity was compared across disease and control treatments by ANOVA in the R statistical system. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as ITIH3, SAA2, SAA1, and FN1 showed increased observation frequency by Chi square (χ2 > 9, p < 0.003) and/or precursor intensity in sepsis. Cellular gene symbols with large Chi square values from tryptic peptides included POTEB, CTNNA1, U2SURP, KIF24, NLGN2, KSR1, GTF2H1, KIT, RPS6KL1, VAV2, HSPA7, SMC2, TCEB3B, ZNF300, SUPV3L1, ADAMTS20, LAMB4, MCCC1, SUPT6H, SCN9A, SBNO1, EPHA1, ABLIM2, cB5E3.2, EPHA10, GRIN2B, HIVEP2, CCL16, TKT, LRP2 and TMF1 amongst others showed increased observation frequency. Similarly, increased frequency of tryptic phosphopeptides were observed from POM121C, SCN8A, TMED8, NSUN7, SLX4, MADD, DNLZ, PDE3B, UTY, DEPDC7, MTX1, MYO1E, RXRB, SYDE1, FN1, PUS7L, FYCO1, USP26, ACAP2, AHI1, KSR2, LMAN1, ZNF280D and SLC8A2 amongst others. Increases in mean precursor intensity in peptides from common plasma proteins such as ITIH3, SAA2, SAA1, and FN1 as well as cellular proteins such as COL24A1, POTEB, KANK1, SDCBP2, DNAH11, ADAMTS7, MLLT1, TTC21A, TSHR, SLX4, MTCH1, and PUS7L among others were associated with sepsis. The processing of SAA1 included the cleavage of the terminal peptide D/PNHFRPAGLPEKY from the most hydrophilic point of SAA1 on the COOH side of the cystatin C binding that was most apparent in ICU-Sepsis patients compared to all other diseases and controls. Additional cleavage of SAA1 on the NH2 terminus side of the cystatin binding site were observed in ICU-Sepsis. Thus there was disease associated variation in the processing of SAA1 in ICU-Sepsis versus ICU controls or other diseases and controls. CONCLUSION: Specific proteins and peptides that vary between diseases might be discovered by the random and independent sampling of multiple disease and control plasma from different hospital and clinics by LC-ESI-MS/MS for storage in a relational SQL Server database and analysis with the R statistical system that will be a powerful tool for clinical research. The processing of SAA1 may play an unappreciated role in the inflammatory response to Sepsis.

The plasma peptides of breast versus ovarian cancer.

BACKGROUND: There is a need to demonstrate a proof of principle that proteomics has the capacity to analyze plasma from breast cancer versus other diseases and controls in a multisite clinical trial design. The peptides or proteins that show a high observation frequency, and/or precursor intensity, specific to breast cancer plasma might be discovered by comparison to other diseases and matched controls. The endogenous tryptic peptides of breast cancer plasma were compared to ovarian cancer, female normal, sepsis, heart attack, Alzheimer's and multiple sclerosis along with the institution-matched normal and control samples collected directly onto ice. METHODS: Endogenous tryptic peptides were extracted from individual breast cancer and control EDTA plasma samples in a step gradient of acetonitrile, and collected over preparative C18 for LC-ESI-MS/MS with a set of LTQ XL linear quadrupole ion traps working together in parallel to randomly and independently sample clinical populations. The MS/MS spectra were fit to fully tryptic peptides or phosphopeptides within proteins using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The observation frequency was subsequently tested by Chi Square analysis. The log10 precursor intensity was compared by ANOVA in the R statistical system. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as APOE, C4A, C4B, C3, APOA1, APOC2, APOC4, ITIH3 and ITIH4 showed increased observation frequency and/or precursor intensity in breast cancer. Many cellular proteins also showed large changes in frequency by Chi Square (χ2 > 100, p < 0.0001) in the breast cancer samples such as CPEB1, LTBP4, HIF-1A, IGHE, RAB44, NEFM, C19orf82, SLC35B1, 1D12A, C8orf34, HIF1A, OCLN, EYA1, HLA-DRB1, LARS, PTPDC1, WWC1, ZNF562, PTMA, MGAT1, NDUFA1, NOGOC, OR1E1, OR1E2, CFI, HSA12, GCSH, ELTD1, TBX15, NR2C2, FLJ00045, PDLIM1, GALNT9, ASH2L, PPFIBP1, LRRC4B, SLCO3A1, BHMT2, CS, FAM188B2, LGALS7, SAT2, SFRS8, SLC22A12, WNT9B, SLC2A4, ZNF101, WT1, CCDC47, ERLIN1, SPFH1, EID2, THOC1, DDX47, MREG, PTPRE, EMILIN1, DKFZp779G1236 and MAP3K8 among others. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. An increase in mean precursor intensity of peptides was observed for QSER1 as well as SLC35B1, IQCJ-SCHIP1, MREG, BHMT2, LGALS7, THOC1, ANXA4, DHDDS, SAT2, PTMA and FYCO1 among others. In contrast, the QSER1 peptide QPKVKAEPPPK was apparently specific to ovarian cancer. CONCLUSION: There was striking agreement between the breast cancer plasma peptides and proteins discovered by LC-ESI-MS/MS with previous biomarkers from tumors, cells lines or body fluids by genetic or biochemical methods. The results indicate that variation in plasma peptides from breast cancer versus ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research. It may be possible to use a battery of sensitive and robust linear quadrupole ion traps for random and independent sampling of plasma from a multisite clinical trial.

Potential biomarkers of tissue hypoxia during acute hemodilutional anemia in cardiac surgery: A prospective study to assess tissue hypoxia as a mechanism of organ injury.

PURPOSE: Hemodilutional anemia is associated with acute kidney injury (AKI) and mortality in patients undergoing cardiac surgery by mechanisms that may include tissue hypoxia. Our hypothesis was to assess if changes in the potential hypoxic biomarkers, including methemoglobin and erythropoietin, correlated with a decrease in hemoglobin (Hb) concentration following hemodilution on cardiopulmonary bypass (CPB). METHODS: Arterial blood samples were taken from patients (n = 64) undergoing heart surgery and CPB at baseline, during CPB, following CPB, and in the intensive care unit (ICU). Potential hypoxic biomarkers were measured, including methemoglobin, plasma Hb, and erythropoietin. Data were analyzed by repeated measures one-way analysis of variance on ranks and linear regression. RESULTS: Hemoglobin levels decreased following CPB and methemoglobin increased in the ICU (P < 0.001 for both). No correlation was observed between the change in Hb and methemoglobin (P = 0.23). By contrast, reduced Hb on CPB correlated with increased lactate, reduced pH, and increased erythropoietin levels following CPB (P ≤ 0.004 for all). Increased plasma Hb (P < 0.001) also correlated with plasma erythropoietin levels (P < 0.001). CONCLUSION: These data support the hypothesis that erythropoietin rather than methemoglobin is a potential biomarker of anemia-induced tissue hypoxia. The observed relationships between decreased Hb during CPB and the increase in lactate, reduced pH, and increase in erythropoietin levels suggest that early changes in plasma erythropoietin may be a pragmatic early biomarker of anemia-induced renal hypoxia. Further study is required to determine if anemia-induced increases in erythropoietin may predict AKI in patients undergoing cardiac surgery. TRIAL REGISTRATION: www.clinicaltrials.gov (NCT01883713). Registered 21 June 2013.

The plasma peptides of ovarian cancer.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation. METHODS: The endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 µl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC-ESI-MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey-Kramer HSD test. CONCLUSION: Here we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC-ESI-MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research.

Acoustic wave biosensor for the detection of the breast and prostate cancer metastasis biomarker protein PTHrP.

There are currently no biosensors that are able to reliably detect the process of cancer metastasis. We describe the first label-free real-time ultra-high frequency acoustic wave biosensor prototype capable of detecting the breast and prostate cancer metastasis biomarker, parathyroid hormone-related peptide (PTHrP). Two different linkers - 11-trichlorosilyl-undecanoic acid pentafluorophenyl ester (PFP) and S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) - were used to immobilize whole anti-PTHrP antibodies and Fab' fragments to surfaces as biorecognition elements. The biosensor surfaces were optimized using X-ray photoelectron spectroscopy (XPS) and the ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS). One optimized whole antibody-based surface (PFP/protein G'/whole antibodies/ethanolamine) and one optimized Fab' fragment-based surface (TUBTS/Fab' fragments) were tested as biosensors. It was determined that an in-line injection of bovine serum albumin prior to analyte injection yielded the most minimally fouling surfaces. Each surface was tested with no mass amplification and with sandwich-type secondary antibody mass amplification. The whole antibody-based mass-amplified biosensor yielded the lowest limit of detection (61 ng/mL), highest sensitivity, and a linear range from 61 ng/mL to 100 µg/mL. However, the Fab' fragment-based biosensor displayed better regenerability as a loss of ~20% of the initial analyte signal intensity was observed with each subsequent injection. The whole antibody-based biosensor was only capable of producing an analyte signal in the first injection.

Endotoxemia Following Multiple Trauma: Risk Factors and Prognostic Implications.

OBJECTIVE: To evaluate the prevalence and time course of systemic endotoxemia following severe multiple trauma, to define its risk factors, and to explore the correlation between post-trauma endotoxemia and organ dysfunction. DESIGN: Prospective single-center cohort study. SETTING: Emergency department and ICU of adult tertiary care level I trauma center. PATIENTS: Forty-eight severely injured (Injury Severity Score ≥ 16) patients, admitted to ICU within 24 hours of injury. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Endotoxemia was not evident on initial presentation, but developed subsequently in 75% of patients, even in the absence of Gram-negative infection. Nonsurviving patients had higher endotoxin levels than survivors on day 1 (endotoxemia, 0.48 vs 0.28; p = 0.048). Shock at admission, or surgery within the first 48 hours after trauma, was associated with higher endotoxin levels and predicted subsequent maximal endotoxemia, after adjusting for other significant covariates. Maximal endotoxemia levels were higher in patients who developed organ dysfunction, reflected in a cumulative Multiple Organ Dysfunction Score greater than 25, and patients with an intermediate endotoxemia level (≥ 0.4) had more cardiovascular dysfunction. CONCLUSIONS: It is the first study to detect increasing levels of endotoxemia following multiple trauma. Shock and early surgery predict the development of endotoxemia; endotoxemia is particularly associated with cardiovascular dysfunction. However, Gram-negative infections are uncommon in these patients, suggesting that the gastrointestinal tract is the dominant reservoir of endotoxin. Endotoxin may be an appropriate therapeutic target in patients who have sustained severe multiple trauma.

In vitro and in vivo cell-capture strategies using cardiac stent technology - A review.

Stenosis is a symptom of coronary artery disease (CAD), and is caused by narrowing of arteries in the heart. Over the last several decades, medical implants such as cardiac stents have been developed to counter stenosis. Upon implantation of a stent to open up a restricted artery, narrowing of the artery can reoccur (restenosis), due to an immune response launched by the body towards the stent. Currently, restenosis is a major health concern for patients who have undergone heart surgery for coronary artery disease. Recently, there have been new methods developed to combat restenosis, which have shown potential signs of success. One proposed method is the use of stents to capture cells, thereby reducing immune response. This review will explore the different methods for cell capture both in vitro and in vivo. Biological modifications of the stent will be surveyed, as well as the use of surface science to immobilize biological probes. Immobilization of proteins and nucleotides, as well as use of magnetic field are all methods that will be further discussed. Finally, concluding remarks and future prospects will be presented.

Adlayer-mediated antibody immobilization to stainless steel for potential application to endothelial progenitor cell capture.

This work describes the straightforward surface modification of 316L stainless steel with BTS, S-(11-trichlorosilylundecanyl)-benzenethiosulfonate, a thiol-reactive trichlorosilane cross-linker molecule designed to form intermediary coatings with subsequent biofunctionalization capability. The strategy is more specifically exemplified with the immobilization of intact antibodies and their Fab' fragments. Both surface derivatization steps are thoroughly characterized by means of X-ray photoelectron spectroscopy. The antigen binding capability of both types of biofunctionalized surfaces is subsequently assessed by fluorescence microscopy. It was determined that BTS adlayers achieve robust immobilization of both intact and fragmented antibodies, while preserving antigen binding activity. Another key finding was the observation that the Fab' fragment immobilization strategy would constitute a preferential option over that involving intact antibodies in the context of in vivo capture of endothelial progenitor cells in stent applications.

Neurofilament medium polypeptide (NFM) protein concentration is increased in CSF and serum samples from patients with brain injury.

BACKGROUND: Brain injury is a medical emergency that needs to be diagnosed and treated promptly. Several proteins have been studied as biomarkers of this medical condition. The aims of this study were to: 1) evaluate the selectivity and precision of a commercial ELISA kit for neurofilament medium polypeptide (NFM) protein; and 2) evaluate the concentration in cerebrospinal fluid (CSF) and serum of healthy individuals and patients with brain damage. METHODS: An ELISA from Elabscience was used. The selectivity was evaluated using size-exclusion chromatography and mass spectrometry. Intra- and inter-batch coefficients of variation (CV) were also studied. Fifty-one CSF samples from 36 age-matched patients with hemorrhagic stroke (HS) (n=30), ischemic stroke (IS) (n=11) and healthy individuals (n=10) were assayed. In addition, serum samples from healthy volunteers (n=47), 68 serum samples from seven patients with HS, 106 serum samples from 12 patients with traumatic brain injury (TBI) and 68 serum samples from 68 patients with mild traumatic brain injury (mTBI) were also analyzed. RESULTS: NFM was identified in the chromatographic fraction with highest immunoreactivity. The intra- and inter-batch CVs were ≤10% and ≤13%, respectively. The CSF-NFM concentration in HS was significantly higher (p<0.0001) than in IS and controls. Serum NFM concentration ranged from 0.26 to 8.57 ng/mL in healthy individuals (median=2.29), from 0.97 to 42.4 ng/mL in HS (median=10.8) and from 3.48 to 45.4 ng/mL in TBI (median=14.7). Finally, 44% of patients with mTBI had increased NFM concentration, with significantly higher levels (p=0.01) in patients with polytrauma. CONCLUSIONS: To our knowledge this is the first study describing increased NFM levels in CSF and serum from patients with brain damage.

Profilin-1 expression is associated with high grade and stage and decreased disease-free survival in renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is associated with high mortality, although individual outcomes are highly variable. Identification of patients with increased risk of disease progression can guide customizing management plan according to disease severity. Profilin-1 (Pfn1) has been recently identified as overexpressed in metastatic ccRCC compared with primary tumors. We examined Pfn1 expression in a tissue microarray of 384 cases of histologically confirmed primary ccRCC with detailed clinical follow-up. Profilin-1 expression showed both cytoplasmic and nuclear staining patterns. The immunoexpression of Pfn1 was scored in a semiquantitative fashion. There was no significant difference in Pfn1 expression between normal kidney and kidney ccRCC. Our results show that strong cytoplasmic Pfn1 expression is associated with high-grade (P < .001) and high-stage (III-IV) (P = .018) disease. Univariate analysis of the data set showed that higher Pfn1 expression is associated with significantly shorter disease-free survival (hazard ratio 7.36, P = .047) and also lower overall survival. Kaplan-Meier analysis showed that high cytoplasmic expression of Pfn1 was also associated with a statistically significant lower disease-free survival (P = .018). It was also associated with lower overall survival, although this was not statistically significant. Profilin-1 lost its prognostic significance in the multivariate analysis when controlling for grade and stage. Profilin-1 expression was not associated with significant prognostic deference in the subgroup of patients with stage 1 disease. Our results suggest that the evaluation of Pfn1 by immunohistochemistry may help to identify patients with an increased risk of disease progression. We validated our results at the messenger RNA level on an independent patient cohort. Higher messenger RNA expression of Pfn1 is associated with significantly lower survival.

A true theranostic approach to medicine: towards tandem sensor detection and removal of endotoxin in blood.

Sepsis is one of the leading causes of death around the world. The condition occurs when a local infection overcomes the host natural defense mechanism and suddenly spreads into the circulatory system, triggering a vigorous, self-injurious inflammatory host response. The pathogenesis of sepsis is relatively well known, one of the most potent immuno-activator being bacterial lipopolysaccharide (LPS) - also known as 'endotoxin'. Tests exist to detect endotoxin in bodily fluids, but are expensive, not necessarily user-friendly and require reporter molecules. In addition, the situation for safe and effective anti-endotoxin therapy is problematical. At the present time, endotoxin removal through cartridge hemoperfusion is one of the better alternatives to combat sepsis. The capability to both measure endotoxemia levels and offer an adapted response treatment in a timely manner is crucial for better management and improved prognosis, but is currently unavailable. In this context, we describe herein preliminary research towards the development of an alternative LPS biosensor and an innovative LPS neutralization cartridge to be eventually combined in an all-integrated configuration for the theranostic, personalized treatment of blood endotoxemia/sepsis. LPS detection is performed in a real-time and label-free manner in full human blood plasma, using ultra-high frequency acoustic wave sensing in combination with ultrathin, oligoethylene glycol-based mixed surface chemistry imposed on piezoelectric quartz discs. Biosensing platforms are functionalized with polymyxin B (PMB), a cyclic peptide antibiotic with high affinity for LPS. Analogous surface modification is used on glass beads for the therapeutic cartridge component of the combined strategy. Incubation of LPS-spiked whole blood with PMB-bead chemistry resulted in a significant decrease in the production of pro-inflammatory TNF-α cytokine. LPS neutralization is discussed in relation to the perturbation of its supramolecular chemistry in solution.

Quantitative proteomic analysis reveals potential diagnostic markers and pathways involved in pathogenesis of renal cell carcinoma.

There are no serum biomarkers for the accurate diagnosis of clear cell renal cell carcinoma (ccRCC). Diagnosis and decision of nephrectomy rely on imaging which is not always accurate. Non-invasive diagnostic biomarkers are urgently required. In this study, we preformed quantitative proteomics analysis on a total of 199 patients including 30 matched pairs of normal kidney and ccRCC using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify differentially expressed proteins. We found 55 proteins significantly dysregulated in ccRCC compared to normal kidney tissue. 54 were previously reported to play a role in carcinogenesis, and 39 are secreted proteins. Dysregulation of alpha-enolase (ENO1), L-lactate dehydrogenase A chain (LDHA), heat shock protein beta-1 (HSPB1/Hsp27), and 10 kDa heat shock protein, mitochondrial (HSPE1) was confirmed in two independent sets of patients by western blot and immunohistochemistry. Pathway analysis, validated by PCR, showed glucose metabolism is altered in ccRCC compared to normal kidney tissue. In addition, we examined the utility of Hsp27 as biomarker in serum and urine. In ccRCC patients, Hsp27 was elevated in the urine and serum and high serum Hsp27 was associated with high grade (Grade 3-4) tumors. These data together identify potential diagnostic biomarkers for ccRCC and shed new light on the molecular mechanisms that are dysregulated and contribute to the pathogenesis of ccRCC. Hsp27 is a promising diagnostic marker for ccRCC although further large-scale studies are required. Also, molecular profiling may help pave the road to the discovery of new therapies.

Identification and validation of dysregulated metabolic pathways in metastatic renal cell carcinoma.

Metastatic renal cell carcinoma (mRCC) is a devastating disease with a 5-year survival rate of approximately 9 % and low response to chemotherapy and radiotherapy. Targeted therapies have slightly improved patient survival, but are only effective in a small subset of patients, who eventually develop resistance. A better understanding of pathways contributing to tumor progression and metastasis will allow for the development of novel targeted therapies and accurate prognostic markers. We performed extensive bioinformatics coupled with experimental validation on proteins dysregulated in mRCC. Gene ontology analysis showed that many proteins are involved in oxidation reduction, metabolic processes, and signal transduction. Pathway analysis showed metabolic pathways are altered in mRCC including glycolysis and pyruvate metabolism, the citric acid cycle, and the pentose phosphate pathway. RT-qPCR analysis showed that genes involved in the citric acid cycle were downregulated in metastatic RCC while genes of the pentose phosphate pathway were overexpressed. Protein-protein interaction analysis showed that most of the 198 proteins altered in mRCC clustered together and many were involved in glycolysis and pyruvate metabolism. We identified 29 reported regions of chromosomal aberrations in metastatic disease that correlate with the direction of protein dysregulation in mRCC. Furthermore, 36 proteins dysregulated in mRCC are predicted to be targets of metastasis-related miRNAs. A more comprehensive understanding of the pathways dysregulated in metastasis can be useful for the development of new therapies and novel prognostic markers. Also, multileveled analyses provide a unique "snapshot" of the molecular "environment" in RCC with prognostic and therapeutic implications.

MicroRNAs in kidney disease: an emerging understanding.

MicroRNAs (miRNAs) are short noncoding RNA molecules that function by negatively regulating the expression of their target genes in a tightly controlled manner. Accumulating evidence, based in part on effects seen after miRNA overexpression and/or knockdown, points to the critical involvement of miRNAs in kidney function in health and disease. In this review, we provide a quick overview of the biogenesis of miRNAs and their potential involvement in kidney development and normal function. We also discuss the current literature that has begun to uncover the role of miRNAs in the pathogenesis of kidney diseases, including diabetic nephropathy, hypertension, glomerulonephritis, and cancer. As such, miRNAs have potential utility in the clinical realm as disease biomarkers. Moreover, miRNAs represent an attractive therapeutic target for a number of kidney diseases. We close by discussing a number of potential challenges that face the field of miRNA research and clinical use.

Quantitative proteomic analysis in metastatic renal cell carcinoma reveals a unique set of proteins with potential prognostic significance.

Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies, and patients have a dismal prognosis, with a <10% five-year survival rate. The identification of markers that can predict the potential for metastases will have a great effect in improving patient outcomes. In this study, we used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify proteins that are differentially expressed in metastatic and primary RCC. We identified 1256 non-redundant proteins, and 456 of these were quantified. Further analysis identified 29 proteins that were differentially expressed (12 overexpressed and 17 underexpressed) in metastatic and primary RCC. Dysregulated protein expressions of profilin-1 (Pfn1), 14-3-3 zeta/delta (14-3-3ζ), and galectin-1 (Gal-1) were verified on two independent sets of tissues by means of Western blot and immunohistochemical analysis. Hierarchical clustering analysis showed that the protein expression profile specific for metastatic RCC can distinguish between aggressive and non-aggressive RCC. Pathway analysis showed that dysregulated proteins are involved in cellular processes related to tumor progression and metastasis. Furthermore, preliminary analysis using a small set of tumors showed that increased expression of Pfn1 is associated with poor outcome and is a potential prognostic marker in RCC. In addition, 14-3-3ζ and Gal-1 also showed higher expression in tumors with poor prognosis than in those with good prognosis. Dysregulated proteins in metastatic RCC represent potential prognostic markers for kidney cancer patients, and a greater understanding of their involved biological pathways can serve as the foundation of the development of novel targeted therapies for metastatic RCC.

Bench-to-bedside review: Clinical experience with the endotoxin activity assay.

Endotoxin detection in human patients has been a difficult challenge, in part due to the fact that the conserved active portion of the molecule (lipid A) is a relatively small epitope only amenable to binding by a single ligand at any one instance and low levels (pg/ml) are capable of stimulating the immune system. The endotoxin activity assay, a bioassay based on neutrophil activation by complement opsonized immune complexes of lipopolysaccharide (LPS), has allowed the specific detection of the lipid A epitope of LPS in a rapid whole blood assay format. This review summarizes diagnostic studies utilizing the endotoxin activity assay in a variety of hospital patient populations in whom endotoxin is postulated to play a significant role in disease etiology. These include ICU patients at risk of developing 'sepsis syndrome', abdominal and cardiovascular surgery patients and patients with serious traumatic injury. Significant features of these studies include the high negative predictive value of the assay (98.6%) for rule out of Gram-negative infection, ability to risk stratify patients progressing to severe sepsis (odds ratio 3.0) and evidence of LPS release in patients with gut hypoperfusion. Preliminary studies have successfully combined the assay with anti-LPS removal strategies to prospectively identify patients who might benefit from this therapy with early evidence of clinical benefit.

Tranexamic acid concentrations associated with human seizures inhibit glycine receptors.

Antifibrinolytic drugs are widely used to reduce blood loss during surgery. One serious adverse effect of these drugs is convulsive seizures; however, the mechanisms underlying such seizures remain poorly understood. The antifibrinolytic drugs tranexamic acid (TXA) and ε-aminocaproic acid (EACA) are structurally similar to the inhibitory neurotransmitter glycine. Since reduced function of glycine receptors causes seizures, we hypothesized that TXA and EACA inhibit the activity of glycine receptors. Here we demonstrate that TXA and EACA are competitive antagonists of glycine receptors in mice. We also showed that the general anesthetic isoflurane, and to a lesser extent propofol, reverses TXA inhibition of glycine receptor-mediated current, suggesting that these drugs could potentially be used to treat TXA-induced seizures. Finally, we measured the concentration of TXA in the cerebrospinal fluid (CSF) of patients undergoing major cardiovascular surgery. Surprisingly, peak TXA concentration in the CSF occurred after termination of drug infusion and in one patient coincided with the onset of seizures. Collectively, these results show that concentrations of TXA equivalent to those measured in the CSF of patients inhibited glycine receptors. Furthermore, isoflurane or propofol may prevent or reverse TXA-induced seizures.

Plasma methemoglobin as a potential biomarker of anemic stress in humans.

PURPOSE: Transfusion of allogeneic red blood cells (RBCs) is one of the main treatments of acute anemia secondary to blood loss and fluid resuscitation within the operating room. Decisions to transfuse blood are based largely on intermediate biological markers (hemoglobin, arterial oxygen saturation, blood pressure, heart rate) which may not accurately reflect inadequacy of tissue oxygen delivery. Based on experimental studies, we hypothesized that anemia-induced tissue hypoxia activates adaptive mechanisms which promote local vascular nitric oxide (NO) production to improve tissue perfusion and survival during acute anemia. Hemoglobin (Hb) oxidation to methemoglobin (MetHb) may be a byproduct of such local NO production. Therefore, we tested the hypothesis that MetHb is a biomarker of hypoxic-anemic stress during acute hemodilution associated with cardiopulmonary bypass. METHODS: With institutional ethics approval, routine laboratory arterial blood gas and co-oximetry values were obtained from 295 patients undergoing heart surgery during February 1 to September 30, 2010, and the values were assessed retrospectively. All samples with an arterial oxygen saturation value ≥ 90% were included (n = 1,421). The maximal change in Hb associated with hemodilution on cardiopulmonary bypass was determined within 48 hr of surgery (n = 180). A chart review was performed to determine the incidence of RBC transfusion and exogenous nitrate administration. All anonymous data were analyzed by linear regression to determine the relationship between Hb and MetHb. A Wilcoxon Signed Rank Test and Student's t test were used to determine changes in Hb, MetHb, and carboxyhemoglobin (CarboxHb) levels. All data are presented as mean and significance was assigned at P < 0.05. RESULTS: A significant decrease in Hb [118 (20) g x L(-1) vs 94 (18) g x L(-1)] was associated with an increase in MetHb [0.88 (0.22)% vs 0.95 (0.24)%] (P < 0.001 for both), but not CarboxHb [1.08 (0.47)% vs 1.08 (0.49)%]. Regression analysis revealed a significant relationship between the change in Hb and MetHb (F = 40.3; P < 0.001) but not between the change in Hb and CarboxyHb (F = 0.2; P = 0.694). This correlation was not influenced by RBC transfusion or exogenous nitrate use. CONCLUSIONS: A negative correlation was observed between the change in Hb and MetHb in patients undergoing cardiac surgery and cardiopulmonary bypass. These data support the previously unreported hypothesis that MetHb may be a marker of anemic stress associated with reduced tissue perfusion during acute hemodilution in humans. Further prospective studies are needed to determine if these changes in MetHb are linked to adverse outcomes in patients undergoing cardiac surgery.

Identification of novel molecular targets for endometrial cancer using a drill-down LC-MS/MS approach with iTRAQ.

BACKGROUND: The number of patients with endometrial carcinoma (EmCa) with advanced stage or high histological grade is increasing and prognosis has not improved for over the last decade. There is an urgent need for the discovery of novel molecular targets for diagnosis, prognosis and treatment of EmCa, which will have the potential to improve the clinical strategy and outcome of this disease. METHODOLOGY AND RESULTS: We used a "drill-down" proteomics approach to facilitate the identification of novel molecular targets for diagnosis, prognosis and/or therapeutic intervention for EmCa. Based on peptide ions identified and their retention times in the first LC-MS/MS analysis, an exclusion list was generated for subsequent iterations. A total of 1529 proteins have been identified below the Proteinpilot® 5% error threshold from the seven sets of iTRAQ experiments performed. On average, the second iteration added 78% new peptides to those identified after the first run, while the third iteration added 36% additional peptides. Of the 1529 proteins identified, only 40 satisfied our criteria for significant differential expression in EmCa in comparison to normal proliferative tissues. These proteins included metabolic enzymes (pyruvate kinase M2 and lactate dehydrogenase A); calcium binding proteins (S100A6, calcyphosine and calumenin), and proteins involved in regulating inflammation, proliferation and invasion (annexin A1, interleukin enhancer-binding factor 3, alpha-1-antitrypsin, macrophage capping protein and cathepsin B). Network analyses revealed regulation of these molecular targets by c-myc, Her2/neu and TNF alpha, suggesting intervention with these pathways may be a promising strategy for the development of novel molecular targeted therapies for EmCa. CONCLUSIONS: Our analyses revealed the significance of drill-down proteomics approach in combination with iTRAQ to overcome some of the limitations of current proteomics strategies. This study led to the identification of a number of novel molecular targets having therapeutic potential for targeted molecular therapies for endometrial carcinoma.

Endotoxemia related to cardiopulmonary bypass is associated with increased risk of infection after cardiac surgery: a prospective observational study.

INTRODUCTION: Previous studies have documented a high frequency of endotoxemia associated with cardiopulmonary bypass (CPB). Endotoxemia may be responsible for some of the complications associated with cardiac surgery. The purpose of the study was to examine the prevalence of endotoxemia during cardiopulmonary bypass supported aortocoronary bypass grafting surgery (ACB) using a new assay, the Endotoxin Activity Assay (EAA), and explore the association between endotoxemia and post-operative infection. METHODS: The study was a single center prospective observational study measuring EAA during the perioperative period for elective ACB. Blood samples were drawn at induction of anesthesia (T1), immediately prior to release of the aortic cross-clamp (T2), and on the first post-operative morning (T3). The primary outcome was the prevalence of endotoxemia. Secondary outcomes assessed included infection rates, intensive care unit (ICU) and hospital length of stay. An EAA of < 0.40 units was interpreted as "low", 0.41 to 0.59 units as "intermediate", and ≥ 0.60 units as "high". RESULTS: A total of 57 patients were enrolled and 54 patients were analyzable. The mean EAA at T1 was 0.38 +/- 0.14, at T2 0.39 +/- 0.18, and at T3 0.33 +/- 0.18. At T2 only 13.5% (7/52) of patients had an EAA in the high range. There was a positive correlation between EAA and duration of surgery (P = 0.02). In patients with EAA ≥ 0.40 at T2, 26.1% (6/23) of patients developed post-operative infections compared to 3.5% (1/29) of those that had a normal EAA (P = 0.0354). Maximum EAA over the first 24 hours was also strongly correlated with risk of post-operative infection (P = 0.0276). CONCLUSIONS: High levels of endotoxin occur less frequently during ACB than previously documented. However, endotoxemia is associated with a significantly increased risk of the development of post-operative infection. Measuring endotoxin levels during ACB may provide a mechanism to identify and target a high risk patient population.