Shotgun glycomics: a microarray strategy for functional glycomics.

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.

Infection, viral dissemination, and antibody responses of rhesus macaques exposed to the human gammaretrovirus XMRV.

Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.

Diabetes cases on the rise: current diagnosis guidelines and research efforts for a cure.

Diabetes is a devastating disease that accounts for more than $132 billion in healthcare costs annually in the U.S., and these costs are predicted to rise as high as $192 billion by the year 2020 (see recent statistics from AHRQ on page 12). For many people with diabetes, the life expectancy is shorter than that of age-matched non-diabetics. This fate is due to both the microvascular and macrovascular complications resulting from prolonged hyperglycemia. Current ADA guidelines for diagnosis include measures of plasma glucose and Alc, a glycated form of hemoglobin that has been used for many years as a marker of average glycemia. To see how Alc affects the overall number of people in the U.S. diagnosed with diabetes as a result of the test's greater practicality will be interesting. Significant progress has been made in diabetes research through the use of stem-cell technology, molecular DNA methods, and discoveries of novel insulin-controlling systems in the body. Several federally funded diabetes-research centers across the United States are currently continuing these efforts and, ultimately, hope for a cure for diabetes and its complications.

Cancer biomarkers: surviving the journey from bench to bedside.

In summary, the abundance of reported candidate-biomarker proteins in the scientific literature compared to the lack of those reaching clinical use indicates that the aforementioned pipeline bottleneck falls in either the verification or validation phases. To stress this point, Polanski and Anderson compiled a list of 1,261 proteins that have been cited in the literature as being differentially expressed in human cancers.¹ Of the 1,261 proteins, 22% are reported to be present in the blood and should be detectable given a sensitive enough assay. Interestingly, only 5% of these candidates have been thoroughly investigated as biomarkers (greater than 500 citations),¹ with 41 (~3%) actually being used in some clinical capacity. The reason behind so few biomarkers reaching the clinic can largely be explained by the inability of current technologies to consistently and quantitatively verify the presence of the candidates in patient samples and the failure, thus far, to identify biomarkers with high specificity for a particular disease.9 As noted above, none of the nine FDA-approved cancer biomarkers demonstrate the specificity required for diagnosis when used alone. Thus, the development of panels of proteins, such as the FDA-approved OVA1 test,57 may be crucial to achieve the specificity required for early cancer diagnosis, and is interesting to speculate that members of such panels are likely to have already been identified but not yet implemented.58

The anticoagulant effect of protamine sulfate is attenuated in the presence of platelets or elevated factor VIII concentrations.

BACKGROUND: Protamine sulfate is the antidote for heparin, but in excess it exerts weak anticoagulation. METHODS: We evaluated the effects of increasing protamine concentrations (0 to 24 microg/mL) on prothrombin time and diluted Russell's viper venom time measurements on thrombin generation in platelet-poor and platelet-rich plasma after activation by tissue factor or actin, and on thromboelastometry in platelet-poor plasma and whole blood from 6 healthy volunteers. The reversibility of excess protamine (24 microg/mL) by recombinant factor VIIa or factor VIII/von Willebrand factor concentrate was also tested. RESULTS: Protamine prolonged prothrombin time and Russell's viper venom time, concentration dependently. Protamine also increased lag time and decreased peak of thrombin generation in platelet-poor plasma after tissue factor and actin activation. In platelet-rich plasma with platelets at 50 to 200 x 10(3)/microL, protamine (24 microg/mL) prolonged the lag time, but had no effect on peak thrombin generation. The addition of factor VIII/von Willebrand factor (1.5-3.0 U/mL) to platelet-poor plasma with protamine (24 microg/mL) decreased lag time and increased peak thrombin generation with actin activation. A therapeutic concentration of recombinant factor VIIa (60 nM) only affected the lag time of thrombin generation triggered with actin. In agreement, protamine increased coagulation time evaluated by thromboelastometry significantly more in platelet-poor plasma than in whole blood. CONCLUSIONS: We demonstrated that protamine affects the propagation of thrombin generation, which is partially reversed by platelets or increased factor VIII/von Willebrand factor concentrations. The present data suggest that excess protamine might potentially increase bleeding in the case of severe thrombocytopenia or low factor VIII.

In vitro comparative study of hemostatic components in warfarin-treated and fibrinogen-deficient plasma.

OBJECTIVE: The authors hypothesized that various hemostatic products may differently affect viscoelastic clot formation depending on their respective procoagulant activity and fibrinogen content. DESIGN: In vitro coagulopathy modeling using warfarin-treated plasma (international normalized ratio, 2.8-3.8) and fibrinogen-deficient plasma evaluated by rotational thromboelastometry (ROTEM; Pentapharm, Munich, Germany). SETTING: A university laboratory. INTERVENTION: Different volumes of cryoprecipitate, fresh frozen plasma (FFP), fibrinogen concentrate, and platelet concentrate were mixed with each abnormal plasma to simulate the in vivo transfusions of 250 mL to 1,000 mL. Three thromboelastometric variables that reflect the rate and extent of clot growth were measured: (1) coagulation time (CT), (2) angle, and (3) maximal clot firmness (MCF). MEASUREMENTS AND MAIN RESULTS: In warfarin-treated plasma, the addition of FFP, cryoprecipitate, and platelets led to a dose-dependent improvement of CT and angle, whereas MCF increased with cryoprecipitate or platelets only. The addition of fibrinogen concentrate improved MCF and angle but not CT. In fibrinogen-deficient plasma, the addition of cryoprecipitate, platelets, and fibrinogen concentrate led to a dose-dependent improvement of ROTEM variables, whereas the addition of FFP resulted in significantly longer CT and lower MCF values compared with other hemostatic products. The addition of platelets in the presence of cytochalasin D (a platelet inhibitor) resulted in improvements of ROTEM variables that were similar to when FFP was added to warfarin-treated and fibrinogen-deficient plasma. CONCLUSIONS: Cryoprecipitate supports clot formation on ROTEM more efficiently than FFP because of the high fibrinogen content. Improved ROTEM variables after platelet addition are presumably caused by increased interaction among thrombin-activated platelets and fibrinogen.

Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.

Ribonuclease L (RNase L) is an important effector of the innate antiviral response. Mutations or variants that impair function of RNase L, particularly R462Q, have been proposed as susceptibility factors for prostate cancer. Given the role of this gene in viral defense, we sought to explore the possibility that a viral infection might contribute to prostate cancer in individuals harboring the R462Q variant. A viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses identified the presence of gammaretroviral sequences in cDNA samples from seven of 11 R462Q-homozygous (QQ) cases, and in one of eight heterozygous (RQ) and homozygous wild-type (RR) cases. An expanded survey of 86 tumors by specific RT-PCR detected the virus in eight of 20 QQ cases (40%), compared with only one sample (1.5%) among 66 RQ and RR cases. The full-length viral genome was cloned and sequenced independently from three positive QQ cases. The virus, named XMRV, is closely related to xenotropic murine leukemia viruses (MuLVs), but its sequence is clearly distinct from all known members of this group. Comparison of gag and pol sequences from different tumor isolates suggested infection with the same virus in all cases, yet sequence variation was consistent with the infections being independently acquired. Analysis of prostate tissues from XMRV-positive cases by in situ hybridization and immunohistochemistry showed that XMRV nucleic acid and protein can be detected in about 1% of stromal cells, predominantly fibroblasts and hematopoietic elements in regions adjacent to the carcinoma. These data provide to our knowledge the first demonstration that xenotropic MuLV-related viruses can produce an authentic human infection, and strongly implicate RNase L activity in the prevention or clearance of infection in vivo. These findings also raise questions about the possible relationship between exogenous infection and cancer development in genetically susceptible individuals.

Selection and cloning of poly(rC)-binding protein 2 and Raf kinase inhibitor protein RNA activators of 2',5'-oligoadenylate synthetase from prostate cancer cells.

The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2',5'-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2.

Targeting HbA1c: standardization and clinical laboratory measurement.

In this article, we have provided a review of the implications of diabetes and HbA1c, the standardization efforts of HbAlc as a long-term monitor of average glycemia, the pathobiology of HbA1c, as well as current measurement pitfalls associated with clinical-laboratory measurements. Long-term studies, including the DCCT and UKPDS, have established a correlation between average blood glucose (HbA1c) and the severity of diabetic complications, thereby providing clinicians and diabetic patients with established HbA1c goals to reduce these problems. This led to an increased need for standardization across all laboratories that perform HbA1c measurements. Through their significant efforts, the NGSP and IFCC have paved the way toward achieving this goal. Despite these advances, the various HbAlc methods that are currently available each have specific limitations associated with the presence of Hb variants. In areas where there is a high prevalence of Hb variants, HbA1c methods must be carefully selected to reduce the inaccuracy of these measurements. Alternative methods of determining average blood-glucose control (e.g., glycated albumin and fructosamine) are recommended in these populations in which HbA1c determinations have limited value. Unfortunately, as of yet there are no established guidelines or goals that can be followed by clinicians or diabetic patients regarding HbA1c or other glycated protein values in these populations. Importantly, clinicians should be cautious when using glycated albumin and fructosamine as an estimate of long-term average blood glucose. First, glycated albumin and fructosamine assess the degree of glycemia over a period of approximately two weeks, as opposed to two to three months, for HbA1c. Second, glycated albumin and fructosamine have not been correlated with the development of long-term complications from diabetes mellitus, as was shown with HbA1c in the DCCT or UKPDS.

An alternative spliced RNASEL variant in peripheral blood leukocytes.

2-5A-Dependent RNase L is an endoribonuclease that catalyzes RNA degradation and promotes apoptosis during the innate antiviral response in mammalian cells. Prior studies showed that RNASEL is widely expressed and suggested the presence of mRNA species of different sizes but lacked a characterization of these variants. Using RT-PCR, we show that RNASEL is expressed in all human tissues examined, whereas an alternatively generated spliced variant lacking the third exon (RNASEL del_Ex3) is solely expressed in peripheral blood leukocytes (PBL). Quantitative RT-PCR measurements of RNA from different PBL cell types separated by fluorescence activated cell sorting (FACS) showed that complete RNASEL mRNA levels were significantly elevated in granulocytes compared with all other PBL cell types, whereas expression was lowest in CD8(+) T cells. The alternatively spliced RNASEL del_Ex3 transcript was present in all PBL cell types examined but at lower levels than the full-length RNASEL mRNA. The presence of high levels of RNase L protein in granulocytes was confirmed by immunohistochemistry. Our findings are the first to demonstrate the presence of an alternatively spliced RNASEL mRNA and to demonstrate the variable expression of RNase L in different leukocytes. Our results suggest that RNase L plays an important role in granulocytes as an innate immunity enzyme that controls viral infections.

Mapping of the human RNASEL promoter and expression in cancer and normal cells.

2',5'-Oligoadenylate (2-5A)-dependent RNase L is a ubiquitous endoribonuclease of higher vertebrates that functions in the interferon (IFN) antiviral response by degrading both viral and cellular single-stranded RNA (ssRNA). In addition, the RNase L gene, RNASEL, was mapped to the hereditary prostate cancer 1 (HPC1) gene. Previous analyses of human RNASEL determined its exon/intron structure but lacked a description of the promoter region. We thus mapped the RNASEL transcriptional start site using 5'-rapid amplification of cDNA ends (5'-RACE) and primer extension methods with RNA from human histiocytic lymphoma U937 cells. The promoter sequence was analyzed for potential transcription factor binding sites. Although a canonical IFN-gamma activation site (GAS) element (TTCCAAGAA) was identified (nucleotides -155 to -147), there was only slight induction of RNASEL promoter-reporter activity or of endogenous RNase L expression in response to IFN-alpha or IFN-gamma. Several sites for tissue-specific and general promoters were observed, however, which could explain the widespread expression of RNase L in mammalian cells. Accordingly, RNase L levels were determined and compared in different human and rodent cancer and normal cell types using a radiolabeled 2-5A derivative. In addition, levels of RNase L were established in various normal human tissues and cell types by immunoblotting and immunohistochemistry. Our findings are the first description of the human RNASEL promoter that allows constituitive expression in a range of normal and neoplastic cell types.

Direct total and free testosterone measurement by liquid chromatography tandem mass spectrometry across two different platforms.

OBJECTIVES: To develop and validate liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the direct measurement of total and free testosterone in patient samples on two different analytical systems. DESIGN AND METHODS: An API 4000 and 5000 triple quadropoles were used and compared; the former is reported to be 3-5 times less sensitive, as was used to set the quantitation limits. Free testosterone was separated from the protein-bound fraction by equilibrium dialysis followed by derivatization. Either free or total testosterone, and a deuterated internal standard (d3-testosterone) were extracted by liquid-liquid extraction. The validation results were compared to two different clinical laboratories. RESULTS: The use of d2-testosterone was found to be unacceptable for our method. The total testosterone LC-MS/MS methods on both systems were linear over a wide concentration range of 1.5-2000ng/dL. Free testosterone was measured directly using equilibrium dialysis coupled LC-MS/MS and linear over the concentration range of 2.5-2500pg/mL. Good correlation (total testosterone, R(2)=0.96; free testosterone, R(2)=0.98) was observed between our LC-MS/MS systems and comparator laboratory. However, differences in absolute values for both free and total testosterone measurements were observed while a comparison to a second published LC-MS/MS method showed excellent correlation. Free and total testosterone measurements correlated well with clinical observations. CONCLUSIONS: To our knowledge, this is the first published validation of free and total testosterone methods across two analytical systems of different analytical sensitivities. A less sensitive system does not sacrifice analytical or clinical sensitivity to directly measure free and total testosterone in patient samples.

Development of a liquid chromatography tandem mass spectrometry method for iothalamate measurement to assess renal function for potential kidney donation.

BACKGROUND: Chronic kidney disease often goes undetected due to the insensitivity of current methods to accurately assess glomerular filtration rate (GFR) in early stages of renal dysfunction. The clearance of exogenously introduced iothalamate, a commonly used radiopaque agent, is an alternative to inulin clearance for the assessment of renal function and its use in calculating GFR can serve as a screening tool for kidney transplant donors. METHODS: A method was developed to measure iothalamate in plasma and urine samples by HPLC combined with electrospray positive ionization tandem mass spectrometry (MS/MS). Iothalamate is isolated from plasma by methanol extraction and urine using a quick-spin filtration approach, then monitored by multiple reaction monitoring using the hydrogen adduct mass transitions. Iohexol was used as an internal standard. RESULTS: Iothalamate was measured within an analytical run time of 5 min, with a lower limit of quantification of 18.75 ng/ml. The intraassay and interassay variations of the plasma and urine iothalamate assays were both <9%. Recovery from plasma and urine samples ranged from 93.6% to 104.1%. GFR was calculated using the patient's urine flow rate and plasma and urine iothalamate values. Linear correlations tested by LC-MS/MS and an accepted capillary electrophoresis (CE) assay showed similar results (GFR, r=0.92, Sy/x=10.3). CONCLUSIONS: We developed and validated an LC-MS/MS method for quantitating iothalamate in plasma and urine to calculate GFR used for screening potential kidney donors in our hospital system. A less sensitive mass spectrometry system does not sacrifice analytical or clinical sensitivity for measuring GFR.