Review of optical reporters of radiation effects in vivo: tools to quantify improvements in radiation delivery technique.

Significance: Radiation damage studies are used to optimize radiotherapy treatment techniques. Although biological indicators of damage are the best assays of effect, they are highly variable due to biological heterogeneity. The free radical radiochemistry can be assayed with optical reporters, allowing for high precision titration of techniques. Aim: We examine the optical reporters of radiochemistry to highlight those with the best potential for translational use in vivo, as surrogates for biological damage assays, to inform on mechanisms. Approach: A survey of the radical chemistry effects from reactive oxygen species (ROS) and oxygen itself was completed to link to DNA or biological damage. Optical reporters of ROS include fluorescent, phosphorescent, and bioluminescent molecules that have a variety of activation pathways, and each was reviewed for its in vivo translation potential. Results: There are molecular reporters of ROS having potential to report within living systems, including derivatives of luminol, 2'7'-dichlorofluorescein diacetate, Amplex Red, and fluorescein. None have unique specificity to singular ROS species. Macromolecular engineered reporters unique to specific ROS are emerging. The ability to directly measure oxygen via reporters, such as Oxyphor and protoporphyrin IX, is an opportunity to quantify the consumption of oxygen during ROS generation, and this translates from in vitro to in vivo use. Emerging techniques, such as ion particle beams, spatial fractionation, and ultra-high dose rate FLASH radiotherapy, provide the motivation for these studies. Conclusions: In vivo optical reporters of radiochemistry are quantitatively useful for comparing radiotherapy techniques, although their use comes at the cost of the unknown connection to the mechanisms of radiobiological damage. Still their lower measurement uncertainty, compared with biological response assay, makes them an invaluable tool. Linkage to DNA damage and biological damage is needed, and measures such as oxygen consumption serve as useful surrogate measures that translate to in vivo use.

Mapping estimates of vascular permeability with a clinical indocyanine green fluorescence imaging system in experimental pancreatic adenocarcinoma tumors.

Significance: Pancreatic cancer tumors are known to be avascular, but their neovascular capillaries are still chaotic leaky vessels. Capillary permeability could have significant value for therapy assessment, and its quantification might be possible with macroscopic imaging of indocyanine green (ICG) kinetics in tissue. Aim: The capacity of using standard fluorescence surgical systems for ICG kinetic imaging as a probe for capillary leakage was evaluated using a clinical surgical fluorescence imaging system, as interpreted through vascular permeability modeling. Approach: Xenograft pancreatic adenocarcinoma models were imaged in mice during bolus injection of ICG to capture the kinetics of uptake. Image analysis included ratiometric data, normalization, and match to theoretical modeling. Kinetic data were converted into the extraction fraction of the capillary leakage. Results: Pancreatic tumors were usually less fluorescent than the surrounding healthy tissues, but still the rate of tumor perfusion could be assessed to quantify capillary extraction. Model simulations showed that flow kinetics stabilized after about 1 min beyond the initial bolus injection and that the relative extraction fraction model estimates matched the experimental data of normalized uptake within the tissue. The kinetics in the time period of 1 to 2 min post-injection provided optimal differential data between AsPC1 and BxPC3 tumors, although high individual variation exists between tumors. Conclusions: ICG kinetic imaging during the initial leakage phase was diagnostic for quantitative vascular permeability within pancreatic tumors. Methods for autogain correction and normalized model-based interpretation allowed for quantification of extraction fraction and difference identification between tumor types in early timepoints.

Identification of mosquito proteins that differentially interact with alphavirus nonstructural protein 3, a determinant of vector specificity.

Chikungunya virus (CHIKV) and the closely related onyong-nyong virus (ONNV) are arthritogenic arboviruses that have caused significant, often debilitating, disease in millions of people. However, despite their kinship, they are vectored by different mosquito subfamilies that diverged 180 million years ago (anopheline versus culicine subfamilies). Previous work indicated that the nonstructural protein 3 (nsP3) of these alphaviruses was partially responsible for this vector specificity. To better understand the cellular components controlling alphavirus vector specificity, a cell culture model system of the anopheline restriction of CHIKV was developed along with a protein expression strategy. Mosquito proteins that differentially interacted with CHIKV nsP3 or ONNV nsP3 were identified. Six proteins were identified that specifically bound ONNV nsP3, ten that bound CHIKV nsP3 and eight that interacted with both. In addition to identifying novel factors that may play a role in virus/vector processing, these lists included host proteins that have been previously implicated as contributing to alphavirus replication.

A mAb for the detection of the antiretroviral drug emtricitabine.

Antibody-based testing for emtricitabine (FTC), a critical component of pre-exposure prophylaxis and antiretroviral therapy, would provide low-cost detection for clinical monitoring to improve adherence. We developed a mAb (5D2) to FTC and demonstrated its high specificity and physiologically relevant linear range of detection in a competitive enzyme immunoassay. Thus, this mAb is a key reagent that will enable simple and low-cost lateral flow assays and enzyme immunoassays for adherence monitoring.

Heterogeneous Ribonucleoprotein A1 (hnRNPA1) Interacts with the Nucleoprotein of the Influenza a Virus and Impedes Virus Replication.

Influenza A virus (IAV), like other viruses, depends on the host cellular machinery for replication and production of progeny. The relationship between a virus and a host is complex, shaped by many spatial and temporal interactions between viral and host proteome, ultimately dictating disease outcome. Therefore, it is imperative to identify host-virus interactions as crucial determinants of disease pathogenies. Heterogeneous ribonucleoprotein A1 (hnRNPA1) is an RNA binding protein involved in the life cycle of many DNA and RNA viruses; however, its role in IAV remains undiscovered. Here we report that human hnRNPA1 physically interacts with the nucleoprotein (NP) of IAV in mammalian cells at different time points of the viral replication cycle. Temporal distribution studies identify hnRNPA1 and NP co-localize in the same cellular milieu in both nucleus and mitochondria in NP-transfected and IAV-infected mammalian cells. Interestingly, hnRNPA1 influenced NP gene expression and affected viral replication. Most importantly, hnRNPA1 knockdown caused a significant increase in NP expression and enhanced viral replication (93.82%) in IAV infected A549 cells. Conversely, hnRNPA1 overexpression reduced NP expression at the mRNA and protein levels and impeded virus replication by (60.70%), suggesting antagonistic function. Taken together, results from this study demonstrate that cellular hnRNPA1 plays a protective role in the host hitherto unknown and may hold potential as an antiviral target to develop host-based therapeutics against IAV.

Development of a Multiplex Bead Assay To Detect Immunoglobulin G Antibodies to Babesia duncani in Human Serum.

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Evaluation of Thyroid Function in Pregnant Women Using Automated Immunoassays.

INTRODUCTION: Automated free thyroxine (FT4) immunoassays are widely available, but professional guidelines discourage their use in pregnant women due to theoretical under-recoveries attributed to increased thyroid hormone binding capacity and instead advocate the use of total T4 (TT4) or free thyroxine index (FTI). The impact of this recommendation on the classification of thyroid status in apparently euthyroid pregnant patients was evaluated. METHODS: After excluding specimens with thyroid autoantibody concentrations above reference limits, thyroid-stimulating hormone (TSH), FT4, TT4, and T-uptake were measured on the Roche Cobas® platform in remnant clinical specimens from at least 147 nonpregnant women of childbearing age and pregnant women at each trimester. Split-sample comparisons of FT4 as measured by the Cobas and equilibrium dialysis were performed. RESULTS: FT4 decreased with advancing gestational age by both immunoassay and equilibrium dialysis. TSH declined during the first trimester, remained constant in the second, and increased throughout the third, peaking just before delivery. Interpretation of TT4 concentrations using 1.5-times the nonpregnant reference interval classified 13.6% of first trimester specimens below the lower reference limit despite TSH concentrations within trimester-specific reference intervals. Five FTI results from 480 pregnant individuals (about 1.0%) fell outside the manufacturer's reference interval. CONCLUSIONS: Indirect FT4 immunoassay results interpreted in the context of trimester-specific reference intervals provide a practical and viable alternative to TT4 or FTI. Declining FT4 and increasing TSH concentrations near term suggest that declining FT4 is not an analytical artifact but represents a true physiological change in preparation for labor and delivery.

Influenza A Virus Nucleoprotein Activates the JNK Stress-Signaling Pathway for Viral Replication by Sequestering Host Filamin A Protein.

Influenza A virus (IAV) poses a major threat to global public health and is known to employ various strategies to usurp the host machinery for survival. Due to its fast-evolving nature, IAVs tend to escape the effect of available drugs and vaccines thus, prompting the development of novel antiviral strategies. High-throughput mass spectrometric screen of host-IAV interacting partners revealed host Filamin A (FLNA), an actin-binding protein involved in regulating multiple signaling pathways, as an interaction partner of IAV nucleoprotein (NP). In this study, we found that the IAV NP interrupts host FLNA-TRAF2 interaction by interacting with FLNA thus, resulting in increased levels of free, displaced TRAF2 molecules available for TRAF2-ASK1 mediated JNK pathway activation, a pathway critical to maintaining efficient viral replication. In addition, siRNA-mediated FLNA silencing was found to promote IAV replication (87% increase) while FLNA-overexpression impaired IAV replication (65% decrease). IAV NP was observed to be a crucial viral factor required to attain FLNA mRNA and protein attenuation post-IAV infection for efficient viral replication. Our results reveal FLNA to be a host factor with antiviral potential hitherto unknown to be involved in the IAV replication cycle thus, opening new possibilities of FLNA-NP interaction as a candidate anti-influenza drug development target.

Acetylation by Eis and Deacetylation by Rv1151c of Mycobacterium tuberculosis HupB: Biochemical and Structural Insight.

Bacterial nucleoid-associated proteins (NAPs) are critical to genome integrity and chromosome maintenance. Post-translational modifications of bacterial NAPs appear to function similarly to their better studied mammalian counterparts. The histone-like NAP HupB from Mycobacterium tuberculosis (Mtb) was previously observed to be acetylated by the acetyltransferase Eis, leading to genome reorganization. We report biochemical and structural aspects of acetylation of HupB by Eis. We also found that the SirT-family NAD+-dependent deacetylase Rv1151c from Mtb deacetylated HupB in vitro and characterized the deacetylation kinetics. We propose that activities of Eis and Rv1151c could regulate the acetylation status of HupB to remodel the mycobacterial chromosome in response to environmental changes.

Molecular Pathogenesis of Chlamydia Disease Complications: Epithelial-Mesenchymal Transition and Fibrosis.

The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor ß (TGF-ß), TGF-ß receptor 1 (TGF-ßR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications.

Role of Epithelial-Mesenchyme Transition in Chlamydia Pathogenesis.

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.

Ethanol Attenuates Histiotrophic Nutrition Pathways and Alters the Intracellular Redox Environment and Thiol Proteome during Rat Organogenesis.

Ethanol (EtOH) is a reactive oxygen-generating teratogen involved in the etiology of structural and functional developmental defects. Embryonic nutrition, redox environment, and changes in the thiol proteome following EtOH exposures (1.56.0 mg/ml) were studied in rat whole embryo culture. Glutathione (GSH) and cysteine (Cys) concentrations with their respective intracellular redox potentials (Eh) were determined using high-performance liquid chromatography. EtOH reduced GSH and Cys concentrations in embryo (EMB) and visceral yolk sac (VYS) tissues, and also in yolk sac and amniotic fluids. These changes produced greater oxidation as indicated by increasingly positive Eh values. EtOH reduced histiotrophic nutrition pathway activities as measured by the clearance of fluorescin isothiocyanate (FITC)-albumin from culture media. A significant decrease in total FITC clearance was observed at all concentrations, reaching approximately 50% at the highest dose. EtOH-induced changes to the thiol proteome were measured in EMBs and VYSs using isotope-coded affinity tags. Decreased concentrations for specific proteins from cytoskeletal dynamics and endocytosis pathways (α-actinin, α-tubulin, cubilin, and actin-related protein 2); nuclear translocation (Ran and RanBP1); and maintenance of receptor-mediated endocytosis (cubilin) were observed. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis also identified a decrease in ribosomal proteins in both EMB and VYS. Results show that EtOH interferes with nutrient uptake to reduce availability of amino acids and micronutrients required by the conceptus. Intracellular antioxidants such as GSH and Cys are depleted following EtOH and Eh values increase. Thiol proteome analysis in the EMB and VYS show selectively altered actin/cytoskeleton, endocytosis, ribosome biogenesis and function, nuclear transport, and stress-related responses.

Comprehensive laboratory evaluation of a specific lateral flow assay for the presumptive identification of abrin in suspicious white powders and environmental samples.

Abrin is a heterodimeric toxin present in the seeds of the Abrus precatorius plant. The easily obtainable seeds can yield a highly toxic product that can be used in various types of biocrimes and terrorism-related activities, including "white-powder" letters. Although the vast majority of these threats are hoaxes, the lack of rapid and reliable detection assays for abrin, such as lateral flow assays (LFAs), can be an impediment to accurate and rapid hazard assessment. One of the complicating factors associated with LFAs is the use of antibodies of poor affinity and specificity that cross-react with near neighbors or that bind to plant lectins, which are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the critical need to promote public safety and public health, we conducted a comprehensive laboratory evaluation of a commercial LFA for the rapid detection of abrin. This study was conducted using comprehensive inclusivity and exclusivity panels of abrin and near-neighbor plant materials, along with panels of lectins, related proteins, white powders, and environmental background material, to determine the sensitivity, specificity, limit of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples for the presumptive presence of abrin.

Inhibition of glutathione biosynthesis alters compartmental redox status and the thiol proteome in organogenesis-stage rat conceptuses.

Developmental signals that control growth and differentiation are regulated by environmental factors that generate reactive oxygen species (ROS) and alter steady-state redox environments in tissues and fluids. Protein thiols are selectively oxidized and reduced in distinct spatial and temporal patterns in conjunction with changes in glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox potentials (E(h)) to regulate developmental signaling. The purpose of this study was to measure compartment-specific thiol redox status in cultured organogenesis-stage rat conceptuses and to evaluate the impact of thiol oxidation on the redox proteome. The visceral yolk sac (VYS) has the highest initial (0 h) total intracellular GSH (GSH+2GSSG) concentration (5.5 mM) and the lowest Eh (-223 mV) as determined by HPLC analysis. Total embryo (EMB) GSH concentrations ranged lower (3.2 mM) and were only slightly more oxidized than the VYS. Total GSH concentrations in yolk sac fluid (YSF) and amniotic fluid (AF) are >500-fold lower than in tissues and are highly oxidized (YSF E(h)=-121 mV and AF E(h)=-49 mV). Steady-state total Cys concentrations (Cys+2CySS) were significantly lower than GSH in tissues but were otherwise equal in VYS and EMB near 0.5 mM. On gestational day 11, total GSH and Cys concentrations in EMB and VYS increase significantly over the 6h time course while E(h) remains relatively constant. The Eh (GSH/GSSG) in YSF and AF become more reduced over time while E(h) (Cys/CySS) become more oxidized. Addition of L-buthionine-S,R-sulfoximine (BS0) to selectively inhibit GSH synthesis and mimic the effects of some GSH-depleting environmental chemicals significantly decreased VYS and EMB GSH and Cys concentrations and increased Eh over the 6h exposure period, showing a greater overall oxidation. In the YSF, BSO caused a significant increase in total Cys concentrations to 1.7 mM but did not significantly change the E(h) for Cys/CySS. A significant net oxidation was seen in the BSO-treated AF compartment after 6 h. Biotinylated iodoacetamide (BIAM) labeling of proteins revealed the significant thiol oxidation of many EMB proteins following BSO treatment. Quantitative changes in the thiol proteome, associated with developmentally relevant pathways, were detected using isotope coded affinity tag (ICAT) labeling and mass spectroscopy. Adaptive pathways were selectively enriched with increased concentrations of proteins involved in mRNA processing (splicesome) and mRNA stabilization (glycolysis, GAPDH), as well as protein synthesis (aminoacyl-tRNA) and protein folding (antigen processing, Hsp70, protein disulfide isomerase). These results show the ability of chemical and environmental modulators to selectively alter compartmental intracellular and extracellular GSH and Cys concentrations and change their corresponding E(h) within the intact viable conceptus. The altered E(h) were also of sufficient magnitude to alter the redox proteome and change relative protein concentrations, suggesting that the mechanistic links through which environmental factors inform and regulate developmental signaling pathways may be discovered using systems developmental biology techniques.

Concurrent serotyping and genotyping of pneumococci by use of PCR and electrospray ionization mass spectrometry.

A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ionization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65- to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single- and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates.

Genomic signature-based identification of influenza A viruses using RT-PCR/electro-spray ionization mass spectrometry (ESI-MS) technology.

BACKGROUND: The emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza "core" genes. Combination of the BC signatures represents a "genomic print" of an influenza A virus. METHODOLOGY/PRINCIPAL FINDINGS: Here, 757 samples collected between 2006 and 2009 were tested, including 302 seasonal H1N1, 171 H3N2, 7 swine triple reassortants, and 277 H1N1pdm viruses. Of the 277 H1N1pdm samples, 209 were clinical specimens (throat, nasal and nasopharyngeal swabs, nasal washes, blood and sputum). BC signatures for the clinical specimen from one of the first cases of the 2009 pandemic, A/California/04/2009, confirmed it as an unusual, previously unrecognized influenza A virus, with "core" genes related to viruses of avian, human and swine origins. Subsequent analysis of additional 276 H1N1pdm samples revealed that they shared the genomic print of A/California/04/2009, which differed from those of North American swine triple reassortant viruses, seasonal H1N1 and H3N2 and other viruses tested. Moreover, this assay allowed distinction between "core" genes of co-circulating groups of seasonal H1N1, such as clades 2B, 2C, and their reassortants with dual antiviral resistance to adamantanes and oseltamivir. CONCLUSIONS/SIGNIFICANCE: The RT-PCR/ESI-MS assay is a broad range influenza identification tool that can be used directly on clinical specimens for rapid and accurate detection of influenza virus genes. The assay differentiates the H1N1pdm from seasonal and other nonhuman hosts viruses. Although not a diagnostic tool, this assay demonstrates its usefulness and robustness in influenza virus surveillance and detection of novel and unusual viruses with previously unseen genomic prints.

Proteomic identification of biomarkers in the cerebrospinal fluid (CSF) of astrocytoma patients.

The monitoring of changes in the protein composition of the cerebrospinal fluid (CSF) can be used as a sensitive indicator of central nervous system (CNS) pathology, yet its systematic application to analysis of CNS neoplasia has been limited. There is a pressing need for both a better understanding of gliomagenesis and the development of reliable biomarkers of the disease. In this report, we used two proteomic techniques, two-dimensional gel electrophoresis (2-DE), and cleavable Isotope-Coded Affinity Tag (cICAT) to compare CSF proteomes to identify tumor- and grade-specific biomarkers in patients bearing brain tumors of differing histologies and grades. Retrospective analyses were performed on 60 samples derived from astrocytomas WHO grade II, III, and IV, schwannomas, metastastic brain tumors, inflammatory samples, and non-neoplastic controls. We identified 103 potential tumor-specific markers of which 20 were high-grade astrocytoma-specific. These investigations allowed us to identify a spectrum of signature proteins that could be used to distinguish CSF derived from control patients versus those with low- (AII) or high-grade (AIV) astrocytoma. These proteins may represent new diagnostic, prognostic, and disease follow-up markers when used alone or in combination. These candidate biomarkers may also have functional properties that play a critical role in the development and malignant progression of human astrocytomas, thus possibly representing novel therapeutic targets for this highly lethal disease.

Cytosolic Arl2 is complexed with cofactor D and protein phosphatase 2A.

Arl2 is a member of the ADP-ribosylation factor family of 20-kDa GTPases that is highly conserved in eukaryotes. Recent results revealed that a portion of cellular Arl2 and its binding partner, BART, localize to mitochondria. Because approximately 90% of cellular Arl2 is cytosolic, we investigated properties of the soluble protein and found that it is stably bound in a complex that migrates in gel filtration medium with a predicted molecular mass of approximately 300 kDa. This complex was purified approximately 500-fold from the soluble fraction of bovine brain. Protein components were identified by mass spectroscopy and revealed the presence of four other proteins that include the tubulin folding cochaperone cofactor D and all three subunits of at least two protein phosphatase 2A (PP2A) protein phosphatase trimers. The presence of more than one PP2A B-type subunit and the low stoichiometry of Arl2 indicate that the purified preparation still contains a mixture of complexes that cannot currently be completely resolved. Thus, although all the soluble Arl2 in bovine brain is in high molecular mass complexes, only a portion of the total cellular cofactor D and PP2A are associated with the Arl2. We further show that the Arl2 in the complex cannot bind GTP and that complexed cofactor D does not efficiently participate in tubulin refolding reactions in a manner comparable with free cofactor D. Our data suggest functional roles for the cytosolic Arl2 complex in modulating tubulin and microtubule behavior as well as a possible role in apoptosis.

Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif.

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.