Bladder cancer leads to approximately 13,000 deaths annually in the United States. Early disease is often treated with minimal morbidity and has good prognosis, while the opposite is true for advanced disease. Currently, no tools exist for early detection of this cancer. Microparticles are small, subcellular particles released by essentially all cells upon activation and are known to be produced constitutively by cancer cells. Since most bladder cancers originate in the urothelial cells lining the lumen of the organ, we hypothesize that these cells will release microparticles into the urine. The goal of this study was to identify potential biomarkers in the urinary microparticles of individuals with bladder cancer. Urine microparticles from five healthy individuals and four individuals with bladder cancer were isolated. Samples were delipidated by PAGE and trypsin-digested, peptides were extracted, and the proteome was examined by LC-MS/MS using a Thermo Finnigan LTQ and LTQ-FT ion trap mass spectrometer. Protein identification was determined by SEQUEST and relative quantitation was assessed by comparing spectral counts. Eight proteins were elevated in the microparticles from individuals with bladder cancer. They include five proteins associated with the epidermal growth factor receptor pathway, the alpha subunit of GsGTP binding protein, resistin, and retinoic acid-induced protein 3. Further studies will be needed to validate these potential biomarkers.

Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the emerging infectious disease human granulocytic anaplasmosis. A. phagocytophilum undergoes a biphasic developmental cycle, transitioning between an infectious dense-cored cell (DC) and a noninfectious reticulate cell (RC). To gain insights into the organism's biology and pathogenesis during human myeloid cell infection, we conducted proteomic analyses on A. phagocytophilum organisms purified from HL-60 cells. A total of 324 proteins were unambiguously identified, thereby verifying 23.7% of the predicted A. phagocytophilum proteome. Fifty-three identified proteins had been previously annotated as hypothetical or conserved hypothetical. The second most abundant gene product, after the well-studied major surface protein 2 (P44), was the hitherto hypothetical protein APH_1235. APH_1235 homologs are found in other Anaplasma and Ehrlichia species but not in other bacteria. The aph_1235 RNA level is increased 70-fold in the DC form relative to that in the RC form. Transcriptional upregulation of and our ability to detect APH_1235 correlate with RC to DC transition, DC exit from host cells, and subsequent DC binding and entry during the next round of infection. Immunoelectron microscopy pronouncedly detects APH_1235 on DC organisms, while detection on RC bacteria minimally, at best, exceeds background. This work represents an extensive study of the A. phagocytophilum proteome, discerns the complement of proteins that is generated during survival within human myeloid cells, and identifies APH_1235 as the first known protein that is pronouncedly upregulated on the infectious DC form.Human granulocytic anaplasmosis (HGA) is an emerging and potentially deadly tick-borne disease (44,53). HGA is an acute febrile illness, the clinical manifestations of which range from subclinical infection to severe disease, including death. Nonspecific symptoms include fever, chills, headache, malaise, and myalgia. Severe complications include prolonged fever, shock, leukopenia, thrombocytopenia, high levels of C-reactive protein and hepatic transaminases, pneumonitis, acute renal failure, and hemorrhages. Immunocompromised and elderly individuals are at greatest risk for fatal opportunistic infections that can be associated with HGA (53). The causative agent of HGA is Anaplasma phagocytophilum, an obligate intracellular bacterium that displays an unusual tropism for granulocytes in humans and several domestic and wild reservoir animal hosts. The annotated genome of the A. phagocytophilum HZ strain, a human isolate, is available (20). The bacterium can be cultivated in the promyelocytic cell line , as well as other cell lines (15,18,37,53), which enables it to be grown in large quantities and has permitted in vitro modeling of infection.Following cellular adhesion, A. phagocytophilum promotes its own uptake into a host cell-derived vacuole that it remodels into a protective safe haven in which it remains and replicates (44,53).A. phagocytoph...

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Background: Suppressor of IB kinase ⑀ (SIKE) inhibits a key innate immune effector molecule, TANK-binding kinase 1 (TBK1), through an undefined mechanism. Results: SIKE is a TBK1 substrate. Conclusion: SIKE controls TBK1 activity by acting as a high affinity substrate. Significance: SIKE attenuates phosphorylation of interferon regulatory factor 3 (IRF3) by serving as an alternative, high affinity substrate for TBK1.

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Traveling wave ion mobility (TWIM) mass spectrometry (MS) is a powerful method for the structural and conformational analysis of proteins and peptides, enabling the differentiation of isomeric peptides (or proteins) that have the same sequence but are modified at different residues. In this study, the TWIM-MS technique was used to separate isomeric Au metallopeptide ions that were formed by Zn displacement from the parent zinc fingers (ZFs). The synthetic gold finger peptides were derived from the C-terminus of the HIV nucleocapsid p7 protein (NCp7-F2) and finger 3 of the Sp1 transcription factor (Sp1-F3). TWIM-MS enabled the acquisition of distinct product ion spectra for each isomer, clearly indicating the binding sites for the major conformers in the presence of multiple coordination possibilities. Collision cross-section measurements showed that the aurated peptide has a slightly more compact structure than the parent zinc compound NCp7-F2, which showed only one conformation.

Background: Males of the cattle tick Rhipicephalus microplus produce salivary immunoglobulin-binding proteins and allotypic variations in IgG are associated with tick loads in bovines. These findings indicate that antibody responses may be essential to control tick infestations. Infestation loads with cattle ticks are heritable: some breeds carry high loads of reproductively successful ticks, in others, few ticks feed and they reproduce inefficiently. Different patterns of humoral immunity against tick salivary proteins may explain these phenotypes.