Precursor Ion Independent Algorithm for Top-Down Shotgun Proteomics

We present a precursor ion independent top-down algorithm (PIITA) for use in automated assignment of protein identifications from tandem mass spectra of whole proteins. To acquire the data, we utilize data-dependent acquisition to select protein precursor ions eluting from a C4-based HPLC column for collision induced dissociation in the linear ion trap of an LTQ-Orbitrap mass spectrometer. Gas-phase fractionation is used to increase the number of acquired tandem mass spectra, all of which are recorded in the Orbitrap mass analyzer. To identify proteins, the PIITA algorithm compares deconvoluted, deisotoped, observed tandem mass spectra to all possible theoretical tandem mass spectra for each protein in a genomic sequence database without regard for measured parent ion mass. Only after a protein is identified, is any difference in measured and theoretical precursor mass used to identify and locate post-translation modifications. We demonstrate the application of PIITA to data generated via our wet-lab approach on a Salmonella typhimurium outer membrane extract and compare these results to bottom-up analysis. From these data, we identify 154 proteins by top-down analysis, 73 of which were not identified in a parallel bottom-up analysis. We also identify 201 unique isoforms of these 154 proteins at a false discovery rate (FDR) of <1%

Microcystic adnexal carcinoma (MAC)-like squamous cell carcinoma as a differential diagnosis to Bell´s palsy: review of guidelines for refractory facial nerve palsy

Background Bell´s palsy is the most common cause of facial paralysis worldwide and the most common disorder of the cranial nerves. It is a diagnosis of exclusion, accounting for 60–75% of all acquired peripheral facial nerve palsies. Our case shows the first case of a microcystic adnexal carcinoma-like squamous cell carcinoma as a cause of facial nerve palsy. Case presentation The patient, a 70-year-old Caucasian male, experienced subsequent functional impairment of the trigeminal and the glossopharyngeal nerve about 1½ years after refractory facial nerve palsy. An extensive clinical work-up and tissue biopsy of the surrounding parotid gland tissue was not able to determine the cause of the paralysis. Primary infiltration of the facial nerve with subsequent spreading to the trigeminal and glossopharyngeal nerve via neuroanastomoses was suspected. After discussing options with the patient, the main stem of the facial nerve was resected to ascertain the diagnosis of MAC-like squamous cell carcinoma, and radiochemotherapy was subsequently started. Conclusion This case report shows that even rare neoplastic etiologies should be considered as a cause of refractory facial nerve palsy and that it is necessary to perform an extended diagnostic work-up to ascertain the diagnosis. This includes high-resolution MRI imaging and, as perilesional parotid biopsies might be inadequate for rare cases like ours, consideration of a direct nerve biopsy to establish the right diagnosis

Qualität in der Befundung von Kopf- und Halssonographien an Universitätskliniken – eine Stichprobe

Background Ultrasound diagnostics are widely used and are standard for radiologists, otolaryngologists, and oral and maxillofacial surgeons in the diagnostic work-up of various pathologies. There is agreement that digital documentation is urgently needed at present to improve and standardize the quality of sonographic documentation. There are more and more publications on the implementation of standardized documentation of findings in imaging diagnostics, including head and neck sonography. Objective The present work aims to determine the quality of routine head and neck sonography findings on a random basis, according to the criteria of the Bavarian Association of Statutory Health Insurance Physicians (KVB) at a selection of German university otolaryngology departments (ENT). Materials and methods A total of 70 randomly selected anonymized written findings including image documentation from seven ENT departments were retrospectively analyzed by an experienced KVB examiner concerning fulfilment of KVB criteria. The data were evaluated descriptively. Results Of the 70 reports, 69 were eligible for evaluation. The average documentation completeness was 80.6%. A total of 9 findings were correctly documented in full (13%). The documentation completeness of the individual departments was sorted in ascending order from 68.1% to 93%. With 88.5% vs. 75%, the hospitals with a structured report showed a higher level of completeness. In 75% of the cases the hospitals with structured reports also had digital solutions for reporting and image archiving. Conclusion In general, there is potential for optimization regarding the completeness and quality of routinely prepared head and neck sonography findings at the selected university ENT departments. The implementation of structured reporting masks and the conversion of analogue documentation into digital solutions as well as digital networking with the hospital information systems, picture archiving and communication systems should be promoted. Supervision by senior doctors is required to ensure the quality of findings of inexperienced colleagues and to help to achieve standards in reporting.Hintergrund Die Ultraschalldiagnostik gilt für den Radiologen, Hals-Nasen-Ohren-Arzt (HNO) oder Mund-Kiefer-Gesichts-Chirurgen als Standard in der Abklärung zahlreicher Pathologien. Es besteht ein Konsens, dass die digitale Dokumentation heute dringend notwendig ist, um die Qualität der sonographischen Dokumentationen zu verbessern und zu standardisieren. Es häufen sich Publikationen zur Implementierung standardisierter Befunddokumentation einschließlich der Kopf- und Halssonographie. Ziel der Arbeit Die vorliegende Arbeit zielt darauf ab, die Qualität von routinemäßig angefertigten Kopf- und Halssonographiebefunden nach Kriterien der Kassenärztlichen Vereinigung (KV) Bayern an einer Auswahl deutscher HNO-Universitätskliniken stichprobenartig zu ermitteln. Material und Methoden Insgesamt wurden retrospektiv 70 zufällig ausgewählte, anonymisierte schriftliche Befunde einschließlich Bildmaterial von insgesamt 7 HNO-Universitätskliniken stichprobenartig nach KV-Kriterien durch einen erfahrenen Prüfer der KV Bayern ausgewertet und deskriptiv analysiert. Ergebnisse Von 70 Befunden konnten 69 ausgewertet werden. Die Dokumentationsvollständigkeit lag im Mittel bei 80,6 %. Neun Befunde waren vollständig korrekt dokumentiert (13 %). Die Dokumentationsvollständigkeit der einzelnen Kliniken lag zwischen 68,1 % und 93 %. Mit 88,5 % vs. 75 % erbrachte eine strukturierte Befundung eine höhere Befundvollständigkeit. In 75 % der Fälle verfügten die Kliniken mit strukturiertem Befund auch über digitale Dokumentationslösungen. Schlussfolgerung Die Vollständigkeit und Qualität von routinemäßig angefertigten Kopf- und Halssonographiebefunden an einer Auswahl von HNO-Universitätskliniken ist insgesamt optimierbar. Die Implementierung strukturierter Befundmasken und die Umstellung der analogen Dokumentation auf digitale Lösungen sowie Vernetzung mit dem Klinikinformationssystem (KIS) und Bildarchivierungs- und Kommunikationssystem (PACS) sollte weiter vorangetrieben werden. Darüber hinaus sind leitende Ärzte dazu angehalten, die Befundqualität unerfahrener Kollegen regelmäßig zu prüfen und im Rahmen der Facharztausbildung auf die Erfüllung entsprechender Standards wie der KV-Ultraschallvereinbarung hinzuarbeiten

Role of complement and antibodies in controlling infection with pathogenic simian immunodeficiency virus (SIV) in macaques vaccinated with replication-deficient viral vectors

<p>Abstract</p> <p>Background</p> <p>We investigated the interplay between complement and antibodies upon priming with single-cycle replicating viral vectors (SCIV) encoding SIV antigens combined with Adeno5-SIV or SCIV pseudotyped with murine leukemia virus envelope boosting strategies. The vaccine was applied via spray-immunization to the tonsils of rhesus macaques and compared with systemic regimens.</p> <p>Results</p> <p>Independent of the application regimen or route, viral loads were significantly reduced after challenge with SIVmac239 (p < 0.03) compared to controls. Considerable amounts of neutralizing antibodies were induced in systemic immunized monkeys. Most of the sera harvested during peak viremia exhibited a trend with an inverse correlation between complement C3-deposition on viral particles and plasma viral load within the different vaccination groups. In contrast, the amount of the observed complement-mediated lysis did not correlate with the reduction of SIV titres.</p> <p>Conclusion</p> <p>The heterologous prime-boost strategy with replication-deficient viral vectors administered exclusively via the tonsils did not induce any neutralizing antibodies before challenge. However, after challenge, comparable SIV-specific humoral immune responses were observed in all vaccinated animals. Immunization with single cycle immunodeficiency viruses mounts humoral immune responses comparable to live-attenuated immunodeficiency virus vaccines.</p

A Distinct Urinary Biomarker Pattern Characteristic of Female Fabry Patients That Mirrors Response to Enzyme Replacement Therapy

Female patients affected by Fabry disease, an X-linked lysosomal storage disorder, exhibit a wide spectrum of symptoms, which renders diagnosis, and treatment decisions challenging. No diagnostic test, other than sequencing of the alpha-galactosidase A gene, is available and no biomarker has been proven useful to screen for the disease, predict disease course and monitor response to enzyme replacement therapy. Here, we used urine proteomic analysis based on capillary electrophoresis coupled to mass spectrometry and identified a biomarker profile in adult female Fabry patients. Urine samples were taken from 35 treatment-naive female Fabry patients and were compared to 89 age-matched healthy controls. We found a diagnostic biomarker pattern that exhibited 88.2% sensitivity and 97.8% specificity when tested in an independent validation cohort consisting of 17 treatment-naive Fabry patients and 45 controls. The model remained highly specific when applied to additional control patients with a variety of other renal, metabolic and cardiovascular diseases. Several of the 64 identified diagnostic biomarkers showed correlations with measures of disease severity. Notably, most biomarkers responded to enzyme replacement therapy, and 8 of 11 treated patients scored negative for Fabry disease in the diagnostic model. In conclusion, we defined a urinary biomarker model that seems to be of diagnostic use for Fabry disease in female patients and may be used to monitor response to enzyme replacement therapy

Dysregulation of Ribosome Biogenesis and Translational Capacity Is Associated with Tumor Progression of Human Breast Cancer Cells

Protein synthesis is a fundamental cell process and ribosomes - particularly through the ribosomal RNA that display ribozyme activity - are the main effectors of this process. Ribosome biogenesis is a very complex process involving transcriptional a

Characterization of colon cancer cells: a functional approach characterizing CD133 as a potential stem cell marker

<p>Abstract</p> <p>Background</p> <p>Isolation and characterization of tumourigenic colon cancer initiating cells may help to develop novel diagnostic and therapeutic procedures.</p> <p>Methods</p> <p>We characterized a panel of fourteen human colon carcinoma cell lines and their corresponding xenografts for the surface expression of potential stem cell markers CD133, CD24, CD44, CDCP1 and CXCR4. In five cell lines and nine xenografts, mRNA expression of these markers was determined. Tumour growth behaviour of CD133+, CD133- and unsorted SW620 cells was evaluated <it>in vivo</it>.</p> <p>Results</p> <p>All five putative stem cell markers showed distinct expression patterns in the tumours examined. Two patient-derived cell lines highly expressed CD133 (> 85% of positive cells) and three other cell lines had an expression level of about 50% whereas in long-term culture based models CD133 expression ranged only from 0 to 20%. In 8/14 cell lines, more than 80% of the cells were positive for CD24 and 11/14 were over 70% positive for CD44. 10/14 cell lines expressed CDCP1 on ≥ 83% of cells. CXCR4 expression was determined solely on 94 L and SW480.</p> <p>Analyses of the corresponding xenografts revealed a significant reduction of cell numbers expressing the investigated surface markers and showed single cell fractions expressing up to three markers simultaneously.</p> <p>Statistical analysis revealed that the CXCR4 mRNA level correlates negatively with the protein expression of CD133, CD44, CD24 and CDCP1 in cell lines and xenografts.</p> <p>A lower differentiation grade of donor material correlated with a higher CDCP1 mRNA expression level in the respective tumour model.</p> <p><it>In vivo </it>growth behaviour studies of SW620 revealed significantly higher take rates and shorter doubling times in the tumour growth of CD133 positive subclones in comparison to the unsorted cell line or CD133 negative subclones.</p> <p>Conclusions</p> <p>Our data revealed correlations in the expression of surface markers CD44 and CD24 as well as CD44 and CDCP1 and strongly suggest that CD133 is a stem cell marker within our colon carcinoma panel. Further studies will elucidate its role as a potential therapeutic target.</p

Proteome Analyses of Cellular Proteins in Methicillin-Resistant Staphylococcus aureus Treated with Rhodomyrtone, a Novel Antibiotic Candidate

The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC) values ranged from 31.25–62.5 µg/ml, and the minimal bactericidal concentration (MBC) was 250 µg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5–125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 µg/ml and 2 µg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 µg/ml) affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections

Nucleolin Inhibits G4 Oligonucleotide Unwinding by Werner Helicase

The Werner protein (WRNp), a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA).These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes