Prostate cancer: to screen or not to screen?

Unfortunately, interim analyses of the long-awaited ERSPC and PLCO trial data have generated conflicting conclusions. Here, two European authors speculate as to the reasons underlying this contradiction, while highlighting clinically relevant points that are supported by both studies. Particular attention is paid to the potential consequences of overdiagnosis and overtreatment.

Secondary arterial hypertension: when, who, and how to screen?

Secondary hypertension refers to arterial hypertension due to an identifiable cause and affects ∼5-10% of the general hypertensive population. Because secondary forms are rare and work up is time-consuming and expensive, only patients with clinical suspicion should be screened. In recent years, some new aspects gained importance regarding this screening. In particular, increasing evidence suggests that 24 h ambulatory blood pressure (BP) monitoring plays a central role in the work up of patients with suspected secondary hypertension. Moreover, obstructive sleep apnoea has been identified as one of the most frequent causes. Finally, the introduction of catheter-based renal denervation for the treatment of patients with resistant hypertension has dramatically increased the interest and the number of patients evaluated for renal artery stenosis. We review the clinical clues of the most common causes of secondary hypertension. Specific recommendations are given as to evaluation and treatment of various forms of secondary hypertension. Despite appropriate therapy or even removal of the secondary cause, BP rarely ever returns to normal with long-term follow-up. Such residue hypertension indicates either that some patients with secondary hypertension also have concomitant essential hypertension or that irreversible vascular remodelling has taken place. Thus, in patients with potentially reversible causes of hypertension, early detection and treatment are important to minimize/prevent irreversible changes in the vasculature and target organs.

Proteomic analysis of the Theileria annulata schizont

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.

Using micropower impulse radar technology to screen for pneumothorax: an international bi-institutional study. Corrigendum

In an article in the December 2012 issue of The Journal of Trauma and Acute Care Surgery, several author names were misprinted.

Characterization of the potential role for RNA-binding protein FUS/TLS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein of the hnRNP family, that has been discovered as fused to transcription factors, through chromosomal translocations, in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis (ALS) [1]. To date, FUS/TLS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS/TLS to genome stability control and DNA damage response. In fact, mice lacking FUS/TLS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and in response to double-strand breaks, FUS/TLS gets phosphorylated by the protein kinase ATM [3,4,5]. Furthermore, the inducible depletion of FUS/TLS in a neuroblastoma cell line (SH-SY5Y FUS/TLS TET-off iKD) subjected to genotoxic stress (IR) resulted in an increased phosphorylation of γH2AX respect to control cells, suggesting an higher activation of the DNA damage response. The study aims to investigate the specific role of FUS/TLS in DNA damage response through the characterization of the proteomic profile of SH-SY5Y FUS/TLS iKD cells subjected to DNA damage stress, by mass spectrometry-based quantitative proteomics (e.g. SILAC). Preliminary results of mass spectrometric identification of FUS/TLS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS/TLS protein, highlighted the interactions with several proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS/TLS is involved in this pathway, even thou its exact role still need to be addressed.

Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

Influence of hormone replacement therapy on proteomic pattern in serum of postmenopausal women

OBJECTIVES: Proteomics approaches to cardiovascular biology and disease hold the promise of identifying specific proteins and peptides or modification thereof to assist in the identification of novel biomarkers. METHOD: By using surface-enhanced laser desorption and ionization time of flight mass spectroscopy (SELDI-TOF-MS) serum peptide and protein patterns were detected enabling to discriminate between postmenopausal women with and without hormone replacement therapy (HRT). RESULTS: Serum of 13 HRT and 27 control subjects was analyzed and 42 peptides and proteins could be tentatively identified based on their molecular weight and binding characteristics on the chip surface. By using decision tree-based Biomarker Patternstrade mark Software classification and regression analysis a discriminatory function was developed allowing to distinguish between HRT women and controls correctly and, thus, yielding a sensitivity of 100% and a specificity of 100%. The results show that peptide and protein patterns have the potential to deliver novel biomarkers as well as pinpointing targets for improved treatment. The biomarkers obtained represent a promising tool to discriminate between HRT users and non-users. CONCLUSION: According to a tentative identification of the markers by their molecular weight and binding characteristics, most of them appear to be part of the inflammation induced acute-phase response

Proteomic analysis of porcine bone-conditioned medium.

PURPOSE Autografts are considered to support bone regeneration. Paracrine factors released from cortical bone might contribute to the overall process of graft consolidation. The aim of this study was to characterize the paracrine factors by means of proteomic analysis. MATERIALS AND METHODS Bone-conditioned medium (BCM) was prepared from fresh bone chips of porcine mandibles and subjected to proteomic analysis. Proteins were categorized and clustered using the bioinformatic tools UNIPROT and PANTHER, respectively. RESULTS Proteomic analysis showed that BCM contains more than 150 proteins, of which 43 were categorized into "secreted" and "extracellular matrix." Growth factors that are not only detectable in BCM, but potentially also target cellular processes involved in bone regeneration, eg, pleiotrophin, galectin-1, transforming growth factor beta (TGF-β)-induced gene (TGFBI), lactotransferrin, insulin-like growth factor (IGF)-binding protein 5, latency-associated peptide forming a complex with TGF-β1, and TGF-β2, were discovered. CONCLUSION The present results demonstrate that cortical bone chips release a large spectrum of proteins with the possibility of modulating cellular aspects of bone regeneration. The data provide the basis for future studies to understand how these paracrine factors may contribute to the complex process of graft consolidation.

Novel prognostic markers revealed by a proteomic approach separating benign from malignant insulinomas.

The prognosis of pancreatic neuroendocrine tumors is related to size, histology and proliferation rate. However, this stratification needs to be refined further. We conducted a proteome study on insulinomas, a well-defined pancreatic neuroendocrine tumor entity, in order to identify proteins that can be used as biomarkers for malignancy. Based on a long follow-up, insulinomas were divided into those with metastases (malignant) and those without (benign). Microdissected cells from six benign and six malignant insulinomas were subjected to a procedure combining fluorescence dye saturation labeling with high-resolution two-dimensional gel electrophoresis. Differentially expressed proteins were identified using nano liquid chromatography-electrospray ionization/multi-stage mass spectrometry and validated by immunohistochemistry on tissue microarrays containing 62 insulinomas. Sixteen differentially regulated proteins were identified among 3000 protein spots. Immunohistochemical validation revealed that aldehyde dehydrogenase 1A1 and voltage-dependent anion-selective channel protein 1 showed significantly stronger expression in malignant insulinomas than in benign insulinomas, whereas tumor protein D52 (TPD52) binding protein was expressed less strongly in malignant insulinomas than in benign insulinomas. Using multivariate analysis, low TPD52 expression was identified as a strong independent prognostic factor for both recurrence-free and overall disease-related survival.

Proteomic characterization of the FUS/TLS interactome

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [1]. To date, FUS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS to genome stability control and DNA damage response. In fact, mice lacking FUS are hypersensitive to ionizing radiation and show high levels of chromosome instability and in response to double-strand breaks, FUS gets phosphorylated by the protein kinase ATM [3, 4, 5]. Moreover, upon DNA damage stress, FUS mediates Ebp1 (ErbB3 receptor-binding protein) SUMOylation, a post-translational modification that is required for its onco-suppressive activity, by acting as SUMO E3 ligase [6]. The study aims to investigate the role of FUS in DNA damage response and SUMOylation, two cellular pathways tightly interconnected to each other. Moreover, we will exploit biochemical and mass spectrometry-based approaches in order to identify other potential substrates of the E3 SUMO ligase activity of FUS. Preliminary results of mass spectrometric identification of FUS interacting proteins, in HEK293 and SHSY5Y cells, highlighted the interaction of FUS with several proteins involved in DNA damage response and many of those have been described already as target of SUMOylation, such as XRCC5, DDX5, PARP1, Nucleophosmin, and others. These evidences strengthen the hypothesis that FUS might represent a link between these pathways, even thou its exact role still needs to be clearly addressed. [1] Vance C. et al. (2009) Science 323(5918): p. 1208-11 [2] Fiesel FC., Kahle PJ. (2011) FEBS J. 278(19): p. 3550-68 [3] Kuroda M. et al. (2000) Embo J. 19(3): p. 453-62 [4] Hicks GG. et al. (2000) Nat Genet. 24(2):p. 175-9 [5] Gardiner M. et al. (2008) Biochem J. 415(2): p. 297-307 [6] Oh SM. et al. (2010) Oncogene 29(7): p. 1017-30

Spatial biases during mental arithmetic: evidence from eye movements on a blank screen

While the influence of spatial-numerical associations in number categorization tasks has been well established, their role in mental arithmetic is less clear. It has been hypothesized that mental addition leads to rightward and upward shifts of spatial attention (along the “mental number line”), whereas subtraction leads to leftward and downward shifts. We addressed this hypothesis by analyzing spontaneous eye movements during mental arithmetic. Participants solved verbally presented arithmetic problems (e.g., 2 + 7, 8–3) aloud while looking at a blank screen. We found that eye movements reflected spatial biases in the ongoing mental operation: Gaze position shifted more upward when participants solved addition compared to subtraction problems, and the horizontal gaze position was partly determined by the magnitude of the operands. Interestingly, the difference between addition and subtraction trials was driven by the operator (plus vs. minus) but was not influenced by the computational process. Thus, our results do not support the idea of a mental movement toward the solution during arithmetic but indicate a semantic association between operation and space.

Comparative proteomic analysis of lung tissue from guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) reveals a decrease in abundance of host proteins involved in cytoskeletal and cellular organization

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a particularly severe form of leptospirosis. LPHS is increasingly recognized in both humans and animals and is characterized by rapidly progressive intra-alveolar haemorrhage leading to high mortality. The pathogenic mechanisms of LPHS are poorly understood which hampers the application of effective treatment regimes. In this study a 2-D guinea pig proteome lung map was created and used to investigate the pathogenic mechanisms of LPHS. Comparison of lung proteomes from infected and non-infected guinea pigs via differential in-gel electrophoresis revealed highly significant differences in abundance of proteins contained in 130 spots. Acute phase proteins were the largest functional group amongst proteins with increased abundance in LPHS lung tissue, and likely reflect a local and/or systemic host response to infection. The observed decrease in abundance of proteins involved in cytoskeletal and cellular organization in LPHS lung tissue further suggests that infection with pathogenic Leptospira induces changes in the abundance of host proteins involved in cellular architecture and adhesion contributing to the dramatically increased alveolar septal wall permeability seen in LPHS. BIOLOGICAL SIGNIFICANCE The recent completion of the complete genome sequence of the guinea pig (Cavia porcellus) provides innovative opportunities to apply proteomic technologies to an important animal model of disease. In this study, the comparative proteomic analysis of lung tissue from experimentally infected guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) revealed a decrease in abundance of proteins involved in cellular architecture and adhesion, suggesting that loss or down-regulation of cytoskeletal and adhesion molecules plays an important role in the pathogenesis of LPHS. A publically available guinea pig lung proteome map was constructed to facilitate future pulmonary proteomics in this species.

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility

BACKGROUND Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. METHODS Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. RESULTS 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. CONCLUSIONS This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.