Human phosphodiesterase 4D7 (PDE4D7) expression is increased in TMPRSS2-ERG positive primary prostate cancer and independently adds to a reduced risk of post-surgical disease progression

background: There is an acute need to uncover biomarkers that reflect the molecular pathologies, underpinning prostate cancer progression and poor patient outcome. We have previously demonstrated that in prostate cancer cell lines PDE4D7 is downregulated in advanced cases of the disease. To investigate further the prognostic power of PDE4D7 expression during prostate cancer progression and assess how downregulation of this PDE isoform may affect disease outcome, we have examined PDE4D7 expression in physiologically relevant primary human samples. methods: About 1405 patient samples across 8 publically available qPCR, Affymetrix Exon 1.0 ST arrays and RNA sequencing data sets were screened for PDE4D7 expression. The TMPRSS2-ERG gene rearrangement status of patient samples was determined by transformation of the exon array and RNA seq expression data to robust z-scores followed by the application of a threshold >3 to define a positive TMPRSS2-ERG gene fusion event in a tumour sample. results: We demonstrate that PDE4D7 expression positively correlates with primary tumour development. We also show a positive association with the highly prostate cancer-specific gene rearrangement between TMPRSS2 and the ETS transcription factor family member ERG. In addition, we find that in primary TMPRSS2-ERG-positive tumours PDE4D7 expression is significantly positively correlated with low-grade disease and a reduced likelihood of progression after primary treatment. Conversely, PDE4D7 transcript levels become significantly decreased in castration resistant prostate cancer (CRPC). conclusions: We further characterise and add physiological relevance to PDE4D7 as a novel marker that is associated with the development and progression of prostate tumours. We propose that the assessment of PDE4D7 levels may provide a novel, independent predictor of post-surgical disease progression

The association of the long prostate cancer expressed PDE4D transcripts to poor patient outcome depends on the tumor’s TMPRSS2-ERG fusion status

Objectives: To investigate the added value of assessing transcripts for the long cAMP phosphodiesterase-4D (PDE4D) isoforms, PDE4D5 and PDE4D9, regarding the prognostic power of the ‘CAPRA & PDE4D7’ combination risk model to predict longitudinal postsurgical biological outcomes in prostate cancer. Patients and Methods: RNA was extracted from both biopsy punches of resected tumours (606 patients; RP cohort) and diagnostic needle biopsies (168 patients; DB cohort). RT-qPCR was performed in order to determine PDE4D5, PDE4D7, and PDE4D9 transcript scores in both study cohorts. By RNA sequencing, we determined the TMPRSS2-ERG fusion status of each tumour sample in the RP cohort. Kaplan-Meier survival analyses were then applied to correlate the PDE4D5, PDE4D7 and PDE4D9 scores with postsurgical patient outcomes. Logistic regression was then used to combine the clinical CAPRA score with PDE4D5, PDE4D7, and PDE4D9 scores in order to build a ‘CAPRA & PDE4D5/7/9’ regression model. ROC and decision curve analysis was used to estimate the net benefit of the ‘CAPRA & PDE4D5/7/9’ risk model. Results: Kaplan-Meier survival analysis, on the RP cohort, revealed a significant association of the PDE4D7 score with postsurgical biochemical recurrence (BCR) in the presence of the TMPRSS2-ERG gene rearrangement (logrank p<0.0001), compared to the absence of this gene fusion event (logrank p=0.08). In contrast, the PDE4D5 score was only significantly associated with BCR in TMPRSS2-ERG fusion negative tumours (logrank p<0.0001 vs. logrank p=0.4 for TMPRSS2-ERG+ tumours). This was similar for the PDE4D9 score although less pronounced compared to that of the PDE4D5 score (TMPRSS2ERG- logrank p<0.0001 vs. TMPRSS2ERG+ logrank p<0.005). In order to predict BCR after primary treatment, we undertook ROC analysis of the logistic regression combination model of the CAPRA score with the PDE4D5, PDE4D7, and PDE4D9 scores. For the DB cohort, this demonstrated significant differences in the AUC between the CAPRA and the PDE4D5/7/9 regression model vs. the CAPRA and PDE4D7 risk model (AUC 0.87 vs. 0.82; p=0.049) vs. the CAPRA score alone (AUC 0.87 vs. 0.77; p=0.005). The CAPRA and PDE4D5/7/9 risk model stratified 19.2% patients of the DB cohort to either ‘no risk of biochemical relapse’ (NPV 100%) or the ‘start of any secondary treatment (NPV 100%)’, over a follow-up period of up to 15 years. Decision curve analysis presented a clear, net benefit for the use of the novel CAPRA & PDE4D5/7/9 risk model compared to the clinical CAPRA score alone or the CAPRA and PDE4D7 model across all decision thresholds. Conclusion: Association of the long PDE4D5, PDE4D7, and PDE4D9 transcript scores to prostate cancer patient outcome, after primary intervention, varies in opposite directions depending on the TMPRSS2-ERG genomic fusion background of the tumour. Adding transcript scores for the long PDE4D isoforms, PDE4D5 and PDE4D9, to our previously presented combination risk model of the combined ‘CAPRA & PDE4D7’ score, in order to generate the CAPRA and PDE4D5/7/9 score, significantly improves the prognostic power of the model in predicting postsurgical biological outcomes in prostate cancer patients

Under-reporting bicycle accidents to police in the COST TU1101 international survey: Cross-country comparisons and associated factors

Police crash reports are often the main source for official data in many countries. However, with the exception of fatal crashes, crashes are often underreported in a biased manner. Consequently, the countermeasures adopted according to them may be inefficient. In the case of bicycle crashes, this bias is most acute and it probably varies across countries, with some of them being more prone to reporting accidents to police than others. Assessing if this bias occurs and the size of it can be of great importance for evaluating the risks associated with bicycling. This study utilized data collected in the COST TU1101 action “Towards safer bicycling through optimization of bicycle helmets and usage”. The data came from an online survey that included questions related to bicyclists' attitudes, behaviour, cycling habits, accidents, and patterns of use of helmets. The survey was filled by 8655 bicyclists from 30 different countries. After applying various exclusion factors, 7015 questionnaires filled by adult cyclists from 17 countries, each with at least 100 valid responses, remained in our sample. The results showed that across all countries, an average of only 10% of all crashes were reported to the police, with a wide range among countries: from a minimum of 0.0% (Israel) and 2.6% (Croatia) to a maximum of a 35.0% (Germany). Some factors associated with the reporting levels were type of crash, type of vehicle involved, and injury severity. No relation was found between the likelihood of reporting and the cyclist's gender, age, educational level, marital status, being a parent, use of helmet, and type of bicycle. The significant under-reporting – including injury crashes that do not lead to hospitalization – justifies the use of self-report survey data for assessment of bicycling crash patterns as they relate to (1) crash risk issues such as location, infrastructure, cyclists' characteristics, and use of helmet and (2) strategic approaches to bicycle crash prevention and injury reduction.Fil: Shinar, D.. Ben Gurion University of the Negev; IsraelFil: Valero Mora, Pedro. Universidad de Valencia; EspañaFil: van Strijp Houtenbos, M.. Institute For Road Safety Research; Países BajosFil: Haworth, N.. Queensland University of Technology; AustraliaFil: Schramm, A.. Queensland University of Technology; AustraliaFil: de Bruyne, G.. Universiteit Antwerp; BélgicaFil: Cavallo, V.. No especifíca;Fil: Chliaoutakis, J.. No especifíca;Fil: Pereira Dias, Joao. Instituto Superior Tecnico; PortugalFil: Ferraro, Ottavia Eleonora. Universita Degli Studi Di Pavia; ItaliaFil: Fyhri, Aslak. No especifíca;Fil: Sajatovic, Anika Hursa. No especifíca;Fil: Kuklane, Kalev. Lund University; SueciaFil: Ledesma, Ruben Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Psicología Básica, Aplicada y Tecnología. Universidad Nacional de Mar del Plata. Facultad de Psicología. Instituto de Psicología Básica, Aplicada y Tecnología.; ArgentinaFil: Calvé Mascarell, Oscar. Ben Gurion University of the Negev; IsraelFil: Morandi, A.. Universita Degli Studi Di Pavia; ItaliaFil: Muser, Markus. No especifíca;Fil: Otte, Diettmar. No especifíca;Fil: Papadakaki, M.. No especifíca;Fil: Sanmartín, J.. Universidad de Valencia; EspañaFil: Dulf, D.. No especifíca;Fil: Saplioglu, M.. No especifíca;Fil: Tzamalouka, Georgia. No especifíca

Complete sequence-based pathway analysis by differential on-chip DNA and RNA extraction from a single cell

Abstract We demonstrate on-chip, differential DNA and RNA extraction from a single cell using a microfluidic chip and a two-stage lysis protocol. This method enables direct use of the whole extract, without additional washing steps, reducing sample loss. Using this method, the tumor driving pathway in individual cells from a colorectal cancer cell line was determined by applying a Bayesian computational pathway model to sequences obtained from the RNA fraction of a single cell and, the mutations driving the pathway were determined by analyzing sequences obtained from the DNA fraction of the same single cell. This combined functional and mutational pathway assessment of a single cell could be of significant value for dissecting cellular heterogeneity in tumors and analyzing single circulating tumor cells

Human phosphodiesterase 4D7 (PDE4D7) expression is increased in TMPRSS2-ERG-positive primary prostate cancer and independently adds to a reduced risk of post-surgical disease progression

Background:There is an acute need to uncover biomarkers that reflect the molecular pathologies, underpinning prostate cancer progression and poor patient outcome. We have previously demonstrated that in prostate cancer cell lines PDE4D7 is downregulated in advanced cases of the disease. To investigate further the prognostic power of PDE4D7 expression during prostate cancer progression and assess how downregulation of this PDE isoform may affect disease outcome, we have examined PDE4D7 expression in physiologically relevant primary human samples.Methods:About 1405 patient samples across 8 publically available qPCR, Affymetrix Exon 1.0 ST arrays and RNA sequencing data sets were screened for PDE4D7 expression. The TMPRSS2-ERG gene rearrangement status of patient samples was determined by transformation of the exon array and RNA seq expression data to robust z-scores followed by the application of a threshold >3 to define a positive TMPRSS2-ERG gene fusion event in a tumour sample.Results:We demonstrate that PDE4D7 expression positively correlates with primary tumour development. We also show a positive association with the highly prostate cancer-specific gene rearrangement between TMPRSS2 and the ETS transcription factor family member ERG. In addition, we find that in primary TMPRSS2-ERG-positive tumours PDE4D7 expression is significantly positively correlated with low-grade disease and a reduced likelihood of progression after primary treatment. Conversely, PDE4D7 transcript levels become significantly decreased in castration resistant prostate cancer (CRPC).Conclusions:We further characterise and add physiological relevance to PDE4D7 as a novel marker that is associated with the development and progression of prostate tumours. We propose that the assessment of PDE4D7 levels may provide a novel, independent predictor of post-surgical disease progression

Влияние фенилгидразина и алкилсульфатов на осмотическую чувствительность эритроцитов млекопитающих

Показано, что чувствительность эритроцитов млекопитающих к гипертоническому стрессу при 37 °С после обработки фенилгидразином зависит от видовой принадлежности клеток. Установлено, что модификация фенилгидразином эритроцитов млекопитающих приводит к снижению антигемолитической активности алкилкилсульфатов в условиях гипертонического стресса.Показано, що чутливість еритроцитів ссавців до гіпертонічного стресу при 37 °С після обробки фенілгідразином залежить від видової приналежності клітин. Встановлено, що модифікація фенілгідразином еритроцитів ссавців призводить до зниження антигемолітичної активності алкілсульфатів в умовах гіпертонічного стресу.It is shown that the sensitivity of mammalian erythrocytes to hypertonic stress at 37 °C after the treatment with phenylhydrazine depends on the species of a cell. We have established that the modification with phenylhydrazine of mammalian erythrocytes leads to a decrease of the alkyl sulfates antihemolytic activity under hypertonic stress

Microenvironment involved in FPR1 expression by human glioblastomas

Formyl peptide receptor 1 (FPR1) activity in U87 glioblastoma (GBM) cells contributes to tumor cell motility. The present study aimed to evaluate the FPR1 expression in human GBM, the possibility to elicit agonist induced FPR1 activation of GBM cells and inhibit this activation with chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Immunohistochemistry was used to assess FPR1 expression in GBM patient samples, which was present in all 178 samples. Also FPR1 mRNA levels measured with quantitative PCR, could be detected in all 25 GBM patient samples tested. Activation of FPR1 in U87 cells, as measured by human mitochondrial-derived agonists, increased calcium mobilization, AKT and ERK1/2 phosphorylation, and ligand-induced migration. Inhibition of all responses could be achieved with CHIPS. Eight early passage human Groningen Glioma (GG) cell lines, isolated from primary GBM tissue were screened for the presence of FPR1. FPR1 mRNA and protein expression as well as receptor activation could not be detected in any of these early passage GG cell lines. However FPR1 was present in ex vivo tumors formed by the same GG cell lines after being implanted in mouse brains. FPR1 is highly expressed in human GBM specimens, it can be activated by human mitochondrial-derived agonists in U87 and inhibited with CHIPS. FPR1 cannot be detected in early passage GG cell lines in vitro, however when engrafted in the mouse brain these cells show FPR1 expression. These results suggest a role of the brain microenvironment in FPR1 expression in GBM.</p

Impact of glycan linkage to staphylococcus aureus wall teichoic acid on langerin recognition and langerhans cell activation

Staphylococcus aureus is the leading cause of skin and soft tissue infections. It remains incompletely understood how skin-resident immune cells respond to invading S. aureus and contribute to an effective immune response. Langerhans cells (LCs), the only professional antigen-presenting cell type in the epidermis, sense S. aureus through their pattern-recognition receptor langerin, triggering a proinflammatory response. Langerin recognizes the β-1,4-linked N-acetylglucosamine (β1,4-GlcNAc) but not α-1,4-linked GlcNAc (α1,4-GlcNAc) modifications, which are added by dedicated glycosyltransferases TarS and TarM, respectively, on the cell wall glycopolymer wall teichoic acid (WTA). Recently, an alternative WTA glycosyltransferase, TarP, was identified, which also modifies WTA with β-GlcNAc but at the C-3 position (β1,3-GlcNAc) of the WTA ribitol phosphate (RboP) subunit. Here, we aimed to unravel the impact of β-GlcNAc linkage position for langerin binding and LC activation. Using genetically modified S. aureus strains, we observed that langerin similarly recognized bacteria that produce either TarS- or TarP-modified WTA, yet tarP-expressing S. aureus induced increased cytokine production and maturation of in vitro-generated LCs compared to tarS-expressing S. aureus. Chemically synthesized WTA molecules, representative of the different S. aureus WTA glycosylation patterns, were used to identify langerin-WTA binding requirements. We established that β-GlcNAc is sufficient to confer langerin binding, thereby presenting synthetic WTA molecules as a novel glycobiology tool for structure-binding studies and for elucidating S. aureus molecular pathogenesis. Overall, our data suggest that LCs are able to sense all β-GlcNAc-WTA producing S. aureus strains, likely performing an important role as first responders upon S. aureus skin invasion.Bio-organic Synthesi

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays

Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30–60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results obtained with the high-temporal-resolution Epic BT, but could only provide end-point data. In contrast, complex, nonmonotonic cell adhesion kinetics measured by the high-throughput optical biosensor is expected to open a window on the hidden background of the immune cell–extracellular matrix interactions

Pseudomonas Evades Immune Recognition of Flagellin in Both Mammals and Plants

The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants