Expression of Protease-Activated Receptor 1 and 2 and Anti-Tubulogenic Activity of Protease-Activated Receptor 1 in Human Endothelial Colony-Forming Cells

Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50–100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25–100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade

The Impact of Duty Hours on Resident Self Reports of Errors

BACKGROUND: Resident duty hour limitations aim, in part, to reduce medical errors. Residents’ perceptions of the impact of duty hours on errors are unknown. OBJECTIVE: To determine residents’ self-reported contributing factors, frequency, and impact of hours worked on suboptimal care practices and medical errors. DESIGN: Cross-sectional survey. SUBJECTS: 164 Internal Medicine Residents at the University of California, San Francisco. MEASUREMENTS AND RESULTS: Residents were asked to report the frequency and contributing factors of suboptimal care practices and medical errors, and how duty hours impacted these practices and aspects of resident work-life. One hundred twenty-five residents (76%) responded. The most common suboptimal care practices were working while impaired by fatigue and forgetting to transmit information during sign-out. In multivariable models, residents who felt overwhelmed with work (p = 0.02) and who reported spending >50% of their time in nonphysician tasks (p = 0.002) were more likely to report suboptimal care practices. Residents reported work-stress (a composite of fatigue, excessive workload, distractions, stress, and inadequate time) as the most frequent contributing factor to medical errors. In multivariable models, only engaging in suboptimal practices was associated with self-report of higher risk for medical errors (p < 0.001); working more than 80 hours per week was not associated with suboptimal care or errors. CONCLUSION: Our findings suggest that administrative load and work stressors are more closely associated with resident reports of medical errors than the number of hours work. Efforts to reduce resident duty hours may also need to address the nature of residents’ work to reduce errors

Congenital Plasmodium falciparum infection in neonates in Muheza District, Tanzania

BACKGROUND\ud \ud Although recent reports on congenital malaria suggest that the incidence is increasing, it is difficult to determine whether the clinical disease is due to parasites acquired before delivery or as a result of contamination by maternal blood at birth. Understanding of the method of parasite acquisition is important for estimating the time incidence of congenital malaria and design of preventive measures. The aim of this study was to determine whether the first Plasmodium falciparum malaria disease in infants is due to same parasites present on the placenta at birth.\ud \ud METHODS\ud \ud Babies born to mothers with P. falciparum parasites on the placenta detected by PCR were followed up to two years and observed for malaria episodes. Paired placental and infant peripheral blood samples at first malaria episode within first three months of life were genotyped (msp2) to determine genetic relatedness. Selected amplifications from nested PCR were sequenced and compared between pairs.\ud \ud RESULTS\ud \ud Eighteen (19.1%) out of 95 infants who were followed up developed clinical malaria within the first three months of age. Eight pairs (60%) out of 14 pairs of sequenced placental and cord samples were genetically related while six (40%) were genetically unrelated. One pair (14.3%) out of seven pairs of sequenced placental and infants samples were genetically related. In addition, infants born from primigravidae mothers were more likely to be infected with P. falciparum (P < 0.001) as compared to infants from secundigravidae and multigravidae mothers during the two years of follow up. Infants from multigravidae mothers got the first P. falciparum infection earlier than those from secundigravidae and primigravidae mothers (RR = 1.43).\ud \ud CONCLUSION\ud \ud Plasmodium falciparum malaria parasites present on the placenta as detected by PCR are more likely to result in clinical disease (congenital malaria) in the infant during the first three months of life. However, sequencing data seem to question the validity of this likelihood. Therefore, the relationship between placental parasites and first clinical disease need to be confirmed in larger studies

Kindlins, Integrin Activation and the Regulation of Talin Recruitment to αIIbβ3

Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment

Optical biosensor differentiates signaling of endogenous PAR1 and PAR2 in A431 cells

<p>Abstract</p> <p>Background</p> <p>Protease activated receptors (PARs) consist of a family of four G protein-coupled receptors. Many types of cells express several PARs, whose physiological significance is mostly unknown.</p> <p>Results</p> <p>Here, we show that non-invasive resonant waveguide grating (RWG) biosensor differentiates signaling of endogenous protease activated receptor subtype 1 (PAR₁) and 2 (PAR₂) in human epidermoid carcinoma A431 cells. The biosensor directly measures dynamic mass redistribution (DMR) resulted from ligand-induced receptor activation in adherent cells. In A431, both PAR₁and PAR₂agonists, but neither PAR₃nor PAR₄agonists, trigger dose-dependent Ca²⁺mobilization as well as G_q-type DMR signals. Both Ca²⁺flux and DMR signals display comparable desensitization patterns upon repeated stimulation with different combinations of agonists. However, PAR₁and PAR₂exhibit distinct kinetics of receptor re-sensitization. Furthermore, both trypsin- and thrombin-induced Ca²⁺flux signals show almost identical dependence on cell surface cholesterol level, but their corresponding DMR signals present different sensitivities.</p> <p>Conclusion</p> <p>Optical biosensor provides an alternative readout for examining receptor activation under physiologically relevant conditions, and differentiates the signaling of endogenous PAR₁and PAR₂in A431.</p

Observation of associated near-side and away-side long-range correlations in √sNN=5.02 TeV proton-lead collisions with the ATLAS detector

Two-particle correlations in relative azimuthal angle (Δϕ) and pseudorapidity (Δη) are measured in √sNN=5.02  TeV p+Pb collisions using the ATLAS detector at the LHC. The measurements are performed using approximately 1  μb-1 of data as a function of transverse momentum (pT) and the transverse energy (ΣETPb) summed over 3.1<η<4.9 in the direction of the Pb beam. The correlation function, constructed from charged particles, exhibits a long-range (2<|Δη|<5) “near-side” (Δϕ∼0) correlation that grows rapidly with increasing ΣETPb. A long-range “away-side” (Δϕ∼π) correlation, obtained by subtracting the expected contributions from recoiling dijets and other sources estimated using events with small ΣETPb, is found to match the near-side correlation in magnitude, shape (in Δη and Δϕ) and ΣETPb dependence. The resultant Δϕ correlation is approximately symmetric about π/2, and is consistent with a dominant cos⁡2Δϕ modulation for all ΣETPb ranges and particle pT

Implications of Storing Urinary DNA from Different Populations for Molecular Analyses

Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days.Urine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4 degrees C, -20 degrees C & -80 degrees C) with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J) and single copy (TLR2) targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis

Linkage disequilibrium pattern of the ATM gene in breast cancer patients and controls; association of SNPs and haplotypes to radio-sensitivity and post-lumpectomy local recurrence

<p>Abstract</p> <p>Background</p> <p>The ATM protein is activated as a result of ionizing radiation, and genetic variants of the <it>ATM </it>gene may therefore affect the level of radiation-induced damage. Individuals heterozygous for <it>ATM </it>mutations have been reported to have an increased risk of malignancy, especially breast cancer.</p> <p>Materials and methods</p> <p>Norwegian breast cancer patients (272) treated with radiation (252 of which were evaluated for radiation-induced adverse side effects), 95 Norwegian women with no known history of cancer and 95 American breast cancer patients treated with radiation (44 of which developed ipsilateral breast tumour recurrence, IBTR) were screened for sequence variations in all exons of the <it>ATM </it>gene as well as known intronic variants by denaturating high performance liquid chromatography (dHPLC) followed by sequencing to determine the nature of the variant.</p> <p>Results and Conclusion</p> <p>A total of 56 variants were identified in the three materials combined. A borderline significant association with breast cancer risk was found for the 1229 T>C (Val>Ala) substitution in exon 11 (P-value 0.055) between the Norwegian controls and breast cancer patients as well as a borderline significant difference in haplotype distribution (P-value 0.06). Adverse side effects, such as: development of costal fractures and telangiectasias, subcutaneous and lung fibrosis, pleural thickening and atrophy were evaluated in the Norwegian patients. Significant associations were found for several of the identified variants such as rs1800058 (Leu > Phe) where a decrease in minor allele frequency was found with increasing level of adverse side effects for the clinical end-points pleural thickening and lung fibrosis, thus giving a protective effect. Overall our results indicate a role for variation in the <it>ATM </it>gene both for risk of developing breast cancer, and in radiation induced adverse side effects. No association could be found between risk of developing ipsilateral breast tumour recurrence and any of the sequence variants found in the American patient material.</p

Implantation Serine Proteinase 1 Exhibits Mixed Substrate Specificity that Silences Signaling via Proteinase-Activated Receptors

Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation