Ligation of protease-activated receptor 1 enhances alpha(v)beta(6) integrin-dependent TGF-beta activation and promotes acute lung injury

Activation of latent TGF-beta by the alpha(v)beta(6) integrin is a critical step in the development of acute lung injury. However, the mechanism by which a alpha(v)beta(6)-mediated TGF-beta activation is regulated has not been identified. We show that thrombin, and other agonists of protease-activated receptor 1(PAR1), activate TGF-beta in an alpha(v)beta(6) integrin-specific manner. This effect is PART specific and is mediated by RhoA and Rho kinase. Intratracheal instillation of the PART-specific peptide TFLLRN increases lung edema during high-tidal-volume ventilation, and this effect is completely inhibited by a blocking antibody against the alpha(v)beta(6) integrin. Instillation of TFLLRN during high-tidal-volume ventilation is associated with increased pulmonary TGF-beta activation; however, this is not observed in Itgb6(-/-) mice. Furthermore, Itgb6(-/-) mice are also protected from ventilator-induced lung edema. We also demonstrate that pulmonary edema and TGF-beta activity are similarly reduced in Par1(-/-) mice following bleomycin-induced lung injury. These results suggest that PART-mediated enhancement of a alpha(v)beta(6)-dependent TGF-beta activation could be one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury, and they identify PART and the alpha(v)beta(6) integrin as potential therapeutic targets in this condition

Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking.

Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans

Altered channel gating mechanism for CFTR inhibition by a high-affinity thiazolidinone blocker

AbstractThe thiazolidinone CFTRinh-172 was identified recently as a potent and selective blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Here, we characterized the CFTRinh-172 inhibition mechanism by patch-clamp and short-circuit analysis using cells stably expressing wild-type and mutant CFTRs. CFTRinh-172 did not alter CFTR unitary conductance (8 pS), but reduced open probability by >90% with Ki≈0.6 μM. This effect was due to increased mean channel closed time without changing mean channel open time. Short-circuit current experiments indicated similar CFTRinh-172 inhibitory potency (Ki≈0.5 μM) for inhibition of Cl− current in wild-type, G551D, and G1349D CFTR; however, Ki was significantly reduced to 0.2 μM for ΔF508 CFTR. Our studies provide evidence for CFTR inhibition by CFTRinh-172 by a mechanism involving altered CFTR gating

Plum pudding random medium model of biological tissue toward remote microscopy from spectroscopic light scattering

Biological tissue has a complex structure and exhibits rich spectroscopic behavior. There is \emph{no} tissue model up to now able to account for the observed spectroscopy of tissue light scattering and its anisotropy. Here we present, \emph{for the first time}, a plum pudding random medium (PPRM) model for biological tissue which succinctly describes tissue as a superposition of distinctive scattering structures (plum) embedded inside a fractal continuous medium of background refractive index fluctuation (pudding). PPRM faithfully reproduces the wavelength dependence of tissue light scattering and attributes the "anomalous" trend in the anisotropy to the plum and the powerlaw dependence of the reduced scattering coefficient to the fractal scattering pudding. Most importantly, PPRM opens up a novel venue of quantifying the tissue architecture and microscopic structures on average from macroscopic probing of the bulk with scattered light alone without tissue excision. We demonstrate this potential by visualizing the fine microscopic structural alterations in breast tissue (adipose, glandular, fibrocystic, fibroadenoma, and ductal carcinoma) deduced from noncontact spectroscopic measurement

The murine natural cytotoxic receptor NKp46/NCR1 controls TRAIL protein expression in NK cells and ILC1

TRAIL is an apoptosis-inducing ligand constitutively expressed on liver resident type 1 innate lymphoid cells (ILC1) and a subset of Natural Killer (NK) cells where it contributes to NK cell anti-tumor, anti-viral and immunoregulatory functions. Yet the intrinsic pathways involved in TRAIL expression in ILC remain unidentified. Here we demonstrate that the murine natural cytotoxic receptor mNKp46/NCR1, expressed on ILC1 and NK cells, controls TRAIL protein expression. Using NKp46-deficient mice, we show that liver ILC1 lack constitutive expression of TRAIL protein and that NK cells activated in vitro and in vivo fail to upregulate cell-surface TRAIL in the absence of NKp46. We show that NKp46 regulates TRAIL expression in a dose-dependent manner and that the reintroduction of NKp46 in mature NK cells deficient for NKp46 is sufficient to restore TRAIL surface expression. These studies uncover a link between NKp46 and TRAIL expression in ILC with potential implications in pathologies involving NKp46-expressing cells

Essential role of CFTR in PKA-dependent phosphorylation, alkalinization, and hyperpolarization during human dperm capacitation

Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3 − is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm, and membrane hyperpolarization. However, how HCO3 − is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work, we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3 −-entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility, and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH, and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation.Fil: Puga Molina, Lis del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pinto, Nicolás Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Torres Rodríguez, Paulina. Universidad Nacional Autónoma de México; MéxicoFil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vicens Sanchez, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Treviño, Claudia L.. Universidad Nacional Autónoma de México; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Jitter Estimation with High Accuracy for Oscillator-Based TRNGs

Ring oscillator-based true random number generators (RO-based TRNGs) are widely used to provide unpredictable random numbers for cryptographic systems. The unpredictability of the output numbers, which can be measured by entropy, is extracted from the jitter of the oscillatory signal. To quantitatively evaluate the entropy, several stochastic models have been proposed, all of which take the jitter as a key input parameter. So it is crucial to accurately estimate the jitter in the process of entropy evaluation. However, several previous methods have estimated the jitter with non-negligible error, which would cause the overestimation of the entropy. In this paper, we propose a jitter estimation method with high accuracy. Our method aims at eliminating the quantization error in previous counter-based jitter estimation methods and finally can estimate the jitter with the error smaller than $1\%$. Furthermore, for the first time, we give a theoretical error bound for our jitter estimation. The error bound confirms the $1\%$ error level of our method. As a consequence, our method will signicantly help to evaluate the entropy of RO-based TRNGs accurately. Finally, we present the application of our jitter estimation method on a practical FPGA device and provide a circuit module diagram for on-chip implementation

General practitioners’ perspectives on campaigns to promote rapid help-seeking behaviour at the onset of rheumatoid arthritis

Objective. To explore general practitioners’ (GPs’ ) perspectives on public health campaigns to encourage people with the early symptoms of rheumatoid arthritis (RA) to seek medical help rapidly. Design. Nineteen GPs participated in four semistructured focus groups. Focus groups were audio-recorded, transcribed verbatim, and analysed using thematic analysis. Results. GPs recognised the need for the early treatment of RA and identified that facilitating appropriate access to care was important. However, not all held the view that a delay in help seeking was a clinically significant issue. Furthermore, many were concerned that the early symptoms of RA were often non-specific, and that current knowledge about the nature of symptoms at disease onset was inadequate to inform the content of a help-seeking campaign. They argued that a campaign might not be able to specifically target those who need to present urgently. Poorly designed campaigns were suggested to have a negative impact on GPs’ workloads, and would “clog up” the referral pathway for genuine cases of RA. Conclusions. GPs were supportive of strategies to improve access to Rheumatological care and increase public awareness of RA symptoms. However, they have identified important issues that need to be considered in developing a public health campaign that forms part of an overall strategy to reduce time to treatment for patients with new onset RA. This study highlights the value of gaining GPs’ perspectives before launching health promotion campaigns

Scalar and vector Slepian functions, spherical signal estimation and spectral analysis

It is a well-known fact that mathematical functions that are timelimited (or spacelimited) cannot be simultaneously bandlimited (in frequency). Yet the finite precision of measurement and computation unavoidably bandlimits our observation and modeling scientific data, and we often only have access to, or are only interested in, a study area that is temporally or spatially bounded. In the geosciences we may be interested in spectrally modeling a time series defined only on a certain interval, or we may want to characterize a specific geographical area observed using an effectively bandlimited measurement device. It is clear that analyzing and representing scientific data of this kind will be facilitated if a basis of functions can be found that are "spatiospectrally" concentrated, i.e. "localized" in both domains at the same time. Here, we give a theoretical overview of one particular approach to this "concentration" problem, as originally proposed for time series by Slepian and coworkers, in the 1960s. We show how this framework leads to practical algorithms and statistically performant methods for the analysis of signals and their power spectra in one and two dimensions, and, particularly for applications in the geosciences, for scalar and vectorial signals defined on the surface of a unit sphere.Comment: Submitted to the 2nd Edition of the Handbook of Geomathematics, edited by Willi Freeden, Zuhair M. Nashed and Thomas Sonar, and to be published by Springer Verlag. This is a slightly modified but expanded version of the paper arxiv:0909.5368 that appeared in the 1st Edition of the Handbook, when it was called: Slepian functions and their use in signal estimation and spectral analysi

Pkc-Mediated Stimulation of Amphibian Cftr Depends on a Single Phosphorylation Consensus Site. Insertion of This Site Confers Pkc Sensitivity to Human Cftr

Mutations of the CFTR, a phosphorylation-regulated Cl− channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain