The matrilins: modulators of extracellular matrix assembly

The matrilins form a family of oligomeric extracellular adaptor proteins that are most strongly expressed in cartilage but also present in many other extracellular matrices. Matrilins bind to different types of collagen fibrils, to other noncollagenous proteins and to aggrecan. They thereby support matrix assembly by connecting fibrillar components and mediating interactions between these and the aggrecan gel. The binding avidity of a matrilin can be varied by alternative splicing, proteolytic processing and formation of homo- and heterooligomers. Such changes in matrilin structure may lead to a modulation of extracellular matrix assembly. Some matrilins bind weakly to α1β1 integrin and cell surface proteoglycans, but even though matrilins play a role in mechanotransduction and matrilin-3 activates the expression of osteoarthritis-associated genes the physiological relevance of matrilin-cell interactions is unclear. Matrilin knockout mice do not display pronounced phenotypes, which points to a redundancy within the protein family or with functionally related proteins. In man, dominant mutations in the von Willebrand factor A like domain of matrilin-3 lead to a protein retention in the endoplasmic reticulum that causes multiple epiphyseal dysplasia by initiating a cell stress response. In contrast, a mutation in an EGF domain of matrilin-3 that is associated with hand osteoarthritis and disc degeneration does not interfere with secretion but instead with extracellular assembly of matrix structures. In this review we summarize such information on matrilin structure and function that we believe is important for the understanding of extracellular matrix assembly and for deciphering pathophysiological mechanisms in diseases causing skeletal malformations or cartilage degeneration.     

EDISON-WMW: Exact Dynamic Programing Solution of the Wilcoxon–Mann–Whitney Test

In many research disciplines, hypothesis tests are applied to evaluate whether findings are statistically significant or could be explained by chance. The Wilcoxon–Mann–Whitney(WMW) test is among the most popular hypothesis tests in medicine and life science to analyze if two groups of samples are equally distributed. This nonparametric statistical homogeneity test is commonly applied in molecular diagnosis. Generally, the solution of the WMW test takes a high combinatorial effort for large sample cohorts containing a significant number of ties. Hence, P value is frequently approximated by a normal distribution. We developed EDISON-WMW, a new approach to calculate the exact permutation of the two-tailed unpaired WMW test without any corrections required and allowing for ties. The method relies on dynamic programing to solve the combinatorial problem of the WMW test efficiently. Beyond a straightforward implementation of the algorithm, we presented different optimization strategies and developed a parallel solution. Using our program,the exact P value for large cohorts containing more than 1000 samples with ties can be calculated within minutes. We demonstrate the performance of this novel approach on randomly-generated data, benchmark it against 13 other commonly-applied approaches and moreover evaluate molecular biomarkers for lung carcinoma and chronic obstructive pulmonary disease(COPD). We foundthat approximated P values were generally higher than the exact solution provided by EDISONWMW. Importantly, the algorithm can also be applied to high-throughput omics datasets, where hundreds or thousands of features are included. To provide easy access to the multi-threaded version of EDISON-WMW, a web-based solution of our algorithm is freely available at http://www.ccb.uni-saarland.de/software/wtest/.     

Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling

Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ET_A and ET_B receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y₁, and adenosine A₁ receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ET_A and ET_B receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.     

The application of the pedigree approach to the distributions foreseen in ecoinvent v3

Purpose

Data used in life cycle inventories are uncertain (Ciroth et al. Int J Life Cycle Assess 9(4):216–226, 2004). The ecoinvent LCI database considers uncertainty on exchange values. The default approach applied to quantify uncertainty in ecoinvent is a semi-quantitative approach based on the use of a pedigree matrix; it considers two types of uncertainties: the basic uncertainty (the epistemic error) and the additional uncertainty (the uncertainty due to using imperfect data). This approach as implemented in ecoinvent v2 has several weaknesses or limitations, one being that uncertainty is always considered as following a lognormal distribution. The aim of this paper is to show how ecoinvent v3 will apply this approach to all types of distributions allowed by the ecoSpold v2 data format.

Methods

A new methodology was developed to apply the semi-quantitative approach to distributions other than the lognormal. This methodology and the consequent formulas were based on (1) how the basic and the additional uncertainties are combined for the lognormal distribution and on (2) the links between the lognormal and the normal distributions. These two points are summarized in four principles. In order to test the robustness of the proposed approach, the resulting parameters for all probability density functions (PDFs) are tested with those obtained through a Monte Carlo simulation. This comparison will validate the proposed approach.

Results and discussion

In order to combine the basic and the additional uncertainties for the considered distributions, the coefficient of variation (CV) is used as a relative measure of dispersion. Formulas to express the definition parameters for each distribution modeling a flow with its total uncertainty are given. The obtained results are illustrated with default values; they agree with the results obtained through the Monte Carlo simulation. Some limitations of the proposed approach are cited.

Conclusions

Providing formulas to apply the semi-quantitative pedigree approach to distributions other than the lognormal will allow the life cycle assessment (LCA) practitioner to select the appropriate distribution to model a datum with its total uncertainty. These data variability definition technique can be applied on all flow exchanges and also on parameters which play an important role in ecoinvent v3.

     

A high-throughput method for liquid chromatography–tandem mass spectrometry determination of plasma alkylresorcinols,biomarkers of whole grain wheat and rye intake

Plasma alkylresorcinols are increasingly analyzed in cohort studies to improve estimates of whole grain intake and their relationship with disease incidence. Current methods require large volumes of solvent (>10 ml/sample) and have relatively low daily sample throughput. We tested five different supported extraction methods for extracting alkylresorcinols from plasma and improved a normal-phase liquid chromatography coupled to a tandem mass spectrometer method to reduce sample analysis time. The method was validated and compared with gas chromatography–mass spectrometry analysis. Sample preparation with HybridSPE supported extraction was most effective for alkylresorcinol extraction, with recoveries of 77–82% from 100 μl of plasma. The use of 96-well plates allowed extraction of 160 samples per day. Using a 5-cm NH₂ column and heptane reduced run times to 3 min. The new method had a limit of detection and limit of quantification equivalent to 1.1–1.8 nmol/L and 3.5–6.1 nmol/L plasma, respectively, for the different alkylresorcinol homologues. Accuracy was 93–105%, and intra- and inter-batch precision values were 4–18% across different plasma concentrations. This method makes it possible to quantify plasma alkylresorcinols in 100 μl of plasma at a rate of at least 160 samples per day without the need for large volumes of organic solvents.     

Economic Appraisal of Ontario's Universal Influenza Immunization Program: A Cost-Utility Analysis

Background

In July 2000, the province of Ontario, Canada, initiated a universal influenza immunization program (UIIP) to provide free seasonal influenza vaccines for the entire population. This is the first large-scale program of its kind worldwide. The objective of this study was to conduct an economic appraisal of Ontario''s UIIP compared to a targeted influenza immunization program (TIIP).

Methods and Findings

A cost-utility analysis using Ontario health administrative data was performed. The study was informed by a companion ecological study comparing physician visits, emergency department visits, hospitalizations, and deaths between 1997 and 2004 in Ontario and nine other Canadian provinces offering targeted immunization programs. The relative change estimates from pre-2000 to post-2000 as observed in other provinces were applied to pre-UIIP Ontario event rates to calculate the expected number of events had Ontario continued to offer targeted immunization. Main outcome measures were quality-adjusted life years (QALYs), costs in 2006 Canadian dollars, and incremental cost-utility ratios (incremental cost per QALY gained). Program and other costs were drawn from Ontario sources. Utility weights were obtained from the literature. The incremental cost of the program per QALY gained was calculated from the health care payer perspective. Ontario''s UIIP costs approximately twice as much as a targeted program but reduces influenza cases by 61% and mortality by 28%, saving an estimated 1,134 QALYs per season overall. Reducing influenza cases decreases health care services cost by 52%. Most cost savings can be attributed to hospitalizations avoided. The incremental cost-effectiveness ratio is Can$10,797/QALY gained. Results are most sensitive to immunization cost and number of deaths averted.

Conclusions

Universal immunization against seasonal influenza was estimated to be an economically attractive intervention. Please see later in the article for the Editors'' Summary     

Accelerated hand bone mineral density loss is associated with progressive joint damage in hands and feet in recent-onset rheumatoid arthritis

Introduction

To investigate whether accelerated hand bone mineral density (BMD) loss is associated with progressive joint damage in hands and feet in the first year of rheumatoid arthritis (RA) and whether it is an independent predictor of subsequent progressive total joint damage after 4 years.

Methods

In 256 recent-onset RA patients, baseline and 1-year hand BMD was measured in metacarpals 2-4 by digital X-ray radiogrammetry. Joint damage in hands and feet were scored in random order according to the Sharp-van der Heijde method at baseline and yearly up to 4 years.

Results

68% of the patients had accelerated hand BMD loss (>-0.003 g/cm²) in the first year of RA. Hand BMD loss was associated with progressive joint damage after 1 year both in hands and feet with odds ratios (OR) (95% confidence intervals [CI]) of 5.3 (1.3-20.9) and 3.1 (1.0-9.7). In univariate analysis, hand BMD loss in the first year was a predictor of subsequent progressive total joint damage after 4 years with an OR (95% CI) of 3.1 (1.3-7.6). Multivariate analysis showed that only progressive joint damage in the first year and anti-citrullinated protein antibody positivity were independent predictors of long-term progressive joint damage.

Conclusions

In the first year of RA, accelerated hand BMD loss is associated with progressive joint damage in both hands and feet. Hand BMD loss in the first year of recent-onset RA predicts subsequent progressive total joint damage, however not independent of progressive joint damage in the first year.     

One Enzyme,Two Functions: PON2 PREVENTS MITOCHONDRIAL SUPEROXIDE FORMATION AND APOPTOSIS INDEPENDENT FROM ITS LACTONASE ACTIVITY*

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O₂^˙̄ dismutase activities and cytochrome c expression unaltered, and it did not oxidize O₂^˙̄ but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His¹¹⁴ and His¹³³ are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys³¹¹, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.     

Identification of Key Residues in Virulent Canine Distemper Virus Hemagglutinin That Control CD150/SLAM-Binding Activity

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by β-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.Paramyxoviruses are enveloped nonsegmented negative-strand RNA viruses that inject their genetic information into target cells by fusing their lipid envelope with the plasma membrane of the host cell at a neutral pH. Plasma membrane fusion activity is achieved by the concerted action of two viral membrane-bound glycoproteins. The attachment protein (hemagglutinin [H], hemagglutinin-neuraminidase [HN], or attachment [G], depending on the viral genus) is thought to bind a host cell surface receptor, in turn activating the fusion (F) protein, which will then undergo large-scale structural rearrangements, leading to plasma membrane fusion activity (9, 10, 19). In addition, both viral surface glycoproteins may mediate fusion activity between two contacting neighboring cells (22, 27). Virus-induced cell-cell fusion activity eventually leads to multinucleated cell formation (also termed syncytium formation) and, ultimately, to cell lysis.The crystal structure of the measles virus hemagglutinin (MeV-H) has recently become available (3, 7, 8). Interestingly, the overall β-propeller structure consisting of six β-sheets was well conserved compared to already determined paramyxovirus HN structures (4, 12, 29). The canine distemper virus H (CDV-H) protein has a short N-terminal cytoplasmic tail followed by a transmembrane domain and a large C-terminal ectodomain (1). It is suggested that the ectodomain consists of a stalk region with an α-helical coiled-coil configuration (13, 28) that supports a globular head domain containing the receptor recognition site and antigenic regions of the protein (11).Recently, site-directed mutagenesis aimed at identifying residues throughout the MeV-H ectodomain that might selectively control membrane fusion activity in a receptor-dependent manner (CD150/SLAM, CD46, or a yet-unidentified putative epithelial cell receptor [EpR]) was conducted. Indeed, four key residues, located in two connected microdomains (site 1 and site 2) on MeV-H globular head β-propeller blade 5, were necessary to uphold SLAM-dependent fusogenicity. Mutations in each one of the four amino acids resulted in a selective inhibition of SLAM-dependent fusion activity (H-SLAM-blind; HSB [25]). Interestingly, the latter quartet of residues were subsequently demonstrated not to be involved in SLAM-binding activity but presumably were involved in controlling SLAM-dependent F triggering (14). An additional residue (isoleucine 194), located within MeV-H β-propeller blade 6 but in contact with site 2, was next shown to govern interaction with the universal morbillivirus SLAM receptor (14). Consequently, the corresponding residues of both microdomains were mutated in the H protein of the virulent CDV strain 5804P and were also demonstrated to control SLAM-dependent fusion activity (24), although for CDV, full ablation of fusion activity required the substitutions in both microdomains and in two additional neighboring amino acids (CDV-H residues in site 1, D526, I527, S528, and R529; in site 2, Y547 and T548). Moreover, using a CDV-H 3D homology model, the two microdomains were demonstrated to be in very close proximity to one another (compared to those of MeV-H) but not in direct contact (24). Subsequently, a recombinant CDV bearing a SLAM-blind H protein was reported to be completely attenuated in ferrets, a phenotype associated with reduced immunosuppression and lack of neurovirulence (26). However, the precise molecular mechanisms sustaining HSB-dependent lack of fusion support activity was not elucidated and remains to be determined.     

Who Counts? Demography of Swidden Cultivators in Southeast Asia

Swidden cultivators are often found as a distinct category of farmers in the literature, but rarely appear in population censuses or other national and regional classifications. This has led to a worldwide confusion on how many people are dependent on this form of agriculture. The most often cited number of 200–300 million dates back to the early 1970s, but the source is obscure. We assess available, published data from nine countries in Southeast Asia and conclude that on this basis it is not possible to provide a firm estimate of the number of swidden cultivators in the region. A conservative range of 14–34 million people engaged in swidden cultivation in the region is suggested, however. We argue that along with improved knowledge of swidden livelihoods, there is an urgent need to develop techniques that will allow for better estimates of swidden populations in order to secure appropriate rural development and poverty reduction in swidden areas.