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We report the effects of quercetin, a flavonoid present in human diet, on early stage beta-amyloid (Aβ) aggregation – a seminal event in Alzheimer’s disease. Molecular level changes in Aβ arrangements are monitored by time-resolved emission spectral (TRES) measurements of the fluorescence of Aβ’s single tyrosine intrinsic fluorophore (Tyr). The results suggest that quercetin binds beta-amyloid oligomers at early stages of their aggregation, which leads to the formation of modified oligomers and hinders the creation of beta-sheet structures, potentially preventing the onset of Alzheimer’s disease.

Sizzled (Szl) is both a secreted frizzled related protein (sFRP) and a naturally occurring inhibitor of the zinc metalloproteinase bone morphogenetic protein-1 (BMP-1), a key regulator of extracellular matrix assembly and growth factor activation. Here we present a new crystal structure for Szl which differs from that previously reported by a large scale (90°) hinge rotation between its cysteine-rich and netrin-like domains. We also present results of a molecular docking analysis showing interactions likely to be involved in the inhibition of BMP-1 activity by Szl. When compared with known structures of BMP-1 in complex with small molecule inhibitors, this reveals features that may be helpful in the design of new inhibitors to prevent the excessive accumulation of extracellular matrix that is the hallmark of fibrotic diseases.

Plankton ecologists ultimately focus on forecasting, both applied and environmental outcomes. We review how appreciating planktonic ciliates has become central to these predictions. We explore the 350-year-old canon on planktonic ciliates and examine its steady progression, which has been punctuated by conceptual insights and technological breakthroughs. By reflecting on this process, we offer suggestions as to where future leaps are needed, with an emphasis on predicting outcomes of global warming. We conclude that in terms of climate change research: (i) climatic hotspots (e.g. polar oceans) require attention; (ii) simply adding ciliate measurements to zooplankton/phytoplankton-based sampling programs is inappropriate; (iii) elucidating the rare biosphere's functional ecology requires culture-independent genetic methods; (iv) evaluating genetic adaptation (microevolution) and population composition shifts is required; (v) contrasting marine and freshwaters needs attention; (vi) mixotrophy needs attention; (vii) laboratory and field studies must couple automated measurements and molecular assessment of functional gene expression; (viii) ciliate trophic diversity requires appreciation; and (ix) marrying gene expression and function, coupled with climate change scenarios is needed. In short, continued academic efforts and financial support are essential to achieve the above; these will lead to understanding how ciliates will respond to climate change, providing tools for forecasting.

The Ehlers–Danlos syndromes (EDS) are a group of rare inherited connective tissue disorders. Vascular EDS (vEDS) is caused by pathogenic variants in COL3A1, most frequently glycine substitutions. We describe the phenotype of the largest series of vEDS patients with glutamic acid to lysine substitutions (Glu>Lys) in COL3A1, which were all previously considered to be variants of unknown significance. Clinical and molecular data for seven families with three different Glu>Lys substitutions in COL3A1 were analyzed. These Glu>Lys variants were reclassified from variants of unknown significance to either pathogenic or likely pathogenic in accordance with American College of Medical Genetics and Genomics guidelines. All individuals with these atypical variants exhibited skin hyperextensibility as seen in individuals with classical EDS and classical-like EDS and evidence of tissue fragility as seen in individuals with vEDS. The clinical data demonstrate the overlap between the different EDS subtypes and underline the importance of next-generation sequencing gene panel analysis. The three different Glu>Lys variants point toward a new variant type in COL3A1 causative of vEDS, which has consistent clinical features. This is important knowledge for COL3A1 variant interpretation. Further follow-up data are required to establish the severity of tissue fragility complications compared with patients with other recognized molecular causes of vEDS.

The procollagen C-propeptides of the fibrillar collagens play key roles in the intracellular assembly of procollagen molecules from their constituent polypeptides chains, and in the extracellular assembly of collagen molecules into fibrils. Here we review recent advances in understanding the molecular mechanisms controlling C-propeptide trimerization which have revealed the importance of inter-chain disulphide bonding and a small number of charged amino acids in the stability and specificity of different types of chain association. We also show how the crystal structure of the complex between the C-propeptide trimer of procollagen III and the active fragment of procollagen C-proteinase enhancer-1 leads to a detailed model for accelerating release of the C-propeptides from procollagen by bone morphogenetic protein-1 and related proteinases. We then discuss the effects of disease-related missense mutations in the C-propeptides in relation to the sites of these mutations in the three-dimensional structure. While in general there is a good correlation between disease severity and structure-based predictions, there are notable exceptions, suggesting new interactions involving the C-propeptides yet to be characterized. Mutations affecting proteolytic release of the C-propeptides from procollagen are discussed in detail. Finally, the roles of recently discovered interaction partners for the C-propeptides are considered during fibril assembly and cross-linking.

Hip pathology is common amongst athletes and the general population. The mechanics of cycling have the potential to exacerbate symptomatic hip pathology and progress articular pathology in patients with morphologic risk factors such as femoroacetabular impingement. A professional fit of the bicycle to the individual which aims to optimize hip joint function can allow patients with hip pathology to exercise in comfort when alternative high impact exercise such as running may not be possible. Conversely improper fit of the bicycle can lead to hip symptoms in otherwise healthy individuals who present with risk factors for hip pain. Accordingly a bike fit can form part of the overall management strategy in a cyclist with hip symptoms. The purpose of this clinical commentary is to discuss hip pathomechanics with respect to cycling, bicycle fitting methodology and the options available to a physical therapist to optimize hip mechanics during the pedaling action.

M13 bacteriophage is a key instrument in phage display applications, as well as a possible antibacterial therapeutic agent due to its highly restrictive bacterial pathogenesis, and other applications. The traditional phage purification process is usually achieved by gradient ultracentrifugation or a combination of precipitation, centrifugation and microfiltration. These approaches easily lead to long process times, high operational costs, phage aggregation and consequent product loss (approximately 60%). This work is thus focused on an alternative potential large-scale process to achieve high yield and purity while minimizing the operational costs. Electrostatic-based separation processes are also common biomolecules purification techniques. Although anion exchange chromatography has been used before to purify several viral particles, this technique has been poorly reported for the purification of M13 phage. In a recent work, our group has demonstrated the use of a predominant anion exchange process, where a polymeric ionic liquid (PIL) was used as an alternative separation matrix for M13 bacteriophage. In this work, a variety of system parameters was studied, including chemical structure of the cation and the anion, the crosslinker nature and its concentration, either in batch adsorption/elution or chromatographic operation mode. The PIL-based chromatographic operation mode revealed to be a suitable separation process for M13 from directly filtered E. coli supernatant, reaching over 70% M13 recovery and 4.6 purification factor in a single step. To our knowledge, this is the first time that PILs have been reported as separation agents for bioproducts from complex mixtures.

M13 is a filamentous, non-lytic bacteriophage that infects Escherichia coli via the F pilus. Currently, phage M13 is widely used in phage display technology and bio-nanotechnology, and is considered a possible antibacterial therapeutic agent, among other applications. Conventional phage purification involves 5-7 operational steps, with high operational costs and significant product loss (approximately 60%). In this work, we propose a scalable purification process for M13 bacteriophage using a novel stationary phase based on a polymeric ionic liquid (PIL) with a positively charged backbone structure. Poly (1-vinyl-3-ethyl imidazolium bis(trifluoromethylsulfonyl) imide) - poly(VEIM-TFSI) predominantly acted as an anion exchanger under binding-elution mode. This revealed to be a rapid and simple method for the recovery of phage M13 with an overall separation yield of over 70% after a single downstream step. To the best of our knowledge, PILs have never been used as separation matrices for biological products and the results obtained, together with the large number of cations and anions available to prepare PILs, illustrate well the large potential of the proposed methodology.

The defence collagens C1q and mannose-binding lectin (MBL) are immune recognition proteins that associate with the serine proteinases C1r/C1s and MBL-associated serine proteases (MASPs) to trigger activation of complement, a major innate immune system. Bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (BTPs) are metalloproteinases with major roles in extracellular matrix assembly and growth factor signalling. Despite their different functions, C1r/C1s/MASPs and BTPs share structural similarities, including a specific CUB-EGF-CUB domain arrangement found only in these enzymes that mediates interactions with collagen-like proteins, suggesting a possible functional relationship. Here we investigated the potential interactions between the defence collagens C1q and MBL and the BTPs BMP-1 and mammalian tolloid-like-1 (mTLL-1). C1q and MBL bound to immobilized BMP-1 and mTLL-1 with nanomolar affinities. These interactions involved the collagen-like regions of the defence collagens and were inhibited by pre-incubation of C1q or MBL with their cognate complement proteinases. Soluble BMP-1 and mTLL-1 did not inhibit complement activation and the defence collagens were neither substrates nor inhibitors of BMP-1. Finally, C1q co-localized with BMP-1 in skin biopsies following melanoma excision and from patients with recessive dystrophic epidermolysis bullosa. The observed interactions provide support for a functional link between complement and BTPs during inflammation and tissue repair.

A non‐invasive intrinsic fluorescence sensing of the early stages of Alzheimer’s beta amyloid peptide aggregation in the presence of copper ions is reported. By using time‐resolved fluorescence techniques the formation of beta amyloid‐copper complexes and the accelerated peptide aggregation are demonstrated. The shifts in the emission spectral peaks indicate that the peptides exhibit different aggregation pathways than in the absence of copper.

During the last years, poly(ionic liquids) (PILs) have been gaining increased attention as materials for analytical chemistry. This success is due to the fact that PILs synthesis is generally based on the polymerization of ionic liquid monomers (ILMs), and thus some of their remarkable properties are shared with their polymeric form. Consequently, the facile design of task-specific ILMs led to an important platform to design new materials for solid phase microextraction (SPME). In this review, a critical evaluation of the main breakthroughs on the use of PILs as extraction phases for SPME. In particular, this critical review covers from ILMs/PILs synthesis, fiber coating processes, surface modifications (silica and metallic supports), to extraction modes, analytes categories, type of equipment ending with a discussion on limitations and future perspectives. PILs are undoubtedly competing in the frontline as inspiring materials for SPME.

Development of a pseudoaneurysm of the ascending aorta is an uncommon complication of aortic surgery. Several nonsurgical techniques are available for treatment of ascending aortic pseudoaneurysms (AAPs). This report outlines a single-center retrospective experience with 14 nonsurgical procedures for treatment of AAPs in 10 patients. Modified stent grafts, septal defect occlusion devices, coil embolics, and liquid embolics were deployed by transthoracic and endovascular approaches. Complete stasis of the AAP was achieved in 7 of 10 patients (70%). Mean postprocedural recoveries occurred within 3.5 days. Nonsurgical techniques for repair of AAPs offer a comparatively safe and effective alternative to open surgical repair.

Hemangiomas are the most common tumor of the liver and distinguishing them from malignancy is important. This is a report of 3 hemangiomas in 2 patients that exhibit transient washout of gadoxetate disodium (Eovist), relative to blood pool and liver parenchyma, a characteristic that is used to diagnose hepatocellular carcinoma in at-risk patients. It is important to recognize that high-flow hemangiomas can exhibit transient washout when using a small volume of injected contrast agent. This finding is unlikely to be present on CT examinations because of the larger volume of contrast administered.

This work explores the widening of properties of cholinium-based ionic liquids (ILs) through their combination with perfluoroalkanoate anions so that higher number of aqueous biphasic systems (ABSs) containing nontoxic cholinium-based ILs is available. For that purpose, six cholinium perfluoroalkanoate ILs were synthesized and their cytotoxicity was evaluated using three different animal cell lines, envisaging biotechnology applications. Ternary phase equilibrium data for ABSs composed of the cholinium perfluoroalkanoate, with fluoroalkyl chains from C2 up to C7, using a strong salting out agent, K3PO4, were determined at 25 °C. The results show the relevant role of the size of fluorinated alkyl chain length in the anion since, contrary to other ABSs containing ILs with increasing alkyl chain length in the anion, the ABSs with cholinium perfluoroalkanoates present well-spaced solubility curves, allowing the conclusion that these ABSs can be tuned by a proper choice of the IL. The phase splitting mechanism...

Familiarity with how MRI sequences can distinguish different tissues when coupled with an understanding of pathology aids in narrowing differentials or making a specific diagnosis. Utilizing specific MRI pulse sequences allows for identification of key tissue substances such as fat, paramagnetic substances, protein, fibrous tissue, or free or bound water. The identification of these tissue substances allows the radiologist to form narrow or specific diagnoses efficiently. A tissue-based approach to understanding MRI sequences allows the radiologist to both systematically and effectively interpret MRIs despite the large number of pulse sequences particularly in basic MRI body protocols.

Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ± PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.

In this work, a facile preparation of SPME fibers with increased surface area is presented. The SPME fibers were prepared by grinding poly(ionic liquids) (PILs) to obtain particles of 1–16 µm and, with the aid of a silicon adhesive, attach these particles to a steel wire support. Three different PILs, poly(1-vinyl-3-benzylimidazolium-co-styrene bromide) [poly(ViBnIm-co-Sty Br)], poly(1-vinyl-3-benzylimidazolium-co-styrene bis(trifluoromethanesulfonyl)imide) [poly(ViBnIm-co-Sty TFSI)] and poly(diallyldimethylamine bis(trifluoromethanesulfonyl)imide) [poly(Pyrr11 TFSI)], were used. The first two PILs were obtained by reversible addition–fragmentation chain transfer polymerization followed by metathesis reactions. The thicknesses of the prepared fibers were found to be 19 ± 4 µm and 85% of the particles used have diameters between 2 and 10 µm. The prepared fibers were tested by performing the headspace extraction of two standard solutions, one containing a mixture of alcohols with different chain lengths, and the other a mixture of aromatic compounds, leading to sorption times of 10 – 15 min due the large surface area attained with this method. PILs with aromatic moieties containing the bromide anion showed high selectivity towards polar compounds, due to the hydrogen basicity of the anion, and also towards aromatic analytes, due to the aromatic nature of styrene moieties and the cation pendant groups. The limits of detection fall in the sub ppb level, while relative standard deviations and reproducibility from fiber-to-fiber found maximums of 16.2% and 22.5%, respectively. Furthermore, the PIL based fibers showed up to 90% higher extraction efficiencies compared to the commercial fibers of polydimethylsiloxane and polyacrylate.

Fibrillar collagens of the extracellular matrix are critical for tissue structure and physiology; however, excessive or abnormal deposition of collagens is a defining feature of fibrosis. Regulatory mechanisms that act on collagen fibril assembly potentially offer new targets for antifibrotic treatments. Tissue weakening, altered collagen fibril morphologies, or both, are shared phenotypes of mice lacking matricellular thrombospondins. Thrombospondin-1 (TSP1) plays an indirect role in collagen homeostasis through interactions with matrix metalloproteinases and transforming growth factor–1 (TGF-1). We found that TSP1 also affects collagen fibril formation directly. Compared to skin from wild-type mice, skin from Thbs1-/- mice had reduced collagen cross-linking and reduced prolysyl oxidase (proLOX) abundance with increased conversion to catalytically active LOX. In vitro, TSP1 bound to both the C-propeptide domain of collagen I and the highly conserved KGHR sequences of the collagen triple-helical domain that participate in cross-linking. TSP1 also bound to proLOX and inhibited proLOX processing by bone morphogenetic protein-1. In human dermal fibroblasts (HDFs), TSP1 and collagen I colocalized in intracellular vesicles and on extracellular collagen fibrils, whereas TSP1 and proLOX colocalized only in intracellular vesicles. Inhibition of LOX-mediated collagen cross-linking did not prevent the extracellular association between collagen and TSP1; however, treatment of HDFs with KGHR-containing, TSP1-binding, triple-helical peptides disrupted the collagen-TSP1 association, perturbed the collagen extracellular matrix, and increased myofibroblastic differentiation in a manner that depended on TGF- receptor 1. Thus, the extracellular KGHR-dependent interaction of TSP1 with fibrillar collagens contributes to fibroblast homeostasis.

The design of poly(ionic liquids) (PILs) and their application as solid phase microextraction (SPME) fibers has been attracting enormous attention mainly due to the need for new SPME coating materials with improved analytical sensitivity. In this work, the tunability of PILs is explored by preparing different imidazolium monomers bearing benzyl, naphtylmethyl or pentyl pending groups that were subsequently co-polymerized, by reversible addition–fragmentation chain transfer (RAFT) polymerization with styrene. The obtained co-polymers showed excellent thermal stability up to 275 °C, with no melting point up to 250 °C. SPME fibers were prepared by an innovative approach based on layer-by-layer spray coating. The thin (

Purpose To evaluate the utility of dynamic, contrast-enhanced magnetic resonance imaging (MRI) in combination with single-shot T2-weighted (ssT2) sequences in the differentiation of lipid-poor adrenal adenomas from non-adenomas. Materials and methods This retrospective study was approved by the institutional review board and is HIPAA compliant. Between January 2007 and December 2010, 46 patients with MRI demonstrating a lipid-poor adrenal lesion who underwent either surgical resection or a minimum of 24 months of imaging follow-up were identified retrospectively. All images were retrospectively reviewed in blinded fashion by two radiologists. Each adrenal lesion was categorized by dynamic enhancement features and qualitative signal on ssT2 images and was categorized as an adenoma if it demonstrated homogenous enhancement in the arterial phase, washout with capsule enhancement in the delayed phase, and T2 signal isointense to normal adrenal tissue. Any lesion that did not fulfill all the criteria was classified as a non-adenoma. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for characterization of adenoma were calculated for each reader with 95% confidence intervals. A κ test assessed level of agreement between readers. Results Application of our criteria lead to an MRI diagnosis of lipid-poor adrenal adenoma with a sensitivity of 84.2–89.5% (16/19–17/19), specificity of 96.3% (26/27), positive predictive value of 94.1–94.4% (16/17–17/18), negative predictive value of 89.7–92.9% (26/29–26/28), and accuracy of 91.3–93.5% (42/46–43/46). Agreement between the two readers showed substantial κ agreement for the differentiation of adenoma from non-adenoma. Conclusions Dynamic, contrast-enhanced T1-weighted three-dimensional gradient echo sequences in combination with ssT2 images can accurately differentiate lipid-poor adrenal adenomas from non-adenomas.

Binary systems composed of water and cholinium carboxylate ionic liquids, namely cholinium lactate ([Ch][Lac]), cholinium propanoate ([Ch][Prop]) and cholinium malonate ([Ch][Mal]) were studied from the neat ionic liquid to very diluted aqueous solutions. Densities and viscosities were measured and atypical behaviors were observed, such as the increasing density of the binary [Ch][Prop] + H2O mixtures with increasing water content. In order to get molecular level insights on the IL + H2O solvation schemes, 1H NMR studies were performed. Large deviations were obtained in the anions resonances when compared to those of the cation suggesting that water interacts preferentially with the anion counter-part of the ionic liquid. The increasing density of [Ch][Prop] + H2O system with increasing water content can be related to the orientation of the alkyl chains, as a result of their nanoscale organization. This behavior was confirmed through the study of the thermophysical properties of [Ch][Hex] + H2O mixtures, where this phenomenon is known to occur.

Vascular Ehlers–Danlos syndrome (vEDS) is a heritable disorder of connective tissue caused by pathological variants in the COL3A1 gene, which encodes the α1 chain of type III collagen. Type III collagen is a major component of skin, arterial walls, and the gastrointestinal tract. Collagen III protein deficiency manifests as an increased risk of rupture, perforation, and dissection of these structures. The most disruptive gene variants affect the collagen helix via glycine substitutions or splice donor site mutations. The C-propeptide region of COL3A1 includes exons 49–52 and has a crucial role in initiating the C-terminal assembly of procollagen monomers in the early stages of collagen biosynthesis. Nineteen COL3A1 variants have previously been reported in these exons, of which four were associated with a severe vEDS phenotype. We identified two novel C-propeptide missense variants; p.Pro1440Leu, p.Arg1432Leu, and a non-stop mutation, c.4400A > T, p. (*1467Leuext*45). These variants produce variable phenotypes ranging from obvious acrogeria to classical or hypermobile EDS. A previously reported variant p.Lys1313Arg is of unknown clinical significance but likely benign, based on this study. Assigning disease pathogenicity remains complex, clinical phenotyping and crystal structure evidence being crucial. We briefly compare reported phenotypes for patients with missense variants in the C-propeptide domain for other human collagen disorders including COL1A1 and COL1A2 (osteogenesis imperfecta). © 2015 Wiley Periodicals, Inc.

Crosslinked pyrrolidinium-based poly(ionic liquids) (Pyrr-PILs) were synthesized through a fast, simple, and solventless photopolymerization scheme, and tested as solid phase microextraction (SPME) sorbents. A series of Pyrr-PILs bearing three different alkyl side chain lengths with two, eight, and fourteen carbons was prepared, characterized, and homogeneously coated on a steel wire by using a very simple procedure. The resulting coatings showed a high thermal stability, with decomposition temperatures above 350 degrees C, excellent film stability, and lifetime of over 100 injections. The performance of these PIL-based SPME fibers was evaluated using a mixture of eleven organic compounds with different molar volumes and chemical functionalities (alcohols, ketones, and monoterpenes). The Pyrr-PIL fibers were obtained as dense film coatings, with 67 mu m thickness, with an overall sorption increase of 90% and 55% as compared to commercial fibers of Polyacrylate (85 mu m) (PA85) and Polydimethylsiloxane (7 mu m) (PDMS7) coatings, respectively. A urine sample doped with the sample mixture was used to study the matrix effect and establish relative recoveries, which ranged from 60.2% to 104.1%. David J. S. Patinha, and Liliana C. Tome are grateful to FCT (Fundacao para a Ciencia e a Tecnologia) for the PhD research grant SFRH/BD/97042/2013 and the Post-Doctoral research grant (SFRH/BPD/101793/2014), respectively. David J. S. Patinha also thanks the financial support from COST-Exil Project 1206. The NMR data was acquired at CERMAX (Centro de Ressonncia Magnetica Antnio Xavier) which is a member of the National NMR network. This work was partially supported by FCT through Research Unit GREEN-it " Bioresources for Sustainability" (UID/Multi/04551/2013) and the Associate Laboratory CICECO Aveiro Institute of materials (UID/CTM/50011/2013).

The objective of our study was to determine patterns and cost of imaging tumor surveillance in patients after a benign fine-needle aspiration (FNA) biopsy of the thyroid in a large teaching hospital as well as the rate of subsequent cancer detection.This cohort study was approved by the appropriate institutional review board and complied with HIPAA. All patients who had a benign thyroid FNA biopsy between January 1, 1999, and December 31, 2003, were identified from an institutional pathology database. We gathered information from electronic medical records on imaging tumor surveillance and subsequent cancer detection. Cost was determined using the facility total relative value unit and the 2014 Hospital Outpatient Prospective Payment System conversion factor.Between January 1, 1999, and December 31, 2003, 1685 patients had a benign thyroid FNA biopsy, 800 (47.5%) of whom underwent follow-up imaging. These patients underwent 2223 thyroid ultrasound examinations, 606 ultrasound-guided thyroid FNA biopsies, 78 thyroid scintigraphy examinations, 168 neck CTs, and 53 neck MRIs at a cost of $529,874, $176,157, $39,622, $80,580, and $53,114, respectively, for a total cost of $879,347 or $1099 per patient. The mean length of follow-up was 7.3 years, during which time 19 (2.4%) patients were diagnosed with thyroid cancer at a cost of $46,281 per cancer. Seventeen (89.5%) were diagnosed with papillary carcinoma and two (10.5%) with Hurthle cell carcinoma.Over a 5-year period, about half of the patients who had a benign thyroid FNA biopsy underwent follow-up imaging at considerable cost with a small rate of subsequent malignancy.

The C propeptides of fibrillar procollagens have crucial roles in tissue growth and repair by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils. Mutations in C propeptides are associated with several, often lethal, genetic disorders affecting bone, cartilage, blood vessels and skin. Here we report the crystal structure of a C-propeptide domain from human procollagen III. It reveals an exquisite structural mechanism of chain recognition during intracellular trimerization of the procollagen molecule. It also gives insights into why some types of collagen consist of three identical polypeptide chains, whereas others do not. Finally, the data show striking correlations between the sites of numerous disease-related mutations in different C-propeptide domains and the degree of phenotype severity. The results have broad implications for understanding genetic disorders of connective tissues and designing new therapeutic strategies.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-β binding protein 1, TGF-β RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-β activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-β signaling as primary targets.

To elucidate causes for false negative magnetic resonance imaging (MRI) exams by identifying imaging characteristics that predict viable hepatocellular carcinoma (HCC) in lesions previously treated with locoregional therapy when obvious findings of recurrence are absent.This retrospective institutional review board-approved and Health Insurance Portability and Accountability Act-compliant study included patients who underwent liver transplantation at our center between 1/1/2000 and 12/31/2012 after being treated for HCC with locoregional therapy. All selected patients had a contrast-enhanced MRI after locoregional therapy within 90 d of transplant that was prospectively interpreted as without evidence of residual or recurrent tumor. Retrospectively, 2 radiologists, blinded to clinical and pathological data, independently reviewed the pre-transplant MRIs for 7 imaging features. Liver explant histopathology provided the reference standard, with clinically significant tumor defined as viable tumor ≥ 1.0 cm in maximum dimension. Fisher's exact test was first performed to identify significant imaging features.Inclusion criteria selected for 42 patients with 65 treated lesions. Fourteen of 42 patients (33%) and 16 of 65 treated lesions (25%) had clinically significant viable tumor on explant histology. None of the 7 imaging findings examined could reliably and reproducibly determine which treated lesion had viable tumor when the exam had been prospectively read as without evidence of viable HCC.After locoregional therapy some treated lesions that do not demonstrate any MRI evidence of HCC will contain viable tumor. As such even patients with a negative MRI following treatment should receive regular short-term imaging surveillance because some have occult viable tumor. The possibility of occult tumor should be a consideration when contemplating any action which might delay liver transplant.

BMP-1/tolloid-like proteinases (BTPs) are major enzymes involved in extracellular matrix assembly and activation of bioactive molecules, both growth factors and anti-angiogenic molecules. Although the control of BTP activity by several enhancing molecules is well established, the possibility that regulation also occurs through endogenous inhibitors is still debated. Secreted frizzled-related proteins (sFRPs) have been studied as possible candidates, with highly contradictory results, after the demonstration that sizzled, a sFRP found in Xenopus and zebrafish, was a potent inhibitor of Xenopus and zebrafish tolloid-like proteases. In this study, we demonstrate that mammalian sFRP-1, -2, and -4 do not modify human BMP-1 activity on several of its known substrates including procollagen I, procollagen III, pN-collagen V, and prolysyl oxidase. In contrast, Xenopus sizzled appears as a tight binding inhibitor of human BMP-1, with a K(i) of 1.5 ± 0.5 nM, and is shown to strongly inhibit other human tolloid isoforms mTLD and mTLL-1. Because sizzled is the most potent inhibitor of human tolloid-like proteinases known to date, we have studied its mechanism of action in detail and shown that the frizzled domain of sizzled is both necessary and sufficient for inhibitory activity and that it acts directly on the catalytic domain of BMP-1. Residues in sizzled required for inhibition include Asp-92, which is shared by sFRP-1 and -2, and also Phe-94, Ser-43, and Glu-44, which are specific to sizzled, thereby providing a rational basis for the absence of inhibitory activity of human sFRPs.

Background: Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular glycoprotein that increases activity of certain zinc metalloproteinases involved in tissue development and repair. Results: PCPE-1 binds uniquely to the C-propeptide region of the procollagen molecule. Conclusion: PCPE-1 enhances proteolysis by binding solely to the procollagen C-propeptides. Significance: These data may lead to future applications in the development of antifibrotic therapies., Bone morphogenetic protein-1 (BMP-1) and the tolloid-like metalloproteinases control several aspects of embryonic development and tissue repair. Unlike other proteinases whose activities are regulated mainly by endogenous inhibitors, regulation of BMP-1/tolloid-like proteinases relies mostly on proteins that stimulate activity. Among these, procollagen C-proteinase enhancers (PCPEs) markedly increase BMP-1/tolloid-like proteinase activity on fibrillar procollagens, in a substrate-specific manner. Here, we performed a detailed quantitative study of the binding of PCPE-1 and of its minimal active fragment (CUB1-CUB2) to three regions of the procollagen III molecule: the triple helix, the C-telopeptide, and the C-propeptide. Contrary to results described elsewhere, we found the PCPE-1-binding sites to be located exclusively in the C-propeptide region. In addition, binding and enhancing activities were found to be independent of the glycosylation state of the C-propeptide. These data exclude previously proposed mechanisms for the action of PCPEs and also suggest new mechanisms to explain how these proteins can stimulate BMP-1/tolloid-like proteinases by up to 20-fold.

We recently showed that the highly organized architecture of the corneal stroma could be reproduced using scaffolds consisting of orthogonally aligned multilayers of collagen fibrils prepared using a high magnetic field. Here we show that such scaffolds permit the reconstruction in vitro of human hemi-corneas (stroma + epithelium), using primary human keratocytes and limbal stem cell derived human keratinocytes. On the surface of these hemi-corneas, a well-differentiated epithelium was formed, as determined both histologically and ultrastructurally and by the expression of characteristic markers. Within the stroma, the keratocytes aligned with the directions of the fibrils in the scaffold and synthesized a new extracellular matrix with typical collagen markers and small, uniform diameter fibrils. Finally, in vivo experiments using a rabbit model showed that these orthogonally oriented multi-layer scaffolds could be used to repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization.

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.

Procollagen C-proteinase enhancers (PCPE-1 and -2) specifically activate bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family during C-terminal processing of fibrillar collagen precursors. PCPEs consist of two CUB domains (CUB1 and CUB2) and one NTR domain separated by one short and one long linker. It was previously shown that PCPEs can strongly interact with procollagen molecules, but the exact mechanism by which they enhance BMP-1 activity remains largely unknown. Here, we used a series of deletion mutants of PCPE-1 and two chimeric constructs with repetitions of the same CUB domain to study the role of each domain and linker. Out of all the forms tested, only those containing both CUB1 and CUB2 were capable of enhancing BMP-1 activity and binding to a mini-procollagen substrate with nanomolar affinity. Both these properties were lost by individual CUB domains, which had dissociation constants at least three orders of magnitude higher. In addition, none of the constructs tested could inhibit PCPE activity, although CUB2CUB2NTR was found to modulate BMP-1 activity through direct complex formation with the enzyme, resulting in a decreased rate of substrate processing. Finally, increasing the length of the short linker between CUB1 and CUB2 was without detrimental effect on both activity and substrate binding. These data support the conclusion that CUB1 and CUB2 bind to the procollagen substrate in a cooperative manner, involving the short linker that provides a flexible tether linking the two binding regions.

Tissue remodelling results from the concerted action of numerous extracellular and cell surface proteases. These act to synchronize the synthesis and degradation of the extracellular matrix with the control of cytokine activity and cell signalling in order to create appropriate environments for cell proliferation, migration and differentiation. Wound healing is a complex example of tissue remodelling that includes several steps occurring either concomitantly or successively during the process of repair: haemostasis, inflammation, angiogenesis, re-epithelialisation, granulation tissue formation, wound contraction and matrix remodelling. The main extracellular and cell surface proteases involved in wound healing are serine proteases, especially plasmin, and metalloproteases of the metzincin family (MMPs, ADAM(TS)s, tolloids, meprins, pappalysins) with cysteine proteases playing less prominent roles. Several regulatory proteins and hundreds of substrates have been identified for these proteases, either in vitro or in vivo. The aim of this review is not to present an exhaustive list of proteases and related molecules but to give an overview of the proteolytic events that are potentially relevant during tissue repair. New developments aimed at approaching a more integrative view of all the molecular events involved in tissue remodelling are also discussed.

Collagen IX is a heterotrimer of three α-chains, which consists of three COL domains (collagenous domains) (COL1–COL3) and four NC domains (non-collagenous domains) (NC1–NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX α-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1–NC1 junction. Our results demonstrate that collagen IX α-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2–NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1–NC1 region is important for chain specificity.

The creation of 3D scaffolds that mimic the structure of physiological tissue required for normal cell function is a major bioengineering challenge. For corneal stroma reconstruction this necessitates the creation of a stroma-like scaffold consisting of a stack of orthogonally disposed sheets of aligned collagen fibrils. This study demonstrates that such a scaffold can be built up using magnetic alignment. By allowing neutralized acid-soluble type I collagen to gel in a horizontal magnetic field (7 T) and by combining a series of gelation-rotation-gelation cycles, a scaffold of orthogonal lamellac composed of aligned collagen fibrils has been formed. Although initially dilute, the gels can be concentrated without noticeable loss in orientation. The gels are translucent but their transparency can be greatly improved by the addition of proteoglycans to the gel-forming solution. Keratocytes align by contact guidance along the direction of collagen fibrils and respect the orthogonal design of the collagen template as they penetrate into the bulk of the 3D matrix. The scaffold is a significant step towards the creation of a corneal substitute with properties resembling those of native corneal stroma. (c) 2007 Elsevier Ltd. All rights reserved.

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.

Bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases, here called BTPs, include the proteases originally identified for their roles in the C-terminal maturation of fibrillar procollagens ("procollagen C-proteinase"). Though numerous other substrates have since been discovered, the BTPs remain the main proteases involved in extracellular matrix assembly with little or no implication in matrix degradation. During the same period however, the BTPs have also become established as important proteases in the activation of growth factors, including TGF-β1, BMP-2/-4, GDF-8/-11 and IGFs, as well as the release of anti-angiogenic fragments from parent proteins. The BTPs are therefore key players in many pathophysiological processes such as morphogenesis, tissue repair and tumor progression. This mini-review summarizes our current knowledge of the functions of BTPs, their substrates and unusual mechanisms of regulation, and discusses their potential as new targets for future therapies.

Members of the bone morphogenetic protein-1/tolloid (BMP-1/Tld) family of metalloproteinases, also known as procollagen C-proteinases (PCPs), control multiple biological events (including matrix assembly, cross-linking, cell adhesion/migration and pattern formation) through enzymatic processing of several extracellular substrates. PCP activities on fibrillar procollagens can be stimulated by another family of extracellular proteins, PCP enhancers (PCPE-1, PCPE-2), which lack intrinsic enzymatic activity. While PCPs have multiple substrates, the extent to which PCPEs is involved in the processing of proteins other than fibrillar procollagens is unknown. In the experiments reported here, PCPE-1 was found to have no effect on the in vitro BMP-1 processing of procollagen VII, the procollagen V N-propeptide, the laminin 5 γ2 chain, osteoglycin, prolysyl oxidase, or chordin. In contrast, PCPE-1 enhanced C-terminal processing of human fibrillar procollagen III but only when this substrate was in its native, disulfide-bonded conformation. Surprisingly, processing of procollagen III continued to be enhanced when essentially all the triple-helical region was removed. These and previous results (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869; Bernocco, S., Steiglitz, B. M., Svergun, D. I., Petoukhov, M. V., Ruggiero, F., Ricard-Blum, S., Ebel, C., Geourjon, C., Deleage, G., Font, B., Eichenberger, D., Greenspan, D. S., and Hulmes, D. J. S. (2003) J. Biol. Chem. 278, 7199-7205) indicate that the mechanism of PCPE-1 action involves recognition sites in both the C-propeptide domain and in the C-telopeptide region of the procollagen molecule. PCPEs therefore define a new class of extracellular adaptor proteins that stimulate proteinase activity in a substrate-specific manner, thereby providing a new target for the selective regulation of PCP activity on fibrillar procollagen substrates.

Alpha-helical coiled-coils are widely occurring protein oligomerization motifs. Here we show that most members of the collagen superfamily contain short, repeating heptad sequences typical of coiled coils. Such sequences are found at the N-terminal ends of the C-propeptide domains in all fibrillar procollagens. When fused C-terminal to a reporter molecule containing a collagen-like sequence that does not spontaneously trimerize, the C-propeptide heptad repeats induced trimerization. C-terminal heptad repeats were also found in the oligomerization domains of the multiplexins (collagens XV and XVIII). N-terminal heptad repeats are known to drive trimerization in transmembrane collagens, whereas fibril-associated collagens with interrupted triple helices, as well as collagens VII, XIII, XXIII, and XXV, were found to contain heptad repeats between collagen domains. Finally, heptad repeats were found in the von Willebrand factor A domains known to be involved in trimerization of collagen VI, as well as in collagen VII. These observations suggest that coiled-coil oligomerization domains are widely used in the assembly of collagens and collagen-like proteins.

International audience; Purpose: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian tolloid (mTLD), and mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring. Methods: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1), and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). Bone morphogenetic protein-1/mTLD and PCPE-1 were also immunolocalized in cases of human corneal scarring following injuries. Results: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding, both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas. Conclusions: Bone morphogenetic protein-1/mTLD and PCPE-1 are upregulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring.

International audience; The type I procollagen carboxyterminal(C-)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype-phenotype correlations have been described. To gain insight in the functional consequences of C-propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C-propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C-propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50-55 nucleotides upstream of the most 3' exon-exon junction lead to nonsense-mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe-to-lethal OI. Proα1(I)-C-propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)-C-propeptide is a reliable tool to predict phenotypic severity for most COL1A1-C-propeptide missense variants, whereas for COL1A2-C-propeptide variants, the phenotypic outcome is milder than predicted.

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Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89-91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitro by bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.

Although the collagen V heterotrimer is known to be involved in the control of fibril assembly, the role of the homotrimer in fibrillar organization has not yet been examined. Here, the production of substantial amounts of recombinant collagen V homotrimer has allowed a detailed study of its role in homotypic and heterotypic fibril formation. After removal of terminal regions by pepsin digestion, both the collagen V heterotrimer and homotrimer formed thin homotypic fibrils, thus showing that diameter limitation is at least in part an intrinsic property of the collagen V triple helix. When mixed with collagen I, however, various complementary approaches indicated that the collagen V heterotrimer and homotrimer exerted different effects in heterotypic fibril formation. Unlike the heterotrimer, which was buried in the fibril interior, the homotrimer was localized as thin filamentous structures at the surface of wide collagen I fibrils and did not regulate fibril assembly. Its localization at the fibril surface suggests that the homotrimer can act as a molecular linker between collagen fibrils or macromolecules in the extracellular matrix or both. Thus, depending on their respective distribution in tissues, the different collagen V isoforms might fulfill specific biological functions.

Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or “crimp”, is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo , fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5–30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 μm 2 domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly.

The morphology and basic biochemical composition of articular cartilage from two strains of fowl were examined. Broiler breeder fowl are considered susceptible to degenerative joint disease (DJD); histological examination of one-year-old broiler breeders showed in some samples, articular cartilage thinning, fibrillation and chondrocyte cluster formation, features considered typical of DJD. Examination of similar samples from laying strain fowl showed only minor age-related changes such as some slight cartilage thinning and very mild fibrillation. The articular cartilage from the broiler breeder birds was significantly more hydrated with a higher uronic acid content than that of the laying strain birds. In addition, unloaded articular surfaces such as the proximal humerus had significantly higher amounts of uronic acid than the loaded cartilage surfaces of the proximal tarsometatarsus and the distal tibiotarsus; this suggested that the joint loading may have a role in any biochemical differences found between joints and between strains of fowl. These findings concur with other reports in mammals that showed increased hydration and uronic acid in association with early DJD and in models of osteoarthritis (OA). Thus, despite some differences between avian and mammalian articular cartilage, studies on avian DJD may give insights into mammalian disease.

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a −/− and Mep1b −/− mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.