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We planned to develop predator-prey models using Paramecium and yeast, but they have not been empirically examined since work by Gause in the 1930s. Therefore, we evaluated if Paramecium aurelia ingests and grows on eight yeasts. Recognising that it ingested yeasts but could not grow, we assessed if it might grow on other yeasts, by empirically parameterising a predator-prey model that relies on ingestion, not growth. Simulations were compared to P. aurelia-yeast time-series data, from Gause. We hypothesised that if the model simulated predator-prey dynamics that mimicked the original data, then possibly P. aurelia could grow on yeast; simulations did not mimic the original data. Reviewing works by Gause exposed two issues: experiments were undoubtedly contaminated with bacteria, allowing growth on bacteria, not yeast; and the population cycle data cannot be considered a self-sustaining time series, as they were manipulated by adding yeast and ciliates. We conclude that past and future work should not rely on this system, for either empirical or theoretical evaluations. Finally, although we show that P. aurelia, P. caudatum, Euplotes patella, and Blepharisma sp. cannot grow on yeast, Tetrahymena pyriformis and Colpidium striatum can; these may provide models to explore predator-prey dynamics. [ABSTRACT FROM AUTHOR]

Summary The Penman–Monteith equation has been widely used to estimate the maximum evaporation rate ( E ) from wet/saturated forest canopies, regardless of canopy cover fraction. Forests are then represented as a big leaf and interception loss considered essentially as a one-dimensional process. With increasing forest sparseness the assumptions behind this big leaf approach become questionable. In sparse forests it might be better to model E and interception loss at the tree level assuming that the individual tree crowns behave as wet bulbs (“wet bulb approach”). In this study, and for five different forest types and climate conditions, interception loss measurements were compared to modelled values (Gash’s interception model) based on estimates of E by the Penman–Monteith and the wet bulb approaches. Results show that the wet bulb approach is a good, and less data demanding, alternative to estimate E when the forest canopy is fully ventilated (very sparse forests with a narrow canopy depth). When the canopy is not fully ventilated, the wet bulb approach requires a reduction of leaf area index to the upper, more ventilated parts of the canopy, needing data on the vertical leaf area distribution, which is seldom-available. In such cases, the Penman–Monteith approach seems preferable. Our data also show that canopy cover does not per se allow us to identify if a forest canopy is fully ventilated or not. New methodologies of sensitivity analyses applied to Gash’s model showed that a correct estimate of E is critical for the proper modelling of interception loss. [ABSTRACT FROM AUTHOR]

Abstract: Beta-amyloid (Aβ) aggregation, believed to be responsible for Alzheimer''s disease, is monitored using its intrinsic fluorescence decay. Alterations in the fluorescence decay of tyrosine correlate with the Aβ aggregation at a much earlier stage than the traditionally used fluorescence intensity of Thioflavin T (ThT). Potentially the finding may underpin progress towards an earlier diagnosis of the onset of Alzheimer''s disease and an improved approach to developing intervention therapies. [Copyright &y& Elsevier]

Abstract: In a previous study, it was shown that an isolated, fully saturated tree-crown behaves like a wet bulb, allowing evaporation of intercepted rainfall to be estimated by a simple diffusion equation for water vapour. This observation was taken as the basis for a new approach in modelling interception loss from savanna-type woodland, whereby the ecosystem evaporation is derived by scaling up the evaporation from individual trees, rather than by considering a homogeneous forest cover. Interception loss from isolated trees was estimated by combining the aforementioned equation for water vapour flux with Gash''s analytical model. A new methodology, which avoids the subjectivity inherent in the Leyton method, was used for estimating the crown storage capacity. Modelling performance was evaluated against data from two Mediterranean savanna-type oak woodlands (montados) in southern Portugal. Interception loss estimates were in good agreement with observations in both sites. The proposed modelling approach is physically based, requires only a limited amount of data and should be suitable for the modelling of interception loss in isolated trees and savanna-type ecosystems. [Copyright &y& Elsevier]

Abstract: A new approach is suggested for estimating evaporation of intercepted rainfall from single trees in sparse forests. It is shown that, theoretically, the surface temperature of a wet tree crown will depend on the available energy and windspeed. But for a fully saturated canopy under rainy conditions, surface temperature will approach the wet bulb temperature when available energy tends to zero. This was confirmed experimentally from measurements of the radiation balance, aerodynamic conductance for water vapour and surface temperature on an isolated tree crown. Net radiation over a virtual cylindrical surface, enclosing the tree crown, was monitored by a set of radiometers positioned around that surface. Aerodynamic conductance for the tree crown was derived by scaling up measurements of leaf boundary layer conductance using the heated leaf replica method. Thermocouples were used to measure the average leaf surface temperature. Results showed that a fully wet single tree crown behaves like a wet bulb, allowing evaporation of intercepted rainfall to be estimated by a simple diffusion equation for water vapour, which is not restricted by the assumptions of one-dimensional transfer models usually used at the stand scale. Using this approach, mean evaporation rate from wet, saturated tree crowns was 0.27 or 0.30mmh−1, when surface temperature was taken equal to the air wet bulb temperature or estimated accounting for the available energy, respectively. [Copyright &y& Elsevier]

Abstract: Brachionus plicatilis is used in aquaculture to feed larval fish and crustaceans. It is well established that different prey species alter rotifer productivity. Isochrysis galbana is one microalgal prey that is commonly fed to rotifers, and there are several strains of this flagellate available to aquaculturists. As microalgae strains may differ in their composition and growth attributes, we rigorously examined if growth and biochemical differences in I. galbana strains elicit differences in the growth and biochemical attributes of B. plicatilis. Four I. galbana strains and one strain of the flagellate Nanochloropsis were grown under standard conditions. Growth rate, cell volume, production, and composition (dry weight, carbohydrate, protein, lipid) were measured. Significant differences occurred between strains in all of these attributes (at times 2 to 3 fold), but no clear pattern emerged that one strain was superior. Of note was that for some measurements, strain differences were significantly greater than differences between species. The strains were then fed to rotifers, and a number of parameters were measured: growth rate, reproductive rate, fecundity attributes, a number of developmental rates, and composition (dry weight, carbohydrate, protein, lipid). There were significant effects of prey strain on some of these attributes, but none was dramatic (rarely more than 10% and occasionally up to 30%), suggesting that aquaculturists need not be too concerned regarding which I. galbana strain they use. However, we do indicate subtle differences, induced by different prey strains and suggest that for maximum productivity these differences should be considered. [Copyright &y& Elsevier]

We monitor early stages of beta-amyloid (Aβ 1–40) aggregation, one of the key processes leading to Alzheimer's disease (AD), in the presence of high glucose concentrations by measuring Aβ 1– 40 intrinsic fluorescence. The multiple peaks and their shifts observed in the time-resolved emission spectra (TRES) reveal the impact of glycation on Aβ 1– 40 oligomerisation. The results show that formation of the advanced glycation end products (AGEs) alters the aggregation pathway. These changes are highly relevant to our understanding of the pathophysiology of AD and the implication of AGE and diabetes in these pathways. [Display omitted] [ABSTRACT FROM AUTHOR]

Protein aggregation can lead to several incurable amyloidosis diseases. The full aggregation pathway is not fully understood, creating the need for new methods of studying this important biological phenomenon. Lysozyme is an amyloidogenic protein which is often used as a model protein for studying amyloidosis. This work explores the potential of employing Lysozyme encapsulated gold nanoclusters (Ly-AuNCs) to study the protein's aggregation. The fluorescence emission properties of Ly-AuNCs were studied in the presence of increasing concentrations of native lysozyme and as a function of pH, of relevance in macromolecular crowding and inflammation-triggered aggregation. AuNC fluorescence was observed to both redshift and increase in intensity as pH is increased or when native lysozyme is added to a solution of Ly-AuNCs at pH 3. The long (μs) fluorescence lifetime component of AuNC emission was observed to decrease under both conditions. Interestingly it was found via Time-Resolved Emission Spectra (TRES) that both AuNC fluorescence components increase in intensity and redshift with increasing pH while only the long lifetime component of AuNC was observed to change when adding native lysozyme to solution; indicating that the underlying mechanisms for the changes observed are fundamentally different for each case. It is possible that the sensitivity of Ly-AuNCs to native lysozyme concentration could be utilized to study early-stage aggregation. • Lysozyme encapsulated gold nanoclusters are a promising new type of fluorescent nanoprobes. • Lysozyme encapsulated gold nanoclusters has a potential to be used to probe early stage aggregation of lysozyme. • Fluorescence lifetime of gold nanoclusters is sensitive to local environment and early stage lysozyme aggregation. • Time resolved emission spectra disclosed mechanism involved in fluorescence lifetime changes. [ABSTRACT FROM AUTHOR]

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. • C-propeptide mutation phenotype severity is modeled on CPI crystal structure. • Procollagen with C-propeptide mutations localizes to ER lumen, instead of ER membrane. • Mutant C-propeptide pericellular processing is defective, with increased pC-collagen and reduced mature collagen. • BMP-1 processing of mutant C-propeptide in vitro is impaired both with and without enhancer. • Incorporation of pC-collagen disorganizes extracellular matrix. [ABSTRACT FROM AUTHOR]

Competitive binding assays based on the lectin Concanavalin A (ConA) have displayed significant potential to serve in continuous glucose monitoring applications. However, to date, this type of fluorescent, affinity-based assay has yet to show the stable, glucose predictive capabilities that are required for such an application. This instability has been associated with the extensive crosslinking between traditionally-used fluorescent ligands (presenting multiple low-affinity moieties) and ConA (presenting multiple binding sites) in free solution. The work herein introduces the design and synthesis of a new type of fluorescent ligand that can avoid this aggregation and allow the assay to be sensitive across the physiologically relevant glucose concentration range. This fluorescent ligand (APTS–MT) presents a single high-affinity trimannose moiety that is recognized by ConA's full binding site and a fluorophore that can effectively track the ligand's equilibrium binding via fluorescent anisotropy. This is confirmed by comparing its measured fluorescent lifetime to experimentally-determined rotational correlation lifetimes of the free and bound populations. Using an assay comprised of 200 nM APTS–MT and 1 µM ConA, the fluorescence anisotropy capably tracks the concentration of monosaccharides that are known to bind to ConA's primary binding site, and the assay displays a MARD of 6.5% across physiologically relevant glucose concentrations. Ultimately, this rationally-designed fluorescent ligand can facilitate the realization of the full potential of ConA-based glucose sensing assays and provide the basis for a new set of competing ligands to be paired with ConA. [ABSTRACT FROM AUTHOR]

Questions How do semi-deciduous and evergreen shrubs exploit environmental resources during summer drought? What is the contribution of the understorey shrubby layer to ecosystem carbon assimilation? To what extent are carbon balance and transpiration impacted by a rain pulse? Location Cork oak open woodland in the Mediterranean region. Methods We used closed dynamic light and dark chambers to measure gas exchange ( CO2 and H2 O) in the dominant shrub understorey species Cistus salviifolius, Cistus crispus (semi-deciduous) and Ulex airensis (evergreen), together with plant physiological and morphological measurements during summer drought and autumn recovery. A hyperbolic light response model constrained by vapour pressure deficits was fitted for up-scaling shrub photosynthesis to the ecosystem level. The data were compared, on a daily and daytime basis, with gross primary productivity estimates from ecosystem eddy-covariance flux measurements. Results The onset of summer drought led to a significant leaf area reduction in semi-deciduous species. A general decrease in photosynthesis in all species was observed, while evapotranspiration and above-ground respiration fluxes contrasted among species during summer progression and autumn recovery. The shallow-rooted C. salviifolius was able to use light more efficiently than the other two species, although with poor stomatal control over water loss and consistently higher above-ground respiration rates, leading to lower water and carbon use efficiencies when compared with C. crispus. The deep-rooted shrub U. airensis maintained higher leaf water potentials and very low photosynthetic rates while decreasing transpiration rates throughout the summer drought. A summer rain pulse showed that shallow-rooted shrubs use water in an opportunistic way, with immediate leaf rehydration and concomitant photosynthesis increments. Conversely, deep-rooted shrubs ( U. airensis) were unresponsive, only recovering photosynthesis with high soil water content. An opportunistic growth response may be disadvantageous to shallow-rooted shrubs in a future climate with extended dry summers and higher probability of rain pulse events. The prominent increase in transpiration rates and plant respiration costs observed during the dry conditions that followed the rain pulse, led to a reduced plant ability to recover after autumn rains. Conclusions The shrubs that naturaly colonized this montado understorey showed contrasting strategies to overcome summer drought, suggesting an efficient mosaic exploitation of seasonal environmental resources. The contribution of these shrubs to total ecosystem CO2 uptake during summer and autumn recovery was 17%. This high contribution implies that shrub density management decisions should consider a carbon balance perspective. [ABSTRACT FROM AUTHOR]

Abstract: Hazardous materials, such as ammonia and microcystin, are released into lakes during cyanobacterial bloom degradation and may severely impact aquatic organisms. To assess the combined effects of ammonia and microcystin on survival, growth, and oxidative stress of larval fish, 14-day-old larvae of bighead carp Hypophthalmythys nobilis were exposed to solutions with different combined concentrations of ammonia (0, 0.06, 0.264mgL−1) and microcystin (0, 2, 10, 30μgL−1) for 10 days. Microcystin significantly decreased body length, while ammonia significantly increased body weight, specific growth rate, and condition factor, but there was no significant interaction between ammonia and microcystin on them. Superoxide dismutase, catalase, and malondialdehyde significantly changed with microcystin concentration, whereas glutathione was not affected by microcystin. Ammonia significantly affected the antioxidant system. There were significant interactions between ammonia and microcystin on superoxide dismutase and malondialdehyde. Our data clearly demonstrate that ammonia and microcystin adversely affect bighead carp larvae. [Copyright &y& Elsevier]

Abstract: Elevated nitrite and microcystin concentrations co-occur during degradation of Microcystis blooms and are toxic to aquatic organisms. We studied the relative and combined effects of these on Daphnia obtusa life-history. Nitrite and microcystin-LR treatments were: 0, 1, 3mgL−1 and 0, 10, 100, 300μgL−1, respectively. Experiments were factorial with 12 treatment combinations. Incubations were 15 d and recorded: moult number; time to first batch of eggs; time to first clutch; size at first batch of eggs; size at first clutch; number of clutches per female; number of offspring per clutch; total offspring per female. Interactive effects of the toxins occurred for time to first batch of eggs and time to first clutch. The remaining traits were negatively affected by nitrite: a significant decrease occurred in number of offspring per clutch and total number of offspring per mother (both decreased by ∼50%); total clutches per mother; number of moults; mother size at first clutch; and first appearance of eggs (primarily at the highest nitrite concentration). We support the literature, recognising nitrite is toxic, and although Microcystis is toxic to zooplankton, the main threat is not from dissolved microcystin but from degradative products such as nitrite. [Copyright &y& Elsevier]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: Dialysis related amyloidosis (DRA) is a progressive and serious complication in patients under long-term hemodialysis and mainly leads to osteo-articular diseases. Although β2-microglobulin (β2-m) is the major structural component of β2-m amyloid fibrils, the initiation of amyloid formation is not clearly understood. Here, we have identified procollagen C-proteinase enhancer-1 (PCPE-1) as a new interacting protein with β2-m by screening a human synovium cDNA library. The interaction of β2-m with full-length PCPE-1 was confirmed by immunoprecipitation, solid-phase binding and pull-down assays. By yeast two-hybrid analysis and pull-down assay, β2-m appeared to interact with PCPE-1 via the NTR (netrin-like) domain and not via the CUB (C1r/C1s, Uegf and BMP-1) domain region. In synovial tissues derived from hemodialysis patients with DRA, β2-m co-localized and formed a complex with PCPE-1. β2-m did not alter the basal activity of bone morphogenetic protein-1/procollagen C-proteinase (BMP-1/PCP) nor BMP-1/PCP activity enhanced by PCPE-1. PCPE-1 did not stimulate β2-m amyloid fibril formation from monomeric β2-m in vitro under acidic and neutral conditions as revealed by thioflavin T fluorescence spectroscopy and electron microscopy. Since PCPE-1 is abundantly expressed in connective tissues rich in type I collagen, it may be involved in the initial accumulation of β2-m in selected tissues such as tendon, synovium and bone. Furthermore, since such preferential deposition of β2-m may be linked to subsequent β2-m amyloid fibril formation, the disruption of the interaction between β2-m and PCPE-1 may prevent β2-m amyloid fibril formation and therefore PCPE-1 could be a new target for the treatment of DRA. [Copyright &y& Elsevier]

Abstract: In this review paper, state-of-the-art observational and numerical modeling methods for small scale turbulence and mixing with applications to coastal oceans are presented in one context. Unresolved dynamics and remaining problems of field observations and numerical simulations are reviewed on the basis of the approach that modern process-oriented studies should be based on both observations and models. First of all, the basic dynamics of surface and bottom boundary layers as well as intermediate stratified regimes including the interaction of turbulence and internal waves are briefly discussed. Then, an overview is given on just established or recently emerging mechanical, acoustic and optical observational techniques. Microstructure shear probes although developed already in the 1970s have only recently become reliable commercial products. Specifically under surface waves turbulence measurements are difficult due to the necessary decomposition of waves and turbulence. The methods to apply Acoustic Doppler Current Profilers (ADCPs) for estimations of Reynolds stresses, turbulence kinetic energy and dissipation rates are under further development. Finally, applications of well-established turbulence resolving particle image velocimetry (PIV) to the dynamics of the bottom boundary layer are presented. As counterpart to the field methods the state-of-the-art in numerical modeling in coastal seas is presented. This includes the application of the Large Eddy Simulation (LES) method to shallow water Langmuir Circulation (LC) and to stratified flow over a topographic obstacle. Furthermore, statistical turbulence closure methods as well as empirical turbulence parameterizations and their applicability to coastal ocean turbulence and mixing are discussed. Specific problems related to the combined wave–current bottom boundary layer are discussed. Finally, two coastal modeling sensitivity studies are presented as applications, a two-dimensional study of upwelling and downwelling and a three-dimensional study for a marginal sea scenario (Baltic Sea). It is concluded that the discussed methods need further refinements specifically to account for the complex dynamics associated with the presence of surface and internal waves. [Copyright &y& Elsevier]

Abstract: 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 (7T) and by combining a series of gelation–rotation–gelation cycles, a scaffold of orthogonal lamellae 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. [Copyright &y& Elsevier]

Abstract: There is an urgent need to develop technology for continuous in vivo glucose monitoring in subjects with diabetes mellitus. Problems with existing devices based on electrochemistry have encouraged alternative approaches to glucose sensing in recent years, and those based on fluorescence intensity and lifetime have special advantages, including sensitivity and the potential for non-invasive measurement when near-infrared light is used. Several receptors have been employed to detect glucose in fluorescence sensors, and these include the lectin concanavalin A (Con A), enzymes such as glucose oxidase, glucose dehydrogenase and hexokinase/glucokinase, bacterial glucose-binding protein, and boronic acid derivatives (which bind the diols of sugars). Techniques include measuring changes in fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor either within a protein which undergoes glucose-induced changes in conformation or because of competitive displacement; measurement of glucose-induced changes in intrinsic fluorescence of enzymes (e.g. due to tryptophan residues in hexokinase) or extrinsic fluorophores (e.g. using environmentally sensitive fluorophores to signal protein conformation). Non-invasive glucose monitoring can be accomplished by measurement of cell autofluorescence due to NAD(P)H, and fluorescent markers of mitochondrial metabolism can signal changes in extracellular glucose concentration. Here we review the principles of operation, context and current status of the various approaches to fluorescence-based glucose sensing. [Copyright &y& Elsevier]

Our newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence nanotomography (FN), is used to determine the morphology and dynamics of some soft materials and bio-molecules by attaching donor (D) fluorophores and acceptors (A) to the investigated structure and using fluorescence lifetime measurements to reveal the D–A distance distribution function ρDA(r). We report the effect of the limited sizes of the donor and acceptor, effect of porous polymer, and molecular structure and phase transition in phospholipid bilayers. [Copyright &y& Elsevier]

The experiment took place at a sparse evergreen oak woodland in southern Portugal. Seasonal courses of sap flow, measured in eight points of the stem of a Quercus rotundifolia tree, were monitored during a 2-year period. Plant water relations (predawn and midday leaf water potential, canopy conductance and whole-plant hydraulic conductance) as well as meteorological variables were also measured during the experimental period (May 1996–August 1998). All evidence showed that the plants remained well watered throughout the observation period. The highest transpiration rates occurred during the summer, when only vestigial amounts of rain fell on the shallow soil with a low water storage capacity. This could only be explained by the direct access of the root system to a 13 m deep water table. Although there was no increase in water stress during the summer drought, the transpiration rates showed an upper limit well below the atmospheric evaporative demand. This was consistent with the occurrence of a maximum limit for the root water uptake capacity determined by the summer value of whole-plant hydraulic conductance and by stomatal control, which prevented leaf water potential from falling below a cavitation threshold. [Copyright &y& Elsevier]

Studies procollagen C-proteinase enhancer (PCPE), an extracellular matrix glycoprotein. Low-resolution structure determination showing that PCPE is an elongated multidomain glycoprotein; Ability to stimulate the action of tolloid metalloproteinasse; Function of PCPE.

• Polymeric ionic liquid (PIL) as separation matrix for M13 phage was studied. • PIL cation and anion, the crosslinker nature and its concentration were evaluated. • M13 phage was successfully separated directly from filtered E. coli. • Batch adsorption/elution and chromatographic operation mode were compared. • The PIL-based chromatography reached over 70% M13 recovery in a single step. 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. [ABSTRACT FROM AUTHOR]

Summary Procollagen C-proteinase enhancer-1 (PCPE-1) is a secreted protein that specifically accelerates proteolytic release of the C-propeptides from fibrillar procollagens, a crucial step in fibril assembly. As such, it is a potential therapeutic target to improve tissue repair and prevent fibrosis, a major cause of mortality worldwide. Here we present the crystal structure of the active CUB1CUB2 fragment of PCPE-1 bound to the C-propeptide trimer of procollagen III (CPIII). This shows that the two CUB domains bind to two different chains of CPIII and that the N-terminal region of one CPIII chain, close to the proteolytic cleavage site, lies in the cleft between CUB1 and CUB2. This suggests that enhancing activity involves unraveling of this chain from the rest of the trimer, thus facilitating the action of the proteinase involved. Support for this hypothesis comes from site-directed mutagenesis, enzyme assays, binding studies, and molecular modeling. Graphical Abstract Highlights • The crystal structure of PCPE-1 bound to the C-propeptides has been determined • The N terminus of one propeptide chain binds to the CUB1CUB2 fragment of PCPE-1 • PCPE-1 seems to unravel the propeptide trimer to enable proteolytic release • Molecular modeling with the proteinase and its substrate supports this hypothesis Collagens are the most abundant proteins in mammals. Fiber-forming collagens are synthesized as procollagens, where the C-propeptides are released by specific proteinases (BMP-1). C-propeptide release is accelerated by another extracellular matrix protein called PCPE-1. Here, based on structural data, the authors present a mechanism for the acceleration of C-propeptide release. [ABSTRACT FROM AUTHOR]