Nutrients and herbivores impact grassland stability across spatial scales through different pathways.

Nutrients and herbivores are well-known drivers of grassland diversity and stability in local communities. However, whether they interact to impact the stability of aboveground biomass and whether these effects depend on spatial scales remain unknown. It is also unclear whether nutrients and herbivores impact stability via different facets of plant diversity including species richness, evenness, and changes in community composition through time and space. We used a replicated experiment adding nutrients and excluding herbivores for 5 years in 34 global grasslands to explore these questions. We found that both nutrient addition and herbivore exclusion alone reduced stability at the larger spatial scale (aggregated local communities; gamma stability), but through different pathways. Nutrient addition reduced gamma stability primarily by increasing changes in local community composition over time, which was mainly driven by species replacement. Herbivore exclusion reduced gamma stability primarily by decreasing asynchronous dynamics among local communities (spatial asynchrony). Their interaction weakly increased gamma stability by increasing spatial asynchrony. Our findings indicate that disentangling the processes operating at different spatial scales may improve conservation and management aiming at maintaining the ability of ecosystems to reliably provide functions and services for humanity.

Nutrient identity modifies the destabilising effects of eutrophication in grasslands.

Nutrient enrichment can simultaneously increase and destabilise plant biomass production, with co-limitation by multiple nutrients potentially intensifying these effects. Here, we test how factorial additions of nitrogen (N), phosphorus (P) and potassium with essential nutrients (K+) affect the stability (mean/standard deviation) of aboveground biomass in 34 grasslands over 7 years. Destabilisation with fertilisation was prevalent but was driven by single nutrients, not synergistic nutrient interactions. On average, N-based treatments increased mean biomass production by 21-51% but increased its standard deviation by 40-68% and so consistently reduced stability. Adding P increased interannual variability and reduced stability without altering mean biomass, while K+ had no general effects. Declines in stability were largest in the most nutrient-limited grasslands, or where nutrients reduced species richness or intensified species synchrony. We show that nutrients can differentially impact the stability of biomass production, with N and P in particular disproportionately increasing its interannual variability.

An experimental investigation into the physical, thermal and mechanical degradation of a polymeric bioresorbable scaffold.

This study presents a comprehensive evaluation of the mechanical, micro-mechanical and physical properties of Reva Medical Fantom Encore Bioresorbable Scaffolds (BRS) subjected to a thermally-accelerated degradation protocol. The Fantom Encore BRS were immersed in phosphate buffered saline solution at 50 °C for 112 days with radial compression testing, nanoindentation, differential scanning calorimetry, gel permeation chromatography and mass loss characterisation performed at consecutive time points. In the initial stages of degradation (Days 0-21), the Fantom Encore BRS showed increases in radial strength and stiffness, despite a substantial reduction in in molecular weight, with a slight increase in the melt temperature also observed. In the second phase (Days 35-54), the radial strength of the BRS samples were maintained despite a continued loss in molecular weight. However, during this phase, the ductility of the stent showed a reduction, with stent fracture occurring earlier in the crimp process and with lower amounts of plastic deformation evident under visual examination post-fracture. In the final phase (Days 63-112), the load-bearing capacity of the Fantom Encore BRS showed continued reduction, with decreases in radial stiffness and strength, and drastic reduction in the work-to-fracture of the devices. Throughout each phase, there was a steady increase in the relative crystallinity, with limited mass loss until day 112 and only minor changes in glass transition and melt temperatures. Limited changes were observed in nano-mechanical properties, with measured local elastic moduli and hardness values remaining largely similar throughout degradation. Given that the thermally-accelerated in vitro conditions represented a four-fold acceleration of physiological conditions, these results suggest that the BRS scaffolds could exhibit substantially brittle behaviour after âˆ¼ one year of implantation.

Physical and mechanical degradation behaviour of semi-crystalline PLLA for bioresorbable stent applications.

This study presents a systematic evaluation of the physical, thermal and mechanical performance of medical-grade semi-crystalline PLLA undergoing thermally-accelerated degradation. Samples were immersed in phosphate-buffered saline solution at 50 °C for 112 days and mass loss, molecular weight, thermal properties, degree of crystallinity, FTIR and Raman spectra, tensile elastic modulus, yield stress and failure stress/strain were evaluated at consecutive time points. Samples showed a consistent reduction in molecular weight and melting temperature, a consistent increase in percent crystallinity and limited changes in glass transition temperature and mass loss. At day 49, a drastic reduction in tensile failure strain was observed, despite the fact that elastic modulus, yield and tensile strength of samples were maintained. Brittleness increase was followed by rapid increase in degradation rate. Beyond day 70, samples became too brittle to test indicating substantial deterioration of their load-bearing capacity. This study also presents a computational micromechanics framework that demonstrates that the elastic modulus of a semi-crystalline polymer undergoing degradation can be maintained, despite a reducing molecular weight through compensatory increases in percent crystallinity. This study presents novel insight into the relationship between physical properties and mechanical performance of medical-grade PLLA during degradation and could have important implications for design and development of bioresorbable stents for vascular applications.

General destabilizing effects of eutrophication on grassland productivity at multiple spatial scales.

Eutrophication is a widespread environmental change that usually reduces the stabilizing effect of plant diversity on productivity in local communities. Whether this effect is scale dependent remains to be elucidated. Here, we determine the relationship between plant diversity and temporal stability of productivity for 243 plant communities from 42 grasslands across the globe and quantify the effect of chronic fertilization on these relationships. Unfertilized local communities with more plant species exhibit greater asynchronous dynamics among species in response to natural environmental fluctuations, resulting in greater local stability (alpha stability). Moreover, neighborhood communities that have greater spatial variation in plant species composition within sites (higher beta diversity) have greater spatial asynchrony of productivity among communities, resulting in greater stability at the larger scale (gamma stability). Importantly, fertilization consistently weakens the contribution of plant diversity to both of these stabilizing mechanisms, thus diminishing the positive effect of biodiversity on stability at differing spatial scales. Our findings suggest that preserving grassland functional stability requires conservation of plant diversity within and among ecological communities.

Ageing affects chondroitin sulfates and their synthetic enzymes in the intervertebral disc.

The depletion of chondroitin sulfates (CSs) within the intervertebral disc (IVD) during degenerative disc disease (DDD) results in a decrease in tissue hydration, a loss of fluid movement, cell apoptosis, a loss of nerve growth inhibition and ultimately, the loss of disc function. To date, little is known with regards to the structure and content of chondroitin sulfates (CSs) during IVD ageing. The behavior of glycosaminoglycans (GAGs), specifically CSs, as well as xylosyltransferase I (XT-I) and glucuronyltransferase I (GT-I), two key enzymes involved in CS synthesis as a primer of glycosaminoglycan (GAG) chain elongation and GAG synthesis in the nucleus pulposus (NP), respectively, were evaluated in a bovine ageing IVD model. Here, we showed significant changes in the composition of GAGs during the disc ageing process (6-month-old, 2-year-old and 8-year-old IVDs representing the immature to mature skeleton). The CS quantity and composition of annulus fibrosus (AF) and NP were determined. The expression of both XT-I and GT-I was detected using immunohistochemistry. A significant decrease in GAGs was observed during the ageing process. CSs are affected at both the structural and quantitative levels with important changes in sulfation observed upon maturity, which correlated with a decrease in the expression of both XT-I and GT-I. A progressive switch of the sulfation profile was noted in both NP and AF tissues from 6 months to 8 years. These changes give an appreciation of the potential impact of CSs on the disc biology and the development of therapeutic approaches for disc regeneration and repair.

A sustained release formulation of novel quininib-hyaluronan microneedles inhibits angiogenesis and retinal vascular permeability in vivo.

Pathologic neovascularisation and ocular permeability are hallmarks of proliferative diabetic retinopathy and age-related macular degeneration. Current pharmacologic interventions targeting VEGF are effective in only 30-60% of patients and require multiple intraocular injections associated with iatrogenic infection. Thus, our goal is to develop novel small molecule drugs that are VEGF-independent are amenable to sustained ocular-release, and which reduce retinal angiogenesis and retinal vascular permeability. Here, the anti-angiogenic drug quininib was formulated into hyaluronan (HA) microneedles whose safety and efficacy was evaluated in vivo. Quininib-HA microneedles were formulated via desolvation from quininib-HA solution and subsequent cross-linking with 4-arm-PEG-amine prior to freeze-drying. Scanning electron microscopy revealed hollow needle-shaped particle ultrastructure, with a zeta potential of -35.5mV determined by electrophoretic light scattering. The incorporation efficiency and pharmacokinetic profile of quininib released in vitro from the microneedles was quantified by HPLC. Quininib incorporation into these microneedles was 90%. In vitro, 20% quininib was released over 4months; or in the presence of increasing concentrations of hyaluronidase, 60% incorporated quininib was released over 4months. Zebrafish hyaloid vasculature assays demonstrated quininib released from these microneedles significantly (p<0.0001) inhibited ocular developmental angiogenesis compared to control. Sustained amelioration of retinal vascular permeability (RVP) was demonstrated using a bespoke cysteinyl leukotriene induced rodent model. Quininib-HA microparticles significantly inhibited RVP in Brown Norway rats one month after administration compared to neat quininib control (p=0.0071). In summary, quininib-HA microneedles allow for sustained release of quininib; are safe in vivo and quininib released from these microneedles effectively inhibits angiogenesis and RVP in vivo.

An injectable elastin-based gene delivery platform for dose-dependent modulation of angiogenesis and inflammation for critical limb ischemia.

Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ-forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 µg) and IL-10 (10 µg)/eNOS (20 µg) and reduced inflammation with IL-10 (10 µg) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELP injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia.

Tunable elastin-like polypeptide hollow sphere as a high payload and controlled delivery gene depot.

Self-assembly driven processes can be utilized to produce a variety of nanostructures useful for various in vitro and in vivo applications. Characteristics such as size, stability, biocompatibility, high therapeutic loading and controlled delivery of these nanostructures are particularly crucial in relation to in vivo applications. In this study, we report the fabrication of tunable monodispersed elastin-like polypeptide (ELP) hollow spheres of 100, 300, 500 and 1000 nm by exploiting the self-assembly property and net positive charge of ELP. The microbial transglutaminase (mTGase) cross-linking provided robustness and stability to the hollow spheres while maintaining surface functional groups for further modifications. The resulting hollow spheres showed a higher loading efficiency of plasmid DNA (pDNA) by using polyplex (~70 µg pDNA/mg of hollow sphere) than that of self-assembled ELP particles and demonstrated controlled release triggered by protease and elastase. Moreover, polyplex-loaded hollow spheres showed better cell viability than polyplex alone and yielded higher luciferase expression by providing protection against endosomal degradation. Overall, the monodispersed, tunable hollow spheres with a capability of post-functionalization can provide an exciting new opportunity for use in a range of therapeutic and diagnostic applications.

IRAP identifies an endosomal compartment required for MHC class I cross-presentation.

Major histocompatibility complex (MHC) class I molecules present peptides, produced through cytosolic proteasomal degradation of cellular proteins, to cytotoxic T lymphocytes. In dendritic cells, the peptides can also be derived from internalized antigens through a process known as cross-presentation. The cellular compartments involved in cross-presentation remain poorly defined. We found a role for peptide trimming by insulin-regulated aminopeptidase (IRAP) in cross-presentation. In human dendritic cells, IRAP was localized to a Rab14+ endosomal storage compartment in which it interacted with MHC class I molecules. IRAP deficiency compromised cross-presentation in vitro and in vivo but did not affect endogenous presentation. We propose the existence of two pathways for proteasome-dependent cross-presentation in which final peptide trimming involves IRAP in endosomes and involves the related aminopeptidases in the endoplasmic reticulum.

Characterizing the N-terminal processing motif of MHC class I ligands.

Most peptide ligands presented by MHC class I molecules are the product of an intracellular pathway comprising protein breakdown in the cytosol, transport into the endoplasmic reticulum, and successive N-terminal trimming events. The efficiency of each of these processes depends on the amino acid sequence of the presented ligand and its precursors. Thus, relating the amino acid composition N-terminal of presented ligands to the sequence specificity of processes in the pathway gives insight into the usage of ligand precursors in vivo. Examining the amino acid composition upstream the true N terminus of MHC class I ligands, we demonstrate the existence of a distinct N-terminal processing motif comprising approximately seven residues and matching the known preferences of proteasome and TAP, two key players in ligand processing. Furthermore, we find that some residues, which are preferred by both TAP and the proteasome, are underrepresented at positions immediately preceding the N terminus of MHC class I ligands. Based on experimentally determined aminopeptidase activities, this pattern suggests trimming next to the final N terminus to take place predominantly in the endoplasmic reticulum.

Expression of endoplasmic reticulum aminopeptidases in EBV-B cell lines from healthy donors and in leukemia/lymphoma, carcinoma, and melanoma cell lines.

Peptide trimming in the endoplasmic reticulum (ER), the final step required for the generation of most HLA class I-binding peptides, implicates the concerted action of two aminopeptidases, ERAP1 and ERAP2. Because defects in the expression of these peptidases could lead to aberrant surface HLA class I expression in tumor cells, we quantitatively assayed 14 EBV-B cell lines and 35 human tumor cell lines of various lineages for: 1) expression and enzymatic activities of ERAP1 and ERAP2; 2) ER peptide-trimming activity in microsomes; 3) expression of HLA class I H chains and TAP1; and 4) surface HLA class I expression. ERAP1 and ERAP2 expression was detectable in all of the EBV-B and tumor cell lines, but in the latter it was extremely variable, sometimes barely detectable, and not coordinated. The expression of the two aminopeptidases corresponded well to the respective enzymatic activities in most cell lines. A peptide-trimming assay in microsomes revealed additional enzymatic activities, presumably contributed by other unidentified aminopeptidases sharing substrate specificity with ERAP2. Interestingly, surface HLA class I expression showed significant correlation with ERAP1 activity, but not with the activity of either ERAP2 or other unidentified aminopeptidases. Transfection with ERAP1 or ERAP2 of two tumor cell lines selected for simultaneous low expression of the two aminopeptidases resulted in the expected, moderate increases of class I surface expression. Thus, low and/or imbalanced expression of ERAP1 and probably ERAP2 may cause improper Ag processing and favor tumor escape from the immune surveillance.

Complexity, contradictions, and conundrums: studying post-proteasomal proteolysis in HLA class I antigen presentation.

The vast majority of the peptides produced during protein degradation by the cytosolic proteasome-ubiquitin system are consecutively hydrolyzed to single amino acids by multiple cytosolic peptidases preferring intermediate length or short substrates. The small fraction of peptides surviving the aggressive cytosolic environment can be recruited for presentation by major histocompatibility complex (MHC) class I molecules. However, such peptides may frequently have to be adapted to the strict MHC class I-binding requirements by one or several N-terminal-trimming steps. A recent model proposes that an initial step, in which peptides of 15 or more residues are shortened by cytosolic tripeptidylpeptidase II, is followed by additional trimming by cytosolic or endoplasmic reticulum (ER) aminopeptidases. In humans, at least two ER resident aminopeptidases, ERAP1 and ERAP2, contribute to trimming of human leukocyte antigen class I ligands. These interferon-gamma-regulated metallopeptidases show distinct substrate preferences and may have to act in a concerted fashion to remove some complex or longer N-terminal extensions and to trim the full spectrum of precursor peptides. This task is likely facilitated by the formation of presumably heterodimeric ERAP1-2 complexes. RNA interference experiments suggest that both enzymes are important for normal antigen presentation, but precise determination of the extent and the cellular context of their requirement will be left to future experimentation.

Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum.

The generation of many HLA class I peptides entails a final trimming step in the endoplasmic reticulum that, in humans, is accomplished by two 'candidate' aminopeptidases. We show here that one of these, ERAP1, was unable to remove several N-terminal amino acids that were trimmed efficiently by the second enzyme, ERAP2. This trimming of a longer peptide required the concerted action of both ERAP1 and ERAP2, both for in vitro digestion and in vivo for cellular antigen presentation. ERAP1 and ERAP2 localized together in vivo and associated physically in complexes that were most likely heterodimeric. Thus, the human endoplasmic reticulum is equipped with a pair of trimming aminopeptidases that have complementary functions in HLA class I peptide presentation.