The Mechano-Ubiquitinome of Articular Cartilage: Differential Ubiquitination and Activation of a Group of ER-Associated DUBs and ER Stress Regulators

Understanding how connective tissue cells respond to mechanical stimulation is important to human health and disease processes in musculoskeletal diseases. Injury to articular cartilage is a key risk factor in predisposition to tissue damage and degenerative osteoarthritis. Recently, we have discovered that mechanical injury to connective tissues including murine and porcine articular cartilage causes a significant increase in lysine-63 polyubiquitination. Here, we identified the ubiquitin signature that is unique to injured articular cartilage tissue upon mechanical injury (the “mechano-ubiquitinome”). A total of 463 ubiquitinated peptides were identified, with an enrichment of ubiquitinated peptides of proteins involved in protein processing in the endoplasmic reticulum (ER), also known as the ER-associated degradation response, including YOD1, BRCC3, ATXN3, and USP5 as well as the ER stress regulators, RAD23B, VCP/p97, and Ubiquilin 1. Enrichment of these proteins suggested an injury-induced ER stress response and, for instance, ER stress markers DDIT3/CHOP and BIP/GRP78 were upregulated following cartilage injury on the protein and gene expression levels. Similar ER stress induction was also observed in response to tail fin injury in zebrafish larvae, suggesting a generic response to tissue injury. Furthermore, a rapid increase in global DUB activity following injury and significant activity in human osteoarthritic cartilage was observed using DUB-specific activity probes. Combined, these results implicate the involvement of ubiquitination events and activation of a set of DUBs and ER stress regulators in cellular responses to cartilage tissue injury and in osteoarthritic cartilage tissues. This link through the ER-associated degradation pathway makes this protein set attractive for further investigation in in vivo models of tissue injury and for targeting in osteoarthritis and related musculoskeletal diseases.     

Interaction between heparan sulphate chains II. Structural characterization of iduronate- and glucuronate-containing sequences in aggregating chains

A heparan sulphate fraction (uronic acid composition: 20% sulphated iduronate, 15% iduronate and 65% glucuronate of total uronate) was separated into aggregating and non-aggregating chains by gel chromatography. ¹³C-NMR analyses revealed that non-aggregating chains had a higher degree of sulphation than did aggregating chains. In aggregating chains, there was more N-acetyl-glucosamine than N-sulphamidoglucosamine; the extent of C-6 sulphation of the latter moiety was low and most of the iduronate residues were non-sulphated. In non-aggregating chains, the N-acetyl-to-N-sulphate ratio was approx. 2 : 1, the N-sulphated glucosamines were also largely C-6 sulphated and the sulphated iduronates were concentrated to these species.Both preparations were subjected to deaminative cleavage which produces fragments like uronate-(N-acetylglucosamine-uronate)_n-anhydromannose. Tetrasaccharides (n = 1) were further fractionated into non-, mono-, di- and trisulphated species by ion-exchange chromatography. The tetrasaccharides have the general carbohydrate structure uronate-N-acetylglucosamine-glucuronate-anhydromannose. Non-reducing terminal glucuronate was removed by β-glucuronidase. The results showed that saccharides containing glucuronate in both positions were more prevalent in the products of aggregating chains. The β-glucuronidase-resistant saccharides (carrying either sulphated or non-sulphated iduronate in non-reducing terminal position) were oxidised with periodate under conditions where non-sulphated residues are degraded, whereas sulphated residues are resistant. Mono-sulphated and di-sulphated tetrasaccharides from aggregating chains were extensively degraded indicating that iduronate-N-acetylglucosamine-glucuronate-anhydromannose was the major sequence.In saccharides from non-aggregating chains iduronate was frequently sulphated. The results of this and previous investigations (Fransson, L.-Å., Nieduszynski, I.A. and Sheehan, J.K. (1980) Biochim. Biophys. Acta 630, 287–300) indicate that an alternating arrangement of iduronate and glucuronate in aggregating chains is present both in N-sulphated block regions and in regionsthat carry alternating N-acetyl- and N-sulphated glucosamine.     

Nitrogen availability and weed invasion in a remnant native woodland in urban Melbourne

Abstract The woodlands of Studley Park, and the greater Yarra Bend Park, are within 4 km of the centre of the city of Melbourne and total more than 200 ha. Weeds are major contributors to the loss of diversity of native species. We examined the hypothesis that an increase in the availability of nitrogen (N) may be responsible for the increase in weed abundance. In a small field trial, nitrogen availability was greater in areas invaded by weeds and weeds had greater nitrate reductase activity than native species. Both invaded and uninvaded areas were well‐supplied with weed seed, although the composition of the seed bank varied greatly between sites. Diaspores of the weed Romulea rosea were abundant in uninvaded sites while those of Ehrharta erecta and Ehrharta longiflora were abundant in invaded sites. The abundance of weed diaspores suggested that increased N availability may be a precondition for invasion. Formation of a dense litter layer by senescent weeds is likely to promote the capture and retention of N added from anthropogenic sources including air pollution and overland flow from nearby roads and drainage channels.     

Murray Catchment habitat restoration: Lessons from landscape‐level research and monitoring

Native vegetation restoration and conservation works have been ongoing for three decades in the South West Slopes Bioregion of NSW's Murray Catchment. Monitoring of extensive areas of protected remnants and revegetation undertaken through a major partnership between ANU researchers and the Murray CMA shows early benefits of restoration efforts to biodiversity and helps to refine future efforts.     

Australia’s Stock Route Network: 2. Representation of fertile landscapes

The Stock Route Network (SRN) of New South Wales (NSW) and Queensland is a large‐scale system of predominantly roadside remnant vegetation, which was established in the 1800s to allow livestock to be moved. Proposed changes to the management of the SRN could result in some portions of it being sold to private landholders, or subjected to long‐term set‐stocking. This may have potentially negative impacts on some of the values of the SRN. One key feature of the SRN is that it covers low‐lying parts of the landscape, which are poorly protected by national parks. To quantify this, we specifically analysed a 41 million hectare portion of the SRN which transects the NSW ‘wheat‐sheep belt’, characterising its representation of woody vegetation cover and topography, and contrasting this with the National Reserve System. Our analysis revealed that 55% of stock routes occur in low‐lying valley portions of the landscape, compared with only 6% of the National Reserve System. The SRN supports a wide range of vegetation types and, unlike the National Reserve System, is not biased towards heavily forested areas. White Box‐Yellow Box‐Blakeley’s Red Gum woodland, which is listed as critically endangered by the Australian Government, was recorded in 803 (or 17.5%) of the 4575 stock routes in our data set. In contrast, only 10 of the 335 reserves within our spatial study region are known to support small occurrences of this community. Our findings suggest that the protection of the SRN and National Reserve System together may fulfil the ‘representation’ goal of systematic conservation planning far better than the National Reserve System on its own. Future research should quantify which stock routes in particular should receive priority for protection.     

Reduction in lipoprotein-associated apoC-III levels following volanesorsen therapy: phase 2 randomized trial results

Elevated apoC-III levels predict increased cardiovascular risk when present on LDL and HDL particles. We developed novel high-throughput chemiluminescent ELISAs that capture apoB, lipoprotein (a) [Lp(a)], and apoA-I in plasma and then detect apoC-III on these individual lipoproteins as apoCIII-apoB, apoCIII-Lp(a), and apoCIII-apoAI complexes, respectively. We assessed the effects on these complexes of placebo or 100–300 mg volanesorsen, a generation 2.0+ antisense drug that targets apoC3 mRNA in patients with hypertriglyceridemia, including familial chylomicronemia syndrome (n = 3), volanesorsen monotherapy (n = 51), and as add-on to fibrate (n = 26), treated for 85 days and followed for 176 days. Compared with placebo, volanesorsen was associated with an 82.3 ± 11.7%, 81.3 ± 15.7%, and 80.8 ± 13.6% reduction in apoCIII-apoB, apoCIII-Lp(a), and apoCIII-apoA-I, respectively (300 mg dose; P < 0.001 for all), at day 92. Strong correlations in all assay measures were noted with total plasma apoC-III, chylomicron-apoC-III, and VLDL-apoC-III. In conclusion, novel high-throughput ELISAs were developed to detect lipoprotein-associated apoC-III, including for the first time on Lp(a). Volanesorsen uniformly lowers apoC-III on apoB-100, Lp(a), and apoA-I lipoproteins, and may be a potent agent to reduce triglycerides and cardiovascular risk mediated by apoC-III.     

A broader ecological context to habitat fragmentation: Why matrix habitat is more important than we thought

Abstract. Consequences of habitat fragmentation have garnered much attention over the past few decades. The resulting literature has been useful for understanding how land‐use changes influence population viability and community structure, but we are still hampered by a major aspect of the conceptual framework within which most fragmentation work arises. Specifically, habitat between fragments (‘matrix’) is usually treated as uniform and ecologically irrelevant. However, recent work on animals shows that matrix habitat can profoundly influence within‐fragment dynamics. We review related evidence for plants. Various matrix types (e.g. clear‐cutting, agriculture, or urbanization) can act in different ways to alter resource availability and movement of pollinators, seed dispersers, and herbivores. Inclusion of matrix qualities in fragmentation studies is further complicated since most matrices are not static; sites in which timber harvesting or agriculture occur develop through succession or change as crops are rotated, respectively, such that their influence on within‐fragment processes vary temporally. Also, many plants are not restricted to remnants of original habitat. Using studies of forest understory plants, we summarize work showing how diversity can change significantly through time in matrix. Understanding the persistence of a species across fragmented landscapes will require more attention to matrix habitat, and to the species utilizing the matrix.     

Riparian vegetation diversity along regulated rivers: contribution of novel and relict habitats

1. The creation and maintenance of spatial and temporal heterogeneity by rivers flowing through floodplain landscapes has been disrupted worldwide by dams and water diversions. Large reservoirs ( novel ecosystems ) now separate and isolate remnant floodplains ( relict ecosystems ). From above, these appear as a string of beads, with beads of different sizes and string connections of varying lengths.
2. Numerous studies have documented or forecast sharp declines in riparian biodiversity in relict ecosystems downstream from dams. Concurrently, novel ecosystems containing species and communities of the former predam ecosystems have arisen along all regulated rivers. These result from the creation of new environments caused by upper reservoir sedimentation, tributary sedimentation and the formation of reservoir shorelines.
3. The contribution of novel habitats to the overall biodiversity of regulated rivers has been poorly studied. Novel ecosystems may become relatively more important in supporting riverine biodiversity if relict ecosystems are not restored to predam levels. The Missouri River of the north-central U.S.A. is used to illustrate existing conditions on a large, regulated river system with a mixture of relict and novel ecosystems.     

Redox index of Cys-thiol residues of serum apolipoprotein E and its diagnostic potential

Background: The redox modulation of Cys-thiol participates in various pathophysiological processes. We explored the proper index for estimating the redox status of Cys-thiol of serum apolipoprotein E (apoE), named “redox-IDX-apoE,” which is necessary to understand the redox biology of age-related diseases.Methods: The fractions of the reduced form (red-), reversible oxidized form (roxi-), and irreversibly oxidized form (oxi-) apoE in serum, obtained from the patients with no apparent disease (controls, n=192) and with atherosclerosis and type 2 diabetes (patients, n=16), were measured by a band-shift assay using a maleimide compound. Redox-IDX-apoE candidates were determined by calculating the values of these fractions and the total apoE concentration.Results: Cys number of apoE significantly increased for the ratio of roxi-apoE to total-apoE (roxi/total) (E2/E3>E3/E3>E3/E4) but decreased for the ratios of red-apoE to roxi-apoE (red/roxi) and [red-apoE + oxi-apoE] to roxi-apoE ([red + oxi]/roxi) (E2/E3<E3/E3<E3/E4). Considering the subjects with apoE3/E3, these ratios were independent of age and sex. Roxi/total showed negative correlations with serum triglyceride (TG) and HbA1c levels, while both red/roxi and [red + oxi]/roxi showed significant positive correlations with them. However, red/roxi and [red + oxi]/roxi in patients were significantly lower than those in controls, although serum TG and HbA1c levels in the patients were significantly higher than those in controls.Conclusion: The redox status of serum apoE-Cys-thiol is closely involved in the metabolism of TG-rich lipoproteins and glucose. The appropriate use of redox-IDX-apoE could be helpful in the diagnosis and prognosis of age-related diseases and in understanding the underlying mechanisms.     

Influence of woodland remnant edges on small skinks (Richmond,New South Wales)

Abstract Habitat fragmentation due to clearing often results in an increase in edge areas compared with overall remnant size, but there are limited data on the influence of increased edge areas on generalist species. Therefore, the abundance of small (<55 mm) generalist scinid lizards in small woodland remnants of the Cumberland Plain (western Sydney, Australia) was compared. Using pitfall trapping and points of transect survey techniques, three species were encountered: Lampropholis guichenoti, Lampropholis delicata and Cryptoblepharus virgatus. The abundance and diversity of these skinks differed significantly between edge and core areas; fewer individuals were encountered on the edge of the remnants than in core sites. Cryptoblepharus virgatus was only observed in edge sites where L. guichenoti made up the greatest skink abundance. In contrast, L. delicata abundance was greatest at core sites. There was also a significant difference between the edge and core with respect to the size structure of the L. guichenoti population. Larger numbers of subadults were observed in the edge sites, whereas there were significantly fewer adults and juveniles in these areas. Habitat characteristics did not account for the skink distribution observed.