Wading through the swamp: what does tropical peatland restoration mean to national‐level stakeholders in Indonesia?

Ecological restoration is considered to play an important role in mitigating climate change, protecting biodiversity, and preventing environmental degradation. Yet, there are often multiple perspectives on what outcomes restoration should be aiming to achieve, and how we should get to that point. In this study we interview a range of policymakers, academics, and non‐governmental organization (NGO) representatives to explore the range of perspectives on the restoration of Indonesia's tropical peatlands—key global ecosystems that have undergone large‐scale degradation. Thematic analysis suggests that participants agreed about the importance of restoration, but had differing opinions on how effective restoration activities to date have been and what a restored peatland landscape should look like. These results exemplify how ecological restoration can mean different things to different people, but also highlight important areas of consensus for moving forward with peatland restoration strategies.     

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.     

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9?? resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.     

Stop and Go – Waves of Tarsier Dispersal Mirror the Genesis of Sulawesi Island

The Indonesian island of Sulawesi harbors a highly endemic and diverse fauna sparking fascination since long before Wallace’s contemplation of biogeographical patterns in the region. Allopatric diversification driven by geological or climatic processes has been identified as the main mechanism shaping present faunal distribution on the island. There is both consensus and conflict among range patterns of terrestrial species pointing to the different effects of vicariant events on once co-distributed taxa. Tarsiers, small nocturnal primates with possible evidence of an Eocene fossil record on the Asian mainland, are at present exclusively found in insular Southeast Asia. Sulawesi is hotspot of tarsier diversity, whereby island colonization and subsequent radiation of this old endemic primate lineage remained largely enigmatic. To resolve the phylogeographic history of Sulawesi tarsiers we analyzed an island-wide sample for a set of five approved autosomal phylogenetic markers (ABCA1, ADORA3, AXIN1, RAG1, and TTR) and the paternally inherited SRY gene. We constructed ML and Bayesian phylogenetic trees and estimated divergence times between tarsier populations. We found that their arrival at the Proto-Sulawesi archipelago coincided with initial Miocene tectonic uplift and hypothesize that tarsiers dispersed over the region in distinct waves. Intra-island diversification was spurred by land emergence and a rapid succession of glacial cycles during the Plio-Pleistocene. Some tarsier range boundaries concur with spatial limits in other taxa backing the notion of centers of faunal endemism on Sulawesi. This congruence, however, has partially been superimposed by taxon-specific dispersal patterns.     

Correlation of C4ST-1 and ChGn-2 expression with chondroitin sulfate chain elongation in atherosclerosis

Subendothelial retention of lipoproteins by proteoglycans (PGs) is the initiating event in atherosclerosis. The elongation of chondroitin sulfate (CS) chains is associated with increased low-density lipoprotein (LDL) binding and progression of atherosclerosis. Recently, it has been shown that 2 Golgi enzymes, chondroitin 4-O-sulfotransferase-1 (C4ST-1) and chondroitin N-acetylgalactosaminyltransferase-2 (ChGn-2), play a critical role in CS chain elongation. However, the roles of C4ST-1 and ChGn-2 during the progression of atherosclerosis are not known. The aim of this study was to analyze the expression of C4ST-1 and ChGn-2 in atherosclerotic lesions in vivo and determine whether their expression correlated with CS chain elongation.Low-density lipoprotein receptor knockout (LDLr KO) mice were fed a western diet for 2, 4, and 8 weeks to stimulate development of atherosclerosis. The binding of LDL and CS PG in this mouse model was confirmed by chondroitinase ABC (ChABC) digestion and apolipoprotein B (apo B) staining. Gel filtration analysis revealed that the CS chains began to elongate as early as 2 weeks after beginning a western diet and continued as the atherosclerosis progressed. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) showed that the mRNA levels of C4ST-1 and ChGn-2 increased after 8 weeks of this diet. In contrast, the mRNA levels of their homologs, C4ST-2 and ChGn-1, were unchanged. In addition, immunohistochemical analysis demonstrated that the expression of C4ST-1 and ChGn-2 appeared to have similar site-specific patterns and coincided with biglycan expression at the aortic root.Our results suggested that C4ST-1 and ChGn-2 may be involved in the elongation of CS chains in the arterial wall during the progression of atherosclerosis. Therefore, modulating their expression and activity might be a novel therapeutic strategy for atherosclerosis.     

Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis)

Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level.     

Utilizing the Dog Genome in the Search for Novel Candidate Genes Involved in Glioma Development—Genome Wide Association Mapping followed by Targeted Massive Parallel Sequencing Identifies a Strongly Associated Locus

Gliomas are the most common form of malignant primary brain tumors in humans and second most common in dogs, occurring with similar frequencies in both species. Dogs are valuable spontaneous models of human complex diseases including cancers and may provide insight into disease susceptibility and oncogenesis. Several brachycephalic breeds such as Boxer, Bulldog and Boston Terrier have an elevated risk of developing glioma, but others, including Pug and Pekingese, are not at higher risk. To identify glioma-associated genetic susceptibility factors, an across-breed genome-wide association study (GWAS) was performed on 39 dog glioma cases and 141 controls from 25 dog breeds, identifying a genome-wide significant locus on canine chromosome (CFA) 26 (p = 2.8 x 10⁻⁸). Targeted re-sequencing of the 3.4 Mb candidate region was performed, followed by genotyping of the 56 SNVs that best fit the association pattern between the re-sequenced cases and controls. We identified three candidate genes that were highly associated with glioma susceptibility: CAMKK2, P2RX7 and DENR. CAMKK2 showed reduced expression in both canine and human brain tumors, and a non-synonymous variant in P2RX7, previously demonstrated to have a 50% decrease in receptor function, was also associated with disease. Thus, one or more of these genes appear to affect glioma susceptibility.     

The haemocytic origin of lymphoid organ spheroid cells in the penaeid prawn Penaeus monodon

Studies on lymphoid organ spheroid (LOS) cells of Penaeus monodon were undertaken. Phenoloxidase and peroxidase assays showed that LOS cells have characteristics similar to semi-granular and, in particular, large granular haemocytes. The mean percentage of LOS cells positive for phenoloxidase and peroxidase was 85 +/- 23 and 82 +/- 23%, respectively. There was no significant difference between the sites of phenoloxidase and peroxidase activity in LOS cells (t = 1.617, df = 29, p > 0.05). The relative sectional area occupied by LOS cells relative to that of the stromal matrix cells from both laboratory-held and farmed prawns was not correlated to increasing weight or total length of the prawns (p > 0.05). An apoptosis detection assay showed that LOS cells were often apoptotic whilst stromal matrix cells were not. There was a significant difference (t = -5.533, df = 58, p < 0.05) in the mean percentage of apoptotic spheroid cells between laboratory-held prawns (52 +/- 24%) and farmed prawns with midcrop mortality syndrome (MCMS) (80 +/- 12%). In conclusion, LOS cells have the characteristics of exocytosed, granular haemocytes that have phagocytosed foreign material, particularly viruses, and probably constitute a major mechanism for penaeid antiviral defense.