Subpopulations,locations and fragmentation: applying IUCN red list criteria to herbarium specimen data

Despite the ecological and economic importance of plants, the majority of plant species and their conservation status are still poorly known. Based on the limited knowledge we have of many plant species, especially those in the tropics, the use of GIS techniques can give us estimates of the degree of population subdivision to be used in conservation assessments of extinction risk. This paper evaluates how best to use the IUCN Red List Categories and Criteria to produce effective and consistent estimates of subpopulation structure based on specimen data available in the herbaria around the world. We assessed population structure through GIS-based analysis of the geographic distribution of collections, using herbarium specimen data for 11 species of Delonix sensu lato. We used four methods: grid adjacency, circular buffer, Rapoport’s mean propinquity and alpha hull, to quantify population structure according to the terms used in the IUCN Red List: numbers of subpopulations and locations, and degree of fragmentation. Based on our findings, we recommend using the circular buffer method, as it is not dependent on collection density and allows points to be added, subtracted and/or moved without altering the buffer placement. The ideal radius of the buffer is debatable; however when dispersal characteristics of the species are unknown then a sliding scale, such as the 1/10th maximum inter-point distance, is the preferred choice, as it is species-specific and not sensitive to collection density. Such quantitative measures of population structure provide a rigorous means of applying IUCN criteria to a wide range of plant species that hitherto were inaccessible to IUCN classification.     

Hypoxia-inducible factor and vascular endothelial growth factor in the neuroretina and retinal blood vessels after retinal ischemia

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1β and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1β immunoreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia.     

The Human Visceral Fat Depot Has a Unique Inflammatory Profile

Obesity can be considered as a low‐grade inflammatory condition, strongly linked to adverse metabolic outcomes. Obesity‐associated adipose tissue inflammation is characterized by infiltration of macrophages and increased cytokine and chemokine production. The distribution of adipose tissue impacts the outcomes of obesity, with the accumulation of fat in visceral adipose tissue (VAT) and deep subcutaneous adipose tissue (SAT), but not superficial SAT, being linked to insulin resistance. We hypothesized that the inflammatory gene expression in deep SAT and VAT is higher than in superficial SAT. A total of 17 apparently healthy women (BMI: 29.3±5.5 kg/m²) were included in the study. Body fat (dual‐energy X‐ray absorptiometry) and distribution (computed tomography) were measured, and insulin sensitivity, blood lipids, and blood pressure were determined. Inflammation‐related differences in gene expression (real‐time PCR) from VAT, superficial and deep SAT biopsies were analyzed using univariate and multivariate data analyses. Using multivariate discrimination analysis, VAT appeared as a distinct depot in adipose tissue inflammation, while the SAT depots had a similar pattern, with respect to gene expression. A significantly elevated (P < 0.01) expression of the CC chemokine receptor 2 (CCR2) and macrophage migration inhibitory factor (MIF) in VAT contributed strongly to the discrimination. In conclusion, the human adipose tissue depots have unique inflammatory patterns, with CCR2 and MIF distinguishing between VAT and the SAT depots.     

One-step fractionation of complex proteomes enables detection of low abundant analytes using antibody-based microarrays

Antibody-based microarray is a novel technology with great promise within high-throughput proteomics. The tremendous complexity of all proteomes will, however, pose major technological challenges, especially when targeting low-abundant analytes that remains to be resolved. In this paper, we have shown that antibody microarrays readily could be used for screening of low-abundant low molecular weight analytes in complex proteomes by optimizing the sample format. Focused antibody microarrays, based on human recombinant single-chain Fv anti-cytokine antibodies on Ni2+-NTA functionalized glass slides or black polymer Maxisorp substrates, and crude cell supernatants from activated dendritic cells, containing low levels of secreted cytokines, was used for evaluation. The proteome was pre-fractionated based on size in a simple one-step procedure using centrifugal filter devices of various molecular weight cutoffs. The results showed that the generation of a nondiluted low molecular weight (LMW) fraction, corresponding to less than 2% of the original protein content, was critical for the successful screening of cytokines in the sub pg/mL range. The reduced complexity of the LMW fraction significantly improved the assay sensitivity, by improving the fluorescent tagging step and/or reducing the nonspecific binding to the substrates.     

A novel histology-directed strategy for MALDI-MS tissue profiling that improves throughput and cellular specificity in human breast cancer

We describe a novel tissue profiling strategy that improves the cellular specificity and analysis throughput of protein profiles obtained by direct MALDI analysis. The new approach integrates the cellular specificity of histology, the accuracy and reproducibility of robotic liquid dispensing, and the speed and objectivity of automated spectra acquisition. Traditional methodologies for preparing and analyzing tissue samples rely heavily on manual procedures, which for various reasons discussed, restrict cellular specificity and sample throughput. Here, a robotic spotter deposits micron-sized droplets of matrix precisely onto foci of normal mammary epithelium, ductal carcinoma in situ, invasive mammary cancer, and peritumoral stroma selected by a pathologist from high resolution histological images of sectioned human breast cancer samples. The location of each matrix spot was then determined and uploaded into the instrument to facilitate automated profile acquisition by MALDI-TOF. In the example shown, the different lesions were clearly differentiated using mass profiling. Further, the workflow permits a visual projection of any information produced from the profile analyses directly on the histological image for a unique combination of proteomic and histological assessment of sample regions. The higher performance characteristics offered by the new workflow promises to be a significant advancement toward the next generation of tissue profiling studies.     

Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5, 6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage lambda DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.     

Exploring the conformational space of membrane protein folds matching distance constraints

Herein we present a computational technique for generating helix-membrane protein folds matching a predefined set of distance constraints, such as those obtained from NMR NOE, chemical cross-linking, dipolar EPR, and FRET experiments. The purpose of the technique is to provide initial structures for local conformational searches based on either energetic considerations or ad-hoc scoring criteria. In order to properly screen the conformational space, the technique generates an exhaustive list of conformations within a specified root-mean-square deviation (RMSD) where the helices are positioned in order to match the provided distances. Our results indicate that the number of structures decreases exponentially as the number of distances increases, and increases exponentially as the errors associated with the distances increases. We also found the number of solutions to be smaller when all the distances share one helix in common, compared to the case where the distances connect helices in a daisy-chain manner. We found that for 7 helices, at least 15 distances with errors up to 8 A are needed to produce a number of solutions that is not too large to be processed by local search refinement procedures. Finally, without energetic considerations, our enumeration technique retrieved the transmembrane domains of Bacteriorhodopsin (PDB entry1c3w), Halorhodopsin (1e12), Rhodopsin (1f88), Aquaporin-1 (1fqy), Glycerol uptake facilitator protein (1fx8), Sensory Rhodopsin (1jgj), and a subunit of Fumarate reductase flavoprotein (1qlaC) with Calpha level RMSDs of 3.0 A, 2.3 A, 3.2 A, 4.6 A, 6.0 A, 3.7 A, and 4.4 A, respectively.