Using mass spectrometry to explore the neglected glycan moieties of the antiophidic proteins DM43 and DM64

The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.     

Proteomic profiling of skeletal muscle in an animal model of overtraining

Excessive training (i.e. overtraining, OT) may result in underperformance, which can be characterized by the time needed to re-establish performance (i.e. functional overreaching (FOR), nonfunctional overreaching, OT syndrome). The present study is an initial screening for proteins presenting altered abundance in the red (RG) and white (WG) portions of the gastrocnemius muscle from rats submitted to an OT protocol that induced FOR. In the RG, compared to the nontrained control, FOR demonstrated an increased abundance of proteins normally related to adaptation to endurance training (e.g. proteins of oxidative phosphorylation complexes, proteins related to lipid metabolism, antioxidants, and chaperones). In the WG, spots identified as mitochondrial aconitase and a component of the succinate dehydrogenase complex were downregulated in FOR, as were proteins related to myofibril stabilization; these latter were upregulated in the RG. This initial study shows that skeletal muscles with different fiber-type compositions respond differently to an OT period. Also, it is likely that actin-interacting proteins have an important role in muscle adaptation to endurance exercise.     

Search engine processor: Filtering and organizing peptide spectrum matches

The search engine processor (SEPro) is a tool for filtering, organizing, sharing, and displaying peptide spectrum matches. It employs a novel three-tier Bayesian approach that uses layers of spectrum, peptide, and protein logic to lead the data to converge to a single list of reliable protein identifications. SEPro is integrated into the PatternLab for proteomics environment, where an arsenal of tools for analyzing shotgun proteomic data is provided. By using the semi-labeled decoy approach for benchmarking, we show that SEPro significantly outperforms a commercially available competitor.     

Assessment of grassland use intensity by remote sensing to support conservation schemes

Grassland is a land cover in the area of conflict between agriculture and conservation, where intensification of land use is a major threat to grassland biodiversity. Grassland use intensity is a key factor for the conservation value of grassland, and detailed spatial data on grassland use intensity is needed to improve strategies for biodiversity conservation. A new remote sensing-based approach using multi-temporal high resolution RapidEye satellite data was developed in the present study that makes a large-scale assessment of grassland use intensity possible. RapidEye is a constellation of five satellites with 6.5 m spatial resolution, which allows frequent and timely image acquisition targeted at specific growing seasons. Semi-natural grassland, extensively used grassland, intensively used grassland and tilled grassland could be reliably differentiated at the management plot level in a study area in southern Germany. Various combinations of images from different observation dates have been tested as classification input and their overall classification accuracies were validated by field data. Best results were achieved using a combination of five multi-temporal scenes with an overall accuracy of 85.7%. A three-scene combination resulted in an overall accuracy of 82.2%. The analysis showed that seasonal aspects are very important when selecting adequate observation dates. Grassland use intensity was also assessed on peatlands using a peat soil map, since land use intensity significantly affects greenhouse gas emissions from peatlands. The results demonstrate the potential of targeted multi-spectral, high spatial resolution remote sensing for the large-scale monitoring of dynamic habitats, which is of vital importance to support various environmental conservation schemes through improved monitoring and reporting capabilities.     

Low charge density cationic polymers for gene delivery: exploring the influence of structural elements on in vitro transfection

A series of end-functionalized poly(trimethylene carbonate) DNA carriers, characterized by low cationic charge density and pronounced hydrophobicity, is used to study structural effects on in vitro gene delivery. As the DNA-binding moieties are identical in all polymer structures, the differences observed between the different polymers are directly related to the functionality and length of the polymer backbone. The transfection efficiency and cytotoxicity of the polymer/DNA complexes are thus found to be dependent on a combination of polymer charge density and functionality, highlighting the importance of such structural considerations in the development of materials for efficient gene delivery.     

Scleral thickness in human eyes

Purpose

To obtain information about scleral thickness in different ocular regions and its associations.

Methods

The histomorphometric study included 238 human globes which had been enucleated because of choroidal melanomas or due to secondary angle-closure glaucoma. Using light microscopy, anterior-posterior pupil-optic nerve sections were measured.

Results

In the non-axially elongated group (axial length ≤26 mm), scleral thickness decreased from the limbus (0.50±0.11 mm) to the ora serrata (0.43±0.14 mm) and the equator (0.42±0.15 mm), and then increased to the midpoint between posterior pole and equator (0.65±0.15 mm) and to the posterior pole (0.94±0.18 mm), from where it decreased to the peri-optic nerve region (0.86±0.21 mm) and finally the peripapillary scleral flange (0.39±0.09 mm). Scleral thickness was significantly lower in the axially elongated group (axial length >26 mm) than in the non-axially elongated group for measurements taken at and posterior to the equator. Scleral thickness measurements of the posterior pole and of the peripapillary scleral flange were correlated with lamina cribrosa thickness measurements. Scleral thickness measurements at any location of examination were not significantly (all P>0.10) correlated with corneal thickness measurements. Scleral thickness was statistically independent of age, gender and presence of glaucoma.

Conclusions

In non-axially elongated eyes, the sclera was thickest at the posterior pole, followed by the peri-optic nerve region, the midpoint between posterior pole and equator, the limbus, the ora serrata, the equator and finally the peripapillary scleral flange. In axially elongated eyes, scleral thinning occurred at and posterior to the equator, being more marked closer to the posterior pole and the longer the axial length was. Within the anterior and posterior segment respectively, scleral thickness measurements were correlated with each other. Posterior scleral thickness was correlated with lamina cribrosa thickness. Scleral thickness measurements at any location of examination were not significantly correlated with corneal thickness or with age, gender and presence of absolute secondary angler-closure glaucoma.     

Age patterns of intra‐pair DNA methylation discordance in twins: Sex difference in epigenomic instability and implication on survival

Aging is a biological process linked to specific patterns and changes in the epigenome. We hypothesize that age‐related variation in the DNA methylome could reflect cumulative environmental modulation to the epigenome which could impact epigenomic instability and survival differentially by sex. To test the hypothesis, we performed sex‐stratified epigenome‐wide association studies on age‐related intra‐pair DNA methylation discordance in 492 twins aged 56–80 years. We identified 3084 CpGs showing increased methylation variability with age (FDR < 0.05, 7 CpGs with p < 1e‐07) in male twins but no significant site found in female twins. The results were replicated in an independent cohort of 292 twins aged 30–74 years with 37% of the discovery CpGs successfully replicated in male twins. Functional annotation showed that genes linked to the identified CpGs were significantly enriched in signaling pathways, neurological functions, extracellular matrix assembly, and cancer. We further explored the implication of discovery CpGs on individual survival in an old cohort of 224 twins (220 deceased). In total, 264 CpGs displayed significant association with risk of death in male twins. In female twins, 175 of the male discovery CpGs also showed non‐random correlation with mortality. Intra‐pair comparison showed that majority of the discovery CpGs have higher methylation in the longer‐lived twins suggesting that loss of DNA methylation during aging contributes to increased risk of death which is more pronounced in male twins. In conclusion, age‐related epigenomic instability in the DNA methylome is more evident in males than in females and could impact individual survival and contribute to sex difference in human lifespan.     

Accumulation and Fate of Microorganisms and Microspheres in Biofilms Formed in a Pilot-Scale Water Distribution System

The accumulation and fate of model microbial “pathogens” within a drinking-water distribution system was investigated in naturally grown biofilms formed in a novel pilot-scale water distribution system provided with chlorinated and UV-treated water. Biofilms were exposed to 1-μm hydrophilic and hydrophobic microspheres, Salmonella bacteriophages 28B, and Legionella pneumophila bacteria, and their fate was monitored over a 38-day period. The accumulation of model pathogens was generally independent of the biofilm cell density and was shown to be dependent on particle surface properties, where hydrophilic spheres accumulated to a larger extent than hydrophobic ones. A higher accumulation of culturable legionellae was measured in the chlorinated system compared to the UV-treated system with increasing residence time. The fate of spheres and fluorescence in situ hybridization-positive legionellae was similar and independent of the primary disinfectant applied and water residence time. The more rapid loss of culturable legionellae compared to the fluorescence in situ hybridization-positive legionellae was attributed to a loss in culturability rather than physical desorption. Loss of bacteriophage 28B plaque-forming ability together with erosion may have affected their fate within biofilms in the pilot-scale distribution system. The current study has demonstrated that desorption was one of the primary mechanisms affecting the loss of microspheres, legionellae, and bacteriophage from biofilms within a pilot-scale distribution system as well as disinfection and biological grazing. In general, two primary disinfection regimens (chlorination and UV treatment) were not shown to have a measurable impact on the accumulation and fate of model microbial pathogens within a water distribution system.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.