Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry

Abstract Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-ProHyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of ±10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined

Unrecognized diversity and distribution of soil algae from Maritime Antarctica (Fildes Peninsula, King George Island)

IntroductionEukaryotic algae in the top few centimeters of fellfield soils of ice-free Maritime Antarctica have many important effects on their habitat, such as being significant drivers of organic matter input into the soils and reducing the impact of wind erosion by soil aggregate formation. To better understand the diversity and distribution of Antarctic terrestrial algae, we performed a pilot study on the surface soils of Meseta, an ice-free plateau mountain crest of Fildes Peninsula, King George Island, being hardly influenced by the marine realm and anthropogenic disturbances. It is openly exposed to microbial colonization from outside Antarctica and connected to the much harsher and dryer ice-free zones of the continental Antarctic. A temperate reference site under mild land use, SchF, was included to further test for the Meseta algae distribution in a contrasting environment.MethodsWe employed a paired-end metabarcoding analysis based on amplicons of the highly variable nuclear-encoded ITS2 rDNA region, complemented by a clone library approach. It targeted the four algal classes, Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Xanthophyceae, representing key groups of cold-adapted soil algae.ResultsA surprisingly high diversity of 830 algal OTUs was revealed, assigned to 58 genera in the four targeted algal classes. Members of the green algal class Trebouxiophyceae predominated in the soil algae communities. The major part of the algal biodiversity, 86.1% of all algal OTUs, could not be identified at the species level due to insufficient representation in reference sequence databases. The classes Ulvophyceae and Xanthophyceae exhibited the most unknown species diversity. About 9% of the Meseta algae species diversity was shared with that of the temperate reference site in Germany.DiscussionIn the small portion of algal OTUs for which their distribution could be assessed, the entire ITS2 sequence identity with references shows that the soil algae likely have a wide distribution beyond the Polar regions. They probably originated from soil algae propagule banks in far southern regions, transported by aeolian transport over long distances. The dynamics and severity of environmental conditions at the soil surface, determined by high wind currents, and the soil algae’s high adaptability to harsh environmental conditions may account for the high similarity of soil algal communities between the northern and southern parts of the Meseta

Global effects of agriculture on fluvial dissolved organic matter

Agricultural land covers approximately 40% of Earth's land surface and affects hydromorphological, biogeochemical and ecological characteristics of fluvial networks. In the northern temperate region, agriculture also strongly affects the amount and molecular composition of dissolved organic matter (DOM), which constitutes the main vector of carbon transport from soils to fluvial networks and to the sea and is involved in a large variety of biogeochemical processes. Here, we provide first evidence about the wider occurrence of agricultural impacts on the concentration and composition of fluvial DOM across climate zones of the northern and southern hemispheres. Both extensive and intensive farming altered fluvial DOM towards a more microbial and less plant-derived composition. Moreover, intensive farming significantly increased dissolved organic nitrogen (DON) concentrations. The DOM composition change and DON concentration increase differed among climate zones and could be related to the intensity of current and historical nitrogen fertilizer use. As a result of agriculture intensification, increased DON concentrations and a more microbial-like DOM composition likely will enhance the reactivity of catchment DOM emissions, thereby fuelling the biogeochemical processing in fluvial networks and resulting in higher ecosystem productivity and CO2 outgassing

Different antibiotic profiles in wild and farmed Chilean salmonids. Which is the main source for antibiotic in fish?

Fish from both aquaculture and wild capture are exposed to veterinary and medicinal antibiotics (ABs). This study explored the occurrence and probable source of 46 antibiotic residues in muscle of farmed salmon and wild trout from Chile. Results showed that at least one AB was detected in all studied samples. Diverse patterns were observed between farmed and wild specimens, with higher ABs concentrations in wild fish. Considering antimicrobial resistance, detected ABs corresponded to the categories B (Restrict), C (Caution) and D (Prudence) established by Antimicrobial Advice Ad Hoc Expert Group (European Medicines Agency). Multivariate statistic was used to verify differences between farmed and wild populations, looking for the probable source of ABs as well. Principal components analysis (PCA) revealed that ciprofloxacin, moxifloxacin, enrofloxacin, amoxicillin, penicillin G, oxolinic acid, sulfamethoxazole, trimethoprim and clarithromycin were associated with wild samples, collected during the cold season. Conversely, norfloxacin, sulfaquinoxaline, sulfadimethoxine, nitrofurantoin, nalidixic acid, penicillin V, doxycycline, flumequine, oxacillin, pipemidic acid and sulfamethizole were associated with wild samples collected during the warm season. All farmed salmon samples were associated with ofloxacin, tetracycline, cephalexin, erythromycin, azithromycin, roxithromycin, sulfabenzamide, sulfamethazine, sulfapyridine, sulfisomidin, and sulfaguanidine. In addition, linear discriminant analysis showed that the AB profile in wild fish differ from farmed ones. Most samples showed ABs levels below the EU regulatory limit for edible fish, except for sulfaquinoxaline in one sample. Additionally, nitrofurantoin (banned in EU) was detected in one aquaculture sample. The differences observed between farmed and wild fish raise questions on the probable source of ABs, either aquaculture or urban anthropic activities. Further research is necessary for linking the ABs profile in wild fish with the anthropic source. However, to our knowledge, this is the first report showing differences in the ABs profile between wild and aquaculture salmonids, which could have both environmental and health consequences.Fil: Carrizo, Juan Cruz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Griboff, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bonansea, Rocío Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Nimptsch, Jorge. Universidad Austral de Chile; ChileFil: Valdés, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Wunderlin, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Amé, María Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Tracing dissolved organic matter (DOM) from land-based aquaculture systems in North Patagonian streams

Chile is the second largest producer of salmonids worldwide. The first step in the production of salmonids takes place in land-based aquacultures. However, the effects of the discharge from these aquacultures on stream dissolved organic matter (DOM) content, molecular composition and degradability are unknown. The aim of this study was thus to investigate the inputs of anthropogenic DOM from land-based aquaculture to the predominantly pristine river systems of North Patagonia. We hypothesized, that i) DOM exported from land-based aquaculture mainly consists of protein-like fluorescence (tyrosine and tryptophan) released from fish feces and food remains, and that ii) this DOM is highly degradable and therefore rapidly turned-over within the receiving streams. In the North Patagonian region we conducted a screening of ten land-based aquacultures and an intensive sampling campaign for one aquaculture. This was combined with longitudinal transects and a degradation experiment in order to couple the composition of DOM exported from land-based aquacultures to its degradability in streams. We measured dissolved organic carbon (DOC) concentration by high-temperature catalytic oxidation and DOM composition by fluorescence spectroscopy and parallel factor analysis. In the effluent of the ten screened aquacultures and in the repeated sampling of one aquaculture, we consistently found an increase of DOC concentrations and a dominance of protein-like fluorescence. The protein-like fluorescence rapidly disappeared downstream of the aquacultures, and in the degradation experiment. 21% of the DOC export from the repeatedly sampled aquaculture resulted from food addition and 76% from fish production. We conclude that large amounts of degradable DOM are exported from land-based aquacultures. This probably has strong effects on the ecological structure and function of North Patagonian streams, and similarly affected streams worldwide. (C) 2015 Elsevier B.V. All rights reserved

Structural analysis of the interleukin-8/glycosaminoglycan interactions by amide hydrogen/deuterium exchange mass spectrometry

The recruitment of different chemokines and growth factors by glycosaminoglycans (GAGs) such as chondroitin sulfate or hyaluronan plays a critical role in wound healing processes. Thus, there is a special interest in the design of artificial extracellular matrices with improved properties concerning GAG interaction with common regulating proteins. In this study, amide hydrogen/deuterium (H/D) exchange mass spectrometry (HDX MS) combined with molecular modeling and docking experiments was used to obtain structural models of proinflammatory chemokine interleukin-8 (IL-8) in complex with hexameric chondroitin sulfate. Experiments on the intact protein showed a difference in deuterium labeling of IL-8 due to chondroitin sulfate binding. The extent of deuteration was reduced from 24% to 13% after 2. min exchange time, which corresponds to a reduced exchange of approximately 10 backbone amides. By local HDX MS experiments, H/D exchange information on the complete sequence of IL-8 could be obtained. A significantly reduced H/D exchange, especially of the C-terminal α-helical region comprising amino acids 70-77 and to the loop comprising amino acids 27-29 was observed in the presence of chondroitin sulfate. HDX MS data were used to model the IL-8/chondroitin sulfate complex. The binding interface of IL-8 and chondroitin sulfate determined this way correlated excellently with the corresponding NMR based atomistic model previously published. Our results demonstrate that HDX-MS in combination with molecular modeling is a valuable approach for the analysis of protein/GAG complexes at physiological pH, temperature, and salt concentration. The fact that HDX-MS requires only micrograms of protein and GAGs makes it a very promising technique to address protein-GAG interactions.</p