Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

Motor network degeneration in amyotrophic lateral sclerosis: a structural and functional connectivity study

Background

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by motor neuron degeneration. How this disease affects the central motor network is largely unknown. Here, we combined for the first time structural and functional imaging measures on the motor network in patients with ALS and healthy controls.

Methodology/Principal Findings

Structural measures included whole brain cortical thickness and diffusion tensor imaging (DTI) of crucial motor tracts. These structural measures were combined with functional connectivity analysis of the motor network based on resting state fMRI. Focal cortical thinning was observed in the primary motor area in patients with ALS compared to controls and was found to correlate with disease progression. DTI revealed reduced FA values in the corpus callosum and in the rostral part of the corticospinal tract. Overall functional organisation of the motor network was unchanged in patients with ALS compared to healthy controls, however the level of functional connectedness was significantly correlated with disease progression rate. Patients with increased connectedness appear to have a more progressive disease course.

Conclusions/Significance

We demonstrate structural motor network deterioration in ALS with preserved functional connectivity measures. The positive correlation between functional connectedness of the motor network and disease progression rate could suggest spread of disease along functional connections of the motor network.     

Pater Dr.h.c. Josef Donner (28.2.1909–8.1.1989) in memoriam

Pater Dr.h.c. Josef Donner (28.2.1909–8.1.1989) in memoriam     

Assessment of biases associated with profiling simple, model communities using terminal-restriction fragment length polymorphism-based analyses

Community profiles based on terminal-restriction fragment length polymorphism (T-RFLP) analyses of amplified ribosomal RNA genes are used to monitor changes in microbial community structure and are sometimes employed for semi-quantitative estimates of species richness and abundance in environmental samples. To assess the accuracy of T-RFLP community profiles representing the relative abundance of bacteria in a sample, five species of ruminal bacteria were used to construct simple "communities". Template DNA for PCR amplification was generated either by mixing equal quantities of genomic DNA from pure cultures or by mixing equal numbers of cells prior to DNA extraction. Pairwise mixtures of Fibrobacter succinogenes S85 with Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49 and Streptococcus bovis JB1 were created and a 5-member community was constructed. With genomic DNA mixes, relative abundance calculations based on T-RFLP patterns did not reflect input ratios. These discrepancies could not be accounted for by differences in genome size and rRNA operon copy number. In cell mixing experiments, easily lysed cells were overrepresented. To determine if a numerical correction factor could be used to compensate for observed discrepancies, we attempted to quantify biases attributed to DNA extraction and PCR amplification. Biases attributable to these factors led to deviations from expected PCR product ratios by 6% to 38%. We found that interactions were so complex that a suitable factor could not be derived. The unsystematic dependence of T-RFLP peak ratios on variability of DNA extraction and PCR amplification prevents accurate quantification of the relative abundance of microorganisms designed to represent simplified natural populations.     

Above-ground biomass references for urban trees from terrestrial laser scanning data

Background and AimsWithin extending urban areas, trees serve a multitude of functions (e.g. carbon storage, suppression of air pollution, mitigation of the ‘heat island’ effect, oxygen, shade and recreation). Many of these services are positively correlated with tree size and structure. The quantification of above-ground biomass (AGB) is of especial importance to assess its carbon storage potential. However, quantification of AGB is difficult and the allometries applied are often based on forest trees, which are subject to very different growing conditions, competition and form. In this article we highlight the potential of terrestrial laser scanning (TLS) techniques to extract highly detailed information on urban tree structure and AGB.MethodsFifty-five urban trees distributed over seven cities in Switzerland were measured using TLS and traditional forest inventory techniques before they were felled and weighed. Tree structure, volume and AGB from the TLS point clouds were extracted using quantitative structure modelling. TLS-derived AGB estimates were compared with AGB estimates based on forest tree allometries dependent on diameter at breast height only. The correlations of various tree metrics as AGB predictors were assessed.Key ResultsEstimates of AGB derived by TLS showed good performance when compared with destructively harvested references, with an R² of 0.954 (RMSE = 556 kg) compared with 0.837 (RMSE = 1159 kg) for allometrically derived AGB estimates. A correlation analysis showed that different TLS-derived wood volume estimates as well as trunk diameters and tree crown metrics show high correlation in describing total wood AGB, outperforming tree height.ConclusionsWood volume estimates based on TLS show high potential to estimate tree AGB independent of tree species, size and form. This allows us to retrieve highly accurate non-destructive AGB estimates that could be used to establish new allometric equations without the need for extensive destructive harvesting.     

A database system for the analysis of biochemical pathways

To provide support for the analysis of biochemical pathways a database system based on a model that represents the characteristics of the domain is needed. This domain has proven to be difficult to model by using conventional data modelling techniques. We are building an ontology for biochemical pathways, which acts as the basis for the generation of a database on the same domain, allowing the definition of complex queries and complex data representation. The ontology is used as a modelling and analysis tool which allows the expression of complex semantics based on a first-order logic representation language. The induction capabilities of the system can help the scientist in formulating and testing research hypotheses that are difficult to express with the standard relational database mechanisms. An ontology representing the shared formalisation of the knowledge in a scientific domain can also be used as data integration tool clarifying the mapping of concepts to the developers of different databases. In this paper we describe the general structure of our system, concentrating on the ontology-based database as the key component of the system.