MALDI imaging mass spectrometry to investigate endogenous peptides in an animal model of Usher's disease

Imaging MS (MSI) has emerged as a valuable tool to study the spatial distribution of biomolecules in the brain. Herein, MALDI‐MSI was used to determine the distribution of endogenous peptides in a rat model of Usher's disease. This rare disease is considered as a leading cause of deaf‐blindness in humans worldwide. Cryosections of brain tissue were analyzed by MALDI‐MSI to differentiate between healthy and diseased rats. MSI results were highly reproducible. Tissue‐specific peptides were identified by MS/MS using LC‐Orbitrap and MALDI‐TOF/TOF analyses. These peptides were proposed for histological classification due to their particular spatial distribution in the brain, for example, substantia nigra, corpus callosum, and hippocampus. Several endogenous peptides showed significantly increased ion densities, particularly in the colliculi superiores and in the substantia nigra of diseased rats, including peptides derived from Fsd1, dystrobrevin‐β, and ProSAAS. Furthermore, several proteolytic degradation products of the myelin basic protein were identified, of which one peptide is most likely mediated by calpain‐2. Our findings contribute to the characterization of this animal model and include possible peptide markers of disease.     

Spring emergence and canopy development strategies in miscanthus hybrids in Mediterranean,continental and maritime European climates

Due to its versatility and storability, biomass is an important resource for renewable materials and energy. Miscanthus hybrids combine high yield potential, low input demand, tolerance of certain marginal land types and several ecosystem benefits. To date, miscanthus breeding has focussed on increasing yield potential by maximising radiation interception through: (1) selection for early emergence, (2) increasing the growth rate to reach canopy closure as fast as possible, and (3) delayed flowering and senescence. The objective of this study is to compare early season re-growth in miscanthus hybrids cultivated across Europe. Determination of differences in early canopy development on end-of-year yield traits is required to provide information for breeding decisions to improve future crop performance. For this purpose, a trial was planted with four miscanthus hybrids (two novel seed-based hybrids M. sinensis × sinensis [M sin × sin] and M. sacchariflorus × sinensis [M sac × sin], a novel rhizome-based M sac × sin and a standard Miscanthus × giganteus [M × g] clone) in the UK, Germany, Croatia and Italy, and was monitored in the third and fourth growing season. We determined differences between the hybrids in base temperature, frost sensitivity and emergence strategy. M × g and M sac × sin mainly emerged from belowground plant organs, producing fewer but thicker shoots at the beginning of the growing season but these shoots were susceptible to air frosts (determined by recording 0°C 2 m above ground surface). By contrast, M sin × sin emerged 10 days earlier, avoiding damage by late spring frosts and producing a high number of thinner shoots from aboveground shoots. Therefore, we recommend cultivating M sac × sin at locations with low risk and M sin × sin at locations with higher risk of late spring frosts. Selecting miscanthus hybrids that produce shoots throughout the vegetation period is an effective strategy to limit the risk of late frost damage and avoid reduction in yield from a shortened growing season.     

Aryl 1,4-diazepane compounds as potent and selective CB2 agonists: optimization of drug-like properties and target independent parameters

A high throughput screening campaign identified aryl 1,4-diazepane compounds as potent and selective cannabinoid receptor 2 agonists as compared to cannabinoid receptor 1. This class of compounds suffered from poor drug-like parameters as well as low microsomal stability and poor solubility. Structure-activity relationships are described with a focus on improving the drug-like parameters resulting in compounds with improved solubility and permeability.     

THE IMPORTANCE OF DIATOM CELL SIZE IN COMMUNITY ANALYSIS1

The large variation in size and shape in diatoms is shown by morphometric measurements of 515 benthic and pelagic diatom species from the Baltic Sea area. The largest mean cell dimension (mostly the apical axis) varied between 4.2 and 653 μm, cell surface area between 55 and 344,000 μm², and cell volume between 21 and 14.2 × 10⁶μm³. The shape‐related index, length to width ratio, was between 1.0 and 63.3 and the shape‐ and size‐related index, surface area to volume ratio, was between 0.02 and 3.13. Diatom community analysis by multivariate statistics is usually based on counts of a fixed number of diatom valves with species scores irrespective of cell size. This procedure underestimates the large species for two reasons. First, the importance of a species with higher cell volume is usually larger in a community. Second, larger species usually have lower abundances and their occurrence in the diatom counts is stochastic. This article shows that co‐occurring small and large diatom species can respond very differently to environmental constraints. Large epiphytic diatoms responded most to macroalgal host species and small epiphytic diatoms most to environmental conditions at the sampling site. Large epilithic diatoms responded strongly to salinity, whereas small epilithic diatoms did so less clearly. The conclusion is that different scale‐dependent responses are possible within one data set. The results from the test data also show that important ecological information from diatom data can be missed when the large species are neglected or underestimated.     

Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)₂ with the catalytic (BchY/BchZ)₂ protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF₄⁻. Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)₂. A second [4Fe-4S] cluster was identified on (BchY/BchZ)₂. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.     

Radiosensitization by Kinase Inhibition Revealed by Phosphoproteomic Analysis of Pancreatic Cancer Cells

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Highlights

• •Proteomes and phosphoproteomes of radiosensitive and radioresistant PDAC cell lines.
• •Common activation of DDR is proven by ATM activity on known and novel substrates.
• •Resistant cells bear raised NQO1 expression, actin dynamics including FAK activity.
• •Inhibitors of CHEK Rabusertib and FAK Defactinib radiosensitize PDAC cells.

     

CD8+ cells protect mice against reinfection with the intestinal parasite Eimeria falciformis

We investigated cellular immune responses of mice infected with the apicomplexan parasite Eimeria falciformis in order to characterise protective immune mechanisms and effector functions. Adoptive transfer experiments with mesenterial lymph node cells (MLNC) from immune donor mice were performed, and the oocyst output monitored after challenge infection. Phenotypical analysis by fluorescence cytometry and T cell proliferation assay showed that already from day four post infection E. falciformis-specific lymphocytes were present in the MLN. The frequency of parasite-specific, IFN-γ producing CD4+ and CD8+ cells increased in this period by 9.8% and 16.4%, respectively. Infection experiments with IFN-γ deficient mice revealed that IFN-γ is involved in resistance to primary and secondary infection. Transfer of total MLNC from immune donors reduced the oocyst output by 65–74%, as compared to the oocyst output of animals transferred with cells from naïve donors. Transfer of CD8+ cells inhibited parasite development resulting in a reduction of oocyst numbers by 42–64%, whereas CD4+ cells showed no influence on resistance to reinfection.