Optofluidic rotation of living cells for single‐cell tomography

Flow cytometry provides a high throughput, multi‐dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z‐resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.

     

Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m³) in Ogg1^{? / ?} and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in Ogg1^{? / ?} mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1^{? / ?} mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1^{? / ?} mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1^{? / ?} mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.     

External morphology of Lightiella monniotae (Crustacea, Cephalocarida) in the light of Cambrian 'Orsten' crustaceans

The species-poor meiofaunal Cephalocarida have played an important role in discussions of the phylogeny and evolution of Crustacea since their discovery in 1955. One reason may be that the morphology of cephalocarids includes some aspects of putatively ancient appearance, such as the simple roof-shaped head shield, the anterior three head appendages resembling those of a nauplius larva, or the trunk-limb-like second maxilla. Cephalocarida have even been suggested to represent the sister taxon to all other Eucrustacea. Presence of possibly plesiomorphic characters, however, does not necessarily point to a basal position in the system. Growing evidence demonstrates that the modification of the fourth post-antennular cephalic appendage, the ‘maxilla’, into a “mouth part” may have occurred independently in the different eucrustacean lineages, so a trunk-limb-like maxilla is an ancient feature that does not hold only for cephalocarids. Retention of its plesiomorphic shape and function in the Cephalocarida remains, however, noteworthy. Cephalocarids are still little studied and incompletely known, especially their external morphology. By examining several adults and one young specimen of Lightiella monniotae Cals and Delamare Deboutteville, 1970 from New Caledonia, we aimed to a) document as many details as possible, and b) compare these data with other species of Cephalocarida. We also aimed to reconstruct aspects of the ground pattern of Cephalocarida, which is a pre-requisite for any comparisons in a broader perspective of crustacean phylogeny. Among the new findings or conclusions are: (1) Lightiella is in need of a revision since several assumed differences between the species are questionable or subject to intra-specific variability; (2) the cuticle of the trunk-limb basipod is sub-divided into a number of smaller sclerotized areas as in various exceptionally 3D preserved fossil crustaceans from Cambrian ‘Orsten’ faunal assemblages; (3) a small transitional portion on the post-maxillulary limbs in the area where the endopod and basipod connect is discussed as either a reduced, proximal endopod segment or as an evolutionary new joint of the basipod to enhance its flexibility; (4) the so-called pseud-epipod is interpreted as an outer branch of the exopod; (5) compared to ‘Orsten’ crustaceans many characters of the Cephalocarida are more modified than previously assumed, including the morphology of the trunk-limb basipod, and the unique, ring-shaped appearance of the abdominal segments. Also the development is not as plesiomorphic as sometimes assumed, at least not compared to that of the strictly anamorphic series of the ‘Orsten’ eucrustacean Rehbachiella kinnekullensis. The application of SEM techniques has again proved to be especially appropriate because of the small size of these animals, and because it permits direct comparisons with other similarly small crustaceans and the ‘Orsten’ crustaceans and their larvae.     

The diet of polar bears (Ursus maritimus) from Svalbard, Norway, inferred from scat analysis

Polar bears (Ursus maritimus) are heavily dependent on marine prey, in particular ice-associated seals, which they hunt on landfast sea ice or free-floating pack ice. Dramatic current (and predicted) losses of sea ice habitat make it increasingly important to gain more knowledge of the relative use by bears of all types of prey from the marine food web as well as from terrestrial sources. This study uses frequency of occurrence of food items in 119 polar bear scats sampled on the sea ice as well as on shore in coastal areas in the Svalbard Archipelago, mainly in spring, between 2003 and 2010 to explore the diet of bears in the region. Ringed seals (Pusa hispida) occurred in 62.2 % (CI 52.8–70.9 %) of the scat samples examined. Various terrestrial plants (32.8 %, CI 24.4–42.0 %) and marine algae (21.8 %, CI 14.8–30.4 %) also occurred frequently in the scats; the significance of this high occurrence of plants and algae is not clear. Bearded seals (Erignathus barbatus) and various bird species constituted only minor components of the diet, while Svalbard reindeer (Rangifer tarandus platyrhynchus) occurred in 9.2 % (CI 4.7–15.9 %) of the scats, indicating that this species may play a more important role than previously reported. The novel combination of genetic analyses of material in the fecal samples along with detailed exploration of the physical–structural properties of prey hairs and plant parts provided a much fuller picture of the diet of polar bears than would have been possible from observational studies of polar bear predation behavior alone. This approach may provide an important tool for monitoring the responses of polar bears to ongoing ecosystem changes that will result from continued warming in the Arctic.     

Examination of functional morphology of dajiid isopods using Arthrophryxus sp. parasitising a mysid shrimp as an example

Parasitic isopods have become specialised to many different host species and therefore show a wide variety of attachment and feeding specialisations. Often such structures are difficult to examine due to their small sizes which makes a more complete understanding of their functional morphology difficult. Here we present a new report and a first time high-resolution, non-SEM documentation of a parasitic epicaridean isopod, a female of the dajiid Arthrophryxus sp. from the Sea of Okhotsk. It was found during the SokhoBio 2015 on its host, a mysid shrimp (Holmsiella sp.). Arthrophryxus has only one formally described species, Arthrophryxus beringanus Richardson, 1908. With high-resolution documentation methods, we reveal new details of the morphological structures of mouthparts and thoracopods of this dajiid. Furthermore, we discuss the functional morphology of attachment to the host and feeding according to our new findings. We suggest that the thoracopods are involved in the attachment process even more than formerly assumed for different dajiids. The piercing-sucking mouthparts and the additional attachment mechanisms strongly indicate a permanent parasitism of the dajiid isopod. Permanent parasites affect the fitness of their hosts; therefore, deep-sea forms of dajiid isopods have direct impact on the deep-sea crustacean communities.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

In‐depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host‐specific post‐translational modifications

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7A^rec) was compared with the native fungal enzyme (TrCel7A^nat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7A^rec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7A^rec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7A^rec had 25% lower catalytic activity than TrCel7A^nat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7A^rec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.