Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets

Endothelial cells release prostacyclin (PGI₂) and nitric oxide (NO) to inhibit platelet functions. PGI₂ and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.     

A Framework for Evaluating Heterogeneity and Landscape-Level Impacts of Non-native Aquatic Species

Non-native species are a major component of global environmental change, and aquatic systems are especially vulnerable to non-native species impacts. Much of the research on aquatic non-native species impact has occurred at the local or site level. In reality, non-native species impacts play out across multiple spatial scales on heterogeneous landscapes. How can we ‘scale up’ our understanding of site-level impacts to the broader landscape scale? To address this disconnect, we synthesize our current understanding of key components of landscape-scale non-native species impacts: geographic range, abundance, and local impacts. Most aquatic non-native species have small ranges, while a few have large ranges. However, aquatic non-native species are often far from saturated on landscapes, and occurrence records are often woefully incomplete. Aquatic non-native species are often at low abundances where they are present, reaching high abundance in a small number of locations. Finally, local-scale impact can be estimated from abundance, but this requires knowledge of the abundance–impact relationship. Considering these multiple components enables understanding of non-native species impacts at broader spatial scales. Although the landscape-level impacts of aquatic non-native species may be high, the spatial distribution of site-level impacts is uneven, and highly impacted sites may be relatively uncommon. This heterogeneity in impacts provides an opportunity to optimize and prioritize non-native species management and prevention efforts.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Development of media for the mixotrophic/heterotrophic culture ofBrachiomonas submarina

The marine algaBrachiomonas submarina var.pulsifera (Droop) CCAP 7/2A, is employed as a food organism in aquaculture; it can be cultured heterotrophically or mixotrophically. Growth rates and productivity under heterotrophic conditions were lower than those achieved under mixotrophic conditions. By reducing the osmotic potential of the medium, whilst simultaneously increasing the levels of nitrogen and phosphorus and using sodium acetate as a carbon source, a 20-fold increase in final yield was attained. This corresponded to a maximum culture of 9.02times 10⁶ cells ml^–1 and a dry weight of 2.51 g l^–1.Author for correspondence     

Effects of predator exposure on baseline and stress-induced glucocorticoid hormone concentrations in pumpkinseed Lepomis gibbosus

We compared baseline and maximal cortisol concentrations between predator exposure and prey blood samples in pumpkinseed Lepomis gibbosus, captured using a standardised fishing event underneath osprey Pandion haliaetus nests and away from osprey nests. We did not detect differences in cortisol or glucose between sites. These findings suggest that predictable sources of predation risk may not confer stress-related costs in teleosts.     

Species sorting and mass effect along forest succession: Evidence from taxonomic,functional, and phylogenetic diversity of amphibian communities

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Investigation of fouling mechanisms of virus filters during the filtration of protein solutions using a high throughput filtration screening device

The downstream process development of novel antibodies (Abs) is often challenged by virus filter fouling making a better understanding of the underlying mechanisms highly desirable. The present study combines the protein characterization of different feedstreams with their virus filtration performance using a novel high throughput filtration screening system. Filtration experiments with Ab concentrations of up to 20 g/L using either low interacting or hydrophobically interacting pre-filters indicate the existence of two different fouling mechanisms, an irreversible and a reversible one. At the molecular level, size exclusion chromatography revealed that the presence of large amount of high molecular weight species—considered as irreversible aggregates—correlates with irreversible fouling that caused reduced Ab throughput. Results using dynamic light scattering show that a concentration dependent increase of the mean hydrodynamic diameter to the range of dimers (17 nm at 20 g/L) together with a negative DLS interaction parameter k_D (−18 mL/g) correlate with the propensity to form reversible aggregates and to cause reversible fouling, probably by a decelerated Ab transport velocity within the virus filter. The two fouling mechanisms are further supported by buffer flush experiments. Finally, concepts for reversible and irreversible fouling mechanisms are discussed together with strategies for respective fouling mitigation. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2776, 2019.     

Ribosomal Tag Pyrosequencing of DNA and RNA Reveals “Rare” Taxa with High Protein Synthesis Potential in the Sediment of a Hypersaline Lake in Western Australia

Little is known about the potential activity of microbial communities in hypersaline sediment ecosystems. Ribosomal tag libraries of DNA and RNA extracted from the sediment of Lake Strawbridge (Western Australia) revealed bacterial and archaeal operational taxonomic units (OTUs) with high RNA/DNA ratios providing evidence for the presence of ‘rare’ but potentially “active” taxa. Among the ‘rare’ bacterial taxa Halomonas, Salinivibrio and Idiomarina showed the highest protein synthesis potential. Rare but ‘active’ archaeal OTUs were related to the KTK 4A cluster and the Marine-Benthic-Groups B and D. We present the first molecular analysis of the microbial diversity and protein synthesis potential of rare microbial taxa in a hypersaline sediment ecosystem.     

Photosystem II reaction centre quenching: mechanisms and physiological role

Dissipation of excess absorbed light energy in eukaryotic photoautotrophs through zeaxanthin- and DeltapH-dependent photosystem II antenna quenching is considered the major mechanism for non-photochemical quenching and photoprotection. However, there is mounting evidence of a zeaxanthin-independent pathway for dissipation of excess light energy based within the PSII reaction centre that may also play a significant role in photoprotection. We summarize recent reports which indicate that this enigma can be explained, in part, by the fact that PSII reaction centres can be reversibly interconverted from photochemical energy transducers that convert light into ATP and NADPH to efficient, non-photochemical energy quenchers that protect the photosynthetic apparatus from photodamage. In our opinion, reaction centre quenching complements photoprotection through antenna quenching, and dynamic regulation of photosystem II reaction centre represents a general response to any environmental condition that predisposes the accumulation of reduced Q(A) in the photosystem II reaction centres of prokaryotic and eukaryotic photoautotrophs. Since the evolution of reaction centres preceded the evolution of light harvesting systems, reaction centre quenching may represent the oldest photoprotective mechanism.     

Pathology of Puumala hantavirus infection in macaques

Hantaviruses are globally important human pathogens that cause hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Capillary leakage is central to hantaviral diseases, but how it develops, has remained unknown. It has been hypothesized that the pathogenesis of hantavirus infection would be a complex interplay between direct viral effects and immunopathological mechanisms. Both of these were studied in the so far best model of mild hemorrhagic fever with renal syndrome, i.e. cynomolgus macaques infected with wild-type Puumala hantavirus. Viral RNA detected by in situ hybridization and nucleocapsid protein detected by immunohistochemical staining were observed in kidney, spleen and liver tissues. Inflammatory cell infiltrations and tubular damage were found in the kidneys, and these infiltrations contained mainly CD8-type T-cells. Importantly, these results are consistent with those obtained from patients with hantaviral disease, thus showing that the macaque model of hantavirus infection mimics human infection also on the tissue level. Furthermore, both the markers of viral replication and the T-cells appeared to co-localize in the kidneys to the sites of tissue damage, suggesting that these two together might be responsible for the pathogenesis of hantavirus infection.