Effect of pseudophosphorylation and cross-linking by lipid peroxidation and advanced glycation end product precursors on tau aggregation and filament formation

Accumulation of hyperphosphorylated Tau protein as paired helical filaments in pyramidal neurons is a major hallmark of Alzheimer disease. Besides hyperphosphorylation, other modifications of the Tau protein, such as cross-linking, are likely to contribute to the characteristic features of paired helical filaments, including their insolubility and resistance against proteolytic degradation. In this study, we have investigated whether the four reactive carbonyl compounds acrolein, malondialdehyde, glyoxal, and methylglyoxal accelerate the formation of Tau oligomers, thioflavin T-positive aggregates, and fibrils using wild-type and seven pseudophosphorylated mutant Tau proteins. Acrolein and methylglyoxal were the most reactive compounds followed by glyoxal and malondialdehyde in terms of formation of Tau dimers and higher molecular weight oligomers. Furthermore, acrolein and methylglyoxal induced the formation of thioflavin T-fluorescent aggregates in a triple pseudophosphorylation-mimicking mutant to a slightly higher degree than wild-type Tau. Analysis of the Tau aggregates by electron microscopy study showed that formation of fibrils using wild-type Tau and several Tau mutants could be observed with acrolein and methylglyoxal but not with glyoxal and malondialdehyde. Our results suggest that reactive carbonyl compounds, particularly methylglyoxal and acrolein, could accelerate tangle formation in vivo and that this process could be slightly accelerated, at least in the case of methylglyoxal and acrolein, by hyperphosphorylation. Interference with the formation or the reaction of these reactive carbonyl compounds could be a promising way of inhibiting tangle formation and neuronal dysfunction in Alzheimer disease and other tauopathies.     

Mapping the interactions between a RUN domain from DENND5/Rab6IP1 and sorting nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.     

Listening to Puns Elicits the Co-Activation of Alternative Homophone Meanings during Language Production

Recent evidence suggests that lexical-semantic activation spread during language production can be dynamically shaped by contextual factors. In this study we investigated whether semantic processing modes can also affect lexical-semantic activation during word production. Specifically, we tested whether the processing of linguistic ambiguities, presented in the form of puns, has an influence on the co-activation of unrelated meanings of homophones in a subsequent language production task. In a picture-word interference paradigm with word distractors that were semantically related or unrelated to the non-depicted meanings of homophones we found facilitation induced by related words only when participants listened to puns before object naming, but not when they heard jokes with unambiguous linguistic stimuli. This finding suggests that a semantic processing mode of ambiguity perception can induce the co-activation of alternative homophone meanings during speech planning.     

Climate‐driven shifts in the distribution of koala‐browse species from the Last Interglacial to the near future

The koala's Phascolarctos cinereus distribution is currently restricted to eastern and south‐eastern Australia. However, fossil records dating from 70 ± 4 ka (ka = 10³ yr) from south‐western Australia and the Nullarbor Plain are evidence of subpopulation extinctions in the southwest at least after the Last Interglacial (~128–116 ka). We hypothesize that koala sub‐population extinctions resulted from the eastward retraction of the koala's main browse species in response to unsuitable climatic conditions. We further posit a general reduction in the distribution of main koala‐browse trees in the near future in response climate change. We modelled 60 koala‐browse species and constructed a set of correlative species distribution models for five time periods: Last Interglacial (~128–116 ka), Last Glacial Maximum (~23–19 ka), Mid‐Holocene (~7–5 ka), present (interpolations of observed data, representative of 1960–1990), and 2070. We based our projections on five hindcasts and one forecast of climatic variables extracted from WorldClim based on two general circulation models (considering the most pessimistic scenario of high greenhouse‐gas emissions) and topsoil clay fraction. We used 17 dates of koala fossil specimens identified as reliable from 70 (± 4) to 535 (± 49) ka, with the last appearance of koalas at 70 ka in the southwest. The main simulated koala‐browse species were at their greatest modelled extent of suitability during the Last Glacial Maximum, with the greatest loss of koala habitat occurring between the Mid‐Holocene and the present. We predict a similar habitat loss between the present and 2070. The spatial patterns of habitat change support our hypothesis that koala extinctions in the southwest, Nullarbor Plain and central South Australia resulted from the eastward retraction of the dominant koala‐browse species in response to long‐term climate changes. Future climate patterns will likely increase the extinction risk of koalas in their remaining eastern ranges.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

Screening drug effects in patient‐derived cancer cells links organoid responses to genome alterations

Cancer drug screening in patient‐derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix‐dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy‐based assay to resolve drug‐induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug‐induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.     

A modified dinucleotide motif specifies tRNA recognition by TLR7

RNA can function as a pathogen-associated molecular pattern (PAMP) whose recognition by the innate immune system alerts the body to an impending microbial infection. The recognition of tRNA as either self or nonself RNA by TLR7 depends on its modification patterns. In particular, it is known that the presence of a ribose methylated guanosine at position 18, which is overrepresented in self-RNA, antagonizes an immune response. Here, we report that recognition extends to the next downstream nucleotide and the effectively recognized molecular detail is actually a methylated dinucleotide. The most efficient nucleobases combination of this motif includes two purines, while pyrimidines diminish the effect of ribose methylation. The constraints of this motif stay intact when transposed to other parts of the tRNA. The results argue against a fixed orientation of the tRNA during interaction with TLR7 and, rather, suggest a processive type of inspection.     

Multiple Stressors in Agricultural Streams: A Mesocosm Study of Interactions among Raised Water Temperature,Sediment Addition and Nutrient Enrichment

Changes to land use affect streams through nutrient enrichment, increased inputs of sediment and, where riparian vegetation has been removed, raised water temperature. We manipulated all three stressors in experimental streamside channels for 30 days and determined the individual and pair-wise combined effects on benthic invertebrate and algal communities and on leaf decay, a measure of ecosystem functioning. We added nutrients (phosphorus+nitrogen; high, intermediate, natural) and/or sediment (grain size 0.2 mm; high, intermediate, natural) to 18 channels supplied with water from a nearby stream. Temperature was increased by 1.4°C in half the channels, simulating the loss of upstream and adjacent riparian shade. Sediment affected 93% of all biological response variables (either as an individual effect or via an interaction with another stressor) generally in a negative manner, while nutrient enrichment affected 59% (mostly positive) and raised temperature 59% (mostly positive). More of the algal components of the community responded to stressors acting individually than did invertebrate components, whereas pair-wise stressor interactions were more common in the invertebrate community. Stressors interacted often and in a complex manner, with interactions between sediment and temperature most common. Thus, the negative impact of high sediment on taxon richness of both algae and invertebrates was stronger at raised temperature, further reducing biodiversity. In addition, the decay rate of leaf material (strength loss) accelerated with nutrient enrichment at ambient but not at raised temperature. A key implication of our findings for resource managers is that the removal of riparian shading from streams already subjected to high sediment inputs, or land-use changes that increase erosion or nutrient runoff in a landscape without riparian buffers, may have unexpected effects on stream health. We highlight the likely importance of intact or restored buffer strips, both in reducing sediment input and in maintaining cooler water temperatures.     

Development,characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Large‐scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2‐Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large‐scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N‐glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large‐scale bioreactors.