Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration

Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.     

Above- and belowground fluxes of methane from boreal dwarf shrubs and Pinus sylvestris seedlings

Plant and Soil - Legume break crops provide a series of agronomic benefits to the following wheat crop in a rotation. Phosphorus-efficient break-crop plants can mobilise P from non-labile pools in...     

CRISPR/Cas9 screening using unique molecular identifiers

Loss‐of‐function screening by CRISPR/Cas9 gene knockout with pooled, lentiviral guide libraries is a widely applicable method for systematic identification of genes contributing to diverse cellular phenotypes. Here, Random Sequence Labels (RSLs) are incorporated into the guide library, which act as unique molecular identifiers (UMIs) to allow massively parallel lineage tracing and lineage dropout screening. RSLs greatly improve the reproducibility of results by increasing both the precision and the accuracy of screens. They reduce the number of cells needed to reach a set statistical power, or allow a more robust screen using the same number of cells.     

Carbon balance and allocation of assimilated CO2 in Scots pine, Norway spruce, and Silver birch seedlings determined with gas exchange measurements and 14C pulse labelling

Carbon dioxide is released from the soil to the atmosphere in heterotrophic respiration when the dead organic matter is used for substrates for soil micro-organisms and soil animals. Respiration of roots and mycorrhiza is another major source of carbon dioxide in soil CO₂ efflux. The partitioning of these two fluxes is essential for understanding the carbon balance of forest ecosystems and for modelling the carbon cycle within these ecosystems. In this study, we determined the carbon balance of three common tree species in boreal forest zone, Scots pine, Norway spruce, and Silver birch with gas exchange measurements conducted in laboratory in controlled temperature and light conditions. We also studied the allocation pattern of assimilated carbon with ¹⁴C pulse labelling experiment. The photosynthetic light responses of the tree species were substantially different. The maximum photosynthetic capacity (P _max) was 2.21 μg CO₂ s⁻¹ g⁻¹ in Scots pine, 1.22 μg CO₂ s⁻¹ g⁻¹ in Norway spruce and 3.01 μg CO₂ s⁻¹ g⁻¹ in Silver birch seedlings. According to the pulse labelling experiments, 43–75% of the assimilated carbon remained in the aboveground parts of the seedlings. The amount of carbon allocated to root and rhizosphere respiration was about 9–26%, and the amount of carbon allocated to root and ectomycorrhizal biomass about 13–21% of the total assimilated CO₂. The ¹⁴CO₂ pulse reached the root system within few hours after the labelling and most of the pulse had passed the root system after 48 h. The transport rate of carbon from shoot to roots was fastest in Silver birch seedlings.     

Phe27Cys Polymorphism Alters the Maturation and Subcellular Localization of the Human δ Opioid Receptor

The human δ opioid receptor (hδOR) is a G-protein-coupled receptor that is mainly involved in the modulation of pain and mood. Only one nonsynonymous single nucleotide polymorphism (T80G) has been described, causing Phe27Cys substitution in the receptor N-terminus and showing association with substance dependence. In this study, we expressed the two hδOR variants in a heterologous expression system with an identical genetic background. They differed greatly during early steps of biosynthesis, displaying a significant difference in the maturation efficiency (50% and 85% for the Cys27 and Phe27 variants, respectively). The Cys27 variant also showed accumulation in pre-Golgi compartments of the secretory pathway and impaired targeting to endoplasmic reticulum (ER)-associated degradation following long-term expression. In addition, the cell surface receptors of the Cys27 variant internalized constitutively. Replacement of phenylalanine with other amino acids revealed that cysteine at position 27 decreased the mature receptor/precursor ratio most extensively, suggesting a thiol-mediated retention of precursors in the ER. However, cysteine did not cause a major folding defect because pharmacological characteristics and the maturation kinetics of the variants were identical, and an opioid antagonist was able to enhance the maturation of both variants. We conclude that, instead of causing loss of function, Phe27Cys polymorphism of the hδOR causes a gain-of-function phenotype, which may have implications for the regulation of receptor expression at the cell surface and possibly also for the susceptibility to pathophysiological states.     

Tricholoma matsutake dominates diverse microbial communities in different forest soils

Fungal and actinobacterial communities were analyzed together with soil chemistry and enzyme activities in order to profile the microbial diversity associated with the economically important mushroom Tricholoma matsutake. Samples of mycelium-soil aggregation (shiro) were collected from three experimental sites where sporocarps naturally formed. PCR was used to confirm the presence and absence of matsutake in soil samples. PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting and direct sequencing were used to identify fungi and actinobacteria in the mineral and organic soil layers separately. Soil enzyme activities and hemicellulotic carbohydrates were analyzed in a productive experimental site. Soil chemistry was investigated in both organic and mineral soil layers at all three experimental sites. Matsutake dominated in the shiro but also coexisted with a high diversity of fungi and actinobacteria. Tomentollopsis sp. in the organic layer above the shiro and Piloderma sp. in the shiro correlated positively with the presence of T. matsutake in all experimental sites. A Thermomonosporaceae bacterium and Nocardia sp. correlated positively with the presence of T. matsutake, and Streptomyces sp. was a common cohabitant in the shiro, although these operational taxonomic units (OTUs) did not occur at all sites. Significantly higher enzyme activity levels were detected in shiro soil. These enzymes are involved in the mobilization of carbon from organic matter decomposition. Matsutake was not associated with a particular soil chemistry compared to that of nearby sites where the fungus does not occur. The presence of a significant hemicellulose pool and the enzymes to degrade it indicates the potential for obtaining carbon from the soil rather than tree roots.     

Archaeal Communities in Boreal Forest Tree Rhizospheres Respond to Changing Soil Temperatures

Temperature has generally great effects on both the activity and composition of microbial communities in different soils. We tested the impact of soil temperature and three different boreal forest tree species on the archaeal populations in the bulk soil, rhizosphere, and mycorrhizosphere. Scots pine, silver birch, and Norway spruce seedlings were grown in forest humus microcosms at three different temperatures, 7–11.5°C (night–day temperature), 12–16°C, and 16–22°C, of which 12–16°C represents the typical mid-summer soil temperature in Finnish forests. RNA and DNA were extracted from indigenous ectomycorrhiza, non-mycorrhizal long roots, and boreal forest humus and tested for the presence of archaea by nested PCR of the archaeal 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing. Methanogenic Euryarchaeota belonging to Methanolobus sp. and Methanosaeta sp. were detected on the roots and mycorrhiza. The most commonly detected archaeal 16S rRNA gene sequences belonged to group I.1c Crenarchaeota, which are typically found in boreal and alpine forest soils. Interestingly, also one sequence belonging to group I.1b Crenarchaeota was detected from Scots pine mycorrhiza although sequences of this group are usually found in agricultural and forest soils in temperate areas. Tree- and temperature-related shifts in the archaeal population structure were observed. A clear decrease in crenarchaeotal DGGE band number was seen with increasing temperature, and correspondingly, the number of euryarchaeotal DGGE bands, mostly methanogens, increased. The greatest diversity of archaeal DGGE bands was detected in Scots pine roots and mycorrhizas. No archaea were detected from humus samples from microcosms without tree seedling, indicating that the archaea found in the mycorrhizosphere and root systems were dependent on the plant host. The detection of archaeal 16S rRNA gene sequences from both RNA and DNA extractions show that the archaeal populations were living and that they may have significant contribution to the methane cycle in boreal forest soil, especially when soil temperatures rise.     

Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low‐cost purification by surfactant‐based aqueous two‐phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY‐2) suspension cell platform and the establishment of pilot‐scale propagation and downstream processing including first‐step purification by ATPS. Green fluorescent protein‐hydrophobin fusion (GFP‐HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY‐2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP‐HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large‐scale hydrophobin‐assisted production of recombinant proteins in tobacco BY‐2 cell suspensions.