Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Diversity and ecology of algae from the Nahal Qishon river,northern Israel

In 59 samples of periphyton and phytoplankton collected in 2002 - 2003 from the Nahal Qishon (Qishon River), northern Israel, we found 178 species from seven divisions of algae and cyanoprocaryotes. Diatoms, clorophytes, and cyanoprocaryotes prevail. Nitzschia and Navicula (Bacillariophyta) are the most abundant. Most of the species are cosmopolitan or widespread, except Lagynion janei (Chrysophyta), which is endemic for the Mediterranean Realm. About 17% of species (26) are new for Israel and five of them represent the first recorded genera: Crinalium endophyticum Crow, Actinocyclus normanii (Gregory) Hustedt, Rhizoclonium hieroglyphicum (Agardh) Kütz (Chlorophyta), Lagynion janei Bourelly, and Stylococcus aureus Chodat. Most of them come from a rare riverine assemblage with red alga Audouinella pygmea, as well as from the estuarine assemblage. Alkaliphiles predominate among the indicators of acidity, with few acidophiles confined to the communities under the impact of industrial wastes. Among the indicators of salinity, most numerous are the oligohalobien-indifferents and species adapted to a moderate salinity level. The relative species richness of ecological groups and the indices of saprobity are correlated with changes in conductivity, pH, and N-nitrate concentration. Indicators of organic pollution fall in the range of betameso- to alfamesosaprobic self-purification grades. Our studies show ecological significance of the Nahal Qishon as a model for a strongly disturbed aquatic ecosystem in the coastal zone of eastern Mediterranean.     

Crystal structures of Trichoderma reesei β-galactosidase reveal conformational changes in the active site

We have determined the crystal structure of Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal) at a 1.2? resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4? resolutions, respectively. Tr-β-gal is a potential enzyme for lactose hydrolysis in the dairy industry and belongs to family 35 of the glycoside hydrolases (GH-35). The high resolution crystal structures of this six-domain enzyme revealed interesting features about the structure of Tr-β-gal. We discovered conformational changes in the two loop regions in the active site, implicating a conformational selection-mechanism for the enzyme. In addition, the Glu200, an acid/base catalyst showed two different conformations which undoubtedly affect the pK(a) value of this residue and the catalytic mechanism. The electron density showed extensive glycosylation, suggesting a structure stabilizing role for glycans. The longest glycan showed an electron density that extends to the eighth monosaccharide unit in the extended chain. The Tr-β-gal structure also showed a well-ordered structure for a unique octaserine motif on the surface loop of the fifth domain.     

Impact of beta-glucan on the faecal microbiota of polypectomized patients: a pilot study

Beta-glucans are polysaccharides present in the cell walls of higher plants, in the seeds of some cereals, and certain yeasts and fungi also produce them. It is suggested that they exhibit, among many other health benefits, protective effects against carcinogenesis in the colon, but there is not enough human data to support this. The aim of the study was to determine the effect of barley-derived beta-glucan in the gut microbiota of polypectomized patients. Subjects were randomly assigned to consume 125 g of bread per day with beta-glucan (3 g/d), or without (placebo group), for 3 months. Thirty-three polypectomized men and women (mean age 57.6 years) were recruited into the study, but only 20 completed. Subjects did not consume any probiotics, prebiotics or antibiotics 2 months prior the intervention, or during the study. Stool samples were collected at baseline, on days 30 and 90 of intervention, as well as 2 weeks after the intervention, for enumeration of total aerobes and anaerobes, coliforms, E. coli, enterococci, Bacteroides spp., Clostridium perfringens, bifidobacteria, lactobacilli and Candida spp. Faecal bacterial enzyme activity (beta-glucuronidase and beta-glucosidase), pH, faecal moisture and the concentration of volatile fatty acids in the faeces were measured. Gastrointestinal symptoms were also recorded. Overall, no significant differences were observed in bacterial viable counts between the two feeding groups. Group specific analysis for β-glucan group revealed significantly decreased total coliform counts on the 30th day of the trial compared to the baseline (p = 0.041). Clostridium perfringens concentration increased without reaching statistical significance, on the 30th day, while it decreased significantly on the 90th day of the intervention compared to the 30th day (p = 0.016). An increase was noted in the molar ratio of acetate on the 90th day of the trial compared to placebo (p = 0.018). The molar ratio of butyrate presented a trend to increase on the 30th day, which decreased (p = 0.013) on the 90th day and then increase 2 weeks after the intervention (p = 0.017) compared to placebo. A decrease was recorded in the β-glucan group in the bloating and abdominal pain score after the 30th day of the intervention (Day 30–37) compared to placebo. During β-glucan administration we did not observe any changes on beta-glucuronidase or beta-glucosidase activity, faecal pH, or on faecal moisture.     

Life-cycle climate impacts of peat fuel: calculation methods and methodological challenges

Purpose

There has been lively debate, especially in Finland and Sweden, on the climate impacts of peat fuel. Previous studies of peat fuel's life-cycle climate impacts were controversial in their interpretation. The aim of this paper is conclusive examination of the issues of LCA methodology, derived from critical review of previous studies and recalculation based on the latest knowledge of greenhouse gas balances related to peat fuel’s utilisation and the radiative forcing impacts of greenhouse gases.

Methods

The most recent findings on emissions and the gas fluxes between soil, vegetation and atmosphere were used in calculation of the life-cycle climate impacts of the various peat fuel utilisation chains by means of LCA methodology. In the main, the calculation methods and rules were the same as in the previous studies, with the aim being to distinguish the impact of peat fuel’s utilisation from that of the natural or semi-natural situation. A dynamic method was employed for assessing changes in radiative forcing. The results of alternative peat fuel utilisation chains were compared to the corresponding result for coal.

Results

There are many steps in peat fuel LCA, where different assumptions lead to different outcomes. Determining the functional unit, reference situations and system boundaries, as well as the emission calculation methods, is important from this point of view. Determination of the initial reference situation emerged as one of the critical points in the calculations. Time scale can strongly affect the final outcomes in a study where effects of long-term land-use change are considered.

Conclusions

Each peatland area is unique. The higher the greenhouse gas emissions in the initial reference situation, the greater is the climate impact of the area and the more suitable the area is for peat extraction. The study showed that more greenhouse gas flux measurements are needed, for better assessment of the climate impacts of different potential peat extraction sites. Climate change mitigation requires quick actions, and uncertainties related to emissions are higher for longer time spans. Therefore, it can be concluded that a perspective spanning more than 100 years is inappropriate in peat fuel's life-cycle climate impact assessments.     

Carbon storage change and δ13C transitions of peat columns in a partially forestry-drained boreal bog

Background and aims

In forestry-drained peatlands, drying leads to changes in C cycling which could affect peat δ¹³C. Furthermore, the δ¹³C profile of the entire peat column may reveal effects of earlier climatic periods.

Methods

We measured peat δ¹³C and C inventories in adjacent peat profiles, two collected from undrained and two from the drained side of a bog that was partially ditch-drained 37 years earlier. The cores were sliced into 10-cm subsamples for analyses; matching of the profiles based on surface levelling, peat stratigraphic correlation and a horizontal ash layer found in both profiles.

Results

Surface subsidence of 30 cm was observed in the dried site and the uppermost 160 cm in the undrained site contained an excess of 5.9 kg m⁻² of C compared with the corresponding strata of the ditch-drained site. The δ¹³C values increased but markedly only in the thin surface layer of the drained site, indicating low δ¹³C of the missing C (ca. –30‰). In the deeper strata, dating to Mid-Holocene, high dry bulk density, C%, N%, humification index and low C/N ratio were connected to low δ¹³C of peat.

Conclusions

Drainage of 37 years increased δ¹³C values in the upper peat profile of the drained bog and led to the selective loss of ¹³C depleted C. Results indicate that C balance studies can be aided by C isotope analyses. Low δ¹³C values in the peat profile indicate the existence of a wet fen stage during the moist and warm period during Mid-Holocene.

     

Germline loss‐of‐function variants in MBD4 are rare in Finnish patients with uveal melanoma

Uveal melanoma (UM) is a rare intraocular cancer with the highest incidence in northern latitudes. Metastases develop in approximately 50% of patients, whereafter the median survival is 13 months. Generally, the mutation burden of these tumors is low. Germline variants predisposing to UM have been previously described in BRCA1‐associated protein 1 (BAP1). Recently, germline and somatic loss‐of‐function (LOF) variants in the methyl‐CpG‐binding domain 4 (MBD4) gene have been found to cause a hypermutated UM, and MBD4 also has been put forward as a gene predisposing to UM. We sequenced for MBD4 germline variants in 440 Finnish patients with UM and identified seven rare exonic missense variants in 16 (3.6%) patients, of which one likely alters MBD4 function. The frequency of likely pathogenic variants in our cohort is 0.23% (1/432; 95% CI, 0.01–1.28). We identified no LOF variants though their frequency in the Finnish population is 0.052%. Thus, our data do not support the suggestion that MBD4 germline variants predispose to UM. Somatic loss of MBD4 might modify the mutational burden in UM and change its response to immune checkpoint inhibitors.     

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer-specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR-binding sites (ARBs) on LNCaP-1F5 cell chromatin that was commensurate with changes in androgen-dependent gene expression signature. We identified three distinct classes of ARBs and androgen-responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid-dependent signalling in LNCaP-1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.     

Different response of two reindeer forage plants to enhanced UV-B radiation: modification of the phenolic composition

The long-term effects of enhanced UV-B radiation on the content and composition of leaf phenolics in Epilobium angustifolium L. and Eriophorum russeolum Fries ex Hartman were studied in northern Finland (68°N) using two UV-B enhancement experiments, both simulating UV-B_CIE radiation and corresponding to a 20% loss of ozone layer. High proportions of hydrolyzable tannins (69%) and condensed tannins (66%) characterized both Epilobium and Eriophorum leaves, respectively. No UV treatment effect was detected in the content or composition of Epilobium leaf soluble phenolics, whereas significant UV effects were detected in Eriophorum leaves in a developmental-specific manner. At the end of the growing season, the proportion of total soluble phenolics was higher in leaves exposed to enhanced UV-A and UV-B radiation than in the control leaves, but the phenolic composition was not significantly modified. This study introduces a new example on plants’ phenolic response to UV radiation being species-specific and detectable only at certain developmental stages. Possible consequences of increased phenolic content in forage plants for selection and digestibility by reindeer are, however, not yet known.