mTOR kinase and the regulatory subunit of protein kinase A (PRKAR1A) spatially and functionally interact during autophagosome maturation

The regulatory subunit 1-alpha (RIalpha) of protein kinase A (PKA) and the mTOR kinase are involved in a common pathway regulating mammalian autophagy. RIalpha was found to localize on Rab7-positive late endosomes and on LC3-positive autophagosomal membranes in cultured cells. RIalpha was also shown to physically interact with mTOR kinase and affect its phosphorylation and activity. In this addendum, we further explore the subcellular distribution of mTOR related to RIalpha and LC3. We present experiments showing that mTOR colocalizes with RIalpha-, Rab7- and LC3-positive membranes in cultured cells. Because RIalpha regulates the phosphorylation and activity of mTOR kinase, which we now show localizes on autophagosomal membranes, the possibility emerges that the RIalpha-mTOR complex acts at the level of autophagosome maturation.     

Process design and optimization of novel wheat-based continuous bioethanol production system

A novel design of a wheat-based biorefinery for bioethanol production, including wheat milling, gluten extraction as byproduct, fungal submerged fermentation for enzyme production, starch hydrolysis, fungal biomass autolysis for nutrient regeneration, yeast fermentation with recycling integrated with a pervaporation membrane for ethanol concentration, and fuel-grade ethanol purification by pressure swing distillation (PSD), was optimized in continuous mode using the equation-based software General Algebraic Modelling System (GAMS). The novel wheat biorefining strategy could result in a production cost within the range of dollars 0.96-0.50 gal(-1) ethanol (dollars 0.25-0.13 L(-1) ethanol) when the production capacity of the plant is within the range of 10-33.5 million gal y(-1) (37.85-126.8 million L y(-1)). The production of value-added byproducts (e.g., bran-rich pearlings, gluten, pure yeast cells) was identified as a crucial factor for improving the economics of fuel ethanol production from wheat. Integration of yeast fermentation with pervaporation membrane could result in the concentration of ethanol in the fermentation outlet stream (up to 40 mol %). The application of a PSD system that consisted of a low-pressure and a high-pressure column and employing heat integration between the high- and low-pressure columns resulted in reduced operating cost (up to 44%) for fuel-grade ethanol production.     

Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA-U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N-terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS-PAGE, and this aberrant migration was thought to result from post-translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafTlU genes resulted in non-motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.     

Physical mapping of the human T-cell receptor beta gene complex,using yeast artificial chromosomes

Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell chain gene complex (TCRB). Variable region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identified, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes (BC) when analyzed by pulse field gel electrophoresis. Restriction enzyme mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragments of 280 110, 90, and 125 kbp and was in accordance with published data. In addition comparison of hybridization results of Sfi I-restricted B3 and genomic DNA from the parental cell line GM1416B revealed identical banding patterns. The data thus showed YAC B3 encoded a complete and unrearranged TCRB gene locus of some 600–620 kbp. The map was further resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene families. Flourescent in situ hybridization of B3 to spreads of human metaphase chromosomes localized B3 to 7q35. However, two additional signals were obtained; one attributable to the TCRBV orphon cluster on 9p21, the second to the long arm of chromosome 2. Polymerase chain reaction amplification of a chromosome 2 somatic cell hybrid, using primers for all 25 TCRBV gene families, revealed that the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCRB locus flanked by chromosome 2 sequences.     

Impact of <Emphasis Type="Italic">Populus</Emphasis> Plantations on Water and Soil Quality

Trees of genus Populus (in our context primarily poplars) are predominantly grown in Sweden in small plantations on arable land in southern and central parts of the country to produce biomass for energy and other purposes. This study evaluated the effects (i) of poplar plantations on groundwater quality, by determining differences in leaching of nitrogen and phosphorus to groundwater, and (ii) of poplar and hybrid aspen plantations on soil quality in terms of carbon in the top- and subsoil. The study was conducted comparing Populus plantations in Sweden with adjacent fields with cereals and grasslands. The experiment concerning the groundwater leaching was conducted in eight poplar plantations along three growing seasons (2012–2015). For the soil carbon experiments, 19 poplar and two hybrid aspen plantations and the respective reference fields were sampled. NO₃-N leaching from poplar plantations was significantly lower than that from reference fields with cereals, but not when compared with grasslands. Spring NO₃-N leaching was significantly lower in poplars than in the reference fields, whereas leaching of NO₃-N in autumn did not differ. Concentrations of PO₄-P in the groundwater of poplar plantations were lower compared to the respective ones of the reference fields. There were no clear trends observed when comparing carbon concentrations in the topsoil of the poplar and hybrid aspen plantations compared to the respective adjacent reference fields. For the subsoil, the average carbon concentrations in the poplar and hybrid aspen plantations were equal to the respective ones of cereals, but were higher when compared to grassland.     

The role of bioenergy and biochemicals in CO2 mitigation through the energy system – a scenario analysis for the Netherlands

Bioenergy as well as bioenergy with carbon capture and storage are key options to embark on cost‐efficient trajectories that realize climate targets. Most studies have not yet assessed the influence on these trajectories of emerging bioeconomy sectors such as biochemicals and renewable jet fuels (RJFs). To support a systems transition, there is also need to demonstrate the impact on the energy system of technology development, biomass and fossil fuel prices. We aim to close this gap by assessing least‐cost pathways to 2030 for a number of scenarios applied to the energy system of the Netherlands, using a cost‐minimization model. The type and magnitude of biomass deployment are highly influenced by technology development, fossil fuel prices and ambitions to mitigate climate change. Across all scenarios, biomass consumption ranges between 180 and 760 PJ and national emissions between 82 and 178 Mt CO₂. High technology development leads to additional 100–270 PJ of biomass consumption and 8–20 Mt CO₂ emission reduction compared to low technology development counterparts. In high technology development scenarios, additional emission reduction is primarily achieved by bioenergy and carbon capture and storage. Traditional sectors, namely industrial biomass heat and biofuels, supply 61–87% of bioenergy, while wind turbines are the main supplier of renewable electricity. Low technology pathways show lower biochemical output by 50–75%, do not supply RJFs and do not utilize additional biomass compared to high technology development. In most scenarios the emission reduction targets for the Netherlands are not met, as additional reduction of 10–45 Mt CO₂ is needed. Stronger climate policy is required, especially in view of fluctuating fossil fuel prices, which are shown to be a key determinant of bioeconomy development. Nonetheless, high technology development is a no‐regrets option to realize deep emission reduction as it also ensures stable growth for the bioeconomy even under unfavourable conditions.