Multiple factors influence substrate oxidation during exercise including exercise duration and intensity, sex, and dietary intake before and during exercise. However, the relative influence and interaction between these factors is unclear.

The simple deletion of nucleotides is common in many organisms. It can be advantageous when it activates genes beneficial to microbial survival in adverse environments, and deleterious when it mutates genes relevant to survival, cancer or degenerative diseases. The classical idea is that simple deletions arise by strand slippage. A prime opportunity for slippage occurs during DNA synthesis, but it remains unclear how slippage is controlled during a polymerization cycle. Here, we report crystal structures and molecular dynamics simulations of mutant derivatives of DNA polymerase lambda bound to a primer-template during strand slippage. Relative to the primer strand, the template strand is in multiple conformations, indicating intermediates on the pathway to deletion mutagenesis. Consistent with these intermediates, the mutant polymerases generate single-base deletions at high rates. The results indicate that dNTP-induced template strand repositioning during conformational rearrangements in the catalytic cycle is crucial to controlling the rate of strand slippage.

It has previously been shown that pretreatment of differentiated human skeletal muscle cells (myotubes) with eicosapentaenoic acid (EPA) promoted increased uptake of fatty acids and increased triacylglycerol accumulation, compared to pretreatment with oleic acid (OA) and palmitic acid (PA). The aim of the present study was to examine whether EPA could affect substrate cycling in human skeletal muscle cells by altering lipolysis rate of intracellular TAG and re-esterification of fatty acids. Fatty acid metabolism was studied in human myotubes using a mixture of fatty acids, consisting of radiolabelled oleic acid as tracer (14C-OA) together with EPA or PA. Co-incubation of myotubes with EPA increased cell-accumulation and incomplete fatty acid oxidation of 14C-OA compared to co-incubation with PA. Lipid distribution showed higher incorporation of 14C-OA into all cellular lipids after co-incubation with EPA relative to PA, with most markedly increases (3 to 4-fold) for diacylglycerol and triacylglycerol. Further, the increases in cellular lipids after co-incubation with EPA were accompanied by higher lipolysis and fatty acid re-esterification rate. Correspondingly, basal respiration, proton leak and maximal respiration were significantly increased in cells exposed to EPA compared to PA. Microarray and Gene Ontology (GO) enrichment analysis showed that EPA, related to PA, significantly changed i.e. the GO terms "Neutral lipid metabolic process" and "Regulation of lipid storage". Finally, an inhibitor of diacylglycerol acyltransferase 1 decreased the effect of EPA to promote fatty acid accumulation. In conclusion, incubation of human myotubes with EPA, compared to PA, increased processes of fatty acid turnover and oxidation suggesting that EPA may activate futile substrate cycling of fatty acids in human myotubes. Increased TAG-FA cycling may be involved in the potentially favourable effects of long-chain polyunsaturated n-3 fatty acids on skeletal muscle and whole-body energy metabolism.

Creatine serves as an ATP buffer and is thus an integral component of cellular energy metabolism. Most cells maintain their creatine levels via uptake by the creatine transporter (CRT-1, SLC6A8). The activity of CRT-1, therefore, is a major determinant of cytosolic creatine concentrations. We determined the kinetics of CRT-1 in real time by relying on electrophysiological recordings of transport-associated currents. Our analysis revealed that CRT-1 harvested the concentration gradient of NaCl and the membrane potential but not the potassium gradient to achieve a very high concentrative power. We investigated the mechanistic basis for the ability of CRT-1 to maintain the forward cycling mode in spite of high intracellular concentrations of creatine: this is achieved by cooperative binding of substrate and co-substrate ions, which, under physiological ion conditions, results in a very pronounced (i.e. about 500-fold) drop in the affinity of creatine to the inward-facing state of CRT-1. Kinetic estimates were integrated into a mathematical model of the transport cycle of CRT-1, which faithfully reproduced all experimental data. We interrogated the kinetic model to examine the most plausible mechanistic basis of cooperativity: based on this systematic exploration, we conclude that destabilization of binary rather than ternary complexes is necessary for CRT-1 to maintain the observed cytosolic creatine concentrations. Our model also provides a plausible explanation why neurons, heart and skeletal muscle cells must express a creatine releasing transporter to achieve rapid equilibration of the intracellular creatine pool.

The mitochondrial Lon protease (LonP1) regulates mitochondrial health by removing redundant proteins from the mitochondrial matrix. We determined LonP1 in eight nucleotide-dependent conformational states by cryoelectron microscopy (cryo-EM). The flexible assembly of N-terminal domains had 3-fold symmetry, and its orientation depended on the conformational state. We show that a conserved structural motif around T803 with a high similarity to the trypsin catalytic triad is essential for proteolysis. We show that LonP1 is not regulated by redox potential, despite the presence of two conserved cysteines at disulfide-bonding distance in its unfoldase core. Our data indicate how sequential ATP hydrolysis controls substrate protein translocation in a 6-fold binding change mechanism. Substrate protein translocation, rather than ATP hydrolysis, is a rate-limiting step, suggesting that LonP1 is a Brownian ratchet with ATP hydrolysis preventing translocation reversal. 3-fold rocking motions of the flexible N-domain assembly may assist thermal unfolding of the substrate protein.

Methanethiol (MeSH) and dimethylsulfide (DMS) are volatile organic sulfur compounds (VOSCs) with important roles in sulfur cycling, signaling and atmospheric chemistry. DMS can be produced from MeSH through a reaction mediated by the methyltransferase MddA. The mddA gene is present in terrestrial and marine metagenomes, being most abundant in soil environments. The substrate for MddA, MeSH, can also be oxidized by bacteria with the MeSH oxidase (MTO) enzyme, encoded by the mtoX gene, found in marine, freshwater and soil metagenomes. Methanethiol-dependent DMS production (Mdd) pathways have been shown to function in soil and marine sediments, but have not been characterized in detail in the latter environments. In addition, few molecular studies have been conducted on MeSH consumption in the environment. Here, we performed process measurements to confirm that Mdd-dependent and Mdd-independent MeSH consumption pathways are active in tested surface saltmarsh sediment when MeSH is available. We noted that appreciable natural Mdd-independent MeSH and DMS consumption processes masked Mdd activity. 16S rRNA gene amplicon sequencing and metagenomics data showed that Methylophaga, a bacterial genus known to catabolise DMS and MeSH, was enriched by the presence of MeSH. Moreover, some MeSH and/or DMS-degrading bacteria isolated from this marine environment lacked known DMS and/or MeSH cycling genes and can be used as model organisms to potentially identify novel genes in these pathways. Thus, we are likely vastly underestimating the abundance of MeSH and DMS degraders in these marine sediment environments. The future discovery and characterization of novel enzymes involved in MeSH and/or DMS cycling is essential to better assess the role and contribution of microbes to global organosulfur cycling.

Triglyceride cycling is the process of continuous degradation and re-synthesis of triglyceride in cellular stores. We show in 3T3-L1 adipocytes that triglycerides are subject to rapid turnover and re-arrangement of fatty acids with an estimated half-life of 2-4 h. We develop a tracing technology that can simultaneously and quantitatively follow the metabolism of multiple fatty acids to study the triglyceride futile substrate cycle directly and with molecular species resolution. Our approach is based on alkyne fatty acid tracers and mass spectrometry. The triglyceride cycling is connected to modification of released fatty acids by elongation and desaturation. Through cycling and modification, saturated fatty acids are slowly converted to monounsaturated fatty acids, and linoleic acid to arachidonic acid. We conclude that triglyceride cycling renders stored fatty acids accessible for metabolic alteration. The overall process facilitates cellular adjustments to the stored fatty acid pool to meet changing needs of the cell.

Interferons (IFNs) are essential in antiviral defense, antitumor effects, and immunoregulatory activities. Although methionine oxidation is associated with various physiological and pathophysiological processes in plants, animals, and humans, its role in immunity remains unclear. We find that the redox cycling of signal transducer and activator of transcription 2 (STAT2) is an intrinsic cellular biological process, and that impairment of the redox status contributes to STAT2 methionine oxidation, inhibiting its activation. IFN protects STAT2 from methionine oxidation through the recruitment of methionine sulfoxide reductase MSRB2, whose enzymatic activity is enhanced by N-acetyltransferase 9 (NAT9), a chaperone of STAT2 defined in this study, upon IFN treatment. Consequently, loss of Nat9 renders mice more susceptible to viral infection. Our study highlights the key function of methionine oxidation in immunity, which provides evidence for the decline of immune function by aging and may provide insights into the clinical applications of IFN in immune-related diseases.

Urbanised environments have been identified as hotspots of anthropogenic methane emissions. Especially urban aquatic ecosystems are increasingly recognised as important sources of methane. However, the microbiology behind these emissions remains unexplored. Here, we applied microcosm incubations and molecular analyses to investigate the methane-cycling community of the Amsterdam canal system in the Netherlands. The sediment methanogenic communities were dominated by Methanoregulaceae and Methanosaetaceae, with co-occurring methanotrophic Methanoperedenaceae and Methylomirabilaceae indicating the potential for anaerobic methane oxidation. Methane was readily produced after substrate amendment, suggesting an active but substrate-limited methanogenic community. Bacterial 16S rRNA gene amplicon sequencing of the sediment revealed a high relative abundance of Thermodesulfovibrionia. Canal wall biofilms showed the highest initial methanotrophic potential under oxic conditions compared to the sediment. During prolonged incubations the maximum methanotrophic rate increased to 8.08 mmol gDW -1  d-1 that was concomitant with an enrichment of Methylomonadaceae bacteria. Metagenomic analysis of the canal wall biofilm lead to the recovery of a single methanotroph metagenome-assembled genome. Taxonomic analysis showed that this methanotroph belongs to the genus Methyloglobulus. Our results underline the importance of previously unidentified and specialised environmental niches at the nexus of the natural and human-impacted carbon cycle.

Isolated hepatocytes from young (4-6mo) and old (24-26mo) F344 rats were exposed to increasing concentrations of menadione, a vitamin K derivative and redox cycling agent, to determine whether the age-related decline in Nrf2-mediated detoxification defenses resulted in heightened susceptibility to xenobiotic insult. An LC50 for each age group was established, which showed that aging resulted in a nearly 2-fold increase in susceptibility to menadione (LC50 for young: 405μM; LC50 for old: 275μM). Examination of the known Nrf2-regulated pathways associated with menadione detoxification revealed, surprisingly, that NAD(P)H: quinone oxido-reductase 1 (NQO1) protein levels and activity were induced 9-fold and 4-fold with age, respectively (p=0.0019 and p=0.018; N=3), but glutathione peroxidase 4 (GPX4) declined by 70% (p=0.0043; N=3). These results indicate toxicity may stem from vulnerability to lipid peroxidation instead of inadequate reduction of menadione semi-quinone. Lipid peroxidation was 2-fold higher, and GSH declined by a 3-fold greater margin in old versus young rat cells given 300µM menadione (p<0.05 and p≤0.01 respectively; N=3). We therefore provided 400µMN-acetyl-cysteine (NAC) to hepatocytes from old rats before menadione exposure to alleviate limits in cysteine substrate availability for GSH synthesis during challenge. NAC pretreatment resulted in a >2-fold reduction in cell death, suggesting that the age-related increase in menadione susceptibility likely stems from attenuated GSH-dependent defenses. This data identifies cellular targets for intervention in order to limit age-related toxicological insults to menadione and potentially other redox cycling compounds.

Similar to plant growth, soil carbon (C) cycling is constrained by the availability of nitrogen (N) and phosphorus (P). We hypothesized that stoichiometric control over soil microbial C cycling may be shaped by functional guilds with distinct nutrient substrate preferences. Across a series of rice fields spanning 5-25% soil C (N:P from 1:12 to 1:70), C turnover was best correlated with P availability and increased with experimental N addition only in lower C (mineral) soils with N:P⩽16. Microbial community membership also varied with soil stoichiometry but not with N addition. Shotgun metagenome data revealed changes in community functions with increasing C turnover, including a shift from aromatic C to carbohydrate utilization accompanied by lower N uptake and P scavenging. Similar patterns of C, N and P acquisition, along with higher ribosomal RNA operon copy numbers, distinguished that microbial taxa positively correlated with C turnover. Considering such tradeoffs in genomic resource allocation patterns among taxa strengthened correlations between microbial community composition and C cycling, suggesting simplified guilds amenable to ecosystem modeling. Our results suggest that patterns of soil C turnover may reflect community-dependent metabolic shifts driven by resource allocation strategies, analogous to growth rate-stoichiometry coupling in animal and plant communities.

Syntrophic bacteria play a key role in the anaerobic conversion of biological matter to methane. They convert short-chain fatty acids or alcohols to H2, formate, and acetate that serve as substrates for methanogenic archaea. Many syntrophic bacteria can also grow with unsaturated fatty acids such as crotonate without a syntrophic partner, and the reducing equivalents derived from the oxidation of one crotonate to two acetate are regenerated by the reduction of a second crotonate. However, it has remained unresolved how the oxidative and reductive catabolic branches are interconnected and how energy may be conserved in the reductive branch. Here, we provide evidence that during axenic growth of the syntrophic model organism Syntrophus aciditrophicus with crotonate, the NAD+-dependent oxidation of 3-hydroxybutyryl-CoA to acetoacetyl-CoA is coupled to the reduction of crotonyl-CoA via formate cycling. In this process, the intracellular formate generated by a NAD+-regenerating CO2 reductase is taken up by a periplasmic, membrane-bound formate dehydrogenase that in concert with a membrane-bound electron-transferring flavoprotein (ETF):methylmenaquinone oxidoreductase, ETF, and an acyl-CoA dehydrogenase reduces intracellular enoyl-CoA to acyl-CoA. This novel type of energy metabolism, referred to as enoyl-CoA respiration, generates a proton motive force via a methylmenaquinone-dependent redox-loop. As a result, the beneficial syntrophic cooperation of fermenting bacteria and methanogenic archaea during growth with saturated fatty acids appears to turn into a competition for formate and/or H2 during growth with unsaturated fatty acids. IMPORTANCE The syntrophic interaction of fermenting bacteria and methanogenic archaea is important for the global carbon cycle. As an example, it accomplishes the conversion of biomass-derived saturated fatty acid fermentation intermediates into methane. In contrast, unsaturated fatty acid intermediates such as crotonate may serve as growth substrate for the fermenting partner alone. Thereby, the reducing equivalents generated during the oxidation of one crotonate to two acetate are regenerated by reduction of a second crotonate to butyrate. Here, we show that the oxidative and reductive branches of this pathway are connected via formate cycling involving an energy-conserving redox-loop. We refer to this previously unknown type of energy metabolism as to enoyl-CoA respiration with acyl-CoA dehydrogenases serving as cytoplasmic terminal reductases.

Soil carbon (C) storage is dependent upon the complex dynamics of fresh and native organic matter cycling, which are regulated by plant and soil-microbial activities. A fundamental challenge exists to link microbial biodiversity with plant-soil C cycling processes to elucidate the underlying mechanisms regulating soil carbon. To address this, we contrasted vegetated grassland soils with bare soils, which had been plant-free for 3 years, using stable isotope ((13)C) labeled substrate assays and molecular analyses of bacterial communities. Vegetated soils had higher C and N contents, biomass, and substrate-specific respiration rates. Conversely, following substrate addition unlabeled, native soil C cycling was accelerated in bare soil and retarded in vegetated soil; indicative of differential priming effects. Functional differences were reflected in bacterial biodiversity with Alphaproteobacteria and Acidobacteria dominating vegetated and bare soils, respectively. Significant isotopic enrichment of soil RNA was found after substrate addition and rates varied according to substrate type. However, assimilation was independent of plant presence which, in contrast to large differences in (13)CO2 respiration rates, indicated greater substrate C use efficiency in bare, Acidobacteria-dominated soils. Stable isotope probing (SIP) revealed most community members had utilized substrates with little evidence for competitive outgrowth of sub-populations. Our findings support theories on how plant-mediated soil resource availability affects the turnover of different pools of soil carbon, and we further identify a potential role of soil microbial biodiversity. Specifically we conclude that emerging theories on the life histories of dominant soil taxa can be invoked to explain changes in soil carbon cycling linked to resource availability, and that there is a strong case for considering microbial biodiversity in future studies investigating the turnover of different pools of soil carbon.

Green roofs provide ecosystem services through evapotranspiration and nutrient cycling that depend, among others, on plant species, substrate type, and substrate depth. However, no study has assessed thoroughly how interactions between these factors alter ecosystem functions and multifunctionality of green roofs. We simulated some green roof conditions in a pot experiment. We planted 20 plant species from 10 genera and five families (Asteraceae, Caryophyllaceae, Crassulaceae, Fabaceae, and Poaceae) on two substrate types (natural vs. artificial) and two substrate depths (10 cm vs. 30 cm). As indicators of major ecosystem functions, we measured aboveground and belowground biomasses, foliar nitrogen and carbon content, foliar transpiration, substrate water retention, and dissolved organic carbon and nitrates in leachates. Interactions between substrate type and depth strongly affected ecosystem functions. Biomass production was increased in the artificial substrate and deeper substrates, as was water retention in most cases. In contrast, dissolved organic carbon leaching was higher in the artificial substrates. Except for the Fabaceae species, nitrate leaching was reduced in deep, natural soils. The highest transpiration rates were associated with natural soils. All functions were modulated by plant families or species. Plant effects differed according to the observed function and the type and depth of the substrate. Fabaceae species grown on natural soils had the most noticeable patterns, allowing high biomass production and high water retention but also high nitrate leaching from deep pots. No single combination of factors enhanced simultaneously all studied ecosystem functions, highlighting that soil-plant interactions induce trade-offs between ecosystem functions. Substrate type and depth interactions are major drivers for green roof multifunctionality.

Dimethyl sulfide (DMS) is the dominant volatile organic sulfur in global oceans. The predominant source of oceanic DMS is the cleavage of dimethylsulfoniopropionate (DMSP), which can be produced by marine bacteria and phytoplankton. Polar oceans, which represent about one fifth of Earth's surface, contribute significantly to the global oceanic DMS sea-air flux. However, a global overview of DMS and DMSP cycling in polar oceans is still lacking and the key genes and the microbial assemblages involved in DMSP/DMS transformation remain to be fully unveiled.

The phenomena of synchronization, rhythmogenesis and coherence observed in brain networks are believed to be a dynamic substrate for cognitive functions such as learning and memory. However, researchers are still debating whether the rhythmic activity emerges from the network morphology that developed during neurogenesis or as a result of neuronal dynamics achieved under certain conditions. In the present study, we observed self-organized spiking activity that converged to long, complex and rhythmically repeated superbursts in neural networks formed by mature hippocampal cultures with a high cellular density. The superburst lasted for tens of seconds and consisted of hundreds of short (50-100 ms) small bursts with a high spiking rate of 139.0 ± 78.6 Hz that is associated with high-frequency oscillations in the hippocampus. In turn, the bursting frequency represents a theta rhythm (11.2 ± 1.5 Hz). The distribution of spikes within the bursts was non-random, representing a set of well-defined spatio-temporal base patterns or motifs. The long superburst was classified into two types. Each type was associated with a unique direction of spike propagation and, hence, was encoded by a binary sequence with random switching between the two "functional" states. The precisely structured bidirectional rhythmic activity that developed in self-organizing cultured networks was quite similar to the activity observed in the in vivo experiments.

Geothermal environments, such as hot springs and hydrothermal vents, are hotspots for carbon cycling and contain many poorly described microbial taxa. Here, we reconstructed 15 archaeal metagenome-assembled genomes (MAGs) from terrestrial hot spring sediments in China and deep-sea hydrothermal vent sediments in Guaymas Basin, Gulf of California. Phylogenetic analyses of these MAGs indicate that they form a distinct group within the TACK superphylum, and thus we propose their classification as a new phylum, 'Brockarchaeota', named after Thomas Brock for his seminal research in hot springs. Based on the MAG sequence information, we infer that some Brockarchaeota are uniquely capable of mediating non-methanogenic anaerobic methylotrophy, via the tetrahydrofolate methyl branch of the Wood-Ljungdahl pathway and reductive glycine pathway. The hydrothermal vent genotypes appear to be obligate fermenters of plant-derived polysaccharides that rely mostly on substrate-level phosphorylation, as they seem to lack most respiratory complexes. In contrast, hot spring lineages have alternate pathways to increase their ATP yield, including anaerobic methylotrophy of methanol and trimethylamine, and potentially use geothermally derived mercury, arsenic, or hydrogen. Their broad distribution and their apparent anaerobic metabolic versatility indicate that Brockarchaeota may occupy previously overlooked roles in anaerobic carbon cycling.

Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.

The commonly-used superstrate configuration (depositing front subcell first and then depositing back subcell) in all-perovskite tandem solar cells is disadvantageous for long-term stability due to oxidizable narrow-bandgap perovskite assembled last and easily exposable to air. Here we reverse the processing order and demonstrate all-perovskite tandems in a substrate configuration (depositing back subcell first and then depositing front subcell) to bury oxidizable narrow-bandgap perovskite deep in the device stack. By using guanidinium tetrafluoroborate additive in wide-bandgap perovskite subcell, we achieve an efficiency of 25.3% for the substrate-configured all-perovskite tandem cells. The unencapsulated devices exhibit no performance degradation after storage in dry air for 1000 hours. The substrate configuration also widens the choice of flexible substrates: we achieve 24.1% and 20.3% efficient flexible all-perovskite tandem solar cells on copper-coated polyethylene naphthalene and copper metal foil, respectively. Substrate configuration offers a promising route to unleash the commercial potential of all-perovskite tandem solar cells.

Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10), a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(P)H:quinone oxidoreductase (NQO) enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to the mitochondrial respiratory chain in both human hepatoma cells (HepG2) and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders.