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Buck, M.

Publications and source records attributed to Buck, M..

At least 55 records · Page 3Linked to original sources

Molecular dynamics simulations and functional studies reveal that hBD-2 binds SARS-CoV-2 spike RBD and blocks viral entry into ACE2 expressing cells

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (KD [~] 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC50 of 2.4{+/-} 0.1 M. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.

biochemistry↗

Comprehensive dataset of shotgun metagenomes from stratified freshwater lakes and ponds

Stratified lakes and ponds featuring steep oxygen gradients are significant net sources of greenhouse gases and hotspots in the carbon cycle. Despite their significant biogeochemical roles, the microbial communities, especially in the oxygen depleted compartments, are poorly known. Here, we present a comprehensive dataset including 267 shotgun metagenomes from 41 stratified lakes and ponds mainly located in the boreal and subarctic regions, but also including one tropical reservoir and one temperate lake. For most lakes and ponds, the data includes a vertical sample set spanning from the oxic surface to the anoxic bottom layer. The majority of the samples were collected during the open water period, but also a total of 29 samples were collected from under the ice. In addition to the metagenomic sequences, the dataset includes environmental variables for the samples, such as oxygen, nutrient and organic carbon concentrations. The dataset is ideal for further exploring the microbial taxonomic and functional diversity in freshwater environments and potential climate change impacts on the functioning of these ecosystems.

ecology↗

Freshwater Chlorobia exhibit metabolic specialization among cosmopolitan and endemic populations

Photosynthetic bacteria from the class Chlorobia (formerly phylum Chlorobi) sustain carbon fixation in anoxic water columns. They harvest light at extremely low intensities and use various inorganic electron donors to fix carbon dioxide into biomass. Until now, most information on their functional ecology and local adaptations came from isolates and merely 26 sequenced genomes that are poor representatives of natural populations. To address these limitations, we analyzed global metagenomes to profile planktonic Chlorobia cells from the oxyclines of 42 freshwater bodies, spanning subarctic to tropical regions and encompassing all four seasons. We assembled and compiled over 500 genomes, including metagenome-assembled genomes (MAGs), single-cell genomes (SAGs), and reference genomes from cultures, clustering them into 71 metagenomic operational taxonomic units (mOTUs) or "species". Of the 71 mOTUs, 57 were classified as genus Chlorobium and these mOTUs varied in relative abundance up to ~60% of the microbial communities in the sampled anoxic waters. Several Chlorobium-associated mOTUs were globally distributed whereas others were endemic to individual lakes. Although most clades encoded the ability to oxidize hydrogen, many were lacking genes for the oxidation of specific sulfur and iron substrates. Surprisingly, one globally distributed Scandinavian Chlorobium clade encoded the ability to oxidize hydrogen, sulfur, and iron, suggesting that metabolic versatility facilitated such widespread colonization. Overall, these findings provide new insights into the biogeography of the Chlorobia and the metabolic traits that facilitate niche specialization within lake ecosystems. ImportanceThe reconstruction of genomes from metagenomes has enabled unprecedented insights into the ecology and evolution of environmental microbiomes. We applied this powerful approach to 274 metagenomes collected from diverse freshwater habitats that spanned oxic and anoxic zones, sampling seasons, and latitudes. We demonstrate widespread and abundant distributions of planktonic Chlorobia-associated bacteria in hypolimnetic waters of stratified freshwater ecosystems and pinpoint nutrients that likely fuel their electron chains. Being photoautotrophs, these Chlorobia organisms also have the potential to serve as carbon sources that support metalimnetic and hypolimnetic food webs.

ecology↗

Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodelling superfamily

Membrane remodelling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, CdvB in TACK archaea and ESCRT-III in eukaryotes. Here, we show that these protein families are homologous and share a common evolutionary origin. Using cryo-electron microscopy we present structures for Vipp1 rings over a range of symmetries. Each ring is built from rungs that stack and spontaneously self-organise to form domes. Rungs are assembled from a polymer that is strikingly similar in structure to ESCRT-III. A tilt between rungs generates the dome-shaped curvature with constricted open ends and an inner membrane-binding lumen. Overall, our results reveal conserved mechanistic principles that underlie Vipp1, PspA and ESCRT-III dependent membrane remodelling across all domains of life. One sentence summaryEvolutionary and structural analyses of Vipp1/IM30 rings reveal ESCRT-III-like polymers that remodel membranes in bacteria.

microbiology↗

Proteome-scale Analysis of Vertebrate Protein Thermoadaptation Modulated by Dynamic Allostery And Protein Solvation

Despite differences in behaviors and living conditions, vertebrate organisms share the great majority of proteins, often with subtle differences in amino acid sequence. Here, we present a simple way to analyze the difference in amino acid occurrence by comparing highly homologous proteins on a sub-proteome level between several vertebrate model organisms. Specifically, we use this method to identify a pattern of amino acid conservation as well as a shift in amino acid occurrence between homeotherms (warm-blooded species) and poikilotherms (cold-blooded species). Importantly, this general analysis and a specific example further establish a correlation, if not likely connection between the thermoadaptation of protein sequences and two of their physical features: a possible change in their protein dynamics and, even more strongly, in their solvation. For poikilotherms, such as frog and fish, the lower body temperature is expected to increase the association of proteins due to a decrease in protein internal dynamics. In order to prevent overly-sticky protein association at low temperatures, the use of amino acids suggests that poikilotherms enhance the solvation of their proteins by favoring polar groups on their proteins surface. This feature appears to dominate over possible changes in dynamics. The results suggest that a general trend for amino acid choice is part of the mechanism for thermoadaptation of vertebrate organisms at the molecular level.

biophysics↗

Raf promotes dimerization of the Ras G-domain with increased allosteric connections

Ras dimerization is critical for Raf activation, yet Ras alone does not dimerize. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics (MD) simulations show robust allosteric connections linking the two Raf-RBD D113 residues, located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 [A] apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.

biophysics↗

Energy efficiency and biological interactions define the core microbiome of deep oligotrophic groundwater

Extremely oligotrophic deep groundwaters host organisms attuned to the low-end of the bioenergetics spectrum. While all domains of life along with viruses are active in this habitat, the evolutionary and ecological constraints on colonization and niche shifts and their consequences for the microbiome convergence are unknown. Here we provide a comparative genome-resolved analysis of the prokaryotic community in disconnected fracture fluids of the Fennoscandian Shield. The data show that the oligotrophic deep groundwaters flowing in similar lithologies offer fixed niches that are occupied by a common deep groundwater core microbiome. Based on this high resolution "multi-omics" enabled understanding of the underlying mechanisms via functional expression analysis, we conclude that deep groundwater ecosystems foster highly diverse, yet cooperative microbial communities adapted to this setting. The fitness of primary energy producers is increased by ecological traits such as aggregate or biofilm formation. This also facilitates reciprocal promiscuous partnerships with diverse and prevalent epi-bionts, alleviating the "tragedy of common goods". Hence, instead of a lifestyle where microbes predominantly invest in functions related to maintenance and survival, an episodic and cooperative lifestyle ensures the subsistence of the deep groundwater microbiome. We suggest the name "halt and catch fire" for this way of life.

microbiology↗

A multiplexed work-flow for absolute quantification of Klebsiella oxytoca Nif proteins

Global imbalances of the nitrogen cycle are increasingly recognized as a major challenge to human development, and are exacerbated by the use of synthetic inorganic fertilizers. Biotechnology alternatives to inorganic fertilizers include biofertilisation from nitrogen fixing bacteria (diazotrophs), expressing nitrogenase and auxiliary genes (nif genes). In order to directly quantify all twenty Nif proteins through multiple reaction monitoring (MRM) MS, we established a high throughput pipeline to generate a set of Klebsiella oxytoca Nif protein QconCATs as quantotypic standards. A stringent validation of the pipeline and QconCATs application with regards to isotopic labelling efficiency (100%), limits of detection and quantification, analyte to internal standard concentration boundaries was used for optimisation. Using three QconCATs for the measurement of 20 likely low, middle and high Nif protein abundances enabled detection of all Nif proteins, 19 of which could be accurately quantified and their variation over time monitored. Stoichiometries between Klebsiella oxytoca Nif proteins and changes between early and late transition into diazotrophy suggest i) a temporal regulation of nif gene cluster expression that may be linked to nitrogenase expression and maturation; ii) vast disparities in Nif protein abundances and iii) high dependency on the nifLA master regulator pair for nif gene expression.

biochemistry↗

Streamlined freshwater bacterioplankton Nanopelagicales (acI) and Ca. Fonsibacter (LD12) thrive in functional cohorts

While fastidious microbes can be abundant and ubiquitous in their natural communities, many fail to grow axenically in laboratories due to auxotrophies or other dependencies. To overcome auxotrophies these microbes rely on their surrounding cohort. A cohort may consist of kin (ecotypes) or more distantly related organisms (community) with the cooperation being reciprocal or non-reciprocal, and expensive (Black Queen hypothesis) or costless (byproduct). These metabolic partnerships (whether at single species population or community level) enable dominance by and coexistence of these lineages in nature. Here we examine the relevance of these cooperation models to explain the abundance and ubiquity of the dominant fastidious bacterioplankton of a dimictic mesotrophic freshwater lake. Using both culture dependent (minimalist mixed cultures) and culture independent (SSU rRNA gene time series and environmental metagenomics) methods we independently identified the primary cohorts of Actinobacterial genera "Ca. Planktophila" (acI-A) and "Ca. Nanopelagicus" (acI-B), and the Proteobacterial genus "Ca. Fonsibacter" (LD12). While "Ca. Planktophila" and "Ca. Fonsibacter" had no correlation in their natural habitat, they have the potential to be complementary in laboratory settings. We also investigated the bi-functional catalase-peroxidase enzyme KatG (a common good which "Ca. Planktophila" is dependent upon) and its most likely providers in the lake. Further we found that while ecotype and community cooperation combined may explain "Ca. Planktophila" population abundance, the success of "Ca. Nanopelagicus" and "Ca. Fonsibacter" is better explained as a community byproduct. Ecotype differentiation of "Ca. Fonsibacter" as a means of escaping predation was supported but not for overcoming auxotrophies. IMPORTANCEThis study examines evolutionary and ecological relationships of three of the most ubiquitous and abundant freshwater bacterial genera: "Ca. Planktophila" (acI-A), "Ca. Nanopelagicus" (acI-B), and "Ca. Fonsibacter" (LD12). Due to high abundance, these genera might have a significant influence on nutrient cycling in freshwaters worldwide and this study adds a layer of understanding to how seemingly competing clades of bacteria can co-exist by having different cooperation strategies. Our synthesis ties together network and ecological theory with empirical evidence and lays out a framework for how the functioning of populations within complex microbial communities can be studied.

microbiology↗

Marine snow as a habitat for microbial mercury methylators in the Baltic Sea

Methylmercury (MeHg), a neurotoxic compound biomagnifying in aquatic food webs, can be a threat to human health via fish consumption. However, the composition and distribution of the microbial communities mediating the methylation of mercury (Hg) to MeHg in marine systems remain largely unknown. In order to fill this gap of knowledge, we used the Baltic Sea Reference Metagenome (BARM) dataset to study the distribution of the genes involved in Hg methylation (the hgcAB gene cluster). We determined the relative abundance of the hgcAB genes and their taxonomic identity in 81 brackish metagenomes that cover spatial, seasonal and redox variability in the Baltic Sea water. The hgcAB genes were predominantly detected in anoxic water, but some hgcAB genes were also detected in hypoxic and normoxic waters. Higher relative quantities of hgcAB genes were found in metagenomes from marine snow compared to free-living communities in anoxic water, suggesting that marine snow are hotspot habitats for Hg methylators in oxygen-depleted seawater. Phylogenetic analysis identified well-characterized Hg methylators such as Deltaproteobacteria in oxygen-depleted water, but also uncovered Hg methylators within the Spirochaetes and Lentisphaerae phyla. Altogether, our work unveils the diversity of the microorganisms mediating MeHg production in the Baltic Sea and pinpoint the ecological niches of these microorganisms within the marine water column.

microbiology↗

Molecular origins of transcriptional heterogeneity in diazotrophic K. oxytoca

Phenotypic heterogeneity in clonal bacterial batch cultures is an important adaptive strategy to changing environments, including in diazotrophs with the unique capacity to convert di-nitrogen into bio-available ammonium. In diazotrophic Klebsiella oxytoca we simultaneously measured mRNA levels of key regulatory (glnK-amtB, nifLA) and structural (nifHDK) operons required for establishing nitrogen fixation, using dual molecule, single cell RNA-FISH. Through stochastic transcription models and mutual information analysis we revealed likely molecular origins for heterogeneity in nitrogenase expression. In wildtype and regulatory variant strains we inferred contributions from intrinsic and extrinsic noise, finding that nifHDK transcription is inherently bursty, but that noise propagation through signalling is also significant. The regulatory gene glnK had the highest discernible effect on nifHDK variance, while noise from factors outside of the regulatory pathway were negligible. Results provide evidence that heterogeneity is a fundamental property of this regulatory system, indicating potential constraints for engineering homogeneous nitrogenase expression.

microbiology↗

Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7

Bacteriophage T7 infects Escherichia coli and evades the host defence system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.\n\nImpact statementDNA mimicry Ocr protein, a well-studied T7 phage protein that inhibits host restriction enzymes, can also inhibit host transcription through competing with sigma factors in binding to RNA polymerase.

biophysics↗

Plexin-Bs enhance their GAP activity with a novel activation switchloop

Plexins receive guidance cues from semaphorin ligands and transmit their signal through the plasma membrane. This family of proteins is unique amongst single-pass transmembrane receptors as their intracellular regions interact directly with several small GTPases, which regulate cytoskeletal dynamics and cell adhesion. Here, we characterize the GTPase Activating Protein (GAP) function of Plexin-B1 and find that a cooperative GAP activity towards the substrate GTPase, Rap1b, is associated with the N-terminal Juxtamembrane region of Plexin-B1. Importantly, we unveil an activation mechanism of Plexin-B1 by identifying a novel functional loop which partially blocks Rap1b entry to the plexin GAP domain. Consistent with the concept of allokairy developed for other systems, Plexin-B activity is increased by an apparent substrate mediated cooperative effect. Simulations and mutagenesis suggest the repositioned JM conformation is stabilized by the new activation switch loop when the active site is occupied, giving rise to faster enzymatic turnover and cooperative behavior. The biological implications, essentially of a threshold behavior for cell migration are discussed.

biophysics↗

Effects of long-term warming and enhanced nitrogen and sulfur deposition on microbial communities in a boreal peatland

With ongoing environmental change, it is important to understand ecosystem responses to multiple perturbations over long time scales at in situ conditions. Here, we investigated the individual and combined effects of 18 years of warming and enhanced nitrogen and sulfate deposition on peat microbial communities in a nutrient-poor boreal mire. The three perturbations individually affected prokaryotic community composition, where nitrogen addition had the most pronounced effect, and its combination with the other perturbations led to additive effects. The functional potential of the community, characterized by shotgun metagenomics, was strongly affected by the interactive effects in the combined treatments. The responses in composition were also partly reflected in the functional gene repertoire and in altered carbon turnover, i.e. an increase of methane production rates as a result of nitrogen addition and a decrease with warming. Long-term nitrogen addition and warming-induced changes caused a shift from Sphagnum-dominated plant communities to vascular plant dominance, which likely transact with many of the observed microbial responses. We conclude that simultaneous perturbations do not always lead to synergistic effects, but can also counteract and even neutralize one another, and thus must be studied in combination when attempting to predict future characteristics and services of peatland ecosystems.

microbiology↗

Computational Design of Myristoylated Cell Penetrating Peptides Targeting Oncogenic K-Ras.G12D at the Effector Binding Membrane Interface

A number of small inhibitors have been developed in the recent years to target the cancer driving protein, K-Ras. In this study we propose and design a novel way of targeting oncogenic K-Ras4B.G12D with myristoylated cell penetrating peptides which become membrane anchored and lock the protein into an inactive state. In all atom molecular dynamics simulations such peptides associate with K-Ras4B exclusively at the effector binding region, which, in turn, expected to hinder the binding of down-stream effector proteins (e.g. C-Raf). The myristoylated R9 (Arg9) peptide strongly locks K-Ras4B.G12D into orientations that are unfavorable for effector binding. After breaking the cyclic structure and myristoylation, a cell penetrating peptide cyclorasin 9A5, which was designed for targeting the Ras: Raf interface, is also found to be effective in targeting the Ras: membrane interface. The myristoylated peptides likely have high cell permeability due to their mixed cationic/hydrophobic character at the N-terminus, while simultaneously the subsequent multiple charges help to maintain a strong association of the peptide with the K-Ras4B.G12D effector binding lobe. Targeting protein-membrane interfaces is starting to attract attention very recently, thanks to our understanding of the signaling mechanism of an increased number of peripheral membrane proteins. The strategy used in this study has potential applications in the design of drugs against K-Ras4B driven cancers. It also provides insights into the general principles of targeting protein-membrane interfaces.

biophysics↗

Metastable contacts and structural disorder in the estrogen receptor transactivation domain

The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins (IDPs), mediates the receptors transactivation function to regulate gene expression. However, an accurate molecular dissection of NTDs structure-function relationships remains elusive. Here, using small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), circular dichroism, and hydrogen exchange mass spectrometry, we show that NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion upon chemical denaturation. By combining SAXS, hydroxyl radical protein footprinting and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map that reveals metastable regional and long-range contacts, including interactions between residues I33 and S118. We show that mutation at S118, a known phosphorylation site, promotes conformational changes and increases coactivator binding. We further demonstrate via fluorine-19 (19F) NMR that mutations near residue I33 alter 19F chemical shifts at residue S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of IDPs structure-function relationship, and how specific metastable contacts mediate critical functions of disordered proteins.\n\nHighlightsO_LIA compact disorder is observed for the N-terminal domain (NTD) of estrogen receptor\nC_LIO_LIMulti-technique modeling elucidates the NTD ensemble structures\nC_LIO_LIEnsemble-based contact map reveals metastable contacts between I33 and S118\nC_LIO_LI19F-NMR data validate the proposed I33-S118 contact in the IDP\nC_LI

biophysics↗

K-Ras G-domain binding with signaling lipid phosphoinositides: PIP2 association, orientation, function

Ras genes are potent drivers of human cancers, with mutated K-Ras4B being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the holy grail of present-day cancer therapy, and recent discoveries of small molecule inhibitors for K-Ras4B greatly benefited from a deeper understanding of the protein structure and dynamics of the GTPase. Since interactions with biological membranes are key for Ras function, the details of Ras - lipid interactions have become a major focus of study, especially since it is becoming clear that such interactions not only involve the Ras C-terminus for lipid anchoring, but also the G-protein domain. Here we investigated the interaction between K-Ras4B with the signaling lipid phosphatidyl inositol (4,5) phosphate (PIP2) using NMR spectroscopy and molecular dynamics simulations, complemented by biophysical and cell biology assays. We discovered that the {beta}2 and {beta}3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2, and that these secondary structural elements employ specific residues for these interactions. These likely occur in two orientation states of the protein relative to the membrane. Importantly, we found that some of these residues, which are known to be oncogenic when mutated (D47K, D92N, K104M and D126N), are critical for K-Ras-mediated transformation of fibroblast cells, while not substantially affecting basal and assisted nucleotide hydrolysis and exchange. We further showed that mutation K104M can indeed abolish localization of mutant K-Ras to the plasma membrane. These findings suggest that specific G-domain residues play an important, previously-unknown role in regulating Ras function by mediating interactions with membrane PIP2 lipids. Thus, a detailed description of the novel K-Ras-PIP2 binding surfaces is likely to inform the future design of therapeutic reagents.

biophysics↗

Novel autotrophic organisms contribute significantly to the internal carbon cycling potential of a boreal lake

The authors declare no conflict of interest\n\nFunding sources: the Academy of Finland, Science for Life Laboratories, Tryggers Foundation, the Swedish Research Council VR and the Swedish Foundation for strategic research\n\nAbstractOxygen stratified lakes are typical for the boreal zone, and also a major source of greenhouse gas emissions in the region. Due to shallow light penetration, restricting the growth of phototrophic organisms, and large allochthonous organic carbon inputs from the catchment area, the lake metabolism is expected to be dominated by heterotrophic organisms. In this study we test this assumption and show that the potential for autotrophic carbon fixation and internal carbon cycling is high throughout the water column. Further, we show that during the summer stratification carbon fixation can exceed respiration in a boreal lake even below the euphotic zone. Metagenome assembled genomes and 16S profiling of a vertical transect of the lake revealed multiple organisms in oxygen depleted compartment belonging to novel or poorly characterized phyla. Many of these organisms were chemolithotrophic, deriving their energy from reactions related to sulfur, iron and nitrogen transformations. The community as well as the functions were stratified following the redox potentials. The autotrophic potential in the lake metagenome below the oxygenic zone was high, pointing towards a need for revising our concepts of internal carbon cycling in boreal lakes. Further, the importance of chemolithoautotrophy for the internal carbon cycling suggests that many predicted climate change associated changes in the physical properties of the lake, such as altered mixing patterns, likely have consequences for the whole lake metabolism even beyond the impact to the phototrophic community.\n\nImportanceAutotrophic organisms at the base of the food web are the only life form capable of turning inorganic carbon into organic form, facilitating the survival of all other organisms. In certain environments the autotrophic production is limited by environmental conditions and the food web is supported by carbon coming from outside the ecosystem. One such environment is stratified boreal lakes, which are one of the biggest sources of greenhouse gas emissions in the boreal region. Thus, carbon cycling in these habitats is of outmost importance for the future climate. Here we demonstrate a high potential for internal carbon cycling via phototrophic and novel chemolithotrophic organisms in the dark and anoxic layers of a boreal lake. Our results significantly increase our knowledge on the microbial communities and their metabolic potential in oxygen depleted freshwaters and help to understand and predict how climate change induced alterations could impact the lake carbon dynamics.

ecology↗