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Understanding the assembly mechanism and function of membrane proteins is a fundamental problem in biochemical research. Among the membrane proteins, G protein-coupled receptors (GPCRs) represent the largest class in the human body and have long been considered to function as monomers.
Nowadays, the oligomeric assembly of GPCRs is widely accepted, although the functional importance and therapeutic intervention remain largely unexplored. This is partly due to difficulties in the heterologous production of membrane proteins.
Cell-free protein synthesis (CFPS) with its endogenous endoplasmic reticulum-derived structures has proven as a technique to address this issue.
In this study, we investigate for the first time the conceptual CFPS of a heteromeric GPCR, the gamma-aminobutyric acid receptor type B (GABA(B)), from its protomers BR1 and BR2 using a eukaryotic cell-free lysate. Using a fluorescence-based proximity ligation assay, we provide evidence for colocalization and thus suggesting heterodimerization.
We prove the heterodimeric assembly by a bioluminescence resonance energy transfer saturation assay providing the manufacturability of a heterodimeric GPCR by CFPS. Additionally, we show the binding of a fluorescent orthosteric antagonist, demonstrating the feasibility of combining the CFPS of GPCRs with pharmacological applications.
These results provide a simple and powerful experimental platform for the synthesis of heteromeric GPCRs and open new perspectives for the modelling of protein-protein interactions.
Accordingly, the presented technology enables the targeting of protein assemblies as a new interface for pharmacological intervention in disease-relevant dimers.
Theoretical and empirical studies show increased diversity in crops, supply chains, and markets helps stabilize food systems. At the same time global commodity markets and industrial agriculture have driven homogenization of local and regional production systems, and consolidated power in fewer larger specialized farms and distributers. This is a global challenge, with no obvious global solutions. An important question therefore, is how individual countries can build their own resilience through maintaining or increasing diversity within their borders. Here we show, using farm level data from Germany, that spreading production risk by growing the same crops across different farms carries stabilizing benefits by allowing for increased spatiotemporal asynchrony within crops. We also find that increasing asynchrony between the year-to-year production of different crops has stabilizing effects on food supply. Importantly, the benefits of increasing crop diversity are lower in specialized landscapes growing the same crop on large patches. Our results illustrate clear benefits of diversified crops, producers, and agricultural landscapes to buffer supply side shocks, and for incorporation in subsidies and other regulatory measures aimed at stabilizing food systems.
Riverine ecosystems provide various ecosystem services. One of these services is the biological control of eutrophication by grazing macroinvertebrates.
However, riverine ecosystems are subject to numerous stressors that affect community structure, functions, and stability properties. To manage rivers in response to these stressors, a better understanding of the ecological functions underlying services is needed.
This requires consideration of local and regional processes, which requires a metacommunity approach that links local food webs through drift and dispersal. This takes into account long-distance interactions that can compensate for local effects of stressors.
Our modular model MASTIFF (Multiple Aquatic STressors In Flowing Food webs) is stage-structured, spatially explicit, and includes coupled food webs consisting of benthic resource-consumer interactions between biofilm and three competing macroinvertebrate functional types. River segments are unidirectionally connected through organismal drift and bidirectionally connected through dispersal. Climate and land use stressors along the river can be accounted for. Biocontrol of biofilm eutrophication is used as an exemplary functional indicator.
We present the model and the underlying considerations, and show in an exemplary application that explicit consideration of drift and dispersal is essential for understanding the spatiotemporal biocontrol of eutrophication.
The combination of drift and dispersal reduced eutrophication events. While dispersal events were linked to specific periods in the species life cycles and therefore had limited potential to control, drift was ubiquitous and thus responded more readily to changing habitat conditions.
This indicates that drift is an important factor for coping with stress situations.
Finally, we outline and discuss the potential and possibilities of MASTIFF as a tool for mechanistic, cross-scale analyses of multiple stressors to advance knowledge of riverine ecosystem functioning.
Paleogenomes reveal a complex evolutionary history of late Pleistocene bison in Northeastern China
(2022)
Steppe bison are a typical representative of the Mid-Late Pleistocene steppes of the northern hemisphere.
Despite the abundance of fossil remains, many questions related to their genetic diversity, population structure and dispersal route are still elusive.
Here, we present both near-complete and partial mitochondrial genomes, as well as a partial nuclear genome from fossil bison samples excavated from Late Pleistocene strata in northeastern China.
Maximum-likelihood and Bayesian trees both suggest the bison clade are divided into three maternal haplogroups (A, B and C), and Chinese individuals fall in two of them. Bayesian analysis shows that the split between haplogroup C and the ancestor of haplogroups A and B dates at 326 ky BP (95% HPD: 397-264 ky BP).
In addition, our nuclear phylogenomic tree also supports a basal position for the individual carrying haplogroup C. Admixture analyses suggest that CADG467 (haplogroup C) has a similar genetic structure to steppe bison from Siberia (haplogroup B).
Our new findings indicate that the genetic diversity of Pleistocene bison was probably even higher than previously thought and that northeastern Chinese populations of several mammalian species, including Pleistocene bison, were genetically distinct.
Plant cell walls are versatile materials that can adopt a wide range of mechanical properties through controlled deposition of cellulose fibrils. Wall integrity requires a sufficiently homogeneous fibril distribution to cope effectively with wall stresses. Additionally, specific conditions, such as the negative pressure in water transporting xylem vessels, may require more complex wall patterns, e.g., bands in protoxylem.
The orientation and patterning of cellulose fibrils are guided by dynamic cortical microtubules.
New microtubules are predominantly nucleated from parent microtubules causing positive feedback on local microtubule density with the potential to yield highly inhomogeneous patterns. Inhomogeneity indeed appears in all current cortical array simulations that include microtubule-based nucleation, suggesting that plant cells must possess an as-yet unknown balancing mechanism to prevent it.
Here, in a combined simulation and experimental approach, we show that a limited local recruitment of nucleation complexes to microtubules can counter the positive feedback, whereas local tubulin depletion cannot.
We observe that nucleation complexes preferentially appear at the plasma membrane near microtubules. By incorporating our experimental findings in stochastic simulations, we find that the spatial behavior of nucleation complexes delicately balances the positive feedback, such that differences in local microtubule dynamics-as in developing protoxylem-can quickly turn a homogeneous array into a banded one. Our results provide insight into how the plant cytoskeleton has evolved to meet diverse mechanical requirements and greatly increase the predictive power of computational cell biology studies.
During a survey of aquatic fungi from Anzali Lagoon in Iran, several fungal specimens were isolated from freshwater habitats. Morphological evidence and comparing sequencing based on rDNA (ITS and LSU) and protein-coding genes (TEF1 and TUB2) showed that some isolates belong to undescribed fungal species.
These isolates belong to Arthrobotrys and Sarocladium, two ascomycetes genera. Arthrobotrys hyrcanus, sp. nov., differs from closely related species such as A. dianchiensis by its larger conidia and septation of primary conidia. Sarocladium pseudokiliense, sp. nov., was similar to S. kiliense, but distinguished by its conidial shape and the absence of adelophialides and chlamydospores.
Morphological descriptions, illustrations and multilocus phylogenetic analysis for both new species are provided.
Meropenem is one of the most frequently used antibiotics to treat life-threatening infections in critically ill patients.
This study aimed to develop a meropenem dosing algorithm for the treatment of Gram-negative infections based on intensive care unit (ICU)-specific resistance data. Antimicrobial susceptibility testing of Gram-negative bacteria obtained from critically ill patients was carried out from 2016 to 2020 at a tertiary care hospital.
Based on the observed MIC distribution, stochastic simulations (n = 1,000) of an evaluated pharmacokinetic meropenem model, and a defined pharmacokinetic/pharmacodynamic target (100%T->4xMIC while minimum concentrations were <44.5 mg/L), dosing recommendations for patients with varying renal function were derived.
Pathogen-specific MIC distributions were used to calculate the cumulative fraction of response (CFR), and the overall MIC distribution was used to calculate the local pathogen-independent mean fraction of response (LPIFR) for the investigated dosing regimens.
A CFR/LPIFR of >90% was considered adequate. The observed MIC distribution significantly differed from the EUCAST database. Based on the 6,520 MIC values included, a three-level dosing algorithm was developed.
If the pathogen causing the infection is unknown (level 1), known (level 2), known to be neither Pseudomonas aeruginosa nor Acinetobacrer baumannii, or classified as susceptible (level 3), a continuous infusion of 1.5 g daily reached sufficient target attainment independent of renal function.
In all other cases, dosing needs to be adjusted based on renal function. ICU-specific susceptibility data should be assessed regularly and integrated into dosing decisions. The presented workflow may serve as a blueprint for other antimicrobial settings.
Quantifying the influence of pollen aging on the adhesive
properties of Hypochaeris radicata pollen
(2022)
Simple Summary Pollination is the transfer of pollen from a plant's male part (anther) to the corresponding female part (stigma). It is a fundamental biological process that ensures plant reproduction. Most studies investigate pollination from a biological perspective, but the underlying physical processes are poorly understood. Many plants rely on insects to transport pollen and the forces with which pollen adhere to insects and floral surfaces are fundamental for successful pollination.
We quantified pollen adhesion by measuring the forces necessary to detach Hypochaeris radicata (catsear, a common insect-pollinated plant) pollen from glass and studied for the first time how the adhesion forces change with pollen aging.
Our results show that newly formed adhesion bonds between H. radicata pollen and glass are stronger for fresh pollen than for old ones. On the other hand, when H. radicata pollen age in contact with glass, the adhesion between pollen and glass strengthens over time. These effects are probably caused by the viscous liquid covering most pollen (pollenkitt) changing its viscoelastic properties as it dries.
Although pollination is one of the most crucial biological processes that ensures plant reproduction, its mechanisms are poorly understood. Especially in insect-mediated pollination, a pollen undergoes several attachment and detachment cycles when being transferred from anther to insect and from insect to stigma. The influence of the properties of pollen, insect and floral surfaces on the adhesion forces that mediate pollen transfer have been poorly studied.
Here, we investigate the adhesive properties of Hypochaeris radicata pollen and their dependence on pollen aging by quantifying the pull-off forces from glass slides using centrifugation and atomic force microscopy. We found that the properties of the pollenkitt-the viscous, lipid liquid on the surface of most pollen grains-influences the forces necessary to detach a pollen from hydrophilic surfaces.
Our results show that aged H. radicata pollen form weaker adhesions to hydrophilic glass than fresh ones. On the other hand, when a pollen grain ages in contact with glass, the adhesion between the two surfaces increases over time.
This study shows for the first time the pollen aging effect on the pollination mechanism.
Volatile organic compounds (VOCs) are involved in microbial interspecies communication and in the mode of action of various antagonistic interactions. They are important for balancing host-microbe interactions and provide the basis for developing biological control strategies to control plant pathogens.
We studied the interactions between the bacterial antagonist Serratia plymuthica HRO-C48 and three fungal plant pathogens Rhizoctonia solani, Leptosphaeria maculans and Verticillium longisporum. Significant differences in fungal growth inhibition by the Serratia-emitted VOCs in pairwise dual culture assays and changes in the transcriptome of the bacterium and in the volatilomes of both interacting partners were observed. Even though the rate of fungal growth inhibition by Serratia was variable, the confrontation of the bacterium with the VOCs of all three fungi changed the levels of expression of the genes involved in stress response, biofilm formation, and the production of antimicrobial VOCs. Pairwise interacting microorganisms switched between defense (downregulation of gene expression) and attack (upregulation of gene expression and metabolism followed by growth inhibition of the interacting partner) modes, subject to the combinations of microorganisms that were interacting.
In the attack mode HRO-C48 significantly inhibited the growth of R. solani while simultaneously boosting its own metabolism; by contrast, its metabolism was downregulated when HRO-C48 went into a defense mode that was induced by the L. maculans and V. longisporum VOCs. L. maculans growth was slightly reduced by the one bacterial VOC methyl acetate that induced a strong downregulation of expression of genes involved in almost all metabolic functions in S. plymuthica.
Similarly, the interaction between S. plymuthica and V. longisporum resulted in an insignificant growth reduction of the fungus and repressed the rate of bacterial metabolism on the transcriptional level, accompanied by an intense volatile dialogue. Overall, our results indicate that VOCs substantially contribute to the highly break species-specific interactions between pathogens and their natural antagonists and thus deserving of increased consideration for pathogen control.
Ciliates represent a crucial link between phytoplankton and bacteria and mesozooplankton in pelagic food webs, but little is known about the processes influencing the dynamics of individual species.
Using long-term, high-frequency observations, we compared the diversity and the temporal variability in biomass and species composition of the ciliate community in large, deep, mesotrophic Lake Constance to that of the phytoplankton and rotifer communities in the same lake.
Furthermore, we used boosted regression trees to evaluate possible environmental predictors (temperature, three prey groups, four predator/competitor groups) influencing ciliate net growth.
The biomass of all ciliate species showed a common, recurrent seasonal pattern, often with peaks in spring and summer.
The ciliate community was more diverse than the rotifer community, exhibited highly synchronous dynamics and its species were regularly encountered during the season. The top-down control by copepods likely contributes to the ciliates' synchronized decline prior to the clear-water phase when food concentration is still high.
The high temporal autocorrelation of the ciliate biomasses together with the inter-annual recurrent seasonal patterns and the low explanatory power of the environmental predictors suggest that the dynamics of individual ciliate species are strictly controlled, yet it remains difficult to determine the responsible factors.
Current evidence suggests that migratory animals extract map information from the geomagnetic field for true navigation. The sensory basis underlying this feat is elusive, but presumably involves magnetic particles.
A common experimental manipulation procedure consists of pre-treating animals with a magnetic pulse, with the aim of re-magnetising particles to alter the internal representation of the external field prior to a navigation task.
Although pulsing provoked deflected bearings in caged songbirds, analogous studies with free-flying songbirds yielded inconsistent results.
Here, we pulsed European robins (Erithacus rubecula) at an offshore stopover site during spring migration and monitored their free-flight behaviour with a regional-scale network of radio-receiving stations.
We found no pulse effect on departure probability, nocturnal departure timing departure direction or consistency of flight direction.
This suggests either no use of the geomagnetic map by our birds, or that magnetic pulses do not affect the sensory system underlying geomagnetic map detection.
Nature conservation is currently shaping many terrestrial ecosystems in Africa.
This is particularly evident in Sub-Saharan Africa (SSA), where conservation is intended to recover wildlife populations, with special focus on elephants.
Rising numbers of elephants induce woody biomass losses but increase soil organic carbon (SOC) stocks from decaying wood and dung.
We hypothesized that these increases under wildlife conservation in SSA go along with rising contents of plant residues in SOC, traceable by the molecular markers lignin and n-alkanes.
In contrast, agricultural intensification would reduce them due to lower C input and faster SOC turnover through tillage.
To test this, we analyzed lignin by the CuO oxidation method and n-alkanes by fast pressurized solvent extraction in topsoils (0-10 cm) of Arenosols and corresponding plant samples (trees, grasses and crops).
Sampling sites followed conservation gradients with low, medium and high elephant densities and intensification gradients with rangeland and cropland in the woodland savanna of the Namibian Zambezi Region.
Patterns of lignin-derived phenols were retained in the soil, whereas n-alkanes showed shifts in chain lengths. n-Alkanes also showed no clear increase or decrease under conservation or intensification, respectively.
Differently, lignin-derived phenols showed lower values under intensification than under conservation. Confirming our hypothesis, rising SOC contents with rising elephant densities (from 4.4 at low to 5.7 g kg(-1) SOC at high elephant densities) went along with an increasing accumulation of lignin-derived phenols (24.4-34.8 g kg(-1) VSCOC).
This increase is associated with the input of woody debris to the soil, as indicated by V-units and carbon isotopes, modulated by clay and woody biomass.
We conclude, that increasing input of woody residues into soil by browsing behaviour of elephants is an important mechanism for controlling SOC supply in the context of wildlife conservation and is traceable with lignin-derived phenols, but not with n-alkanes.
Rhizophagus irregularis is one of the most extensively studied arbuscular mycorrhizal fungi (AMF) that forms symbioses with and improves the performance of many crops. Lack of transformation protocol for R. irregularis renders it challenging to investigate molecular mechanisms that shape the physiology and interactions of this AMF with plants.
Here, we used all published genomics, transcriptomics, and metabolomics resources to gain insights into the metabolic functionalities of R. irregularis by reconstructing its high-quality genome-scale metabolic network that considers enzyme constraints. Extensive validation tests with the enzyme-constrained metabolic model demonstrated that it can be used to (i) accurately predict increased growth of R. irregularis on myristate with minimal medium; (ii) integrate enzyme abundances and carbon source concentrations that yield growth predictions with high and significant Spearman correlation (rS = 0.74) to measured hyphal dry weight; and (iii) simulate growth rate increases with tighter association of this AMF with the host plant across three fungal structures.
Based on the validated model and system-level analyses that integrate data from transcriptomics studies, we predicted that differences in flux distributions between intraradical mycelium and arbuscles are linked to changes in amino acid and cofactor biosynthesis.
Therefore, our results demonstrated that the enzyme-constrained metabolic model can be employed to pinpoint mechanisms driving developmental and physiological responses of R. irregularis to different environmental cues.
In conclusion, this model can serve as a template for other AMF and paves the way to identify metabolic engineering strategies to modulate fungal metabolic traits that directly affect plant performance.
IMPORTANCE Mounting evidence points to the benefits of the symbiotic interactions between the arbuscular mycorrhiza fungus Rhizophagus irregularis and crops; however, the molecular mechanisms underlying the physiological responses of this fungus to different host plants and environments remain largely unknown.
We present a manually curated, enzyme-constrained, genome-scale metabolic model of R. irregularis that can accurately predict experimentally observed phenotypes.
We show that this high-quality model provides an entry point into better understanding the metabolic and physiological responses of this fungus to changing environments due to the availability of different nutrients.
The model can be used to design metabolic engineering strategies to tailor R. irregularis metabolism toward improving the performance of host plants.
Cell division and the resulting changes to the cell organization affect the shape and functionality of all tissues.
Thus, understanding the determinants of the tissue-wide changes imposed by cell division is a key question in developmental biology.
Here, we use a network representation of live cell imaging data from shoot apical meristems (SAMs) in Arabidopsis thaliana to predict cell division events and their consequences at the tissue level.
We show that a support vector machine classifier based on the SAM network properties is predictive of cell division events, with test accuracy of 76%, which matches that based on cell size alone.
Furthermore, we demonstrate that the combination of topological and biological properties, including cell size, perimeter, distance and shared cell wall between cells, can further boost the prediction accuracy of resulting changes in topology triggered by cell division.
Using our classifiers, we demonstrate the importance of microtubule-mediated cell-to-cell growth coordination in influencing tissue-level topology.
Together, the results from our network-based analysis demonstrate a feedback mechanism between tissue topology and cell division in A. thaliana SAMs.
In plants, the trehalose biosynthetic pathway plays key roles in the regulation of carbon allocation and stress adaptation.
Engineering of the pathway holds great promise to increase the stress resilience of crop plants.
The synthesis of trehalose proceeds by a two-step pathway in which a trehalose-phosphate synthase (TPS) uses UDP-glucose and glucose-6-phosphate to produce trehalose-6 phosphate (T6P) that is subsequently dephosphorylated by trehalose-6 phosphate phosphatase (TPP).
While plants usually do not accumulate high amounts of trehalose, their genome encodes large families of putative trehalose biosynthesis genes, with many members lacking obvious enzymatic activity.
Thus, the function of putative trehalose biosynthetic proteins in plants is only vaguely understood. To gain a deeper insight into the role of trehalose biosynthetic proteins in crops, we assessed the enzymatic activity of the TPS/TPP family from tomato (Solanum lycopersicum L.) and investigated their expression pattern in different tissues as well as in response to temperature shifts. From the 10 TPS isoforms tested, only the 2 proteins belonging to class I showed enzymatic activity, while all 5 TPP isoforms investigated were catalytically active.
Most of the TPS/TPP family members showed the highest expression in mature leaves, and promoter-reporter gene studies suggest that the two class I TPS genes have largely overlapping expression patterns within the vasculature, with only subtle differences in expression in fruits and flowers.
The majority of tomato TPS/TPP genes were induced by heat stress, and individual family members also responded to cold.
This suggests that trehalose biosynthetic pathway genes could play an important role during temperature stress adaptation.
In summary, our study represents a further step toward the exploitation of the TPS and TPP gene families for the improvement of tomato stress resistance.
Pichia pastoris (syn. Komagataella phaffii) represents a commonly used expression system in the biotech industry. High clonal variation of transformants, however, typically results in a broad range of specific productivities for secreted proteins. To isolate rare clones with exceedingly high product titers, an extensive number of clones need to be screened.
In contrast to high-throughput screenings of P. pastoris clones in microtiter plates, secrete-and -capture methodologies have the potential to efficiently isolate high-producer clones among millions of cells through fluorescence-activated cell sorting (FACS).Here, we describe a novel approach for the non-covalent binding of fragment antigen-binding (Fab) proteins to the cell surface for the isolation of high-producing clones.
Eight different single-chain variable fragment (scFv)-based capture matrices specific for the constant part of the Fabs were fused to the Saccharomyces cerevisiae alpha -agglutinin (SAG1) anchor protein for surface display in P. pastoris. By encoding the capture matrix on an episomal plasmid harboring inherently unstable autonomously replicating sequences (ARS), this secrete-and -capture system offers a switchable scFv display.
Efficient plasmid clearance upon removal of selective pres-sure enabled the direct use of isolated clones for subsequent Fab production. Flow-sorted clones (n = 276) displaying high amounts of Fabs showed a significant increase in median Fab titers detected in the cell-free supernatant (CFS) compared to unsorted clones (n = 276) when cells were cultivated in microtiter plates (fac-tor in the range of-21-49). Fab titers of clones exhibiting the highest product titer observed for each of the two approaches were increased by up to 8-fold for the sorted clone.
Improved Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask cultivation of selected candidates (factor in the range of-2-3). Hence, the developed display-based selection method proved to be a valuable tool for efficient clone screening in the early stages of our bioprocess development.
Many proteins are anchored to the cell surface of eukaryotes using a unique family of glycolipids called glycosylphosphatidylinositol (GPI) anchors.
These glycolipids also exist without a covalently bound protein, in particular on the cell surfaces of protozoan parasites where they are densely populated.
GPIs and GPI-anchored proteins participate in multiple cellular processes such as signal transduction, cell adhesion, protein trafficking and pathogenesis of Malaria, Toxoplasmosis, Trypanosomiasis and prion diseases, among others.
All GPIs share a common conserved glycan core modified in a cell-dependent manner with additional side glycans or phosphoethanolamine residues. Here, we use atomistic molecular dynamic simulations and perform a systematic study to evaluate the structural properties of GPIs with different side chains inserted in lipid bilayers.
Our results show a flop-down orientation of GPIs with respect to the membrane surface and the presentation of the side chain residues to the solvent.
This finding agrees well with experiments showing the role of the side residues as active epitopes for recognition of GPIs by macrophages and induction of GPI-glycan-specific immune responses.
Protein-GPI interactions were investigated by attaching parasitic GPIs to Green Fluorescent Protein. GPIs are observed to recline on the membrane surface and pull down the attached protein close to the membrane facilitating mutual contacts between protein, GPI and the lipid bilayer.
This model is efficient in evaluating the interaction of GPIs and GPI-anchored proteins with membranes and can be extended to study other parasitic GPIs and proteins and develop GPI-based immunoprophylaxis to treat infectious diseases.
The debate whether single large or several small (SLOSS) patches benefit biodiversity has existed for decades, but recent literature provides increasing evidence for the importance of small habitats.
Possible beneficial mechanisms include reduced presence of preda-tors and competitors in small habitat areas or specific functions such as stepping stones for dispersal.
Given the increasing amount of studies highlighting individual behavioral differences that may influence these functions, we hypothesize that the advantage of small versus large habitat patches not only depends on patch functionality but also on the presence of animal personalities (i.e., risk-tolerant vs. risk-averse). Using an individual-based, spatially-explicit community model, we analyzed the diversity of mammal communities in landscapes consisting of a few large habitat islands interspersed with different amounts and sizes of small habitat patches.
Within these heterogeneous environments, individuals compete for resources and form home-ranges, with only risk-tolerant individuals using habitat edges. Results show that when risk-tolerant individuals exist, small patches increase species diversity. A strong peak occurs at approximately 20% habitat cover in small patches when those small habitats are only used for foraging but not for breeding and home-range core position. Additional usage as stepping stones for juvenile dispersal further increases species persistence. Over-all, our results reveal that a combination of a few large and several small habitat patches promotes biodiversity by enhancing land-scape heterogeneity.
Here, heterogeneity is created by pronounced differences in habitat functionality, increasing edge density, and variability in habitat use by different behavioral types. The finding that a combination of single large AND several small (SLASS) patches is needed for effective biodiversity preservation has implications for advancing landscape conservation.
Particularly in struc-turally poor agricultural areas, modern technology enables precise management with the opportunity to create small foraging habitats by excluding less profitable agricultural land from cultivation.
The deficiency of a (bio)chemical reaction network can be conceptually interpreted as a measure of its ability to support exotic dynamical behavior and/or multistationarity. The classical definition of deficiency relates to the capacity of a network to permit variations of the complex formation rate vector at steady state, irrespective of the network kinetics. However, the deficiency is by definition completely insensitive to the fine details of the directionality of reactions as well as bounds on reaction fluxes. While the classical definition of deficiency can be readily applied in the analysis of unconstrained, weakly reversible networks, it only provides an upper bound in the cases where relevant constraints on reaction fluxes are imposed. Here we propose the concept of effective deficiency, which provides a more accurate assessment of the network’s capacity to permit steady state variations at the complex level for constrained networks of any reversibility patterns. The effective deficiency relies on the concept of nonstoichiometric balanced complexes, which we have already shown to be present in real-world biochemical networks operating under flux constraints. Our results demonstrate that the effective deficiency of real-world biochemical networks is smaller than the classical deficiency, indicating the effects of reaction directionality and flux bounds on the variation of the complex formation rate vector at steady state.
Introduction Flux phenotypes from different organisms and growth conditions allow better understanding of differential metabolic networks functions. Fluxes of metabolic reactions represent the integrated outcome of transcription, translation, and post-translational modifications, and directly affect growth and fitness. However, fluxes of intracellular metabolic reactions cannot be directly measured, but are estimated via metabolic flux analysis (MFA) that integrates data on isotope labeling patterns of metabolites with metabolic models. While the application of metabolomics technologies in photosynthetic organisms have resulted in unprecedented data from 13CO2-labeling experiments, the bottleneck in flux estimation remains the application of isotopically nonstationary MFA (INST-MFA). INST-MFA entails fitting a (large) system of coupled ordinary differential equations, with metabolite pools and reaction fluxes as parameters. Here, we focus on the Calvin-Benson cycle (CBC) as a key pathway for carbon fixation in photosynthesizing organisms and ask if approaches other than classical INST-MFA can provide reliable estimation of fluxes for reactions comprising this pathway.
Methods First, we show that flux estimation with the labeling patterns of all CBC intermediates can be formulated as a single constrained regression problem, avoiding the need for repeated simulation of time-resolved labeling patterns.
Results We then compare the flux estimates of the simulation-free constrained regression approach with those obtained from the classical INST-MFA based on labeling patterns of metabolites from the microalgae Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii under different growth conditions.
Discussion Our findings indicate that, in data-rich scenarios, simulation-free regression-based approaches provide a suitable alternative for flux estimation from classical INST-MFA since we observe a high qualitative agreement (rs=0.89) to predictions obtained from INCA, a state-of-the-art tool for INST-MFA.