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Plants are often challenged by an array of unfavorable environmental conditions. During cold exposure, many changes occur that include, for example, the stabilization of cell membranes, alterations in gene expression and enzyme activities, as well as the accumulation of metabolites. In the presented study, the carbohydrate metabolism was analyzed in the very early response of plants to a low temperature (2 degrees C) in the leaves of 5-week-old potato plants of the Russet Burbank cultivar during the first 12 h of cold treatment (2 h dark and 10 h light). First, some plant stress indicators were examined and it was shown that short-term cold exposure did not significantly affect the relative water content and chlorophyll content (only after 12 h), but caused an increase in malondialdehyde concentration and a decrease in the expression of NDA1, a homolog of the NADH dehydrogenase gene. In addition, it was shown that the content of transitory starch increased transiently in the very early phase of the plant response (3-6 h) to cold treatment, and then its decrease was observed after 12 h. In contrast, soluble sugars such as glucose and fructose were significantly increased only at the end of the light period, where a decrease in sucrose content was observed. The availability of the monosaccharides at constitutively high levels, regardless of the temperature, may delay the response to cold, involving amylolytic starch degradation in chloroplasts. The decrease in starch content, observed in leaves after 12 h of cold exposure, was preceded by a dramatic increase in the transcript levels of the key enzymes of starch degradation initiation, the alpha-glucan, water dikinase (GWD-EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD-EC 2.7.9.5). The gene expression of both dikinases peaked at 9 h of cold exposure, as analyzed by real-time PCR. Moreover, enhanced activities of the acid invertase as well as of both glucan phosphorylases during exposure to a chilling temperature were observed. However, it was also noticed that during the light phase, there was a general increase in glucan phosphorylase activities for both control and cold-stressed plants irrespective of the temperature. In conclusion, a short-term cold treatment alters the carbohydrate metabolism in the leaves of potato, which leads to an increase in the content of soluble sugars.
Large-scale biochemical models are of increasing sizes due to the consideration of interacting organisms and tissues. Model reduction approaches that preserve the flux phenotypes can simplify the analysis and predictions of steady-state metabolic phenotypes. However, existing approaches either restrict functionality of reduced models or do not lead to significant decreases in the number of modelled metabolites. Here, we introduce an approach for model reduction based on the structural property of balancing of complexes that preserves the steady-state fluxes supported by the network and can be efficiently determined at genome scale. Using two large-scale mass-action kinetic models of Escherichia coli, we show that our approach results in a substantial reduction of 99% of metabolites. Applications to genome-scale metabolic models across kingdoms of life result in up to 55% and 85% reduction in the number of metabolites when arbitrary and mass-action kinetics is assumed, respectively. We also show that predictions of the specific growth rate from the reduced models match those based on the original models. Since steady-state flux phenotypes from the original model are preserved in the reduced, the approach paves the way for analysing other metabolic phenotypes in large-scale biochemical networks.
Synthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized hirudin showed a considerably higher anti-thrombin activity compared to recombinant hirudin produced in bacterial cells.
Transposable elements (TEs) are loci that can replicate and multiply within the genome of their host. Within the host, TEs through transposition are responsible for variation on genomic architecture and gene regulation across all vertebrates. Genome assemblies have increased in numbers in recent years. However, to explore in deep the variations within different genomes, such as SNPs (single nucleotide polymorphism), INDELs (Insertion-deletion), satellites and transposable elements, we need high-quality genomes. Studies of molecular markers in the past 10 years have limitations to correlate with biological differences because molecular markers rely on the accuracy of the genomic resources. This has generated that a substantial part of the studies of TE in recent years have been on high quality genomic resources such as Drosophila, zebrafinch and maize. As testudine have a slow mutation rate lower only to crocodilians, with more than 300 species, adapted to different environments all across the globe, the testudine clade can help us to study variation. Here we propose Testudines as a clade to study variation and the abundance of TE on different species that diverged a long time ago. We investigated the genomic diversity of sea turtles, identifying key genomic regions associated to gene family duplication, specific expansion of particular TE families for Dermochelyidae and that are important for phenotypic differentiation, the impact of environmental changes on their populations, and the dynamics of TEs within different lineages. In chapter 1, we identify that despite high levels of genome synteny within sea turtles, we identified that regions of reduced collinearity and microchromosomes showed higher concentrations of multicopy gene families, as well as genetic distances between species, indicating their potential importance as sources of variation underlying phenotypic differentiation. We found that differences in the ecological niches occupied by leatherback and green turtles have led to contrasting evolutionary paths for their olfactory receptor genes. We identified in leatherback turtles a long-term low population size. Nonetheless, we identify no correlation between the regions of reduced collinearity with abundance of TEs or an accumulation of a particular TE group. In chapter 2, we identified that sea turtle genomes contain a significant proportion of TEs, with differences in TE abundance between species, and the discovery of a recent expansion of Penelope-like elements (PLEs) in the highly conserved sea turtle genome provides new insights into the dynamics of TEs within Testudines. In chapter 3, we compared the proportion of TE across the Testudine clade, and we identified that the proportion of transposable elements within the clade is stable, regardless of the quality of the assemblies. However, we identified that the proportion of TEs orders has correlation with genome quality depending of their expanded abundancy. For retrotransposon, a highly abundant element for this clade, we identify no correlation. However, for DNA elements a rarer element on this clade, correlate with the quality of the assemblies.
Here we confirm that high-quality genomes are fundamental for the study of transposable element evolution and the conservation within the clade. The detection and abundance of specific orders of TEs are influenced by the quality of the genomes. We identified that a reduction in the population size on D. coriacea had left signals of long-term low population sizes on their genomes. On the same note we identified an expansion of TE on D. coriacea, not present in any other member of the available genomes of Testudines, strongly suggesting that it is a response of deregulation of TE on their genomes as consequences of the low population sizes.
Here we have identified important genomic regions and gene families for phenotypic differentiation and highlighted the impact of environmental changes on the populations of sea turtles. We stated that accurate classification and analysis of TE families are important and require high-quality genome assemblies. Using TE analysis we manage to identify differences in highly syntenic species. These findings have significant implications for conservation and provide a foundation for further research into genome evolution and gene function in turtles and other vertebrates. Overall, this study contributes to our understanding of evolutionary change and adaptation mechanisms.
Reaction lumping in metabolic networks for application with thermodynamic metabolic flux analysis
(2021)
Thermodynamic metabolic flux analysis (TMFA) can narrow down the space of steady-state flux distributions, but requires knowledge of the standard Gibbs free energy for the modelled reactions. The latter are often not available due to unknown Gibbs free energy change of formation ,Delta fG0, of metabolites. To optimize the usage of data on thermodynamics in constraining a model, reaction lumping has been proposed to eliminate metabolites with unknown Delta fG0. However, the lumping procedure has not been formalized nor implemented for systematic identification of lumped reactions. Here, we propose, implement, and test a combined procedure for reaction lumping, applicable to genome-scale metabolic models. It is based on identification of groups of metabolites with unknown Delta fG0 whose elimination can be conducted independently of the others via: (1) group implementation, aiming to eliminate an entire such group, and, if this is infeasible, (2) a sequential implementation to ensure that a maximal number of metabolites with unknown Delta fG0 are eliminated. Our comparative analysis with genome-scale metabolic models of Escherichia coli, Bacillus subtilis, and Homo sapiens shows that the combined procedure provides an efficient means for systematic identification of lumped reactions. We also demonstrate that TMFA applied to models with reactions lumped according to the proposed procedure lead to more precise predictions in comparison to the original models. The provided implementation thus ensures the reproducibility of the findings and their application with standard TMFA.
There has been a growing awareness that graphing is an essential part of the science curriculum. While much research has focused on student conceptions and abilities regarding graphical representations, only few studies have investigated what teachers think about them and how they use graphs in science class. The purpose of this study is to explore educational beliefs, motivation, and teaching practices of German secondary biology teachers regarding graph construction. Via questionnaire surveys, 71 teachers from different regions in Germany rated their beliefs and motivation as well as the frequency of different graph construction activities in biology class. The teachers surveyed in this study were quite motivated in their teaching of graph construction. Furthermore, they tended to believe that graph construction should be practiced explicitly in biology class and that students should learn clear strategies for constructing graphs. We found that teaching subjects and own research experience make a difference in teachers' beliefs and motivation regarding graph construction in biology class. The self-report on classroom practices revealed that participants may provide limited opportunities for students to experience graphing as a social and iterative practice. Implications are drawn for teacher education and professional development as well as for further research in teacher education contexts.
The genetic structure of Bryde's whale (Balaenoptera brydei) on the central and western North Pacific feeding grounds was investigated using a total of 1195 mitochondrial control region sequences and 1182 microsatellite genotypes at 17 loci in specimens collected from three longitudinal areas, 1W (135 degrees E-165 degrees E), 1E (165 degrees E-180 degrees), and 2 (180 degrees-155 degrees W). Genetic diversities were similar among areas and a haplotype network did not show any geographic structure, while an analysis of molecular variance found evidence of genetic structure in this species. Pairwise FST and G'ST estimates and heterogeneity tests attributed this structure to weak but significant differentiation between areas 1W/1E and 2. A Mantel test and a high-resolution analysis of genetic diversity statistics showed a weak spatial cline of genetic differentiation. These findings could be reconciled by two possible stock structure scenarios: (1) a single population with kin-association affecting feeding ground preference and (2) two populations with feeding ground preference for either area 1W or area 2. An estimated dispersal rate between areas 1W and 2 indicates that both scenarios should be considered as a precautionary principle in stock assessments.
There is an urgent need for screening of patients with a communicable viral disease to cut infection chains. Recently, we demonstrated that ion mobility spectrometry coupled with a multicapillary column (MCC-IMS) is able to identify influenza-A infections in patients' breath. With a decreasing influenza epidemic and upcoming SARS-CoV-2 infections we proceeded further and analyzed patients with suspected SARS-CoV-2 infections. In this study, the nasal breath of 75 patients (34 male, 41 female, aged 64.4 +/- 15.4 years) was investigated by MCC-IMS for viral infections. Fourteen were positively diagnosed with influenza-A infection and sixteen with SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) of nasopharyngeal swabs. In one patient RT-PCR was highly suspicious of SARS-CoV-2 but initially inconclusive. The remaining 44 patients served as controls. Breath fingerprints for specific infections were assessed by a combination of cluster analysis and multivariate statistics. There were no significant differences in gender or age according to the groups. In the cross validation of the discriminant analysis 72 of the 74 clearly defined patients could be correctly classified to the respective group. Even the inconclusive patient could be mapped to the SARS-CoV-2 group by applying the discrimination functions. Conclusion: SARS-CoV-2 infection and influenza-A infection can be detected with the help of MCC-IMS in breath in this pilot study. As this method provides a fast non-invasive diagnosis it should be further developed in a larger cohort for screening of communicable viral diseases. A validation study is ongoing during the second wave of COVID-19.
Trial registration: ClinicalTrial.gov, NCT04282135 Registered 20 February 2020-Retrospectively registered,
Floral volatiles and reward traits are major drivers for the behavior of mutualistic as well as antagonistic flower visitors, i.e., pollinators and florivores. These floral traits differ tremendously between species, but intraspecific differences and their consequences on organism interactions remain largely unknown. Floral volatile compounds, such as terpenoids, function as cues to advertise rewards to pollinators, but should at the same time also repel florivores. The reward composition, e.g., protein and lipid contents in pollen, differs between individuals of distinct plant families. Whether the nutritional value of rewards within the same plant species is linked to their chemotypes, which differ in their pattern of specialized metabolites, has yet not been investigated. In the present study, we compared Tanacetum vulgare plants of five terpenoid chemotypes with regard to flower production, floral headspace volatiles, pollen macronutrient and terpenoid content, and floral attractiveness to florivorous beetles. Our analyses revealed remarkable differences between the chemotypes in the amount and diameter of flower heads, duration of bloom period, and pollen nutritional quality. The floral headspace composition of pollen-producing mature flowers, but not of premature flowers, was correlated to that of pollen and leaves in the same plant individual. For two chemotypes, florivorous beetles discriminated between the scent of mature and premature flower heads and preferred the latter. In semi-field experiments, the abundance of florivorous beetles and flower tissue miners differed between T. vulgare chemotypes. Moreover, the scent environment affected the choice and beetles were more abundant in homogenous plots composed of one single chemotype than in plots with different neighboring chemotypes. In conclusion, flower production, floral metabolic composition and pollen quality varied to a remarkable extend within the species T. vulgare, and the attractiveness of floral scent differed also intra-individually with floral ontogeny. We found evidence for a trade-off between pollen lipid content and pollen amount on a per-plant-level. Our study highlights that chemotypes which are more susceptible to florivory are less attacked when they grow in the neighborhood of other chemotypes and thus gain a benefit from high overall chemodiversity.
Biofilms are heterogeneous structures made of microorganisms embedded in a self-secreted extracellular matrix. Recently, biofilms have been studied as sustainable living materials with a focus on the tuning of their mechanical properties. One way of doing so is to use metal ions. In particular biofilms have been shown to stiffen in presence of some metal cations and to soften in presence of others. However, the specificity and the determinants of those interactions vary between species. While Escherichia coli is a widely studied model organism, little is known concerning the response of its biofilms to metal ions. In this work, we aimed at tuning the mechanics of E. coli biofilms by acting on the interplay between matrix composition and metal cations. To do so, we worked with E. coli strains producing a matrix composed of curli amyloid fibres or phosphoethanolamine-cellulose (pEtN-cellulose) fibres or both. The viscoelastic behaviour of the resulting biofilms was investigated with rheology after incubation with one of the following metal ion solutions: FeCl3, AlCl3, ZnCl2 and CaCl2 or ultrapure water. We observed that the strain producing both fibres stiffen by a factor of two when exposed to the trivalent metal cations Al(III) and Fe(III) while no such response is observed for the bivalent cations Zn(II) and Ca(II). Strains producing only one matrix component did not show any stiffening in response to either cation, but even a small softening. In order to investigate further the contribution of each matrix component to the mechanical properties, we introduced additional bacterial strains producing curli fibres in combination with non-modified cellulose, non-modified cellulose only or neither component. We measured biofilms produced by those different strains with rheology and without any solution. Since rheology does not preserve the architecture of the matrix, we compared those results to the mechanical properties of biofilms probed with the non-destructive microindentation. The microindentation results showed that biofilm stiffness is mainly determined by the presence of curli amyloid fibres in the matrix. However, this clear distinction between biofilm matrices containing or not containing curli is absent from the rheology results, i.e. following partial destruction of the matrix architecture. In addition, rheology also indicated a negative impact of curli on biofilm yield stress and flow stress. This suggests that curli fibres are more brittle and therefore more affected by the mechanical treatments. Finally, to examine the molecular interactions between the biofilms and the metal cations, we used Attenuated total reflectance - Fourier transform infrared spectroscopy (ATR-FTIR) to study the three E.coli strains producing a matrix composed of curli amyloid fibres, pEtN-cellulose fibres or both. We measured biofilms produced by those strains in presence of each of the aforementioned metal cation solutions or ultrapure water. We showed that the three strains cannot be distinguished based on their FTIR spectra and that metal cations seem to have a non-specific effect on bacterial membranes in absence of pEtN-cellulose. We subsequently conducted similar experiments on purified curli or pEtN-cellulose fibres. The spectra of the pEtN-cellulose fibres revealed a non-valence-specific interaction between metal cations and the phosphate of the pEtN-modification. Altogether, these results demonstrate that the mechanical properties of E. coli biofilms can be tuned via incubation with metal ions. While the mechanism involving curli fibres remains to be determined, metal cations seem to adsorb onto pEtN-cellulose and this is not valence-specific. This work also underlines the importance of matrix architecture to biofilm mechanics and emphasises the specificity of each matrix composition.