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- Institut für Biochemie und Biologie (337) (entfernen)
Aldol reactions play an important role in organic synthesis, as they belong to the class of highly beneficial C-C-linking reactions. Aldol-type reactions can be efficiently and stereoselectively catalyzed by the enzyme 2-deoxy-D-ribose-5-phosphate aldolase (DERA) to gain key intermediates for pharmaceuticals such as atorvastatin. The immobilization of DERA would open the opportunity for a continuous operation mode which gives access to an efficient, large-scale production of respective organic intermediates. In this contribution, we synthesize and utilize DERA/polymer conjugates for the generation and fixation of a DERA bearing thin film on a polymeric membrane support. The conjugation strongly increases the tolerance of the enzyme toward the industrial relevant substrate acetaldehyde while UV-cross-linkable groups along the conjugated polymer chains provide the opportunity for covalent binding to the support. First, we provide a thorough characterization of the conjugates followed by immobilization tests on representative, nonporous cycloolefinic copolymer supports. Finally, immobilization on the target supports constituted of polyacrylonitrile (PAN) membranes is performed, and the resulting enzymatically active membranes are implemented in a simple membrane module setup for the first assessment of biocatalytic performance in the continuous operation mode using the combination hexanal/acetaldehyde as the substrate.
Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1(-/-) zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.
Diagnostics of Autoimmune Diseases involve screening of patient samples for containing autoantibodies against various antigens. To ensure quality of diagnostic assays a calibrator is needed in each assay system. Different calibrators as recombinant human monoclonal antibodies as well as chimeric antibodies against the autoantigens of interest are described. A less cost-intensive and also more representative possibility covering different targets on the antigens is the utilization of polyclonal sera from other species. Nevertheless, the detection of human autoantibodies as well as the calibration reagent containing antibodies from other species in one assay constitutes a challenge in terms of assay calibration. We therefore developed a cross-reactive monoclonal antibody which binds human as well as rabbit sera with similar affinities in the nanomolar range. We tested our monoclonal antibody S38CD11B12 successfully in the commercial Serazym (R) Anti-Cardiolipin-beta 2-GPI IgG/IgM assay and could thereby prove the eligibility of S38CD11B12 as detection antibody in autoimmune diagnostic assays using rabbit derived sera as reference material.
A familial congenital heart disease with a possible multigenic origin involving a mutation in BMPR1A
(2019)
The genetics of many congenital heart diseases (CHDs) can only unsatisfactorily be explained by known chromosomal or Mendelian syndromes. Here, we present sequencing data of a family with a potentially multigenic origin of CHD. Twelve of nineteen family members carry a familial mutation [NM_004329.2:c.1328 G > A (p.R443H)] which encodes a predicted deleterious variant of BMPR1A. This mutation co-segregates with a linkage region on chromosome 1 that associates with the emergence of severe CHDs including Ebstein’s anomaly, atrioventricular septal defect, and others. We show that the continuous overexpression of the zebrafish homologous mutation bmpr1aap.R438H within endocardium causes a reduced AV valve area, a downregulation of Wnt/ß-catenin signalling at the AV canal, and growth of additional tissue mass in adult zebrafish hearts. This finding opens the possibility of testing genetic interactions between BMPR1A and other candidate genes within linkage region 1 which may provide a first step towards unravelling more complex genetic patterns in cardiovascular disease aetiology.
Background: The outcrossing rate is a key determinant of the population-genetic structure of species and their long-term evolutionary trajectories. However, determining the outcrossing rate using current methods based on PCRgenotyping individual offspring of focal plants for multiple polymorphic markers is laborious and time-consuming.
Results: We have developed an amplicon-based, high-throughput enabled method for estimating the outcrossing rate and have applied this to an example of scented versus non-scented Capsella (Shepherd’s Purse) genotypes. Our results show that the method is able to robustly capture differences in outcrossing rates. They also highlight potential biases in the estimates resulting from differential haplotype sharing of the focal plants with the pollen-donor population at individual amplicons.
Conclusions: This novel method for estimating outcrossing rates will allow determining this key population-genetic parameter with high-throughput across many genotypes in a population, enabling studies into the genetic determinants of successful pollinator attraction and outcrossing.
Background: The outcrossing rate is a key determinant of the population-genetic structure of species and their long-term evolutionary trajectories. However, determining the outcrossing rate using current methods based on PCRgenotyping individual offspring of focal plants for multiple polymorphic markers is laborious and time-consuming.
Results: We have developed an amplicon-based, high-throughput enabled method for estimating the outcrossing rate and have applied this to an example of scented versus non-scented Capsella (Shepherd’s Purse) genotypes. Our results show that the method is able to robustly capture differences in outcrossing rates. They also highlight potential biases in the estimates resulting from differential haplotype sharing of the focal plants with the pollen-donor population at individual amplicons.
Conclusions: This novel method for estimating outcrossing rates will allow determining this key population-genetic parameter with high-throughput across many genotypes in a population, enabling studies into the genetic determinants of successful pollinator attraction and outcrossing.
A Metabarcoding Analysis of the Mycobiome of Wheat Ears Across a Topographically Heterogeneous Field
(2019)
By using an integrative approach, we describe a new species of mayfly, Bungona (Chopralla) pontica sp. n., from Turkey. The discovery of a representative of the tropical mayfly genus Bungona in the Middle East is rather unexpected. The new species shows all the main morphological characters of the subgenus Chopralla, which has its closest related species occurring in southeastern Asia. Barcoding clearly indicated that the new species represents an independent lineage isolated for a very long time from other members of the complex. The claw is equipped with two rows of three or four flattened denticles. This condition is a unique feature of Bungona (Chopralla) pontica sp. n. among West Palaearctic mayfly species. Within the subgenus Chopralla, the species can be identified by the presence of a simple, not bifid right prostheca (also present only in Bungona (Chopralla) liebenauae (Soldan, Braasch & Muu, 1987)), the shape of the labial palp, and the absence of protuberances on pronotum.
Most biochemical reactions depend on the pH value of the aqueous environment and some are strongly favoured to occur in an acidic environment. A non-invasive control of pH to tightly regulate such reactions with defined start and end points is a highly desirable feature in certain applications, but has proven difficult to achieve so far. We report a novel optical approach to reversibly control a typical biochemical reaction by changing the pH and using acid phosphatase as a model enzyme. The reversible photoacid G-acid functions as a proton donor, changing the pH rapidly and reversibly by using high power UV LEDs as an illumination source in our experimental setup. The reaction can be tightly controlled by simply switching the light on and off and should be applicable to a wide range of other enzymatic reactions, thus enabling miniaturization and parallelization through non-invasive optical means.
Objectives: The objectives of this study were to determine the interrater reliability (IRR) of assessment of multiple systematic reviews (AMSTAR) 2 for reviews of pharmacological or psychological interventions for the treatment of major depression, to compare it to that of AMSTAR and risk of bias in systematic reviews (ROBIS), and to assess the convergent validity between the appraisal tools. Results: The median kappa values as a measure of IRR indicated a moderate agreement for AMSTAR 2 (median = 0.51), a substantial agreement for AMSTAR (median = 0.62), and a fair agreement for ROBIS (median = 0.27). Validity results showed a positive association for AMSTAR and AMSTAR 2 (r = 0.91) as well as ROBIS and AMSTAR 2 (r = 0.84). For the overall rating, AMSTAR 2 showed a high concordance with ROBIS and a lower concordance with AMSTAR. Conclusion: The IRR of AMSTAR 2 was found to be slightly lower than the IRR of AMSTAR and higher than the IRR of ROBIS. Validity measurements indicate that AMSTAR 2 is closely related to both ROBIS and AMSTAR. (C) 2019 Elsevier Inc. All rights reserved.
As sessile life forms, plants are repeatedly confronted with adverse environmental conditions, which can impair development, growth, and reproduction. During evolution, plants have established mechanisms to orchestrate the delicate balance between growth and stress tolerance, to reset cellular biochemistry once stress vanishes, or to keep a molecular memory, which enables survival of a harsher stress that may arise later. Although there are several examples of memory in diverse plants species, the molecular machinery underlying the formation, duration, and resetting of stress memories is largely unknown so far. We report here that autophagy, a central self-degradative process, assists in resetting cellular memory of heat stress (HS) in Arabidopsis thaliana. Autophagy is induced by thermopriming (moderate HS) and, intriguingly, remains high long after stress termination. We demonstrate that autophagy mediates the specific degradation of heat shock proteins at later stages of the thermorecovery phase leading to the accumulation of protein aggregates after the second HS and a compromised heat tolerance. Autophagy mutants retain heat shock proteins longer than wild type and concomitantly display improved thermomemory. Our findings reveal a novel regulatory mechanism for HS memory in plants.
The plasma membrane (PM) is at the interface of plant-pathogen interactions and, thus, many bacterial type-III effector (T3E) proteins target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E HopZ1a is a host cell PM-localized effector protein that has several immunity-associated host targets but also activates effector-triggered immunity in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated PM-associated HopZ1a target protein has been identified until now. Here, we show that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 relocalizes to membrane nanodomains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense-related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase avrPphB-susceptible 1 (PBS1) and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a-NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed.
Salinity is a significant factor for structuring microbial communities, but little is known for aquatic fungi, particularly in the pelagic zone of brackish ecosystems. In this study, we explored the diversity and composition of fungal communities following a progressive salinity decline (from 34 to 3 PSU) along three transects of ca. 2000 km in the Baltic Sea, the world’s largest estuary. Based on 18S rRNA gene sequence analysis, we detected clear changes in fungal community composition along the salinity gradient and found significant differences in composition of fungal communities established above and below a critical value of 8 PSU. At salinities below this threshold, fungal communities resembled those from freshwater environments, with a greater abundance of Chytridiomycota, particularly of the orders Rhizophydiales, Lobulomycetales, and
Gromochytriales. At salinities above 8 PSU, communities were more similar to those from marine environments and, depending on the season, were dominated by a strain of the LKM11 group (Cryptomycota) or by members of Ascomycota and Basidiomycota. Our results highlight salinity as an important environmental driver also for pelagic fungi, and thus should be taken into account to better understand fungal diversity and ecological function in the aquatic realm.
Salinity is a significant factor for structuring microbial communities, but little is known for aquatic fungi, particularly in the pelagic zone of brackish ecosystems. In this study, we explored the diversity and composition of fungal communities following a progressive salinity decline (from 34 to 3 PSU) along three transects of ca. 2000 km in the Baltic Sea, the world’s largest estuary. Based on 18S rRNA gene sequence analysis, we detected clear changes in fungal community composition along the salinity gradient and found significant differences in composition of fungal communities established above and below a critical value of 8 PSU. At salinities below this threshold, fungal communities resembled those from freshwater environments, with a greater abundance of Chytridiomycota, particularly of the orders Rhizophydiales, Lobulomycetales, and
Gromochytriales. At salinities above 8 PSU, communities were more similar to those from marine environments and, depending on the season, were dominated by a strain of the LKM11 group (Cryptomycota) or by members of Ascomycota and Basidiomycota. Our results highlight salinity as an important environmental driver also for pelagic fungi, and thus should be taken into account to better understand fungal diversity and ecological function in the aquatic realm.
Being at the western fringe of Europe, Iberia had a peculiar prehistory and a complex pattern of Neolithization. A few studies, all based on modern populations, reported the presence of DNA of likely African origin in this region, generally concluding it was the result of recent gene flow, probably during the Islamic period. Here, we provide evidence of much older gene flow from Africa to Iberia by sequencing whole genomes from four human remains from northern Portugal and southern Spain dated around 4000 years BP (from the Middle Neolithic to the Bronze Age). We found one of them to carry an unequivocal sub-Saharan mitogenome of most probably West or West-Central African origin, to our knowledge never reported before in prehistoric remains outside Africa. Our analyses of ancient nuclear genomes show small but significant levels of sub-Saharan African affinity in several ancient Iberian samples, which indicates that what we detected was not an occasional individual phenomenon, but an admixture event recognizable at the population level. We interpret this result as evidence of an early migration process from Africa into the Iberian Peninsula through a western route, possibly across the Strait of Gibraltar.
Zinc is an essential trace element, making it crucial to have a reliable biomarker for evaluating an individual’s zinc status. The total serum zinc concentration, which is presently the most commonly used biomarker, is not ideal for this purpose, but a superior alternative is still missing. The free zinc concentration, which describes the fraction of zinc that is only loosely bound and easily exchangeable, has been proposed for this purpose, as it reflects the highly bioavailable part of serum zinc. This report presents a fluorescence-based method for determining the free zinc concentration in human serum samples, using the fluorescent probe Zinpyr-1. The assay has been applied on 154 commercially obtained human serum samples. Measured free zinc concentrations ranged from 0.09 to 0.42 nM with a mean of 0.22 ± 0.05 nM. It did not correlate with age or the total serum concentrations of zinc, manganese, iron or selenium. A negative correlation between the concentration of free zinc and total copper has been seen for sera from females. In addition, the free zinc concentration in sera from females (0.21 ± 0.05 nM) was significantly lower than in males (0.23 ± 0.06 nM). The assay uses a sample volume of less than 10 µL, is rapid and cost-effective and allows us to address questions regarding factors influencing the free serum zinc concentration, its connection with the body’s zinc status, and its suitability as a future biomarker for an individual’s zinc status.
Recent investigations propose the acid sphingomyelinase (ASM)/ceramide system as a novel target for antidepressant action. ASM catalyzes the breakdown of the abundant membrane lipid sphingomyelin to the lipid messenger ceramide. This ASM‐induced lipid modification induces a local shift in membrane properties, which influences receptor clustering and downstream signaling. Canonical transient receptor potential channels 6 (TRPC6) are non‐selective cation channels located in the cell membrane that play an important role in dendritic growth, synaptic plasticity and cognition in the brain. They can be activated by hyperforin, an ingredient of the herbal remedy St. John’s wort for treatment of depression disorders. Because of their role in the context of major depression, we investigated the crosstalk between the ASM/ceramide system and TRPC6 ion channels in a pheochromocytoma cell line 12 neuronal cell model (PC12 rat pheochromocytoma cell line). Ca2+ imaging experiments indicated that hyperforin‐induced Ca2+ influx through TRPC6 channels is modulated by ASM activity. While antidepressants, known as functional inhibitors of ASM activity, reduced TRPC6‐mediated Ca2+ influx, extracellular application of bacterial sphingomyelinase rebalanced TRPC6 activity in a concentration‐related way. This effect was confirmed in whole‐cell patch clamp electrophysiology recordings. Lipidomic analyses revealed a decrease in very long chain ceramide/sphingomyelin molar ratio after ASM inhibition, which was connected with changes in the abundance of TRPC6 channels in flotillin‐1–positive lipid rafts as visualized by western blotting. Our data provide evidence that the ASM/ceramide system regulates TRPC6 channels likely by controlling their recruitment to specific lipid subdomains and thereby fine‐tuning their physical properties.
Farber disease is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments for Farber disease are clinically available, and affected patients have a severely shortened lifespan. We have recently reported a novel acid ceramidase deficiency model that mirrors the human disease closely. Acid sphingomyelinase is the enzyme that generates ceramide upstream of acid ceramidase in the lysosomes. Using our acid ceramidase deficiency model, we tested if acid sphingomyelinase could be a potential novel therapeutic target for the treatment of Farber disease. A number of functional acid sphingomyelinase inhibitors are clinically available and have been used for decades to treat major depression. Using these as a therapeutic for Farber disease, thus, has the potential to improve central nervous symptoms of the disease as well, something all other treatment options for Farber disease can’t achieve so far. As a proof-of-concept study, we first cross-bred acid ceramidase deficient mice with acid sphingomyelinase deficient mice in order to prevent ceramide accumulation. Double-deficient mice had reduced ceramide accumulation, fewer disease manifestations, and prolonged survival. We next targeted acid sphingomyelinase pharmacologically, to test if these findings would translate to a setting with clinical applicability. Surprisingly, the treatment of acid ceramidase deficient mice with the acid sphingomyelinase inhibitor amitriptyline was toxic to acid ceramidase deficient mice and killed them within a few days of treatment. In conclusion, our study provides the first proof-of-concept that acid sphingomyelinase could be a potential new therapeutic target for Farber disease to reduce disease manifestations and prolong survival. However, we also identified previously unknown toxicity of the functional acid sphingomyelinase inhibitor amitriptyline in the context of Farber disease, strongly cautioning against the use of this substance class for Farber disease patients.
The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.
The importance of cryptic diversity in rotifers is well understood regarding its ecological consequences, but there remains an in depth comprehension of the underlying molecular mechanisms and forces driving speciation. Temperature has been found several times to affect species spatio-temporal distribution and organisms’ performance, but we lack information on the mechanisms that provide thermal tolerance to rotifers. High cryptic diversity was found recently in the freshwater rotifer “Brachionus calyciflorus”, showing that the complex comprises at least four species: B. calyciflorus sensu stricto (s.s.), B. fernandoi, B. dorcas, and B. elevatus. The temporal succession among species which have been observed in sympatry led to the idea that temperature might play a crucial role in species differentiation.
The central aim of this study was to unravel differences in thermal tolerance between species of the former B. calyciflorus species complex by comparing phenotypic and gene expression responses. More specifically, I used the critical maximum temperature as a proxy for inter-species differences in heat-tolerance; this was modeled as a bi-dimensional phenotypic trait taking into consideration the intention and the duration of heat stress. Significant differences on heat-tolerance between species were detected, with B. calyciflorus s.s. being able to tolerate higher temperatures than B. fernandoi.
Based on evidence of within species neutral genetic variation, I further examined adaptive genetic variability within two different mtDNA lineages of the heat tolerant B. calyciflorus s.s. to identify SNPs and genes under selection that might reflect their adaptive history. These analyses did not reveal adaptive genetic variation related to heat, however, they show putatively adaptive genetic variation which may reflect local adaptation. Functional enrichment of putatively positively selected genes revealed signals of adaptation in genes related to “lipid metabolism”, “xenobiotics biodegradation and metabolism” and “sensory system”, comprising candidate genes which can be utilized in studies on local adaptation. An absence of genetically-based differences in thermal adaptation between the two mtDNA lineages, together with our knowledge that B. calyciflorus s.s. can withstand a broad range of temperatures, led to the idea to further investigate shared transcriptomic responses to long-term exposure to high and low temperatures regimes. With this, I identified candidate genes that are involved in the response to temperature imposed stress. Lastly, I used comparative transcriptomics to examine responses to imposed heat-stress in heat-tolerant and heat-sensitive Brachionus species. I found considerably different patterns of gene expression in the two species. Most striking are patterns of expression regarding the heat shock proteins (hsps) between the two species. In the heat-tolerant, B. calyciflorus s.s., significant up-regulation of hsps at low temperatures was indicative of a stress response at the cooler end of the temperature regimes tested here. In contrast, in the heat-sensitive B. fernandoi, hsps generally exhibited up-regulation of these genes along with rising temperatures. Overall, identification of differences in expression of genes suggests suppression of protein biosynthesis to be a mechanism to increase thermal tolerance. Observed patterns in population growth are correlated with the hsp gene expression differences, indicating that this physiological stress response is indeed related to phenotypic life history performance.
Biological responses to climate change have been widely documented across taxa and regions, but it remains unclear whether species are maintaining a good match between phenotype and environment, i.e. whether observed trait changes are adaptive. Here we reviewed 10,090 abstracts and extracted data from 71 studies reported in 58 relevant publications, to assess quantitatively whether phenotypic trait changes associated with climate change are adaptive in animals. A meta-analysis focussing on birds, the taxon best represented in our dataset, suggests that global warming has not systematically affected morphological traits, but has advanced phenological traits. We demonstrate that these advances are adaptive for some species, but imperfect as evidenced by the observed consistent selection for earlier timing. Application of a theoretical model indicates that the evolutionary load imposed by incomplete adaptive responses to ongoing climate change may already be threatening the persistence of species.
While Africa’s lacustrine gastropod fauna, in particular of Lake Tanganyika, has received much attention, the continent’s riverine malacofauna has long been neglected. Pseudocleopatra is a relatively poorly known paludomid gastropod genus with species found throughout the lower reaches of the West African Volta and Congo rivers. In the course of ongoing systematic revisions of African paludomids, we present here a morphometric analysis and revision of the recent species assigned to the genus, i.e., P. togoensis, P. voltana, P. dartevellei and P. bennikei, to improve taxonomic acuity for this group. We use available museum material for geometric morphometrics, multivariate ratio analysis and comparisons of radular and opercular characters. Our results demonstrate that the four recent species of Pseudocleopatra are clearly distinguishable on the basis of ratios of shell measurements as well as radular and opercular characters. Pseudocleopatra has generic-level synapomorphies including: concentric opercula with relatively large paucispiral nuclei, and rachidian radular teeth with usually 13–20 cusps. On the basis of this characterisation, the nominal species Cleopatra broecki is transferred to Pseudocleopatra. Additionally, the nominal taxon P. bennikei is synonymized with P. broecki n. comb. The phylogenetic relationships of Pseudocleopatra are currently unknown, but the observed tendency of some fossil taxa assigned to Pseudocleopatra towards thalassoidism, i.e., the resemblance to marine gastropods, has led to the hypothesis that some of the thalassoid endemics in Lake Tanganyika may have originated from or be related to Pseudocleopatra. Should this hypothesis be correct, which is in need of testing by molecular genetic methods when suitable samples become available, Pseudocleopatra may play a crucial role in understanding of the evolution of thalassoidism in African Paludomidae.
There is an increasing need for an assessment of the impacts of land use and land use change (LUCC). In this context, simulation models are valuable tools for investigating the impacts of stakeholder actions or policy decisions. Agricultural landscape generators (ALGs), which systematically and automatically generate realistic but simplified representations of land cover in agricultural landscapes, can provide the input for LUCC models. We reviewed existing ALGs in terms of their objectives, design and scope. We found eight ALGs that met our definition. They were based either on generic mathematical algorithms (pattern-based) or on representations of ecological or land use processes (process-based). Most ALGs integrate only a few landscape metrics, which limits the design of the landscape pattern and thus the range of applications. For example, only a few specific farming systems have been implemented. We conclude that existing ALGs contain useful approaches that can be used for specific purposes, but ideally generic modular ALGs are developed that can be used for a wide range of scenarios, regions and model types. We have compiled features of such generic ALGs and propose a possible software architecture. Considerable joint efforts are required to develop such generic ALGs, but the benefits in terms of a better understanding and development of more efficient agricultural policies would be high.
Plants are in permanent contact with various microorganisms and are always impacted by them. To better understand the first steps of a plant’s recognition of soil-borne microorganisms, the early release of volatile organic compounds (VOCs) emitted from roots of Medicago truncatula in response to the symbiont Rhizophagus irregularis or the pathogenic oomycete Aphanomyces euteiches was analysed. More than 90 compounds were released from roots as detected by an untargeted gas chromatography-mass spectrometry approach. Principal component analyses clearly distinguished untreated roots from roots treated with either R. irregularis or A. euteiches. Several VOCs were found to be emitted specifically in response to each of the microorganisms. Limonene was specifically emitted from wild-type roots after contact with R. irregularis spores but not from roots of the mycorrhiza-deficient mutant does not make infections3. The application of limonene to mycorrhizal roots, however, did not affect the mycorrhization rate. Inoculation of roots with A. euteiches zoospores resulted in the specific emission of several sesquiterpenes, such as nerolidol, viridiflorol and nerolidol-epoxyacetate but application of nerolidol to zoospores of A. euteiches did not affect their vitality. Therefore, plants discriminate between different microorganisms at early stages of their interaction and respond differently to the level of root-emitted volatiles.
Global warming has profound effects on plant growth and fitness. Plants have evolved sophisticated epigenetic machinery to respond quickly to heat, and exhibit transgenerational memory of the heat-induced release of post-transcriptional gene silencing (PTGS). However, how thermomemory is transmitted to progeny and the physiological relevance are elusive. Here we show that heat-induced HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2) directly activates the H3K27me3 demethylase RELATIVE OF EARLY FLOWERING 6 (REF6), which in turn derepresses HSFA2. REF6 and HSFA2 establish a heritable feedback loop, and activate an E3 ubiquitin ligase, SUPPRESSOR OF GENE SILENCING 3 (SGS3)-INTERACTING PROTEIN 1 (SGIP1). SGIP1-mediated SGS3 degradation leads to inhibited biosynthesis of trans-acting siRNA (tasiRNA). The REF6-HSFA2 loop and reduced tasiRNA converge to release HEAT-INDUCED TAS1 TARGET 5 (HTT5), which drives early flowering but attenuates immunity. Thus, heat induces transmitted phenotypes via a coordinated epigenetic network involving histone demethylases, transcription factors, and tasiRNAs, ensuring reproductive success and transgenerational stress adaptation.
The human intestinal anaerobe Eubacterium ramulus is known for its ability to degrade various dietary flavonoids. In the present study, we demonstrate the cleavage of the heterocyclic C-ring of flavanones and flavanonols by an oxygen-sensitive NADH-dependent reductase, previously described as enoate reductase, from E. ramulus. This flavanone- and flavanonol-cleaving reductase (Fcr) was purified following its heterologous expression in Escherichia coli and further characterized. Fcr cleaved the flavanones naringenin, eriodictyol, liquiritigenin, and homoeriodictyol. Moreover, the flavanonols taxifolin and dihydrokaempferol served as substrates. The catalyzed reactions were stereospecific for the (2R)-enantiomers of the flavanone substrates and for the (25,35)-configured flavanonols. The enantioenrichment of the nonconverted stereoisomers allowed for the determination of hitherto unknown flavanone racemization rates. Fcr formed the corresponding dihydrochalcones and hydroxydihydrochalcones in the course of an unusual reductive cleavage of cyclic ether bonds. Fcr did not convert members of other flavonoid subclasses, including flavones, flavonols, and chalcones, the latter indicating that the reaction does not involve a chalcone intermediate. This view is strongly supported by the observed enantiospecificity of Fcr. Cinnamic acids, which are typical substrates of bacterial enoate reductases, were also not reduced by Fcr. Based on the presence of binding motifs for dinucleotide cofactors and a 4Fe-4S cluster in the amino acid sequence of Fcr, a cofactor-mediated hydride transfer from NADH onto C-2 of the respective substrate is proposed. IMPORTANCE Gut bacteria play a crucial role in the metabolism of dietary flavonoids, thereby contributing to their activation or inactivation after ingestion by the human host. Thus, bacterial activities in the intestine may influence the beneficial health effects of these polyphenolic plant compounds. While an increasing number of flavonoid-converting gut bacterial species have been identified, knowledge of the responsible enzymes is still limited. Here, we characterized Fcr as a key enzyme involved in the conversion of flavonoids of several subclasses by Eubacterium ramulus, a prevalent human gut bacterium. Sequence similarity of this enzyme to hypothetical proteins from other flavonoid-degrading intestinal bacteria in databases suggests a more widespread occurrence of this enzyme. Functional characterization of gene products of human intestinal microbiota enables the assignment of metagenomic sequences to specific bacteria and, more importantly, to certain activities, which is a prerequisite for targeted modulation of gut microbial functionality.
Analysis of supramolecular assemblies of NE81, the first lamin protein in a non-metazoan organism
(2019)
Nuclear lamins are nucleus-specific intermediate filaments forming a network located at the inner nuclear membrane of the nuclear envelope. They form the nuclear lamina together with proteins of the inner nuclear membrane regulating nuclear shape and gene expression, among others. The amoebozoan Dictyostelium NE81 protein is a suitable candidate for an evolutionary conserved lamin protein in this non-metazoan organism. It shares the domain organization of metazoan lamins and is fulfilling major lamin functions in Dictyostelium. Moreover, field-emission scanning electron microscopy (feSEM) images of NE81 expressed on Xenopus oocytes nuclei revealed filamentous structures with an overall appearance highly reminiscent to that of metazoan Xenopus lamin B2. For the classification as a lamin-like or a bona fide lamin protein, a better understanding of the supramolecular NE81 structure was necessary. Yet, NE81 carrying a large N-terminal GFP-tag turned out as unsuitable source for protein isolation and characterization; GFP-NE81 expressed in Dictyostelium NE81 knock-out cells exhibited an abnormal distribution, which is an indicator for an inaccurate assembly of GFP-tagged NE81. Hence, a shorter 8×HisMyc construct was the tag of choice to investi-gate formation and structure of NE81 assemblies. One strategy was the structural analysis of NE81 in situ at the outer nuclear membrane in Dictyostelium cells; NE81 without a func-tional nuclear localization signal (NLS) forms assemblies at the outer face of the nucleus. Ultrastructural feSEM pictures of NE81ΔNLS nuclei showed a few filaments of the expected size but no repetitive filamentous structures. The former strategy should also be established for metazoan lamins in order to facilitate their structural analysis. However, heterologously expressed Xenopus and C. elegans lamins showed no uniform localization at the outer nucle-ar envelope of Dictyostelium and hence, no further ultrastructural analysis was undertaken. For in vitro assembly experiments a Dictyostelium mutant was generated, expressing NE81 without the NLS and the membrane-anchoring isoprenylation site (HisMyc-NE81ΔNLSΔCLIM). The cytosolic NE81 clusters were soluble at high ionic strength and were purified from Dictyostelium extracts using Ni-NTA Agarose. Widefield immunofluorescence microscopy, super-resolution light microscopy and electron microscopy images of purified NE81 showed its capability to form filamentous structures at low ionic strength, as described previously for metazoan lamins. Introduction of a phosphomimetic point mutation (S122E) into the CDK1-consensus sequence of NE81 led to disassembled NE81 protein in vivo, which could be reversibly stimulated to form supramolecular assemblies by blue light exposure.
The results of this work reveal that NE81 has to be considered a bona fide lamin, since it is able to form filamentous assemblies. Furthermore, they highlight Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope containing all rele-vant protein components known in animal cells.
The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the L-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm(5)s(2) U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm(5)s(2)U thio-modified tRNAs were not detectable. Because the L-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.
Multidrug resistant (MDR) Pseudomonas aeruginosa having strong biofilm potential and virulence factors are a serious threat for hospitalized patients having compromised immunity In this study, 34 P. aeruginosa isolates of human origin (17 MDR and 17 non-MDR clinical isolates) were checked for biofilm formation potential in enriched and minimal media. The biofilms were detected using crystal violet method and a modified software package of the automated VideoScan screening method. Cytotoxic potential of the isolates was also investigated on HepG2, LoVo and T24 cell lines using automated VideoScan technology. Pulse field gel electrophoresis revealed 10 PFGE types in MDR and 8 in non-MDR isolates. Although all isolates showed biofilm formation potential, strong biofilm formation was found more in enriched media than in minimal media. Eight MDR isolates showed strong biofilm potential in both enriched and minimal media by both detection methods. Strong direct correlation between crystal violet and VideoScan methods was observed in identifying strong biofilm forming isolates. High cytotoxic effect was observed by 4 isolates in all cell lines used while 6 other isolates showed high cytotoxic effect on T24 cell line only. Strong association of multidrug resistance was found with biofilm formation as strong biofilms were observed significantly higher in MDR isolates (p-value < 0.05) than non-MDR isolates. No significant association of cytotoxic potential with multidrug resistance or biofilm formation was found (p-value > 0.05). The MDR isolates showing significant cytotoxic effects and strong biofilm formation impose a serious threat for hospitalized patients with weak immune system.
Premise Hybridization is a key process in plant speciation. Despite its importance, there is no detailed study of hybridization rates in fern populations. A proper estimate of hybridization rates is needed to understand factors regulating hybridization. Methods We studied hybridization in the European Dryopteris carthusiana group, represented by one diploid and two tetraploid species and their hybrids. We sampled 100 individuals per population in 40 mixed populations of the D. carthusiana group across Europe. All plants were identified by measuring genome size (DAPI staining) using flow cytometry. To determine the maternal parentage of hybrids, we sequenced the chloroplast region trnL-trnF of all taxa involved. Results We found hybrids in 85% of populations. Triploid D. xambroseae occurred in every population that included both parent species and is most abundant when the parent species are equally abundant. By contrast, tetraploid D. xdeweveri was rare (15 individuals total) and triploid D. xsarvelae was absent. The parentage of hybrid taxa is asymmetric. Despite expectations from previous studies, tetraploid D. dilatata is the predominant male parent of its triploid hybrid. Conclusions This is a thorough investigation of hybridization rates in natural populations of ferns. Hybridization rates differ greatly even among closely related fern taxa. In contrast to angiosperms, our data suggest that hybridization rates are highest in balanced parent populations and support the notion that some ferns possess very weak barriers to hybridization. Our results from sequencing cpDNA challenge established notions about the correlation of ploidy level and mating tendencies.
The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.
The endosperm is an ephemeral tissue that nourishes the developing embryo, similar to the placenta in mammals. In most angiosperms, endosperm development starts as a syncytium, in which nuclear divisions are not followed by cytokinesis. The timing of endosperm cellularization largely varies between species, and the event triggering this transition remains unknown. Here we show that increased auxin biosynthesis in the endosperm prevents its cellularization, leading to seed arrest. Auxin-overproducing seeds phenocopy paternal-excess triploid seeds derived from hybridizations of diploid maternal plants with tetraploid fathers. Concurrently, auxin-related genes are strongly overexpressed in triploid seeds, correlating with increased auxin activity. Reducing auxin biosynthesis and signaling reestablishes endosperm cellularization in triploid seeds and restores their viability, highlighting a causal role of increased auxin in preventing endosperm cellularization. We propose that auxin determines the time of endosperm cellularization, and thereby uncovered a central role of auxin in establishing hybridization barriers in plants.
The structure of leaf veins is typically described by a hierarchical scheme (e.g. midrib, 1(st) order, 2nd order), which is used to predict variation in conduit diameter from one order to another whilst overlooking possible variation within the same order. We examined whether xylem conduit diameter changes within the same vein order, with resulting consequences for resistance to embolism. We measured the hydraulic diameter (D-h), and number of vessels (V-N) along the midrib and petioles of leaves of Acer pseudoplatanus, and estimated the leaf area supplied (A(leaf-sup)) at different points of the midrib and how variation in anatomical traits affected embolism resistance. We found that D-h scales with distance from the midrib tip (path length, L) with a power of 0.42, and that V-N scales with A(leaf-sup) with a power of 0.66. Total conductive area scales isometrically with A(leaf-sup). Embolism events along the midrib occurred first in the basipetal part and then at the leaf tip where vessels are narrower. The distance from the midrib tip is a good predictor of the variation in vessel diameter along the 1st order veins in A. pseudoplatanus leaves and this anatomical pattern seems to have an effect on hydraulic integrity since wider vessels at the leaf base embolize first.
The whereabouts of the Balaenoptera borealis holotype, the skeleton of a 1819 stranded specimen, have been unknown since the World War II (WWII). Due to nomenclatural confusion, deficient documentation, and finally WWII bombing, which destroyed predominantly cetacean material in the Museum fib Naturkunde Berlin (MfN), the type skeleton of the sei whale sank into oblivion. Construction activities enabled a recent search and study on the remaining whale material. Here, we provide evidence that the type specimen was not destroyed. On the basis of species-wide and individual characters of the type material such as the shape of cranial elements and the pattern of the maxillary foramina, we show that the skull and mandibles, the vertebral column (except the atlas), and the ribs of the holotype remain intact. Further evidence that these skeletal remains belong to the previously missing holotype is provided by the characteristics of the spine. In addition, we analyzed ancient DNA from bone samples and confirm they are B. borealis, and the occurrence of same mitochondrial haplotypes indicate that the bones belong to the same individual. Additionally, a blue inscription was discovered at the caudal epiphysis of a thoracic vertebra; historical research matched this inscription with the material belonging to the former Anatomical-Zootomical Museum, from which the holotype was once bought.
Intra-organ communication guides morphogenetic processes that are essential for an organ to carry out complex physiological functions. In the heart, the growth of the myocardium is tightly coupled to that of the endocardium, a specialized endothelial tissue that lines its interior. Several molecular pathways have been implicated in the communication between these tissues including secreted factors, components of the extracellular matrix, or proteins involved in cell-cell communication. Yet, it is unknown how the growth of the endocardium is coordinated with that of the myocardium. Here, we show that an increased expansion of the myocardial atrial chamber volume generates higher junctional forces within endocardial cells. This leads to biomechanical signaling involving VE-cadherin, triggering nuclear localization of the Hippo pathway transcriptional regulator Yap1 and endocardial proliferation. Our work suggests that the growth of the endocardium results from myocardial chamber volume expansion and ends when the tension on the tissue is relaxed.
A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5‐isomerase gene from Comamonas testosteronii, (c) a mutated steroid‐5β‐reductase gene from Arabidopsis thaliana, and (d) a steroid 21‐hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed “CARD II yeast”, was capable of producing 5β‐pregnane‐3β,21‐diol‐20‐one, a central intermediate in 5β‐cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast.
Blood Flow Suppresses Vascular Anomalies in a Zebrafish Model of Cerebral Cavernous Malformations
(2019)
RATIONALE: Pathological biomechanical signaling induces vascular anomalies including cerebral cavernous malformations (CCM), which are caused by a clonal loss of CCM1/KRIT1 (Krev interaction trapped protein 1), CCM2/MGC4607, or CCM3/PDCD10. Why patients typically experience lesions only in lowly perfused venous capillaries of the cerebrovasculature is completely unknown. OBJECTIVE: In contrast, animal models with a complete loss of CCM proteins lack a functional heart and blood flow and exhibit vascular anomalies within major blood vessels as well. This finding raises the possibility that hemodynamics may play a role in the context of this vascular pathology. METHODS AND RESULTS: Here, we used a genetic approach to restore cardiac function and blood flow in a zebrafish model of CCM1. We find that blood flow prevents cardiovascular anomalies including a hyperplastic expansion within a large Ccm1-deficient vascular bed, the lateral dorsal aorta. CONCLUSIONS: This study identifies blood flow as an important physiological factor that is protective in the cause of this devastating vascular pathology.
Background: Multiple linear correlations between parameters can be shown in correlation matrices. Correlations can be ranked, but can also be visualized in network graphs. Yet, translating a correlation matrix into a network graph is not trivial. In view of a popular child game, we propose to name this method St. Nicolas House Analysis. Material and methods: We present a new method (St. Nicolas House Analysis) that helps translating correlation matrices into network graphs. The performance of this and other network reconstruction methods was tested in randomly created virtual scale-free networks, networks consisting of bands or hubs, using balanced classification rate and the F1-Score for correctly predicting existing and non-existing edges. Thereafter we analyzed anthropometric data and information on parental education, obtained from an anthropometric survey in 908 Indonesian boys and 808 Indonesian girls. Seven parameters were analyzed: child height standard deviation score (hSDS), child BMI standard deviation scores (BMI_SDS), mid-upper-arm circumference (MUAC), mean thickness of subscapular and triceps skinfold (mean SF), and elbow breadth; as well as maternal and paternal education (years of schooling). The parameters were considered as the nodes of the network; the edges represent the correlations between the nodes. Results: Performance measures, balanced classification rate and the F1-score, showed that St. Nicolas’ House Analysis was superior to methods using sophisticated correlation value thresholds and methods based on partial correlations for analyzing bands and hubs. We applied this method also in an Indonesia data set. Ranking correlations showed the direct association between parental education and child growth. Conclusion: St. Nicolas House Analysis confirmed that growth of Indonesian school children directly depends on maternal education, with no evidence that this effect is mediated by the state of nutrition.
The selection of a nest site is crucial for successful reproduction of birds. Animals which re-use or occupy nest sites constructed by other species often have limited choice. Little is known about the criteria of nest-stealing species to choose suitable nesting sites and habitats. Here, we analyze breeding-site selection of an obligatory "nest-cleptoparasite", the Amur Falcon Falco amurensis. We collected data on nest sites at Muraviovka Park in the Russian Far East, where the species breeds exclusively in nests of the Eurasian Magpie Pica pica. We sampled 117 Eurasian Magpie nests, 38 of which were occupied by Amur Falcons. Nest-specific variables were assessed, and a recently developed habitat classification map was used to derive landscape metrics. We found that Amur Falcons chose a wide range of nesting sites, but significantly preferred nests with a domed roof. Breeding pairs of Eurasian Hobby Falco subbuteo and Eurasian Magpie were often found to breed near the nest in about the same distance as neighboring Amur Falcon pairs. Additionally, the occurrence of the species was positively associated with bare soil cover, forest cover, and shrub patches within their home range and negatively with the distance to wetlands. Areas of wetlands and fallow land might be used for foraging since Amur Falcons mostly depend on an insect diet. Additionally, we found that rarely burned habitats were preferred. Overall, the effect of landscape variables on the choice of actual nest sites appeared to be rather small. We used different classification methods to predict the probability of occurrence, of which the Random forest method showed the highest accuracy. The areas determined as suitable habitat showed a high concordance with the actual nest locations. We conclude that Amur Falcons prefer to occupy newly built (domed) nests to ensure high nest quality, as well as nests surrounded by available feeding habitats.
Pollen/ovule (P/O) ratios are often used as proxy for breeding systems. Here, we investigate the relations between breeding systems and P/O ratios, pollination syndromes, life history and climate zone in Balsaminaceae. We conducted controlled breeding system experiments (autonomous and active self-pollination and outcrossing tests) for 65 Balsaminaceae species, analysed pollen grain and ovule numbers and evaluated the results in combination with data on pollination syndrome, life history and climate zone on a phylogenetic basis. Based on fruit set, we assigned three breeding systems: autogamy, self-compatibility and self-incompatibility. Self-pollination led to lower fruit set than outcrossing. We neither found significant P/O differences between breeding systems nor between pollination syndromes. However, the numbers of pollen grains and ovules per flower were significantly lower in autogamous species, but pollen grain and ovule numbers did not differ between most pollination syndromes. Finally, we found no relation between breeding system and climate zone, but a relation between climate zone and life history. In Balsaminaceae reproductive traits can change under resource or pollinator limitation, leading to the evolution of autogamy, but are evolutionary rather constant and not under strong selection pressure by pollinator guild and geographic range changes. Colonisation of temperate regions, however, is correlated with transitions towards annual life history. Pollen/ovule-ratios, commonly accepted as good indicators of breeding system, have a low predictive value in Balsaminaceae. In the absence of experimental data on breeding system, additional floral traits (overall pollen grain and ovule number, traits of floral morphology) may be used as proxies.
Canavanine (CAN) is a nonproteinogenic amino acid synthesized in legumes. In mammalians, as arginine analogue, it is an inhibitor of nitric oxide synthase (NOS) activity. The aim of this study was to investigate the impact of CAN-induced nitric oxide level limitation on the antioxidant system and S-nitrosoglutathione (GSNO) metabolism in roots of tomato seedlings. Treatment with CAN (10 or 50 mu M) for 24-72 h led to restriction in root growth. Arginine-dependent NOS-like activity was almost completely inhibited, demonstrating direct effect of CAN action. CAN increased total antioxidant capacity and the level of sulphydryl groups. Catalase (CAT) and superoxide dismutase (SOD) activity decreased in CAN exposed roots. CAN supplementation resulted in the decrease of transcript levels of genes coding CAT (with the exception of CAT1). Genes coding SOD (except MnSOD and CuSOD) were upregulated by CAN short treatment; prolonged exposition to 50-mu M CAN resulted in downregulation of FeSOD, CuSOD, and SODP-2. Activity of glutathione reductase dropped down after short-term (10-mu M CAN) supplementation, while glutathione peroxidase activity was not affected. Transcript levels of glutathione reductase genes declined in response to CAN. Genes coding glutathione peroxidase were upregulated by 50-mu M CAN, while 10-mu M CAN downregulated GSHPx1. Inhibition of NOS-like activity by CAN resulted in lower GSNO accumulation in root tips. Activity of GSNO reductase was decreased by short-term supplementation with CAN. In contrast, GSNO reductase protein abundance was higher, while transcript levels were slightly altered in roots exposed to CAN. This is the first report on identification of differentially nitrated proteins in response to supplementation with nonproteinogenic amino acid. Among nitrated proteins differentially modified by CAN, seed storage proteins (after short-term CAN treatment) and components of the cellular redox system (after prolonged CAN supplementation) were identified. The findings demonstrate that due to inhibition of NOS-like activity, CAN leads to modification in antioxidant system. Limitation in GSNO level is due to lower nitric oxide formation, while GSNO catabolism is less affected. We demonstrated that monodehydroascorbate reductase, activity of which is inhibited in roots of CAN-treated plants, is the protein preferentially modified by tyrosine nitration.
Achromatium oxaliferum is a large sulfur bacterium easily recognized by large intracellular calcium carbonate bodies. Although these bodies often fill major parts of the cells' volume, their role and specific intracellular location are unclear. In this study, we used various microscopy and staining techniques to identify the cell compartment harboring the calcium carbonate bodies. We observed that Achromatium cells often lost their calcium carbonate bodies, either naturally or induced by treatments with diluted acids, ethanol, sodium bicarbonate and UV radiation which did not visibly affect the overall shape and motility of the cells (except for UV radiation). The water-soluble fluorescent dye fluorescein easily diffused into empty cavities remaining after calcium carbonate loss. Membranes (stained with Nile Red) formed a network stretching throughout the cell and surrounding empty or filled calcium carbonate cavities. The cytoplasm (stained with FITC and SYBR Green for nucleic acids) appeared highly condensed and showed spots of dissolved Ca2+ (stained with Fura-2). From our observations, we conclude that the calcium carbonate bodies are located in the periplasm, in extra-cytoplasmic pockets of the cytoplasmic membrane and are thus kept separate from the cell's cytoplasm. This periplasmic localization of the carbonate bodies might explain their dynamic formation and release upon environmental changes.
Achromatium oxaliferum is a large sulfur bacterium easily recognized by large intracellular calcium carbonate bodies. Although these bodies often fill major parts of the cells' volume, their role and specific intracellular location are unclear. In this study, we used various microscopy and staining techniques to identify the cell compartment harboring the calcium carbonate bodies. We observed that Achromatium cells often lost their calcium carbonate bodies, either naturally or induced by treatments with diluted acids, ethanol, sodium bicarbonate and UV radiation which did not visibly affect the overall shape and motility of the cells (except for UV radiation). The water-soluble fluorescent dye fluorescein easily diffused into empty cavities remaining after calcium carbonate loss. Membranes (stained with Nile Red) formed a network stretching throughout the cell and surrounding empty or filled calcium carbonate cavities. The cytoplasm (stained with FITC and SYBR Green for nucleic acids) appeared highly condensed and showed spots of dissolved Ca2+ (stained with Fura-2). From our observations, we conclude that the calcium carbonate bodies are located in the periplasm, in extra-cytoplasmic pockets of the cytoplasmic membrane and are thus kept separate from the cell's cytoplasm. This periplasmic localization of the carbonate bodies might explain their dynamic formation and release upon environmental changes.
Wastewater treatment is crucial to environmental hygiene in urban environments. However, wastewater treatment plants (WWTPs) collect chemicals, organic matter, and microorganisms including pathogens and multi-resistant bacteria from various sources which may be potentially released into the environment via WWTP effluent. To better understand microbial dynamics in WWTPs, we characterized and compared the bacterial community of the inflow and effluent of a WWTP in Berlin, Germany using full-length 16S rRNA gene sequences, which allowed for species level determination in many cases and generally resolved bacterial taxa. Significantly distinct bacterial communities were identified in the wastewater inflow and effluent samples. Dominant operational taxonomic units (OTUs) varied both temporally and spatially. Disease associated bacterial groups were efficiently reduced in their relative abundance from the effluent by the WWTP treatment process, except for Legionella and Leptospira species which demonstrated an increase in relative proportion from inflow to effluent. This indicates that WWTPs, while effective against enteric bacteria, may enrich and release other potentially pathogenic bacteria into the environment. The taxonomic resolution of full-length 16S rRNA genes allows for improved characterization of potential pathogenic taxa and other harmful bacteria which is required to reliably assess health risk.
ATP-binding cassette (ABC) transporters are present in all kingdoms of life and enable active transport of various different molecules across biological membranes. They all share an overall architecture of two lipophilic transmembrane spanning domains (TMDs) traversing the membrane and two hydrophilic nucleotide binding domains (NBDs) usually lacking sequence identity. The multiplicity in transported molecules is accompanied by extreme diversity in TMDs. Human mitochondria harbor four ABC transporters, namely ABCB6, ABCB7, ABCB8 and ABCB10 with functional homologues in yeast and plants. Except the ones found in Rickettsiae and related bacteria mitochondrial ABC transporters are absent in bacteria. In addition to converting energy mitochondria are important platforms for biosynthesizing various cofactors as iron sulfur clusters, molybdenum cofactor (Moco) or heme. ABCB7 (Atm1 in yeast) has been shown to connect mitochondrial with cytosolic iron sulfur cluster assembly by exporting a yet unknown sulfur containing molecule. In addition, TMDs of Atm1 display a glutathione binding pocket accessible from the matrix which has been identified in all ABCB7-like transporters and also exists in a bacterial ABC transporter homologue of Atm1 in Novosphingobium aromaticivorans. In addition, ATM3, a plant mitochondrial homologous ABC transporter to human ABCB7, has been associated with biosynthesizing Moco.
In this study we used the α-proteobacterium Rhodobacter capsulatus as a model organism to characterize mitochondrial ABC transporter homologues. R. capsulatus contains two homologues to mitochondrial ABC transporters with the corresponding gene loci rcc03139 and rcc02305. They share 38 to 47 % sequence identities to human mitochondrial ABC transporters ABCB8/ABCB10 and ABCB7/ABCB6, respectively. We created interposon mutants lacking either rcc03139 or rcc02305, analyzed the physiological effects on R. capsulatus and compared the findings especially to eukaryotic deletion studies. A viable bacterial double mutant strain lacking both mitochondrial ABC transporters was constructed to investigate possible overlapping functions. Both R. capsulatus single mutants showed a severe accumulation of intracellular reactive oxygen species (ROS) in comparison to ∆nifDK which revealed to be additive in the double mutant. In the proteome of ∆rcc03139I abundancies of tetrapyrrole related proteins were significantly increased in comparison to the proteome of parental strain, which was further validated by reduced amounts of tetrapyrrole intermediates in ∆rcc03139. In contrast, in ∆rcc02305I total glutathione (GSH) was elevated when endogenous GSH biosynthesis was inhibited. In conjunction with proteomic studies we uncovered misbalanced sulfur distribution in ∆rcc02305I. Furthermore, strains lacking Rcc02305 accumulated cyclic pyranopterin monophosphate (cPMP), an intermediate of Moco biosynthesis, as it was already shown for the deletion strain of the eukaryotic counterpart ATM3 in plants. In contrast single mutant strain Δrcc03139I neither accumulated cPMP nor glutathione.
Bioinformatic analysis of the amino acid sequence of Rcc02305 revealed a pyridoxal 5´phosphate (PLP) binding site which overlaps with Walker A within the NBDs of Rcc02305 and other ABCB7-like transporters. The PLP cofactor is well studied in C-DES (L-cysteine/cystine lyase from Synechocystis) for persulfide production and in L-cysteine desulfurases such as IscS and NFS1 for its role in formation of protein-bound persulfides. Based on our findings we are able to propose a new modality for the transport of the sulfur containing molecule: first of all, the transporter produces a highly reactive persulfide which is then subsequently trapped by glutathione polysulfide, already bound within the binding pocket in TMDs. Walker A becomes accessible for ATP and after hydrolysis the mixed polysulfide is released.
Based on our studies we are convinced that both mitochondrial ABC transporter homologues fulfil distinct roles in R. capsulatus: Rcc02305 is a representative of Atm1/ABCB7-like transporters and important for proper sulfur distribution by exporting persulfides. In contrast Rcc03139 is a representative of ABCB6/ABCB10 related transporters and involved in biosynthesizing tetrapyrroles.
Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.
Racemic and highly enantioenriched 3-methoxycarbonyl, 3-carboxy, and 3-hydroxymethyl derivatives of dibenzo[6]helicene were prepared. The Langmuir layers of these helicenes were formed at the air-water interface and transferred onto solid substrates to afford Langmuir-Blodgett films, which were then studied by ambient atomic force microscopy and (chir)optical spectroscopy. Significant differences were found in the behaviour of the Langmuir layers as well as in the morphology, UV/Vis, electronic circular dichroism (ECD), and fluorescence spectra of the Langmuir-Blodgett thin films depending on the molecular chirality and nature of the polar group. The experimental results were supported by molecular dynamics simulations.
Larix populations at the tundra-taiga ecotone in northern Siberia are highly under-represented in population genetic studies, possibly due to the remoteness of these regions that can only be accessed at extraordinary expense. The genetic signatures of populations in these boundary regions are therefore largely unknown. We aim to generate organelle reference genomes for the detection of single nucleotide polymorphisms (SNPs) that can be used for paleogenetic studies. We present 19 complete chloroplast genomes and mitochondrial genomic sequences of larches from the southern lowlands of the Taymyr Peninsula (northernmost range of Larix gmelinii (Rupr.) Kuzen.), the lower Omoloy River, and the lower Kolyma River (both in the range of Larix cajanderi Mayr). The genomic data reveal 84 chloroplast SNPs and 213 putatively mitochondrial SNPs. Parsimony-based chloroplast haplotype networks show no spatial structure of individuals from different geographic origins, while the mitochondrial haplotype network shows at least a slight spatial structure with haplotypes from the Omoloy and Kolyma populations being more closely related to each other than to most of the haplotypes from the Taymyr populations. Whole genome alignments with publicly available complete chloroplast genomes of different Larix species show that among official plant barcodes only the rcbL gene contains sufficient polymorphisms, but has to be sequenced completely to distinguish the different provenances. We provide 8 novel mitochondrial SNPs that are putatively diagnostic for the separation of L. gmelinii and L. cajanderi, while 4 chloroplast SNPs have the potential to distinguish the L. gmelinii/ L. cajanderi group from other Larix species. Our organelle references can be used for a targeted primer and probe design allowing the generation of short amplicons. This is particularly important with regard to future investigations of, for example, the biogeographic history of Larix by screening ancient sedimentary DNA of Larix.
Study Design. A nonrandomized, prospective, and single-center clinical trial. Objective. The aim of this study was to investigate the clinical and radiographic efficacy of ProDisc Vivo cervical total disc replacement (cTDR) in patients with clinical and radiographic documented cervical spondylotic myelopathy (CSM), due to degenerative changes at the index level. Summary of Background Data. Decompression and fusion is still the gold standard in patients with cervical myelopathy. Very limited data are available regarding the application of cTDR in patients with clinical and radiological documented CSM in context of clinical and radiographic outcomes. Methods. Clinical outcome scores included the Neck Disability Index (NDI), Visual Analogue Scale (VAS), arm and neck pain self-assessment questionnaires as well as the Nurick grade and the Japanese Orthopaedic Association (JOA) score. The radiological outcome included the range of motion (ROM), the segmental and global (C2-C7) lordosis, and the occurrence of heterotopic ossifications. Results. Eighteen consecutive patients (10 males, 8 females) with documented clinical and radiological signs of myelopathy were included in this investigation. The study population had a mean age of 52.4 years and a follow-up period of 20.3 months in average (range 3-48 months). The mean range ROM of the index level stayed consistent with 6.8 degrees preoperatively and 7.2 degrees (P = 0.578) at the last follow-up; the global lordosis in neutral position changed from 3.5 degrees to 14.2 degrees significantly (P = 0.005) in mean. The JOA score improved from 11.3 to 16.6 (P < 0.001) as well as the NDI 36.7 to 10.3 (P < 0.001) and the VAS score from 5.7/6.1 (arm/neck) to 1.3/2.0 (P P < 0.001). The mean Nurick grade was 1.33 preoperatively and dropped down in all cases to Nurick grade of 0 (P < 0.001). Conclusion. cTDR (with ProDisc Vivio) in patients with CSM yielded good clinical and radiographic outcomes and found as a reliable, safe, and motion-preserving surgical treatment option, although its indication is very limited due to numerous exclusion criteria.
Coherent network partitions
(2019)
Graph clustering is widely applied in the analysis of cellular networks reconstructed from large-scale data or obtained from experimental evidence. Here we introduce a new type of graph clustering based on the concept of coherent partition. A coherent partition of a graph G is a partition of the vertices of G that yields only disconnected subgraphs in the complement of G. The coherence number of G is then the size of the smallest edge cut inducing a coherent partition. A coherent partition of G is optimal if the size of the inducing edge cut is the coherence number of G. Given a graph G, we study coherent partitions and the coherence number in connection to (bi)clique partitions and the (bi)clique cover number. We show that the problem of finding the coherence number is NP-hard, but is of polynomial time complexity for trees. We also discuss the relation between coherent partitions and prominent graph clustering quality measures.
The natural abundance of Coiled Coil (CC) motifs in cytoskeleton and extracellular matrix proteins suggests that CCs play an important role as passive (structural) and active (regulatory) mechanical building blocks. CCs are self-assembled superhelical structures consisting of 2-7 α-helices. Self-assembly is driven by hydrophobic and ionic interactions, while the helix propensity of the individual helices contributes additional stability to the structure. As a direct result of this simple sequence-structure relationship, CCs serve as templates for protein design and sequences with a pre-defined thermodynamic stability have been synthesized de novo. Despite this quickly increasing knowledge and the vast number of possible CC applications, the mechanical function of CCs has been largely overlooked and little is known about how different CC design parameters determine the mechanical stability of CCs. Once available, this knowledge will open up new applications for CCs as nanomechanical building blocks, e.g. in biomaterials and nanobiotechnology.
With the goal of shedding light on the sequence-structure-mechanics relationship of CCs, a well-characterized heterodimeric CC was utilized as a model system. The sequence of this model system was systematically modified to investigate how different design parameters affect the CC response when the force is applied to opposing termini in a shear geometry or separated in a zipper-like fashion from the same termini (unzip geometry). The force was applied using an atomic force microscope set-up and dynamic single-molecule force spectroscopy was performed to determine the rupture forces and energy landscape properties of the CC heterodimers under study. Using force as a denaturant, CC chain separation is initiated by helix uncoiling from the force application points. In the shear geometry, this allows uncoiling-assisted sliding parallel to the force vector or dissociation perpendicular to the force vector. Both competing processes involve the opening of stabilizing hydrophobic (and ionic) interactions. Also in the unzip geometry, helix uncoiling precedes the rupture of hydrophobic contacts.
In a first series of experiments, the focus was placed on canonical modifications in the hydrophobic core and the helix propensity. Using the shear geometry, it was shown that both a reduced core packing and helix propensity lower the thermodynamic and mechanical stability of the CC; however, with different effects on the energy landscape of the system. A less tightly packed hydrophobic core increases the distance to the transition state, with only a small effect on the barrier height. This originates from a more dynamic and less tightly packed core, which provides more degrees of freedom to respond to the applied force in the direction of the force vector. In contrast, a reduced helix propensity decreases both the distance to the transition state and the barrier height. The helices are ‘easier’ to unfold and the remaining structure is less thermodynamically stable so that dissociation perpendicular to the force axis can occur at smaller deformations.
Having elucidated how canonical sequence modifications influence CC mechanics, the pulling geometry was investigated in the next step. Using one and the same sequence, the force application points were exchanged and two different shear and one unzipping geometry were compared. It was shown that the pulling geometry determines the mechanical stability of the CC. Different rupture forces were observed in the different shear as well as in the unzipping geometries, suggesting that chain separation follows different pathways on the energy landscape. Whereas the difference between CC shearing and unzipping was anticipated and has also been observed for other biological structures, the observed difference for the two shear geometries was less expected. It can be explained with the structural asymmetry of the CC heterodimer. It is proposed that the direction of the α-helices, the different local helix propensities and the position of a polar asparagine in the hydrophobic core are responsible for the observed difference in the chain separation pathways. In combination, these factors are considered to influence the interplay between processes parallel and perpendicular to the force axis.
To obtain more detailed insights into the role of helix stability, helical turns were reinforced locally using artificial constraints in the form of covalent and dynamic ‘staples’. A covalent staple bridges to adjacent helical turns, thus protecting them against uncoiling. The staple was inserted directly at the point of force application in one helix or in the same terminus of the other helix, which did not experience the force directly. It was shown that preventing helix uncoiling at the point of force application reduces the distance to the transition state while slightly increasing the barrier height. This confirms that helix uncoiling is critically important for CC chain separation. When inserted into the second helix, this stabilizing effect is transferred across the hydrophobic core and protects the force-loaded turns against uncoiling. If both helices were stapled, no additional increase in mechanical stability was observed. When replacing the covalent staple with a dynamic metal-coordination bond, a smaller decrease in the distance to the transition was observed, suggesting that the staple opens up while the CC is under load.
Using fluorinated amino acids as another type of non-natural modification, it was investigated how the enhanced hydrophobicity and the altered packing at the interface influences CC mechanics. The fluorinated amino acid was inserted into one central heptad of one or both α-helices. It was shown that this substitution destabilized the CC thermodynamically and mechanically. Specifically, the barrier height was decreased and the distance to the transition state increased. This suggests that a possible stabilizing effect of the increased hydrophobicity is overruled by a disturbed packing, which originates from a bad fit of the fluorinated amino acid into the local environment. This in turn increases the flexibility at the interface, as also observed for the hydrophobic core substitution described above. In combination, this confirms that the arrangement of the hydrophobic side chains is an additional crucial factor determining the mechanical stability of CCs.
In conclusion, this work shows that knowledge of the thermodynamic stability alone is not sufficient to predict the mechanical stability of CCs. It is the interplay between helix propensity and hydrophobic core packing that defines the sequence-structure-mechanics relationship. In combination, both parameters determine the relative contribution of processes parallel and perpendicular to the force axis, i.e. helix uncoiling and uncoiling-assisted sliding as well as dissociation. This new mechanistic knowledge provides insight into the mechanical function of CCs in tissues and opens up the road for designing CCs with pre-defined mechanical properties. The library of mechanically characterized CCs developed in this work is a powerful starting point for a wide spectrum of applications, ranging from molecular force sensors to mechanosensitive crosslinks in protein nanostructures and synthetic extracellular matrix mimics.
Force plays a fundamental role in the regulation of biological processes. Cells can sense the mechanical properties of the extracellular matrix (ECM) by applying forces and transmitting mechanical signals. They further use mechanical information for regulating a wide range of cellular functions, including adhesion, migration, proliferation, as well as differentiation and apoptosis. Even though it is well understood that mechanical signals play a crucial role in directing cell fate, surprisingly little is known about the range of forces that define cell-ECM interactions at the molecular level.
Recently, synthetic molecular force sensor (MFS) designs have been established for measuring the molecular forces acting at the cell-ECM interface. MFSs detect the traction forces generated by cells and convert this mechanical input into an optical readout. They are composed of calibrated mechanoresponsive building blocks and are usually equipped with a fluorescence reporter system. Up to date, many different MFS designs have been introduced and successfully used for measuring forces involved in the adhesion of mammalian cells. These MFSs utilize different molecular building blocks, such as double-stranded deoxyribonucleic acid (dsDNA) molecules, DNA hairpins and synthetic polymers like polyethylene glycol (PEG). These currently available MFS designs lack ECM mimicking properties.
In this work, I introduce a new MFS building block for cell biology applications, derived from the natural ECM. It combines mechanical tunability with the ability to mimic the native cellular microenvironment. Inspired by structural ECM proteins with load bearing function, this new MFS design utilizes coiled coil (CC)-forming peptides. CCs are involved in structural and mechanical tasks in the cellular microenvironment and many of the key protein components of the cytoskeleton and the ECM contain CC structures. The well-known folding motif of CC structures, an easy synthesis via solid phase methods and the many roles CCs play in biological processes have inspired studies to use CCs as tunable model systems for protein design and assembly. All these properties make CCs ideal candidates as building blocks for MFSs. In this work, a series of heterodimeric CCs were designed, characterized and further used as molecular building blocks for establishing a novel, next-generation MFS prototype.
A mechanistic molecular understanding of their structural response to mechanical load is essential for revealing the sequence-structure-mechanics relationships of CCs. Here, synthetic heterodimeric CCs of different length were loaded in shear geometry and their mechanical response was investigated using a combination of atomic force microscope (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations. SMFS showed that the rupture forces of short heterodimeric CCs (3-5 heptads) lie in the range of 20-50 pN, depending on CC length, pulling geometry and the applied loading rate (dF/dt). Upon shearing, an initial rise in the force, followed by a force plateau and ultimately strand separation was observed in SMD simulations. A detailed structural analysis revealed that CC response to shear load depends on the loading rate and involves helix uncoiling, uncoiling-assisted sliding in the direction of the applied force and uncoiling-assisted dissociation perpendicular to the force axis.
The application potential of these mechanically characterized CCs as building blocks for MFSs has been tested in 2D cell culture applications with the goal of determining the threshold force for cell adhesion. Fully calibrated, 4- to 5-heptad long, CC motifs (CC-A4B4 and CC-A5B5) were used for functionalizing glass surfaces with MFSs. 3T3 fibroblasts and endothelial cells carrying mutations in a signaling pathway linked to cell adhesion and mechanotransduction processes were used as model systems for time-dependent adhesion experiments. A5B5-MFS efficiently supported cell attachment to the functionalized surfaces for both cell types, while A4B4-MFS failed to maintain attachment of 3T3 fibroblasts after the first 2 hours of initial cell adhesion. This difference in cell adhesion behavior demonstrates that the magnitude of cell-ECM forces varies depending on the cell type and further supports the application potential of CCs as mechanoresponsive and tunable molecular building blocks for the development of next-generation protein-based MFSs.This novel CC-based MFS design is expected to provide a powerful new tool for observing cellular mechanosensing processes at the molecular level and to deliver new insights into the mechanisms and forces involved. This MFS design, utilizing mechanically tunable CC building blocks, will not only allow for measuring the molecular forces acting at the cell-ECM interface, but also yield a new platform for the development of mechanically controlled materials for a large number of biological and medical applications.
Biochar is being discussed as a soil amendment to improve soil fertility and mitigate climate change. While biochar interactions with soil microbial biota have been frequently studied, interactions with soil mesofauna are understudied. We here present an experiment in which we tested if the collembolan Folsomia candida I) can transport biochar particles, II) if yes, how far the particles are distributed within 10 days, and III) if it shows a preference among biochars made from different feedstocks, i.e. pine wood, pine bark and spelt husks. In general, biochar particles based on pine bark and pine wood were consistently distributed significantly more than those made of spelt husks, but all types were transported more than 4cm within 10 days. Additionally, we provide evidence that biochar particles can become readily attached to the cuticle of collembolans and hence be transported, potentially even over large distances. Our study shows that the soil mesofauna can indeed act as a vector for the transport of biochar particles and show clear preferences depending on the respective feedstock, which would need to be studied in more detail in the future.
Biochar is being discussed as a soil amendment to improve soil fertility and mitigate climate change. While biochar interactions with soil microbial biota have been frequently studied, interactions with soil mesofauna are understudied. We here present an experiment in which we tested if the collembolan Folsomia candida I) can transport biochar particles, II) if yes, how far the particles are distributed within 10 days, and III) if it shows a preference among biochars made from different feedstocks, i.e. pine wood, pine bark and spelt husks. In general, biochar particles based on pine bark and pine wood were consistently distributed significantly more than those made of spelt husks, but all types were transported more than 4cm within 10 days. Additionally, we provide evidence that biochar particles can become readily attached to the cuticle of collembolans and hence be transported, potentially even over large distances. Our study shows that the soil mesofauna can indeed act as a vector for the transport of biochar particles and show clear preferences depending on the respective feedstock, which would need to be studied in more detail in the future.
Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing n-carotene and co-producing p-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.
The adaptation of plants to future climatic conditions is crucial for their survival. Not surprisingly, phenotypic responses to climate change have already been observed in many plant populations. These responses may be due to evolutionary adaptive changes or phenotypic plasticity. Especially plant species with a wide geographic range are either expected to show genetic differentiation in response to differing climate conditions or to have a high phenotypic plasticity. We investigated phenotypic responses and plasticity as an estimate of the adaptive potential in the widespread species Silene vulgaris. In a greenhouse experiment, 25 European populations covering a geographic range from the Canary Islands to Sweden were exposed to three experimental precipitation and two temperature regimes mimicking a possible climate-change scenario for central Europe. We hypothesized that southern populations have a better performance under high temperature and drought conditions, as they are already adapted to a comparable environment. We found that our treatments significantly influenced the plants, but did not reveal a latitudinal difference in response to climate treatments for most plant traits. Only flower number showed a stronger plasticity in northern European populations (e.g. Swedish populations) where numbers decreased more drastically with increased temperature and decreased precipitation treatment. Synthesis. The significant treatment response in Silene vulgaris, independent of population origin - except for the number of flowers produced - suggests a high degree of universal phenotypic plasticity in this widely distributed species. This reflects the likely adaptation strategy of the species and forms the basis for a successful survival strategy during upcoming climatic changes. However, as flower number, a strongly fitness-related trait, decreased more strongly in northern populations under a climate-change scenario, there might be limits to adaptation even in this widespread, plastic species.
Successfully designed and implemented plant-specific synthetic metabolic pathways hold promise to increase crop yield and nutritional value. Advances in synthetic biology have already demonstrated the capacity to design artificial biological pathways whose behavior can be predicted and controlled in microbial systems. However, the transfer of these advances to model plants and crops faces the lack of characterization of plant cellular pathways and increased complexity due to compartmentalization and multicellularity. Modern computational developments provide the means to test the feasibility of plant synthetic metabolic pathways despite gaps in the accumulated knowledge of plant metabolism. Here, we provide a succinct systematic review of optimization-based and retrobiosynthesis approaches that can be used to design and in silico test synthetic metabolic pathways in large-scale plant context-specific metabolic models. In addition, by surveying the existing case studies, we highlight the challenges that these approaches face when applied to plants. Emphasis is placed on understanding the effect that metabolic designs can have on native metabolism, particularly with respect to metabolite concentrations and thermodynamics of biochemical reactions. In addition, we discuss the computational developments that may help to transform the identified challenges into opportunities for plant synthetic biology.
Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly alpha-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil-helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze-thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of alpha-helical structure and the increased alpha-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil-helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of alpha-helical structure during freezing induced dehydration.
Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly alpha-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil-helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze-thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of alpha-helical structure and the increased alpha-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil-helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of alpha-helical structure during freezing induced dehydration.
The facilitation of species coexistence has been a central theme in ecological research for years, highlighting two key aspects: ecological niches and competition between species. According to the competitive exclusion principle, the overlap of species niches predicts the amount of shared resources and therefore competition between species, determining their ability to coexist. Only if niches of two species are sufficiently different, thus niche overlap is low, competition within species is higher than competition between species and stable coexistence is possible. Thereby, differences in species mean traits are focused on and conspecific individuals are assumed to be interchangeable. This approach might be outdated since behaviour, as a key aspect mediating niche differentiation between species, is individual based. Individuals from one species consistently differ across time and situations in their behavioural traits. Causes and consequences of consistent behavioural differences have been thoroughly investigated stimulating their recent incorporation into ecological interactions and niche theory. Spatial components have so far been largely overlooked, although animal movement is strongly connected to several aspects of ecological niches and interactions between individuals. Furthermore, numerous movement aspects haven been proven to be crucially influenced by consistent individual differences. Considering spatial parameters could therefore crucially broaden our understanding of how individual niches are formed and ecological interactions are shaped. Furthermore, extending established concepts on species interactions by an individual component could provide new insights into how species coexistence is facilitated and local biodiversity is maintained.
The main aim of this thesis was to test whether consistent inter-individual differences can facilitate the coexistence of ecological similar species. Therefore, the effects of consistent inter-individual differences on the spatial behaviour of two rodent species, the bank vole (Myodes glareolus) and the striped field mouse (Apodemus agrarius), were investigated and put in the context of: (i) individual spatial niches, (ii) interactions between species, and (iii) the importance of different levels of behavioural variation within species for their interactions. Consistent differences of study animals in boldness and exploration were quantified with the same tests in all presented studies and always combined with observations of movement and space use via automated VHF radio telemetry. Consequently, results are comparable throughout the thesis and the methods provide a common denominator for all chapters. The first two chapters are based on observations of free-ranging rodents in natural populations, while chapter III represents an experimental approach under semi-natural conditions.
Chapter I focusses on the effect of consistent differences in boldness and exploration on movement and space use of bank voles and their contribution to individual spatial niche separation. Results show boldness to be the dominating predictor for spatial parameters in bank voles. Irrespective of sex, bolder individuals had larger home ranges, moved longer distances, had less spatial interactions with conspecifics and occupied different microhabitats compared to shy individuals. The same boldness-dependent spatial patterns could be observed in striped field mice which is reported in chapter II. Therefore, both study species showed individual spatial niche occupation.
Chapter II builds on findings from the first chapter, investigating the effect of boldness driven individual spatial niche occupation on the interactions between species. Irrespective of species and sex, bolder individuals had more interspecific spatial interactions, but less intraspecific interactions, compared to shy individuals. Due to individual niches occupation the competitive environment individuals experience is not random. Interactions are restricted to individuals of similar behavioural type with presumably similar competitive ability, which could balance differences on the species level and support coexistence.
In chapter III the experimental populations were either comprised of only shy or only bold bank voles, while striped field mice varied, creating either a shy- or bold-biased competitive community. Irrespective of behavioural type, striped field mice had more intraspecific interactions in bold-biased competitive communities. Only in a shy-biased competitive community, bolder striped field mice had less interspecific interactions compared to shy individuals. Bank voles showed no difference in intra- or interspecific interactions between populations. Chapter III highlights, that not only consistent inter-individual differences per se are important for interactions within and between species, but also the amount of behavioural variation within coexisting species.
Overall, this thesis highlights the importance of considering consistent inter-individual differences in a spatial context and their connection to individual spatial niche occupation, as well as the resulting effects on interactions within and between species. Individual differences are discussed in the context of similarity of individuals, individual and species niche width, and individual and species niche overlap. Thereby, this thesis makes one step further from the existing research on individual niches towards integrating consistent inter-individual differences into the larger framework of species coexistence.
Recent discovery of oxic methane production in sea and lake waters, as well as wetlands, demands re-thinking of the global methane cycle and re-assessment of the contribution of oxic waters to atmospheric methane emission. Here we analysed system-wide sources and sinks of surface-water methane in a temperate lake. Using a mass balance analysis, we show that internal methane production in well-oxygenated surface water is an important source for surface-water methane during the stratified period. Combining our results and literature reports, oxic methane contribution to emission follows a predictive function of littoral sediment area and surface mixed layer volume. The contribution of oxic methane source(s) is predicted to increase with lake size, accounting for the majority (>50%) of surface methane emission for lakes with surface areas >1 km(2).
The oligotrophic subtropical gyre covers a vast area of the Atlantic Ocean. Decades of time-series monitoring have generated detailed temporal information about zooplankton species and abundances at fixed locations within the gyre, but their live/dead status is often omitted, especially in the dynamic subtropical convergence zone (STCZ) where the water column stratification pattern can change considerably across the front as warm and cold water masses converge. We conducted a detailed survey in the North Atlantic STCZ and showed that over 85% of the copepods were typically concentrated in the upper 200 m. Copepod carcasses were present in all samples and their proportional numerical abundances increased with depth, reaching up to 91% at 300-400 m. Overall, 14-19% of the copepods within the upper 200 m were carcasses. Shipboard experiments showed that during carcass decomposition, microbial respiration increased, and the bacterial community associated with the carcasses diverged from that in the ambient water. Combining field and experimental data, we estimated that decomposing copepod carcasses constitute a negligible oxygen sink in the STCZ, but sinking carcasses may represent an overlooked portion of the passive carbon sinking flux and should be incorporated in future studies of carbon flux in this area.
Between-individual differences in coping with stress encompass neurophysiological, cognitive and behavioural reactions. The coping style model proposes two alternative response patterns to challenges that integrate these types of reactions. The “proactive strategy” combines a general fight-or-flight response and inflexibility in learning with a relatively low HPA (hypothalamic–pituitary–adrenal) response. The “reactive strategy” includes risk aversion, flexibility in learning and an enhanced HPA response. Although numerous studies have investigated the possible covariance of cognitive, behavioural and physiological responses, findings are still mixed. In the present study, we tested the predictions of the coping style model in an unselected population of bank voles (Myodes glareolus) (N = 70). We measured the voles’ boldness, activity, speed and flexibility in learning and faecal corticosterone metabolite levels under three conditions (holding in indoor cages, in outdoor enclosures and during open field test). Individuals were moderately consistent in their HPA response across situations. Proactive voles had significantly lower corticosterone levels than reactive conspecifics in indoor and outdoor conditions. However, we could not find any co-variation between cognitive and behavioural traits and corticosterone levels in the open field test. Our results partially support the original coping style model but suggest a more complex relationship between cognitive, behavioural and endocrine responses than was initially proposed.
One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO2 and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO2 in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.
Global change threatens the maintenance of ecosystem functions that are shaped by the persistence and dynamics of populations. It has been shown that the persistence of species increases if they possess larger trait adaptability. Here, we investigate whether trait adaptability also affects the robustness of population dynamics of interacting species and thereby shapes the reliability of ecosystem functions that are driven by these dynamics. We model co‐adaptation in a predator–prey system as changes to predator offense and prey defense due to evolution or phenotypic plasticity. We investigate how trait adaptation affects the robustness of population dynamics against press perturbations to environmental parameters and against pulse perturbations targeting species abundances and their trait values. Robustness of population dynamics is characterized by resilience, elasticity, and resistance. In addition to employing established measures for resilience and elasticity against pulse perturbations (extinction probability and return time), we propose the warping distance as a new measure for resistance against press perturbations, which compares the shapes and amplitudes of pre‐ and post‐perturbation population dynamics. As expected, we find that the robustness of population dynamics depends on the speed of adaptation, but in nontrivial ways. Elasticity increases with speed of adaptation as the system returns more rapidly to the pre‐perturbation state. Resilience, in turn, is enhanced by intermediate speeds of adaptation, as here trait adaptation dampens biomass oscillations. The resistance of population dynamics strongly depends on the target of the press perturbation, preventing a simple relationship with the adaptation speed. In general, we find that low robustness often coincides with high amplitudes of population dynamics. Hence, amplitudes may indicate the robustness against perturbations also in other natural systems with similar dynamics. Our findings show that besides counteracting extinctions, trait adaptation indeed strongly affects the robustness of population dynamics against press and pulse perturbations.
Trace elements, like Cu, Zn, Fe, or Se, are important for the proper functioning of antioxidant enzymes. However, in excessive amounts, they can also act as pro-oxidants. Accordingly, trace elements influence redox-modulated signaling pathways, such as the Nrf2 pathway. Vice versa, Nrf2 target genes belong to the group of transport and metal binding proteins. In order to investigate whether Nrf2 directly regulates the systemic trace element status, we used mice to study the effect of a constitutive, whole-body Nrf2 knockout on the systemic status of Cu, Zn, Fe, and Se. As the loss of selenoproteins under Se-deprived conditions has been described to further enhance Nrf2 activity, we additionally analyzed the combination of Nrf2 knockout with feeding diets that provide either suboptimal, adequate, or supplemented amounts of Se. Experiments revealed that the Nrf2 knockout partially affected the trace element concentrations of Cu, Zn, Fe, or Se in the intestine, liver, and/or plasma. However, aside from Fe, the other three trace elements were only marginally modulated in an Nrf2-dependent manner. Selenium deficiency mainly resulted in increased plasma Zn levels. One putative mediator could be the metal regulatory transcription factor 1, which was up-regulated with an increasing Se supply and downregulated in Se-supplemented Nrf2 knockout mice.
Trace elements, like Cu, Zn, Fe, or Se, are important for the proper functioning of antioxidant enzymes. However, in excessive amounts, they can also act as pro-oxidants. Accordingly, trace elements influence redox-modulated signaling pathways, such as the Nrf2 pathway. Vice versa, Nrf2 target genes belong to the group of transport and metal binding proteins. In order to investigate whether Nrf2 directly regulates the systemic trace element status, we used mice to study the effect of a constitutive, whole-body Nrf2 knockout on the systemic status of Cu, Zn, Fe, and Se. As the loss of selenoproteins under Se-deprived conditions has been described to further enhance Nrf2 activity, we additionally analyzed the combination of Nrf2 knockout with feeding diets that provide either suboptimal, adequate, or supplemented amounts of Se. Experiments revealed that the Nrf2 knockout partially affected the trace element concentrations of Cu, Zn, Fe, or Se in the intestine, liver, and/or plasma. However, aside from Fe, the other three trace elements were only marginally modulated in an Nrf2-dependent manner. Selenium deficiency mainly resulted in increased plasma Zn levels. One putative mediator could be the metal regulatory transcription factor 1, which was up-regulated with an increasing Se supply and downregulated in Se-supplemented Nrf2 knockout mice.
Ecological effects of alien species can be dramatic, but management and prevention of negative impacts are often hindered by crypticity of the species or their ecological functions. Ecological functions can change dramatically over time, or manifest after long periods of an innocuous presence. Such cryptic processes may lead to an underestimation of long-term impacts and constrain management effectiveness. Here, we present a conceptual framework of crypticity in biological invasions. We identify the underlying mechanisms, provide evidence of their importance, and illustrate this phenomenon with case studies. This framework has potential to improve the recognition of the full risks and impacts of invasive species.
Understanding the strategies employed by plant species that live in extreme environments offers the possibility to discover stress tolerance mechanisms. We studied the physiological, antioxidant and metabolic responses to three temperature conditions (4, 15, and 23 degrees C) of Colobanthus quitensis (CQ), one of the only two native vascular species in Antarctica. We also employed Dianthus chinensis (DC), to assess the effects of the treatments in a non-Antarctic species from the same family. Using fused LASSO modelling, we associated physiological and biochemical antioxidant responses with primary metabolism. This approach allowed us to highlight the metabolic pathways driving the response specific to CQ. Low temperature imposed dramatic reductions in photosynthesis (up to 88%) but not in respiration (sustaining rates of 3.0-4.2 mu mol CO2 m(-2) s(-1)) in CQ, and no change in the physiological stress parameters was found. Its notable antioxidant capacity and mitochondrial cytochrome respiratory activity (20 and two times higher than DC, respectively), which ensure ATP production even at low temperature, was significantly associated with sulphur-containing metabolites and polyamines. Our findings potentially open new biotechnological opportunities regarding the role of antioxidant compounds and respiratory mechanisms associated with sulphur metabolism in stress tolerance strategies to low temperature.
A new isoflavone, 4′-prenyloxyvigvexin A (1) and a new pterocarpan, (6aR,11aR)-3,8-dimethoxybitucarpin B (2) were isolated from the leaves of Lonchocarpus bussei and the stem bark of Lonchocarpus eriocalyx, respectively. The extract of L. bussei also gave four known isoflavones, maximaisoflavone H, 7,2′-dimethoxy-3′,4′-methylenedioxyisoflavone, 6,7,3′-trimethoxy-4′,5′-methylenedioxyisoflavone, durmillone; a chalcone, 4-hydroxylonchocarpin; a geranylated phenylpropanol, colenemol; and two known pterocarpans, (6aR,11aR)-maackiain and (6aR,11aR)-edunol. (6aR,11aR)-Edunol was also isolated from the stem bark of L. eriocalyx. The structures of the isolated compounds were elucidated by spectroscopy. The cytotoxicity of the compounds was tested by resazurin assay using drug-sensitive and multidrug-resistant cancer cell lines. Significant antiproliferative effects with IC50 values below 10 μM were observed for the isoflavones 6,7,3′-trimethoxy-4′,5′-methylenedioxyisoflavone and durmillone against leukemia CCRF-CEM cells; for the chalcone, 4-hydroxylonchocarpin and durmillone against its resistant counterpart CEM/ADR5000 cells; as well as for durmillone against the resistant breast adenocarcinoma MDA-MB231/BCRP cells and resistant gliobastoma U87MG.ΔEGFR cells.
Since half a century, cytometry has been a major scientific discipline in the field of cytomics - the study of system’s biology at single cell level. It enables the investigation of physiological processes, functional characteristics and rare events with proteins by analysing multiple parameters on an individual cell basis. In the last decade, mass cytometry has been established which increased the parallel measurement to up to 50 proteins. This has shifted the analysis strategy from conventional consecutive manual gates towards multi-dimensional data processing. Novel algorithms have been developed to tackle these high-dimensional protein combinations in the data. They are mainly based on clustering or non-linear dimension reduction techniques, or both, often combined with an upstream downsampling procedure. However, these tools have obstacles either in comprehensible interpretability, reproducibility, computational complexity or in comparability between samples and groups.
To address this bottleneck, a reproducible, semi-automated cytometric data mining workflow PRI (pattern recognition of immune cells) is proposed which combines three main steps: i) data preparation and storage; ii) bin-based combinatorial variable engineering of three protein markers, the so called triploTs, and subsequent sectioning of these triploTs in four parts; and iii) deployment of a data-driven supervised learning algorithm, the cross-validated elastic-net regularized logistic regression, with these triploT sections as input variables. As a result, the selected variables from the models are ranked by their prevalence, which potentially have discriminative value. The purpose is to significantly facilitate the identification of meaningful subpopulations, which are most distinguish between two groups. The proposed workflow PRI is exemplified by a recently published public mass cytometry data set. The authors found a T cell subpopulation which is discriminative between effective and ineffective treatment of breast carcinomas in mice. With PRI, that subpopulation was not only validated, but was further narrowed down as a particular Th1 cell population. Moreover, additional insights of combinatorial protein expressions are revealed in a traceable manner. An essential element in the workflow is the reproducible variable engineering. These variables serve as basis for a clearly interpretable visualization, for a structured variable exploration and as input layers in neural network constructs.
PRI facilitates the determination of marker levels in a semi-continuous manner. Jointly with the combinatorial display, it allows a straightforward observation of correlating patterns, and thus, the dominant expressed markers and cell hierarchies. Furthermore, it enables the identification and complex characterization of discriminating subpopulations due to its reproducible and pseudo-multi-parametric pattern presentation. This endorses its applicability as a tool for unbiased investigations on cell subsets within multi-dimensional cytometric data sets.
The great auk was once abundant and distributed across the North Atlantic. It is now extinct, having been heavily exploited for its eggs, meat, and feathers. We investigated the impact of human hunting on its demise by integrating genetic data, GPS-based ocean current data, and analyses of population viability. We sequenced complete mitochondrial genomes of 41 individuals from across the species' geographic range and reconstructed population structure and population dynamics throughout the Holocene. Taken together, our data do not provide any evidence that great auks were at risk of extinction prior to the onset of intensive human hunting in the early 16th century. In addition, our population viability analyses reveal that even if the great auk had not been under threat by environmental change, human hunting alone could have been sufficient to cause its extinction. Our results emphasise the vulnerability of even abundant and widespread species to intense and localised exploitation.
Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo
(2019)
We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce similar to 100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.
Light-switchable proteins are being used increasingly to understand and manipulate complex molecular systems. The success of this approach has fueled the development of tailored photo-switchable proteins, to enable targeted molecular events to be studied using light. The development of novel photo-switchable tools has to date largely relied on rational design. Complementing this approach with directed evolution would be expected to facilitate these efforts. Directed evolution, however, has been relatively infrequently used to develop photo-switchable proteins due to the challenge presented by high-throughput evaluation of switchable protein activity. This thesis describes the development of two genetic circuits that can be used to evaluate libraries of switchable proteins, enabling optimization of both the on- and off-states. A screening system is described, which permits detection of DNA-binding activity based on conditional expression of a fluorescent protein. In addition, a tunable selection system is presented, which allows for the targeted selection of protein-protein interactions of a desired affinity range. This thesis additionally describes the development and characterization of a synthetic protein that was designed to investigate chromophore reconstitution in photoactive yellow protein (PYP), a promising scaffold for engineering photo-controlled protein tools.
Effective antibiotic dosing is vital for therapeutic success in critically ill patients. This work aimed to develop an algorithm to identify appropriate meropenem dosing in critically ill patients. Population pharma-cokinetic (PK) modelling was performed in NONMEM (R) 7.3 based on densely sampled meropenem serum samples (n(patients) = 48; n(samples) =1376) and included a systematic analysis of 27 pre-selected covariates to identify factors influencing meropenem exposure. Using Monte Carlo simulations newly considering the uncertainty of PK parameter estimates, standard meropenem dosing was evaluated with respect to attainment of the pharmacokinetic/pharmacodynamic (PK/PD) target and was compared with alternative infusion regimens (short-term, prolonged, continuous; daily dose, 2000-6000 mg). Subsequently, a dosing algorithm was developed to identify appropriate dosing regimens. The two-compartment population PK model included three factors influencing meropenem pharmacokinetics: the Cockcroft-Gault creatinine clearance (CLCRCG ) on meropenem clearance; and body weight and albumin on the central and peripheral volume of distribution, respectively; of these, only CLCRCG was identified as a vital influencing factor on PK/PD target attainment. A three-level dosing algorithm was developed (considering PK parameter uncertainty), suggesting dosing regimens depending on renal function and the level (L) of knowledge about the infecting pathogen (L1, pathogen unknown; L2, pathogen known; L3((-MIC)), pathogen and susceptibility known; L3((+MIC)), MIC known). Whereas patients with higher CLCRCG and lower pathogen susceptibility required mainly intensified dosing regimens, lower than standard doses appeared sufficient for highly susceptible pathogens. In conclusion, a versatile meropenem dosing algorithm for critically ill patients is proposed, indicating appropriate dosing regimens based on patient- and pathogen-specific information. (C) 2019 Published by Elsevier B.V.
Domestic Bactrian camel (Camelus bactrianus) used to be one of the most important livestock species in Chinese history, as well as the major transport carrier on the ancient Silk Road. However, archeological studies on Chinese C. bactrianus are still limited, and molecular biology research on this species is mainly focused on modern specimens. In this study, we retrieved the complete mitochondrial genome from a C. bactrianus specimen, which was excavated from northwestern China and dated at 1290-1180 cal. Phylogenetic analyses using 18 mitochondrial genomes indicated that the C. bactrianus clade was divided into two maternal lineages. The majority of samples originating from Iran to Japan and Mongolia belong to subclade A1, while our sample together with two Mongolian individuals formed the much smaller subclade A2. Furthermore, the divergence time of these two maternal lineages was estimated as 165 Kya (95% credibility interval 117-222 Kya), this might indicate that several different evolutionary lineages were incorporated into the domestic gene pool during the initial domestication process. Bayesian skyline plot (BSP) analysis a slow increase in female effective population size of C. bactrianus from 5000 years ago, which to the beginning of domestication of C. bactrianus. The present study also revealed that there were extensive exchanges of genetic information among C. bactrianus populations in regions along the Silk Road.
For the early detection of bacterial infection, there is a need for rapid, sensitive, and label-free assays. Thus, in this study, nanostrucured microbial electrochemical platform is designed to monitor the viability and cell growth of S. aureus. Using multi-walled carbon nanotube modified screen-printed electrodes (MWCNTs/SPE), the cyclic voltammetric measurements showed only one irreversible oxidation peak at 600 mV vs Ag/AgCl that accounts for the viable and metabolically active bacterial cells. The assay was optimized and the secreted metabolites, in the extracellular matrix, were directly detected. The peak current showed a positive correlation with viable cell numbers ranging from OD600 nm of 0.1 to 1.1, indicating that the activity of live cells can be quantified. Consequently, responses of viable and non-viable cells of S. aureus to the effects of antibiotic and respiratory chain inhibitors were determined. Thus, the proposed nanostructure-based bacterial sensor provides a reasonable and reliable way for real-time monitoring of live-dead cell functions, and antibacterial profiling.
A strong temperature increase in the Arctic is expected to lead to latitudinal treeline shift. This tundra-taiga turnover would cause a positive vegetation-climate feedback due to albedo decrease. However, reliable estimates of tree migration rates are currently lacking due to the complex processes involved in forest establishment, which depend strongly on seed dispersal. We aim to fill this gap using LAVESI, an individual-based and spatially explicit Larix vegetation simulator. LAVESI was designed to simulate plots within homogeneous forests. Here, we improve the implementation of the seed dispersal function via field-based investigations. We inferred the effective seed dispersal distances of a typical open-forest stand on the southern Taymyr Peninsula (northern central Siberia) from genetic parentage analysis using eight nuclear microsatellite markers. The parentage analysis gives effective seed dispersal distances (median similar to 10 m) close to the seed parents. A comparison between simulated and observed effective seed dispersal distances reveals an overestimation of recruits close to the releasing tree and a shorter dispersal distance generally. We thus adapted our model and used the newly parameterised version to simulate south-to-north transects; a slow-moving treeline front was revealed. The colonisation of the tundra areas was assisted by occasional long-distance seed dispersal events beyond the treeline area. The treeline (similar to 1 tree ha(-1)) advanced by similar to 1.6 m yr(-1), whereas the forest line (similar to 100 trees ha(-1)) advanced by only similar to 0.6 m yr(-1). We conclude that the treeline in northern central Siberia currently lags behind the current strong warming and will continue to lag in the near future.
Determining the relationship between genotype and phenotype is the key to understand the plasticity and robustness of phenotypes in nature. While the directly observable plant phenotypes (e.g. agronomic, yield and stress resistance traits) have been well-investigated, there is still a lack in our knowledge about the genetic basis of intermediate phenotypes, such as metabolic phenotypes. Dissecting the links between genotype and phenotype depends on suitable statistical models. The state-of-the-art models are developed for directly observable phenotypes, regardless the characteristics of intermediate phenotypes. This thesis aims to fill the gaps in understanding genetic architecture of intermediate phenotypes, and how they tie to composite traits, namely plant growth. The metabolite levels and reaction fluxes, as two aspects of metabolic phenotypes, are shaped by the interrelated chemical reactions formed in genome-scale metabolic network. Here, I attempt to answer the question: Can the knowledge of underlying genome-scale metabolic network improve the model performance for prediction of metabolic phenotypes and associated plant growth? To this end, two projects are investigated in this thesis. Firstly, we propose an approach that couples genomic selection with genome-scale metabolic network and metabolic profiles in Arabidopsis thaliana to predict growth. This project is the first integration of genomic data with fluxes predicted based on constraint-based modeling framework and data on biomass composition. We demonstrate that our approach leads to a considerable increase of prediction accuracy in comparison to the state-of-the-art methods in both within and across environment predictions. Therefore, our work paves the way for combining knowledge on metabolic mechanisms in the statistical approach underlying genomic selection to increase the efficiency of future plant breeding approaches. Secondly, we investigate how reliable is genomic selection for metabolite levels, and which single nucleotide polymorphisms (SNPs), obtained from different neighborhoods of a given metabolic network, contribute most to the accuracy of prediction. The results show that the local structure of first and second neighborhoods are not sufficient for predicting the genetic basis of metabolite levels in Zea mays. Furthermore, we find that the enzymatic SNPs can capture most the genetic variance and the contribution of non-enzymatic SNPs is in fact small. To comprehensively understand the genetic architecture of metabolic phenotypes, I extend my study to a local Arabidopsis thaliana population and their hybrids. We analyze the genetic architecture in primary and secondary metabolism as well as in growth. In comparison to primary metabolites, compounds from secondary metabolism were more variable and show more non-additive inheritance patterns which could be attributed to epistasis. Therefore, our study demonstrates that heterozygosity in local Arabidopsis thaliana population generates metabolic variation and may impact several tasks directly linked to metabolism. The studies in this thesis improve the knowledge of genetic architecture of metabolic phenotypes in both inbreed and hybrid population. The approaches I proposed to integrate genome-scale metabolic network with genomic data provide the opportunity to obtain mechanistic insights about the determinants of agronomically important polygenic traits.
Distinct cellular roles for PDCD10 define a gut-brain axis in cerebral cavernous malformation
(2019)
Cerebral cavernous malformation (CCM) is a genetic, cerebrovascular disease. Familial CCM is caused by genetic mutations in KRIT1, CCM2, or PDCD10. Disease onset is earlier and more severe in individuals with PDCD10 mutations. Recent studies have shown that lesions arise from excess mitogen-activated protein kinase kinase kinase 3 (MEKK3) signaling downstream of Toll-like receptor 4 (TLR4) stimulation by lipopolysaccharide derived from the gut microbiome. These findings suggest a gut-brain CCM disease axis but fail to define it or explain the poor prognosis of patients with PDCD10 mutations. Here, we demonstrate that the gut barrier is a primary determinant of CCM disease course, independent of microbiome configuration, that explains the increased severity of CCM disease associated with PDCD10 deficiency. Chemical disruption of the gut barrier with dextran sulfate sodium augments CCM formation in a mouse model, as does genetic loss of Pdcd10, but not Krit1, in gut epithelial cells. Loss of gut epithelial Pdcd10 results in disruption of the colonic mucosal barrier. Accordingly, loss of Mucin-2 or exposure to dietary emulsifiers that reduce the mucus barrier increases CCM burden analogous to loss of Pdcd10 in the gut epithelium. Last, we show that treatment with dexamethasone potently inhibits CCM formation in mice because of the combined effect of action at both brain endothelial cells and gut epithelial cells. These studies define a gut-brain disease axis in an experimental model of CCM in which a single gene is required for two critical components: gut epithelial function and brain endothelial signaling.
Distributions of mammals in Southeast Asia: The role of the legacy of climate and species body mass
(2019)
Aim Current species distributions are shaped by present and past biotic and abiotic factors. Here, we assessed whether abiotic factors (habitat availability) in combination with past connectivity and a biotic factor (body mass) can explain the unique distribution pattern of Southeast Asian mammals, which are separated by the enigmatic biogeographic transition zone, the Isthmus of Kra (IoK), for which no strong geophysical barrier exists. Location Southeast Asia. Taxon Mammals. Methods We projected habitat suitability for 125 mammal species using climate data for the present period and for two historic periods: mid-Holocene (6 ka) and last glacial maximum (LGM 21 ka). Next, we employed a phylogenetic linear model to assess how present species distributions were affected by the suitability of areas in these different periods, habitat connectivity during LGM and species body mass. Results Our results show that cooler climate during LGM provided suitable habitat south of IoK for species presently distributed north of IoK (in mainland Indochina). However, the potentially suitable habitat for these Indochinese species did not stretch very far southwards onto the exposed Sunda Shelf. Instead, we found that the emerged landmasses connecting Borneo and Sumatra provided suitable habitat for forest dependent Sundaic species. We show that for species whose current distribution ranges are mainly located in Indochina, the area of the distribution range that is located south of IoK is explained by the suitability of habitat in the past and present in combination with the species body mass. Main conclusions We demonstrate that a strong geophysical barrier may not be necessary for maintaining a biogeographic transition zone for mammals, but that instead a combination of abiotic and biotic factors may suffice.
The aim of the present work was to check whether carbohydrate metabolism and partitioning contribute to the higher salt tolerance of the facultative halophyte Hordeum marinum compared to the glycophyte Hordeum vulgare. Seedlings with the same size from the two species were hydroponically grown at 0 (control), 150, and 300 mM NaCl for 3 weeks. H. marinum maintained higher relative growth rate, which was concomitant with a higher aptitude to maintain better shoot tissue hydration and membrane integrity under saline conditions compared to H. vulgare. Gas exchanges were reduced in the two species under saline conditions, but an increase in their water use efficiency was recorded. H. marinum exhibited an increase in leaf soluble sugar concentrations under saline conditions together with an enhancement in the transglucosidase DPE2 (EC 2.4.1.25) activity at 300 mM NaCl. However, H. vulgare showed a high increase in starch phosphorylase (EC 2.4.1.1) activity under saline conditions together with a decrease in leaf glucose and starch concentrations at 300 mM NaCl. In roots, both species accumulated glucose and fructose at 150 mM NaCl, but H. marinum exhibited a marked decrease in soluble sugar concentrations and an increase in starch concentration at 300 mM NaCl. Our data constitute an initiation to the involvement of carbohydrate metabolism and partitioning in salt responses of barley species and further work is necessary to elucidate how their flexibility confers higher tolerance to H. marinum compared to H. vulgare.
The effects of habitat fragmentation and isolation on plant species richness have been verified for a wide range of anthropogenically fragmented habitats, but there is currently little information about their effects in naturally small and isolated habitats. We tested whether habitat area, heterogeneity, and isolation affect the richness of wetland vascular plant species in kettle holes, i.e., small glacially created wetlands, in an agricultural landscape of 1 km(2) in NE Germany. We compared fragmentation effects with those of forest fragments in the same landscape window. Since wetland and forest species might differ in their tolerance to isolation, and because isolation effects on plant species may be trait dependent, we asked which key life history traits might foster differences in isolation tolerance between wetland and forest plants. We recorded the flora and vegetation types in 83 isolated sites that contained 81 kettle holes and 25 forest fragments. Overall, the number of wetland species increased with increasing area and heterogeneity, i.e., the number of vegetation types, while area was not a surrogate for heterogeneity in these naturally fragmented systems. Isolation did not influence the number of wetland species but decreased the number of forest species. We also found that seeds of wetland species were on average lighter, more persistent and better adapted to epizoochory, e.g., by waterfowl, than seeds of forest species. Therefore, we suggest that wetland species are more tolerant to isolation than forest species due to their higher dispersal potential in space and time, which may counterbalance the negative effects of isolation.
Animal personality may affect an animal’s mobility in a given landscape, influencing its propensity to take risks in an unknown environment. We investigated the mobility of translocated common voles in two corridor systems 60 m in length and differing in width (1 m and 3 m). Voles were behaviorally phenotyped in repeated open field and barrier tests. Observed behavioral traits were highly repeatable and described by a continuous personality score. Subsequently, animals were tracked via an automated very high frequency (VHF) telemetry radio tracking system to monitor their movement patterns in the corridor system. Although personality did not explain movement patterns, corridor width determined the amount of time spent in the habitat corridor. Voles in the narrow corridor system entered the corridor faster and spent less time in the corridor than animals in the wide corridor. Thus, landscape features seem to affect movement patterns more strongly than personality. Meanwhile, site characteristics, such as corridor width, could prove to be highly important when designing corridors for conservation, with narrow corridors facilitating faster movement through landscapes than wider corridors.
Animal personality may affect an animal’s mobility in a given landscape, influencing its propensity to take risks in an unknown environment. We investigated the mobility of translocated common voles in two corridor systems 60 m in length and differing in width (1 m and 3 m). Voles were behaviorally phenotyped in repeated open field and barrier tests. Observed behavioral traits were highly repeatable and described by a continuous personality score. Subsequently, animals were tracked via an automated very high frequency (VHF) telemetry radio tracking system to monitor their movement patterns in the corridor system. Although personality did not explain movement patterns, corridor width determined the amount of time spent in the habitat corridor. Voles in the narrow corridor system entered the corridor faster and spent less time in the corridor than animals in the wide corridor. Thus, landscape features seem to affect movement patterns more strongly than personality. Meanwhile, site characteristics, such as corridor width, could prove to be highly important when designing corridors for conservation, with narrow corridors facilitating faster movement through landscapes than wider corridors.
In ecological communities, especially the pelagic zones of aquatic ecosystems, certain bodysize ranges are often over-represented compared to others. Community size spectra, the distributions of community biomass over the logarithmic body-mass axis, tend to exhibit regularly spaced local maxima, called "domes", separated by steep troughs. Contrasting established theory, we explain these dome patterns as manifestations of top-down trophic cascades along aquatic food chains. Compiling high quality size-spectrum data and comparing these with a size-spectrum model introduced in this study, we test this theory and develop a detailed picture of the mechanisms by which bottom-up and top-down effects interact to generate dome patterns. Results imply that strong top-down trophic cascades are common in freshwater communities, much more than hitherto demonstrated, and may arise in nutrient rich marine systems as well. Transferring insights from the general theory of nonlinear pattern formation to domes patterns, we provide new interpretations of past lake-manipulation experiments.