Biology subjects

Toth, K.

Publications and source records attributed to Toth, K..

35 records · Page 2Linked to original sources

bioRxiv2022

Chloroplast genome assemblies and comparative analyses of major Vaccinium berry crops

BackgroundVaccinium is an economically important genus of berry crops in the family Ericaceae. Given the numerous hybridizations and polyploidization events among Vaccinium species, the taxonomy of this genus has remained uncertain and the subject of long debate. Therefore, the availability of more genomic resources for Vaccinium can provide useful tools for phylogenetic resolution, species identification, authentication of berry food products, and a framework for genetic engineering. ResultsIn this study, we assembled five Vaccinium chloroplast sequences representing the following berry types: northern highbush blueberry (V. corymbosum), southern highbush blueberry (V. corymbosum hybrids), rabbiteye blueberry (V. virgatum), lowbush blueberry (V. angustifolium), and bilberry (V. myrtillus). Two complete plastid genomes were achieved using long-read PacBio sequencing, while three draft sequences were obtained using short-read Illumina sequencing. Comparative analyses also included other previously available Vaccinium chloroplast sequences, especially the commercially important species V. macrocarpon (cranberry). The Vaccinium chloroplast genomes exhibited a circular quadripartite structure, with an overall highly conserved synteny and sequence identity among them. Despite their high similarity, we identified some polymorphic regions in terms of expansion/contraction of inverted repeats, gene copy number variation, simple sequence repeats, and single nucleotide polymorphisms. Phylogenetic analysis revealed multiple origins of highbush blueberry plastomes, likely due to the hybridization events during northern and southern highbush blueberry domestication. ConclusionsOur results enrich the genomic data availability for new Vaccinium species by sequencing and assembling the chloroplast DNA of major economically important berry types. Additional whole plastome analyses including more samples and wild species will be useful to obtain a refined knowledge of the maternal breeding history of blueberries and increase phylogenetic resolution at low taxonomic levels.

plant biology↗

bioRxiv2021

Effect of A-tract-mediated linker histone orientation on trinucleosome compaction

Linker histones (LH) have been shown to preferentially bind to AT-rich DNA, particularly A-tracts, contiguous stretches of adenines. Using spFRET (single pair Forster/Fluorescence Resonance Energy Transfer), we recently found that the globular domain (gH) of Xenopus laevis H1.0b LH orients towards A-tracts on the linker-DNA (L-DNA) while binding on-dyad in LH:mononucleosome complexes. Here, we investigate the impact of this A-tract-mediated orientation of the gH on the compaction of higher-order structures by studying trinucleosomes as minimal models for chromatin. Two 600 bp DNA sequences were constructed, each containing three consecutive Widom 601 core sequences connected by about 40 bp L-DNA but differing in the positioning of A-tracts on either the outer or the inner L-DNAs flanking the first and third Widom 601 sequences. The two inner L-DNAs were fluorescently labelled at their midpoints. Trinucleosomes were reconstituted using the doubly labelled DNA, core histone octamers and H1.0b. SpFRET was performed for a range of NaCl concentrations to measure the compaction and whether gH orientations affected the stability of the trinucleosomes to salt-induced dissociation. While the LH compacted the trinucleosomes, the extent of compaction and the stability were similar for the two DNA sequences. Modeling constrained by the measured FRET efficiency suggests that the structures adopted by the trinucleosomes correspond to the standard zig-zagged two-helical start arrangement with the first and third nucleosomes stacked on top of each other. In this arrangement, the first and third LHs are insufficiently close to interact and affect compaction. Thus, despite differences in the positioning of the A-tracts in the sequences studied, LH binding compacts the corresponding trinucleosomes similarly. Why it mattersThe compaction and three-dimensional structure of chromatin affect the exposure of the DNA and thus regulate gene expression. Linker histone proteins bind to nucleosomes and thereby contribute to chromatin compaction. We here investigated whether the DNA A-tract-mediated orientation of a linker histone globular domain affects chromatin structure by using a trinucleosome as a minimal model for chromatin. Our observations suggest that the trinucleosome structure and compaction are robust against differences in linker histone globular domain orientations. eTOC blurbWe investigate whether DNA sequences, such as adenine-tracts, and sequence-induced linker histone reorientation affect chromatin structure. Using trinucleosomes as model systems for chromatin, we demonstrate that the chromatin structure and compaction are robust to the studied DNA sequence differences and sequence-induced linker histone orientation.

biophysics↗

bioRxiv2021

A Promising Chemical Series of Positive Allosteric Modulators of the μ-Opioid Receptor that Enhance the Antinociceptive Efficacy of Opioids but not their Adverse Effects

Positive allosteric modulators (PAMs) of the {micro}-opioid receptor (MOR) have been proposed to exhibit therapeutic potential by maximizing the analgesic properties of clinically used opioid drugs while limiting their adverse effects or risk of overdose as a result of using lower drug doses. We herein report in vitro and in vivo characterization of two small molecules from a chemical series of MOR PAMs that exhibit: (i) MOR PAM activity and receptor subtype selectivity in vitro, (ii) a differential potentiation of the antinociceptive effect of oxycodone, morphine, and methadone in mouse models of pain that roughly correlates with in vitro activity, and (iii) a lack of potentiation of adverse effects associated with opioid administration, such as somatic withdrawal, respiratory depression, and analgesic tolerance. This series of MOR PAMs holds promise for the development of adjuncts to opioid therapy to mitigate against overdose and opioid use disorders.

neuroscience↗

bioRxiv2021

Microglial NKCC1 shapes microglial phenotype, cerebral inflammatory responses and brain injury

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to osmotic stress in a cell-autonomous manner via regulating membrane potential and chloride fluxes. In addition, microglial NKCC1 deficiency results in increased expression of the D subunit of volume regulated anion channel (VRAC), NLRP3 inflammasome priming and production of interleukin-1{beta} (IL-1{beta}), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels, whereas systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

neuroscience↗

bioRxiv2020

DNA-sequence dependent positioning of the linker histone in a chromatosome: a single-pair FRET study

Linker histones (LH) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed, with 64% AT. The labelled gH consistently exhibited higher FRET efficiency with the labelled L-DNA containing the A-tract, than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on-dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizingff AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences. Statement of SignificanceLinker histones (LHs) associate with the smallest repeat unit of chromatin, the nucleosome. They have been observed to have affinity for AT-rich DNA, which is found in constitutive heterochromatin and scaffold-associated regions (SAR), which could explain how the LHs can compact such parts of the chromatin. How the LH recognizes such sequences is poorly understood. Using single-pair FRET and modelling, we provide experimental evidence of DNA-sequence-induced changes in the orientation of a LH bound to a nucleosome, and thereby reveal a new mechanism by which the LH can recognize A-tract sequences that are abundantly present in the SAR. Our results show that, depending on how the LH associates with the nucleosome, it can employ more than one mechanism to recognize AT-rich DNA.

biophysics↗

bioRxiv2020

Establishment of Multi-stage Models of Drug Taking and Seeking in Mice

A genetically tractable animal model would provide a needed strategy to resolve the biological basis of drug addiction. Intravenous self-administration (IVSA) is the gold standard for modeling cocaine and opioid addiction in animals, but technical limitations have precluded the widespread use of IVSA in mice. Here, we describe the first IVSA paradigms for mice that capture the multi-stage nature of the disease and permit predictive modeling. Mice with long-standing indwelling jugular catheters engaged in cocaine or opioid-associated lever responding that was fixed ratio- and dose-dependent, extinguished by the withholding of drug, and reinstated by the presentation of paired cues. Machine learning revealed that vulnerability to drug seeking and relapse were predicted by a mouses a priori response to novelty, sensitivity to drug-induced locomotion, and drug-taking behavior. Application of this behavioral and analysis approach to genetically-engineered mice will facilitate the identification of the neural circuits driving addiction susceptibility and relapse and focused therapeutic development.

neuroscience↗

bioRxiv2020

Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

pathology↗

bioRxiv2020

Synaptic dynamics as convolutional units

Short-term synaptic dynamics differ markedly across connections and strongly regulate how action potentials communicate information. To model the range of synaptic dynamics observed in experiments, we have developed a flexible mathematical framework based on a linear-nonlinear operation. This model can capture various experimentally observed features of synaptic dynamics and different types of heteroskedasticity. Despite its conceptual simplicity, we show that it is more adaptable than previous models. Combined with a standard maximum likelihood approach, synaptic dynamics can be accurately and efficiently characterized using naturalistic stimulation patterns. These results make explicit that synaptic processing bears algorithmic similarities with information processing in convolutional neural networks. Author summaryUnderstanding how information is transmitted relies heavily on knowledge of the underlying regulatory synaptic dynamics. Existing computational models for capturing such dynamics are often either very complex or too restrictive. As a result, effectively capturing the different types of dynamics observed experimentally remains a challenging problem. Here, we propose a mathematically flexible linear-nonlinear model that is capable of efficiently characterizing synaptic dynamics. We demonstrate the ability of this model to capture different features of experimentally observed data.

neuroscience↗

bioRxiv2020

Establishment gaps in species-poor grasslands: artificial biodiversity hotspots to support the colonization of target species

Sowing of grass seed mixtures is a feasible and cost-effective method for landscape-scale grassland restoration. However, sowing only grasses usually leads to species-poor and dense grass sward, where the establishment of target forbs is hampered both by microsite and propagule limitation. To overcome these limitations and increase the diversity of species-poor sown grasslands we developed a novel method by creating establishment gaps. We used tillage to open gaps of 1 m2, 4 m2 and 16 m2 size in the dense grass sward of six species-poor sown grasslands in the Great Hungarian Plain. We sowed high-diversity seed mixtures of 35 native species into all gaps. We analyzed vegetation development during the first five years after setting up the trial. We also studied the colonization dynamics of the sown species along four 20-m transects around each gap, resulting in a total of 1440 plots of 1 m2 size that were studied. Our results indicated that most of the sown species were able to establish permanently in the establishment gaps. The total cover and the cover of perennial sown species increased independently of gap size. Meanwhile the cover of short-lived sown species decreased during the five years. There was only a moderate level of weed abundance in the gaps, and weed cover decreased over the years. The sown target species started to colonize the species-poor grasslands surrounding the establishment gaps within five years. The highest number of species and individuals dispersed from the 4 m2-sized gaps, as they had a more stable development than small gaps and were exposed to lower grazing pressure than large ones. Implications for practiceO_LIEstablishment gaps are widely applicable tools to increase the diversity of species-poor grasslands. Gaps of 4 m2 represent a more feasible solution compared to larger openings also for the farmers, because there is only a moderate level of weed encroachment and smaller soil disturbance occurs during their creation. C_LIO_LIWe recommend sowing high-diversity seed mixtures containing both short-lived species that can establish in the first year and perennial species, which guarantee a high cover of target species later on. C_LIO_LIGaps sown with high-diversity seed mixture are highly resistant to unfavorable climatic conditions: increasing grass abundance in dry years does not hamper the recovery of target grassland species in the following years. C_LI

ecology↗

bioRxiv2020

Consumption rate and dietary choice of cattle in species-rich mesic grasslands

For the improvement and maintenance of the desirable ecological value of grasslands it is necessary to manage them in a way which maintains their structure and their long-term functioning. Extensive grazing plays a crucial role in the seasonal biomass removal, thereby it prevents litter accumulation and shrub encroachment. Defoliation and biomass removal are among the most important effects of grazing on the vegetation, while the sufficient quantity and quality of plant biomass is an important ecosystem service for animal husbandry. In order to maintain the long term functioning of pastures it is important to gather information about the amount of consumed biomass and the dietary choice of the grazers. Therefore, we studied the direct effects of grazing on species-rich meadow steppes in Central-Hungary and the underlying mechanisms of dietary choice of cattle using trait-based approach. We asked the following questions: (i) What are the direct effects of grazing on the main biomass fractions (litter, moss, forbs and graminoids)? (ii) Which traits distinguish the preferred and non-preferred vascular plant species? The studied pastures were divided into two adjacent units, which were managed differently in the study year: the grazed units were managed by grazing for three months before the sampling date, while the control units remained ungrazed until the sampling. We collected above-ground biomass samples, measured leaf traits and shoot nitrogen content of plants. The consumption of the litter and moss biomass was negligible, while the reduction of the live biomass of vascular plants was 65%. Grazing significantly decreased the flowering success of plants. Cattle consumed species characterized by high specific leaf area and high nitrogen content. Based on our results we emphasize that, in order to ensure the reproduction of most plant species in the long term, it is unfavourable to graze an area every year in the same period. Instead, it is recommended to use grazing in a mosaic spatial and temporal pattern. The livestock carrying capacity of an area and the long-term management of grasslands can be carefully planned based on biomass measurements and the nutritional value of plants, which is well indicated by some easily measurable plant properties such as specific leaf area and the nitrogen content of species.

ecology↗

bioRxiv2020

Vegetation and seed bank dynamics highlight the importance of post-restoration management in sown grasslands

Sowing grass seeds generally supports the rapid development of a closed perennial vegetation, which makes the method universally suitable for fast and effective landscape-scale restoration of grasslands. However, sustaining the recovered grasslands, and increasing their diversity is a challenging task. Understanding the role of seed bank compositional changes and vegetation dynamics contributes to designating management regimes that support the establishment of target species and suppress weeds. Our aim was to reveal the effect of post-restoration management on the vegetation and seed bank dynamics in grasslands restored in one of the largest European landscape-scale restoration projects. Eight years after restoration we sampled the vegetation and seed bank in a total of 96 plots located in 12 recovered grasslands in the Great Hungarian Plain. In each recovered grassland stand we designated a mown (mown from Year 1 to Year 8) and an abandoned sample site (mown from Year 1 to Year 3 then abandoned from Year 4 to Year 8). Mown and abandoned sites showed divergent vegetation and seed bank development. Abandonment led to the decline of sown grasses and higher cover of weeds, especially in the alkaline grasslands. Our study confirmed that seed bank has a limited contribution to the maintenance of biodiversity in both grassland types. We found that five years of abandonment had a larger effect on the seed bank than on the vegetation. We stress that long-term management is crucial for controlling the emergence of the weeds from their dense seed bank in restored grasslands. Implications for practiceO_LISeed sowing of grass mixtures can be a feasible tool for restoring grasslands at large scales. However, the developed vegetation usually has low biodiversity and a high seed density of weeds is typical in the soil seed bank even several years after the restoration. Therefore, post-restoration management is necessary for suppressing weeds both aboveground and belowground. C_LIO_LIWe recommend to design the long-term management of the sites subjected to grassland restoration already in the planning phase of the restoration projects and ensure that the management plan is ecologically and economically feasible. C_LIO_LIWe recommend to complement the monitoring of vegetation with the analysis of soil seed bank for evaluating restoration success. C_LI

ecology↗

bioRxiv2020

Germination response of invasive plants to soil burial depth and litter accumulation is species specific

QuestionsPlant invasions are considered among the biggest threats to biodiversity worldwide. In a full-factorial greenhouse experiment we analysed the effect of soil burial depth and litter cover on the germination of invasive plants. We hypothesised that (i) burial depth and litter cover affect the germination of the studied species, (ii) the effects of burial and litter cover interact with each other, and (iii) the effects are species-specific, but dependent on seed size. MethodsWe tested the germination and seedling growth of 11 herbaceous invasive species in a full-factorial experiment using four levels of seed burial depths and litter cover. We analysed the effect of burial, litter cover, and their interactions on germination, seedling length and biomass across species and at the species level. ResultsSoil burial depth and litter cover had a significant effect on the germination of the studied species, but there were considerable differences between species. We observed a general trend of species with bigger seeds being not or less seriously affected by soil burial and litter cover than smaller-seeded species. Correlations between seed weight and effect sizes mostly confirmed this general trend, but not in the case of soil burial. ConclusionsOur findings confirmed that seed size is a major driver of species response to litter cover and to the combined effects of litter cover and soil burial, but there is no general trend regarding the response to soil burial depth. Despite its very small seeds the germination of Cynodon dactylon was not affected by soil burial. The germination of Ambrosia artemisiifolia was hampered by both soil burial and litter cover despite its relatively large seeds. Thus, specific information on species response to burial depth and litter accumulation is crucial when planning management or restoration in areas threatened by plant invasions.

ecology↗

bioRxiv2019

Fxr1 regulates sleep and synaptic homeostasis

The fragile X autosomal homolog 1 (Fxr1) has been GWAS-associated to schizophrenia and insomnia but its contributions to brain functions are unclear. Homeostatic regulation of synaptic strength is essential for the maintenance of brain functions and engages both global and cell autonomous level processes. We used Crispr/Cas9-mediated somatic knockouts, overexpression, neuronal activity recordings and translatome sequencing, to examine the contribution of Fxr1 to cell-autonomous homeostatic synaptic scaling and global-level sleep homeostasis. Our findings indicate that Fxr1 is downregulated during scaling and sleep deprivation via a Gsk3{beta} dependent mechanism. In both conditions, downregulation of Fxr1 is essential for the homeostatic modulation of synaptic strength. Furthermore, overexpression of Fxr1 during sleep deprivation results in altered EEG signatures and reverts changes of translatome profiles. These findings indicate that Fxr1 represents a shared signaling hub linking cell autonomous homeostatic plasticity and system level sleep homeostasis with potential implications for neuropsychiatric illnesses.

neuroscience↗

bioRxiv2019

GIT2 is dispensable for normal learning and memory function due to a predominant brain GIT2 splice variant that evades GIT/PIX complexes.

G protein-coupled receptor kinase-interacting protein 2 (GIT2) and GIT1 are highly similar, sharing the same domain structure and many binding partners. The most important GIT partners are the p21-activated protein kinase-interacting exchange factor (PIX) proteins, since through homomeric and heteromeric interactions, GIT and PIX proteins form oligomeric GIT/PIX complexes. Oligomeric GIT/PIX complexes function both as regulators of small GTP-binding proteins and as scaffolds for signalling molecules, including p21-activated protein kinases (PAKs). Deficits in learning and memory have been demonstrated in GIT1 knockout mice, and it has been assumed that GIT2 also would affect learning and memory. Unexpectedly, we find that GIT2-deficient mice respond normally in multiple tests of learning and memory, and have normal hippocampal long-term potentiation. Further, we find no evidence that GIT2 regulates ADHD-like phenotypes. To investigate why GIT2 and GIT1 differ so markedly in the brain, we identified the major isoform of GIT2 in the brain as a previously uncharacterized splice variant, GIT2({Delta}BCE). This variant cannot dimerize or form oligomeric complexes with PIX proteins, and is thus incapable of regulating PAK in synapses, compared to oligomeric GIT1/PIX complexes. Because localized activation of PAK in synapses is required for structural plasticity underlying cognitive performance, loss of monomeric GIT2({Delta}BCE) in the brain does not influence these responses.

animal behavior and cognition↗

bioRxiv2018

Fast two-photon volumetric imaging of an improved voltage indicator reveals electrical activity in deeply located neurons in the awake brain

Imaging of transmembrane voltage deep in brain tissue with cellular resolution has the potential to reveal information processing by neuronal circuits in living animals with minimal perturbation. Multi-photon voltage imaging in vivo, however, is currently limited by speed and sensitivity of both indicators and imaging methods. Here, we report the engineering of an improved genetically encoded voltage indicator, ASAP3, which exhibits up to 51% fluorescence responses in the physiological voltage range, sub-millisecond activation kinetics, and full responsivity under two-photon illumination. We also introduce an ultrafast local volume excitation (ULOVE) two-photon scanning method to sample ASAP3 signals in awake mice at kilohertz rates with increased stability and sensitivity. ASAP3 and ULOVE allowed continuous single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution, and with repeated sampling over multiple days. By imaging voltage in visual cortex neurons, we found evidence for cell type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULOVE enable continuous high-speed high-resolution imaging of electrical activity in deeply located genetically defined neurons during awake behavior.

neuroscience↗

bioRxiv2017

Action potential counting at giant mossy fiber terminals gates information transfer in the hippocampus

Hippocampal mossy fibers have long been recognized as conditional detonators owing to prominent short-term facilitation, but the patterns of activity required to fire postsynaptic CA3 pyramidal neurons remain poorly understood. We show that mossy fibers count the number of spikes to transmit information to CA3 pyramidal cells through a distinctive interplay between presynaptic calcium dynamics, buffering and vesicle replenishment. This identifies a previously unexplored information coding mechanism in the brain.

neuroscience↗

bioRxiv2017

Revealing The Distribution Of Transmembrane Currents Along The Dendritic Tree Of A Neuron With Known Morphology From Extracellular Recordings

Revealing the membrane current source distribution of neurons is a key step on the way to understanding neural computations, however, the experimental and theoretical tools to achieve sufficient spatiotemporal resolution for the estimation remain to be established. Here we address this problem using extracellularly recorded potentials with arbitrarily distributed electrodes in a neuron of known morphology. We use simulations of models with varying complexity to validate the proposed method and to give recommendations for experimental applications. The method is applied to in vitro data from rat hippocampus.

neuroscience↗