Biology subjects

Urban, P.

Publications and source records attributed to Urban, P..

12 recordsLinked to original sources

bioRxiv2024

Expanding the diversity of nitroxide-based paramagnetic probes conjugated to non-canonical amino acids for SDSL-EPR applications

Understanding protein structure requires studying its dynamics, which is critical to elucidating its functional role. Over time, biophysical techniques have revolutionized this field, offering remarkable insights into the structure-function relationship. Among these, Site-Directed Spin Labelling (SDSL) combined with Electron Paramagnetic Resonance (EPR) is a powerful method delivering structural data at the residue level, irrespective of protein size or environment. Traditional nitroxide labels, which target cysteine residues, often face limitations when these residues are essential for protein structure or function. To address this, alternatives have been proposed as the use of non-canonical amino acids (ncaa) coupled with specific nitroxide labels. This study introduces 14N-HO-5223, a novel nitroxide label specific to the pAzPhe ncaa, alongside its 15N-derivative. These labels were grafted at two sites of the model protein, the diflavin Cytochrome P450 reductase. For comparative purpose, two already reported labels were also used. Continuous wave (cw) EPR spectroscopy validated the HO-5223 label as an effective reporter of protein dynamics. Additionally, Double Electron-Electron Resonance (DEER) measurements provided distance distributions between the semi-quinone FMNH* state of the CPR and all nitroxide labels. These results expand the toolkit of the ncaa-nitroxide pairs, enabling EPR-based structural studies of proteins where cysteine modification is impractical, further advancing our ability to decode protein dynamics and function.

biophysics↗

bioRxiv2024

Bridging species boundaries: eDNA and genetic analysis reveal hybrid settlement at the very end of American eel distribution

Documenting species distributions and hybridization patterns is paramount for elucidating biogeography and understanding speciation processes. Here we combined genetic specimen analysis and environmental DNA (eDNA) to investigate the presence of American eel (Anguilla rotrata) and American x European eel (Anguilla anguilla) hybrids in Greenland freshwater. We further tested the use of eDNA to document hybridization by using European eel mtDNA as a proxy for hybrid occurrence. Overall, eDNA analysis detected mainly American eel but also European eel mtDNA. This finding was validated by DNA sequencing, which identified 3 out of 26 captured eels (14.3%) carrying European eel mtDNA. Five eels (19.2%), including all three with European mtDNA, were heterozygous for species-specific nuclear gene variants, supporting a hybrid ancestry. Further, eDNA successfully identified eels in lakes where they were caught by fyke net fishing, extending their confirmed northern range by 40 km, and indicated eel presence >200km further north. The study provides an empirical demonstration of the use of eDNA to document hybrid occurrence and extends the reported northern distribution of eels in Greenland significantly beyond previous observations. Further, the existence of hybrid eels in Greenland may be key for understanding the complex mechanisms of hybridization between American and European eels.

genetics↗

bioRxiv2024

Unraveling Morphogenesis, Starvation, and Light Responses in a Mushroom-Forming Fungus, Coprinopsis cinerea, Using Long Read Sequencing and Extensive Expression Profiling

Mushroom-forming fungi (Agaricomycetes) are emerging as pivotal players in several fields, as drivers of nutrient cycling, sources of novel applications, and the group includes some of the most morphologically complex multicellular fungi. Genomic data for Agaricomycetes are accumulating at a steady pace, however, this is not paralleled by improvements in the quality of genome sequence and associated functional gene annotations, which leaves gene function notoriously poorly understood in comparison with other fungi and model eukaryotes. We set out to improve our functional understanding of the model mushroom Coprinopsis cinerea by integrating a new, chromosome-level assembly with high-quality gene predictions and functional information derived from gene-expression profiling data across 67 developmental, stress, and light conditions. The new annotation has considerably improved quality metrics and includes 5- and 3-untranslated regions (UTRs), polyadenylation sites (PAS), upstream ORFs (uORFs), splicing isoforms, conserved sequence motifs (e.g., TATA and Kozak boxes) and microexons. We found that alternative polyadenylation is widespread in C. cinerea, but that it is not specifically regulated across the various conditions used here. Transcriptome profiling allowed us to delineate core gene sets corresponding to carbon starvation, light-response, and hyphal differentiation, and uncover new aspects of the light-regulated phases of life cycle. As a result, the genome of C. cinerea has now become the most comprehensively annotated genome among mushroom-forming fungi, which will contribute to multiple rapidly expanding fields, including research on their life history, light and stress responses, as well as multicellular development.

genomics↗

bioRxiv2023

Cooperativity, information gain, and energy cost during early LTP in dendritic spines

We investigate a mutual relationship between information and energy during early phase of LTP induction and maintenance in a large-scale system of mutually coupled dendritic spines with discrete internal states and probabilistic dynamics within the framework of nonequilibrium stochastic thermodynamics. In order to analyze this computationally intractable stochastic multidimensional system, we introduce a pair approximation, which allows us to reduce the spine dynamics into a lower dimensional manageable system of closed equations. It is found that the rates of information gain and energy attain their maximal values during an initial period of LTP (i.e. during stimulation), and after that they recover to their baseline low values, as opposed to a memory trace that lasts much longer. This suggests that learning phase is much more energy demanding than the memory phase. We show that positive correlations between neighboring spines increase both a duration of memory trace and energy cost during LTP, but the memory time per invested energy increases dramatically for very strong positive synaptic cooperativity, suggesting a beneficial role of synaptic clustering on memory duration. In contrast, information gain after LTP is the largest for negative correlations, and energy efficiency of that information generally declines with increasing synaptic cooperativity. We also find that dendritic spines can use sparse representations for encoding of long-term information, as both energetic and structural efficiencies of retained information and its lifetime exhibit maxima for low fractions of stimulated synapses during LTP. Moreover, we find that such efficiencies drop significantly with increasing the number of spines. In general, our stochastic thermodynamics approach provides a unifying framework for studying, from first principles, information encoding and its energy cost during learning and memory in stochastic systems of interacting synapses.

neuroscience↗

bioRxiv2022

Confrontation of AlphaFold2 models with cryo-EM and crystal structures enlightens alternate geometries of the CYP102A1 multidomain protein

Large range structural dynamics plays a critical role for the function of electron transfer proteins. This information is generally not available from crystallographic structures, while cryo-electron microscopy (cryo-EM) can provide some elements but frequently with a degraded spatial resolution. Recently, AlphaFold-based structural modelling was extended to the prediction of protein complexes. In this work, bacterial CYP102A1 from Priestia megaterium was used as a test case to evaluate the capability of AlphaFold2 to predict alternative structures critical for catalysis. CYP102A1 monooxygenase, a NADPH-supported fatty acid hydroxylase, works as a soluble homodimer, each monomer harboring two flavins (FAD and FMN) and one heme cofactors. Large conformational changes are required during catalytic cycle to allow successive electron transfers from FAD to FMN and finally heme iron. We used the recently released AlphaFold2_advanced notebook (AF2A), to predict the possible alternate conformations supporting electron transfers in CYP102A1 homodimer. Challenging AF2A-derived models with previously reported experimental data revealed an unforeseen domain connectivity of the diflavin reductase part of the enzyme. Intermolecular crossed complex constitutes a novel type of structural organization never previously described. The predicted formation within the dimer of a stable complex between the heme containing domains was challenged and found consistent with uninterpreted features of reported crystallographic structures and cryo-EM imaging. The particularly efficient CYP102A1 catalytic mechanism was revisited to the light of the new evidenced connectivity in which the FMN-binding domain of each monomer oscillates on themselves to alternatively receive and transfer electrons without needing large structural change in the dimer. Such model was found explanatory for previously contradictory reported biochemical data. Possibility to mimic CYP102A1 structural organization into bicomponent eukaryotic P450 systems was evaluated by designing and modeling in silico synthetic reductase domains built from composite sequence segments from P. megaterium and human origins. More generally, this work illustrates how the ability of AF2A to predict alternate complex structures can enlighten and explain conformational changes critical for bio-assemblies.

biochemistry↗

bioRxiv2022

What drives wolf preference towards wild ungulates? Insights from a multi-prey system in the Slovak Carpathians

The wolf is a generalist-opportunistic predator that displays diverse and remarkably adaptable feeding strategies across its range with local adaptations to certain prey species depending on their availability and vulnerability. The multi-prey system of the Slovak Carpathians supports important portion of the European wolf population; however, it has been markedly understudied. We evaluated winter diet composition and prey selection of Slovak wolves based on 321 scat samples collected between September - February within four different study areas during 2015 - 2017. The winter diet of wolves in the Slovak Carpathians was characterized by a 98% occurrence of wild large-sized and medium-sized ungulates with red deer occurring in wolf scats most often, consistent with their highest density among other wild ungulates. However, by comparing the consumption with availability of wild prey, we found that wolves in fact selected for wild boar especially in areas with higher altitudinal range, while selected for red deer in areas with low altitudinal range where this prey species was more spatially predictable. Although wolves showed the potential to switch between red deer and wild boar when their density increases, we found that this variation can be rather linked to changing prey vulnerability, which is dependent on particular environmental conditions at local scale such as topography and snow accumulation. The present study provides valuable insights into the winter foraging ecology of Slovak wolves in a multi-prey system of the Carpathians and allows for practical implications in the management of the rapidly increasing populations of wild ungulates across Europe.

zoology↗

bioRxiv2021

Information encoded in volumes and areas of dendritic spines is nearly maximal across mammalian brains

Many experiments suggest that long-term information associated with neuronal memory resides collectively in dendritic spines. However, spines can have a limited size due to metabolic and neuroanatomical constraints, which should effectively limit the amount of encoded information in excitatory synapses. This study investigates how much information can be stored in the population of sizes of dendritic spines, and whether it is optimal in any sense. It is shown here, using empirical data for several mammalian brains across different regions and physiological conditions, that dendritic spines nearly maximize entropy contained in their volumes and surface areas for a given mean size in cortical and hippocampal regions. Although both short- and heavy-tailed fitting distributions approach 90 - 100% of maximal entropy in the majority of cases, the best maximization is obtained primarily for short-tailed gamma distribution. We find that most empirical ratios of standard deviation to mean for spine volumes and areas are in the range 1.0 {+/-} 0.3, which is close to the theoretical optimal ratios coming from entropy maximization for gamma and lognormal distributions. On average, the highest entropy is contained in spine length (4-5 bits per spine), and the lowest in spine volume and area (2 - 3 bits), although the latter two are closer to optimality. In contrast, we find that entropy density (entropy per spine size) is always suboptimal. Our results suggest that spine sizes are almost as random as possible given the constraint on their size, and moreover the general principle of entropy maximization is applicable and potentially useful to information and memory storing in the population of cortical and hippocampal excitatory synapses, and to predicting their morphological properties. Significance statementIt is believed that information related to long-term memory is stored collectively in the parts of excitatory synapses called dendritic spines. But how efficient is the information capacity given synaptic size variability? Generally, the larger this variability the higher entropy (information capacity) of spine population. However, this process comes at some cost, as larger synapses use more metabolic energy and brain tissue, suggesting a benefit-cost trade-off for storing long-term information. We show that volumes and areas of actual spines in many different parts of the brain across different mammals and conditions adjust simultaneously their variable mean and standard deviation values to nearly maximize entropy of their distributions. This suggests that storing capacity of dendritic spines is nearly maximal, despite large variability in their sizes.

neuroscience↗

bioRxiv2021

Aedes koreicus, a vector on the rise: pan-European genetic patterns, mitochondrial and draft genome sequencing

BackgroundThe mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species biology, behavior, and dispersal patterns were poorly investigated to date. Methodology/Principal FindingsTo understand the species population relationships and dispersal patterns within Europe, a fragment of the COI gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering clearly indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. Conclusions/SignificanceOur results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of Single Nucleotide Polymorphism markers. Considering its public health importance, it is crucial to further investigate the species population genetic dynamic, including a larger sampling and additional genomic markers. Author SummaryIn the present context of globalization and changing environment, the rapid spread of Invasive Mosquito Species (IMS) across Europe represents a serious public health threat because some species are competent vectors for several pathogens. A better knowledge of the IMS population relationships, demographic trends, and dispersal patterns can help the relevant authorities mitigating further spread. Aedes koreicus is an IMS that invaded the continent and has been expanding its geographic range over the last decade. In the present study, one of the most popular DNA marker (COI) was used to investigate the pan-European haplotype diversity and phylogenetic relatedness within and between Ae. koreicus populations. Also, the first complete mitochondrial genome and draft nuclear genome of Ae. koreicus were generated using combined high-throughput sequencing techniques (Oxford Nanopore, Illumina). This provides a significant leap in the general understanding of this species and opens the possibility for future genomic studies.

genetics↗

bioRxiv2020

Multiple SARS-CoV-2 introductions shaped the early outbreak in Central Eastern Europe: comparing Hungarian data to a worldwide sequence data-matrix

Severe Acute Respiratory Syndrome Coronavirus 2 is the third highly pathogenic human coronavirus in history. Since the emergence in Hubei province, China, during late 2019 the situation evolved to pandemic level. Following China, Europe was the second epicenter of the pandemic. To better comprehend the detailed founder mechanisms of the epidemic evolution in Central-Eastern Europe, particularly in Hungary, we determined the full-length SARS-CoV-2 genomes from 32 clinical samples collected from laboratory confirmed COVID-19 patients over the first month of disease in Hungary. We applied a haplotype network analysis on all available complete genomic sequences of SARS-CoV-2 from GISAID database as of the 21th of April, 2020. We performed additional phylogenetic and phylogeographic analyses to achieve the recognition of multiple and parallel introductory events into our region. Here we present a publicly available network imaging of the worldwide haplotype relations of SARS-CoV-2 sequences and conclude the founder mechanisms of the outbreak in Central-Eastern Europe.

microbiology↗

bioRxiv2020

Identification, presence, and possible multifunctional regulatory role of invertebrate gonadotropin-releasing hormone/corazonin molecule in the great pond snail (Lymnaea stagnalis).

In the last years, the interpretation of gonadotropin-releasing hormone (GnRH) neuropeptide superfamily has changed tremendously. One main driver is the investigation of functions and evolutionary lineage of previously identified molluscan GnRH molecules. Emerging evidence suggests not only reproductive, but also diverse biological effects of these molecules and proposes they should most likely be called corazonin (CRZ). Clearly, a more global understanding necessitates further exploration of species-specific functions and structure of invGnRH/CRZ peptides. Towards this goal, we have identified the full-length cDNA of invGnRH/CRZ peptide in an invertebrate model species, the great pond snail Lymnaea stagnalis, termed ly-GnRH/CRZ, and characterized the transcript and peptide distribution in the central nervous system (CNS) and peripheral organs. Our results are consistent with previous data that molluscan GnRHs are more related to CRZs and serve diverse functions. For this, our findings support the notion that peptides originally termed molluscan GnRH are multifunctional modulators and that nomenclature change should be taken into consideration.

neuroscience↗

bioRxiv2020

Spreading depressions and periinfarct spreading depolarizations in the context of cortical plasticity

Studies of cortical function-recovery require a comparison between normal and post-stroke conditions that lead to changes in cortical metaplasticity. Focal cortical stroke impairs experience-dependent plasticity (ExDP) in the neighboring somatosensory cortex and usually evokes periinfarct depolarizations (PiDs) - a spreading depression-like waves. Experimentally induced spreading depressions (SDs) affect gene expression and some of these changes persist for at least 30 days. However, such changes are not stroke-specific: migraine patients had prolonged protein changes after a single SD episode during migraine aura. This study investigates whether non-stroke depolarizations impair cortical ExDP similarly to the stroke. ExDP was induced in rates with SDs or PiDs by a month of contralateral partial whiskers deprivation. Cortical activity was mapped by [14C]-2-deoxy-D-glucose (2DG) incorporation during stimulation of spared and contralateral homotopic whiskers. We found that whiskers deprivation after SDs resulted in normal cortical representation enlargement suggesting that SDs and PiDs depolarization have no influence on ExDP cortical map reorganization. PiDs and the MMP-9, -3, -2 or COX-2 proteins which are assumed to influence metaplasticity in rats after stroke were compared between the SDs induced by high osmolarity KCl solution and the PiDs following cortical photothrombotic stroke (PtS). We found that none of these factors directly caused cortical post-stroke metaplasticity changes. The only significant difference between stoke and induced SD was a greater imbalance in interhemispheric activity equilibrium after stroke. The interhemispheric interactions modified by stroke may therefore be a promising target for future studies of post-stroke ExDP and for convalescence studies. HighlightsO_LIPost-stroke metaplasticity changes in an injured hemisphere are not a consequence of spreading depolarizations C_LIO_LINone of the monitored proteins (MMP-2, -3, -9; COX-2) cause modifications in poststroke cortical plasticity C_LIO_LISpreading depressions have a prolonged, global influence on the functioning of both hemispheres and of the thalamus C_LIO_LIImpaired interhemispheric interactions may underlie the post-stroke metaplasticity changes in the injured hemisphere C_LI

neuroscience↗

bioRxiv2018

Evaluating analgesic efficacy and administration route following craniotomy in mice using the grimace scale

Most research laboratories abide by guidelines and mandates set by their research institution regarding the administration of analgesics to control pain during the postoperative period. Unfortunately, measuring pain originating from the head is difficult, making adequate decisions regarding pain control following stereotaxic surgery problematic. In addition, most postsurgical analgesia protocols require multiple injections over several days, which may cause stress and distress during a critical recovery period. Here we sought to 1) assess the degree of postoperative pain following craniotomy in mice, 2) compare the efficacy of three common rodent analgesics (carprofen, meloxicam and buprenorphine) for reducing this pain and 3) determine whether the route of administration (injected or self-administered through the water supply) influenced pain relief post-craniotomy. Using the mouse grimace scale (MGS), we found that injectable analgesics were significantly more effective at relieving post-craniotomy pain, however, both routes of administration decreased pain scores in the first 24 h post-surgery. Specifically, buprenorphine administered independently of administration route was the most effective at reducing MGS scores, however, female mice showed greater sensitivity to carprofen when administered through the water supply. Although it is necessary to provide laboratory animals with analgesics after an invasive procedure, there remains a gap in the literature regarding the degree of craniotomy-related pain in rodents and the efficacy of alternative routes of administration. Our study highlights the limitations of administering drugs through the water supply, even at doses that are considered to be higher than those currently recommended by most research institutions for treating pain of mild to moderate severity.

neuroscience↗