Biology subjects

Schmidt, D.

Publications and source records attributed to Schmidt, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Multiparametric domain insertional profiling of Adeno-Associated Virus VP1

Evolved properties of Adeno-Associated Virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Previous efforts to re-engineer these properties have focused on variable regions near AAVs 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of large, structured protein domains into the entire AAV-DJ capsid protein VP1. This is the largest and most comprehensive AAV domain insertion dataset to date. Our data revealed a surprising robustness of AAV capsids to accommodate large domain insertions. There was strong positional, domain-type, and fitness phenotype dependence of insertion permissibility, which clustered into correlated structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified new engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.

biochemistry↗

bioRxiv2022

PADAPT 1.0 - the Pannonian Database of Plant Traits

We present PADAPT 1.0, the Pannonian Database of Plant Traits which relies on regional data sources and integrates existing data and new measurements on a wide range of traits and attributes of the plant species of the Pannonian Biogeographical Region and makes it freely accessible at www.padapt.eu. The current version covers the species of the region occurring in Hungary (cc. 90% of the regions flora) and consists of 126,337 records on 2745 taxa. There are 53 plant attributes in PADAPT 1.0 organised in six major groups: (i) Habitus and strategy, (ii) Reproduction, (iii) Kariology, (iv) Distribution and conservation, (v) Ecological indicator values, and (vi) Leaf traits. By including species of the eastern part of Europe not covered by other databases, PADAPT can facilitate studying the flora and vegetation of the eastern part of the continent. Data collection will continue in the future and the PADAPT team welcomes researchers interested in contributing with data. The main task before an updated version of the database is to include species of the Pannonian region not covered by the current version. In conclusion, although data coverage is far from complete, PADAPT meets the longstanding need for a regional database of the Pannonian flora.

ecology↗

bioRxiv2022

BioImage Model Zoo: A Community-Driven Resource for Accessible Deep Learning in BioImage Analysis

Deep learning-based approaches are revolutionizing imaging-driven scientific research. However, the accessibility and reproducibility of deep learning-based workflows for imaging scientists remain far from sufficient. Several tools have recently risen to the challenge of democratizing deep learning by providing user-friendly interfaces to analyze new data with pre-trained or fine-tuned models. Still, few of the existing pre-trained models are interoperable between these tools, critically restricting a models overall utility and the possibility of validating and reproducing scientific analyses. Here, we present the BioImage Model Zoo (https://bioimage.io): a community-driven, fully open resource where standardized pre-trained models can be shared, explored, tested, and downloaded for further adaptation or direct deployment in multiple end user-facing tools (e.g., ilastik, deepImageJ, QuPath, StarDist, ImJoy, ZeroCostDL4Mic, CSBDeep). To enable everyone to contribute and consume the Zoo resources, we provide a model standard to enable cross-compatibility, a rich list of example models and practical use-cases, developer tools, documentation, and the accompanying infrastructure for model upload, download and testing. Our contribution aims to lay the groundwork to make deep learning methods for microscopy imaging findable, accessible, interoperable, and reusable (FAIR) across software tools and platforms.

bioinformatics↗

bioRxiv2022

Determinants of trafficking, conduction, and disease within a K+ channel revealed through multiparametric deep mutational scanning

A longstanding goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships and delineate the mechanisms by which mutations cause disease. Deep Mutational Scanning (DMS) is a promising strategy to map how amino acids contribute to protein structure and function and to advance clinical variant interpretation. Here, we introduce 7,429 single residue missense mutation into the Inward Rectifier K+ channel Kir2.1 and determine how this affects folding, assembly, and trafficking, as well as regulation by allosteric ligands and ion conduction. Our data provides high-resolution information on a cotranslationally{-}folded biogenic unit, trafficking and quality control signals, and segregated roles of different structural elements in fold-stability and function. We show that Kir2.1 trafficking mutants are underrepresented in variant effect databases, which has implications for clinical practice. By comparing fitness scores with expert-reviewed variant effects, we can predict the pathogenicity of variants of unknown significance and disease mechanisms of know pathogenic mutations. Our study in Kir2.1 provides a blueprint for how multiparametric DMS can help us understand the mechanistic basis of genetic disorders and the structure-function relationships of proteins.

biochemistry↗

bioRxiv2021

Labkit: Labeling and Segmentation Toolkit for Big Image Data

We present LO_SCPLOWABKITC_SCPLOW, a user-friendly Fiji plugin for the segmentation of microscopy image data. It offers easy to use manual and automated image segmentation routines that can be rapidly applied to single- and multi-channel images as well as to timelapse movies in 2D or 3D. LO_SCPLOWABKITC_SCPLOW is specifically designed to work efficiently on big image data and enables users of consumer laptops to conveniently work with multiple-terabyte images. This efficiency is achieved by using ImgLib2 and BigDataViewer as the foundation of our software. Furthermore, memory efficient and fast random forest based pixel classification inspired by the Waikato Environment for Knowledge Analysis (Weka) is implemented. Optionally we harness the power of graphics processing units (GPU) to gain additional runtime performance. LO_SCPLOWABKITC_SCPLOW is easy to install on virtually all laptops and workstations. Additionally, LO_SCPLOWABKITC_SCPLOW is compatible with high performance computing (HPC) clusters for distributed processing of big image data. The ability to use pixel classifiers trained in LO_SCPLOWABKITC_SCPLOW via the ImageJ macro language enables our users to integrate this functionality as a processing step in automated image processing workflows. Last but not least, LO_SCPLOWABKITC_SCPLOW comes with rich online resources such as tutorials and examples that will help users to familiarize themselves with available features and how to best use LO_SCPLOWABKITC_SCPLOW in a number of practical real-world use-cases.

bioinformatics↗

bioRxiv2021

Electrocorticography based monitoring of anaesthetic depth in mice

To improve animal welfare and data quality and reproducibility during research conducted under anaesthesia, anaesthetic depth in laboratory animals must be precisely monitored and controlled. While a variety of methods have been developed to estimate the depth of anaesthesia in humans, such tools for monitoring anaesthetic depth in laboratory animals remain limited. Here we propose an epidural electrocorticogram-based monitoring system that accurately tracks the depth of anesthesia in mice receiving inhalable isoflurane anaesthesia. Several features of the electrocorticogram signals exhibit robust modulation by the concentration of the administered anesthetic, notably, corticocortical coherence serves as an excellent indicator of anaesthetic depth. We developed a gradient boosting regressor framework that utilizes the extracted features to accurately estimate the depth of anaesthesia. Our method for feature extraction and estimation is conducted with a latency of only ten seconds, establishing a system for the real-time tracking of anaesthetic depth in mice.

neuroscience↗

bioRxiv2021

Sand dropseed (Sporobolus cryptandrus) - A new pest in Eurasian sand areas?

For the effective control of an invasive species, gathering as much information as possible on its ecology, establishment and persistence in the subjected communities is of utmost importance. We aimed to review the current distribution and characteristics of Sporobolus cryptandrus (sand dropseed), an invasive C4 grass species of North American origin recently discovered in Hungary. We aimed to provide information on (i) its current distribution paying special attention to its invasion in Eurasia; (ii) the characteristics of the invaded habitats in Central Europe; (iii) seed bank formation and germination characteristics, crucial factors in early establishment; and (iv) the effects of its increasing cover on vegetation composition. Finally, we aimed to (v) point out further research directions that could enable us to understand the invasion success of this potential invasive species. Field surveys uncovered large stands of the species in Central and Eastern Hungary with most of the locations in the former, especially the Kiskunsag region. The species invaded disturbed stands of dry and open sand grasslands, closed dune slack grasslands and it also penetrates into natural open sand grasslands from neighbouring disturbed habitats. Increasing cover of Sporobolus cryptandrus caused a decline in species richness and abundance of subordinate species both in the vegetation and seed banks, but a low density of Sporobolus cryptandrus can even have a weak positive effect on these characteristics. Viable seeds of Sporobolus were detected from all soil layers (2.5 cm layers measured from the surface to 10 cm in depth), which indicates that the species is able to form a persistent seed bank (1,114 to 3,077 seeds/m2 with increasing scores towards higher abundance of the species in vegetation). Germination of Sporobolus cryptandrus was negatively affected by both litter cover and 1 cm deep soil burial. To sum up, Sporobolus cryptandrus can be considered as a transformer invasive species, whose spread forms a high risk for dry sand and steppe grasslands in Eurasia. We can conclude that for the effective suppression of the species it is necessary: (i) to clarify the origin of the detected populations; (ii) to assess its competitive ability including its potential allelopathic effects; (iii) to assess its seed bank formation potential in habitats with different abiotic conditions; and (iv) to assess the possibility of its suppression by natural enemies and management techniques such as mowing or livestock grazing.

ecology↗

bioRxiv2021

Split Staphylococcus aureus prime editor for AAV delivery

Prime editing brings immense promise to correct a large number of human pathogenic mutations and enact diverse edit types without introducing widespread undesired editing events. Delivery of prime editors in vivo would enable such edits to be introduced in a clinical setting. The coding sequence for prime editor, however, is too large to fit within the size-constrained adeno-associated virus (AAV) genome. Herein, we describe a split Staphylococcus aureus prime editor capable of being delivered by dual AAVs. We characterize the editing ability of plasmid-based versions of an S. aureus prime editor in vitro at a variety of loci with diverse edit types. We investigate various split prime editor architectures and alternative dimerization domains. Finally, we demonstrate the capacity of prime editor to be co-delivered by dual AAVs in vitro. While editing rates are lower than desired, this approach presents an important step to translate prime editing for in vivo delivery.

molecular biology↗

bioRxiv2021

Multivariate analysis of strawberry experiments: where are we now and where can we go?

The multi-trait genotype-ideotype distance index (MGIDI) was used to select superior treatments in experiments with strawberries. Twenty-three productive, qualitative, physiological, and phenological traits with negative and positive desired gains were accessed in 16 treatments, a combination of two cultivars (Albion-neutral days, and Camarosa-short days), two transplants origins (National and Imported), and four organic substrates mixes (Crushed sugarcane bagasse, burnt rice husk, organic substrate, and Carolina commercial substrate). Our results suggest that most of the strawberry traits are influenced by the cultivar, transplant origin, cultivation substrates, as well as by the interaction between cultivar and transplant origin. The MGIDI index indicated that the Albion cultivar originated from imported transplants grown in substrates where the main component (70%) is burnt rice husk provides desired values for 20 of a total of 22 traits, which represents a success rate of ~ 91% in selecting traits with desired values. The strengths and weakness view provided by the MGIDI index revealed that the looking for an ideal treatment should direct the efforts on improving the water efficiency use and reducing total acidy of fruits. On the other hand, the strengths of selected treatments are mainly related to productive precocity, total soluble solids, and flesh firmness. The MGIDI index provides a unique, robust, and easy-to-handle process, standing out as a powerful tool to develop better treatment recommendations for experiments with strawberries when multiple traits are assessed.

plant biology↗

bioRxiv2020

The biophysical basis of protein domain compatibility in ion channels

Understanding the biophysical mechanisms that govern the combination of protein domains into viable proteins is essential for advancing synthetic biology and biomedical engineering. Here, we use massively parallel genotype/phenotype assays to determine cell surface expression of over 300,000 variants of the inward rectifier K+ channel Kir2.1 recombined with hundreds of protein motifs. We use machine learning to derive a quantitative biophysical model and practical rules for domain recombination. Insertional fitness depends on nonlinear interactions between the biophysical properties of inserted motifs and the recipient protein, which adds a new dimension to the rational design of fusion proteins. Insertion maps reveal a generalizable hierarchical organization of Kir2.1 and several other ion channels that balances stability needed for folding and dynamics required for function. SummaryMassively parallel assays reveal interactions between donor domains and recipient proteins govern domain compatibility

biophysics↗

bioRxiv2020

Three-dimensional FIB-SEM reconstruction ofmicrotubule-organelle interaction in wholeprimary mouse beta cells

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is currently under debate. Here, we use FO_SCPLOWIBC_SCPLOW-SO_SCPLOWEMC_SCPLOW to image islet beta cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules and micro-tubules of seven beta cells, and generate a comprehensive spatial map of microtubule-organelle interactions. We find that micro-tubules form non-radial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus supportive role in insulin secretion.

cell biology↗

bioRxiv2020

MATHEMATICAL MODELING OF RETINAL DEGENERATION: AEROBIC GLYCOLYSIS IN A SINGLE CONE

Cell degeneration, including that resulting in retinal diseases, is linked to metabolic issues. In the retina, photoreceptor degeneration can result from imbalance in lactate production and consumption as well as disturbances to pyruvate and glucose levels. To identify the key mechanisms in metabolism that may be culprits of this degeneration, we use a nonlinear system of differential equations to mathematically model the metabolic pathway of aerobic glycolysis in a healthy cone photoreceptor. This model allows us to analyze the levels of lactate, glucose, and pyruvate within a single cone cell. We perform numerical simulations, use available metabolic data to estimate parameters and fit the model to this data, and conduct a sensitivity analysis using two different methods (LHS/PRCC and eFAST) to identify pathways that have the largest impact on the system. Using bifurcation techniques, we find that the system has a bistable regime, biologically corresponding to a healthy versus a pathological state. The system exhibits a saddle node bifurcation and hysteresis. This work confirms the necessity for the external glucose concentration to sustain the cell even at low initial internal glucose levels. It also validates the role of {beta}-oxidation of fatty acids which fuel oxidative phosphorylation under glucose- and lactate-depleted conditions, by showing that the rate of {beta}-oxidation of ingested outer segment fatty acids in a healthy cone cell must be low. Model simulations reveal the modulating effect of external lactate in bringing the system to steady state; the bigger the difference between external lactate and initial internal lactate concentrations, the longer the system takes to achieve steady state. Parameter estimation for metabolic data demonstrates the importance of rerouting glucose and other intermediate metabolites to produce glycerol 3-phosphate (G3P), thus increasing lipid synthesis (a precursor to fatty acid production) to support their high growth rate. While a number of parameters are found to be significant by one or both of the methods for sensitivity analysis, the rate of {beta}-oxidation of ingested outer segment fatty acids is shown to consistently play an important role in the concentration of glucose, G3P, and pyruvate, whereas the extracellular lactate level is shown to consistently play an important role in the concentration of lactate and acetyl coenzyme A. The ability of these mechanisms to affect key metabolites variability and levels (as revealed in our analyses) signifies the importance of inter-dependent and inter-connected feedback processes modulated by and affecting both the RPEs and cones metabolism.

systems biology↗

bioRxiv2020

A survey of pairwise epistasis supports an outside-in hierarchy of clade-specifying and function-defining residues in PSD95 PDZ3

Deep mutational scanning enables data-driven models of protein structure and function. Here, we adapted Saturated Programmable Insertion Engineering as an economical and programmable deep mutational scanning technique. We validate this approach with an existing single mutant dataset in the PSD95 PDZ3 domain, and further characterize most pairwise double mutants to study how a mutation’s phenotype depends on mutations at other sites, a phenomenon called epistasis. We observe wide-spread proximal negative epistasis, which we attribute to mutations affecting thermodynamic stability, and strong long-range positive epistasis, which is enriched in an evolutionarily conserved and function-defining network of ‘sector’ and clade-specifying residues. Conditional neutrality of mutations in clade-specifying residues compensates for deleterious mutations in sector positions. This suggests an outside-in hierarchy of interactions through which positive epistasis between clade-specifying residues and the PDZ sector facilitated the evolutionary expansion and specialization of PDZ domains.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

The thrombopoietin receptor agonist eltrombopag inhibits human cytomegalovirus replication via iron chelation

The thrombopoietin receptor agonist eltrombopag was successfully used against human cytomegalovirus (HCMV)-associated thrombocytopenia refractory to immunomodulatory and antiviral drugs. These effects were ascribed to effects of eltrombopag on megakaryocytes. Here, we tested whether eltrombopag may also exert direct antiviral effects. Therapeutic eltrombopag concentrations inhibited HCMV replication in human fibroblasts and adult mesenchymal stem cells infected with six different virus strains and drug-resistant clinical isolates. Eltrombopag also synergistically increased the anti-HCMV activity of the mainstay drug ganciclovir. Time-of-addition experiments suggested that eltrombopag interferes with HCMV replication after virus entry. Eltrombopag was effective in thrombopoietin receptor-negative cells, and addition of Fe3+ prevented the anti-HCMV effects, indicating that it inhibits HCMV replication via iron chelation. This may be of particular interest for the treatment of cytopenias after haematopoietic stem cell transplantation, as HCMV reactivation is a major reason for transplantation failure. Since therapeutic eltrombopag concentrations are effective against drug-resistant viruses and synergistically increase the effects of ganciclovir, eltrombopag is also a drug repurposing candidate for the treatment of therapy-refractory HCMV disease.

pharmacology and toxicology↗

bioRxiv2019

CLIJ: Enabling GPU-accelerated image processing in Fiji

Graphics processing units (GPU) allow image processing at unprecedented speed. We present CLIJ, a Fiji plugin enabling end-users with entry level experience in programming to benefit from GPU-accelerated image processing. Freely programmable workflows can speed up image processing in Fiji by factor 10 and more using high-end GPU hardware and on affordable mobile computers with built-in GPUs.

bioinformatics↗

bioRxiv2019

Smoking and blood DNA methylation: novel associations, replication of previous findings and assessment of reversibility

We conducted a genome-wide association study of blood DNA methylation and smoking, attempted replication of previously discovered associations, and assessed the reversibility of smoking-associated methylation changes. DNA methylation was measured in baseline peripheral blood samples for 5,044 participants in the Melbourne Collaborative Cohort Study. For 1,032 participants, these measures were repeated using blood samples collected at follow-up, a median of 11 years later. A cross-sectional analysis of the association between smoking and DNA methylation and a longitudinal analysis of changes in smoking status and changes in DNA methylation were conducted. We used our cross-sectional analysis to replicate previously reported associations for current (N=3,327) and former (N=172) smoking. A comprehensive smoking index accounting for the bioactivity of smoking and several aspects of smoking history was constructed to assess the reversibility of smoking-induced methylation changes. We identified 4,496 cross-sectional associations at P<10-7, including 3,296 that were novel. We replicated the majority (90%) of previously reported associations for current and former smokers. In our data, we observed for former smokers a substantial degree of return to the methylation levels of never smokers, compared with current smokers (median: 74%, IQR=63-86%). Consistent with this, we found wide-ranging estimates for the half-life parameter of the comprehensive smoking index. Longitudinal analyses identified 368 sites at which methylation changed upon smoking cessation. Our study provides evidence of many novel associations between smoking and DNA methylation at CpGs across the genome, replicates the vast majority of previously reported associations, and indicates wide-ranging reversibility rates for smoking-induced methylation changes.

genomics↗

bioRxiv2019

Effect of Antimicrobial Stewardship with Rapid MALDI-TOF Identification and Vitek 2 Antimicrobial Susceptibility Testing on Hospitalization Outcome

IntroductionRapid organism identification (ID) and antimicrobial susceptibility testing (AST) along with antibiotic stewardship (ASP) are critical to appropriate treatment. We sought to capture time for bacterial culture and initiation of appropriate therapy for patients, from 2017 (without MALDI-TOF/Vitek 2 and ASP) and 2018 (with MALDI-TOF/Vitek 2 and ASP).\n\nMethodsEligible patients admitted to our hospital with a positive sputum, blood, or urine culture. Sequential patients were retrospectively obtained from March 1 to May 31, 2017. Seventy-seven patients from 2017 were compared to 77 patients from 2018. A time-in-motion study was performed to compare time to identification (ID), AST results, and ASP team intervention for the two periods. Data were entered into SPSS (ver 25) for analysis. Results are reported as mean ({+/-} SD) or percentage.\n\nResultsTime to organism ID was significantly faster in 2018 (2018 24.9 {+/-} 14.4, 2017 33.8 {+/-} 17 h, p=0.001). Time to AST results was also significantly faster for patients in 2018 compared to 2017 (18.2 {+/-} 14 compared to 28.5 {+/-} 14.9 h, p<0.001). ASP team recommended significantly more adjustments to empiric antimicrobial therapy in 2018 (28% of 2018 vs. 2% in 2017, p< 0.001). Length of hospital stay was significantly shorter in 2018 compared to 2017 (2018 10.7 {+/-} 11.1 days and 2017 15.5 {+/-} 18.1 days, p=0.05).\n\nConclusionsUse of MALDI-TOF/Vitek 2 leads to an average 21.5 h faster ID and AST results that can be acted upon by ASP for appropriate antimicrobial recommendations.

microbiology↗

bioRxiv2019

Efficient mesoscale phenotypic screening in cultured primary neuron culture

A longstanding question in neuroscience is how the activity of ion channels shapes neuronal activity and, as a result, computation in circuits and networks. Optogenetic reagents are tools to answer this question by enabling precise and dynamic perturbation of cellular states. However, development of these reagents can be hampered by low-throughput assays in non-physiological contexts. Here, we develop an all optical phenotypic screen in cultured primary hippocampal neurons that enables the functional assessment of large libraries of genetically encoded optogenetic actuators. Combining real-time analysis and data reduction methods allows for continuous observation of several thousand neurons for several days without onerous data storage overhead. This screening system may be useful in a diversity of research questions that can be coupled to optical perturbation and sensing.

neuroscience↗