Molecular dynamics simulations serve as a prevalent approach for investigating the dynamic behaviour of proteins and protein-ligand complexes. Due to its versatility and speed, GROMACS stands out as a commonly utilized software platform for executing molecular dynamics simulations. However, its effective utilization requires substantial expertise in configuring, executing, and interpreting molecular dynamics trajectories. Existing automation tools are constrained in their capability to conduct simulations for large sets of compounds with minimal user intervention, or in their ability to distribute simulations across multiple servers. To address these challenges, we developed a Python-based tool that streamlines all phases of molecular dynamics simulations, encompassing preparation, execution, and analysis. This tool minimizes the required knowledge for users engaging in molecular dynamics simulations and can efficiently operate across multiple servers within a network or a cluster. Notably, the tool not only automates trajectory simulation but also facilitates the computation of free binding energies for protein-ligand complexes and generates interaction fingerprints across the trajectory. Our study demonstrated the applicability of this tool on several benchmark datasets. Additionally, we provided recommendations for end-users to effectively utilize the tool.Scientific contributionThe developed tool, StreaMD, is applicable to different systems (proteins, ligands and their complexes including co-factors) and requires a little user knowledge to setup and run molecular dynamics simulations. Other features of StreaMD are seamless integration with calculation of MM-GBSA/PBSA binding free energies and protein-ligand interaction fingerprints, and running of simulations within distributed environments. All these will facilitate routine and massive molecular dynamics simulations.

• Keywords
• Distributed simulations, GROMACS, High-throughput molecular dynamics, Molecular dynamics,
• Publication type
• Journal Article MeSH

Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.

• Keywords
• 5′ss, SMN1, U1 snRNA, cryptic splice sites, splicing-affecting mutation,
• MeSH
• Alternative Splicing * MeSH
• Cell Line MeSH
• Exons * MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• RNA Splice Sites MeSH
• Mutation * MeSH
• Mutagenesis MeSH
• Survival of Motor Neuron 1 Protein chemistry   genetics   metabolism   MeSH
• RNA, Small Nuclear chemistry   genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Computational Biology methods   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA Splice Sites MeSH
• Survival of Motor Neuron 1 Protein MeSH
• RNA, Small Nuclear MeSH
• SMN1 protein, human MeSH Browser
• U1 small nuclear RNA MeSH Browser

• Publication type
• Published Erratum MeSH

Apple viruses pose significant threat to global apple production. In this study, HTS technology was used to investigate the apple virome in the Czech Republic. Previously reported viruses, including ACLSV, ASPV, ASGV, ApMV, AGCaV, and CCGaV, were confirmed, and near-complete genomes were assembled. Additionally, two novel viruses, ARWV1 and ARWV2 were identified for the first time in the Czech Republic. Phylogenetic analyses showed low genetic variability among ARWV2 isolates, suggesting a possible recent introduction or limited diversification. In contrast, ARWV1 isolates displayed distinct clustering in the coat protein coding region, separating symptomatic and asymptomatic samples, indicating a potential involvement of genetic determinants in symptom expression. Mixed infections were prevalent, with multiple molecular variants of ACLSV, ASPV, and AGCaV detected within individual samples, along with co-infections involving viruses from different families. Recombination analysis identified frequent recombination events in ACLSV and ASPV, often involving non-apple parental sequences, suggesting their potential for cross-host infections. Additionally, an interspecific recombination event was detected in an almond ApMV isolate, with PNRSV as a minor parent. These findings highlight the impact of agricultural practices on viral evolution and host adaptation. This study demonstrates the utility of HTS as a powerful tool for uncovering viral diversity, recombination events, and evolutionary dynamics.

• Keywords
• ARWV1, ARWV2, HTS, apple, coinfection, molecular variants, recombination,
• MeSH
• Phylogeny MeSH
• Genetic Variation * MeSH
• Genome, Viral MeSH
• Malus * virology   MeSH
• Plant Diseases * virology   MeSH
• Recombination, Genetic MeSH
• Plant Viruses * genetics   classification   isolation & purification   MeSH
• Virome * genetics   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH

Repeated sequences and polyploidy play a central role in plant genome dynamics. Here, we analyze the evolutionary dynamics of repeats in tetraploid and hexaploid Spartina species that diverged during the last 10 million years within the Chloridoideae, one of the poorest investigated grass lineages. From high-throughput genome sequencing, we annotated Spartina repeats and determined what sequence types account for the genome size variation among species. We examined whether differential genome size evolution correlated with ploidy levels and phylogenetic relationships. We also examined the tempo of repeat sequence dynamics associated with allopatric speciation over the last 3-6 million years between hexaploid species that diverged on the American and European Atlantic coasts and tetraploid species from North and South America. The tetraploid S. spartinae, whose phylogenetic placement has been debated, exhibits a similar repeat content as hexaploid species, suggesting common ancestry. Genome expansion or contraction resulting from repeat dynamics seems to be explained mostly by the contrasting divergence times between species, rather than by genome changes triggered by ploidy level change per se. One 370 bp satellite may be exhibiting 'meiotic drive' and driving chromosome evolution in S. alterniflora. Our results provide crucial insights for investigating the genetic and epigenetic consequences of such differential repeat dynamics on the ecology and distribution of the meso- and neopolyploid Spartina species.

• Keywords
• Genome dynamics, Polyploidy, Satellite DNA, Spartina, Transposable elements,
• MeSH
• Phylogeny MeSH
• Genome, Plant genetics   MeSH
• Poaceae genetics   MeSH
• Evolution, Molecular * MeSH
• Polyploidy * MeSH
• DNA, Satellite genetics   MeSH
• Blotting, Southern MeSH
• DNA Transposable Elements genetics   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Satellite MeSH
• DNA Transposable Elements MeSH

The partitioning behavior of drug-like molecules into biomembranes has a crucial impact on the design and efficacy of therapeutic drugs. Thermodynamic properties connected with the interaction of molecules with membranes can be evaluated by calculating free-energy profiles normal to the membrane surface. We calculated the free-energy profiles of 25 drug-like molecules in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane and free energies of solvation in water and heptane using two methods, molecular dynamics (MD) simulations with the Berger lipid force field and COSMOmic, based on a continuum conductor-like screening model for realistic solvation (COSMO-RS). The biased MD simulations (in total ∼22 μs long) were relatively computationally expensive, whereas the COSMOmic approach offered a significantly less expensive alternative. Both methods provided similar results and showed that the studied amphiphilic drug-like molecules accumulate in the membrane, with the majority localized below the head group region. The MD simulations were more lipophilic and gave free-energy profiles that were systematically deeper than those calculated by COSMOmic. To investigate the physical nature of the increased lipophilicity, we analyzed a water/heptane system and identified that it is most likely caused by overestimation of the attractive term of the Lennard-Jones potential in lipid tails. We concluded that COSMOmic can be successfully used for high-throughput computations of global thermodynamic properties, for example, partition coefficients and energy barrier heights, in phosphocholine membranes. In contrast, MD is better for investigating local properties like molecular positioning and orientation in the membrane because they more accurately reflect the complex structure of lipid bilayers. MD is also useful for studies of highly complex systems, for example, drug-membrane-protein interactions.

• MeSH
• Phosphatidylcholines chemistry   MeSH
• Pharmaceutical Preparations chemistry   MeSH
• Molecular Structure MeSH
• Surface-Active Agents chemistry   MeSH
• Surface Properties MeSH
• High-Throughput Screening Assays MeSH
• Molecular Dynamics Simulation MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• 1,2-oleoylphosphatidylcholine MeSH Browser
• Phosphatidylcholines MeSH
• Pharmaceutical Preparations MeSH
• Surface-Active Agents MeSH

Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 de novo; P5, in turn, play important role in immune homeostasis and regulation. However, P5's biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5's mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5's biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5-protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.

• Keywords
• TH2, chemoproteomics, click chemistry, lymphosteroid, pregnenolone,
• MeSH
• Pregnenolone * metabolism   pharmacology   MeSH
• Molecular Dynamics Simulation MeSH
• Steroids MeSH
• Th2 Cells * metabolism   MeSH
• Carrier Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Pregnenolone * MeSH
• Steroids MeSH
• Carrier Proteins MeSH

UNLABELLED: To find and calibrate a robust and reliable computational protocol for mapping conformational space of medium-sized molecules, exhaustive conformational sampling has been carried out for a series of seven macrocyclic compounds of varying ring size and one acyclic analogue. While five of them were taken from the MD/LLMOD/force field study by Shelley and co-workers ( Watts , K. S. ; Dalal , P. ; Tebben , A. J. ; Cheney , D. L. ; Shelley , J. C. Macrocycle Conformational Sampling with MacroModel . J. Chem. Inf. MODEL: 2014 , 54 , 2680 - 2696 ), three represent potential macrocyclic inhibitors of human cyclophilin A. The free energy values (GDFT/COSMO-RS) for all of the conformers of each compound were obtained by a composite protocol based on in vacuo quantum mechanics (DFT-D3 method in a large basis set), standard gas-phase thermodynamics, and the COSMO-RS solvation model. The GDFT/COSMO-RS values were used as the reference for evaluating the performance of conformational sampling algorithms: standard and extended MD/LLMOD search (simulated-annealing molecular dynamics with low-lying eigenvector following algorithms, employing the OPLS2005 force field plus GBSA solvation) available in MacroModel and plain molecular dynamics (MD) sampling at high temperature (1000 K) using the semiempirical quantum mechanics (SQM) potential SQM(PM6-D3H4/COSMO) followed by energy minimization of the snapshots. It has been shown that the former protocol (MD/LLMOD) may provide a more complete set of initial structures that ultimately leads to the identification of a greater number of low-energy conformers (as assessed by GDFT/COSMO-RS) than the latter (i.e., plain SQM MD). The CPU time needed to fully evaluate one medium-sized compound (∼100 atoms, typically resulting in several hundred or a few thousand conformers generated and treated quantum-mechanically) is approximately 1,000-100,000 CPU hours on today's computers, which transforms to 1-7 days on a small-sized computer cluster with a few hundred CPUs. Finally, our data sets based on the rigorous quantum-chemical approach allow us to formulate a composite conformational sampling protocol with multiple checkpoints and truncation of redundant structural data that offers superior insights at affordable computational cost.

• MeSH
• Algorithms MeSH
• Calibration MeSH
• Crystallography MeSH
• Quantum Theory MeSH
• Macrocyclic Compounds chemistry   MeSH
• Molecular Conformation * MeSH
• High-Throughput Screening Assays MeSH
• Molecular Dynamics Simulation MeSH
• Thermodynamics MeSH
• Hot Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Macrocyclic Compounds MeSH

OBJECTIVES: Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome. METHODS: Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays. RESULTS: Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype. CONCLUSIONS: We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.

• Keywords
• colorectal cancer, high-throughput expression profiling, long non-coding RNAs, size exclusion chromatography, small extracellular vesicles, transcriptome,
• MeSH
• Extracellular Vesicles * genetics   metabolism   MeSH
• Chromatography, Gel MeSH
• Humans MeSH
• RNA MeSH
• Gene Expression Profiling * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA MeSH

In order to identify novel lead structures for human toll-like receptor 4 (hTLR4) modulation virtual high throughput screening by a peta-flops-scale supercomputer has been performed. Based on the in silico studies, a series of 12 compounds related to tryptamine was rationally designed to retain suitable molecular geometry for interaction with the hTLR4 binding site as well as to satisfy general principles of drug-likeness. The proposed compounds were synthesized, and tested by in vitro and ex vivo experiments, which revealed that several of them are capable to stimulate hTLR4 in vitro up to 25% activity of Monophosphoryl lipid A. The specific affinity of the in vitro most potent substance was confirmed by surface plasmon resonance direct-binding experiments. Moreover, two compounds from the series show also significant ability to elicit production of interleukin 6.

• Keywords
• PRR, TLR4, adjuvants, innate immunity, molecular dynamics, virtual screening,
• MeSH
• Adjuvants, Immunologic chemistry   metabolism   pharmacology   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Inhibitory Concentration 50 MeSH
• Interleukin-6 blood   MeSH
• Humans MeSH
• Ligands MeSH
• Computer Simulation MeSH
• Surface Plasmon Resonance MeSH
• High-Throughput Screening Assays methods   MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Toll-Like Receptor 4 agonists   metabolism   MeSH
• Tryptamines chemistry   MeSH
• Vaccines MeSH
• Binding Sites MeSH
• Structure-Activity Relationship * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adjuvants, Immunologic MeSH
• IL6 protein, human MeSH Browser
• Interleukin-6 MeSH
• Ligands MeSH
• TLR4 protein, human MeSH Browser
• Toll-Like Receptor 4 MeSH
• tryptamine MeSH Browser
• Tryptamines MeSH
• Vaccines MeSH