Association of circulating biomarkers with illness severity measures differentiates myalgic encephalomyelitis/chronic fatigue syndrome and post-COVID-19 condition: a prospective pilot cohort study.

BACKGROUND: Accumulating evidence suggests that autonomic dysfunction and persistent systemic inflammation are common clinical features in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID. However, there is limited knowledge regarding their potential association with circulating biomarkers and illness severity in these conditions. METHODS: This single-site, prospective, cross-sectional, pilot cohort study aimed to distinguish between the two patient populations by using self-reported outcome measures and circulating biomarkers of endothelial function and systemic inflammation status. Thirty-one individuals with ME/CFS, 23 individuals with long COVID, and 31 matched sedentary healthy controls were included. All study participants underwent non-invasive cardiovascular hemodynamic challenge testing (10 min NASA lean test) for assessment of orthostatic intolerance. Regression analysis was used to examine associations between outcome measures and circulating biomarkers in the study participants. Classification across groups was based on principal component and discriminant analyses. RESULTS: Four ME/CFS patients (13%), 1 with long COVID (4%), and 1 healthy control (3%) presented postural orthostatic tachycardia syndrome (POTS) using the 10-min NASA lean test. Compared with matched healthy controls, ME/CFS and long COVID subjects showed higher levels of ET-1 (p < 0.05) and VCAM-1 (p < 0.001), and lower levels of nitrites (NOx assessed as NO2- + NO3-) (p < 0.01). ME/CFS patients also showed higher levels of serpin E1 (PAI-1) and E-selectin than did both long COVID and matched control subjects (p < 0.01 in all cases). Long COVID patients had lower TSP-1 levels than did ME/CFS patients and matched sedentary healthy controls (p < 0.001). As for inflammation biomarkers, both long COVID and ME/CFS subjects had higher levels of TNF-α than did matched healthy controls (p < 0.01 in both comparisons). Compared with controls, ME/CFS patients had higher levels of IL-1ß (p < 0.001), IL-4 (p < 0.001), IL-6 (p < 0.01), IL-10 (p < 0.001), IP-10 (p < 0.05), and leptin (p < 0.001). Principal component analysis supported differentiation between groups based on self-reported outcome measures and biomarkers of endothelial function and inflammatory status in the study population. CONCLUSIONS: Our findings revealed that combining biomarkers of endothelial dysfunction and inflammation with outcome measures differentiate ME/CFS and Long COVID using robust discriminant analysis of principal components. Further research is needed to provide a more comprehensive characterization of these underlying pathomechanisms, which could be promising targets for therapeutic and preventive strategies in these conditions.

The pseudotorsional space of RNA.

The characterization of the conformational landscape of the RNA backbone is rather complex due to the ability of RNA to assume a large variety of conformations. These backbone conformations can be depicted by pseudotorsional angles linking RNA backbone atoms, from which Ramachandran-like plots can be built. We explore here different definitions of these pseudotorsional angles, finding that the most accurate ones are the traditional η (eta) and θ (theta) angles, which represent the relative position of RNA backbone atoms P and C4'. We explore the distribution of η - θ in known experimental structures, comparing the pseudotorsional space generated with structures determined exclusively by one experimental technique. We found that the complete picture only appears when combining data from different sources. The maps provide a quite comprehensive representation of the RNA accessible space, which can be used in RNA-structural predictions. Finally, our results highlight that protein interactions lead to significant changes in the population of the η - θ space, pointing toward the role of induced-fit mechanisms in protein-RNA recognition.

Sequence-Dependent Properties of the RNA Duplex.

Sequence-dependent properties of the DNA duplex have been accurately described using extensive molecular dynamics simulations. The RNA duplex meanwhile─which is typically represented as a sequence-averaged rigid rod─does not benefit from having equivalent molecular dynamics simulations. In this paper, we present a massive simulation effort using a set of ABC-optimized duplexes from which we derived tetramer-resolution properties of the RNA duplex and a simple mesoscopic model that can represent elastic properties of long RNA duplexes. Despite the extreme chemical similarity between DNA and RNA, the local and global elastic properties of the duplexes are very different. DNA duplexes show a complex and nonelastic pattern of flexibility, for instance, while RNA duplexes behave as an elastic system whose deformations can be represented by simple harmonic potentials. In RNA duplexes (RNA2), not only are intra- and interbase pair parameters (equilibrium and mechanical) different from those in the equivalent DNA duplex sequences (DNA2) but the correlations between movements also differ. Simple statements on the relative flexibility or stability of both polymers are meaningless and should be substituted by a more detailed description depending on the sequence and the type of deformation considered.

Molecular dynamics modelling of the interaction of a synthetic zinc-finger miniprotein with DNA.

We report the modelling of the DNA complex of an artificial miniprotein composed of two zinc finger modules and an AT-hook linking peptide. The computational study provides for the first time a structural view of these types of complexes, dissecting interactions that are key to modulate their stability. The relevance of these interactions was validated experimentally. These results confirm the potential of this type of computational approach for studying peptide-DNA complexes and suggest that they could be very useful for the rational design of non-natural, DNA binding miniproteins.

Structural analysis of the Candida albicans mitochondrial DNA maintenance factor Gcf1p reveals a dynamic DNA-bridging mechanism.

The compaction of mitochondrial DNA (mtDNA) is regulated by architectural HMG-box proteins whose limited cross-species similarity suggests diverse underlying mechanisms. Viability of Candida albicans, a human antibiotic-resistant mucosal pathogen, is compromised by altering mtDNA regulators. Among them, there is the mtDNA maintenance factor Gcf1p, which differs in sequence and structure from its human and Saccharomyces cerevisiae counterparts, TFAM and Abf2p. Our crystallographic, biophysical, biochemical and computational analysis showed that Gcf1p forms dynamic protein/DNA multimers by a combined action of an N-terminal unstructured tail and a long helix. Furthermore, an HMG-box domain canonically binds the minor groove and dramatically bends the DNA while, unprecedentedly, a second HMG-box binds the major groove without imposing distortions. This architectural protein thus uses its multiple domains to bridge co-aligned DNA segments without altering the DNA topology, revealing a new mechanism of mtDNA condensation.

Correlated motions in DNA: beyond base-pair step models of DNA flexibility.

Traditional mesoscopic models of DNA flexibility use a reductionist-local approach, which assumes that the flexibility of DNA can be expressed as local harmonic movements (at the base-pair step level) in the helical space, ignoring multimodality and correlations in DNA movements, which have in reality a large impact in modulating DNA movements. We present a new multimodal-harmonic correlated model, which takes both contributions into account, providing, with a small computational cost, results of an unprecedented local and global quality. The accuracy of this method and its computational efficiency make it an alternative to explore the dynamics of long segments of DNA, approaching the chromatin range.

DNA double-strand break-derived RNA drives TIRR/53BP1 complex dissociation.

Tudor-interacting repair regulator (TIRR) is an RNA-binding protein and a negative regulator of the DNA-repair factor p53-binding protein 1 (53BP1). In non-damage conditions, TIRR is bound to 53BP1. After DNA damage, TIRR and 53BP1 dissociate, and 53BP1 binds the chromatin at the double-strand break (DSB) to promote non-homologous end joining (NHEJ)-mediated repair. However, the exact mechanistic details of this dissociation after damage are unknown. Increasing evidence has implicated RNA as a crucial factor in the DNA damage response (DDR). Here, we show that RNA can separate TIRR/53BP1. Specifically, RNA with a hairpin secondary structure, transcribed at the DSB by RNA polymerase II (RNAPII), promotes TIRR/53BP1 complex separation. This hairpin RNA binds to the same residues on TIRR as 53BP1. Our results uncover a role of DNA-damage-derived RNA in modulating a protein-protein interaction and contribute to our understanding of DSB repair.

DNAffinity: a machine-learning approach to predict DNA binding affinities of transcription factors.

We present a physics-based machine learning approach to predict in vitro transcription factor binding affinities from structural and mechanical DNA properties directly derived from atomistic molecular dynamics simulations. The method is able to predict affinities obtained with techniques as different as uPBM, gcPBM and HT-SELEX with an excellent performance, much better than existing algorithms. Due to its nature, the method can be extended to epigenetic variants, mismatches, mutations, or any non-coding nucleobases. When complemented with chromatin structure information, our in vitro trained method provides also good estimates of in vivo binding sites in yeast.

Molecular basis of Arginine and Lysine DNA sequence-dependent thermo-stability modulation.

We have used a variety of theoretical and experimental techniques to study the role of four basic amino acids-Arginine, Lysine, Ornithine and L-2,4-Diaminobutyric acid-on the structure, flexibility and sequence-dependent stability of DNA. We found that the presence of organic ions stabilizes the duplexes and significantly reduces the difference in stability between AT- and GC-rich duplexes with respect to the control conditions. This suggests that these amino acids, ingredients of the primordial soup during abiogenesis, could have helped to equalize the stability of AT- and GC-rich DNA oligomers, facilitating a general non-catalysed self-replication of DNA. Experiments and simulations demonstrate that organic ions have an effect that goes beyond the general electrostatic screening, involving specific interactions along the grooves of the double helix. We conclude that organic ions, largely ignored in the DNA world, should be reconsidered as crucial structural elements far from mimics of small inorganic cations.

The Impact of the HydroxyMethylCytosine epigenetic signature on DNA structure and function.

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) ➔ d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.

Sequence-dependent structural properties of B-DNA: what have we learned in 40 years?

The structure of B-DNA, the physiological form of the DNA molecule, has been a central topic in biology, chemistry and physics. Far from uniform and rigid, the double helix was revealed as a flexible and structurally polymorphic molecule. Conformational changes that lead to local and global changes in the helix geometry are mediated by a complex choreography of base and backbone rearrangements affecting the ability of the B-DNA to recognize ligands and consequently on its functionality. In this sense, the knowledge obtained from the sequence-dependent structural properties of B-DNA has always been thought crucial to rationalize how ligands and, most notably, proteins recognize B-DNA and modulate its activity, i.e. the structural basis of gene regulation. Honouring the anniversary of the first high-resolution X-ray structure of a B-DNA molecule, in this contribution, we present the most important discoveries of the last 40 years on the sequence-dependent structural and dynamical properties of B-DNA, from the early beginnings to the current frontiers in the field.

Colibactin DNA-damage signature indicates mutational impact in colorectal cancer.

The mucosal epithelium is a common target of damage by chronic bacterial infections and the accompanying toxins, and most cancers originate from this tissue. We investigated whether colibactin, a potent genotoxin1 associated with certain strains of Escherichia coli2, creates a specific DNA-damage signature in infected human colorectal cells. Notably, the genomic contexts of colibactin-induced DNA double-strand breaks were enriched for an AT-rich hexameric sequence motif, associated with distinct DNA-shape characteristics. A survey of somatic mutations at colibactin target sites of several thousand cancer genomes revealed notable enrichment of this motif in colorectal cancers. Moreover, the exact double-strand-break loci corresponded with mutational hot spots in cancer genomes, reminiscent of a trinucleotide signature previously identified in healthy colorectal epithelial cells3. The present study provides evidence for the etiological role of colibactin in human cancer.

Nucleosome Dynamics: a new tool for the dynamic analysis of nucleosome positioning.

We present Nucleosome Dynamics, a suite of programs integrated into a virtual research environment and created to define nucleosome architecture and dynamics from noisy experimental data. The package allows both the definition of nucleosome architectures and the detection of changes in nucleosomal organization due to changes in cellular conditions. Results are displayed in the context of genomic information thanks to different visualizers and browsers, allowing the user a holistic, multidimensional view of the genome/transcriptome. The package shows good performance for both locating equilibrium nucleosome architecture and nucleosome dynamics and provides abundant useful information in several test cases, where experimental data on nucleosome position (and for some cases expression level) have been collected for cells under different external conditions (cell cycle phase, yeast metabolic cycle progression, changes in nutrients or difference in MNase digestion level). Nucleosome Dynamics is a free software and is provided under several distribution models.

How B-DNA Dynamics Decipher Sequence-Selective Protein Recognition.

The rules governing sequence-specific DNA-protein recognition are under a long-standing debate regarding the prevalence of base versus shape readout mechanisms to explain sequence specificity and of the conformational selection versus induced fit binding paradigms to explain binding-related conformational changes in DNA. Using a combination of atomistic simulations on a subset of representative sequences and mesoscopic simulations at the protein-DNA interactome level, we demonstrate the prevalence of the shape readout model in determining sequence-specificity and of the conformational selection paradigm in defining the general mechanism for binding-related conformational changes in DNA. Our results suggest that the DNA uses a double mechanism to adapt its structure to the protein: it moves along the easiest deformation modes to approach the bioactive conformation, while final adjustments require localized rearrangements at the base-pair step and backbone level. Our study highlights the large impact of B-DNA dynamics in modulating DNA-protein binding.

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region.

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compaction.

A multifunctional toolkit for target-directed cancer therapy.

Here we present 2shRNA, a shRNA-based nanobinder, which can simultaneously attack two therapeutic targets involved in drug resistance pathways and can additionally bind accessory molecules such as cell targeting peptides or fluorophores. We create 2shRNAs designed to specifically kill HER2+ breast cancer cells in the absence of a transfecting agent.

Protein Flexibility and Synergy of HMG Domains Underlie U-Turn Bending of DNA by TFAM in Solution.

Human mitochondrial transcription factor A (TFAM) distorts DNA into a U-turn, as shown by crystallographic studies. The relevance of this U-turn is associated with transcription initiation at the mitochondrial light strand promoter (LSP). However, it has not been yet discerned whether a tight U-turn or an alternative conformation, such as a V-shape, is formed in solution. Here, single-molecule FRET experiments on freely diffusing TFAM/LSP complexes containing different DNA lengths show that a DNA U-turn is induced by progressive and cooperative binding of the two TFAM HMG-box domains and the linker between them. SAXS studies further show compaction of the protein upon complex formation. Finally, molecular dynamics simulations reveal that TFAM/LSP complexes are dynamic entities, and the HMG boxes induce the U-turn against the tendency of the DNA to adopt a straighter conformation. This tension is resolved by reversible unfolding of the linker, which is a singular mechanism that allows a flexible protein to stabilize a tight bending of DNA.

Repair of UV-Induced DNA Damage Independent of Nucleotide Excision Repair Is Masked by MUTYH.

DNA lesions caused by UV damage are thought to be repaired solely by the nucleotide excision repair (NER) pathway in human cells. Patients carrying mutations within genes functioning in this pathway display a range of pathologies, including an increased susceptibility to cancer, premature aging, and neurological defects. There are currently no curative therapies available. Here we performed a high-throughput chemical screen for agents that could alleviate the cellular sensitivity of NER-deficient cells to UV-induced DNA damage. This led to the identification of the clinically approved anti-diabetic drug acetohexamide, which promoted clearance of UV-induced DNA damage without the accumulation of chromosomal aberrations, hence promoting cellular survival. Acetohexamide exerted this protective function by antagonizing expression of the DNA glycosylase, MUTYH. Together, our data reveal the existence of an NER-independent mechanism to remove UV-induced DNA damage and prevent cell death.

DNA structure directs positioning of the mitochondrial genome packaging protein Abf2p.

The mitochondrial genome (mtDNA) is assembled into nucleo-protein structures termed nucleoids and maintained differently compared to nuclear DNA, the involved molecular basis remaining poorly understood. In yeast (Saccharomyces cerevisiae), mtDNA is a ∼80 kbp linear molecule and Abf2p, a double HMG-box protein, packages and maintains it. The protein binds DNA in a non-sequence-specific manner, but displays a distinct 'phased-binding' at specific DNA sequences containing poly-adenine tracts (A-tracts). We present here two crystal structures of Abf2p in complex with mtDNA-derived fragments bearing A-tracts. Each HMG-box of Abf2p induces a 90° bend in the contacted DNA, causing an overall U-turn. Together with previous data, this suggests that U-turn formation is the universal mechanism underlying mtDNA compaction induced by HMG-box proteins. Combining this structural information with mutational, biophysical and computational analyses, we reveal a unique DNA binding mechanism for Abf2p where a characteristic N-terminal flag and helix are crucial for mtDNA maintenance. Additionally, we provide the molecular basis for A-tract mediated exclusion of Abf2p binding. Due to high prevalence of A-tracts in yeast mtDNA, this has critical relevance for nucleoid architecture. Therefore, an unprecedented A-tract mediated protein positioning mechanism regulates DNA packaging proteins in the mitochondria, and in combination with DNA-bending and U-turn formation, governs mtDNA compaction.

Long-timescale dynamics of the Drew-Dickerson dodecamer.

We present a systematic study of the long-timescale dynamics of the Drew-Dickerson dodecamer (DDD: d(CGCGAATTGCGC)2) a prototypical B-DNA duplex. Using our newly parameterized PARMBSC1 force field, we describe the conformational landscape of DDD in a variety of ionic environments from minimal salt to 2 M Na(+)Cl(-) or K(+)Cl(-) The sensitivity of the simulations to the use of different solvent and ion models is analyzed in detail using multi-microsecond simulations. Finally, an extended (10 µs) simulation is used to characterize slow and infrequent conformational changes in DDD, leading to the identification of previously uncharacterized conformational states of this duplex which can explain biologically relevant conformational transitions. With a total of more than 43 µs of unrestrained molecular dynamics simulation, this study is the most extensive investigation of the dynamics of the most prototypical DNA duplex.