Targeting the SARS-CoV-2 RNA Translation Initiation Element SL1 by Molecules of Low Molecular Weight.

We present the development of low molecular weight inhibitors that target the 5'-terminal RNA stem-loop 1 (SL1) of the SARS-CoV-2 genome. SL1 is crucial for allowing viral protein synthesis in the context of global translational repression in infected cells. We applied compound- and RNA-detected nuclear magnetic resonance spectroscopy (NMR) experiments to guide a fragment-growth strategy based on two primary NMR screening hits from a diverse fragment library poised for follow-up chemistry.

H, C and N chemical shift assignment for stem-loop 5a from the 5'UTR of HCoV-229E.

Due to the emergence of the SARS-CoV-2 virus, research on coronaviruses has been massively accelerated. In addition to SARS-CoV-2, there are other human coronaviruses, including HCoV-229E. In all coronaviruses, secondary structure predictions indicate the presence of conserved structural elements in the 5'-untranslated region (5'-UTR).

Structural heterogeneity and dynamics in the apical stem loop of s2m from SARS-CoV-2 Delta by an integrative NMR spectroscopy and MD simulation approach.

In structured RNAs, helical elements are often capped by apical loops that are integral structural elements, ranging from 3 to >20 nts of size on average, and display a highly heterogeneous energy landscape profile, rendering structural characterization particularly challenging. We here provide a characterization of the SARS-CoV-2 Delta s2m element containing a highly dynamic nonaloop using an integrative approach of nuclear magnetic resonance spectroscopy (NMR), small angle X-ray scattering (SAXS), and molecular dynamics simulations (MD). We further explored the conformational space in the s2m nonaloop and its transient closing 5'-G-U-3' base pair by MD simulations weighted by experimental NMR observables, leading to a comprehensive representation of the s2m nonaloop motif.

The research beamlines at the Dresden proton therapy facility: available infrastructure and experimental capabilities.

The proton therapy facility in Dresden, Germany, has one treatment room equipped with a rotating gantry where patients are treated and an experimental room equipped with two horizontal beamlines for translational research. The present work describes the technical characteristics and provides measured beam data of these two complementary beamlines, one delivering scanned beams with quasi-clinical parameters and the other one stationary continuous and pulsed pencil beams with parameters exceeding the clinically used range. Features of the facility are the large scale of the experimental room enabling the development and installation of large devices and the parallel beam operation with the clinical room allowing irradiation experiments on weekdays and during daytime.

Feasibility of prompt gamma verification for cone-beam computed tomography-based online adaptive proton therapy.

Background And Purpose: Prompt-gamma based treatment verification, such as prompt-gamma imaging (PGI), is crucial for detecting anatomical changes and serving as safety net during proton therapy treatments. This is especially important in an online-adaptive setting, when imaging will be based on cone-beam computed tomography (CBCT). This study investigated whether PGI, proven effective to detect relevant anatomical changes in clinical settings, can also verify treatment plans adapted on CBCTs, particularly the reliability of CBCT-based PGI-simulations of expected prompt-gamma distributions, a key requirement for PGI-based verification.

Partial adaptation for online-adaptive proton therapy triggered by during-delivery treatment verification: Feasibility study on prostate cancer treatments.

Background And Purpose: Online treatment verification during proton therapy delivery may detect deviations due to anatomical changes occurring along the treatment course and trigger immediate intervention, if necessary. We investigated the potential of partial plan adaptation in two-field prostate cancer treatments as a solution for online-adaptive proton therapy (OAPT) after the detection of relevant treatment deviations during the first field delivery.

Materials And Methods: In a retrospective study, ten fractions from eight prostate cancer patients with prompt gamma imaging (PGI) detected treatment deviations, which were confirmed on respective in-room control computed tomography (cCT) scans, were considered.

NMR characterisation of the antibiotic resistance-mediating 32mer RNA from the 23S ribosomal RNA.

The increasing prevalence of antibiotic resistance represents a significant public health concern, underscoring the urgent need for the development of novel therapeutic strategies. The antibiotic effects of macrolides, the second most widely used class of antibiotics, are counteracted by Erm proteins through the methylation of adenosine 2058 of the 23S ribosomal RNA (rRNA) (~ 2900 nucleotides), yielding either monomethylated or dimethylated A2058. This methylation is the molecular basis for preventing macrolides from binding and leads to the development of resistance of bacteria including Staphylococcus, Streptococcus and Enterococcus.

Accuracy of a helium-beam radiography system based on thin pixel detectors for an anthropomorphic head phantom.

Background: Ion-beam radiography is a promising technique to verify the range of ion-beam radiotherapy treatments regularly. To detect and quantify the water-equivalent thickness (WET) of potential anatomical changes, ion-beam radiographs must provide a sufficient WET accuracy on the level of 1%.

Purpose: In this work, we show an energy-painted helium-beam radiograph of an anthropomorphic head phantom acquired with thin silicon pixel detectors for the first time.

Optimization of Structure-Guided Development of Chemical Probes for the Pseudoknot RNA of the Frameshift Element in SARS-CoV-2.

Targeting the RNA genome of SARS-CoV-2 is a viable option for antiviral drug development. We explored three ligand binding sites of the core pseudoknot RNA of the SARS-CoV-2 frameshift element. We iteratively optimized ligands, based on improved affinities, targeting these binding sites and report on structural and dynamic properties of the three identified binding sites.

Validity of one-time phantomless patient-specific quality assurance in proton therapy with regard to the reproducibility of beam delivery.

Background: Patient-specific quality assurance (PSQA) is a crucial yet resource-intensive task in proton therapy, requiring special equipment, expertise and additional beam time. Machine delivery log files contain information about energy, position and monitor units (MU) of all delivered spots, allowing a reconstruction of the applied dose. This raises the prospect of phantomless, log file-based QA (LFQA) as an automated replacement of current phantom-based solutions, provided that such an approach guarantees a comparable level of safety.

Inter-center comparison of proton range verification prototypes with an anthropomorphic head phantom.

. To compare in reproducible and equalized conditions the performance of two independent proton range verification systems based on prompt gamma-ray detectors from two different proton therapy centers..

Dissecting the Conformational Heterogeneity of Stem-Loop Substructures of the Fifth Element in the 5'-Untranslated Region of SARS-CoV-2.

Throughout the family of coronaviruses, structured RNA elements within the 5' region of the genome are highly conserved. The fifth stem-loop element from SARS-CoV-2 (5_SL5) represents an example of an RNA structural element, repeatedly occurring in coronaviruses. It contains a conserved, repetitive fold within its substructures SL5a and SL5b.

Differential effects of the N-terminal helix of FGF8b on the activity of a small-molecule FGFR inhibitor in cell culture and for the extracellular domain of FGFR3c in solution.

SSR128129E (SSR) is a unique small-molecule inhibitor of fibroblast growth factor receptors (FGFRs). SSR is a high-affinity allosteric binder that selectively blocks one of the two major FGFR-mediated pathways. The mechanisms of SSR activity were studied previously in much detail, allowing the identification of its binding site, located in the hydrophobic groove of the receptor D3 domain.

Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane.

Total internal reflection fluorescence (TIRF) microscopy offers powerful means to uncover the functional organization of proteins in the plasma membrane with very high spatial and temporal resolution. Traditional TIRF illumination, however, shows a Gaussian intensity profile, which is typically deteriorated by overlaying interference fringes hampering precise quantification of intensities-an important requisite for quantitative analyses in single-molecule localization microscopy (SMLM). Here, we combine flat-field illumination by using a standard πShaper with multi-angular TIR illumination by incorporating a spatial light modulator compatible with fast super-resolution structured illumination microscopy (SIM).

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data.

The partial adaptation strategy for online-adaptive proton therapy: A proof of concept study in head and neck cancer patients.

Background: The accuracy of intensity-modulated proton therapy (IMPT) is greatly affected by anatomy variations that might occur during the treatment course. Online plan adaptations have been proposed as a solution to intervene promptly during a treatment session once the anatomy changes are detected. The implementation of online-adaptive proton therapy (OAPT) is still hindered by time-consuming tasks in the workflow.

mA Methylation of Transcription Leader Sequence of SARS-CoV-2 Impacts Discontinuous Transcription of Subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be mA methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity.

Design, quality and validation of the EU-OPENSCREEN fragment library poised to a high-throughput screening collection.

The EU-OPENSCREEN (EU-OS) European Research Infrastructure Consortium (ERIC) is a multinational, not-for-profit initiative that integrates high-capacity screening platforms and chemistry groups across Europe to facilitate research in chemical biology and early drug discovery. Over the years, the EU-OS has assembled a high-throughput screening compound collection, the European Chemical Biology Library (ECBL), that contains approximately 100 000 commercially available small molecules and a growing number of thousands of academic compounds crowdsourced through our network of European and non-European chemists. As an extension of the ECBL, here we describe the computational design, quality control and use case screenings of the European Fragment Screening Library (EFSL) composed of 1056 mini and small chemical fragments selected from a substructure analysis of the ECBL.

NMR characterization and ligand binding site of the stem-loop 2 motif from the Delta variant of SARS-CoV-2.

The stem-loop 2 motif (s2m) in SARS-CoV-2 (SCoV-2) is located in the 3'-UTR. Although s2m has been reported to display characteristics of a mobile genomic element that might lead to an evolutionary advantage, its function has remained unknown. The secondary structure of the original SCoV-2 RNA sequence (Wuhan-Hu-1) was determined by NMR in late 2020, delineating the base-pairing pattern and revealing substantial differences in secondary structure compared to SARS-CoV-1 (SCoV-1).

Biophysical Investigation of RNA ⋅ DNA : DNA Triple Helix and RNA : DNA Heteroduplex Formation by the lncRNAs MEG3 and Fendrr.

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2.

NMR H,F-based screening of the four stem-looped structure 5_SL1-SL4 located in the 5'-untranslated region of SARS-CoV 2 RNA.

Development of new antiviral medication against the beta-coronavirus SARS-CoV-2 (SCoV2) is actively being pursued. Both NMR spectroscopy and crystallography as structural screening technologies have been utilised to screen the viral proteome for binding to fragment libraries. Here, we report on NMR screening of elements of the viral RNA genome with two different ligand libraries using H-NMR-screening experiments and H and F NMR-screening experiments for fluorinated compounds.

Targeting the Main Protease (M, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies.

The main protease M, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding.

The DRESDEN PLATFORM is a research hub for ultra-high dose rate radiobiology.

The recently observed FLASH effect describes the observation of normal tissue protection by ultra-high dose rates (UHDR), or dose delivery in a fraction of a second, at similar tumor-killing efficacy of conventional dose delivery and promises great benefits for radiotherapy patients. Dedicated studies are now necessary to define a robust set of dose application parameters for FLASH radiotherapy and to identify underlying mechanisms. These studies require particle accelerators with variable temporal dose application characteristics for numerous radiation qualities, equipped for preclinical radiobiological research.