Molecular Biology Consortium

BACKGROUNDIn the Netherlands, locally acquired animal and human cases of West Nile virus (WNV) were first identified in 2020 via multidisciplinary WNV monitoring and research activities focusing on mosquitoes, birds, horses, and humans.AIMWe investigated how different activities contributed to WNV detection and response in the Netherlands between 2020 and 2023, to determine best practices, challenges, and opportunities for improvement.METHODSWe identified WNV monitoring and research activities in the Netherlands from 2020 to 2023 and analysed their timeliness to detect and react to WNV circulation. An after-action review (AAR) was conducted with national WNV experts to assess best practices and challenges in the multidisciplinary approach.RESULTSIn 2020, WNV circulation was discovered in an infected bird through a wild live bird research survey and subsequently through mosquito research and monitoring. Thirty-five days after finding the WNV-PCR-positive bird, the first autochthonous human case was uncovered. Between 2021 and 2023, research projects in animals, including sentinel chickens, detected ongoing local enzootic WNV circulation. The AAR highlighted rapid information sharing and interpretation, enabled by multidisciplinary collaborations, as best practice. However, differing institute priorities could sometimes lead to diverging views on follow-up actions.CONCLUSIONResearch and monitoring activities in mosquitoes and animals, particularly wild birds and sentinel chickens, enabled early detection of WNV circulation. Real-time testing could provide early warning of human cases, enabling timely responses. Therefore, these research and monitoring activities should be maintained. Multidisciplinary collaboration enabled rapid detection and response, and addressing remaining challenges could further strengthen effectiveness.

<p dir="ltr">To discover novel treatments, a variety of methods are used. In our laboratory we focus on the discovery of novel drug candidates through chemical high- throughput phenotypic screening. After identifying new compounds with therapeutic potential, the characterization of the properties of these drugs as well as elucidate their mechanism of action (MoA) becomes an important task. In addition to identifying novel therapies, and with the help of genome-wide CRISPR- Cas9 screens, we also try to discover mutations that modulate its effects, which can help to identify patients most likely to respond, or resist, to the drug.</p><p dir="ltr">In <b>Paper I</b>, we conducted a high-throughput image based chemical screen to identify compounds which could upregulate human leukocyte antigen I (HLA-I) on the surface of cancer cells and thus improve the recognition of tumor cells by cytotoxic T cells. In the screen we utilized cells with an inactive IFNy signaling pathway achieved by knocking out STAT1, since mutations in this pathway are one of the main causes of resistance to cancer immunotherapies. We thus explored whether it is possible to upregulate HLA-I expression with drugs independently of the JAK/STAT signaling pathway. The compound library comprised of more than 5.000 chemicals, including medically approved drugs and several agents in advanced preclinical development, targeting several different cell signaling pathways. Unfortunately, we could not identify any significant hit compound, indicating that drug-dependent modulation of HLA-I expression is strictly dependent on intact IFNy signaling.</p><p dir="ltr">In <b>Paper II</b>, we performed a genome-wide CRISPR screen to identify genetic modulators of the toxicity of RNA Polymerase I (Pol I) inhibitors, which are being explored as novel agents in the treatment of cancer. To do so, we studied the antibiotic Actinomycin D (ActD), which is commonly used in cancer therapy, and the pre-clinical tested small molecule BMH21, both known to inhibit Pol I and induce nucleolar stress. While mutations in genes known to regulate the nucleolar stress checkpoint such as TP53 and RB led to resistance to both compounds, mutations that enhanced PI3K/mTOR signalling lead to an increased sensitivity to Pol I inhibition. Interestingly, we could identify mutations which lead to resistance to one drug but sensitivity to the other, suggesting that the drugs have distinct effects on the cells despite both being used as Pol I inhibitors. Overall, our research provides an overview on genetic modulators of nucleolar stress inducing compounds and could therefore help to identify patients who could benefit the most from Pol I targeted therapies based on their tumor genetics.</p><p dir="ltr">In <b>Paper III</b>, we discovered a novel molecule with antiviral properties by conducting a high-throughput phenotypic screen using a pseudotype virus expressing the SARS-CoV-2 spike protein. Subsequence experiments using the molecule, which we named virapinib, demonstrated its ability to limit an infection by SARS-CoV-2 as well as other viruses, such as mpox and tick-borne encephalitis virus (TBEV). Additional mechanistic studies showed that virapinib inhibits macropinocytosis, limiting the entry for viruses using this route of infection of host cells. Virapinib presented no significant toxic effect on the host cells which further highlights its potential as an alternative treatment to prevent viral infections.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Myriam Barz</b>*, Bartlomiej Porebski*, Pranauti Panshikar, Maria Häggbladd, Daniela Hühn, Oscar Fernandez-Capetillo. 2023. 'A chemical screen underscores the essential role of STAT1- dependent IFNy signaling to regulate HLA-I expression in cancer cells'. microPublication Biology. <a href="https://doi.org/10.17912/micropub.biology.000697" rel="noreferrer" target="_blank">https://doi.org/10.17912/micropub.biology.000697</a>.</p><p dir="ltr">II. <b>Myriam Barz</b>*, Alba Corman*, Maria Häggbladd, Alicia González- Serrano, Louise Lidemalm, Matilde Murga, Daniela Hühn, Oscar Fernandez-Capetillo. 2025. 'The PI3K/mTOR axis modulates the response to nucleolar stress'. [Manuscript]</p><p dir="ltr">III. Bartlomiej Porebski, Wanda Christ, Alba Corman, Martin Haraldsson, <b>Myriam Barz</b>, Louise Lidemalm, Maria Häggbladd, Juliana Ilmain, Shane C Wright, Matilde Murga, Jan Schlegel, Malin Jarvius, Maris Lapins, Erdinc Sezgin, Gira Bhabha, Volker M Lauschke, Jordi Carreras-Puigvert, Miguel Lafarga, Jonas Klingström, Daniela Hühn, Oscar Fernandez-Capetillo. 2024. 'Discovery of a novel inhibitor of macropinocytosis with antiviral activity' Mol Ther. 2024 Jul 2:S1525-0016(24)00455-6. <a href="https://doi.org/10.1016/j.ymthe.2024.06.038" target="_blank">https://doi.org/10.1016/j.ymthe.2024.06.038</a><br><br>*These authors contributed equally</p>

ALS-ENABLE is an integrated NIH P30 resource at the Advanced Light Source synchrotron at Lawrence Berkeley National Laboratory in Berkeley, California, USA. The resource provides a single portal to the combined mature structural biology technologies of macromolecular crystallography, small-angle X-ray scattering and X-ray footprinting mass spectrometry, and includes beamlines 2.0.1, 3.3.1, 4.2.2, 5.0.1, 5.0.2, 5.0.3, 8.2.1, 8.2.2, 8.3.1 and 12.3.1. This paper describes the organizational structure and the technologies of ALS-ENABLE. A case study showcasing the main technologies of the resource applied to the characterization of the SpyCatcher-SpyTag protein system is presented.

Large-scale genetic association studies have identified thousands of trait-associated risk loci, establishing the polygenic basis for common complex traits and diseases. Although prior studies suggest that many trait-associated loci are pleiotropic, the extent to which this pleiotropy reflects shared causal variants or confounding by linkage disequilibrium remains poorly characterized. To define a set of candidate loci with potentially pleiotropic associations, we performed genome-wide association study (GWAS) meta-analyses of up to 1,167 clinically relevant traits and diseases across 1,789,365 diverse individuals genetically similar to Admixed American (AMR, NMax = 60,756), African (AFR, NMax = 128,361), East Asian (EAS, NMax = 307,465), European (EUR, NMax = 1,283,907), and South Asian (SAS, NMax = 8,876) reference populations from the VA Million Veteran Program (MVP), UK Biobank (UKB), FinnGen, Biobank Japan (BBJ), Tohoku Medical Megabank (ToMMo), and Korean Genome and Epidemiology Study (KoGES). We identified 27,193 genome-wide significant locus-trait pairs (1MB region with PGWAMA < 5 × 10−8) in within-population analysis and 29,139 in multi-population analysis (PMR-MEGA < 5 × 10−8). Among these, 11.5% (n = 3,149) of locus-trait pairs in population-wise and 6.4% (n = 1,875) in multi-population analyses did not reach genome-wide significance in previously published GWAS. In aggregate, the genome-wide significant loci fell within 2,624 non-overlapping autosomal genomic windows on average ~600kb in size. Each locus contained genome-wide significant signals for a median of 6 traits (IQR 2 to 18), including 2,110 (80%) pleiotropic loci associated with >1 trait. Multi-trait colocalization identified 1,902 (72%) loci with high-confidence (posterior probability > 0.9) evidence of a shared causal variant across two or more traits. Variants in pleiotropic loci were significantly enriched for a broad spectrum of functional annotations compared to non-pleiotropic counterparts. Polygenic scores (PGS) developed from these data generally improved prediction compared to existing PGS, and were broadly associated with both primary and pleiotropic phenotypes. These results provide a contemporary map of genetic pleiotropy across the spectrum of human traits/diseases and diverse genetic backgrounds.

Urinary alkalinization is a key intervention for salicylate poisoning, and clinical guidelines recommend serial urinary pH measurements during treatment. How frequently are urine pH measurements obtained, and how often is alkalinization achieved when managing salicylate poisoning with sodium bicarbonate therapy? This is a retrospective poison center study. Salicylate cases managed by a regional poison center for a 5-year period (2019-2023) were analyzed. Inclusion criteria included all patients receiving bicarbonate therapy for salicylate concentrations >30 mg/dL that were managed in a health care facility. Patients requiring hemodialysis were excluded to eliminate confounding factors. The primary outcome was the documentation of a urine pH in the poison center chart. Secondary outcomes included the frequency of urine pH measurements and the achievement of a urinary pH ≥7.5. Data were analyzed using descriptive statistics. Multivariable logistic regression was performed to determine variables associated with increased odds of urine pH measurement. Among 352 cases, urine pH was documented in 144 (41%). Median urine pH values ranged from 6.0 to 8.5 across serial measurements. Urinary alkalinization (pH ≥7.5) was achieved in 34% of cases with documented urine pH. Higher peak salicylate concentrations were associated with increased odds of urine pH measurement (OR = 1.04, P < 0.001). Verbal recommendations significantly increased the likelihood of pH measurement (OR = 3.43, P < 0.001). Formal guidelines faxed or emailed to clinicians, ICU admission, and toxicology consultation were not significant predictors of urine pH testing. Urine pH was measured in fewer than half of cases, indicating poor adherence to guidelines recommending urinary pH monitoring during alkalinization therapy. Verbal recommendations were more effective than written guidelines in promoting pH measurement. Further investigation into the clinical utility and adherence barriers of urine pH measurement is warranted.

We here update the distribution and provide a checklist of the water boatmen (Hemiptera: Heteroptera: Corixoidea: Corixidae) of Romania based on country-wide sampling, a literature survey, and private and museum collections. The Romanian fauna currently comprises 25 water boatmen species and subspecies in eight genera and two subfamilies. The occurrence of Glaenocorisa propinqua (Fieber, 1860), Arctocorisa carinata carinata (Sahlberg, 1819), and Corixa panzeri Fieber, 1848, all known from old and single records, is here confirmed. The first record of an intermediate shape of the pala between Sigara falleni (Fieber, 1848) (triangular) and S. iactans Jansson, 1983 (trapezoidal) is reported from the southeastern European population of the latter, suggesting possible hybridization.

The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and β isoforms, either standalone or within an β holoenzyme, coexist as 12 and 14 subunit assemblies. Single-molecule fluorescence microscopy of Venus-tagged holoenzymes detects intermediate assemblies and progressive dimer loss due to intrinsic holoenzyme lability, and holoenzyme disassembly into dimers upon mutagenesis of a conserved inter-domain contact. Molecular dynamics (MD) simulations show the flexibility of 4-subunit precursors, extracted in-silico from the β hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the β holoenzyme sample could be created by dimer loss and analysis of its cryo-EM dataset reveals how the gap could open further. An assembly model, considering dimer concentration dependence and strain differences between polymorphs, proposes a mechanism for intrinsic hub lability to fine-tune the stoichiometry of αβ heterooligomers for their dynamic localization within synapses in neurons.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII autophosphorylation. The autophosphorylation in turn increases the CaMKII affinity for CAM. Here, we studied the ATP dependence of CAM association with the actin-binding CaMKIIβ isoform using single-molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface-immobilized Venus-CaMKIIβ were resolved with 0.5 s resolution from video records, batch-processed with a custom algorithm. CAM occupancy was determined simultaneously with spot-photobleaching measurement of CaMKII holoenzyme stoichiometry. We show the ATP-dependent increase of the CAM association requires dimer formation for both the α and β isoforms. The study of mutant β holoenzymes revealed that the ATP-dependent increase in CAM affinity results in two distinct states. The phosphorylation-defective (T287.306-307A) holoenzyme resides only in the low-affinity state. CAM association is further reduced in the T287A holoenzyme relative to T287.306-307A. In the absence of ATP, the affinity of CAM for the T287.306-307A mutant and the wild-type monomer are comparable. The affinity of the ATP-binding impaired (K43R) mutant is even weaker. In ATP, the K43R holoenzyme resides in the low-affinity state. The phosphomimetic mutant (T287D) resides only in a 1000-fold higher-affinity state, with mean CAM occupancy of more than half of the 14-mer holoenzyme stoichiometry in picomolar CAM. ATP promotes T287D holoenzyme disassembly but does not elevate CAM occupancy. Single Poisson distributions characterized the ATP-dependent CAM occupancy of mutant holoenzymes. In contrast, the CAM occupancy of the wild-type population had a two-state distribution with both low- and high-affinity states represented. The low-affinity state was the dominant state, a result different from published in vitro assays. Differences in assay conditions can alter the balance between activating and inhibitory autophosphorylation. Bound ATP could be sufficient for CaMKII structural function, while antagonistic autophosphorylations may tune CaMKII kinase-regulated action-potential frequency decoding in vivo.