BUSCADOR RECOLECTA

In recent years, instrumental improvements have enabled the spread of mass spectrometry-based lipidomics platforms in biomedical research. In mass spectrometry, the reliability of generated data varies for each compound, contingent on, among other factors, the availability of labeled internal standards. It is challenging to evaluate the data for lipids without specific labeled internal standards, especially when dozens to hundreds of lipids are measured simultaneously. Thus, evaluation of the performance of these platforms at the individual lipid level in interlaboratory studies is generally not feasible in a time-effective manner. Herein, using a focused subset of sphingolipids, we present an in-house validation methodology for individual lipid reliability assessment, tailored to the statistical analysis to be applied. Moreover, this approach enables the evaluation of various methodological aspects, including discerning coelutions sharing identical selected reaction monitoring transitions, pinpointing optimal labeled internal standards and their concentrations, and evaluating different extraction techniques. While the full validation according to analytical guidelines for all lipids included in a lipidomics method is currently not possible, this process shows areas to focus on for subsequent method development iterations as well as the robustness of data generated across diverse methodologies., Open access funding provided by Karolinska Institute. CEW received support from the Swedish Heart and Lung Foundation (HLF 20230463 and HLF 20210519) and the Swedish Research Council (2022-00796). BZ is supported by the H2020 ITN consortium ArthritisHeal (#812890), the Konung Gustaf V:s 80-årsfond (FAI-2020-0732), and the Swedish Heart and Lung Foundation (HLF20230363). AW received support from the Swedish Heart and Lung Foundation (HLF 20190017) and the Swedish Research Council (2018-00520). JJ received support from the Spanish Ministry of Science and Innovation MCIU/AEI/10.13039/501100011033 and Severo Ochoa Excelencia Grant CEX2018-000794-S., Peer reviewed

Figure S1. No differences in body weight, gross cerebellar architecture, and motorcoordination between wild-type and GluK4 KO mice, Related to Figures 1 and 2. -- Figure S2. Basic CF- and PF-EPSC properties in wild-type and GluK4 KO mice, Related to Figure 1. -- Figure S3. The KAR components are absent in CF-EPSCs of GluK1 KO and GluK4 KO mice, and CF-EPSCs are reduced in PC-specific GluK4 KO mice, Related to Figure 1. -- Figure S4. The GluK1 KO cerebellum exhibited a reduction in CFPC synapses, Related to Figure 3. -- Figure S5. PF/ΔV-stim failed to induce LTD in C1ql1 KO and Bai3 KO PCs, Related to Figure 2. -- Figure S6. Mono CF innervation to single PC in adult GluK4 KO mice. -- Figure S7. MAP allows immunodetection of endogenous GluK4 at CFPC synapse. -- Figure S8. GluK4 and C1ql1 signals on vGluT2-positive area in GluK4 KO, C1ql1 KO and Bai3 KO mice. -- Figure S9. C1ql1 binds to the ATD of GluK4 but not GluK1 in vitro. -- Figure S10. The ATD of GluK4 indirectly binds to Bai3 via C1ql1 in vitro. -- Figure S11. KAR-mediated currents in GluK4 KO PCs expressing HA-GluK4. -- Figure S12. No KAR component at PF-EPSCs in GluK4 KO PCs overexpressing GluK4. -- Figure S13. Transduction efficiency of PCs by retro-orbital injection of AAV(PHP.eB. -- Figure S14. A proposed model illustrating the KAR−C1ql1−Bai3 complex on CF−PC synapses for synaptic integrity, LTD and motor learning, Peer reviewed

Supplemental figures and table - spectrum.00254-24-s0001.doc Fig. S1-S5; Table S7., Tables S8 and S9 - spectrum.00254-24-s0002.xlsx Table S8: Klebsiella phages isolated in K. pneumoniae clinical isolates. Table S9: Functional annotation of Klebsiella phages., Supplemental tables - spectrum.00254-24-s0003.xlsx Tables S1, S3, S5, and S6., Table S2 - spectrum.00254-24-s0004.xlsx Table S2: Functional annotation of Klebsiella phages., Table S4 - spectrum.00254-24-s0005.docx Table S4: Amino acid sequences of detected depolymerase proteins., Peer reviewed

Native chemical ligation (NCL) ligates two unprotected peptides in an aqueous buffer. One of the fragments features a C-terminal α-thioester functional group, and the second bears an N-terminal cysteine. The reaction mechanism depicts two steps: an intermolecular thiol-thioester exchange resulting in a transient thioester, followed by an intramolecular S-to-N acyl shift to yield the final native peptide bond. Although this mechanism is well established, the direct observation of the transient thioester has been elusive because the fast intramolecular rearrangement prevents its accumulation. Here, the use of α-selenoester peptides allows a faster first reaction and an early buildup of the intermediate, enabling its quantification and the kinetic monitoring of the first and second steps. The results show a correlation between the steric hindrance in the α-thioester residue and the rearrangement rate. In bulky residues, the S-to-N acyl shift has a significant contribution to the overall reaction rate. This is particularly notable for valine and likely for other similar β-branched amino acids., This work has been funded by the Spanish Ministerio de Ciencia, Innovación y Universidades (PDI2021-128902OB-I00). I. S.-C. acknowledges AGAUR (Generalitat de Catalunya) for a fellowship (2021 FI-B 00142)., Peer reviewed

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the coupling of G proteins with GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit, the rate-limiting step during G-protein activation. Using molecular dynamics simulations, we investigate the temporal progression of structural rearrangements of GDP-bound Gs protein (Gs·GDP; hereafter GsGDP) upon coupling to the β2-adrenergic receptor (β2AR) in atomic detail. The binding of GsGDP to the β2AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release: the opening of α1-αF helices, the displacement of the αG helix and the opening of the α-helical domain. Signal propagation to the Gs occurs through an extended receptor interface, including a lysine-rich motif at the intracellular end of a kinked transmembrane helix 6, which was confirmed by site-directed mutagenesis and functional assays. From this β2AR-GsGDP intermediate, Gs undergoes an in-plane rotation along the receptor axis to approach the β2AR-Gsempty state. The simulations shed light on how the structural elements at the receptor-G-protein interface may interact to transmit the signal over 30 Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G-protein coupling specificity., This work was funded by German Research Foundation (DFG) through CRC1423, project number 421152132, subproject C01 (to P.W.H.) and subprojects A01, A05 and Z03 (to P.S.), Stiftung Charité and the Einstein Center Digital for Future to P.W.H. P.S. is further supported through CRC 1078–Project ID 221545957–SFB 1078, subproject B06; through the cluster of excellence ‘UniSysCat‘ (under Germany’s Excellence Strategy-EXC2008/1-390540038 and through the European Union’s Horizon 2020 MSCA Program under grant agreement 956314 (ALLODD). This work was also funded by National Institutes of Health grant R01NS028471 (to B.K.K.), by National Natural Science Foundation of China (Grant 32122041 to X.L.) and by Tsinghua University Initiative Scientific Research Program (to X.L.). We are grateful to A. Inoue (Tohoku University, Japan) for providing the CRISPR–Cas9-edited triple knockout barr1/barr2/β2AR HEK293A cells and to H. Schihada (Philipps-Universität Marburg, Germany) for providing the Gs-CASE sensor DNA material and advice for the BRET dissociation assay. We thank B. Bauer (Charité–Universitätsmedizin Berlin, Germany) for assistance in molecular biology and purifying reagents. B.K.K. and P.W.H. acknowledge the Einstein Foundation and the Berlin Institute of Health for their support. We are grateful to M. Heck (Charité–Universitätsmedizin Berlin, Germany) for advice on the statistical analysis of the BRET 2 assay and M. Heck and K. P. Hofmann (Charité–Universitätsmedizin Berlin, Germany) for helpful discussions. P.F.S. also holds external affiliations with the Institute of Theoretical Chemistry, University of Vienna, Austria, the Universidad Nacional de Colombia, Bogotá, Colombia, the Center for noncoding RNA in Technology and Health at the University of Copenhagen and the Santa Fe Institute, Santa Fe, New Mexico, USA. We gratefully acknowledge the scientific support and HPC resources provided by the Erlangen National High Performance Computing Center (NHR@FAU) of the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) under the NHR project p101ae. NHR funding is provided by federal and Bavarian state authorities. NHR@FAU hardware is partially funded by DFG (440719683)., Peer reviewed

With the aim of improving the uncertainties associated with the correct diagnosis of seronegative rheumatoid arthritis (RA) and identifying those at risk of developing interstitial lung disease (ILD), we have designed new peptide antigens bearing three post-translational modifications (PTMs) (citrulline, homocitrulline and acetyl-lysine) related to RA that could complement existing tests based on anti-citrullinated peptide/protein antibodies (ACPAs). Several chimeric peptides were synthesized and comparatively tested as antigens in ELISAs with two cohorts of sera: 178 RAs and 110 healthy blood donors. The results indicated that although chimeric peptides containing all three PTMs and vimentin and enolase domains do not significantly outperform existing ACPA tests in terms of sensitivity and specificity, they show potential to complement current assays, especially when detecting antibodies in some seronegative patients. Furthermore, the presence of these autoantibodies significantly identified patients with RA and ILD. We can conclude that the identification of specific autoantibody profiles using synthetic antigens containing peptide domains derived from proteins present in the human joint could help in the early detection of the risk of ILD in patients with RA and be useful for adapting follow-up strategies and guiding decisions during treatment., This research was funded by the Spanish Ministry of Science, Innovation and Universities and the European Regional Development Fund (Grant No PID2021-122216OB-I00)., Peer reviewed

Water recycling and reuse are cornerstones of water management, which can be compromised by the presence of pollutants. Among these, pharmaceuticals can overcome standard water treatments and require sophisticated approaches to remove them. Sorption is an economically viable alternative limited by the need for sorbents with a sorption coefficient (Kd) higher than 500 L/kg. The cross-linking of dextrin (Dx) with divinyl sulfone (DVS) in the presence of 1 mmol or 5 mmol of ibuprofen (IBU) yields the insoluble polymers pDx1 and pDx5 with improved affinity for IBU and high selectivity towards erythromycin (ERY) and ERY Kd higher than 4 × 103 L/kg, when tested against a cocktail of six drugs. Characterization of the polymers shows that both pDx1 and pDx5 have similar properties, fast sorption kinetics, and ERY Kd of 13.3 × 103 for pDx1 and 6.4 × 103 for pDx5, representing 26.6 and 12.0 times the 500 L/kg threshold. The fact that new affinities and improvements in Kd can be achieved by cross-linking Dx in the presence of other molecules that promote pre-organization expands the applications of DVS cross-linked polysaccharides as sustainable, scalable, and environmentally friendly sorbents with a potential application in wastewater treatment plants (WTPs)., This research was funded by Junta de Andalucía through the research project B-FQM-316-UGR20 funded by the Programa Operativo FEDER Andalucía., Peer reviewed

The small-molecule iododiflunisal (IDIF) is a transthyretin (TTR) tetramer stabilizer and acts as a chaperone of the TTR-Amyloid beta interaction. Oral administration of IDIF improves Alzheimer's Disease (AD)-like pathology in mice, although the mechanism of action and pharmacokinetics remain unknown. Radiolabeling IDIF with positron or gamma emitters may aid in the in vivo evaluation of IDIF using non-invasive nuclear imaging techniques. In this work, we report an isotopic exchange reaction to obtain IDIF radiolabeled with 18F. [19F/18F]exchange reaction over IDIF in dimethyl sulfoxide at 160 °C resulted in the formation of [18F]IDIF in 7 ± 3% radiochemical yield in a 20 min reaction time, with a final radiochemical purity of >99%. Biodistribution studies after intravenous administration of [18F]IDIF in wild-type mice using positron emission tomography (PET) imaging showed capacity to cross the blood-brain barrier (ca. 1% of injected dose per gram of tissue in the brain at t > 10 min post administration), rapid accumulation in the liver, long circulation time, and progressive elimination via urine. Our results open opportunities for future studies in larger animal species or human subjects., The work was supported by MCIN/AEI/10.13039/501100011033 (PID2020-117656RB-I00), European Commission (FP7- PEOPLE-2012-ITN; ID: 316882). Part of this work was performed under the Maria de Maeztu Units of Excellence Programme–Grant MDM-2017-0720 funded by MCIN/AEI/10.13039/501100011033., Peer reviewed

Dihydroceramide desaturase 1 (Des1) catalyzes the formation of a CC double bond in dihydroceramide to furnish ceramide. Inhibition of Des1 is related to cell cycle arrest and programmed cell death. The lack of the Des1 crystalline structure, as well as that of a close homologue, hampers the detailed understanding of its inhibition mechanism and difficults the design of new inhibitors, thus making Des1 a strategic target. Based on previous structure-activity studies, different ceramides containing rigid scaffolds were designed. The synthesis and evaluation of these compounds as Des1 inhibitors allowed the identification of PR280 as a better Des 1 inhibitor in vitro (IC50 = 700 nM) than GT11 and XM462, the current reference inhibitors. This cyclopropenone ceramide was obtained in a 6-step synthesis with a 24 % overall yield. The highly confident 3D structure of Des1, recently predicted by AlphaFold2, served as the basis for conducting docking studies of known Des1 inhibitors and the ceramide derivatives synthesized by us in this study. For this purpose, a complete holoprotein structure was previously constructed. This study has allowed a better knowledge of key ligand-enzyme interactions for Des1 inhibitory activity. Furthermore, it sheds some light on the inhibition mechanism of GT11., We thank Grant PID2021-125923OB-I00 funded by Ministerio de Ciencia e Innovación, Spain (MCIN) and Agencia Estatal de Investigación, Spain (AEI), MCIN/AEI/10.13039/501100011033, and by “European Regional Development Fund (ERDF), A way of making Europe”. P.R. acknowledges Universitat Rovira i Virgili, Spain, for a predoctoral fellowship (2019PMF-PIPF-36), as well as Generalitat de Catalunya (Secretaria d'Universitats i Recerca), Spain, and European Social Found (ESF), for a predoctoral FI fellowship (FI2021/B00241)., Peer reviewed

The triplet excited states of sulfur dioxide can be accessed in the UV region and have a lifetime large enough that they can react with atmospheric trace gases. In this work, we report high level ab initio calculations for the reaction of the a3B1 and b3A2 excited states of SO2 with weak and strong acidic species such as HCOOH and HNO3, aimed to extend the chemistry reported in previous studies with nonacidic H atoms (water and alkanes). The reactions investigated in this work are very versatile and follow different kinds of mechanisms, namely, proton-coupled electron transfer (pcet) and conventional hydrogen atom transfer (hat) mechanisms. The study provides new insights into a general and very important class of excited-state-promoted reactions, opening up interesting chemical perspectives for technological applications of photoinduced H-transfer reactions. It also reveals that atmospheric triplet chemistry is more significant than previously thought., JMA thanks the Ministerio de Ciencia e Inovación (Grant PID2022-138040OB-I00) for financial support, and the Consorci de Serveis Universitaris de Catalunya (CSUC) and the Centro de Supercomputación de. Galicia (CESGA) for providing computational resources., Peer reviewed