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The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity. Metal ions play essential roles in myriads of biological processes, from catalytic co-factors to supporting protein and nucleic acid structures. Here the authors use long-wavelength X-ray diffraction to locate hundreds of potassium ions taking part in the formation of rRNA tertiary structure, mediating rRNA–protein interactions and supporting ribosomal protein structures and function.

Translation of the genetic code into proteins is realized through repetitions of synchronous translocation of messenger RNA (mRNA) and transfer RNAs (tRNA) through the ribosome. In eukaryotes translocation is ensured by elongation factor 2 (eEF2), which catalyses the process and actively contributes to its accuracy 1 . Although numerous studies point to critical roles for both the conserved eukaryotic posttranslational modification diphthamide in eEF2 and tRNA modifications in supporting the accuracy of translocation, detailed molecular mechanisms describing their specific functions are poorly understood. Here we report a high-resolution X-ray structure of the eukaryotic 80S ribosome in a translocation-intermediate state containing mRNA, naturally modified eEF2 and tRNAs. The crystal structure reveals a network of stabilization of codon–anticodon interactions involving diphthamide 1 and the hypermodified nucleoside wybutosine at position 37 of phenylalanine tRNA, which is also known to enhance translation accuracy 2 . The model demonstrates how the decoding centre releases a codon–anticodon duplex, allowing its movement on the ribosome, and emphasizes the function of eEF2 as a ‘pawl’ defining the directionality of translocation 3 . This model suggests how eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs undergo large-scale molecular reorganizations to ensure maintenance of the mRNA reading frame during the complex process of translocation. Structural analysis of the Saccharomyces cerevisiae 80S ribosome trapped in an intermediate translocation state shows stabilization of codon–anticodon interactions by eukaryote-specific elements of the 80S ribosome, eEF2 and tRNA and demonstrates a major role for eEF2 in maintaining the directionality of translocation.

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNA Lys UUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine–pyrimidine mismatches. We show that mnm 5 s 2 U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism. The anticodon loops of almost all tRNAs contain modifications known to be important for their function. Here the authors use crystallography to provide new mechanistic insights into how the modification at the wobble position of the E. coli tRNALys UUU assists in discrimination between cognate and near-cognate codons.

The decoding of mRNA on the ribosome is the least accurate process during genetic information transfer. Here we propose a unified decoding mechanism based on 11 high-resolution X-ray structures of the 70S ribosome that explains the occurrence of missense errors during translation. We determined ribosome structures in rare states where incorrect tRNAs were incorporated into the peptidyl-tRNA-binding site. These structures show that in the codon–anticodon duplex, a G·U mismatch adopts the Watson–Crick geometry, indicating a shift in the tautomeric equilibrium or ionization of the nucleobase. Additional structures with mismatches in the 70S decoding centre show that the binding of any tRNA induces identical rearrangements in the centre, which favours either isosteric or close to the Watson–Crick geometry codon–anticodon pairs. Overall, the results suggest that a mismatch escapes discrimination by preserving the shape of a Watson–Crick pair and indicate that geometric selection via tautomerism or ionization dominates the translational infidelity mechanism. Translation of mRNA into proteins is the least accurate process during genetic information transfer. Here the authors suggest—based on 11 high-resolution ribosome crystal structures—that the origin of protein missense errors involves molecular mimicry via tautomerism or ionization.

A foot placement of inertial sensors is commonly used for heel-strike (HS) and toe-off (TO) event detection. However, in clinical practice, such sensor placement may be difficult or even impossible due to the deformity of patients’ feet. The first contribution of this paper is a new algorithm for HS and TO event detection for cases when the sensors are placed on the lateral malleolus. Such sensor placement allows gait analysis in patients with foot deformities. In addition, the placement of the sensor directly on the wide bone surface of the lateral malleolus ensures secure fixation of the sensor during walking. The proposed algorithm is based on deep neural networks, which can be easily adapted (by retraining the neural networks) for analysis of various pathological gait patterns. It is especially important in clinical practice when the number of possible pathological gait patterns is very large. The algorithm proposed in this paper was implemented in a new wearable system for the clinical gait analysis. The second contribution is a validation of this new wearable system. The performance of both proposed algorithm and gait analysis system was evaluated against a reference treadmill system where a capacitance–based pressure platform was used. A total of 117 healthy volunteers participated in the comparison (62 males and 55 females, age 24–55 years, height 162–183 cm). They were asked to perform 2 min walking trials with different speed. Mean accuracy±precision was –0.021±0.091 s for gait cycle, 0.589±1.144 steps/min for cadence, –0.051±0.544% for stance phase, –0.37±0.649% for single support, 0.296±0.711% for double support, 0.132±0.561% for load response, and 0.106±0.661% for preswing. Limitations of the proposed algorithm and its compassion with state-of-the-art algorithms were discussed.

Precursor‐messenger RNA (pre‐mRNA) splicing encompasses two sequential transesterification reactions in distinct active sites of the spliceosome that are transiently established by the interplay of small nuclear (sn) RNAs and spliceosomal proteins. Protein Prp8 is an active site component but the molecular mechanisms, by which it might facilitate splicing catalysis, are unknown. We have determined crystal structures of corresponding portions of yeast and human Prp8 that interact with functional regions of the pre‐mRNA, revealing a phylogenetically conserved RNase H fold, augmented by Prp8‐specific elements. Comparisons to RNase H–substrate complexes suggested how an RNA encompassing a 5′‐splice site (SS) could bind relative to Prp8 residues, which on mutation, suppress splice defects in pre‐mRNAs and snRNAs. A truncated RNase H‐like active centre lies next to a known contact region of the 5′SS and directed mutagenesis confirmed that this centre is a functional hotspot. These data suggest that Prp8 employs an RNase H domain to help assemble and stabilize the spliceosomal catalytic core, coordinate the activities of other splicing factors and possibly participate in chemical catalysis of splicing.

Cholinergic septohippocampal projections originating from the medial septal area (MSA) play a critical role in regulating attention, memory formation, stress responses, and synaptic plasticity. Cholinergic axons from the MSA extensively innervate all hippocampal regions, providing a structural basis for the simultaneous release of acetylcholine (ACh) across the entire hippocampus. However, this widespread release appears inconsistent with the specific functional roles that ACh is thought to serve during distinct behaviors. A key unresolved question is how the dynamics of ACh tissue concentrations determine its ability to activate different receptor types and coordinate individual synaptic pathways. Here, we highlight several debated issues, including the potential intrinsic source of ACh within the hippocampus – such as cholinergic interneurons – and the co-release of ACh with GABA. Furthermore, we discuss recent findings on in vivo ACh concentration dynamics, which present a new dilemma for understanding ACh signaling in the hippocampus: the contrast between “global” ACh release, driven by synchronous activation of MSA neurons, and “local” release, which may be influenced by yet unidentified factors.

Aims Insulin‐like growth factor binding protein‐4 (IGFBP‐4) fragments have been shown to predict the risk of major adverse cardiovascular events, including segment‐elevation myocardial infarction, in patients with acute coronary syndrome. We evaluated the prognostic value of the carboxy‐terminal fragment of IGFBP‐4 (CT‐IGFBP‐4) for all‐cause mortality in emergency room patients with acute heart failure (AHF). Methods and results CT‐IGFBP‐4, N‐terminal pro brain natriuretic peptide (NT‐proBNP), and C‐reactive protein (CRP) were measured at admission from the lithium‐heparin plasma of 156 patients with AHF. All‐cause mortality was recorded for 1 year. Receiver operator characteristic (ROC) curves, Kaplan–Meier, and Cox proportional hazard ratio analyses were performed to evaluate the prognostic value of the various clinical variables, CT‐IGFBP‐4, NT‐proBNP, CRP, and their combinations. During 1 year of follow‐up, 52 (33.3%) patients died. CT‐IGFBP‐4 only weakly correlated with NT‐proBNP (Pearson correlation coefficient r = 0.16, P = 0.044) and did not correlate with CRP (r = 0.08, P = 0.35), emphasizing the different nature of these biomarkers. The receiver operator characteristic area under the curve (ROC AUC) of CT‐IGFBP‐4 for the prediction of all‐cause mortality (0.727) was significantly higher than that of NT‐proBNP (0.680, P = 0.045) and CRP (0.669, P = 0.016). The combination of CT‐IGFBP‐4, NT‐proBNP, and CRP predicted mortality significantly better (ROC AUC = 0.788) than any of the biomarkers alone (P < 0.01 for all). The addition of CT‐IGFBP‐4 to a clinical prediction model that included age, gender, systolic blood pressure, creatinine, and sodium levels, as well as the history of previous heart failure, coronary artery disease, and hypertension significantly improved the mortality risk prediction (ROC AUC 0.774 vs. 0.699, P = 0.025). Cox hazard analysis indicated that elevated CT‐IGFBP‐4 was independently associated with 1 year mortality (hazard ratio 3.26, P = 0.0008) after adjustment for age, gender, history of previous heart failure, coronary artery disease, hypertension, chronic kidney failure, history of diabetes, heart rate, haemoglobin, plasma sodium, NT‐proBNP, CRP, cystatin C, and elevated cardiac troponin I or T. Patients with increased levels of either two or three of the biomarkers CT‐IGFBP‐4, NT‐proBNP, and CRP had significantly higher mortality risk (adjusted hazard ratio 10.04, P < 0.0001) than patients with increased levels of one or none of the biomarkers. Conclusions CT‐IGFBP‐4 was independently associated with all‐cause mortality in patients with AHF. Compared with single biomarkers, the combination of CT‐IGFBP‐4, NT‐proBNP, and CRP improved the prediction of all‐cause mortality in patients with AHF.

BackgroundBoth ganglioside GD2-targeted immunotherapy and antibody-drug conjugates (ADCs) have demonstrated clinical success as solid tumor therapies in recent years, yet no research has been carried out to develop anti-GD2 ADCs against solid tumors. This is the first study to analyze cytotoxic activity of clinically relevant anti-GD2 ADCs in a wide panel of cell lines with varying GD2 expression and their effects in mouse models of GD2-positive solid cancer.MethodsAnti-GD2 ADCs were generated based on the GD2-specific antibody ch14.18 approved for the treatment of neuroblastoma and commonly used drugs monomethyl auristatin E (MMAE) or F (MMAF), conjugated via a cleavable linker by thiol-maleimide chemistry. The antibody was produced in a mammalian expression system, and its specific binding to GD2 was analyzed. Antigen-binding properties and biodistribution of the ADCs in mice were studied in comparison with the parent antibody. Cytotoxic effects of the ADCs were evaluated in a wide panel of GD2-positive and GD2-negative tumor cell lines of neuroblastoma, glioma, sarcoma, melanoma, and breast cancer. Their antitumor effects were studied in the B78-D14 melanoma and EL-4 lymphoma syngeneic mouse models.ResultsThe ch14.18-MMAE and ch14.18-MMAF ADCs retained antigen-binding properties of the parent antibody. Direct dependence of the cytotoxic effect on the level of GD2 expression was observed in cell lines of different origin for both ADCs, with IC50 below 1 nM for the cells with high GD2 expression and no cytotoxic effect for GD2-negative cells. Within the analyzed cell lines, ch14.18-MMAF was more effective in the cells overexpressing GD2, while ch14.18-MMAE had more prominent activity in the cells expressing low GD2 levels. The ADCs had a similar biodistribution profile in the B78-D14 melanoma model compared with the parent antibody, reaching 7.7% ID/g in the tumor at 48 hours postinjection. The average tumor size in groups treated with ch14.18-MMAE or ch14.18-MMAF was 2.6 times and 3.8 times smaller, respectively, compared with the control group. Antitumor effects of the anti-GD2 ADCs were also confirmed in the EL-4 lymphoma model.ConclusionThese findings validate the potential of ADCs targeting ganglioside GD2 in treating multiple GD2-expressing solid tumors.

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties–minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.