Unexpected Mode Research Articles

Lactate metabolism is essential in early-onset mitochondrial myopathy.

Myopathies secondary to mitochondrial electron transport chain (ETC) dysfunction can result in devastating disease. While the consequences of ETC defects have been extensively studied in culture, little in vivo data are available. Using a mouse model of severe, early-onset mitochondrial myopathy, we characterized the proteomic, transcriptomic, and metabolic characteristics of disease progression. Unexpectedly, ETC dysfunction in muscle results in reduced expression of glycolytic enzymes in our animal model and patient muscle biopsies. The decrease in glycolysis was mediated by loss of constitutive Hif1α signaling, down-regulation of the purine nucleotide cycle enzyme AMPD1, and activation of AMPK. In vivo isotope tracing experiments indicated that myopathic muscle relies on lactate import to supply central carbon metabolites. Inhibition of lactate import reduced steady-state levels of tricarboxylic acid cycle intermediates and compromised the life span of myopathic mice. These data indicate an unexpected mode of metabolic reprogramming in severe mitochondrial myopathy that regulates disease progression.

Publicly funded exome sequencing for outpatients with neurodevelopmental disorders demonstrates a high rate of unexpected findings impacting medical management

PurposeExome sequencing (ES) is a powerful tool that facilitates the diagnosis of patients with rare Mendelian syndromes. In 2018 the Israeli Ministry of Health initiated a national pilot program that funds ES for outpatients with global developmental delay (GDD). Here, we describe the 3-year impact of this program on patient care in a single tertiary hospital. MethodsFrom 2018 to 2020, trio ES was performed on 170 participants fulfilling Israeli Ministry of Health criteria: (1) moderate to severe GDD and (2) mild GDD with epilepsy or a major congenital anomaly. We retrospectively analyzed this cohort. ResultsA diagnosis was achieved in 74 individuals (43%). There were 82 clinically significant variants, the majority being novel. Consanguinity was reported in 22% and was not associated with a higher diagnostic rate. The presence of autism spectrum was associated with a lower diagnostic rate of 8/33 (24%). Autosomal dominant inheritance was identified in 14% of participants, and the parental phenotype ranged between fully affected and asymptomatic. Among the diagnosed participants, 16% had an unexpected diagnosis that did not fit the typical clinical presentation. In 9%, the diagnosis changed short-term active clinical management, in 19%, the surveillance recommendations, and in 23%, the family-focused outcomes. ConclusionThe introduction of a national program that funds ES for GDD has transformed patient care, leading to a significant effect on medical management and treatment. The high rate of an unexpected inheritance mode and variable phenotypes emphasizes the diagnostic complexity of neurodevelopmental disorders and the strength of a non-targeted approach.

Discovery of a new class of reversible TEA domain transcription factor inhibitors with a novel binding mode.

The TEA domain (TEAD) transcription factor forms a transcription co-activation complex with the key downstream effector of the Hippo pathway, YAP/TAZ. TEAD-YAP controls the expression of Hippo-responsive genes involved in cell proliferation, development, and tumorigenesis. Hyperactivation of TEAD-YAP activities is observed in many human cancers and is associated with cancer cell proliferation, survival, and immune evasion. Therefore, targeting the TEAD-YAP complex has emerged as an attractive therapeutic approach. We previously reported that the mammalian TEAD transcription factors (TEAD1-4) possess auto-palmitoylation activities and contain an evolutionarily conserved palmitate-binding pocket (PBP), which allows small-molecule modulation. Since then, several reversible and irreversible inhibitors have been reported by binding to PBP. Here, we report a new class of TEAD inhibitors with a novel binding mode. Representative analog TM2 shows potent inhibition of TEAD auto-palmitoylation both in vitro and in cells. Surprisingly, the co-crystal structure of the human TEAD2 YAP-binding domain (YBD) in complex with TM2 reveals that TM2 adopts an unexpected binding mode by occupying not only the hydrophobic PBP, but also a new side binding pocket formed by hydrophilic residues. RNA-seq analysis shows that TM2 potently and specifically suppresses TEAD-YAP transcriptional activities. Consistently, TM2 exhibits strong antiproliferation effects as a single agent or in combination with a MEK inhibitor in YAP-dependent cancer cells. These findings establish TM2 as a promising small-molecule inhibitor against TEAD-YAP activities and provide new insights for designing novel TEAD inhibitors with enhanced selectivity and potency.

Analysis of Different Binding Modes for Tiagabine within the GAT-1 Transporter.

The recently obtained cryo-electron microscopy structure (PDB code: 7SK2) of the human γ-aminobutyric acid transporter type 1 (hGAT-1) in complex with the antiepileptic drug, tiagabine, revealed a rather unexpected binding mode for this inhibitor in an inward-open state of the transporter. The simultaneously released crystal structures of the modified dopamine transporter with mutations mimicking hGAT-1 indicated an alternative binding mode for the tiagabine analogues that were found to block the transporter in an outward-open state, which is more consistent with the results of previous biological and molecular modeling studies. In view of the above discrepancies, our study compares different hypothetical tiagabine binding modes using classical and accelerated molecular dynamics simulations, as well as MM-GBSA free binding energy (dG) calculations. The results indicate that the most stable and energetically favorable binding mode of tiagabine is the one where the nipecotic acid fragment is located in the main binding site (S1) and the aromatic rings are arranged within the S2 site of the hGAT-1 transporter in an outward-open state, confirming the previous molecular modelling findings. The position of tiagabine bound to hGAT-1 in an inward-open state, partially within the intracellular release pathway, was significantly less stable and the dG values calculated for this complex were higher. Furthermore, analysis of the cryo-electron map for the 7SK2 structure shows that the model does not appear to fit into the map optimally at the ligand binding site. These findings suggest that the position of tiagabine found in the 7SK2 structure is rather ambiguous and requires further experimental verification. The identification of the main, high-affinity binding site for tiagabine and its analogues is crucial for the future rational design of the GABA transporter inhibitors.

Selection Rules for Resonant Longitudinal Injector-Coupling in Experimental Rocket Combustors

This paper investigates different types of longitudinal mode coupling in subscale rocket combustion chambers using experimental data and numerical simulations. Based on a one-dimensional planar wave acoustic model of coupled cavity resonators with two acoustic inlet boundary conditions, mode selection rules are derived, providing a simple way of predicting which injector and combustion chamber modes have matching frequencies. Longitudinal mode coupling of an injector with an acoustically open inlet boundary condition has been reported in the literature for the start-up transient of a research combustor experiment. In this experiment, every third injector mode couples to a corresponding chamber longitudinal mode, which is explained in terms of the selection rules derived in this paper. Numerical simulation results for a different combustor experiment show an unexpected mode coupling behavior when an acoustically closed injector inlet is used. Theoretical analysis by using the one-dimensional wave model and applying the derived selection rules shows that in this setup, the injector acoustic mode can accommodate two different acoustic boundary conditions at the injector-chamber interface simultaneously. This results in different acoustic mode shapes in the injector, explaining the unexpected behavior for the resonant coupling with an acoustically closed injector inlet.

Indolin-2-one Nitroimidazole Antibiotics Exhibit an Unexpected Dual Mode of Action.

Nitroimidazoles such as metronidazole are used as anti-infective drugs against anaerobic bacteria. Upon in vivo reduction of the nitro group, reactive radicals damage DNA and proteins in the absence of oxygen. Unexpectedly, a recent study of nitroimidazoles linked to an indolin-2-one substituent revealed potent activities against aerobic bacteria. This suggests a different, yet undiscovered mode of action (MoA). To decipher this MoA, we first performed whole proteome analysis of compound-treated cells, revealing an upregulation of bacteriophage-associated proteins, indicative of DNA damage. Since DNA binding of the compound was not observed, we applied activity-based protein profiling (ABPP) for direct target discovery. Labeling studies revealed topoisomerase IV, an essential enzyme for DNA replication, as the most enriched hit in pathogenic Staphylococcus aureus cells. Subsequent topoisomerase assays confirmed the inhibition of DNA decatenation in the presence of indolin-2-one nitroimidazole with an activity comparable to ciprofloxacin, a known inhibitor of this enzyme. Furthermore, we determined significantly increased redox potentials of indolin-2-one nitroimidazoles compared to classic 5-nitroimidazoles such as metronidazole, which facilitates in vivo reduction. Overall, this study unravels that indolin-2-one-functionalized nitroimidazoles feature an unexpected dual MoA: first, the direct inhibition of the topoisomerase IV and second the classic nitroimidazole MoA of reductive bioactivation leading to damaging reactive species. Importantly, this dual MoA impairs resistance development. Given the clinical application of this compound class, the new mechanism could be a starting point to mitigate resistance.

Elevated intracellular Na+ and osmolarity stimulate catalytic activity of the ubiquitin ligase Nedd4-2

Regulation of catalytic activity of E3 ubiquitin ligases is critical for their cellular functions. We identified an unexpected mode of regulation of E3 catalytic activity by ions and osmolarity; enzymatic activity of the HECT family E3 Nedd4-2/Nedd4L is enhanced by increased intracellular Na+ ([Na+]i) and by hyperosmolarity. This stimulated activity is mediated by activation of p38-MAPK and is inhibited by WNKs. Moreover, protease (Furin)-mediated activation of the epithelial Na+ channel ENaC (a bona fide Nedd4-2 substrate), which leads to increased [Na+]i and osmolarity, results in enhanced Nedd4-2 catalytic activity. This enhancement is inhibited by a Furin inhibitor, by a protease-resistant ENaC mutant, or by treatment with the ENaC inhibitor amiloride. Moreover, WNK inhibition, which stimulates catalytic activity of Nedd4-2, leads to reduced levels of cell-surface ENaC and reduced channel activity. ENaC activity does not affect Nedd4-2:ENaC binding. Therefore, these results demonstrate activation of a ubiquitin ligase by Na+ and osmotic changes. Importantly, they reveal a negative feedback loop in which active ENaC leads to stimulation of catalytic activity of its own suppressor, Nedd4-2, to protect cells from excessive Na+ loading and hyperosmotic stress and to protect the animal from hypertension.

Measurement properties of electric field intensity distribution of whispering gallery mode with near-field optical probe

The distribution of the external electric field intensity of the whispering gallery mode (WGM) can be measured using a scanning near-field optical microscopy (SNOM) probe. As probing the resonator influences the optical resonance state, the measurement properties of the SNOM probe are worth studying. In this study, the measurement mechanism of WGMs using a glass SNOM probe was analyzed numerically. A probe with a nanometric-diameter is generally preferable. The results showed that the high-contrast measurement was possible; however, the signal was weak. Using a tip with a diameter equal to half the resonant wavelength, the signal strength was maximized with the same high contrast level as the nanoprobe. Interestingly, the measurement mechanism was different depending on tip size. With the nanoprobe, the interaction with the WGMs varied depending on the sensing locations; therefore, the resonant states were modulated during the measurements, which may have induced unexpected mode hopes. The resonance state was steady during measurements using a probe tip with a diameter of half the resonance wavelength. Although the mechanisms were different, the measured electric field intensity distributions were the same for both tip diameters.

A novel heterozygous missense variant of the ARID4A gene identified in Han Chinese families with schizophrenia-diagnosed siblings that interferes with DNA-binding activity.

ARID4A plays an important role in regulating gene expression and cell proliferation. ARID4A belongs to the AT-rich interaction domain (ARID)-containing family, and a PWWP domain immediately precedes its ARID region. The molecular mechanism and structural basis of ARID4A are largely unknown. Whole-exome sequencing (WES) revealed that a novel heterozygous missense variant, ARID4A c.1231 C > G (p.His411Asp), was associated with schizophrenia (SCZ) in this study. We determined the crystal structure of the PWWP-ARID tandem at 2.05 Å, revealing an unexpected mode in which ARID4A assembles with its PWWP and ARID from a structural and functional supramodule. Our results further showed that compared with the wild type, the p.His411Asp ARID mutant protein adopts a less compact conformation and exhibits a weaker dsDNA-binding ability. The p.His411Asp mutation decreased the number of cells that were arrested in the G0-G1 phase and caused more cells to progress to the G2-M phase. In addition, the missense mutation promoted the proliferation of HEK293T cells. In conclusion, our data provide evidence that ARID4A p.His411Asp could cause a conformational change in the ARID4A ARID domain, influence the DNA binding function, and subsequently disturb the cell cycle arrest in the G1 phase. ARID4A is likely a susceptibility gene for SCZ; thus, these findings provide new insight into the role of ARID4A in psychiatric disorders.

STING Agonists/Antagonists: Their Potential as Therapeutics and Future Developments.

The cGAS STING pathway has received much attention in recent years, and it has been recognized as an important component of the innate immune response. Since the discovery of STING and that of cGAS, many observations based on preclinical models suggest that the faulty regulation of this pathway is involved in many type I IFN autoinflammatory disorders. Evidence has been accumulating that cGAS/STING might play an important role in pathologies beyond classical immune diseases, as in, for example, cardiac failure. Human genetic mutations that result in the activation of STING or that affect the activity of cGAS have been demonstrated as the drivers of rare interferonopathies affecting young children and young adults. Nevertheless, no data is available in the clinics demonstrating the therapeutic benefit in modulating the cGAS/STING pathway. This is due to the lack of STING/cGAS-specific low molecular weight modulators that would be qualified for clinical exploration. The early hopes to learn from STING agonists, which have reached the clinics in recent years for selected oncology indications, have not yet materialized since the initial trials are progressing very slowly. In addition, transforming STING agonists into potent selective antagonists has turned out to be more challenging than expected. Nevertheless, there has been progress in identifying novel low molecular weight compounds, in some cases with unexpected mode of action, that might soon move to clinical trials. This study gives an overview of some of the potential indications that might profit from modulation of the cGAS/STING pathway and a short overview of the efforts in identifying STING modulators (agonists and antagonists) suitable for clinical research and describing their potential as a “drug”.

Acriflavine, a clinically approved drug, inhibits SARS-CoV-2 and other betacoronaviruses

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

Two different kinds of interaction modes of deaminase APOBEC3A with single-stranded DNA in solution detected by nuclear magnetic resonance.

APOBEC3A (A3A) deaminates deoxycytidine in target motif TC in a single-stranded DNA (we termed it as TC DNA), which mortally mutates viral pathogens and immunoglobulins, and leads to the diversification and lethality of cancers. The crystal structure of A3A-DNA revealed a unique U-shaped recognition mode of target base dC0 . However, when TC DNA was titrated into 15 N-labeled A3A solution, we observed two sets of 1 H-15 N cross-peaks of A3A in HSQC spectra, and two sets of 1 H-1 H cross-peaks of DNA in two-dimensional 13 C,15 N-filtered TOCSY spectra, indicating two different kinds of conformers of either A3A or TC DNA existing in solution. Here, mainly by NMR, we demonstrated that one DNA conformer interacted with one A3A conformer, forming a specific complex A3AS -DNAS in a way almost similar to that observed in the reported crystal A3A-DNA structure, where dC0 inserted into zinc ion binding center. While the other DNA conformer bound with another A3A conformer, but dC0 did not extend into the zinc-binding pocket, forming a nonspecific A3ANS -DNANS complex. The NMR solution structure implied three sites Asn61 , His182 and Arg189 were necessary to DNA recognition. These observations indicate a distinctive way from that reported in X-ray crystal structure, suggesting an unexpected mode of deaminase APOBEC3A to identify target motif TC in DNA in solution.

A new and unexpected coordination mode of a bis-phosphine monoxide (BPMO) ligand in a palladacycle complex

A new bis-phosphine monoxide ligand of the formula [P(O)Ph2CH2PPh2C(H)C(O)C6H4-p-NO2] (1) was synthesized and its coordination chemistry explored. The reaction of this ligand with the dinuclear complex [Pd(bzq)(μ-Cl)]2 yields a mononuclear complex of formula [Pd(bzq)(P(O)Ph2C(H)PPh2C(H)C(O)C6H4-p-NO2)] (2) by means of bridge splitting reactions. The synthesized compounds have been characterized by elemental analysis, spectroscopic and crystallographic methods. A novel coordination mode for phosphine monoxide ligand (1) was found in cyclopalladated benzo[h]quinolinate complex, the ligand is coordinating to the palladium metal center through the carbon atom of methine group lying between the two phosphorus atoms and the oxygen atom of carbonyl group, acting as a chelating C,O-donor ligand. The 31P{1H} NMR spectrum of (1) contains two doublets centered at 11.45 and 29.54 ppm, due to the phosphonium and phosphine oxide groups, respectively, while in (2) the corresponding peaks are centered at 27.01 and 42.58 ppm, resulting in a 14 ppm downfield shift on complexation of the ligand to the palladium center. Overall, these types of ligands display interesting reactivity due to their diverse coordination modes and ability to attract considerable attention as an active research field.

Higher-Order Core-Like Modes in Double-Clad Fiber Contribute to Multipath Artifacts in Optical Coherence Tomography

Double-clad fiber (DCF) has enabled the combination of endoscopic optical coherence tomography (OCT) with secondary optical modalities. While DCF offers an additional optical channel, it is widely understood that its use reduces the quality of OCT owing to the introduction of multipath artifacts. We show here that an unexpected higher-order mode (HOM) with its energy confined to the DCF core can contribute to these artifacts. The existence of this HOM is confirmed using the spatially and spectrally (S <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> ) resolved imaging method. The group delay difference of the HOM is shown to be consistent with the delay of the diffuse ghost artifact in DCF-acquired OCT images.

A phase I/IIa trial of the RNA oligonucleotide STNM01 by EUS-FNI to investigate the safety and efficacy in patients with first-line refractory, unresectable pancreatic cancer.

4120 Background: Carbohydrate sulfotransferase 15 (CHST15) is an enzyme that synthesizes heavily sulfated matrix glycosaminoglycan and shown to promote tumor invasion and correlate with poor prognosis in pancreatic ductal adenocarcinoma (PDAC). To explore the role of CHST15 blockage, we conducted a Phase I/IIa (P I/IIa) study to investigate the safety and efficacy of EUS guided locoregional injection of STNM01, a synthetic RNA oligonucleotide, in patients with unresectable PDAC, refractory to first-line chemotherapy. Methods: This was an open-label and dose-escalation study of STNM01 as second-line (2L) therapy in unresectable PDAC patients at 4 centers in Japan. One cycle consists of locoregional injection with STNM01 three times at 2 week-interval in 4 weeks (Days 0, 14 and 28 of dosing) in combination with systemic 2L chemotherapy. A 3+3 dose cohort escalation design initiated with 250 nM (n = 3) followed by 1,000 nM (n = 3), 2,500 nM (n = 3) and 10,000 nM (n = 4) was used for P I part. P IIa part (n = 9) was subsequently conducted with MTD or the highest dose level determined by P I. The primary outcome was incidence of DLT at the end of cycle 1. The secondary outcomes included overall survival (OS), local tumor response, histology and safety. This study was registered with jRCT (jRCT2031190055). Results: A total of 22 patients across 4 doses were enrolled in the study of which 21 were evaluable as per protocol population. All patients received S-1 as a systemic 2L chemotherapy and total 3 cycles were repeated at maximum. The most common AEs were abdominal pain and pyrexia. There were 9 grade 3 AEs. No drug-related SAE as well as DLT was observed. Since MTD was not reached in P I, P IIa was conducted with the highest dose, 10,000 nM. The 6-month survival rate in the entire population (n = 21) and in the highest dose population (n = 12; 3 for P I and 9 for P IIa) was 66.7 and 83.3%, respectively. In histological analyses, the % positive area of CHST15 tended to show dose-dependent suppression at the end of cycle 1, especially with 70.0% reduction from baseline in the highest dose population. Increased tumoral infiltrations of CD3+, CD8+ and CD20+ cells were observed during study period in the highest dose population. Local tumor response for the entire population showed 14 patients (66.7%) with stable disease. Notably, one patient showed complete disappearance on the CT image of both primary and metastatic tumors in the regional lymph node. Conclusions: Repeated locoregional injection of STNM01 is safe and well tolerated in patients with unresectable PDAC as combined 2L therapy. The 6-month survival rate of 10,000 nM of STNM01 was 83.3%, which is remarkable compared with previously reported data. Unexpected mode of action for tumoral lymphocyte infiltration also suggests the promising potential of locoregional injection of STNM01 as a new therapeutic option for PDAC. Clinical trial information: jRCT2031190055.

Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

Bioactivity Profiles of Cytoprotective Short-Chain Quinones.

Short-chain quinones (SCQs) have been investigated as potential therapeutic candidates against mitochondrial dysfunction, which was largely thought to be associated with the reversible redox characteristics of their active quinone core. We recently reported a library of SCQs, some of which showed potent cytoprotective activity against the mitochondrial complex I inhibitor rotenone in the human hepatocarcinoma cell line HepG2. To better characterize the cytoprotection of SCQs at a molecular level, a bioactivity profile for 103 SCQs with different compound chemistries was generated that included metabolism related markers, redox activity, expression of cytoprotective proteins and oxidative damage. Of all the tested endpoints, a positive correlation with cytoprotection by SCQs in the presence of rotenone was only observed for the NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent reduction of SCQs, which also correlated with an acute rescue of ATP levels. The results of this study suggest an unexpected mode of action for SCQs that appears to involve a modification of NQO1-dependent signaling rather than a protective effect by the reduced quinone itself. This finding presents a new selection strategy to identify and develop the most promising compounds towards their clinical use.

BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.

Mutations in BRCA1/BARD1’s E3 ubiquitin ligase RING domains predispose carriers to breast and ovarian cancers. We present the first structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125/127/129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in cancer patients. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.

Structure of SRSF1 RRM1 bound to RNA reveals an unexpected bimodal mode of interaction and explains its involvement in SMN1 exon7 splicing

The human prototypical SR protein SRSF1 is an oncoprotein that contains two RRMs and plays a pivotal role in RNA metabolism. We determined the structure of the RRM1 bound to RNA and found that the domain binds preferentially to a CN motif (N is for any nucleotide). Based on this solution structure, we engineered a protein containing a single glutamate to asparagine mutation (E87N), which gains the ability to bind to uridines and thereby activates SMN exon7 inclusion, a strategy that is used to cure spinal muscular atrophy. Finally, we revealed that the flexible inter-RRM linker of SRSF1 allows RRM1 to bind RNA on both sides of RRM2 binding site. Besides revealing an unexpected bimodal mode of interaction of SRSF1 with RNA, which will be of interest to design new therapeutic strategies, this study brings a new perspective on the mode of action of SRSF1 in cells.

Transverse seismic response and failure mode of towers of a cable-stayed bridge full-model: Tests and simulations

Severe damage to towers of cable-stayed bridges have been recorded in past actual earthquakes. Therefore, increasing attention has been devoted to the seismic failure of cable-stayed bridges under strong earthquakes. However, few experimental studies focus on the seismic failure of long-span cable-stayed bridges including both superstructures and substructures under strong earthquakes. In this paper, a 1/70-scaled bridge full-model was designed and assembled following a 1400 m-span cable-stayed bridge, which was composed of the towers, girder, cables, piers, pile groups, and artificial soil placed in four shear boxes. Shaking table tests were conducted to study the seismic response and potential failure mode of towers of the bridge full-model under transverse earthquake excitation. Test results show that the tower exhibits an unexpected failure mode with the double plastic hinges, which reveals a new feature for the seismic failure of the tower of super long-span cable-stayed bridges. The plastic hinges are firstly observed at near the junction region between the tower and lower beam, then at the proximal joint portion between the tower and upper beam. The tower firstly suffers from severe damage or even local failure for the bridge full-model under strong earthquakes, but it could carry the vertical load. The seismic damage evaluated by the curvature criteria or residual displacements are in good agreement with the observed damage under earthquake excitation with low shaking amplitudes. Moreover, numerical modeling of the bridge full-model could effectively predict the dynamic properties and seismic response of the bridge full-model, and capture the character of the failure mode of the towers compared to the test results.