Cryo-EM Structure of Human Dicer and Its Complexes with a Pre-miRNA Substrate.

Human Dicer (hDicer) is a multi-domain protein belonging to the RNase III family. It plays pivotal roles in small RNA biogenesis during the RNA interference (RNAi) pathway by processing a diverse range of double-stranded RNA (dsRNA) precursors to generate ∼22 nt microRNA (miRNA) or small interfering RNA (siRNA) products for sequence-directed gene silencing. In this work, we solved the cryoelectron microscopy (cryo-EM) structure of hDicer in complex with its cofactor protein TRBP and revealed the precise spatial arrangement of hDicer's multiple domains. We further solved structures of the hDicer-TRBP complex bound with pre-let-7 RNA in two distinct conformations. In combination with biochemical analysis, these structures reveal a property of the hDicer-TRBP complex to promote the stability of pre-miRNA's stem duplex in a pre-dicing state. These results provide insights into the mechanism of RNA processing by hDicer and illustrate the regulatory role of hDicer's N-terminal helicase domain.

Uniform thin ice on ultraflat graphene for high-resolution cryo-EM.

Cryo-electron microscopy (cryo-EM) visualizes the atomic structure of macromolecules that are embedded in vitrified thin ice at their close-to-native state. However, the homogeneity of ice thickness, a key factor to ensure high image quality, is poorly controlled during specimen preparation and has become one of the main challenges for high-resolution cryo-EM. Here we found that the uniformity of thin ice relies on the surface flatness of the supporting film, and developed a method to use ultraflat graphene (UFG) as the support for cryo-EM specimen preparation to achieve better control of vitreous ice thickness. We show that the uniform thin ice on UFG improves the image quality of vitrified specimens. Using such a method we successfully determined the three-dimensional structures of hemoglobin (64 kDa), α-fetoprotein (67 kDa) with no symmetry, and streptavidin (52 kDa) at a resolution of 3.5 Å, 2.6 Å and 2.2 Å, respectively. Furthermore, our results demonstrate the potential of UFG for the fields of cryo-electron tomography and structure-based drug discovery.

Single hormone or synthetic agonist induces Gs/Gi coupling selectivity of EP receptors via distinct binding modes and propagating paths.

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001, re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402, 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390, 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor signaling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that Gs-biased signaling, but not Gi activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-Gi, EP4-Gs, and EP4-Gi in complex with endogenous prostaglandin E2 (PGE2)or two synthetic agonists and comparing with PGE2-EP2-Gs structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the Gs/Gi transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cytoplasmic structural features. In particular, the orientation of the PGE2 ω-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their Gs/Gi coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for Gs/Gi coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.

A p21-ATD mouse model for monitoring and eliminating senescent cells and its application in liver regeneration post injury.

Cellular senescence associates with pathological aging and tissue dysfunctions. Studies utilizing mouse models for cell lineage tracings have emphasized the importance of senescence heterogeneity in different organs and cell types. Here, we constructed a p21- (Akaluc - tdTomato - Diphtheria Toxin Receptor [DTR]) (ATD) mouse model to specifically study the undefined mechanism for p21-expressing senescent cells in the aged and liver injury animals. The successful expressions of these genes enabled in vitro flow cytometric sorting, in vivo tracing, and elimination of p21-expressing senescent cells. During the natural aging process, p21-expressing cells were found in various tissues of p21-ATD mice. Eliminating p21-expressing cells in the aged p21-ATD mice recovered their multiple biological functions. p21-ATD/Fah-/- mice, bred from p21-ATD mice and fumarylacetoacetate hydrolase (Fah)-/- mice of liver injury, showed that the majority of their senescent hepatocytes were the phenotype of p21+ rather than p16+. Furthermore, eliminating the p21-expressing hepatocytes significantly promoted the engraftment of grafted hepatocytes and facilitated liver repopulation, resulting in significant recovery from liver injury. Our p21-ATD mouse model serves as an optimal model for studying the pattern and function of p21-expressing senescent cells under the physical and pathological conditions during aging.

Abnormal global alternative RNA splicing in COVID-19 patients.

Viral infections can alter host transcriptomes by manipulating host splicing machinery. Despite intensive transcriptomic studies on SARS-CoV-2, a systematic analysis of alternative splicing (AS) in severe COVID-19 patients remains largely elusive. Here we integrated proteomic and transcriptomic sequencing data to study AS changes in COVID-19 patients. We discovered that RNA splicing is among the major down-regulated proteomic signatures in COVID-19 patients. The transcriptome analysis showed that SARS-CoV-2 infection induces widespread dysregulation of transcript usage and expression, affecting blood coagulation, neutrophil activation, and cytokine production. Notably, CD74 and LRRFIP1 had increased skipping of an exon in COVID-19 patients that disrupts a functional domain, which correlated with reduced antiviral immunity. Furthermore, the dysregulation of transcripts was strongly correlated with clinical severity of COVID-19, and splice-variants may contribute to unexpected therapeutic activity. In summary, our data highlight that a better understanding of the AS landscape may aid in COVID-19 diagnosis and therapy.

Spatiotemporal Heterogeneity of Temperature and Catalytic Activation within Individual Catalyst Particles.

Temperature is a critical parameter in chemical conversion, significantly affecting the reaction kinetics and thermodynamics. Measuring temperature inside catalyst particles of industrial interest (∼micrometers to millimeters), which is crucial for understanding the evolution of chemical dynamics at catalytic active sites during reaction and advancing catalyst designs, however, remains a big challenge. Here, we propose an approach combining two-photon confocal microscopy and state-of-the-art upconversion luminescence (UL) imaging to measure the spatiotemporal-resolved temperature within individual catalyst particles in the industrially significant methanol-to-hydrocarbons reaction. Specifically, catalyst particles containing zeolites and functional nanothermometers were fabricated using microfluidic chips. Our experimental results directly demonstrate that the zeolite density and particle size can alter the temperature distribution within a single catalyst particle. Importantly, the observed temperature heterogeneity plays a decisive role in the activation of the reaction intermediate and the utilization of active sites. We expect that this work opens a venue for unveiling the reaction mechanism and kinetics within industrial catalyst particles by considering temperature heterogeneity.

Unraveling a Stable 16-Ring Aluminophosphate DNL-11 through Three-Dimensional Electron Diffraction for Atmospheric Water Harvesting.

Sorption-based atmospheric water harvesting (AWH) is a promising solution for addressing water scarcity. Developing cost-effective and stable water adsorbents with high water uptake capacity and a low-temperature regeneration requirement is a crucially important procedure. In this Communication, we present a novel and stable aluminophosphate (AlPO) molecular sieve (MS) named DNL-11 with 16-ring channels synthesized by using an affordable and commercialized organic structure directing agent (OSDA), whose crystallographic structure is elucidated by three-dimensional electron diffraction (3D ED). DNL-11 exhibits a significant water uptake capacity (189 mg/g) at a very low vapor pressure (5% relative humidity at 30 °C). In addition, most of the adsorbed water can be effortlessly removed by purging N2 at 25 °C under ambient pressure conditions. This may expand the possibility of AWH under extreme drought conditions.

Combined Strategies Enable Highly Selective Light Olefins and para-Xylene Production on Single Catalyst Bed.

Achieving multiple high-value-added chemical production through novel reaction processes and shape-selective catalytic strategy is the key to realizing efficient low-carbon catalytic processes. In this work, a methanol-toluene coreaction system was developed, and combined control strategies of reaction pathway guidance and shape-selective catalysis were applied for the successful production of light olefins and para-xylene on single HZSM-5 catalyst bed. Cofeeding toluene additionally provides reactive and flowing aromatic hydrocarbon pool species that change the dominant reaction pathway in the complex network of the methanol reaction on HZSM-5 and promote the formation of ethylene. For the first time, the key reaction intermediates methylmethylenecyclodiene are directly captured and identified by experimental and theoretical techniques. This helps to propose the catalytic cycle for the dominant generation of ethylene and, more importantly, enriches the methanol-to-hydrocarbons (MTH) chemistry and hydrocarbon pool mechanism. Furthermore, 0.4HZSM-5@S-1-CLD, an optimized HZSM-5 catalyst modified by the silicalite-1 epitaxial growth followed by silanization approach, realizes highly selective production of light olefins (especially ethylene) and para-xylene, while excellent reactant activity is maintained. This highly efficient coreaction route gives an important leading significance in synthesizing the raw materials for the polyolefin and polyester industries. The establishment of the combined control strategies provides a model for the joint production of multiple target chemicals in complex catalytic processes.

Dynamic Catalytic Mechanism of the Methanol-to-Hydrocarbons Reaction over Zeolites.

ConspectusThe methanol-to-hydrocarbons (MTH) process has provided a new route to obtaining basic chemicals without relying on an oil resource. Acidity and shape selectivity endow the zeolite with a decisive role in MTH catalysis. However, the inherent reaction characteristics of the MTH reaction over zeolites, such as the complexity of catalytic reaction kinetics, the diversity of catalytic reaction modes, and even the limitations of catalytic and diffusive decoupling, have all confused people with respect to obtaining a comprehensive mechanistic understanding. By examining the zeolite-catalyzed MTH reaction from the perspective of chemical bonding, one would realize that this reaction reflects the dynamic assembly process of C-C bonds from C1 components to multicarbon products. The key to understanding the MTH reaction lies in the mechanism by which C-C bonds are formed and rearranged in the confined microenvironment of the channel or cage structures of zeolite catalysts to achieve shape-selective production.The applications of advanced in situ spectroscopy as well as computational chemistry provide tremendous opportunities for capturing and identifying the details of the structure and properties of reactants, intermediates, and products in the confined reaction space of zeolite channels or cages, observing the real-time dynamic evolution of the catalytic surface, and modeling the elementary reaction steps at the molecular and atomic levels.In this Account, the dynamic catalytic mechanism of the zeolite-catalyzed MTH reaction will be outlined based on decades of continuous research and in-depth understanding. The combination of advanced in situ spectroscopy and theoretical methods allowed us to observe and simulate the formation, growth, and aging process on the working catalyst surface and thus map the dynamical evolution of active sites from a Brønsted acid site (BAS) to an organic-inorganic hybrid supramolecule (OIHS) in the MTH reaction. Moreover, the ever-evolving dynamic succession of the OIHS from surface methoxy species (SMS) to active ion-pair complexes (AIPC) to inert complexes (IC) guided the dynamic autocatalytic process from initiation to sustaining and then to termination, resulting in a complex interlaced hypercycle reaction network. The concept of dynamic catalysis will provide deep insight into the complex catalytic mechanisms as well as the structure-activity relationships in MTH chemistry. More importantly, we are now getting closer to the nature of zeolite catalysis beyond the traditional view of BAS catalysis.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Axis in CD4+ T Cells.

BACKGROUND: Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Ang II (angiotensin II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. METHODS: TF (transcription factor) profiling was performed in peripheral blood mononuclear cells of hypertensive patients. CD4-targeted KLF10 (Kruppel like factor 10)-deficient (Klf10fl/flCD4Cre+; [TKO]) and CD4-Cre (Klf10+/+CD4Cre+; [Cre]) control mice were subjected to Ang II infusion. End point characterization included cardiac echocardiography, aortic imaging, multiorgan histology, flow cytometry, cytokine analysis, aorta and fibroblast transcriptomic analysis, and aortic single-cell RNA-sequencing. RESULTS: TF profiling identified increased KLF10 expression in hypertensive human subjects and in CD4+ T cells in Ang II-treated mice. TKO mice showed enhanced perivascular fibrosis, but not interstitial fibrosis, in aorta, heart, and kidney in response to Ang II, accompanied by alterations in global longitudinal strain, arterial stiffness, and kidney function compared with Cre control mice. However, blood pressure was unchanged between the 2 groups. Mechanistically, KLF10 bound to the IL (interleukin)-9 promoter and interacted with HDAC1 (histone deacetylase 1) inhibit IL-9 transcription. Increased IL-9 in TKO mice induced fibroblast intracellular calcium mobilization, fibroblast activation, and differentiation and increased production of collagen and extracellular matrix, thereby promoting the progression of perivascular fibrosis and impairing target organ function. Remarkably, injection of anti-IL9 antibodies reversed perivascular fibrosis in Ang II-infused TKO mice and C57BL/6 mice. Single-cell RNA-sequencing revealed fibroblast heterogeneity with activated signatures associated with robust ECM (extracellular matrix) and perivascular fibrosis in Ang II-treated TKO mice. CONCLUSIONS: CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for treatment of vascular or fibrotic diseases.

Differences in Kondo Splitting of Surface Quantum Systems Induced by Two Distinct Magnetic Tips: A Joint Method of DFT and HEOM.

The interactions between a magnetic tip and local spin impurities initiate unconventional Kondo phenomena, such as asymmetric suppression or even splitting of the Kondo peak. However, a lack of realistic theoretical models and comprehensive explanations for this phenomenon persists due to the complexity of the interactions. This research employs a joint method of density functional theory (DFT) and hierarchical equation of motion (HEOM) to simulate and contrast the modulation of the spin state and Kondo behavior in the Fe/Cu(100) system with two distinct magnetic tips. A cobalt tip, possessing a larger magnetic moment, incites greater atomic displacement of the iron atom, more notable alterations in electronic structure, and enhanced charge transfer with the environment compared with the control process utilizing a nickel tip. Furthermore, the Kondo resonance undergoes asymmetric splitting as a result of the ferromagnetic correlation between the iron atom and the magnetic tip. The Co tip's higher spin polarization results in a wider spacing between the splitting peaks. This investigation underscores the precision of the DFT + HEOM approach in predicting complex quantum phenomena and explaining the underlying physical principles. This provides valuable theoretical support for developing more sophisticated quantum regulation experiments.

Mutual regulation between GDF11 and TET2 prevents senescence of mesenchymal stem cells.

Growth differentiation factor 11 (GDF11) is a putative systemic rejuvenation factor. In this study, we characterized the mechanism by which GDF11 reversed aging of mesenchymal stem cells (MSCs). In culture, aged MSCs proliferate slower and are positive for senescence markers senescence-associated ß-galactosidase and P16ink4a . They have shortened telomeres, decreased GDF11 expression, and reduced osteogenic potential. GDF11 can block MSC aging in vitro and reverse age-dependent bone loss in vivo. The antiaging effect of GDF11 is via activation of the Smad2/3-PI3K-AKT-mTOR pathway. Unexpectedly, GDF11 also upregulated a DNA demethylase Tet2, which served as a key mediator for GDF11 to autoregulate itself via demethylation of the GDF11 promoter. Mutation of Tet2 facilitates MSC aging by blocking GDF11 expression. Mutagenesis of Tet2-regulated CpG sites also blocks GDF11 expression, leading to MSC aging. Together, a novel mutual regulatory relationship between GDF11 and an epigenetic factor Tet2 unveiled their antiaging roles.

Fluoride- and Seed-Free Synthesis of Pure-Silica Zeolite Adsorbent and Matrix Using OSDA-Mismatch Approach.

The pure-silica zeolite plays a crucially important role in the gas separation of alkane/alkene, the low-k dielectric material, and the robust matrix for confining metal species during catalysis. However, the environmentally friendly synthesis of pure-silica zeolites is still challenging since (1) the toxic fluoride or dealuminum seeds are inevitably utilized through the hydrothermal synthesis and (2) it will also take a longer crystallization time. Herein, we present an efficient method called the OSDA-mismatch approach for the fluoride- and seed-free synthesis of pure-silica zeolites using Si-SOD (enriched 4-rings) as the sole silica source. This approach allows for the rapid and green synthesis of 15 pure-silica zeolites (CHA, *BEA, EUO, SFF, STF, -SVR, *-SVY, DOH, MTN, NON, *MRE, MEL, MFI, MTW, and *STO). Furthermore, distinct crystallization mechanisms of two significant pure-silica CHA- and *BEA-type zeolites (denoted as Si-CHA and Si-BEA) are investigated in detail by advanced characterization techniques such as FIB, 3D ED, 4D-STEM, HRTEM, Raman, and 29Si MAS NMR. More importantly, Si-CHA displays promising propane/propylene separation performance even better than the one synthesized in the presence of toxic HF. In addition, the incorporation of Zn species within Si-BEA fabricated by this approach also renders superior performance on propane dehydrogenation.

Pure Silica with Ordered Silanols for Propylene/Propane Adsorptive Separation Unraveled by Three-Dimensional Electron Diffraction.

Adsorptive separation of propylene (C3H6) from propane (C3H8), which could deal with energy-intensive cryogenic distillation technologies, remains challenging due to their similar physiochemical properties. Herein, we present a pure silica zeolite with ordered silanols (OSs), whose crystallographic structure was unraveled by the advanced three-dimensional electron diffraction (3D ED), displaying the highly efficient separation of propylene from propane under ambient conditions. The 3D ED technique enables us to investigate its 8-ring pore opening transformation from the round one to the elliptical one during the removal of organic structure-directing agents. Such unique elliptical 8-ring pore openings can exclude larger-size propane and only adsorb propylene. Its C3H6/C3H8 separation performance is also confirmed by column breakthrough experiments, showing a high dynamic adsorption capacity of 53.36 cm3 g-1 for C3H6 but negligible C3H8 under ambient conditions. The dynamic capacity for C3H6 is superior to that of the well-known pure silica DDR-type zeolite (31.07 cm3 g-1). The density functional theory calculation demonstrates that the adsorbed propylene is distributed in the heart-shaped cavity and has a weak interaction with the OSs.

iDMer: an integrative and mechanism-driven response system for identifying compound interventions for sudden virus outbreak.

Emerging viral infections seriously threaten human health globally. Several challenges exist in identifying effective compounds against viral infections: (1) at the initial stage of a new virus outbreak, little information, except for its genome information, may be available; (2) although the identified compounds may be effective, they may be toxic in vivo and (3) cytokine release syndrome (CRS) triggered by viral infections is the primary cause of mortality. Currently, an integrative tool that takes all those aspects into consideration for identifying effective compounds to prevent viral infections is absent. In this study, we developed iDMer, as an integrative and mechanism-driven response system for addressing these challenges during the sudden virus outbreaks. iDMer comprises three mechanism-driven compound identification modules, that is, a virus-host interaction-oriented module, an autophagy-oriented module and a CRS-oriented module. As a one-stop integrative platform, iDMer incorporates compound toxicity evaluation and compound combination identification for virus treatment with clear mechanisms. iDMer was successfully tested on five viruses, including the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results indicated that, for all five tested viruses, compounds that were reported in the literature or experimentally validated for virus treatment were enriched at the top, demonstrating the generalized effectiveness of iDMer. Finally, we demonstrated that combinations of the individual modules successfully identified combinations of compounds effective for virus intervention with clear mechanisms.

Advances in Development of mRNA-Based Therapeutics.

Recently, mRNA-based therapeutics have been greatly boosted since the development of novel technologies of both mRNA synthesis and delivery system. Promising results were showed in both preclinical and clinical studies in the field of cancer vaccine, tumor immunotherapy, infectious disease prevention and protein replacement therapy. Recent advancements in clinical trials also encouraged scientists to attempt new applications of mRNA therapy such as gene editing and cell programming. These studies bring mRNA therapeutics closer to real-world application. Herein, we provide an overview of recent advances in mRNA-based therapeutics.

Recognizing the Minimum Structural Units Driving the Crystallization of SAPO-34 in a Top-Down Process.

Recognizing the structure and nature of the nuclei for zeolites crystallization on an atomic level is of great importance, which can provide guidance on the control of crystallization kinetics and the rational synthesis of zeolites. However, it remains a long-standing challenge due to the difficulty in characterization of amorphous precursor with limited crystal nuclei. Herein, a top-down synthesis system was designed for SAPO-34 molecular sieve and well investigated. A clear precursor solution with abundant SAPO-34 crystal nuclei was obtained under a depolymerization-dominant condition. The species in the liquid precursor were identified by FT-ICR MS, solid-state MAS NMR and atomic pair distribution function analyses. In combination with various designed experiments, it is revealed that both the formation of small species containing Si-O-Al bonds and reaching a certain concentration, is crucial for driving the crystallization of SAPO-34, rather than structural units with specific spatial conformation. This work provides an important understanding on the (pre)nucleation of SAPO-34 and sheds light on the synthesis control of SAPO molecular sieves.

Experiments on the Capillary Force of a Clam-Shell Droplet on Fibers by 3-D Model Reconstruction.

The capillary force is critical to the moving and breaking of droplets on fibers. This study brings forward a 3-D model reconstruction method for a clam-shell droplet on fibers and obtains the capillary force by the surface integral of Laplace pressure on the whole droplet. The capillary force results are verified by the droplet gravity and axial drag force, respectively. Moreover, the tensile tangential stresses are analyzed to illustrate the top limits of Laplace pressure against droplet breaking or sliding on the fiber. The experiment shows that the capillary force obtained by the 3-D model accurately describes the vertical and tangential forces of the clam-shell droplet on the fiber. Sharp shrinking of the cross-section on the droplet's upper part results in an exponential increase in tensile and tangential stresses, which makes the droplet break or move on the fiber.