Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy.

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.

Structure and Degradation of Circular RNAs Regulate PKR Activation in Innate Immunity.

Circular RNAs (circRNAs) produced from back-splicing of exons of pre-mRNAs are widely expressed, but current understanding of their functions is limited. These RNAs are stable in general and are thought to have unique structural conformations distinct from their linear RNA cognates. Here, we show that endogenous circRNAs tend to form 16-26 bp imperfect RNA duplexes and act as inhibitors of double-stranded RNA (dsRNA)-activated protein kinase (PKR) related to innate immunity. Upon poly(I:C) stimulation or viral infection, circRNAs are globally degraded by RNase L, a process required for PKR activation in early cellular innate immune responses. Augmented PKR phosphorylation and circRNA reduction are found in peripheral blood mononuclear cells (PBMCs) derived from patients with autoimmune disease systemic lupus erythematosus (SLE). Importantly, overexpression of the dsRNA-containing circRNA in PBMCs or T cells derived from SLE can alleviate the aberrant PKR activation cascade, thus providing a connection between circRNAs and SLE.

Synergistic Coordination of Chromatin Torsional Mechanics and Topoisomerase Activity.

DNA replication in eukaryotes generates DNA supercoiling, which may intertwine (braid) daughter chromatin fibers to form precatenanes, posing topological challenges during chromosome segregation. The mechanisms that limit precatenane formation remain unclear. By making direct torque measurements, we demonstrate that the intrinsic mechanical properties of chromatin play a fundamental role in dictating precatenane formation and regulating chromatin topology. Whereas a single chromatin fiber is torsionally soft, a braided fiber is torsionally stiff, indicating that supercoiling on chromatin substrates is preferentially directed in front of the fork during replication. We further show that topoisomerase II relaxation displays a strong preference for a single chromatin fiber over a braided fiber. These results suggest a synergistic coordination-the mechanical properties of chromatin inherently suppress precatenane formation during replication elongation by driving DNA supercoiling ahead of the fork, where supercoiling is more efficiently removed by topoisomerase II. VIDEO ABSTRACT.

Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular ß sheet with the N-terminal ß strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to ß-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.

Epithelial STAT6 O-GlcNAcylation drives a concerted anti-helminth alarmin response dependent on tuft cell hyperplasia and Gasdermin C.

The epithelium is an integral component of mucosal barrier and host immunity. Following helminth infection, the intestinal epithelial cells secrete "alarmin" cytokines, such as interleukin-25 (IL-25) and IL-33, to initiate the type 2 immune responses for helminth expulsion and tolerance. However, it is unknown how helminth infection and the resulting cytokine milieu drive epithelial remodeling and orchestrate alarmin secretion. Here, we report that epithelial O-linked N-Acetylglucosamine (O-GlcNAc) protein modification was induced upon helminth infections. By modifying and activating the transcription factor STAT6, O-GlcNAc transferase promoted the transcription of lineage-defining Pou2f3 in tuft cell differentiation and IL-25 production. Meanwhile, STAT6 O-GlcNAcylation activated the expression of Gsdmc family genes. The membrane pore formed by GSDMC facilitated the unconventional secretion of IL-33. GSDMC-mediated IL-33 secretion was indispensable for effective anti-helminth immunity and contributed to induced intestinal inflammation. Protein O-GlcNAcylation can be harnessed for future treatment of type 2 inflammation-associated human diseases.

Host adaptation of a bacterial toxin from the human pathogen Salmonella Typhi.

Salmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here, we report that typhoid toxin binds to and is toxic toward cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid toxin. The atomic structure of typhoid toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi's host specificity and may help the development of therapies for typhoid fever.

Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.

Fibroblast growth factor 13 is a microtubule-stabilizing protein regulating neuronal polarization and migration.

Secretory fibroblast growth factors (FGFs) and their receptors are known for their regulatory function in the early stages of neural development. FGF13, a nonsecretory protein of the FGF family, is expressed in cerebral cortical neurons during development and is a candidate gene for syndromal and nonspecific forms of X-chromosome-linked mental retardation (XLMR). However, its function during development remains unclear. We show that FGF13 acts intracellularly as a microtubule-stabilizing protein required for axon and leading process development and neuronal migration in the cerebral cortex. FGF13 is enriched in axonal growth cones and interacts directly with microtubules. Furthermore, FGF13 polymerizes tubulins and stabilizes microtubules. The loss of FGF13 impairs neuronal polarization and increases the branching of axons and leading processes. Genetic deletion of FGF13 in mice results in neuronal migration defects in both the neocortex and the hippocampus. FGF13-deficient mice also exhibit weakened learning and memory, which is correlated to XLMR patients' intellectual disability.

Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.

Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.

Correlating local structure and migration dynamics in Na/Li dual ion conductor Na5YSi4O12.

Na5YSi4O12 (NYSO) is demonstrated as a promising electrolyte with high ionic conductivity and low activation energy for practical use in solid Na-ion batteries. Solid-state NMR was employed to identify the six types of coordination of Na+ ions and migration pathway, which is vital to master working mechanism and enhance performance. The assignment of each sodium site is clearly determined from high-quality 23Na NMR spectra by the aid of Density Functional Theory calculation. Well-resolved 23Na exchangespectroscopy and electrochemical tracer exchange spectra provide the first experimental evidence to show the existence of ionic exchange between sodium at Na5 and Na6 sites, revealing that Na transport route is possibly along three-dimensional chain of open channel-Na4-open channel. Variable-temperature NMR relaxometry is developed to evaluate Na jump rates and self-diffusion coefficient to probe the sodium-ion dynamics in NYSO. Furthermore, NYSO works well as a dual ion conductor in Na and Li metal batteries with Na3V2(PO4)3 and LiFePO4 as cathodes, respectively.

Targeting the PHF8/YY1 axis suppresses cancer cell growth through modulation of ROS.

High levels of mitochondrial reactive oxygen species (mROS) are linked to cancer development, which is tightly controlled by the electron transport chain (ETC). However, the epigenetic mechanisms governing ETC gene transcription to drive mROS production and cancer cell growth remain to be fully characterized. Here, we report that protein demethylase PHF8 is overexpressed in many types of cancers, including colon and lung cancer, and is negatively correlated with ETC gene expression. While it is well known to demethylate histones to activate transcription, PHF8 demethylates transcription factor YY1, functioning as a co-repressor for a large set of nuclear-coded ETC genes to drive mROS production and cancer development. In addition to genetically ablating PHF8, pharmacologically targeting PHF8 with a specific chemical inhibitor, iPHF8, is potent in regulating YY1 methylation, ETC gene transcription, mROS production, and cell growth in colon and lung cancer cells. iPHF8 exhibits potency and safety in suppressing tumor growth in cell-line- and patient-derived xenografts in vivo. Our data uncover a key epigenetic mechanism underlying ETC gene transcriptional regulation, demonstrating that targeting the PHF8/YY1 axis has great potential to treat cancers.

Salt stress-induced chloroplastic hydrogen peroxide stimulates pdTPI sulfenylation and methylglyoxal accumulation.

High salinity, an adverse environmental factor affecting about 20% of irrigated arable land worldwide, inhibits plant growth and development by causing oxidative stress, damaging cellular components, and disturbing global metabolism. However, whether and how reactive oxygen species disturb the metabolism of salt-stressed plants remain elusive. Here, we report that salt-induced hydrogen peroxide (H2O2) inhibits the activity of plastid triose phosphate isomerase (pdTPI) to promote methylglyoxal (MG) accumulation and stimulates the sulfenylation of pdTPI at cysteine 74. We also show that MG is a key factor limiting the plant growth, as a decrease in MG levels completely rescued the stunted growth and repressed salt stress tolerance of the pdtpi mutant. Furthermore, targeting CATALASE 2 into chloroplasts to prevent salt-induced overaccumulation of H2O2 conferred salt stress tolerance, revealing a role for chloroplastic H2O2 in salt-caused plant damage. In addition, we demonstrate that the H2O2-mediated accumulation of MG in turn induces H2O2 production, thus forming a regulatory loop that further inhibits the pdTPI activity in salt-stressed plants. Our findings, therefore, illustrate how salt stress induces MG production to inhibit the plant growth.

Revisiting Solar Energy Flow in Nanomaterial-Microorganism Hybrid Systems.

Nanomaterial-microorganism hybrid systems (NMHSs), integrating semiconductor nanomaterials with microorganisms, present a promising platform for broadband solar energy harvesting, high-efficiency carbon reduction, and sustainable chemical production. While studies underscore its potential in diverse solar-to-chemical energy conversions, prevailing NMHSs grapple with suboptimal energy conversion efficiency. Such limitations stem predominantly from an insufficient systematic exploration of the mechanisms dictating solar energy flow. This review provides a systematic overview of the notable advancements in this nascent field, with a particular focus on the discussion of three pivotal steps of energy flow: solar energy capture, cross-membrane energy transport, and energy conversion into chemicals. While key challenges faced in each stage are independently identified and discussed, viable solutions are correspondingly postulated. In view of the interplay of the three steps in affecting the overall efficiency of solar-to-chemical energy conversion, subsequent discussions thus take an integrative and systematic viewpoint to comprehend, analyze and improve the solar energy flow in the current NMHSs of different configurations, and highlighting the contemporary techniques that can be employed to investigate various aspects of energy flow within NMHSs. Finally, a concluding section summarizes opportunities for future research, providing a roadmap for the continued development and optimization of NMHSs.

Senp7 deficiency impairs lipid droplets maturation in white adipose tissues via Plin4 deSUMOylation.

Lipid metabolism is important for the maintenance of physiological homeostasis. Several members of the small ubiquitin-like modifier (SUMO)-specific protease (SENP) family have been reported as the regulators of lipid homeostasis. However, the function of Senp7 in lipid metabolism remains unclear. In this study, we generated both conventional and adipocyte-specific Senp7 KO mice to characterize the role of Senp7 in lipid metabolism homeostasis. Both Senp7-deficient mice displayed reduced white adipose tissue mass and decreased size of adipocytes. By analyzing the lipid droplet morphology, we demonstrated that the lipid droplet size was significantly smaller in Senp7-deficient adipocytes. Mechanistically, Senp7 could deSUMOylate the perilipin family protein Plin4 to promote the lipid droplet localization of Plin4. Our results reveal an important role of Senp7 in the maturation of lipid droplets via Plin4 deSUMOylation.

Biparental graph strategy to represent and analyze hybrid plant genomes.

Hybrid plants are found extensively in the wild, and they often demonstrate superior performance of complex traits over their parents and other selfing plants. This phenomenon, known as heterosis, has been extensively applied in plant breeding for decades. However, the process of decoding hybrid plant genomes has seriously lagged due to the challenges associated with genome assembly and the lack of appropriate methodologies for their subsequent representation and analysis. Here, we present the assembly and analysis of 2 hybrids, an intraspecific hybrid between 2 maize (Zea mays ssp. mays) inbred lines and an interspecific hybrid between maize and its wild relative teosinte (Z. mays ssp. parviglumis), utilizing a combination of PacBio High Fidelity sequencing and chromatin conformation capture sequencing data. The haplotypic assemblies are well phased at chromosomal scale, successfully resolving the complex loci with extensive parental structural variations (SVs). By integrating into a biparental genome graph, the haplotypic assemblies can facilitate downstream short-read-based SV calling and allele-specific gene expression analysis, demonstrating outstanding advantages over a single linear genome. Our work offers a comprehensive workflow that aims to facilitate the decoding of numerous hybrid plant genomes, particularly those with unknown or inaccessible parentage, thereby enhancing our understanding of genome evolution and heterosis.

The Complexity of Volatile Terpene Biosynthesis in Roses: Particular Insights into ß-Citronellol Production.

The fascinating scent of rose (Rosa genus) flowers has captivated human senses for centuries, making them one of the most popular and widely used floral fragrances. Despite much progress over the last decade, many biochemical pathways responsible for rose scents remain unclear. We analyzed the floral scent compositions from various rose varieties and selected the modern cultivar Rosa hybrida 'Double Delight' as a model system to unravel the formation of rose dominant volatile terpenes, which contribute substantially to the rose fragrance. Key genes involved in rose terpene biosynthesis were functionally characterized. Cytosolic geranyl diphosphate (GPP) generated by geranyl/farnesyl diphosphate synthase (G/FPPS1) catalysis, played a pivotal role in rose scent production, and terpene synthases (TPSs) in roses play an important role in the formation of most volatile terpenes, but not for geraniol, citral or ß-citronellol. Subsequently, a series of enzymes, including geraniol dehydrogenase (GeDH), geranial reductase (GER), 12-oxophytodienoate reductase (OPR) and citronellal reductase (CAR), were characterized as involved in the transformation of geraniol to ß-citronellol in roses through three successive steps. Interestingly, the ß-citronellol biosynthesis pathway appears to be conserved in other horticultural plants like Lagerstroemia caudata and Paeonia lactiflora. Our findings provide valuable insights into the biosynthesis of rose volatile terpenoid compounds and offer essential gene resources for future breeding and molecular modification efforts.

Pilocarpine mediated excessive calcium accumulation leads to ciliary muscle cell senescence and apoptosis.

The ciliary muscle constitutes a crucial element in refractive regulation. Investigating the pathophysiological mechanisms within the ciliary muscle during excessive contraction holds significance in treating ciliary muscle dysfunction. A guinea pig model of excessive contraction of the ciliary muscle induced by drops pilocarpine was employed, alongside the primary ciliary muscle cells was employed in in vitro experiments. The results of the ophthalmic examination showed that pilocarpine did not significantly change refraction and axial length during the experiment, but had adverse effects on the regulatory power of the ciliary muscle. The current data reveal notable alterations in the expression profiles of hypoxia inducible factor 1 (HIF-1α), ATP2A2, P53, α-SMA, Caspase-3, and BAX within the ciliary muscle of animals subjected to pilocarpine exposure, alongside corresponding changes observed in cultured cells treated with pilocarpine. Augmented levels of ROS were detected in both tissue specimens and cells, culminating in a significant increase in cell apoptosis in in vivo and in vitro experiments. Further examination revealed that pilocarpine induced an increase in intracellular Ca2+ levels and disrupted MMP, as evidenced by mitochondrial swelling and diminished cristae density compared to control conditions, concomitant with a noteworthy decline in antioxidant enzyme activity. However, subsequent blockade of Ca2+ channels in cells resulted in downregulation of HIF-1α, ATP2A2, P53, α-SMA, Caspase-3, and BAX expression, alongside ameliorated mitochondrial function and morphology. The inhibition of Ca2+ channels presents a viable approach to mitigate ciliary cells damage and sustain proper ciliary muscle function by curtailing the mitochondrial damage induced by excessive contractions.

Exosomal long non-coding RNA AU020206 alleviates macrophage pyroptosis in atherosclerosis by suppressing CEBPB-mediated NLRP3 transcription.

Recent studies have suggested exosomes (EXO) as potential therapeutic tools for cardiovascular diseases, including atherosclerosis (AS). This study investigates the function of bone marrow stem cell (BMSC)-derived exosomes (EXO) on macrophage pyroptosis in AS and explores the associated mechanism. BMSC-EXO were isolated from healthy mice and identified. RAW264.7 cells (mouse macrophages) were exposed to oxLDL to simulate an AS condition. BMSC-EXO treatment enhanced viability and reduced lactate dehydrogenase release of macrophages. An animal model of AS was established using ApoE-/- mice. BMSC-EXO treatment suppressed plaque formation as well as macrophage and lipid infiltration in mouse aortic tissues. Moreover, BMSC-EXO decreased concentrations of pyroptosis-related markers interleukin (IL)-1ß, IL-18, cleaved-caspase-1 and gasdermin D in vitro and in vivo. Long non-coding RNA AU020206 was carried by the BMSC-EXO, and it bound to CCAAT enhancer binding protein beta (CEBPB) to block CEBPB-mediated transcriptional activation of NLR family pyrin domain containing 3 (NLRP3). Functional assays revealed that silencing of AU020206 aggravated macrophage pyroptosis and exacerbated AS symptoms in mice. These exacerbations were blocked upon CEBPB silencing but then restored after NLRP3 overexpression. In conclusion, this study demonstrates that AU020206 delivered by BMSC-EXO alleviates macrophage pyroptosis in AS by blocking CEBPB-mediated transcriptional activation of NLRP3.

CK2 promotes jasmonic acid signaling response by phosphorylating MYC2 in Arabidopsis.

Jasmonic acid (JA) signaling plays a pivotal role in plant development and defense. MYC2 is a master transcription factor in JA signaling, and was found to be phosphorylated and negatively regulated by MAP kinase and receptor-like kinase. However, the kinases that positively regulate MYC2 through phosphorylation and promote MYC2-mediated activation of JA response have not been identified. Here, we identified CK2 as a kinase that phosphorylates MYC2 and thus regulates the JA signaling. CK2 holoenzyme can interact with MYC2 using its regulatory subunits and phosphorylate MYC2 at multiple sites with its catalytic subunits. Inhibition of CK2 activity in a dominant-negative plant line, CK2mut, repressed JA response. On the other hand, increasing CK2 activity by overexpression of CKB4, a regulatory subunit gene of CK2, enhanced JA response in a MYC2-dependent manner. Substitution of the Ser and Thr residues at phosphorylation sites of MYC2 by CK2 with Ala impaired MYC2 function in activating JA response. Further investigations evidenced that CK2 facilitated the JA-induced increase of MYC2 binding to the promoters of JA-responsive genes in vivo. Our study demonstrated that CK2 plays a positive role in JA signaling, and reveals a previously undiscovered mechanism that regulates MYC2 function.