QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Infection with wild-type SARS-CoV-2 elicits broadly neutralizing and protective antibodies against omicron subvariants.

The omicron variants of SARS-CoV-2 have substantial ability to escape infection- and vaccine-elicited antibody immunity. Here, we investigated the extent of such escape in nine convalescent patients infected with the wild-type SARS-CoV-2 during the first wave of the pandemic. Among the total of 476 monoclonal antibodies (mAbs) isolated from peripheral memory B cells, we identified seven mAbs with broad neutralizing activity to all variants tested, including various omicron subvariants. Biochemical and structural analysis indicated the majority of these mAbs bound to the receptor-binding domain, mimicked the receptor ACE2 and were able to accommodate or inadvertently improve recognition of omicron substitutions. Passive delivery of representative antibodies protected K18-hACE2 mice from infection with omicron and beta SARS-CoV-2. A deeper understanding of how the memory B cells that produce these antibodies could be selectively boosted or recalled can augment antibody immunity against SARS-CoV-2 variants.

Host-Viral Infection Maps Reveal Signatures of Severe COVID-19 Patients.

Viruses are a constant threat to global health as highlighted by the current COVID-19 pandemic. Currently, lack of data underlying how the human host interacts with viruses, including the SARS-CoV-2 virus, limits effective therapeutic intervention. We introduce Viral-Track, a computational method that globally scans unmapped single-cell RNA sequencing (scRNA-seq) data for the presence of viral RNA, enabling transcriptional cell sorting of infected versus bystander cells. We demonstrate the sensitivity and specificity of Viral-Track to systematically detect viruses from multiple models of infection, including hepatitis B virus, in an unsupervised manner. Applying Viral-Track to bronchoalveloar-lavage samples from severe and mild COVID-19 patients reveals a dramatic impact of the virus on the immune system of severe patients compared to mild cases. Viral-Track detects an unexpected co-infection of the human metapneumovirus, present mainly in monocytes perturbed in type-I interferon (IFN)-signaling. Viral-Track provides a robust technology for dissecting the mechanisms of viral-infection and pathology.

Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19.

Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14LowCD16High monocytes, accumulation of HLA-DRLow classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation.

Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from coronavirus disease 2019 (COVID-19)-infected individuals, followed by the Brazilian variant P.1 and the United Kingdom variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K, and N501Y mutations in the receptor-binding domain (RBD) of SARS-CoV-2. Crystal structure analysis of the B.1.351 triple mutant (417N-484K-501Y) RBD complexed with the monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptors for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which poses serious challenges to current antibody therapies and vaccine protection.

Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling.

Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2-9. Here we report photobiocatalytic asymmetric sp3-sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5'-phosphate-dependent enzymes, threonine aldolases10-12, for the α-C-H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13-15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox-pyridoxal biocatalysis provides a platform for sp3-sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.

The 3D Topography of Mitotic Chromosomes.

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.

Hypothetical chloroplast reading frame 51 encodes a photosystem I assembly factor in cyanobacteria.

Hypothetical chloroplast open reading frames (ycfs) are putative genes in the plastid genomes of photosynthetic eukaryotes. Many ycfs are also conserved in the genomes of cyanobacteria, the presumptive ancestors of present-day chloroplasts. The functions of many ycfs are still unknown. Here, we generated knock-out mutants for ycf51 (sll1702) in the cyanobacterium Synechocystis sp. PCC 6803. The mutants showed reduced photoautotrophic growth due to impaired electron transport between photosystem II (PSII) and PSI. This phenotype results from greatly reduced PSI content in the ycf51 mutant. The ycf51 disruption had little effect on the transcription of genes encoding photosynthetic complex components and the stabilization of the PSI complex. In vitro and in vivo analyses demonstrated that Ycf51 cooperates with PSI assembly factor Ycf3 to mediate PSI assembly. Furthermore, Ycf51 interacts with the PSI subunit PsaC. Together with its specific localization in the thylakoid membrane and the stromal exposure of its hydrophilic region, our data suggest that Ycf51 is involved in PSI complex assembly. Ycf51 is conserved in all sequenced cyanobacteria, including the earliest branching cyanobacteria of the Gloeobacter genus, and is also present in the plastid genomes of glaucophytes. However, Ycf51 has been lost from other photosynthetic eukaryotic lineages. Thus, Ycf51 is a PSI assembly factor that has been functionally replaced during the evolution of oxygenic photosynthetic eukaryotes.

Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling.

The origins and developmental mechanisms of coronary arteries are incompletely understood. We show here by fate mapping, clonal analysis, and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.

Dancr-BRG1 regulates Nfatc1 transcription and Pgc1ß-dependent metabolic shifts in osteoclastogenesis.

Long non-coding RNA (lncRNA) serves as a vital regulator of bone metabolism, but its role in pathologically overactive osteoclast differentiation remains elusive. Here, we identify lncRNA Dancr (Differentiation Antagonizing Non-protein Coding RNA) as a critical suppressor of osteoclastogenesis and bone resorption, which is down-regulated in response to estrogen deficiency. Global or osteoclast-specific Dancr Knockout mice display significant trabecular bone deterioration and enhanced osteoclast activity, but minimal alteration of bone formation. Moreover, the bone-targeted delivery of Dancr by Adeno-associated viral remarkably attenuates ovariectomy-induced osteopenia in mice. Mechanistically, Dancr establishes a direct interaction with Brahma-related gene 1 to prevent its binding and preserve H3K27me3 enrichment at the nuclear factor of activated T cells 1 and proliferator-activated receptor gamma coactivator 1-beta promoters, thereby maintaining appropriate expression of osteoclastic genes and metabolic programs during osteoclastogenesis. These results demonstrate that Dancr is a key molecule maintaining proper osteoclast differentiation and bone homeostasis under physiological conditions, and Dancr overexpression constitutes a potential strategy for treating osteoporosis.

Hypoxia inducible factor 2α promotes tolerogenic macrophage development during cardiac transplantation through transcriptional regulation of colony stimulating factor 1 receptor.

Solid organ transplantation mobilizes myeloid cells, including monocytes and macrophages, which are central protagonists of allograft rejection. However, myeloid cells can also be functionally reprogrammed by perioperative costimulatory blockade to promote a state of transplantation tolerance. Transplantation tolerance holds promise to reduce complications from chronic immunosuppression and promote long-term survival in transplant recipients. We sought to identify different mediators of transplantation tolerance by performing single-cell RNA sequencing of acute rejecting or tolerized cardiac allografts. This led to the unbiased identification of the transcription factor, hypoxia inducible factor (HIF)-2α, in a subset of tolerogenic monocytes. Using flow cytometric analyses and mice with conditional loss or gain of function, we uncovered that myeloid cell expression of HIF-2α was required for costimulatory blockade-induced transplantation tolerance. While HIF-2α was dispensable for mobilization of tolerogenic monocytes, which were sourced in part from the spleen, it promoted the expression of colony stimulating factor 1 receptor (CSF1R). CSF1R mediates monocyte differentiation into tolerogenic macrophages and was found to be a direct transcriptional target of HIF-2α in splenic monocytes. Administration of the HIF stabilizer, roxadustat, within micelles to target myeloid cells, increased HIF-2α in splenic monocytes, which was associated with increased CSF1R expression and enhanced cardiac allograft survival. These data support further exploration of HIF-2α activation in myeloid cells as a therapeutic strategy for transplantation tolerance.

Human neutralizing antibodies elicited by SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global health emergency that is in urgent need of intervention1-3. The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2)2,4-6. Here we report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells from 8 individuals infected with SARS-CoV-2. We identified antibodies that potently neutralize SARS-CoV-2; this activity correlates with competition with ACE2 for binding to RBD. Unexpectedly, the anti-SARS-CoV-2 antibodies and the infected plasma did not cross-react with the RBDs of SARS-CoV or Middle East respiratory syndrome-related coronavirus (MERS-CoV), although there was substantial plasma cross-reactivity to their trimeric spike proteins. Analysis of the crystal structure of RBD-bound antibody revealed that steric hindrance inhibits viral engagement with ACE2, thereby blocking viral entry. These findings suggest that anti-RBD antibodies are largely viral-species-specific inhibitors. The antibodies identified here may be candidates for development of clinical interventions against SARS-CoV-2.

GDPF: a data resource for the distribution of prokaryotic protein families across the global biosphere.

Microorganisms encode most of the functions of life on Earth. However, conventional research has primarily focused on specific environments such as humans, soil and oceans, leaving the distribution of functional families throughout the global biosphere poorly comprehended. Here, we present the database of the global distribution of prokaryotic protein families (GDPF, http://bioinfo.qd.sdu.edu.cn/GDPF/), a data resource on the distribution of functional families across the global biosphere. GDPF provides global distribution information for 36 334 protein families, 19 734 superfamilies and 12 089 KEGG (Kyoto Encyclopedia of Genes and Genomes) orthologs from multiple source databases, covering typical environments such as soil, oceans, animals, plants and sediments. Users can browse, search and download the distribution data of each entry in 10 000 global microbial communities, as well as conduct comparative analysis of distribution disparities among multiple entries across various environments. The GDPF data resource contributes to uncovering the geographical distribution patterns, key influencing factors and macroecological principles of microbial functions at a global level, thereby promoting research in Earth ecology and human health.

A point mutation in the meiotic crossover formation gene HEI10/TFS2 leads to thermosensitive genic sterility in rice.

Thermosensitive genic female sterility (TGFS) is a promising property to be utilized for hybrid breeding. Here, we identified a rice TGFS line, tfs2, through an ethyl methyl sulfone (EMS) mutagenesis strategy. This line showed sterility under high temperature and became fertile under low temperature. Few seeds were produced when the tfs2 stigma was pollinated, indicating that tfs2 is female sterile. Gene cloning and genetic complementation showed that a point mutation from leucine to phenylalanine in HEI10 (HEI10tfs2), a crossover formation protein, caused the TGFS trait of tfs2. Under high temperature, abnormal univalents were formed, and the chromosomes were unequally segregated during meiosis, similar to the reported meiotic defects in oshei10. Under low temperature, the number of univalents was largely reduced, and the chromosomes segregated equally, suggesting that crossover formation was restored in tfs2. Yeast two-hybrid assays showed that HEI10 interacted with two putative protein degradation-related proteins, RPT4 and SRFP1. Through transient expression in tobacco leaves, HEI10 were found to spontaneously aggregate into dot-like foci in the nucleus under high temperature, but HEI10tfs2 failed to aggregate. In contrast, low temperature promoted HEI10tfs2 aggregation. This result suggests that protein aggregation at the crossover position contributes to the fertility restoration of tfs2 under low temperature. In addition, RPT4 and SRFP1 also aggregated into dot-like foci, and these aggregations depend on the presence of HEI10. These findings reveal a novel mechanism of fertility restoration and facilitate further understanding of HEI10 in meiotic crossover formation.

RefTM: reference-guided topic modeling of single-cell chromatin accessibility data.

Single-cell analysis is a valuable approach for dissecting the cellular heterogeneity, and single-cell chromatin accessibility sequencing (scCAS) can profile the epigenetic landscapes for thousands of individual cells. It is challenging to analyze scCAS data, because of its high dimensionality and a higher degree of sparsity compared with scRNA-seq data. Topic modeling in single-cell data analysis can lead to robust identification of the cell types and it can provide insight into the regulatory mechanisms. Reference-guided approach may facilitate the analysis of scCAS data by utilizing the information in existing datasets. We present RefTM (Reference-guided Topic Modeling of single-cell chromatin accessibility data), which not only utilizes the information in existing bulk chromatin accessibility and annotated scCAS data, but also takes advantage of topic models for single-cell data analysis. RefTM simultaneously models: (1) the shared biological variation among reference data and the target scCAS data; (2) the unique biological variation in scCAS data; (3) other variations from known covariates in scCAS data.

Role and mechanism of PVN-sympathetic-adipose circuit in depression and insulin resistance induced by chronic stress.

Chronic stress induces depression and insulin resistance, between which there is a bidirectional relationship. However, the mechanisms underlying this comorbidity remain unclear. White adipose tissue (WAT), innervated by sympathetic nerves, serves as a central node in the interorgan crosstalk through adipokines. Abnormal secretion of adipokines is involved in mood disorders and metabolic morbidities. We describe here a brain-sympathetic nerve-adipose circuit originating in the hypothalamic paraventricular nucleus (PVN) with a role in depression and insulin resistance induced by chronic stress. PVN neurons are labelled after inoculation of pseudorabies virus (PRV) into WAT and are activated under restraint stress. Chemogenetic manipulations suggest a role for the PVN in depression and insulin resistance. Chronic stress increases the sympathetic innervation of WAT and downregulates several antidepressant and insulin-sensitizing adipokines, including leptin, adiponectin, Angptl4 and Sfrp5. Chronic activation of the PVN has similar effects. ß-adrenergic receptors translate sympathetic tone into an adipose response, inducing downregulation of those adipokines and depressive-like behaviours and insulin resistance. We finally show that AP-1 has a role in the regulation of adipokine expression under chronic stress.

PAT: a comprehensive database of prokaryotic antimicrobial toxins.

Antimicrobial toxins help prokaryotes win competitive advantages in intraspecific or interspecific conflicts and are also a critical factor affecting the pathogenicity of many pathogens that threaten human health. Although many studies have revealed that antagonism based on antimicrobial toxins plays a central role in prokaryotic life, a database on antimicrobial toxins remains lacking. Here, we present the prokaryotic antimicrobial toxin database (PAT, http://bioinfo.qd.sdu.edu.cn/PAT/), a comprehensive data resource collection on experimentally validated antimicrobial toxins. PAT has organized information, derived from the reported literature, on antimicrobial toxins, as well as the corresponding immunity proteins, delivery mechanisms, toxin activities, structural characteristics, sequences, etc. Moreover, we also predict potential antimicrobial toxins in prokaryotic reference genomes and show the taxonomic information and environmental distribution of typical antimicrobial toxins. These details have been fully incorporated into the PAT database, where users can browse, search, download, analyse and view informative statistics and detailed information. PAT resources have already been used in our prediction and identification of prokaryotic antimicrobial toxins and may contribute to promoting the efficient investigation of antimicrobial toxin functions, the discovery of novel antimicrobial toxins, and an improved understanding of the biological roles and significance of these toxins.

Sister chromatids separate during anaphase in a three-stage program as directed by interaxis bridges.

During mitosis, from late prophase onward, sister chromatids are connected along their entire lengths by axis-linking chromatin/structure bridges. During prometaphase/metaphase, these bridges ensure that sister chromatids retain a parallel, paranemic relationship, without helical coiling, as they undergo compaction. Bridges must then be removed during anaphase. Motivated by these findings, the present study has further investigated the process of anaphase sister separation. Morphological and functional analyses of mammalian mitoses reveal a three-stage pathway in which interaxis bridges play a prominent role. First, sister chromatid axes globally separate in parallel along their lengths, with concomitant bridge elongation, due to intersister chromatin pushing forces. Sister chromatids then peel apart progressively from a centromere to telomere region(s), step-by-step. During this stage, poleward spindle forces dramatically elongate centromere-proximal bridges, which are then removed by a topoisomerase IIα­dependent step. Finally, in telomere regions, widely separated chromatids remain invisibly linked, presumably by catenation, with final separation during anaphase B. During this stage increased separation of poles and/or chromatin compaction appear to be the driving force(s). Cohesin cleavage licenses these events, likely by allowing bridges to respond to imposed forces. We propose that bridges are not simply removed during anaphase but, in addition, play an active role in ensuring smooth and synchronous microtubule-mediated sister separation. Bridges would thereby be the topological gatekeepers of sister chromatid relationships throughout all stages of mitosis.

Hypoxic environment promotes angiogenesis and bone bridge formation by activating Notch/RBPJ signaling pathway in HUVECs.

After epiphyseal fracture, the epiphyseal plate is prone to ischemia and hypoxia, leading to the formation of bone bridge and deformity. However, the exact mechanism controlling the bone bridge formation remains unclear. Notch/RBPJ signaling axis has been indicated to regulate angiogenesis and osteogenic differentiation. Our study aims to investigate the mechanism of bone bridge formation after epiphyseal plate injury, and to provide a theoretical basis for new therapeutic approaches to prevent the bone bridge formation. The expression of DLL4 and RBPJ was significantly up-regulated in HUVECs after ischemia and hypoxia treatment. Notch/RBPJ pathway positively regulated the osteogenic differentiation of BMSCs. HUVECs can induce osteogenic differentiation of BMSCs under ischemia and hypoxia. Notch/RBPJ pathway is involved in the regulation of the trans-epiphyseal bridge formation. Notch/RBPJ in HUVECs is associated with osteogenic differentiation of BMSCs and may participate in the regulation of the bone bridge formation across the epiphyseal plate.