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Global dissemination of SARS-CoV-2 Omicron sublineages has provided a sufficient opportunity for natural selection, thus enabling beneficial mutations to emerge. Characterisation of these mutations uncovers the underlying machinery responsible for the fast transmission of Omicron variants and guides vaccine development for combating the COVID-19 pandemic. Through systematic bioinformatics analysis of 496,606 sequences of Omicron variants, we obtained 40 amino acid substitutions that occurred with high frequency in the S protein. Utilising pseudoviruses and a trans -complementation system of SARS-CoV-2, we identified the effect of high-frequency mutations on viral infectivity and elucidated the molecular mechanisms. Finally, we evaluated the impact of a key emerging mutation on the immune protection induced by the SARS-CoV-2 VLP mRNA vaccine in a murine model. We identified a proline-to-leucine substitution at the 1263rd residue of the Spike protein, and upon investigating the relative frequencies across multiple Omicron sublineages, we found a trend of increasing frequency for P1263L. The substitution significantly enhances the capacity for S-mediated viral entry and improves the immunogenicity of a virus-like particle mRNA vaccine. Mechanistic studies showed that this mutation is located in the FERM binding motif of the cytoplasmic tail and impairs the interaction between the S protein and the Ezrin/Radixin/Moesin proteins. Additionally, this mutation facilitates the incorporation of S proteins into SARS-CoV-2 virions. This study offers mechanistic insight into the constantly increasing transmissibility of SARS-CoV-2 Omicron variants and provides a meaningful optimisation strategy for vaccine development against SARS-CoV-2. This study was supported by grants from the National Key Research and Development Plan of China (2021YFC2302405, 2022YFC2303200, 2021YFC2300200 and 2022YFC2303400), the National Natural Science Foundation of China (32188101, 32200772, 82422049, 82241082, 32270182, 82372254, 82271872, 82341046, 32100755 and 82102389), Shenzhen Medical Research Fund (B2404002, A2303036), the Shenzhen Bay Laboratory Startup Fund (21330111), Shenzhen San-Ming Project for Prevention and Research on Vector-borne Diseases (SZSM202211023), Yunnan Provincial Science and Technology Project at Southwest United Graduate School (202302AO370010). The New Cornerstone Science Foundation through the New Cornerstone Investigator Program, and the Xplorer Prize from Tencent Foundation.

A comprehensive understanding of the cardio-spleen-bone marrow immune cell axis is essential for elucidating the alterations occurring during the pathogenesis of diabetes mellitus (DM). This study investigates the dynamics of immune cell kinetics in DM after myocardial infarction (MI) over time. MI was induced in diabetic and healthy control groups using C57BL/N6 mice, with sacrifices occurring at days 1, 3, 7, and 28 post-MI to collect heart, peripheral blood (PB), spleen, and bone marrow (BM) samples. Cell suspensions from each organ were isolated and analyzed via flow cytometry. Additionally, the endothelial progenitor cell-colony-forming assay (EPC-CFA) was performed using mononuclear cells derived from BM, PB, and the spleen. The results indicated that, despite normal production in BM and the spleen, CD45+ cells were lower in the PB of DM mice at days 1 to 3. Further analysis revealed a reduction in total and pro-inflammatory neutrophils (N1s) in PB at days 1 to 3 and in the spleen at days 3 to 7 in DM mice, suggesting that DM-induced alterations in splenic neutrophils fail to meet the demand in PB and ischemic tissues. Infiltrating macrophages (total, M1, M2) were reduced at day 3 in the DM-ischemic heart, with total and M1 (days 1–3) and M2 (days 3–7) macrophages being significantly decreased in DM-PB compared to controls, indicating impaired macrophage recruitment and polarization in DM. Myeloid dendritic cells (mDCs) in the heart were higher from days 1 to 7, which corresponded with the enhanced recruitment of CD8+ cells from days 1 to 28 in the DM-infarcted myocardium. Total CD4+ cells decreased in DM-PB at days 1 to 3, suggesting a delayed adaptive immune response to MI. B cells were reduced in PB at days 1 to 3, in myocardium at day 3, and in the spleen at day 7, indicating compromised mobilization from BM. EPC-CFA results showed a marked decrease in definitive EPC colonies in the spleen and BM from days 1 to 28 in DM mice compared to controls in vitro, highlighting that DM severely impairs EPC colony-forming activity by limiting the differentiation of EPCs from primitive to definitive forms. Taking together, this study underscores significant disruptions in the cardio-spleen-bone marrow immune cell axis following MI in DM, revealing delayed innate and adaptive immune responses along with impaired EPC differentiation.

NAD(P)-dependent steroid dehydrogenase-like protein (NSDHL), which is involved in breast tumor growth and metastasis, has been implicated in the maintenance of cancer stem cells. However, its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line, MCF-7. NSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2, P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids, tumor initiation and growth were strongly suppressed compared with those in the control. BCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls, along with decreased secretion of TGF-β1 and 3, phosphorylation of Smad2/3, and expression of SOX2. In RNA-sequencing data from The (TCGA) database, a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens ( n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids, suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs, thus preventing breast cancer initiation and progression. Our findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors. The online version contains supplementary material available at 10.1186/s12885-024-13143-3.

Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines, we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers, while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing.

The COVID-19 pandemic has created a global health crisis, with challenges arising from the ongoing evolution of the SARS-CoV-2 virus, the emergence of new strains, and the long-term effects of COVID-19. Aiming to overcome the development of viral resistance, our study here focused on developing broad-spectrum pan-coronavirus antiviral therapies by targeting host protein quality control mechanisms essential for viral replication. Screening an in-house compound library led to the discovery of three candidate compounds targeting cellular proteostasis. The three compounds are (1) the nucleotide analog cordycepin, (2) a benzothiozole analog, and (3) an acyldepsipeptide analog initially developed as part of a campaign to target the mitochondrial ClpP protease. These compounds demonstrated dose-dependent efficacy against multiple coronaviruses, including SARS-CoV-2, effectively inhibiting viral replication in vitro as well as in lung organoids. Notably, the compounds also showed efficacy against SARS-CoV-2 delta and omicron strains. As part of this work, we developed a BSL2-level cell-integrated SARS-CoV-2 replicon, which could serve as a valuable tool for high-throughput screening and studying intracellular viral replication. Our study should aid in the advancement of antiviral drug development efforts. Subject terms: High-throughput screening, Small molecules

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Induction of fetal hemoglobin (HbF) has been shown to be a viable therapeutic approach to treating sickle cell disease and potentially other β-hemoglobinopathies. To identify targets and target-modulating small molecules that enhance HbF expression, we engineered a human umbilical-derived erythroid progenitor reporter cell line (HUDEP2_HBG1_HiBiT) by genetically tagging a HiBiT peptide to the carboxyl (C)-terminus of the endogenous HBG1 gene locus, which codes for γ-globin protein, a component of HbF. Employing this reporter cell line, we performed a chemogenomic screen of approximately 5000 compounds annotated with known targets or mechanisms that have achieved clinical stage or approval by the US Food and Drug Administration (FDA). Among them, 10 compounds were confirmed for their ability to induce HbF in the HUDEP2 cell line. These include several known HbF inducers, such as pomalidomide, lenalidomide, decitabine, idoxuridine, and azacytidine, which validate the translational nature of this screening platform. We identified avadomide, autophinib, triciribine, and R574 as novel HbF inducers from these screens. We orthogonally confirmed HbF induction activities of the top hits in both parental HUDEP2 cells as well as in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs). Further, we demonstrated that pomalidomide and avadomide, but not idoxuridine, induced HbF expression through downregulation of several transcriptional repressors such as BCL11A, ZBTB7A, and IKZF1. These studies demonstrate a robust phenotypic screening workflow that can be applied to large-scale small molecule profiling campaigns for the discovery of targets and pathways, as well as novel therapeutics for sickle cell disease and other β-hemoglobinopathies.

The lack of a permissive cell culture system has limited high-resolution structures of parvovirus B19 (B19V) to virus-like particles (VLPs). In this study, we present the atomic resolution structure (2.2 Å) of authentic B19V purified from a patient blood sample. There are significant differences compared to non-infectious VLPs. Most strikingly, two host protease inhibitors (PIs), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and serpinA3, were identified in complex with the capsids in all patient samples tested. The ITIH4 binds specifically to the icosahedral fivefold axis and serpinA3 occupies the twofold axis. The protein-coated virions remain infectious, and the capsid-associated PIs retain activity; however, upon virion interaction with target cells, the PIs dissociate from the capsid prior to viral entry. Our finding of an infectious virion shielded by bound host serum proteins suggests an evolutionarily favored phenomenon to evade immune surveillance and escape host protease activity. Subject terms: Cryoelectron microscopy, Virology

The application of organoids has been limited by the lack of methods for producing uniformly mature organoids at scale. This study introduces an organoid culture platform, called UniMat, which addresses the challenges of uniformity and maturity simultaneously. UniMat is designed to not only ensure consistent organoid growth but also facilitate an unrestricted supply of soluble factors by a 3D geometrically-engineered, permeable membrane-based platform. Using UniMat, we demonstrate the scalable generation of kidney organoids with enhanced uniformity in both structure and function compared to conventional methods. Notably, kidney organoids within UniMat show improved maturation, showing increased expression of nephron transcripts, more in vivo-like cell-type balance, enhanced vascularization, and better long-term stability. Moreover, UniMat’s design offers a more standardized organoid model for disease modeling and drug testing, as demonstrated by polycystic-kidney disease and acute kidney injury modeling. In essence, UniMat presents a valuable platform for organoid technology, with potential applications in organ development, disease modeling, and drug screening. Subject terms: Nanofabrication and nanopatterning, Biomaterials, Stem-cell biotechnology

Cancer cell plasticity contributes significantly to the failure of chemo- and targeted therapies in triple-negative breast cancer (TNBC). Molecular mechanisms of therapy-induced tumor cell plasticity and associated resistance are largely unknown. Using a genome-wide CRISPR-Cas9 screen, we investigated escape mechanisms of NOTCH-driven TNBC treated with a gamma-secretase inhibitor (GSI) and identified SOX2 as a target of resistance to Notch inhibition. We describe a novel reciprocal inhibitory feedback mechanism between Notch signaling and SOX2. Specifically, Notch signaling inhibits SOX2 expression through its target genes of the HEY family, and SOX2 inhibits Notch signaling through direct interaction with RBPJ. This mechanism shapes divergent cell states with NOTCH positive TNBC being more epithelial-like, while SOX2 expression correlates with epithelial-mesenchymal transition, induces cancer stem cell features and GSI resistance. To counteract monotherapy-induced tumor relapse, we assessed GSI-paclitaxel and dasatinib-paclitaxel combination treatments in NOTCH inhibitor-sensitive and -resistant TNBC xenotransplants, respectively. These distinct preventive combinations and second-line treatment option dependent on NOTCH1 and SOX2 expression in TNBC are able to induce tumor growth control and reduce metastatic burden.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for many diverse blood and immune diseases. However, HSCT regimens currently commonly utilize genotoxic chemotherapy and/or total body irradiation (TBI) conditioning which causes significant morbidity and mortality through inducing broad tissue damage triggering infections, graft vs. host disease, infertility, and secondary cancers. We previously demonstrated that targeted monoclonal antibody (mAb)-based HSC depletion with anti(α)-CD117 mAbs could be an effective alternative conditioning approach for HSCT without toxicity in severe combined immunodeficiency (SCID) mouse models, which has prompted parallel clinical αCD117 mAbs to be developed and tested as conditioning agents in clinical trials starting with treatment of patients with SCID. Subsequent efforts have built upon this work to develop various combination approaches, though none are optimal and how any of these mAbs fully function is unknown. To improve efficacy of mAb-based conditioning as a stand-alone conditioning approach for all HSCT settings, it is critical to understand the mechanistic action of αCD117 mAbs on HSCs. Here, we compare the antagonistic properties of αCD117 mAb clones including ACK2, 2B8, and 3C11 as well as ACK2 fragments in vitro and in vivo in both SCID and wildtype (WT) mouse models. Further, to augment efficacy, combination regimens were also explored. We confirm that only ACK2 inhibits SCF binding fully and prevents HSC proliferation in vitro. Further, we verify that this corresponds to HSC depletion in vivo and donor engraftment post HSCT in SCID mice. We also show that SCF-blocking αCD117 mAb fragment derivatives retain similar HSC depletion capacity with enhanced engraftment post HSCT in SCID settings, but only full αCD117 mAb ACK2 in combination with αCD47 mAb enables enhanced donor HSC engraftment in WT settings, highlighting that the Fc region is not required for single-agent efficacy in SCID settings but is required in immunocompetent settings. This combination was the only non-genotoxic conditioning approach that enabled robust donor engraftment post HSCT in WT mice. These findings shed new insights into the mechanism of αCD117 mAb-mediated HSC depletion. Further, they highlight multiple approaches for efficacy in SCID settings and optimal combinations for WT settings. This work is likely to aid in the development of clinical non-genotoxic HSCT conditioning approaches that could benefit millions of people world-wide. The online version contains supplementary material available at 10.1186/s13287-024-03981-0.

To enable transmission of information in the brain, synaptic vesicles fuse to presynaptic membranes, liberating their content and exposing transiently a myriad of vesicular transmembrane proteins. However, versatile methods for quantifying the synaptic translocation of endogenous proteins during neuronal activity remain unavailable, as the fast dynamics of synaptic vesicle cycling difficult specific isolation of trafficking proteins during such a transient surface exposure. Here, we developed a novel approach using synaptic cleft proximity labeling to capture and quantify activity-driven trafficking of endogenous synaptic proteins at the synapse. We show that accelerating cleft biotinylation times to match the fast dynamics of vesicle exocytosis allows capturing endogenous proteins transiently exposed at the synaptic surface during neural activity, enabling for the first time the study of the translocation of nearly every endogenous synaptic protein. As proof-of-concept, we further applied this technology to obtain direct evidence of the surface translocation of noncanonical trafficking proteins, such as ATG9A and NPTX1, which had been proposed to traffic during activity but for which direct proof had not yet been shown. The technological advancement presented here will facilitate future studies dissecting the molecular identity of proteins exocytosed at the synapse during activity, helping to define the molecular machinery that sustains neurotransmission in the mammalian brain.