A potent pan-sarbecovirus neutralizing antibody resilient to epitope diversification.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has resulted in viral escape from clinically authorized monoclonal antibodies (mAbs), creating a need for mAbs that are resilient to epitope diversification. Broadly neutralizing coronavirus mAbs that are sufficiently potent for clinical development and retain activity despite viral evolution remain elusive. We identified a human mAb, designated VIR-7229, which targets the viral receptor-binding motif (RBM) with unprecedented cross-reactivity to all sarbecovirus clades, including non-ACE2-utilizing bat sarbecoviruses, while potently neutralizing SARS-CoV-2 variants since 2019, including the recent EG.5, BA.2.86, and JN.1. VIR-7229 tolerates extraordinary epitope variability, partly attributed to its high binding affinity, receptor molecular mimicry, and interactions with RBM backbone atoms. Consequently, VIR-7229 features a high barrier for selection of escape mutants, which are rare and associated with reduced viral fitness, underscoring its potential to be resilient to future viral evolution. VIR-7229 is a strong candidate to become a next-generation medicine.

Chaperone-mediated autophagy modulates Snail protein stability: implications for breast cancer metastasis.

Breast cancer remains a significant health concern, with triple-negative breast cancer (TNBC) being an aggressive subtype with poor prognosis. Epithelial-mesenchymal transition (EMT) is important in early-stage tumor to invasive malignancy progression. Snail, a central EMT component, is tightly regulated and may be subjected to proteasomal degradation. We report a novel proteasomal independent pathway involving chaperone-mediated autophagy (CMA) in Snail degradation, mediated via its cytosolic interaction with HSC70 and lysosomal targeting, which prevented its accumulation in luminal-type breast cancer cells. Conversely, Snail predominantly localized to the nucleus, thus evading CMA-mediated degradation in TNBC cells. Starvation-induced CMA activation downregulated Snail in TNBC cells by promoting cytoplasmic translocation. Evasion of CMA-mediated Snail degradation induced EMT, and enhanced metastatic potential of luminal-type breast cancer cells. Our findings elucidate a previously unrecognized role of CMA in Snail regulation, highlight its significance in breast cancer, and provide a potential therapeutic target for clinical interventions.

Molecular basis of convergent evolution of ACE2 receptor utilization among HKU5 coronaviruses.

DPP4 was considered a canonical receptor for merbecoviruses until the recent discovery of African bat-borne MERS-related coronaviruses using ACE2. The extent and diversity with which merbecoviruses engage ACE2 and their receptor species tropism remain unknown. Here, we reveal that HKU5 enters host cells utilizing Pipistrellus abramus (P.abr) and several non-bat mammalian ACE2s through a binding mode distinct from that of any other known ACE2-using coronaviruses. These results show that several merbecovirus clades independently evolved ACE2 utilization, which appears to be a broadly shared property among these pathogens, through an extraordinary diversity of ACE2 recognition modes. We show that MERS-CoV and HKU5 have markedly distinct antigenicity, due to extensive genetic divergence, and identified several HKU5 inhibitors, including two clinical compounds. Our findings profoundly alter our understanding of coronavirus evolution and pave the way for developing countermeasures against viruses poised for human emergence.

A pan-variant miniprotein inhibitor protects against SARS-CoV-2 variants.

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement.

Human coronavirus HKU1 recognition of the TMPRSS2 host receptor.

The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. We designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2, providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among orthologous proteases. We identified TMPRSS2 orthologs from five mammalian orders promoting HKU1 S-mediated entry into cells along with key residues governing host receptor usage. Our data show that the TMPRSS2 binding motif is a site of vulnerability to neutralizing antibodies and suggest that HKU1 uses S conformational masking and glycan shielding to balance immune evasion and receptor engagement.

Toll-like receptor 13-mediated signaling protects against the development of colon cancer.

Appropriate host-microbiota interactions are essential for maintaining intestinal homeostasis; hence, an imbalance in these interactions leads to inflammation-associated intestinal diseases. Toll-like receptors (TLRs) recognize microbial ligands and play a key role in host-microbe interactions in health and disease. TLR13 has a well-established function in enhancing host defenses against pathogenic bacteria. However, its role in maintaining intestinal homeostasis and controlling colitis-associated colon cancer (CAC) is largely unknown. This study aimed to investigate the involvement of TLR13-mediated signaling in intestinal homeostasis and colonic tumorigenesis using ex vivo cell and in vivo CAC animal model. Tlr13-deficient mice were prone to dextran sodium sulfate (DSS)-induced colitis. During the early stages of the CAC regimen (AOM/DSS-treated), Tlr13 deficiency led to severe ulcerative colitis. Moreover, Tlr13-deficient mice exhibited increased intestinal permeability, as evidenced by elevated levels of fluorescein isothiocyanate (FITC)-dextran, endotoxins, and bacterial translocation. Enhanced cell survival and proliferation of colonic intestinal cells were observed in Tlr13-deficient mice. A transcriptome analysis revealed that Tlr13 deficiency is associated with substantial changes in gene expression profile of colonic tumor tissue. Tlr13-deficient mice were more susceptible to CAC, with increased production of interleukin (IL)-6, IL-12, and TNF-α cytokines and enhanced STAT3, NF-κB, and MAPK signaling in colon tissues. These findings suggest that TLR13 plays a protective role in maintaining intestinal homeostasis and controlling CAC. Our study provides a novel perspective on intestinal health via TLR13-mediated signaling, which is crucial for deciphering the role of host-microbiota interactions in health and disease.

A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines.

Evolution of SARS-CoV-2 alters the antigenicity of the immunodominant spike (S) receptor-binding domain and N-terminal domain, undermining the efficacy of vaccines and antibody therapies. To overcome this challenge, we set out to develop a vaccine focusing antibody responses on the highly conserved but metastable S2 subunit, which folds as a spring-loaded fusion machinery. We describe a strategy for prefusion-stabilization and high yield recombinant production of SARS-CoV-2 S2 trimers with native structure and antigenicity. We demonstrate that our design strategy is broadly generalizable to sarbecoviruses, as exemplified with the SARS-CoV-1 (clade 1a) and PRD-0038 (clade 3) S2 subunits. Immunization of mice with a prefusion-stabilized SARS-CoV-2 S2 trimer elicits broadly reactive sarbecovirus antibodies and neutralizing antibody titers of comparable magnitude against Wuhan-Hu-1 and the immune evasive XBB.1.5 variant. Vaccinated mice were protected from weight loss and disease upon challenge with XBB.1.5, providing proof-of-principle for fusion machinery sarbecovirus vaccines.

Glutathione Dynamics in the Tumor Microenvironment: A Potential Target of Cancer Stem Cells and T Cells.

Glutathione (GSH), the main cellular antioxidant, dynamically influences tumor growth, metastasis, and resistance to therapy in the tumor microenvironment (TME), which comprises cancer cells, immune cells, stromal cells, and non-cellular components, including the extracellular matrix, metabolites, hypoxia, and acidity. Cancer stem cells (CSCs) and T cells are minor but significant cell subsets of the TME. GSH dynamics influences the fate of CSCs and T cells. Here, we explored GSH dynamics in CSCs and T cells within the TME, as well as therapeutic approaches that could target these dynamics.

Streptomyces umbrella toxin particles block hyphal growth of competing species.

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

The effect of data transformation on low-dimensional integration of single-cell RNA-seq.

BACKGROUND: Recent developments in single-cell RNA sequencing have opened up a multitude of possibilities to study tissues at the level of cellular populations. However, the heterogeneity in single-cell sequencing data necessitates appropriate procedures to adjust for technological limitations and various sources of noise when integrating datasets from different studies. While many analysis procedures employ various preprocessing steps, they often overlook the importance of selecting and optimizing the employed data transformation methods. RESULTS: This work investigates data transformation approaches used in single-cell clustering analysis tools and their effects on batch integration analysis. In particular, we compare 16 transformations and their impact on the low-dimensional representations, aiming to reduce the batch effect and integrate multiple single-cell sequencing data. Our results show that data transformations strongly influence the results of single-cell clustering on low-dimensional data space, such as those generated by UMAP or PCA. Moreover, these changes in low-dimensional space significantly affect trajectory analysis using multiple datasets, as well. However, the performance of the data transformations greatly varies across datasets, and the optimal method was different for each dataset. Additionally, we explored how data transformation impacts the analysis of deep feature encodings using deep neural network-based models, including autoencoder-based models and proto-typical networks. Data transformation also strongly affects the outcome of deep neural network models. CONCLUSIONS: Our findings suggest that the batch effect and noise in integrative analysis are highly influenced by data transformation. Low-dimensional features can integrate different batches well when proper data transformation is applied. Furthermore, we found that the batch mixing score on low-dimensional space can guide the selection of the optimal data transformation. In conclusion, data preprocessing is one of the most crucial analysis steps and needs to be cautiously considered in the integrative analysis of multiple scRNA-seq datasets.

Broadly neutralizing antibodies against emerging delta-coronaviruses.

Porcine deltacoronavirus (PDCoV) spillovers were recently detected in children with acute undifferentiated febrile illness, underscoring recurrent zoonoses of divergent coronaviruses. To date, no vaccines or specific therapeutics are approved for use in humans against PDCoV. To prepare for possible future PDCoV epidemics, we isolated human spike (S)-directed monoclonal antibodies from transgenic mice and found that two of them, designated PD33 and PD41, broadly neutralized a panel of PDCoV variants. Cryo-electron microscopy structures of PD33 and PD41 in complex with the PDCoV receptor-binding domain and S ectodomain trimer provide a blueprint of the epitopes recognized by these mAbs, rationalizing their broad inhibitory activity. We show that both mAbs inhibit PDCoV by competitively interfering with host APN binding to the PDCoV receptor-binding loops, explaining the mechanism of viral neutralization. PD33 and PD41 are candidates for clinical advancement, which could be stockpiled to prepare for possible future PDCoV outbreaks.

Structure and design of Langya virus glycoprotein antigens.

Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse, and hamster target cells using a different, yet unknown, receptor than Nipah virus (NiV) and Hendra virus (HeV) and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryoelectron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing their conformational landscape and distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.

Fine particulate matter aggravates smoking induced lung injury via NLRP3/caspase-1 pathway in COPD.

BACKGROUND: Exposure to noxious particles, including cigarette smoke and fine particulate matter (PM2.5), is a risk factor for chronic obstructive pulmonary disease (COPD) and promotes inflammation and cell death in the lungs. We investigated the combined effects of cigarette smoking and PM2.5 exposure in patients with COPD, mice, and human bronchial epithelial cells. METHODS: The relationship between PM2.5 exposure and clinical parameters was investigated in patients with COPD based on smoking status. Alveolar destruction, inflammatory cell infiltration, and pro-inflammatory cytokines were monitored in the smoking-exposed emphysema mouse model. To investigate the mechanisms, cell viability and death and pyroptosis-related changes in BEAS-2B cells were assessed following the exposure to cigarette smoke extract (CSE) and PM2.5. RESULTS: High levels of ambient PM2.5 were more strongly associated with high Saint George's respiratory questionnaire specific for COPD (SGRQ-C) scores in currently smoking patients with COPD. Combined exposure to cigarette smoke and PM2.5 increased mean linear intercept and TUNEL-positive cells in lung tissue, which was associated with increased inflammatory cell infiltration and inflammatory cytokine release in mice. Exposure to a combination of CSE and PM2.5 reduced cell viability and upregulated NLRP3, caspase-1, IL-1ß, and IL-18 transcription in BEAS-2B cells. NLRP3 silencing with siRNA reduced pyroptosis and restored cell viability. CONCLUSIONS: PM2.5 aggravates smoking-induced airway inflammation and cell death via pyroptosis. Clinically, PM2.5 deteriorates quality of life and may worsen prognosis in currently smoking patients with COPD.

Development of pluripotent stem cell-derived epidermal organoids that generate effective extracellular vesicles in skin regeneration.

Cellular skin substitutes such as epidermal constructs have been developed for various applications, including wound healing and skin regeneration. These cellular models are mostly derived from primary cells such as keratinocytes and fibroblasts in a two-dimensional (2D) state, and further development of three-dimensional (3D) cultured organoids is needed to provide insight into the in vivo epidermal phenotype and physiology. Here, we report the development of epidermal organoids (EpiOs) generated from induced pluripotent stem cells (iPSCs) as a novel epidermal construct and its application as a source of secreted biomolecules recovered by extracellular vesicles (EVs) that can be utilized for cell-free therapy of regenerative medicine. Differentiated iPSC-derived epidermal organoids (iEpiOs) are easily cultured and expanded through multiple organoid passages, while retaining molecular and functional features similar to in vivo epidermis. These mature iEpiOs contain epidermal stem cell populations and retain the ability to further differentiate into other skin compartment lineages, such as hair follicle stem cells. By closely recapitulating the epidermal structure, iEpiOs are expected to provide a more relevant microenvironment to influence cellular processes and therapeutic response. Indeed, iEpiOs can generate high-performance EVs containing high levels of the angiogenic growth factor VEGF and miRNAs predicted to regulate cellular processes such as proliferation, migration, differentiation, and angiogenesis. These EVs contribute to target cell proliferation, migration, and angiogenesis, providing a promising therapeutic tool for in vivo wound healing. Overall, the newly developed iEpiOs strategy as an organoid-based approach provides a powerful model for studying basic and translational skin research and may also lead to future therapeutic applications using iEpiOs-secreted EVs.

The mechanism underlying correlation of particulate matter-induced ferroptosis with inflammasome activation and iron accumulation in macrophages.

Particulate matter (PM) is a global environmental hazard, which affects human health through free radical production, cell death induction, and immune responses. PM activates inflammasomes leading to excessive inflammatory responses and induces ferroptosis, a type of cell death. Despite ongoing research on the correlation among PM-induced ferroptosis, immune response, and inflammasomes, the underlying mechanism of this relationship has not been elucidated. In this study, we demonstrated the levels of PM-induced cell death and immune responses in murine macrophages, J774A.1 and RAW264.7, depending on the size and composition of particulate matter. PM2.5, with extraction ions, induced significant levels of cell death and immune responses; it induces lipid peroxidation, iron accumulation, and reactive oxygen species (ROS) production, which characterize ferroptosis. In addition, inflammasome-mediated cell death occurred owing to the excessive activation of inflammatory responses. PM-induced iron accumulation activates ferroptosis and inflammasome formation through ROS production; similar results were observed in vivo. These results suggest that the link between ferroptosis and inflammasome formation induced by PM, especially PM2.5 with extraction ions, is established through the iron-ROS axis. Moreover, this study can effectively facilitate the development of a new therapeutic strategy for PM-induced immune and respiratory diseases.

Species-agnostic transfer learning for cross-species transcriptomics data integration without gene orthology.

Novel hypotheses in biomedical research are often developed or validated in model organisms such as mice and zebrafish and thus play a crucial role. However, due to biological differences between species, translating these findings into human applications remains challenging. Moreover, commonly used orthologous gene information is often incomplete and entails a significant information loss during gene-id conversion. To address these issues, we present a novel methodology for species-agnostic transfer learning with heterogeneous domain adaptation. We extended the cross-domain structure-preserving projection toward out-of-sample prediction. Our approach not only allows knowledge integration and translation across various species without relying on gene orthology but also identifies similar GO among the most influential genes composing the latent space for integration. Subsequently, during the alignment of latent spaces, each composed of species-specific genes, it is possible to identify functional annotations of genes missing from public orthology databases. We evaluated our approach with four different single-cell sequencing datasets focusing on cell-type prediction and compared it against related machine-learning approaches. In summary, the developed model outperforms related methods working without prior knowledge when predicting unseen cell types based on other species' data. The results demonstrate that our novel approach allows knowledge transfer beyond species barriers without the dependency on known gene orthology but utilizing the entire gene sets.

Optimization of gearbox housing shape for agricultural UTV using structural-acoustic coupled analysis.

In this study, gearbox radiated noise was successfully reduced through housing shape optimization. First, dynamic and structural-acoustic coupled analysis models of an agricultural UTV gearbox were developed. Then, the test equipment was configured to match that of the simulation model, and a test was conducted. The analysis and test results showed errors within 0.1 dB for vibration and 0.2 dB(A) for noise, indicating that the models were reliable. The housing design was then optimized using topology optimization based on the developed structural-acoustic coupling analysis model. The sound pressure level around the housing was used as an objective function for topology optimization. The optimal distribution of materials for the housing was also derived to reduce the radiated noise. Lastly, the housing rib was designed based on the optimization result, and an improvement in the radiated noise by approximately 2.43 dB(A) was confirmed in the operation area.

IL-27-induced PD-L1highSca-1+ innate lymphoid cells suppress contact hypersensitivity in an IL-10-dependent manner.

Innate lymphoid cells (ILCs) play an important role in maintaining tissue homeostasis and various inflammatory responses. ILCs are typically classified into three subsets, as is the case for T-cells. Recent studies have reported that IL-10-producing type 2 ILCs (ILC210s) have an immunoregulatory function dependent on IL-10. However, the surface markers of ILC210s and the role of ILC210s in contact hypersensitivity (CHS) are largely unknown. Our study revealed that splenic ILC210s are extensively included in PD-L1highSca-1+ ILCs and that IL-27 amplifies the development of PD-L1highSca-1+ ILCs and ILC210s. Adoptive transfer of PD-L1highSca-1+ ILCs suppressed oxazolone-induced CHS in an IL-10-dependent manner Taken together, our results demonstrate that ILC210s are critical for the control of CHS and suggest that ILC210s can be used as target cells for the treatment of CHS.

AMPK Alchemy: Therapeutic Potentials in Allergy, Aging, and Cancer.

All cells are equipped with intricate signaling networks to meet the energy demands and respond to the nutrient availability in the body. AMP-activated protein kinase (AMPK) is among the most potent regulators of cellular energy balance. Under ATP -deprived conditions, AMPK phosphorylates substrates and affects various biological processes, such as lipid/glucose metabolism and protein synthesis. These actions further affect the cell growth, death, and functions, altering the cellular outcomes in energy-restricted environments. AMPK plays vital roles in maintaining good health. AMPK dysfunction is observed in various chronic diseases, making it a promising target for preventing and alleviating such diseases. Herein, we highlight the different AMPK functions, especially in allergy, aging, and cancer, to facilitate the development of new therapeutic approaches in the future.

Human coronavirus HKU1 recognition of the TMPRSS2 host receptor.

The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. Here, we designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2 providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among human type 2 transmembrane serine proteases. We found that human, rat, hamster and camel TMPRSS2 promote HKU1 S-mediated entry into cells and identified key residues governing host receptor usage. Our data show that serum antibodies targeting the HKU1 RBD TMPRSS2 binding-site are key for neutralization and that HKU1 uses conformational masking and glycan shielding to balance immune evasion and receptor engagement.