Morphologically defined substages of tail morphogenesis in C. elegans males.

BACKGROUND: Sex-specific morphogenesis occurs in Caenorhabditis elegans in the vulva of the hermaphrodite and in the male tail during the last larval stage. Temporal progression of vulva morphogenesis has been described in fine detail. However, a similar precise description of male tail morphogenesis was lacking. RESULTS: We here describe morphogenesis of the male tail at time points matching vulva development with special focus on morphogenesis of the tail tip. Using fluorescent reporters, we follow changes in cell shapes, cell fusions, nuclear migration, modifications in the basement membrane, and formation of a new apical extracellular matrix at the end of the tail. CONCLUSION: Our analysis answers two open questions about tail tip morphogenesis (TTM) by showing that one of the four tail tip cells, hyp11, remains largely separate, while the other cells fully fuse with each other and with two additional tail cells to form a ventral tail syncytium. This merger of cells begins at the apical surface early during TTM but is only completed toward the end of the process. This work provides a framework for future investigations of cell biological factors that drive male tail morphogenesis.

Human cytomegalovirus glycoprotein variants governing viral tropism and syncytium formation in epithelial cells and macrophages.

Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814-specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans.

Single-nucleus multi-omic profiling of polyploid heart nuclei identifies fusion-derived cardiomyocytes in the human heart.

Understanding the mechanisms of polyploidization in cardiomyocytes is crucial for advancing strategies to stimulate myocardial regeneration. Although endoreplication has long been considered the primary source of polyploid human cardiomyocytes, recent animal work suggests the potential for cardiomyocyte fusion. Moreover, the effects of polyploidization on the genomic-transcriptomic repertoire of human cardiomyocytes have not been studied previously. We applied single-nuclei whole genome sequencing, single nuclei RNA sequencing, and multiome ATAC + gene expression (from the same nuclei) techniques to nuclei isolated from 11 healthy hearts. Utilizing post-zygotic non-inherited somatic mutations occurring during development as "endogenous barcodes," to reconstruct lineage relationships of polyploid cardiomyocytes. Of 482 cardiomyocytes from multiple healthy donor hearts 75.7% can be sorted into several developmental clades marked by one or more somatic single-nucleotide variants (SNVs). At least ~10% of tetraploid cardiomyocytes contain cells from distinct clades, indicating fusion of lineally distinct cells, whereas 60% of higher-ploidy cardiomyocytes contain fused cells from distinct clades. Combined snRNA-seq and snATAC-seq revealed transcriptome and chromatin landscapes of polyploid cardiomyocytes distinct from diploid cardiomyocytes, and show some higher-ploidy cardiomyocytes with transcriptional signatures suggesting fusion between cardiomyocytes and endothelial and fibroblast cells. These observations provide the first evidence for cell and nuclear fusion of human cardiomyocytes, raising the possibility that cell fusion may contribute to developing or maintaining polyploid cardiomyocytes in the human heart.

How Much Do You Fuse? A Comparison of Cell Fusion Assays in a Breast Cancer Model.

Cell fusion is a biological process that is crucial for the development and homeostasis of different tissues, but it is also pathophysiologically associated with tumor progression and malignancy. The investigation of cell fusion processes is difficult because there is no standardized marker. Many studies therefore use different systems to observe and quantify cell fusion in vitro and in vivo. The comparability of the results must be critically questioned, because both the experimental procedure and the assays differ between studies. The comparability of the fluorescence-based fluorescence double reporter (FDR) and dual split protein (DSP) assay was investigated as part of this study, in which general conditions were kept largely constant. In order to be able to induce both a high and a low cell fusion rate, M13SV1 breast epithelial cells were modified with regard to the expression level of the fusogenic protein Syncytin-1 and its receptor ASCT2 and were co-cultivated for 72 h with different breast cancer cell lines. A high number of fused cells was found in co-cultures with Syncytin-1-overexpressing M13SV1 cells, but differences between the assays were also observed. This shows that the quantification of cell fusion events in particular is highly dependent on the assay selected, but the influence of fusogenic proteins can be visualized very well.

Comprehensive analytics for virus-cell and cell-cell multinucleation system.

Cell-fusion mediated generation of multinucleated syncytia represent critical feature during viral infection and in development. Efficiency of syncytia formation is usually illustrated as fusion efficiency under given condition by quantifying total number of nuclei in syncytia normalized to total number of nuclei (both within syncytia and unfused cell nuclei) in unit field of view. However heterogeneity in multinucleated syncytia sizes poses challenge in quantification of cell-fusion multinucleation under diverse conditions. Taking in-vitro SARS-CoV-2 spike-protein variants mediated virus-cell fusion model and placenta trophoblast syncytialization as cell-cell fusion model; herein we emphasize wide application of simple unbiased detailed measure of virus-cell and cell-cell multinucleation using experiential cumulative distribution function (CDF) and fusion number events (FNE) approaches illustrating comprehensive metrics for syncytia interpretation.

MARCH8 inhibits pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in the trans-Golgi network.

The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.

MARCH1 and MARCH2 inhibit pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in trans-Golgi network.

The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.

Urine-derived stem cells genetically modified with IGF1 improve muscle regeneration.

OBJECTIVE: In this study we aimed to determine the impact of human urine derived stem cells (USC) and genetically modified USC that were designed to overexpress myogenic growth factor IGF1 (USCIGF), on the regenerative capacity of cardiotoxin (CTX)-injured murine skeletal muscle. METHODS: We overexpressed IGF1 in USC and investigated the alterations in myogenic capacity and regenerative function in cardiotoxin-injured muscle tissues. RESULTS: Compared with USC alone, USCIGF1 activated the IGF1-Akt-mTOR signaling pathway, significantly improved myogenic differentiation capacity in vitro, and enhanced the secretion of myogenic growth factors and cytokines. In addition, IGF1 overexpression increased the ability of USC to fuse with skeletal myocytes to form myotubes, regulated the pro-regenerative immune response and inflammatory cytokines, and increased myogenesis in an in vivo model of skeletal muscle injury. CONCLUSION: Overall, USC genetically modified to overexpress IGF1 significantly enhanced skeletal muscle regeneration by regulating myogenic differentiation, paracrine effects, and cell fusion, as well as by modulating immune responses in injured skeletal muscles in vivo. This study provides a novel perspective for evaluating the myogenic function of USC as a nonmyogenic cell source in skeletal myogenesis. The combination of USC and IGF1 expression has the potential to provide a novel efficient therapy for skeletal muscle injury and associated muscular defects in patients with urinary incontinence.

Two mutations on S2 subunit were critical for Vero cell tropism expansion of infectious bronchitis virus HV80.

Infectious bronchitis virus (IBV) restricts cell tropism. Except for the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered IBV vaccine development and research on the mechanisms of IBV infection. A novel Vero cell-adapted strain, HV80, has been previously reported. In this study, we constructed recombinants expressing the chimeric S glycoprotein, S1 or S2 subunit of strain H120 and demonstrated that mutations on S2 subunit are associated with the strain HV80 Vero cell adaptation. R687P or P687R substitution recombinants were constructed with the genome backbone of strains HV80 or H120. We found that the RRRR690/S motif at the S2' cleavage site is crucial to the Vero cell adaptation of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that the Q855H mutation induced syncytium formation. A transient transfection assay demonstrated the S glycoprotein with the PRRR690/S motif at the S2' cleavage site induced low-level cell-cell fusion, while H855Q substitution hindered cell-cell fusion and blocked cleavage event with S20 product. This study provides a basis for the construction of IBV recombinants capable of replicating in Vero cells, thus contributing to the advancement in the development of genetically engineered cell-based IBV vaccines.

Downregulation of SPARC Expression Enhances the Fusion of BeWo Choriocarcinoma Cells.

Syncytiotrophoblasts, which are formed by the fusion of villous cytotrophoblasts, play an essential role in maintaining a successful pregnancy. Secreted protein acidic and rich in cysteine (SPARC) is a non-structural Ca2+-binding extracellular matrix glycoprotein involved in tissue remodeling and cell proliferation, differentiation, and migration. Previous studies have revealed that SPARC is expressed in villous and extravillous cytotrophoblasts in the first trimester and that RNA interference targeted at SPARC significantly inhibited invasion of human extravillous trophoblast HTR8/SVneo cells. However, the involvement of SPARC in cytotrophoblast fusion remains unknown. This study aimed to investigate the role of SPARC in cytotrophoblast fusion, using the BeWo choriocarcinoma cell line as a model of villous cytotrophoblasts. Immunohistochemical analysis was conducted to assess SPARC expression in normal human placentas using placental tissues obtained during the first and third trimesters of pregnancy. We investigated the effects of SPARC knockdown on trophoblast differentiation markers and cell fusion in BeWo cells using small interfering RNA. Immunohistochemical analysis revealed that SPARC expression was high in the early gestational chorionic villi and low in the late gestational chorionic villi. SPARC knockdown increased the expressions of human chorionic gonadotropin and Ovo-like transcriptional repressor 1; however, glial cells missing transcription factor 1, syncytin-1, and syncytin-2 showed no significant changes. The assessment revealed that SPARC knockdown significantly enhanced cell fusion compared to the non-silencing control. Our data suggest that SPARC plays a vital role in regulating trophoblast fusion and differentiation during placental development.

Cell type-specific adaptation of the SARS-CoV-2 spike.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can infect various human tissues and cell types, principally via interaction with its cognate receptor angiotensin-converting enzyme-2 (ACE2). However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After twenty passages of a spike-expressing recombinant vesicular stomatitis virus (VSV), cell-type-specific phenotypic changes were observed and sequencing allowed the identification of sixteen adaptive spike mutations. We used VSV pseudotyping to measure the entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, and entry pathway usage of all the mutants, alone or in combination. The fusogenicity of the mutant spikes was assessed with a cell-cell fusion assay. Finally, mutant recombinant VSVs were used to measure the fitness advantage associated with selected mutations. We found that the effects of these mutations varied across cell types, both in terms of viral entry and replicative fitness. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.

Distinct patterns of SARS-CoV-2 BA.2.87.1 and JN.1 variants in immune evasion, antigenicity, and cell-cell fusion.

The rapid evolution of SARS-CoV-2 variants presents a constant challenge to the global vaccination effort. In this study, we conducted a comprehensive investigation into two newly emerged variants, BA.2.87.1 and JN.1, focusing on their neutralization resistance, infectivity, antigenicity, cell-cell fusion, and spike processing. Neutralizing antibody (nAb) titers were assessed in diverse cohorts, including individuals who received a bivalent mRNA vaccine booster, patients infected during the BA.2.86/JN.1-wave, and hamsters vaccinated with XBB.1.5-monovalent vaccine. We found that BA.2.87.1 shows much less nAb escape from WT-BA.4/5 bivalent mRNA vaccination and JN.1-wave breakthrough infection sera compared to JN.1 and XBB.1.5. Interestingly, BA.2.87.1 is more resistant to neutralization by XBB.1.5-monovalent-vaccinated hamster sera than BA.2.86/JN.1 and XBB.1.5, but efficiently neutralized by a class III monoclonal antibody S309, which largely fails to neutralize BA.2.86/JN.1. Importantly, BA.2.87.1 exhibits higher levels of infectivity, cell-cell fusion activity, and furin cleavage efficiency than BA.2.86/JN.1. Antigenically, we found that BA.2.87.1 is closer to the ancestral BA.2 compared to other recently emerged Omicron subvariants including BA.2.86/JN.1 and XBB.1.5. Altogether, these results highlight immune escape properties as well as biology of new variants and underscore the importance of continuous surveillance and informed decision-making in the development of effective vaccines. IMPORTANCE: This study investigates the recently emerged SARS-CoV-2 variants, BA.2.87.1 and JN.1, in comparison to earlier variants and the parental D614G. Varied infectivity and cell-cell fusion activity among these variants suggest potential disparities in their ability to infect target cells and possibly pathogenesis. BA.2.87.1 exhibits lower nAb escape from bivalent mRNA vaccinee and BA.2.86/JN.1-infected sera than JN.1 but is relatively resistance to XBB.1.5-vaccinated hamster sera, revealing distinct properties in immune reason and underscoring the significance of continuing surveillance of variants and reformulation of vaccines. Antigenic differences between BA.2.87.1 and other earlier variants yield critical information not only for antibody evasion but also for viral evolution. In conclusion, this study furnishes timely insights into the spike biology and immune escape of the emerging variants BA.2.87.1 and JN.1, thus guiding effective vaccine development and informing public health interventions.

Distinct Patterns of SARS-CoV-2 BA.2.87.1 and JN.1 Variants in Immune Evasion, Antigenicity and Cell-Cell Fusion.

The rapid evolution of SARS-CoV-2 variants presents a constant challenge to the global vaccination effort. In this study, we conducted a comprehensive investigation into two newly emerged variants, BA.2.87.1 and JN.1, focusing on their neutralization resistance, infectivity, antigenicity, cell-cell fusion, and spike processing. Neutralizing antibody (nAb) titers were assessed in diverse cohorts, including individuals who received a bivalent mRNA vaccine booster, patients infected during the BA.2.86/JN.1-wave, and hamsters vaccinated with XBB.1.5-monovalent vaccine. We found that BA.2.87.1 shows much less nAb escape from WT-BA.4/5 bivalent mRNA vaccination and JN.1-wave breakthrough infection sera compared to JN.1 and XBB.1.5. Interestingly. BA.2.87.1 is more resistant to neutralization by XBB.15-monovalent-vaccinated hamster sera than BA.2.86/JN.1 and XBB.1.5, but efficiently neutralized by a class III monoclonal antibody S309, which largely fails to neutralize BA.2.86/JN.1. Importantly, BA.2.87.1 exhibits higher levels of infectivity, cell-cell fusion activity, and furin cleavage efficiency than BA.2.86/JN.1. Antigenically, we found that BA.2.87.1 is closer to the ancestral BA.2 compared to other recently emerged Omicron subvariants including BA.2.86/JN.1 and XBB.1.5. Altogether, these results highlight immune escape properties as well as biology of new variants and underscore the importance of continuous surveillance and informed decision-making in the development of effective vaccines.

Measurement of Myonuclear Accretion In Vitro and In Vivo.

Adult skeletal muscle stem cells (MuSC) are the regenerative precursors of myofibers and also have an important role in myofiber growth, adaptation, and maintenance by fusing to the myofibers-a process referred to as "myonuclear accretion." Due to a focus on MuSC function during regeneration, myofibers remain a largely overlooked component of the MuSC niche influencing MuSC fate. Here, we describe a method to directly measure the rate of myonuclear accretion in vitro and in vivo using ethynyl-2'-deoxyuridine (EdU)-based tracing of MuSC progeny. This method supports the dissection of MuSC intrinsic and myofiber-derived factors influencing myonuclear accretion as an alternative fate of MuSCs supporting myofiber homeostasis and plasticity.

Co-Expression of Niemann-Pick Type C1-Like1 (NPC1L1) with ACE2 Receptor Synergistically Enhances SARS-CoV-2 Entry and Fusion.

The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into human embryonic kidney (HEK293T) cells has been shown to be a cholesterol-rich, lipid raft-dependent process. In this study, we investigated if the presence of a cholesterol uptake receptor Niemann-pick type c1-like1 (NPC1L1) impacts SARS-CoV-2 cell entry. Initially, we utilized reporter-based pseudovirus cell entry assays and a spike (S) glycoprotein-mediated cell-to-cell fusion assay. Using Chinese hamster ovary (CHO-K1) cells, which lack endogenous receptors for SARS-CoV-2 entry, our data showed that the co-expression of NPC1L1 together with the ACE2 receptor synergistically increased SARS-CoV-2 pseudovirus entry even more than the cells expressing ACE-2 receptor alone. Similar results were also found with the HEK293T cells endogenously expressing the ACE2 receptor. Co-cultures of effector cells expressing S glycoprotein together with target cells co-expressing ACE-2 receptor with NPC1L1 significantly promoted quantitative cell-to-cell fusion, including syncytia formation. Finally, we substantiated that an elevated expression of NPC1L1 enhanced entry, whereas the depletion of NPC1L1 resulted in a diminished SARS-CoV-2 entry in HEK293T-ACE2 cells using authentic SARS-CoV-2 virus in contrast to their respective control cells. Collectively, these findings underscore the pivotal role of NPC1L1 in facilitating the cellular entry of SARS-CoV-2. Importance: Niemann-Pick type C1-like1 (NPC1L1) is an endosomal membrane protein that regulates intracellular cholesterol trafficking. This protein has been demonstrated to play a crucial role in the life cycle of several clinically important viruses. Although SARS-CoV-2 exploits cholesterol-rich lipid rafts as part of its viral entry process, the role of NPC1L1 in SARS-CoV-2 entry remains unclear. Our research represents the first-ever demonstration of NPC1L1's involvement in facilitating SARS-CoV-2 entry. The observed role of NPC1L1 in human kidney cells is not only highly intriguing but also quite relevant. This relevance stems from the fact that NPC1L1 exhibits high expression levels in several organs, including the kidneys, and the fact that kidney damages are reported during severe cases of SARS-CoV-2. These findings may help us understand the new functions and mechanisms of NPC1L1 and could contribute to the identification of new antiviral targets.

Spontaneous Fusion with Transformed Mesenchymal Stromal Cells Results in Complete Heterogeneity in Prostate Cancer Cells.

Tumor cells gain advantages in growth and survival by acquiring genotypic and phenotypic heterogeneity. Interactions with bystander cells in the tumor microenvironment contribute to the progression of heterogeneity. We have shown that fusion between tumor and bystander cells is one form of interaction, and that tumor-bystander cell fusion has contrasting effects. By trapping fusion hybrids in the heterokaryon or synkaryon state, tumor-bystander cell fusion prevents the progression of heterogeneity. However, if trapping fails, fusion hybrids will resume replication to form derivative clones with diverse genomic makeups and behavioral phenotypes. To determine the characteristics of bystander cells that influence the fate of fusion hybrids, we co-cultured prostate mesenchymal stromal cell lines and their spontaneously transformed sublines with LNCaP as well as HPE-15 prostate cancer cells. Subclones derived from cancer-stromal fusion hybrids were examined for genotypic and phenotypic diversifications. Both stromal cell lines were capable of fusing with cancer cells, but only fusion hybrids with the transformed stromal subline generated large numbers of derivative subclones. Each subclone had distinct cell morphologies and growth behaviors and was detected with complete genomic hybridization. The health conditions of the bystander cell compartment play a crucial role in the progression of tumor cell heterogeneity.

Cell fusion between tumor cells and macrophages promotes the metastasis of OSCC patient through the activation of the chemokine signaling pathway.

BACKGROUND: Tumor metastasis is responsible for the high mortality rate of patients with oral squamous cell carcinoma (OSCC). Although many hypotheses have been proposed to elucidate the mechanism of tumor metastasis, the origin of the metastatic tumor cells remains unclear. In this study, we explored the role of cell fusion in the formation of OSCC metastatic tumor cells. METHODS: Murine OSCC tumor cells and macrophages were fused in vitro, and the cell proliferation, migration, and phagocytosis abilities of hybrid cells and parental cells were compared. Subsequently, we compared the transcriptome differences between hybrid and parental cells. RESULTS: Murine OSCC tumor cells and macrophages were successfully fused in vitro. The cytological and molecular experimental results revealed that OSCC tumor cells obtained a migration-related phenotype after fusion with macrophages, and the migration ability of hybrid cells was related to the activation of the "chemokine signal pathway". CONCLUSION: After fusion with macrophages, the chemokine signaling pathway in OSCC tumor cells was activated, leading to metastasis.

Real-time analysis of osteoclast resorption and fusion dynamics in response to bone resorption inhibitors.

Cathepsin K (CatK), an essential collagenase in osteoclasts (OCs), is a potential therapeutic target for the treatment of osteoporosis. Using live-cell imaging, we monitored the bone resorptive behaviour of OCs during dose-dependent inhibition of CatK by an ectosteric (Tanshinone IIA sulfonate) and an active site inhibitor (odanacatib). CatK inhibition caused drastic reductions in the overall resorption speed of OCs. At IC50 CatK-inhibitor concentration, OCs reduced about 40% of their trench-forming capacity and at fourfold IC50 concentrations, a > 95% reduction was observed. The majority of CatK-inhibited OCs (~ 75%) were involved in resorption-migration-resorption episodes forming adjacent pits, while ~ 25% were stagnating OCs which remained associated with the same excavation. We also observed fusions of OCs during the resorption process both in control and inhibitor-treated conditions, which increased their resorption speeds by 30-50%. Inhibitor IC50-concentrations increased OC-fusion by twofold. Nevertheless, more fusion could not counterweigh the overall loss of resorption activity by inhibitors. Using an activity-based probe, we demonstrated the presence of active CatK at the resorbing front in pits and trenches. In conclusion, our data document how OCs respond to CatK-inhibition with respect to movement, bone resorption activity, and their attempt to compensate for inhibition by activating fusion.

Temperature impacts SARS-CoV-2 spike fusogenicity and evolution.

SARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33°C and 37°C, respectively). In addition, fever is a common COVID-19 symptom. SARS-CoV-2 has been shown to replicate more efficiently at low temperatures, but the effect of temperature on different viral proteins remains poorly understood. Here, we investigate how temperature affects the SARS-CoV-2 spike function and evolution. We first observed that increasing temperature from 33°C to 37°C or 39°C increased spike-mediated cell-cell fusion. We then experimentally evolved a recombinant vesicular stomatitis virus expressing the SARS-CoV-2 spike at these different temperatures. We found that spike-mediated cell-cell fusion was maintained during evolution at 39°C but was lost in a high proportion of viruses that evolved at 33°C or 37°C. Consistently, sequencing of the spikes evolved at 33°C or 37°C revealed the accumulation of mutations around the furin cleavage site, a region that determines cell-cell fusion, whereas this did not occur in spikes evolved at 39°C. Finally, using site-directed mutagenesis, we found that disruption of the furin cleavage site had a temperature-dependent effect on spike-induced cell-cell fusion and viral fitness. Our results suggest that variations in body temperature may affect the activity and diversification of the SARS-CoV-2 spike. IMPORTANCE: When it infects humans, SARS-CoV-2 is exposed to different temperatures (e.g., replication site and fever). Temperature has been shown to strongly impact SARS-CoV-2 replication, but how it affects the activity and evolution of the spike protein remains poorly understood. Here, we first show that high temperatures increase the SARS-CoV-2 spike fusogenicity. Then, we demonstrate that the evolution of the spike activity and variants depends on temperature. Finally, we show that the functional effect of specific spike mutations is temperature-dependent. Overall, our results suggest that temperature may be a factor influencing the activity and adaptation of the SARS-CoV-2 spike in vivo, which will help understanding viral tropism, pathogenesis, and evolution.

Production of Neutralizing Antibody.

Techniques employing monoclonal antibodies (mAbs) are widely used in the initial development phase of biologics. The usefulness of mAbs in basic RA research has been established based on their characteristics, including specificity of binding, homogeneity, and ability to be produced on a large scale. MAb immunoglobulins are the starting material for the generation of smaller antibody fragments and other engineered immunomodulatory antibodies. In this chapter, the basic hybridoma technique, which is a well-established and feasible method for the production of mAbs involving animal immunization, cell fusion, hybridoma screening, expanding positive hybridomas, and purification, is introduced. Aiming at specific affinity to a membrane protein, synthetic proteoliposomes are used in the immunization and screening steps.