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During human development, a temporary organ is formed, the placenta, which invades the uterine wall to support nutrient, oxygen, and waste exchange between the mother and fetus until birth. Most of the human placenta is formed by a syncytial villous structure lined by syncytialized trophoblasts, a specialized cell type that forms via cell-cell fusion of underlying progenitor cells. Genetic and functional studies have characterized the membrane protein fusogens Syncytin-1 and Syncytin-2, both of which are necessary and sufficient for human trophoblast cell-cell fusion. However, identification and characterization of upstream transcriptional regulators regulating their expression have been limited. Here, using CRISPR knockout in an in vitro cellular model of syncytiotrophoblast development (BeWo cells), we found that the transcription factor TFEB, mainly known as a regulator of autophagy and lysosomal biogenesis, is required for cell-cell fusion of syncytiotrophoblasts. TFEB translocates to the nucleus, exhibits increased chromatin interactions, and directly binds the Syncytin-1 and Syncytin-2 promoters to control their expression during differentiation. Although TFEB appears to play a critical role in syncytiotrophoblast differentiation, ablation of TFEB largely does not affect lysosomal gene expression or lysosomal biogenesis in differentiating BeWo cells, suggesting a previously uncharacterized role for TFEB in controlling the expression of human syncytins.

A colloidal gold immunochromatographic assay (ICA) based on a dual-antibody sandwich method was developed for the rapid and convenient detection of Orientia tsutsugamushi (O. tsutsugamushi) antigens in the early stages of infection. Monoclonal antibodies designed as 5B3 targeting the conserved region of 56 kDa outer membrane protein in various strains of O. tsutsugamushi were generated through cell fusion and screening techniques and combined with previously prepared polyclonal antibodies as detection antibodies to establish the ICA. Colloidal gold and polyclonal antibody-colloidal gold complexes were synthesized under optimized conditions. The nitrocellulose membrane was treated with 5B3 monoclonal antibody and goat anti-mouse antibody as the test and control lines, respectively. The ICA demonstrated robust sensitivity, with a minimum detection limit of 70.5 ng for the 56 kDa recombinant of the Gilliam strain. Furthermore, a detection limit of 1 × 106 copies/μL DNA of O. tsutsugamushi was determined for both PT and SJ infected cell strains by constructing a relationship between cell number and copy number of the pathogen using a quantitative PCR-based standard curve. The assay also exhibited exceptional specificity, with no false positives observed against other bacterial species, including Escherichia coli , Salmonella , Staphylococcus aureus , and Listeria monocytogenes. In summary, an ICA which is sensitive, specific, and easy to operate was successfully established for the detection of O. tsutsugamushi in scrub typhus, potentially enabling early rapid point-of-care diagnosis of scrub typhus. [ABSTRACT FROM AUTHOR]

Paramyxoviruses are significant human and animal pathogens that include mumps virus (MuV), Newcastle disease virus (NDV) and the murine parainfluenza virus Sendai (SeV). Despite their importance, few host factors implicated in paramyxovirus infection are known. Using a recombinant SeV expressing destabilized eGFP (rSeVCdseGFP) in a loss-of-function CRISPR screen, we identified the CMP-sialic acid transporter (CST) gene SLC35A1 and the UDP-galactose transporter (UGT) gene SLC35A2 as essential for paramyxovirus infection. As expected, SLC35A1 knockout (KO) cells showed drastic reduction in infections with SeV, NDV and MuV due to the lack of cell surface sialic acids receptors. However, SLC35A2 KO cells revealed unknown critical roles for this factor in virus-cell and cell-to-cell fusion events for the different paramyxoviruses. While UGT was essential for virus-cell fusion during SeV entry to the cell, it was not required for NDV or MuV entry. Importantly, UGT promoted the formation of syncytia during MuV infection, suggesting a role in cell-to-cell virus spread. Our findings demonstrate that paramyxoviruses can bind to or enter A549 cells in the absence of canonical galactose-bound sialic-acid decorations and show that UGT facilitates paramyxovirus fusion processes involved in entry and spread. Author summary: Paramyxovirus entry, the first step in establishing an infection, involves two key processes: attachment and virus-cell fusion. Paramyxoviruses use their attachment protein to bind to sialic acid-containing molecules on the cell surface, which serve as receptors, followed by fusion of the viral and cellular membranes mediated by the viral Fusion protein. Few host factors involved in the attachment and, particularly, the fusion process have been identified. Using CRISPR/Cas9 screening, we identified SLC35A2, a gene encoding the UDP-galactose transporter, as essential for Sendai virus-mediated virus-cell fusion, but not for Newcastle disease virus or mumps virus, which are from the same family. This is the first report of a gene specifically involved in the virus-cell fusion process on paramyxoviruses. Furthermore, we found that the UDP-galactose transporter promotes mumps virus-induced cell-cell fusion, suggesting a difference in the host galactoproteins involved in virus-cell versus cell-cell fusion. Our study provides new insights into the molecular mechanisms of fusion during viral infection. [ABSTRACT FROM AUTHOR]

Monitoring subcellular organelle dynamics in real time and precisely assessing membrane heterogeneity in living cells are very important for studying fundamental biological mechanisms and gaining a comprehensive understanding of cellular processes. However, there remains a shortage of effective tools for these purposes. Herein, we propose a strategy to develop the exchangeable water-sensing probeAPBD for time-lapse imaging of dynamics in cellular membrane-bound organelle morphology with structured illumination microscopy at the nanoscale. In this work, our results reveal mitochondria as the first organelle to undergo morphological changes through swelling, fission, and fusion in cell necrosis, leading to the rupture of the endoplasmic reticulum (ER) sheet adhered to the mitochondria. Meanwhile, the ER tubules are then reconstructed by stretching and fusion of autophagosomes. Moreover, APBD allows us to directly visualize spatially resolved distribution of biomembranes vs. water inside single mammalian cells. Our findings show that the renal ischemia-reperfusion injury (IRI) model results in the increased biomembrane to cytoplasmic water ratio in the tissue. This reveals intracellular water heterogeneity between the nucleus and the cytoplasm during the IRI process. Overall, this study presents a strategy for development of the molecular tools for cellular water heterogeneity and organelle dynamics. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests that fusion of cancer cells with leucocytes, such as macrophages, plays a significant role in cancer metastasis and results in tumor hybrid cells that acquire resistance to chemo- and radiation therapy. However, the precise mechanisms behind the leukocyte-cancer cell fusion remain unclear. The present in vitro study explores the presence of fusion between the monocyte cell line (THP-1) and the breast cancer cell line (MCF-7) in relation to the expression of CD36 and phosphatidylserine with and without treatment of these cells with ionizing radiation. The study reveals that spontaneous THP-1/MCF-7 cell fusion increases significantly from 2.8% to 6% after irradiation. The interaction between CD36 and phosphatidylserine plays a pivotal role in THP-1/MCF-7 cell fusion, as inhibiting this interaction using anti-CD36 antibodies significantly reduces cell fusion. While irradiation leads to a dose-dependent escalation in phosphatidylserine expression in MCF-7 cells, it does not impact the expression of CD36 in either THP-1 or MCF-7 cells. To the best of our knowledge, this is the first study to demonstrate the involvement of the CD36-phosphatidylserine interaction in the fusion between monocytes and cancer cells, shedding light on a novel explanatory mechanism for the roles of CD36 and phosphatidylserine in tumor progression. [ABSTRACT FROM AUTHOR]

In mammalian reproduction, testis-specific protein IZUMO1 and its receptor JUNO on the oocyte surface are essential for sperm-oocyte recognition, binding, and membrane fusion. However, these factors alone are insufficient to accomplish cytoplasmic membrane fusion. It is believed that other gametic proteins interact with them to facilitate sperm-oocyte interaction on the head and mid-tail of rat spermatozoa as well as on the surface of oocytes. In this study, Copper Transport Protein 2 (CTR2) has been identified on the head and mid-tail of rat spermatozoa as well as on the surface of oocytes. CTR2 directly interacts with both IZUMO1 and JUNO, colocalizing with IZUMO1 on the sperm head and with JUNO on the oocyte membrane. Treatment of the capacitated sperm and zona pellucida-free oocytes with anti -CTR2 antibody resulted in a significant decrease in fertilization rates in IVF experiments. These findings suggest that CTR2 plays an important role in mammalian fertilization by interacting with IZUMO1 and JUNO, providing new insights into the molecular mechanisms of mammalian sperm-oocyte adhesion and fusion. [ABSTRACT FROM AUTHOR]

Combined removal of Crb1 and Crb2 from the developing optic vesicle evokes cellular and laminar disorganization by disrupting the apical cell-cell adhesion in developing retinal epithelium. As a result, at postnatal stages, affected mouse retinas show temporarily thickened, coarsely laminated retinas in addition to functional deficits, including a severely abnormal electroretinogram and decreased visual acuity. These features are reminiscent of Leber congenital amaurosis 8, which is caused in humans by subsets of Crb1 mutations. However, the cellular basis of the abnormalities in retinal progenitor cells (RPCs) that lead to retinal disorganization is largely unknown. In this study, we analyze specific features of RPCs in mutant retinas, including maintenance of the progenitor pool, cell cycle progression, cell cycle phase-dependent nuclear positioning, cell survival, and generation of mature retinal cell types. We find crucial defects in the mutant RPCs. Upon removal of CRB1 and CRB2, apical structures of the RPCs, determined by markers of cilia and centrosomes, are basally shifted. In addition, the positioning of the somata of the M-phase cells, normally localized at the apical surface of the retinal epithelium, is basally shifted in a nearly randomized pattern along the apico-basal axis. Consequently, we propose that positioning of RPCs is desynchronized from cell cycle phase and largely randomized during embryonic development at E17.5. Because the resultant postmitotic cells inevitably lose positional information, the outer and inner nuclear layers (ONL and INL) fail to form from ONBL during neonatal development and retinal cells become mixed locally and globally. Additional results of the lost tissue polarity in Crb1/Crb2 dKO retinas include atypical formation of heterotopic cell patches containing photoreceptor cells in the ganglion cell layer and acellular patches filled with neural processes. Collectively, these changes lead to a mouse model of LCA8-like pathology. LCA8-like pathology differs substantially from the well-characterized, broad range of degeneration phenotypes that arise during the differentiation of photoreceptor and Muller glial cells in retinitis pigmentosa 12, a closely related disease caused by mutated human Crb1. Importantly, the present results suggest that Crb1/Crb2 serve indispensable functions in maintaining cell-cycle phase-dependent positioning of RPCs along the apico-basal axis, regulating cell cycle progression, and maintaining structural laminar integrity without significantly affecting the size of the RPC pools, generation of the subsets of the retinal cell types, or the distribution of cell cycle phases during RPC division. Taken together, these findings provide the crucial cellular basis of the thickening and severely disorganized lamination that are the unique features of the retinal abnormalities in LCA8 patients. [Display omitted] • Crb1/2 mutations induce defective apical adhesion and shrink retinal progenitors. • Aberrant adhesion alters cell cycle phase-dependent positioning and migration. • Mispositioning of retinal cells induces fusion and thickening of retinal layers. • Retinal progenitors lacking CRB1/2 undergo delayed cell cycle progression. • Loss of CRB1/2 in retinal progenitors underlies key features of human LCA8. [ABSTRACT FROM AUTHOR]

The ingestion of food contaminated with citrinin (CIT) poses a variety of health risks to humans and animals. The immunogens (CIT-COOH-BSA, CIT-H-BSA) and detection antigen (CIT-COOH-OVA, CIT-H-OVA) were synthesised using the active ester method (-COOH) and formaldehyde addition method (-H). A hybridoma cell line (3G5) that secretes anti-CIT monoclonal antibodies (mAbs) was screened via CIT-H-BSA immunisation of mice, cell fusion, and ELISA screening technology. The cell line was injected intraperitoneally to prepare ascites. The reaction conditions for the indirect competitive ELISA (ic-ELISA) were optimised, and an ic-ELISA method for detecting CIT was preliminarily established. The results revealed that the IC50 of CIT from optimised ic-ELISA was 37 pg/mL, the linear detection range was 5.9~230 pg/mL, and the cross-reaction (CR) rate with other analogues was less than 0.01%. The intra-assay and interassay sample recovery rates of CIT were 84.7~92.0% and 83.6~91.6%, and the coefficients of variation (CVs) were less than 10%. The ic-ELISA of CIT established in this study was not significantly different from the HPLC results and is rapid, highly sensitive and strongly specific, providing technical support for the detection of CIT. [ABSTRACT FROM AUTHOR]

Parainfluenza virus 3 (PIV3) infection poses a substantial risk to vulnerable groups including infants, the elderly, and immunocompromised individuals, and lacks effective treatments or vaccines. This study focuses on targeting the hemagglutinin-neuraminidase (HN) protein, a structural glycoprotein of PIV3 critical for viral infection and egress. With the objective of targeting these activities of HN, we identified eight neutralizing human monoclonal antibodies (mAbs) with potent effects on viral neutralization, cell-cell fusion inhibition, and complement deposition. Three epitopes on PIV3 HN were delineated and one epitope, Site 2, elicits a mAb with cross-neutralizing ability against PIV1 and PIV3. Cryo-EM revealed the cross-neutralizing mAb utilizes a long CDR3 loop to bind inside the pocket of the sialic acid binding site. Additionally, we resolved the structure of a non-protective mAb binding to Site 1 near the HN:F-interaction site. The potent Site 2-directed mAb demonstrated clinical efficacy in hamsters, reducing viral replication prophylactically and therapeutically. These findings advance our understanding of PIV3 immunity and underscore the significance of targeting HN for clinical therapeutic development against PIV3. Effective treatments and vaccines are lacking for parainfluenza virus 3 (PIV3). Here the authors isolate human monoclonal antibodies (mAb) targeting the hemagglutinin-neuraminidase protein of PIV3 and, using cryo-EM, describe the epitope of a cross-neutralizing and protective mAb. [ABSTRACT FROM AUTHOR]

The role of myeloid cells in the pathogenesis of SARS-CoV-2 is well established, in particular as drivers of cytokine production and systemic inflammation characteristic of severe COVID-19. However, the potential for myeloid cells to act as bona fide targets of productive SARS-CoV-2 infection, and the specifics of entry, remain unclear. Using a panel of anti-SARS-CoV-2 monoclonal antibodies (mAbs) we performed a detailed assessment of antibody-mediated infection of monocytes/macrophages. mAbs with the most consistent potential to mediate infection were those targeting a conserved region of the receptor binding domain (RBD; group 1/class 4). Infection was closely related to the neutralising concentration of the mAbs, with peak infection occurring below the IC50, while pre-treating cells with remdesivir or FcγRI-blocking antibodies inhibited infection. Studies performed in primary macrophages demonstrated high-level and productive infection, with infected macrophages appearing multinucleated and syncytial. Infection was not seen in the absence of antibody with the same quantity of virus. Addition of ruxolitinib significantly increased infection, indicating restraint of infection through innate immune mechanisms rather than entry. High-level production of pro-inflammatory cytokines directly correlated with macrophage infection levels. We hypothesise that infection via antibody-FcR interactions could contribute to pathogenesis in primary infection, systemic virus spread or persistent infection. Macrophages drive inflammation associated with severe COVID-19 but it is less clear whether they can be infected. Here, the authors show efficient antibody-mediated infection of primary macrophages by SARS-CoV-2, leading to cell fusion, de novo virus production and a potent cytokine response. [ABSTRACT FROM AUTHOR]

The giant striated muscle protein titin integrates into the developing sarcomere to form a stable myofilament system that is extended as myocytes fuse. The logistics underlying myofilament assembly and disassembly have started to emerge with the possibility to follow labeled sarcomere components. Here, we generated the mCherry knock-in at titin's Z-disk to study skeletal muscle development and remodeling. We find titin's integration into the sarcomere tightly regulated and its unexpected mobility facilitating a homogeneous distribution of titin after cell fusion - an integral part of syncytium formation and maturation of skeletal muscle. In adult mCherry-titin mice, treatment of muscle injury by implantation of titin-eGFP myoblasts reveals how myocytes integrate, fuse, and contribute to the continuous myofilament system across cell boundaries. Unlike in immature primary cells, titin proteins are retained at the proximal nucleus and do not diffuse across the whole syncytium with implications for future cell-based therapies of skeletal muscle disease. [ABSTRACT FROM AUTHOR]

Introduction: The ongoing emergence of SARS-CoV-2 variants poses significant challenges to existing therapeutics. The spike (S) glycoprotein is central to both viral entry and cell-to-cell transmission via syncytia formation, a process that confers resistance to neutralizing antibodies. The mechanisms underlying this resistance, particularly in relation to spike-mediated fusion, remain poorly understood. Methods: We analyzed two clinical SARS-CoV-2 isolates differing by a single amino acid substitution in the S protein. Using biochemical and cell-based assays, we evaluated entry kinetics, syncytia formation, and the neutralizing efficacy of convalescent sera. These parameters were further correlated with S-mediated cell-cell fusion activity. Results: The single amino acid substitution significantly altered entry kinetics and enhanced syncytia formation. This modification did not diminished the neutralizing capacity of convalescent sera, but it increased the efficiency of S-induced cell-cell fusion. These findings highlight the mutation's impact on viral transmissibility and immune evasion. Discussion: Our study demonstrates that even minor changes in the S protein can profoundly influence SARS-CoV-2 transmissibility and resistance to antibody-mediated neutralization. Understanding the molecular basis of S-mediated cell-cell fusion is crucial for anticipating the impact of emerging variants and developing next-generation therapeutic strategies. These insights provide a framework for predicting variant fitness and optimizing treatment approaches against future SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

The document from Lab on a Chip, published by The Royal Society of Chemistry, features various papers on micro- and nanoscale devices and applications. Topics covered include a self-actuated microfluidic chiplet for nucleic acid amplification, utilization of microdroplets for surface-enhanced Raman spectroscopy, nanofluidic energy harvesting, real-time myocardial infarction diagnosis, and more. The journal showcases cutting-edge research in the field of microfluidics and nanotechnology, providing valuable insights for researchers and scientists in the chemistry community. [Extracted from the article]

Somatic cell nuclear transfer (SCNT), referred to as somatic cell cloning, is a pivotal biotechnological technique utilized across various applications. Although robotic SCNT is currently available, the subsequent oocyte electrical activation/reconstructed embryo electrofusion is still manually completed by skilled operators, presenting challenges in efficient manipulation due to the uncontrollable positioning of the reconstructed embryo. This study introduces a robotic SCNT-electrofusion system to enable high-precision batch SCNT cloning. The proposed system integrates optical tweezers and microfluidic technologies. An optical tweezer is employed to facilitate somatic cells in precisely reaching the fusion site, and a specific polydimethylsiloxane (PDMS) chip is designed to assist in positioning and pairing oocytes and somatic cells. Enhancement in the electric field distribution between two parallel electrodes by PDMS pillars significantly reduces the required external voltage for electrofusion/electrical activation. We employed porcine oocytes and porcine fetal fibroblasts for SCNT experiments. The experimental results show that 90.56% of oocytes successfully paired with somatic cells to form reconstructed embryos, 76.43% of the reconstructed embryos successfully fused, and 70.55% of these embryos underwent cleavage. It demonstrates that the present system achieves the robotic implementation of oocyte electrical activation/reconstructed embryo electrofusion. By leveraging the advantages of batch operations using microfluidics, it proposes an innovative robotic cloning procedure that scales embryo cloning. [ABSTRACT FROM AUTHOR]

Host-cell entry of the highly pathogenic rabies virus (RABV) is mediated by glycoprotein (G) spikes, which also comprise the primary target for the humoral immune response. RABV glycoprotein (RABV-G) displays several antigenic sites that are targeted by neutralizing monoclonal antibodies (mAbs). In this study, we determined the epitope of a potently neutralizing human mAb, CR57, which we engineered into a diabody format to facilitate crystallization. We report the crystal structure of the CR57 diabody alone at 2.38 Å resolution, and in complex with RABV-G domain III at 2.70 Å resolution. The CR57−RABV-G structure reveals critical interactions at the antigen interface, which target the conserved "KLCGVL" peptide and residues proximal to it on RABV-G. Structural analysis combined with a cell-cell fusion assay demonstrates that CR57 effectively inhibits RABV-G-mediated fusion by obstructing the fusogenic transitions of the spike protein. Altogether, this investigation provides a structural perspective on RABV inhibition by a potently neutralizing human antibody. [Display omitted] • Anti-rabies human mAb CR57 was engineered into a diabody format • Crystal structures of the diabody CR57 and diabody-RABV-G domain III complex were determined • Key interactions at the interface target the RABV-G peptide sequence "KLCGVL" • CR57 binding inhibits RABV-G-mediated fusion Kedari et al. report the crystal structures of a diabody based on a neutralizing anti-rabies mAb CR57 alone and in complex with antigenic domain III from the rabies virus glycoprotein spike. Structural and functional analyses show that CR57 sterically hinders spike-mediated fusion, which is crucial for host-cell entry. [ABSTRACT FROM AUTHOR]

Cell-based therapies hold great potential for cancer immunotherapy. This approach is based on manipulation of dendritic cells to activate immune system against specific cancer antigens. For the development of an effective cell vaccine platform, gene transfer, and cell fusion have been used for modification of dendritic or tumor cells to express immune (co)stimulatory signals and to load dendritic cells with tumor antigens. Both, gene transfer and cell fusion can be achieved by single technique, a cell membrane electroporation. The cell membrane exposed to external electric field becomes temporarily permeable, enabling introduction of genetic material, and also fusogenic, enabling the fusion of cells in the close contact. We tested the feasability of combining gene electrotransfer and electrofusion into a single-step technique and evaluated the effects of electroporation buffer, pulse parameters, and cell membrane fluidity for single or combined method of gene delivery or cell fusdion. We determined the percentage of fused cells expressing green fluorescence protein (GFP) in a murine cell model of melanoma B16F1, cell line used in our previous studies. Our results suggest that gene electrotransfer and cell electrofusion can be applied in a single step. The percentage of viable hybrid cells expressing GFP depends on electric pulse parameters and the composition of the electroporation buffer. Furthermore, our results suggest that cell membrane fluidity is not related to the efficiency of the gene electrotransfer and electrofusion. The protocol is compatible with microfluidic devices, however further optimization of electric pulse parameters and buffers is still needed. [ABSTRACT FROM AUTHOR]

Congenital hypothyroidism (CH) is a major health issue that can lead to intellectual disability if not detected and treated earlier. The preliminary screening program for neonatal CH in Indonesia gave a provisional incidence of 1:2513. Newborn screening using a dried blood spot sample is the standard method for CH detection, but it has limitations. Despite the proven benefits of CH screening, Indonesia still faces significant challenges in implementing a nationwide program. This study aimed to develop a more sensitive and accessible screening method by creating monoclonal antibodies (mAbs) against the thyroid-stimulating hormone (TSH).TSH protein was isolated from newborn cord blood and confirmed by Western blot analysis. Mice were immunized with purified TSH, and hybridoma cell lines were generated through cell fusion. Hybridoma supernatants were screened for TSH-specific antibodies using ELISA. The mAb with the highest titer was purified by dialysis. Western blot analysis confirmed the presence of TSH in the isolated protein fraction at 28 kDa. Immunized mice showed a significant increase in antibody titer compared with the control group. Hybridoma clones secreting high-titer antibodies against TSH were identified. This research successfully isolated TSH and produced mAbs against it. They enable the development of rapid, point-of-care diagnostic tests, such as lateral flow immunoassays, which can provide results within minutes. It will lay the groundwork for the development of innovative CH screening tools that can significantly improve the early diagnosis and treatment of this condition, particularly in resource-limited settings. [ABSTRACT FROM AUTHOR]

The respiratory syncytial virus (RSV) fusion protein (F) facilitates virus-cell membrane fusion, which is critical for viral entry, and cell-cell fusion. In contrast to many type I fusion proteins, RSV F must be proteolytically cleaved at two distinct sites to be fusogenic. Cleavage at both sites results in the release of a 27 amino-acid fragment, termed Pep27. We examined proteolytic processing and the role of Pep27 for RSV F from both RSV A2 and RSV B9320 laboratory-adapted strains, allowing important comparisons between A and B clade F proteins. F from both clades was cleaved at both sites, and pulse-chase analysis indicated that cleavage at both sites occurs early after synthesis, most likely within the secretory pathway. Mutation of either site to alter the furin recognition motif blocked cell-cell fusion activity. To assess the role of Pep27 in F processing and expression, we deleted the Pep27 fragment, but preserved the cleavage sites. Deletion of Pep27 reduced F surface expression and cell-cell fusion. Two conserved N-linked glycosylation sites within Pep 27 are present in both the RSV A2 and RSV B9320 F. Randomization of the Pep27 sequence, while conserving the two N-liked glycosylation sites, did not significantly change surface expression, and only modestly reduced cell-cell fusion. However, the disruption of either Pep27 glycosylation site reduced cell-cell fusion. This work clarifies the timing of RSV F proteolytic cleavage and offers insight into the crucial role the N-linked glycosylation sites within Pep27 play in the biological function of F. [ABSTRACT FROM AUTHOR]

Human coronavirus (CoV) HKU1 infection typically causes common cold but can lead to pneumonia in children, older people, and immunosuppressed individuals. Recently, human transmembrane serine protease 2 (hTMPRSS2) was identified as the functional receptor for HKU1, but its region and residues critical for HKU1 S binding remain elusive. In this study, we find that HKU1 could utilize human and hamster, but not rat, mouse, or bat TMPRSS2 for virus entry, displaying a narrow host range. Using human-bat TMPRSS2 chimeras, we show that the serine peptidase (SP) domain of TMPRSS2 is essential for entry of HKU1. Further extensive mutagenesis analyses of the C-terminal regions of SP domains of human and bat TMPRSS2s identify residues 417 and 469 critical for entry of HKU1. Replacement of either D417 or Y469 with asparagine in hTMPRSS2 abolishes its abilities to mediate entry of HKU1 S pseudovirions and cell-cell fusion, whereas substitution of N417 with D or N469 with Y in bat TMPRSS2 (bTMPRSS2) renders it supporting HKU1 entry. Our findings contribute to a deeper understanding of coronavirus-receptor interactions and cross-species transmission. [ABSTRACT FROM AUTHOR]

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. Key Findings: We characterize in fine detail the morpogenesis of the male tail in C. elegans and match its progression to that of the vulva in hermaphrodites.Using fluorescent reporters for cellular components, we follow cell shape changes, modifications of the basement membrane, cell fusion, secretion of an apical extracellular matrix and nuclear migration.This work forms the basis for future investigations of the process of tail tip development as a model for post‐embryonic sexual dimorphic morphogenesis. [ABSTRACT FROM AUTHOR]

The present study investigates the species boundaries, evolutionary relationships, and host-parasite interfaces of dimorphic mycoparasites that were previously assigned to Achroomyces soranus, Occultifur internus, and Platygloea mycophila based on morphological similarities. Our comparison of recently collected and cultivated samples with the type specimens of A. soranus and P. mycophila shows that both groups are species complexes, of which the taxa can be differentiated based on morphological and ecological characters. By integrating the results of a seven-locus dataset (SSU, LSU, ITS, RPB1, RPB2, TEF1, and mitochondrial CYT-B) and detailed micromorphological comparisons of the investigated specimens, we show for the first time that these three groups of mycoparasites belong to Cystobasidiomycetes (Pucciniomycotina). We applied a polyphasic species concept involving morphology, phylogeny, and ecology to delineate and circumscribe these and new genera. The genus Occultifur comprises six species. Occultifur internus and the newly proposed O. cerinomycicola are intrahymenial mycoparasites producing haustorial cells and establishing fusion pore interaction with their Dacrymycetous host. Based on microscopical examination, we show that Achroomyces soranus is a member of the genus Occultifur. Based on the molecular phylogenetic reconstruction, we found that three lichen-associated fungi which are only known from a yeast morph are nested within Occultifur, i.e. Lichenozyma pisutiana, Microsporomyces cladoniae, and M. wangii. The genus Obvidator is newly introduced for three mycoparasitic species inhabiting members of the corticioid genus Peniophora (Russulales, Agaricomycetes) and causing gall-like malformations of the host basidiome. Microscopic investigation shows that Platygloea mycophila is a member of this genus. Obvidator species display a yet undiscovered type of host-parasite interface, in which the mycoparasites produce short protrusions on their hyphae adjacent to the host hyphae. The lysis of the host cell wall takes place at points of contact with parasite protrusions, but no rupture of the host plasma membrane or nanometer-fusion pore formation was observed. The updated Cystobasidiomycetes phylogeny obtained in this study by including mycoparasites showed that the genera Occultifur and Microsporomyces as currently circumscribed are polyphyletic. To resolve this polyphyly, we introduce two new genera, i.e. Cystastrum and Millanizyma, and recombine species comprising the Occultifur externus clade and a clade consisting of Microsporomyces bloemfonteinensis and M. cladoniophilus, respectively. [ABSTRACT FROM AUTHOR]

The placenta plays a critical role in nutrient and oxygen exchange during pregnancy, yet the effects of medicinal drugs on this selective barrier remain poorly understood. To overcome this, this study presents a cost-effective bioimpedance spectroscopy (BIS) system to assess tight junction integrity and monolayer formation in BeWo b30 cells, a widely used model of the multinucleated maternal–fetal exchange surface of the placental barrier. Cells were cultured on collagen-coated porous membranes and treated with forskolin to induce controlled syncytialization. Electrical impedance was measured using an entry level impedance analyzer, while immunofluorescence staining was used to confirm monolayer formation and syncytialization. The measurements and staining confirmed the formation of a confluent monolayer on day 4. In fact, the electrical resistance tripled for treated samples indicating a more electrically restrictive barrier. This resistance remained constant for treated samples reflecting the intact barrier's integrity over the next 3 days. The measurements show that, on day 4, the electrical capacitance of the cells decreased for the treated samples as opposed to the untreated samples. This reflects that the surface area of the BeWo b30 cells decreased when the samples were treated with forskolin. Finally, a COMSOL model was developed to explore the effects of electrode positioning, depth, and distance on TEER measurements, explaining discrepancies in the literature. In fact, there was a substantial 97% and 39.4% difference in the obtained TEER values. This study demonstrates the AD2 device's feasibility for monitoring placental barrier integrity and emphasizes the need for standardized setups for comparable results. The system can hence be used to analyze drug effects and nutrient transfer across the placental barrier. [ABSTRACT FROM AUTHOR]

Müller glia, as prominent glial cells within the retina, plays a significant role in maintaining retinal homeostasis in both healthy and diseased states. In lower vertebrates like zebrafish, these cells assume responsibility for spontaneous retinal regeneration, wherein endogenous Müller glia undergo proliferation, transform into Müller glia-derived progenitor cells, and subsequently regenerate the entire retina with restored functionality. Conversely, Müller glia in the mouse and human retina exhibit limited neural reprogramming. Müller glia reprogramming is thus a promising strategy for treating neurodegenerative ocular disorders. Müller glia reprogramming in mice has been accomplished with remarkable success, through various technologies. Advancements in molecular, genetic, epigenetic, morphological, and physiological evaluations have made it easier to document and investigate the Müller glia programming process in mice. Nevertheless, there remain issues that hinder improving reprogramming efficiency and maturity. Thus, understanding the reprogramming mechanism is crucial toward exploring factors that will improve Müller glia reprogramming efficiency, and for developing novel Müller glia reprogramming strategies. This review describes recent progress in relatively successful Müller glia reprogramming strategies. It also provides a basis for developing new Müller glia reprogramming strategies in mice, including epigenetic remodeling, metabolic modulation, immune regulation, chemical small-molecules regulation, extracellular matrix remodeling, and cell-cell fusion, to achieve Müller glia reprogramming in mice.

Abstract Cell–cell fusion is a vital biological process where the membranes of two or more cells merge to form a syncytium. This phenomenon is critical in various physiological and pathological contexts, including embryonic development, tissue repair, immune responses, and the progression of several diseases. Osteoclasts, which are cells from the monocyte/macrophage lineage responsible for bone resorption, have enhanced functionality due to cell fusion. Additionally, other multinucleated giant cells (MGCs) also arise from the fusion of monocytes and macrophages, typically during chronic inflammation and reactions to foreign materials such as prostheses or medical devices. Foreign body giant cells (FBGCs) and Langhans giant cells (LGCs) emerge only under pathological conditions and are involved in phagocytosis, antigen presentation, and the secretion of inflammatory mediators. This review provides a comprehensive overview of the mechanisms underlying the formation of multinucleated cells, with a particular emphasis on macrophages and osteoclasts. Elucidating the intracellular structures, signaling cascades, and fusion-mediating proteins involved in cell–cell fusion enhances our understanding of this fundamental biological process and helps identify potential therapeutic targets for disorders mediated by cell fusion.

Ting-ting Li,1 Jin-hua Yang,1 Meng-jie Jiang,1 Hao-peng Cui,1 Xiao-mei Yang,2 Xiao-ling Lu,2 Ai-qun Liu1 1Department of Gastroenterology and Respiratory Medicine & Endoscopy Center, Guangxi Medical University Cancer Hospital, Nanning, Guangxi, 530021, People’s Republic of China; 2Guangxi Key Laboratory of Nanobody Research, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of ChinaCorrespondence: Ai-qun Liu; Xiao-ling Lu, Email Liuaiqun_2004@163.com; luxiaoling@gxmu.edu.cnBackground: Our previously described a nanobody precisely targeting CTLA-4 and demonstrated that it can promote the antitumor response of adoptive T cells. Here we examined whether fusing it to the IgG1 Fc region would induce stronger, longer-lasting T-cell immune responses after exposure to the dendritic-tumor cell fusions.Methods: The fusion of nanobody to Fc region was overexpressed in E. coli. Next, the proliferation, activation and cytotoxicity of the CD8+ T cells stimulated by the fusion protein and dendritic-tumor cell fusions was assessed in vitro, and the antitumor activity was evaluated in nude mice bearing xenografts of each type of solid tumor.Results: Proliferation, activation and cytotoxicity of CD8+ T cells in vitro were significantly greater in the presence of the fusion protein than those in other groups. Consistently, different types of xenografts growth were significantly slower and animal survival significantly longer when the injected CD8+ T cells had been activated in vitro in the presence of the fusion protein.Conclusion: Fusing our anti-CTLA-4 nanobody to the IgG1 Fc region potentiates its ability to induce strong, persistent CD8+ T cell responses against solid tumors in mice. This fusion strategy has the potential to realize clinical transformation and application to clinical treatment.Keywords: nanobody, CTLA-4, therapeutic antibody derivative, cell fusion, adoptive immunotherapy

Abstract Background The syncytiotrophoblast (SCT) layer in the placenta serves as a crucial physical barrier separating maternal-fetal circulation, facilitating essential signal and substance exchange between the mother and fetus. Any abnormalities in its formation or function can result in various maternal syndromes, such as preeclampsia. The transition of proliferative villous cytotrophoblasts (VCT) from the mitotic cell cycle to the G0 phase is a prerequisite for VCT differentiation and their fusion into SCT. The imprinting gene P57Kip2, specifically expressed in intermediate VCT capable of fusion, plays a pivotal role in driving this key event. Moreover, aberrant expression of P57Kip2 has been linked to pathological placental conditions and adverse fetal outcomes. Methods Validation of STK40 interaction with P57Kip2 using rigid molecular simulation docking and co-immunoprecipitation. STK40 expression was modulated by lentivirus in BeWo cells, and the effect of STK40 on trophoblast fusion was assessed by real-time quantitative PCR, western blot, immunofluorescence, and cell viability and proliferation assays. Co-immunoprecipitation, transcriptome sequencing, and western blot were used to determine the potential mechanisms by which STK40 regulates P57Kip2. Results In this study, STK40 has been identified as a novel interacting protein with P57Kip2, and its expression is down-regulated during the fusion process of trophoblast cells. Overexpressing STK40 inhibited cell fusion in BeWo cells while stimulating mitotic cell cycle activity. Further experiments indicated that this effect is attributed to its specific binding to the CDK-binding and the Cyclin-binding domains of P57Kip2, mediating the E3 ubiquitin ligase COP1-mediated ubiquitination and degradation of P57Kip2. Moreover, abnormally high expression of STK40 might significantly contribute to the occurrence of preeclampsia. Conclusions This study offers new insights into the role of STK40 in regulating the protein-level homeostasis of P57Kip2 during placental development.

Human adenovirus (HAdV)-based oncolytic vectors, which are designed to preferentially replicate in and kill cancer cells, have shown modest efficacy in human clinical trials in part due to poor viral distribution throughout the tumor mass. Previously, we showed that expression of the p14 fusion-associated small transmembrane (FAST) fusogenic protein could enhance oncolytic HAdV efficacy and reduce tumor growth rate in a human xenograft mouse model of cancer. We now explore whether co-expression of the adenovirus death protein (ADP) with p14 FAST protein could synergize to further enhance oncolytic vector efficacy. ADP is naturally encoded within the early region 3 (E3) of HAdV, a region which is frequently removed from HAdV-based vectors, and functions to enhance cell lysis and progeny release. We evaluated a variety of approaches to achieve optimal expression of the two proteins, the most efficient method being insertion of an expression cassette within the E3 deletion, consisting of the coding sequences for p14 FAST protein and ADP separated by a self-cleaving peptide derived from the porcine teschovirus-1 (P2A). However, the quantities of p14 FAST protein and ADP produced from this vector were reduced approximately 10-fold compared to a similar vector-expressing only p14 FAST protein and wildtype HAdV, respectively. Compared to our original oncolytic vector-expressing p14 FAST protein alone, reduced expression of p14 FAST protein and ADP from the P2A construct reduced cell-cell fusion, vector spread, and cell-killing activity in human A549 adenocarcinoma cells in culture. These studies show that a self-cleaving peptide can be used to express two different transgenes in an armed oncolytic HAdV vector, but also highlight the challenges in maintaining adequate transgene expression when modifying vector design. [ABSTRACT FROM AUTHOR]

As life extended into eukaryota, a great host of strategies emerged in the pursuit of cellular life. Some cells have been successful in solitude, some moved into cooperatives (i.e., multicellular organisms), but one additional strategy emerged. Throughout eukaryotes, many of the diverse multicellular cooperatives took life in partnership one step further. These cells came together and lost their singularity in the expanse of syncytial life. Recently in our search for this elusive “how”, we discovered the intriguing peculiarity of a nuclear, RNA‐binding protein living a second life as a fusion manager at the surface of developing osteoclasts, ushering them into syncytia 1. It is from here that we will develop several thoughts about the advantages of multinucleated cells and discuss how these fusing cells pass through several hallmarks of cell death. We will propose that cell fusion shares much with cell death because cell fusion is a death of sorts for the cells that undergo it – a death of the life that was and the beginning of new life in a community without borders. [ABSTRACT FROM AUTHOR]

Cell nucleus segmentation plays a significant role in Computer-Aided systems for cancer diagnosis. However, the nuclear images are characterized by different sizes, overlap, adhesion, and similarities between nuclei and other structures, making this task challenging. Aiming to adjust and enhance the feature learning ability of the network, this paper proposes a FourierFilter Irregular Attention U-Net (FFIA-UNet), which contains FourierFilter Irregular Attention (FFIA) and multi-receptive filed fusion (MRF) module. FFIA module seeks to learn deeper characteristics by taking advantage of frequency information and deformable convolution. MRF module improve the learning capacity of fuzzy edges and irregular forms via multiple dilated convolution. Experiments on three datasets show that the proposed FFIA-UNet achieves state-of-the-art. Dice-Score and mIoU reached 0.929 and 0.885 respectively on DSB2018. Furthermore, numerous ablation experiments have demonstrated the module's efficacy. [ABSTRACT FROM AUTHOR]

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages. [ABSTRACT FROM AUTHOR]

Syncytin-1, a human fusogenic protein of retroviral origin, is crucial for placental syncytiotrophoblast formation. To mediate cell-to-cell fusion, Syncytin-1 requires specific interaction with its cognate receptor. Two trimeric transmembrane proteins, Alanine, Serine, Cysteine Transporters 1 and 2 (ASCT1 and ASCT2), were suggested and widely accepted as Syncytin-1 cellular receptors. To quantitatively assess the individual contributions of human ASCT1 and ASCT2 to the fusogenic activity of Syncytin-1, we developed a model system where the ASCT1 and ASCT2 double knockout was rescued by ectopic expression of either ASCT1 or ASCT2. We demonstrated that ASCT2 was required for Syncytin-1 binding, cellular entry, and cell-to-cell fusion, while ASCT1 was not involved in this receptor interaction. We experimentally validated the ASCT1-ASCT2 heterotrimers as a possible explanation for the previous misidentification of ASCT1 as a receptor for Syncytin-1. This redefinition of receptor specificity is important for proper understanding of Syncytin-1 function in normal and pathological pregnancy. [ABSTRACT FROM AUTHOR]

UBR5 E3 ligase has been associated with cancer susceptibility and neuronal integrity, with functions in chromatin regulation and proteostasis. However, the functions of ubr5 within animals remain unclear due to lethality in both mammals and flies when disrupted. Using Caenorhabditis elegans, we show that UBR-5 E3 ligase is required for timely exit of stem fate and complete transition into multiple cell type descendants in an ectodermal blast lineage. Animals lacking intact UBR-5 function simultaneously exhibit both stem fate and differentiated fate in the same descendant cells. A functional screen of UBR-5 physical interactors allowed us to identify the UBE2D2/3 E2 conjugase LET-70 working with UBR-5 to exit stem fate. Strikingly, we revealed that another UBR-5 physical interactor, namely the nuclear poly(A)-binding protein PABPN1 ortholog PABP-2, worked antagonistically to UBR-5 and LET-70. Lowering pabp-2 levels restored normal transition of cell state out of stemness and promoted normal cell fusion when either ubr-5 or let-70 UBE2D function was compromised. The UBR-5-LET-70 and PABP-2 switch works independently of the stem pool size determined by pluripotency factors like lin-28. UBR-5 limits PABP-2 protein and reverses the PABP-2-dependent gene expression program including developmental, proteostasis, and innate immunity genes. Loss of ubr-5 rescues the developmental stall when pabp-2 is compromised. Disruption of ubr-5 elevates PABP-2 levels and prolongs expression of ectodermal and muscle stem markers at the transition to adulthood. Additionally, ubr-5 mutants exhibit an extended period of motility during aging and suppress pabp-2-dependent early onset of immobility [ABSTRACT FROM AUTHOR]

Background: We previously developed a nanobody targeting CTLA-4 and demonstrated that it can boost antitumour T-cell responses in vitro; however, the resulting responses after the injection of T cells into cancer models are usually weak and transient. Here, we explored whether fusing our nanobody to IL-12 would enable it to induce stronger, longer-lasting T-cell immune responses after exposure to immature dendritic cell and tumour cell fusions. Results: The fusion protein enhanced the response of CD8+ T cells to tumour antigens in vitro and led to stronger, more persistent immune responses after the T cells were injected into mice bearing different types of xenografts. Conclusion: Our in vitro and in vivo results suggest the anticancer potential of our nanobody-interleukin fusion system and support the clinical application of this fusion approach for various nanobodies. [ABSTRACT FROM AUTHOR]

Duchenne Muscular Dystrophy (DMD) is a lethal, X-linked disorder leading to muscle degeneration and premature death due to cardiopulmonary complications. Currently, there is no cure for DMD. We previously confirmed the efficacy of human Dystrophin-Expressing Chimeric (DEC) cells created via the fusion of myoblasts from normal and DMD-affected donors. The current study aimed to optimize the development of DEC therapy via the polyethylene glycol (PEG)-mediated fusion protocol of human myoblasts derived from normal, unrelated donors. The optimization of cell fusion assessed different factors influencing fusion efficacy, including myoblast passage number, the efficacy of PKH myoblast staining, the ratio of the single-stained myoblasts in the MIX, and PEG administration time. Additionally, the effect of PEG fusion procedure on cell viability was assessed. A correlation was found between the number of cells used for PKH staining and staining efficacy. Furthermore, the ratio of single-stained myoblasts in the MIX and PEG administration time correlated with fusion efficacy. There was no correlation found between the myoblast passage number and fusion efficacy. This study successfully optimized the myoblast fusion protocol for creation of human DEC cells, introducing DEC as a new Advanced Therapy Medicinal Product (ATMP) for DMD patients. [ABSTRACT FROM AUTHOR]

External constraints, such as development, disease, and environment, can induce changes in epigenomic patterns that may profoundly impact the health trajectory of fetuses and neonates into adulthood, influencing conditions like obesity. Epigenetic modifications encompass processes including DNA methylation, covalent histone modifications, and RNA-mediated regulation. Beyond forward cellular differentiation (cell programming), terminally differentiated cells are reverted to a pluripotent or even totipotent state, that is, cellular reprogramming. Epigenetic modulators facilitate or erase histone and DNA modifications both in vivo and in vitro during programming and reprogramming. Noticeably, obesity is a complex metabolic disorder driven by both genetic and environmental factors. Increasing evidence suggests that epigenetic modifications play a critical role in the regulation of gene expression involved in adipogenesis, energy homeostasis, and metabolic pathways. Hence, we discuss the mechanisms by which epigenetic interventions influence obesity, focusing on DNA methylation, histone modifications, and non-coding RNAs. We also analyze the methodologies that have been pivotal in uncovering these epigenetic regulations, i.e., Large-scale screening has been instrumental in identifying genes and pathways susceptible to epigenetic control, particularly in the context of adipogenesis and metabolic homeostasis; Single-cell RNA sequencing (scRNA-seq) provides a high-resolution view of gene expression patterns at the individual cell level, revealing the heterogeneity and dynamics of epigenetic regulation during cellular differentiation and reprogramming; Chromatin immunoprecipitation (ChIP) assays, focused on candidate genes, have been crucial for characterizing histone modifications and transcription factor binding at specific genomic loci, thereby elucidating the epigenetic mechanisms that govern cellular programming; Somatic cell nuclear transfer (SCNT) and cell fusion techniques have been employed to study the epigenetic reprogramming accompanying cloning and the generation of hybrid cells with pluripotent characteristics, etc. These approaches have been instrumental in identifying specific epigenetic marks and pathways implicated in obesity, providing a foundation for developing targeted therapeutic interventions. Understanding the dynamic interplay between epigenetic regulation and cellular programming is crucial for advancing mechanism and clinical management of obesity. [ABSTRACT FROM AUTHOR]

Following the Goldilocks principle, mitochondria size must be "just right." Mitochondria balance division and fusion to avoid becoming too big or too small. Defects in this balance produce dysfunctional mitochondria in human diseases. Mitochondrial safeguard (MitoSafe) is a defense mechanism that protects mitochondria against extreme enlarging by suppressing fusion in mammalian cells. In MitoSafe, hyperfused mitochondria elicit flickering—short pulses of mitochondrial depolarization. Flickering activates an inner membrane protease, Oma1, which in turn proteolytically inactivates a mitochondrial fusion protein, Opa1. The mechanisms underlying flickering are unknown. Using a live-imaging screen, we identified Slc25a3 (a mitochondrial carrier transporting phosphate and copper) as necessary for flickering and Opa1 cleavage. Remarkably, copper, but not phosphate, is critical for flickering. Furthermore, we found that two copper-containing mitochondrial enzymes, superoxide dismutase 1 and cytochrome c oxidase, regulate flickering. Our data identify an unforeseen mechanism linking copper, redox homeostasis, and membrane flickering in mitochondrial defense against deleterious fusion. [Display omitted] • Flickering prevents harmful extreme mitochondrial fusion by inducing Opa1 cleavage • Mitochondrial copper, transported by Slc25a3, regulates flickering • Copper-containing SOD1 suppresses flickering • Copper-containing cytochrome c oxidase promotes flickering Murata et al. report that mitochondrial copper, transported by Slc25a3, regulates the balance between mitochondrial fusion and division by suppressing fusion when mitochondria become hyperfused. This function of copper is mediated by two copper-containing enzymes: superoxide dismutase 1 and cytochrome c oxidase. [ABSTRACT FROM AUTHOR]

The antigen-mediated B cell isolation method, based on the detection of surface IgG (sIgG), has increased the efficiency of therapeutic antibody (Ab) discovery. However, the reduction in sIgG expression on B cells during plasma cell differentiation presents challenges as it enables Ab production from only a small subset of B cells (e.g., memory B cells). The present study aimed to addressed this problem by developing a workflow to isolate human-IgG-secreting hybridoma cells produced by cell fusion, the majority of which express sIgG. We showed that our sIgG-based antigen-coated bead separation method efficiently enriched hybridoma cells expressing antigen-specific Abs with a yield of 83.5% (from the cell fusion pool) and a positive rate of 73.2%. Furthermore, because the separation could be performed after only a short (1–2-day) culture period following cell fusion, diverse hybridoma clones could be obtained, minimizing clonal selection and the incidence of duplicates. Given that the expression of membrane-bound IgG and sIgG are regulated by different splicing mechanisms, we speculate that the cell fusion step potentially attenuated the suppression of human sIgG expression. Overall, our proposed method is expected to markedly improve the efficiency of therapeutic Ab candidate production, which will have important clinical implications. [ABSTRACT FROM AUTHOR]

The bone-resorbing activity of osteoclasts plays a critical role in the life-long remod- eling of our bones that is perturbed in many bone loss diseases. Multinucleated osteoclasts are formed by the fusion of precursor cells, and larger cells -- generated by an increased number of cell fusion events -- have higher resorptive activity. We find that osteoclast fusion and bone resorption are promoted by reactive oxygen species (ROS) signaling and by an unconventional low molecular weight species of La protein, located at the osteoclast surface. Here, we develop the hypothesis that La's unique regulatory role in osteoclast multinucleation and function is controlled by an ROS switch in La trafficking. Using antibodies that recognize reduced or oxidized species of La, we find that differentiating osteoclasts enrich an oxidized species of La at the cell surface, which is distinct from the reduced La species conventionally localized within cell nuclei. ROS signaling triggers the shift from reduced to oxidized La species, its dephosphorylation and delivery to the surface of osteoclasts, where La promotes multinucleation and resorptive activity. Moreover, intracellular ROS signaling in differentiating osteoclasts oxidizes critical cysteine residues in the C-terminal half of La, producing this unconventional La species that promotes osteoclast fusion. Our findings suggest that redox signaling induces changes in the location and function of La and may represent a promising target for novel skeletal therapies. [ABSTRACT FROM AUTHOR]

Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits. [ABSTRACT FROM AUTHOR]

Purpose: Chordomas are malignant tumors that destroy bones, compress surrounding nerve tissues and exhibit phenotypes that recapitulate notochordal differentiation in the axial skeleton. Chordomas recur frequently, as they resist radio-chemotherapy and are difficult to completely resect, leading to repeated bone destruction and local expansion via unknown mechanisms. Here, using chordoma specimens and JHC7 chordoma cells, we asked whether chordoma cells possess bone-dissolving activity. Methods: CT imaging and histological analysis were performed to evaluate the structure and mineral density of chordoma-invaded bone and osteolytic marker expression. JHC7 cells were subjected to immunocytochemistry, imaging of cell fusion, calcium dynamics and acidic vacuoles, and bone lysis assays. Results: In patients, we found that the skull base invaded by chordoma was highly porous, showed low mineral density and contained brachyury-positive chordoma cells and conventional osteoclasts both expressing the osteolytic markers tartrate-resistant acid phosphatase (TRAP) and collagenases. JHC7 cells expressed TRAP and cathepsin K, became multinucleated via cell-cell fusion, showed spontaneous calcium oscillation, and were partly responsive to the osteoclastogenic cytokine RANKL. JHC7 cells exhibited large acidic vacuoles, and nonregulatory bone degradation without forming actin rings. Finally, bone-derived factors, calcium ions, TGF-β1, and IGF-1 enhanced JHC7 cell proliferation. Conclusion: In chordoma, we propose that in addition to conventional bone resorption by osteoclasts, chordoma cells possess bone-dissolving activity at the tumor-bone boundary. Furthermore, bone destruction and tumor expansion may occur in a positive feedback loop. [ABSTRACT FROM AUTHOR]

The book review discusses the Chikungunya Virus, a mosquito-transmitted alphavirus first isolated in Tanzania in 1952. It covers the clinical symptoms, impact on public health systems, and long-term effects of the virus. The book delves into molecular virology, immunopathogenesis, vaccine development, and small-molecule inhibitors targeting the virus. While providing valuable insights, the book may become outdated due to rapid advancements in virology and may contain technical terms that could be challenging for readers without a background in related fields. [Extracted from the article]

Syncytins are envelope genes of retroviral origin that play a critical role in the formation of a syncytial structure at the fetomaternal interface via their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one observed in all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryonic development. The cellular receptor for SynA has been identified as the membrane protein LY6E and is not the receptor for SynB. Here, by combining a cell-cell fusion assay with the screening of a human ORFeome-derived expression library, we identified the transmembrane multipass sodium-dependent phosphate transporter 1 PiT1/SLC20A1 as the receptor for SynB. Transfection of cells with the cloned receptor, but not the closely related PiT2/SLC20A2, leads to their fusion with cells expressing SynB, with no cross-reactive fusion activity with SynA. The interaction between the two partners was further demonstrated by immunoprecipitation. PiT1/PiT2 chimera and truncation experiments identified the PiT1 N-terminus as the major determinant for SynB-mediated fusion. RT-qPCR analysis of PiT1 expression on a panel of mouse adult and fetal tissues revealed a concomitant increase of PiT1 and SynB specifically in the developing placenta. Finally, electron microscopy analysis of the placenta of PiT1 null embryo before they die (E11.5) disclosed default of ST-II formation with lack of syncytialization, as previously observed in cognate SynB null placenta, and consistent with the present identification of PiT1 as the SynB partner. [ABSTRACT FROM AUTHOR]

Nipah virus (NiV) is a highly pathogenic paramyxovirus capable of causing severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, underscoring the urgent need for the development of countermeasures. The NiV surface-displayed glycoproteins, NiV-G and NiV-F, mediate host cell attachment and fusion, respectively, and are heavily targeted by host antibodies. Here, we describe a vaccination-derived neutralizing monoclonal antibody, mAb92, that targets NiV-F. Structural characterization of the Fab region bound to NiV-F (NiV-F-Fab92) by cryo-electron microscopy analysis reveals an epitope in the DIII domain at the membrane distal apex of NiV-F, an established site of vulnerability on the NiV surface. Further, prophylactic treatment of hamsters with mAb92 offered complete protection from NiV disease, demonstrating beneficial activity of mAb92 in vivo. This work provides support for targeting NiV-F in the development of vaccines and therapeutics against NiV. [ABSTRACT FROM AUTHOR]

Simple Summary: Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor, and the interaction between fibrinogen-like protein 1 and LAG3 is regarded as a new immune escape mechanism. Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease seriously impacting the porcine industry worldwide. In this study, monoclonal antibodies (mAbs) against porcine LAG3 (pLAG3) were developed, and one mAb (1C2) showed good reactivity with pLAG3 on PHA-activated porcine lymphocytes. The epitope recognized by mAb 1C2 was located at amino acid residues 214–435 of pLAG3. The LAG3 expression in the tissues of PRRSV-infected pigs increased remarkably. Interference of LAG3 expression on PHA-activated lymphocytes promoted PRRSV replication in the co-culture system of monocyte-derived dendritic cells and lymphocytes, whereas overexpression of LAG3 or blocking of the LAG3 signal with mAb 1C2 inhibited PRRSV replication, indicating that PRRSV infection activates the LAG3-signaling pathway, and this pathway plays an important role in PRRSV pathogenesis. Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor and the interaction between fibrinogen-like protein 1 and LAG3 can inhibit the anti-tumor effect of T cells both in vivo and in vitro, which was regarded as a new immune evasion mechanism. Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, is an infectious disease characterized by reproductive disorders in pregnant sows and gilts and respiratory problems in pigs of all ages, seriously impacting the pig industry worldwide. In this study, monoclonal antibodies (mAbs) against porcine LAG3 (pLAG3) were developed, and one mAb (1C2) showed good reactivity with pLAG3 on PHA-activated porcine peripheral blood lymphocytes. Epitope mapping showed the epitope recognized by mAb 1C2 was located at amino acid residues 214–435 of pLAG3. LAG3 expression in the tissues of PRRSV-infected pigs was detected, using mAb 1C2 as the primary antibody, and the results revealed that PRRSV infection caused a marked increase in LAG3 expression compared to the control group. Interference of LAG3 expression on PHA-activated lymphocytes promoted PRRSV replication in the co-culture system of monocyte-derived dendritic cells and lymphocytes, whereas overexpression of LAG3 or blocking of the LAG3 signal with mAb 1C2 inhibited PRRSV replication, indicating that PRRSV infection activates the LAG3-signaling pathway, suggesting that this pathway plays an important role in PRRSV pathogenesis. The results obtained lay the foundation for subsequent research on the role of LAG3 in PRRS and other diseases with persistent infection characteristics. [ABSTRACT FROM AUTHOR]

Cleft lip and/or primary palate (CL/P) represent a prevalent congenital malformation, the aetiology of which is highly intricate. Although it is generally accepted that the condition arises from failed fusion between the upper lip and primary palate, the precise mechanism underlying this fusion process remains enigmatic. In this study, we utilized transposase‐accessible chromatin sequencing (scATAC‐seq) and single‐cell RNA sequencing (scRNA‐seq) to interrogate lambdoidal junction tissue derived from C57BL/6J mouse embryos at critical stages of embryogenesis (10.5, 11.5 and 12.5 embryonic days). We successfully identified distinct subgroups of mesenchymal and ectodermal cells involved in the fusion process and characterized their unique transcriptional profiles. Furthermore, we conducted cell differentiation trajectory analysis, revealing a dynamic repertoire of genes that are sequentially activated or repressed during pseudotime, facilitating the transition of relevant cell types. Additionally, we employed scATAC data to identify key genes associated with the fusion process and demonstrated differential chromatin accessibility across major cell types. Finally, we constructed a dynamic intercellular communication network and predicted upstream transcriptional regulators of critical genes involved in important signalling pathways. Our findings provide a valuable resource for future studies on upper lip and primary palate development, as well as congenital defects. [ABSTRACT FROM AUTHOR]

Notably, clear cell renal cell carcinoma (ccRCC) is characterized by a distinct metabolic tumor phenotype that involves the reprogramming of multiple metabolic pathways. Although there is increasing evidence linking FUCA1 to malignancies, its specific role and downstream signaling pathways in ccRCC remain poorly understood. Here we found that FUCA1 expression was significantly downregulated in ccRCC tissues, which also predicts poor prognosis of ccRCCpatients. Moreover, enhancing FUCA1 expression resulted in reduced invasion and migration of ccRCC cells, further indicating its protective role. CHIP-qPCR and luciferase assays showed that CTCF was an upstream transcription factor of FUCA1 and could reverse the effects caused by FUCA1 inactivation. The change in FUCA1 led to changes in the results of various autophagy-related proteins and the mRFP-GFP-LC3 dual fluorescence system, indicating that it may play a role in the fusion stage of autophagy. Protein-protein interaction analysis revealed that FUCA2 exhibited the closest interaction with FUCA1 and strongly predicted the prognosis of ccRCC patients. Additionally, serum AFU encoded by FUCA2 could serve as a valuable predictor for survival in ccRCC patients. FUCA1 suppresses invasion and migration of ccRCC cells, with its activity being modulated by CTCF. FUCA1 regulates the autophagy process in ccRCC cells by influencing the fusion between autophagosomes and lysosomes. FUCA2 shares similarities with FUCA1, and elevated serum AFU levels along with increased expression of FUCA2 are indicative of a favorable prognosis in ccRCC. [ABSTRACT FROM AUTHOR]

Soluble host factors in the upper respiratory tract can serve as the first line of defense against SARS-CoV-2 infection. In this study, we described the identification and function of a human airway trypsin–like protease (HAT), capable of reducing the infectivity of ancestral SARS-CoV-2. Further, in mouse models, HAT analogue expression was upregulated by SARS-CoV-2 infection. The antiviral activity of HAT functioned through the cleavage of the SARS-CoV-2 spike glycoprotein at R682. This cleavage resulted in inhibition of the attachment of ancestral spike proteins to host cells, which inhibited the cell-cell membrane fusion process. Importantly, exogenous addition of HAT notably reduced the infectivity of ancestral SARS-CoV-2 in vivo. However, HAT was ineffective against the Delta variant and most circulating Omicron variants, including the BQ.1.1 and XBB.1.5 subvariants. We demonstrate that the P681R mutation in Delta and P681H mutation in the Omicron variants, adjacent to the R682 cleavage site, contributed to HAT resistance. Our study reports what we believe to be a novel soluble defense factor against SARS-CoV-2 and resistance of its actions in the Delta and Omicron variants. [ABSTRACT FROM AUTHOR]

Background: The syncytiotrophoblast (SCT) layer in the placenta serves as a crucial physical barrier separating maternal-fetal circulation, facilitating essential signal and substance exchange between the mother and fetus. Any abnormalities in its formation or function can result in various maternal syndromes, such as preeclampsia. The transition of proliferative villous cytotrophoblasts (VCT) from the mitotic cell cycle to the G0 phase is a prerequisite for VCT differentiation and their fusion into SCT. The imprinting gene P57Kip2, specifically expressed in intermediate VCT capable of fusion, plays a pivotal role in driving this key event. Moreover, aberrant expression of P57Kip2 has been linked to pathological placental conditions and adverse fetal outcomes. Methods: Validation of STK40 interaction with P57Kip2 using rigid molecular simulation docking and co-immunoprecipitation. STK40 expression was modulated by lentivirus in BeWo cells, and the effect of STK40 on trophoblast fusion was assessed by real-time quantitative PCR, western blot, immunofluorescence, and cell viability and proliferation assays. Co-immunoprecipitation, transcriptome sequencing, and western blot were used to determine the potential mechanisms by which STK40 regulates P57Kip2. Results: In this study, STK40 has been identified as a novel interacting protein with P57Kip2, and its expression is down-regulated during the fusion process of trophoblast cells. Overexpressing STK40 inhibited cell fusion in BeWo cells while stimulating mitotic cell cycle activity. Further experiments indicated that this effect is attributed to its specific binding to the CDK-binding and the Cyclin-binding domains of P57Kip2, mediating the E3 ubiquitin ligase COP1-mediated ubiquitination and degradation of P57Kip2. Moreover, abnormally high expression of STK40 might significantly contribute to the occurrence of preeclampsia. Conclusions: This study offers new insights into the role of STK40 in regulating the protein-level homeostasis of P57Kip2 during placental development. [ABSTRACT FROM AUTHOR]