Further Characterization of the Antiviral Transmembrane Protein MARCH8.

The cellular transmembrane protein MARCH8 impedes the incorporation of various viral envelope glycoproteins, such as the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G-glycoprotein (VSV-G), into virions by downregulating them from the surface of virus-producing cells. This downregulation significantly reduces the efficiency of virus infection. In this study, we aimed to further characterize this host protein by investigating its species specificity and the domains responsible for its antiviral activity, as well as its ability to inhibit cell-to-cell HIV-1 infection. We found that the antiviral function of MARCH8 is well conserved in the rhesus macaque, mouse, and bovine versions. The RING-CH domains of these versions are functionally important for inhibiting HIV-1 Env and VSV-G-pseudovirus infection, whereas tyrosine motifs are crucial for the former only, consistent with findings in human MARCH8. Through analysis of chimeric proteins between MARCH8 and non-antiviral MARCH3, we determined that both the N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, of MARCH8 are critical for its antiviral activity. Notably, we found that MARCH8 is unable to block cell-to-cell HIV-1 infection, likely due to its insufficient downregulation of Env. These findings offer further insights into understanding the biology of this antiviral transmembrane protein.

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12.

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

Finite Element Analysis of Changes in Deformation of Intraocular Segments by Airbag Impact in Eyes of Various Axial Lengths.

Background: We studied the kinetic phenomenon of an airbag impact on eyes with different axial lengths using finite element analysis (FEA) to sequentially determine the physical and mechanical responses of intraocular segments at various airbag deployment velocities. Methods: The human eye model we created was used in simulations with the FEA program PAM-GENERISTM. The airbag was set to impact eyes with axial lengths of 21.85 mm (hyperopia), 23.85 mm (emmetropia) and 25.85 mm (myopia), at initial velocities of 20, 30, 40, 50 and 60 m/s. The deformation rate was calculated as the ratio of the length of three segments, anterior chamber, lens and vitreous, to that at the baseline from 0.2 ms to 2.0 ms after the airbag impact. Results: Deformation rate of the anterior chamber was greater than that of other segments, especially in the early phase, 0.2-0.4 ms after the impact (P < 0.001), and it reached its peak, 80%, at 0.8 ms. A higher deformation rate in the anterior chamber was found in hyperopia compared with other axial length eyes in the first half period, 0.2-0.8 ms, followed by the rate in emmetropia (P < 0.001). The lens deformation rate was low, its peak ranging from 40% to 75%, and exceeded that of the anterior chamber at 1.4 ms and 1.6 ms after the impact (P < 0.01). The vitreous deformation rate was lower throughout the simulation period than that of the other segments and ranged from a negative value (elongation) in the later phase. Conclusion: Airbag impact on the eyeball causes evident deformation, especially in the anterior chamber. The results obtained in this study, such as the time lag of the peak deformation between the anterior chamber and lens, suggest a clue to the pathophysiological mechanism of airbag ocular injury.

Membrane-Associated Ubiquitin Ligase RING Finger Protein 152 Orchestrates Melanogenesis via Tyrosinase Ubiquitination.

Lysosomal degradation of tyrosinase, a pivotal enzyme in melanin synthesis, negatively impacts melanogenesis in melanocytes. Nevertheless, the precise molecular mechanisms by which lysosomes target tyrosinase have remained elusive. Here, we identify RING (Really Interesting New Gene) finger protein 152 (RNF152) as a membrane-associated ubiquitin ligase specifically targeting tyrosinase for the first time, utilizing AlphaScreen technology. We observed that modulating RNF152 levels in B16 cells, either via overexpression or siRNA knockdown, resulted in decreased or increased levels of both tyrosinase and melanin, respectively. Notably, RNF152 and tyrosinase co-localized at the trans-Golgi network (TGN). However, upon treatment with lysosomal inhibitors, both proteins appeared in the lysosomes, indicating that tyrosinase undergoes RNF152-mediated lysosomal degradation. Through ubiquitination assays, we found the indispensable roles of both the RING and transmembrane (TM) domains of RNF152 in facilitating tyrosinase ubiquitination. In summary, our findings underscore RNF152 as a tyrosinase-specific ubiquitin ligase essential for regulating melanogenesis in melanocytes.

Classification of Subtypes of Vernal Keratoconjunctivitis by Cluster Analysis Based on Clinical Features.

Background: Vernal keratoconjunctivitis (VKC) is a refractory ocular allergic disorder that mainly affects boys. A few studies have attempted to develop a classification of subtypes of VKC. In this study, we investigated a computational approach called cluster analysis to separate VKC cases into groups based on clinically relevant characteristics. Methods: In total, 41 consecutive patients clinically diagnosed with VKC at the Department of Ophthalmology of Fukuoka University Hospital were included. Patients were treated with immunosuppressive eye drops without simultaneous corticosteroid eye drops, except for the occurrence of exacerbations. Collated variables were age at onset, clinical score of ocular lesions at baseline, clinical score of ocular lesions at final visit, clinical score of atopic dermatitis (AD) at baseline, frequency of exacerbations of VKC, serum total IgE level and peripheral blood eosinophil count. Results: VKC patients were grouped into three clusters by cluster analysis, and cluster 1, 2, and 3 comprised 25, 9 and 7 cases, respectively. There were differences in the incidence of complications of AD and age at onset among the clusters; therefore, we named the three clusters for better understanding as traditional VKC (cluster 1), early-onset atopic keratoconjunctivitis (AKC)/VKC (cluster 2) and puberty-onset AKC (cluster 3). Conclusion: We found in this study that VKC in childhood has three phenotypes which were previously unknown. Our findings may help to establish precision medicine by focusing on the phenotype of each case to develop individualized medicine to prevent exacerbations.

Single-molecule tracking of Nanog and Oct4 in living mouse embryonic stem cells uncovers a feedback mechanism of pluripotency maintenance.

Nanog and Oct4 are core transcription factors that form part of a gene regulatory network to regulate hundreds of target genes for pluripotency maintenance in mouse embryonic stem cells (ESCs). To understand their function in the pluripotency maintenance, we visualised and quantified the dynamics of single molecules of Nanog and Oct4 in a mouse ESCs during pluripotency loss. Interestingly, Nanog interacted longer with its target loci upon reduced expression or at the onset of differentiation, suggesting a feedback mechanism to maintain the pluripotent state. The expression level and interaction time of Nanog and Oct4 correlate with their fluctuation and interaction frequency, respectively, which in turn depend on the ESC differentiation status. The DNA viscoelasticity near the Oct4 target locus remained flexible during differentiation, supporting its role either in chromatin opening or a preferred binding to uncondensed chromatin regions. Based on these results, we propose a new negative feedback mechanism for pluripotency maintenance via the DNA condensation state-dependent interplay of Nanog and Oct4.

Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation.

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Here, we established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, our study demonstrated herein would be of help in the risk assessment of future heart failure.

Progress of Endogenous and Exogenous Nanoparticles for Cancer Therapy and Diagnostics.

The focus of this brief review is to describe the application of nanoparticles, including endogenous nanoparticles (e.g., extracellular vesicles, EVs, and virus capsids) and exogenous nanoparticles (e.g., organic and inorganic materials) in cancer therapy and diagnostics. In this review, we mainly focused on EVs, where a recent study demonstrated that EVs secreted from cancer cells are associated with malignant alterations in cancer. EVs are expected to be used for cancer diagnostics by analyzing their informative cargo. Exogenous nanoparticles are also used in cancer diagnostics as imaging probes because they can be easily functionalized. Nanoparticles are promising targets for drug delivery system (DDS) development and have recently been actively studied. In this review, we introduce nanoparticles as a powerful tool in the field of cancer therapy and diagnostics and discuss issues and future prospects.

Finite element analysis of changes in tensile strain and deformation by airbag impact in eyes of various axial lengths.

PURPOSE: Airbags have substantially reduced mortality and morbidity, while ocular injuries caused by airbags have been reported. We applied a three-dimensional finite element analysis (FEA) model we have established for evaluation of the deformation of an intact eyeball of various axial lengths induced by an airbag impact at various impact velocities. METHODS: A model human eye we have created was used in simulations with an FEA program, PAM-GENERIS™ (Nihon ESI, Tokyo, Japan). The airbag was set to impact eyes with various axial lengths of 21.85 mm (hyperopia), 23.85 mm (emmetropia) and 25.85 mm (myopia), at initial velocities of 30, 40, 50 and 60 m/s. Changes in the shape of the eye and the strain induced were calculated. Deformation of the eye in a cross-sectional view was displayed sequentially in slow motion. RESULTS: We found that considerable damage, such as corneal or scleral lacerations, was observed especially at higher impact velocities, such as 50 or 60 m/s, in eyes with any axial length. Deformation was most evident in the anterior segment. The decrease rate of axial length was greatest in the hyperopic eye, followed by the myopic eye, and the emmetropic eye. CONCLUSIONS: It was shown that hyperopic eyes are most susceptible to deformation by an airbag impact in this simulation. The considerable deformation by an airbag impact on the eye during a traffic accident shown in this study might indicate the necessity of ocular protection to avoid permanent eye damage.

Application of the Dynamical Network Biomarker Theory to Raman Spectra.

The dynamical network biomarker (DNB) theory detects the early warning signals of state transitions utilizing fluctuations in and correlations between variables in complex systems. Although the DNB theory has been applied to gene expression in several diseases, destructive testing by microarrays is a critical issue. Therefore, other biological information obtained by non-destructive testing is desirable; one such piece of information is Raman spectra measured by Raman spectroscopy. Raman spectroscopy is a powerful tool in life sciences and many other fields that enable the label-free non-invasive imaging of live cells and tissues along with detailed molecular fingerprints. Naïve and activated T cells have recently been successfully distinguished from each other using Raman spectroscopy without labeling. In the present study, we applied the DNB theory to Raman spectra of T cell activation as a model case. The dataset consisted of Raman spectra of the T cell activation process observed at 0 (naïve T cells), 2, 6, 12, 24 and 48 h (fully activated T cells). In the DNB analysis, the F-test and hierarchical clustering were used to detect the transition state and identify DNB Raman shifts. We successfully detected the transition state at 6 h and related DNB Raman shifts during the T cell activation process. The present results suggest novel applications of the DNB theory to Raman spectra ranging from fundamental research on cellular mechanisms to clinical examinations.

Recent Advances in Raman Spectral Imaging in Cell Diagnosis and Gene Expression Prediction.

Normal and tumor regions within cancer tissue can be distinguished using various methods, such as histological analysis, tumor marker testing, X-ray imaging, or magnetic resonance imaging. Recently, new discrimination methods utilizing the Raman spectra of tissues have been developed and put into practical use. Because Raman spectral microscopy is a non-destructive and non-labeling method, it is potentially compatible for use in the operating room. In this review, we focus on the basics of Raman spectroscopy and Raman imaging in live cells and cell type discrimination, as these form the bases for current Raman scattering-based cancer diagnosis. We also review recent attempts to estimate the gene expression profile from the Raman spectrum of living cells using simple machine learning. Considering recent advances in machine learning techniques, we speculate that cancer type discrimination using Raman spectroscopy will be possible in the near future.

Analysis of Cytokine Production Profiles of Local and Systemic Lymphocytes in Sick Building Syndrome Compared with Ocular Allergy.

Purpose: We have previously studied clinical and allergological aspects of sick building syndrome (SBS) cases with ocular disorders and found that SBS is suggested to be partially induced by an allergic response. We analyzed the cytokine production profiles of conjunctival and peripheral blood lymphocytes in patients with SBS with ocular manifestations to further evaluate the pathophysiology of SBS from an immunological standpoint. Methods: We obtained conjunctival samples and peripheral blood mononuclear cells (PBMC) from 15 cases of SBS with ocular findings, 49 cases of allergic conjunctival diseases (ACD) (allergic conjunctivitis (AC), atopic keratoconjunctivitis (AKC), and vernal keratoconjunctivitis (VKC)), and normal controls. Frequencies of cytokine-producing T cells were analyzed by flow cytometry based on an intracellular cytokine staining method. Results: Although no significant difference was observed in the percentage of interferon (IFN)-γ-producing CD4+ T cells in PBMC between patients with SBS and controls, the percentage of interleukin (IL)-4-producing PBMC CD4+ T cells in patients with SBS was significantly higher than that in controls. The percentage of IL-4-producing CD4+ T cells in the conjunctiva in patients with SBS was significantly higher than that in controls, whereas it was significantly lower than that in AKC and VKC. A significant correlation was observed between the percentage of IL-4-producing CD4+ T cells in the conjunctiva and clinical score. Conclusion: These results suggest that SBS may be a kind of allergic disorder and that IL-4 plays a role in the development of allergic disorders in SBS ocular lesions.

Recent advances in animal cell technologies for industrial and medical applications.

The industrial use of living organisms for bioproduction of valued substances has been accomplished mostly using microorganisms. To produce high-value bioproducts such as antibodies that require glycosylation modification for better performance, animal cells have been recently gaining attention in bioengineering because microorganisms are unsuitable for producing such substances. Furthermore, animal cells are now classified as products because a large number of cells are required for use in regenerative medicine. In this article, we review animal cell technologies and the use of animal cells, focusing on useable cell generation and large-scale production of animal cells. We review recent advance in mammalian cell line development because this is the first step in the production of recombinant proteins, and it largely affects the efficacy of the production. We next review genetic engineering technology focusing on CRISPR-Cas system as well as surrounding technologies as these methods have been gaining increasing attention in areas that use animal cells. We further review technologies relating to bioreactors used in the context of animal cells because they are essential for the mass production of target products. We also review tissue engineering technology because tissue engineering is one of the main exits for mass-produced cells; in combination with genetic engineering technology, it can prove to be a promising treatment for patients with genetic diseases after the establishment of induced pluripotent stem cell technology. The technologies highlighted in this review cover brief outline of the recent animal cell technologies related to industrial and medical applications.

MARCH8 Targets Cytoplasmic Lysine Residues of Various Viral Envelope Glycoproteins.

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoprotein but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we show the antiviral spectrum of MARCH8 using viruses pseudotyped with a variety of viral envelope glycoproteins. Infection experiments revealed that viral envelope glycoproteins derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelope glycoproteins by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor. IMPORTANCE A member of the MARCH E3 ubiquitin ligase family, MARCH8, downregulates many different kinds of host transmembrane proteins, resulting in the regulation of cellular homeostasis. On the other hands, MARCH8 acts as an antiviral factor when it binds to and downregulates HIV-1 envelope glycoprotein and vesicular stomatitis virus G-glycoprotein that are viral transmembrane proteins. This study reveals that, as in the case of cellular membrane proteins, MARCH8 shows broad-spectrum inhibition against various viral envelope glycoproteins by recognizing their cytoplasmic lysine residues, resulting in lysosomal degradation.

MARCH8: the tie that binds to viruses.

Membrane-associated RING-CH (MARCH) family member proteins are RING-finger E3 ubiquitin ligases that are known to downregulate cellular transmembrane proteins. MARCH8 is a novel antiviral factor that inhibits HIV-1 envelope glycoprotein and vesicular stomatitis virus G by downregulating these envelope glycoproteins from the cell surface, resulting in their reduced incorporation into virions. More recently, we have found that MARCH8 reduces viral infectivity via two different mechanisms. Additionally, several groups have reported further antiviral or virus-supportive functions of the MARCH8 protein and its other cellular mechanisms. In this review, we summarize the current knowledge about the molecular mechanisms by which MARCH8 can regulate cellular homeostasis and inhibit and occasionally support enveloped virus infection.

Microarray profiling of gene expression in C2C12 myotubes trained by electric pulse stimulation.

Electric pulse-stimulated C2C12 myotubes are gaining interest in the field of muscle physiology and biotechnology because electric pulse stimulation (EPS) enhances sarcomere structure development and active tension generation capability. Recently, we found that termination of EPS results in the rapid loss of active tension generation accompanied by disassembly of the sarcomere structure, which may represent an in vitro muscle atrophy model. To elucidate the molecular mechanism underlying this rapid loss of active tension generation and sarcomere structure disassembly after termination of EPS, we performed transcriptomic analysis using microarray. After termination of EPS, 74 genes were upregulated and 120 genes were downregulated after 30 min; however, atrophy-related genes were not found among these genes. To further assess the effect of EPS on gene expression, we re-applied EPS after its termination for 8 h and searched for genes whose expression was reversed. Four genes were upregulated by termination of EPS and downregulated by the re-application of EPS, whereas two genes were downregulated by termination of EPS and upregulated by the re-application of EPS. Although none of these genes were atrophy- or hypertrophy-related, the results presented in this study will contribute to the understanding of gene expression changes that mediate rapid loss of active tension generation and sarcomere structure disassembly following termination of EPS in C2C12 myotubes.

Pressure-induced changes on the morphology and gene expression in mammalian cells.

We evaluated the effect of high hydrostatic pressure on mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (ES) cells. Hydrostatic pressures of 15, 30, 60, and 90 MPa were applied for 10 min, and changes in gene expression were evaluated. Among genes related to mechanical stimuli, death-associated protein 3 was upregulated in MEF subjected to 90 MPa pressure; however, other genes known to be upregulated by mechanical stimuli did not change significantly. Genes related to cell differentiation did not show a large change in expression. On the other hand, genes related to pluripotency, such as Oct4 and Sox2, showed a twofold increase in expression upon application of 60 MPa hydrostatic pressure for 10 min. Although these changes did not persist after overnight culture, cells that were pressurized to 15 MPa showed an increase in pluripotency genes after overnight culture. When mouse ES cells were pressurized, they also showed an increase in the expression of pluripotency genes. These results show that hydrostatic pressure activates pluripotency genes in mammalian cells. This article has an associated First Person interview with the first author of the paper.

Glycine insertion modulates the fluorescence properties of Aequorea victoria green fluorescent protein and its variants in their ambient environment.

The green fluorescent protein (GFP) derived from Pacific Ocean jellyfish is an essential tool in biology. GFP-solvent interactions can modulate the fluorescent property of GFP. We previously reported that glycine insertion is an effective mutation in the yellow variant of GFP, yellow fluorescent protein (YFP). Glycine insertion into one of the ß-strands comprising the barrel structure distorts its structure, allowing water molecules to invade near the chromophore, enhancing hydrostatic pressure or solution hydrophobicity sensitivity. However, the underlying mechanism of how glycine insertion imparts environmental sensitivity to YFP has not been elucidated yet. To unveil the relationship between fluorescence and ß-strand distortion, we investigated the effects of glycine insertion on the dependence of the optical properties of GFP variants named enhanced-GFP (eGFP) and its yellow (eYFP) and cyan (eCFP) variants with respect to pH, temperature, pressure, and hydrophobicity. Our results showed that the quantum yield decreased depending on the number of inserted glycines in all variants, and the dependence on pH, temperature, pressure, and hydrophobicity was altered, indicating the invasion of water molecules into the ß-barrel. Peak shifts in the emission spectrum were observed in glycine-inserted eGFP, suggesting a change of the electric state in the excited chromophore. A comparative investigation of the spectral shift among variants under different conditions demonstrated that glycine insertion rearranged the hydrogen bond network between His148 and the chromophore. The present results provide important insights for further understanding the fluorescence mechanism in GFPs and suggest that glycine insertion could be a potent approach for investigating the relationship between water molecules and the intra-protein chromophore.