Instantaneous Inactivation of Herpes Simplex Virus by Silicon Nitride Bioceramics.

Hydrolytic reactions taking place at the surface of a silicon nitride (Si3N4) bioceramic were found to induce instantaneous inactivation of Human herpesvirus 1 (HHV-1, also known as Herpes simplex virus 1 or HSV-1). Si3N4 is a non-oxide ceramic compound with strong antibacterial and antiviral properties that has been proven safe for human cells. HSV-1 is a double-stranded DNA virus that infects a variety of host tissues through a lytic and latent cycle. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of HSV-1 DNA after instantaneous contact with Si3N4 showed that ammonia and its nitrogen radical byproducts, produced upon Si3N4 hydrolysis, directly reacted with viral proteins and fragmented the virus DNA, irreversibly damaging its structure. A comparison carried out upon testing HSV-1 against ZrO2 particles under identical experimental conditions showed a significantly weaker (but not null) antiviral effect, which was attributed to oxygen radical influence. The results of this study extend the effectiveness of Si3N4's antiviral properties beyond their previously proven efficacy against a large variety of single-stranded enveloped and non-enveloped RNA viruses. Possible applications include the development of antiviral creams or gels and oral rinses to exploit an extremely efficient, localized, and instantaneous viral reduction by means of a safe and more effective alternative to conventional antiviral creams. Upon incorporating a minor fraction of micrometric Si3N4 particles into polymeric matrices, antiherpetic devices could be fabricated, which would effectively impede viral reactivation and enable high local effectiveness for extended periods of time.

Raman Multi-Omic Snapshot and Statistical Validation of Structural Differences between Herpes Simplex Type I and Epstein-Barr Viruses.

Raman spectroscopy was applied to study the structural differences between herpes simplex virus Type I (HSV-1) and Epstein-Barr virus (EBV). Raman spectra were first collected with statistical validity on clusters of the respective virions and analyzed according to principal component analysis (PCA). Then, average spectra were computed and a machine-learning approach applied to deconvolute them into sub-band components in order to perform comparative analyses. The Raman results revealed marked structural differences between the two viral strains, which could mainly be traced back to the massive presence of carbohydrates in the glycoproteins of EBV virions. Clear differences could also be recorded for selected tyrosine and tryptophan Raman bands sensitive to pH at the virion/environment interface. According to the observed spectral differences, Raman signatures of known biomolecules were interpreted to link structural differences with the viral functions of the two strains. The present study confirms the unique ability of Raman spectroscopy for answering structural questions at the molecular level in virology and, despite the structural complexity of viral structures, its capacity to readily and reliably differentiate between different virus types and strains.

Significant Inactivation of SARS-CoV-2 In Vitro by a Green Tea Catechin, a Catechin-Derivative, and Black Tea Galloylated Theaflavins.

Potential effects of tea and its constituents on SARS-CoV-2 infection were assessed in vitro. Infectivity of SARS-CoV-2 was decreased to 1/100 to undetectable levels after a treatment with black tea, green tea, roasted green tea, or oolong tea for 1 min. An addition of (-) epigallocatechin gallate (EGCG) significantly inactivated SARS-CoV-2, while the same concentration of theasinensin A (TSA) and galloylated theaflavins including theaflavin 3,3'-di-O-gallate (TFDG) had more remarkable anti-viral activities. EGCG, TSA, and TFDG at 1 mM, 40 µM, and 60 µM, respectively, which are comparable to the concentrations of these compounds in tea beverages, significantly reduced infectivity of the virus, viral RNA replication in cells, and secondary virus production from the cells. EGCG, TSA, and TFDG significantly inhibited interaction between recombinant ACE2 and RBD of S protein. These results suggest potential usefulness of tea in prevention of person-to-person transmission of the novel coronavirus.

Hypoxia Potentiates Anabolic Effects of Exogenous Hyaluronic Acid in Rat Articular Cartilage.

Hyaluronic acid (HA) is used clinically to treat osteoarthritis (OA), but its pharmacological effects under hypoxic conditions remain unclear. Articular chondrocytes in patients with OA are exposed to a hypoxic environment. This study investigated whether hypoxia could potentiate the anabolic effects of exogenous HA in rat articular cartilage and whether these mechanisms involved HA receptors. HA under hypoxic conditions significantly enhanced the expression of extracellular matrix genes and proteins in explant culture, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and dimethylmethylene blue (DMMB) assays. Staining with Safranin-O and immunohistochemical staining with antibody to type II collagen were also enhanced in pellet culture. The expression of CD44 was increased by hypoxia and significantly suppressed by transfection with siRNAs targeting hypoxia-inducible factor 1 alpha (siHIF-1α). These findings indicate that hypoxia potentiates the anabolic effects of exogenous HA by a mechanism in which HIF-1α positively regulates the expression of CD44, enhancing the binding affinity for exogenous HA. The anabolic effects of exogenous HA may increase as OA progresses.

Cycloamylose-nanogel drug delivery system-mediated intratumor silencing of the vascular endothelial growth factor regulates neovascularization in tumor microenvironment.

RNAi enables potent and specific gene silencing, potentially offering useful means for treatment of cancers. However, safe and efficient drug delivery systems (DDS) that are appropriate for intra-tumor delivery of siRNA or shRNA have rarely been established, hindering clinical application of RNAi technology to cancer therapy. We have devised hydrogel polymer nanoparticles, or nanogel, and shown its validity as a novel DDS for various molecules. Here we examined the potential of self-assembled nanogel of cholesterol-bearing cycloamylose with spermine group (CH-CA-Spe) to deliver vascular endothelial growth factor (VEGF)-specific short interfering RNA (siVEGF) into tumor cells. The siVEGF/nanogel complex was engulfed by renal cell carcinoma (RCC) cells through the endocytotic pathway, resulting in efficient knockdown of VEGF. Intra-tumor injections of the complex significantly suppressed neovascularization and growth of RCC in mice. The treatment also inhibited induction of myeloid-derived suppressor cells, while it decreased interleukin-17A production. Therefore, the CH-CA-Spe nanogel may be a feasible DDS for intra-tumor delivery of therapeutic siRNA. The results also suggest that local suppression of VEGF may have a positive impact on systemic immune responses against malignancies.

Milk Casein Inhibits Effect of Black Tea Galloylated Theaflavins to Inactivate SARS-CoV-2 In Vitro.

Continuing caution is required against the potential emergence of SARS-CoV-2 novel mutants that could pose the next global health and socioeconomical threats. If virus in saliva can be inactivated by a beverage, such a beverage may be useful because the saliva of infected persons is the major origin of droplets and aerosols that mediate human-to-human viral transmission. We previously reported that SARS-CoV-2 was significantly inactivated by treatment in vitro with tea including green tea and black tea. Catechins and its derived compounds galloylated theaflavins (gTFs) bound to the receptor-binding domain (RBD) of the S-protein and blocked interaction between RBD and ACE2. Black tea is often consumed with sugar, milk, lemon juice, etc., and it remains unclarified whether these ingredients may influence the anti-SARS-CoV-2 effect of black tea. Here, we examined the effect of black tea on Omicron subvariants in the presence of these ingredients. The infectivity of Omicron subvariants was decreased to 1/100 or lower after treatment with black tea for 10 s. One or two teaspoons of milk (4~8 mL) completely blocked the anti-viral effect of a cup of tea (125 mL), whereas an addition of sugar or lemon juice failed to do so. The suppressive effect was dose-dependently exerted by milk casein but not whey proteins. gTFs were coprecipitated with casein after acidification of milk-supplemented black tea, strongly suggesting the binding of gTFs to casein. The present study demonstrates for the first time that an addition of milk cancelled the anti-SARS-CoV-2 effect of black tea due to binding of casein to gTFs.

Effects of tea, catechins and catechin derivatives on Omicron subvariants of SARS-CoV-2.

The Omicron subvariants of SARS-CoV-2 have multiple mutations in the S-proteins and show high transmissibility. We previously reported that tea catechin (-)-epigallocatechin gallate (EGCG) and its derivatives including theaflavin-3,3'-di-O-digallate (TFDG) strongly inactivated the conventional SARS-CoV-2 by binding to the receptor binding domain (RBD) of the S-protein. Here we show that Omicron subvariants were effectively inactivated by green tea, Matcha, and black tea. EGCG and TFDG strongly suppressed infectivity of BA.1 and XE subvariants, while effect on BA.2.75 was weaker. Neutralization assay showed that EGCG and TFDG inhibited interaction between BA.1 RBD and ACE2. In silico analyses suggested that N460K, G446S and F490S mutations in RBDs crucially influenced the binding of EGCG/TFDG to the RBDs. Healthy volunteers consumed a candy containing green tea or black tea, and saliva collected from them immediately after the candy consumption significantly decreased BA.1 virus infectivity in vitro. These results indicate specific amino acid substitutions in RBDs that crucially influence the binding of EGCG/TFDG to the RBDs and different susceptibility of each Omicron subvariant to EGCG/TFDG. The study may suggest molecular basis for potential usefulness of these compounds in suppression of mutant viruses that could emerge in the future and cause next pandemic.

Raman Fingerprints of SARS-CoV-2 Omicron Subvariants: Molecular Roots of Virological Characteristics and Evolutionary Directions.

The latest RNA genomic mutation of SARS-CoV-2 virus, termed the Omicron variant, has generated a stream of highly contagious and antibody-resistant strains, which in turn led to classifying Omicron as a variant of concern. We systematically collected Raman spectra from six Omicron subvariants available in Japan (i.e., BA.1.18, BA.2, BA.4, BA.5, XE, and BA.2.75) and applied machine-learning algorithms to decrypt their structural characteristics at the molecular scale. Unique Raman fingerprints of sulfur-containing amino acid rotamers, RNA purines and pyrimidines, tyrosine phenol ring configurations, and secondary protein structures clearly differentiated the six Omicron subvariants. These spectral characteristics, which were linked to infectiousness, transmissibility, and propensity for immune evasion, revealed evolutionary motifs to be compared with the outputs of genomic studies. The availability of a Raman "metabolomic snapshot", which was then translated into a barcode to enable a prompt subvariant identification, opened the way to rationalize in real-time SARS-CoV-2 activity and variability. As a proof of concept, we applied the Raman barcode procedure to a nasal swab sample retrieved from a SARS-CoV-2 patient and identified its Omicron subvariant by coupling a commercially available magnetic bead technology with our newly developed Raman analyses.

Hypoxia promotes differentiation of pure cartilage from human induced pluripotent stem cells.

While cartilage can be produced from induced pluripotent stem cells (iPSCs), challenges such as long culture periods and compromised tissue purity continue to prevail. The present study aimed to determine whether cartilaginous tissue could be produced from iPSCs under hypoxia and, if so, to evaluate its effects on cellular metabolism and purity of the produced tissue. Human iPSCs (hiPSCs) were cultured for cartilage differentiation in monolayers under normoxia or hypoxia (5% O2), and chondrocyte differentiation was evaluated using reverse transcription­quantitative PCR and fluorescence­activated cell sorting. Subsequently, cartilage differentiation of hiPSCs was conducted in 3D culture under normoxia or hypoxia (5% O2), and the formed cartilage­like tissues were evaluated on days 28 and 56 using histological analyses. Hypoxia suppressed the expression levels of the immature mesodermal markers brachyury (T) and forkhead box protein F1; however, it promoted the expression of the chondrogenic markers Acan and CD44. The number of sex­determining region Y­box 9­positive cells and the percentages of safranin O­positive and type 2 collagen­positive tissues increased under hypoxic conditions. Moreover, upon hypoxia­inducible factor (HIF)­1α staining, nuclei of tissues cultured under hypoxia stained more deeply compared with those of tissues cultured under normoxia. Overall, these findings indicated that hypoxia not only enhanced cartilage matrix production, but also improved tissue purity by promoting the expression of HIF­1α gene. Potentially, pure cartilage­like tissues could be produced rapidly and conveniently using this method.

Senescent endothelial cells are predisposed to SARS-CoV-2 infection and subsequent endothelial dysfunction.

The coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains to spread worldwide. COVID-19 is characterized by the striking high mortality in elderly; however, its mechanistic insights remain unclear. Systemic thrombosis has been highlighted in the pathogenesis of COVID-19, and lung microangiopathy in association with endothelial cells (ECs) injury has been reported by post-mortem analysis of the lungs. Here, we experimentally investigated the SARS-CoV-2 infection in cultured human ECs, and performed a comparative analysis for post-infection molecular events using early passage and replicative senescent ECs. We found that; (1) SARS-CoV-2 infects ECs but does not replicate and disappears in 72 hours without causing severe cell damage, (2) Senescent ECs are highly susceptible to SARS-CoV-2 infection, (3) SARS-CoV-2 infection alters various genes expression, which could cause EC dysfunctions, (4) More genes expression is affected in senescent ECs by SARS-CoV-2 infection than in early passage ECs, which might causes further exacerbated dysfunction in senescent ECs. These data suggest that sustained EC dysfunctions due to SARS-CoV-2 infection may contribute to the microangiopathy in the lungs, leading to deteriorated inflammation and thrombosis in COVID-19. Our data also suggest a possible causative role of EC senescence in the aggravated disease in elder COVID-19 patients.

Raman Molecular Fingerprints of SARS-CoV-2 British Variant and the Concept of Raman Barcode.

The multiple mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have created variants with structural differences in both their spike and nucleocapsid proteins. While the functional relevance of these mutations is under continuous scrutiny, current findings have documented their detrimental impact in terms of affinity with host receptors, antibody resistance, and diagnostic sensitivity. Raman spectra collected on two British variant sub-types found in Japan (QK002 and QHN001) are compared with that of the original Japanese isolate (JPN/TY/WK-521), and found bold vibrational differences. These included: i) fractions of sulfur-containing amino acid rotamers, ii) hydrophobic interactions of tyrosine phenol ring, iii) apparent fractions of RNA purines and pyrimidines, and iv) protein secondary structures. Building upon molecular scale results and their statistical validations, the authors propose to represent virus variants with a barcode specially tailored on Raman spectrum. Raman spectroscopy enables fast identification of virus variants, while the Raman barcode facilitates electronic recordkeeping and translates molecular characteristics into information rapidly accessible by users.

Raman Fingerprints of the SARS-CoV-2 Delta Variant and Mechanisms of Its Instantaneous Inactivation by Silicon Nitride Bioceramics.

Raman spectroscopy uncovered molecular scale markers of the viral structure of the SARS-CoV-2 Delta variant and related viral inactivation mechanisms at the biological interface with silicon nitride (Si3N4) bioceramics. A comparison of Raman spectra collected on the TY11-927 variant (lineage B.1.617.2; simply referred to as the Delta variant henceforth) with those of the JPN/TY/WK-521 variant (lineage B.1.617.1; referred to as the Kappa variant or simply as the Japanese isolate henceforth) revealed the occurrence of key mutations of the spike receptor together with profound structural differences in the molecular structure/symmetry of sulfur-containing amino acid and altered hydrophobic interactions of the tyrosine residue. Additionally, different vibrational fractions of RNA purines and pyrimidines and dissimilar protein secondary structures were also recorded. Despite mutations, hydrolytic reactions at the surface of silicon nitride (Si3N4) bioceramics induced instantaneous inactivation of the Delta variant at the same rate as that of the Kappa variant. Contact between virions and micrometric Si3N4 particles yielded post-translational deimination of arginine spike residues, methionine sulfoxidation, tyrosine nitration, and oxidation of RNA purines to form formamidopyrimidines. Si3N4 bioceramics proved to be a safe and effective inorganic compound for instantaneous environmental sanitation.

Rapid Inactivation In Vitro of SARS-CoV-2 in Saliva by Black Tea and Green Tea.

Saliva plays major roles in the human-to-human transmission of SARS-CoV-2. If the virus in saliva in SARS-CoV-2-infected individuals can be rapidly and efficiently inactivated by a beverage, the ingestion of the beverage may attenuate the spread of virus infection within a population. Recently, we reported that SARS-CoV-2 was significantly inactivated by treatment with black tea, green tea, roasted green tea and oolong tea, as well as their constituents, (-) epigallocatechin gallate (EGCG), theasinensin A (TSA), and galloylated theaflavins. However, it remains unclear to what extent tea inactivates the virus present in saliva, because saliva contains various proteins, nitrogenous products, electrolytes, and so on, which could influence the antivirus effect of tea. Here, we assessed whether tea inactivated the SARS-CoV-2 which was added in human saliva. A virus was added in healthy human saliva in vitro, and after treatment with black tea or green tea, the infectivity of the virus was evaluated by TCID50 assays. The virus titer fell below the detectable level or less than 1/100 after treatment with black tea or green tea for 10 s. The black tea-treated virus less remarkably replicated in cells compared with the untreated virus. These findings suggest the possibility that the ingestion of tea may inactivate SARS-CoV-2 in saliva in infected individuals, although clinical studies are required to determine the intensity and duration of the anti-viral effect of tea in saliva in humans.

Mechanical stimulation of chondrocytes regulates HIF-1α under hypoxic conditions.

We investigated the effects of hypoxia-inducible factor (HIF)-1α on articular cartilage under mechanical stimulation and the associated mechanisms. Chondrocytes, isolated from articular cartilage from the knee, hip, and shoulder joints of Wistar rats, were subjected to 20 % tensile stress under hypoxic (5% O2) conditions for 24 h. HIF-1α and aggrecan expression was significantly enhanced with mechanical stimulation under hypoxia but not significantly altered with mechanical stimulation under normoxia. The nuclear translocation of HIF-1α was enhanced by mechanical stress under hypoxia. Under both normoxia and hypoxia, a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS) 5 expression was significantly reduced with mechanical stimulation compared to that in the group without mechanical stimulation. However, HIF-1α knockdown mitigated changes in aggrecan and ADAM-TS5 expression mediated by mechanical stimulation under hypoxia. The effects of treadmill running on HIF-1α production in the articular cartilage of rat knee joints were also analyzed. HIF-1α production increased in the moderate running group and decreased to the same levels as those in the control group in the excessive running group. This suggests that HIF-1α regulates aggrecan and ADAM-TS5 expression in response to mechanical stimulation under hypoxia and general mechanical stimulation in articular cartilage under hypoxia, while controlling cartilage homeostasis.

Involvement of IL-17A in the pathogenesis of DSS-induced colitis in mice.

To investigate the etiological implication of IL-17A in inflammatory bowel disease (IBD), dextran sodium sulfate (DSS) was administered to the mice deficient for the IL-17A gene. They showed only faint manifestations of colitis, as revealed by body weight loss, shrinkage in the colon length, serum haptoglobin concentration, and disease activity index. Although the mortality rate of WT mice reached approximately 60%, more than 90% of the IL-17A KO mice survived the DSS treatment. Histological change was also marginal in the IL-17A KO intestine, in which epithelial damage and inflammatory infiltrates were not obvious and the myeloperoxidase activity elevated only slightly. G-CSF and MCP-1 were abundantly produced in WT mouse intestine, whereas the production of these chemokines was drastically hampered in IL-17A-null intestine. The present results show that IL-17A plays a pivotal role in the pathogenesis of DSS-induced colitis, while MCP-1 and G-CSF may be crucially involved in the IL-17A-induced inflammation.

Pleiotrophic functions of Epstein-Barr virus nuclear antigen-1 (EBNA-1) and oriP differentially contribute to the efficiency of transfection/expression of exogenous gene in mammalian cells.

The EBNA1 gene and oriP sequence, originally derived from the EBV genome, provide plasmid vectors with artificial chromosome (AC)-like characteristics, including cytoplasm-to-nuclear transport, nuclear retention, replication and segregation of the DNA, while transcriptional up-regulation has been suggested as another activity of the EBNA1/oriP. Transfection as well as expression rates of various nonviral delivery vehicles are highly improved by inserting these genetic elements into plasmid DNA constructs. Here we differentially analyzed the contribution of each function of the EBNA1/oriP to the efficacy of electroporation-mediated genetic delivery and expression in mammalian cells. It was found that the EBNA1/oriP-mediated acceleration of genetic delivery and expression was predominantly due to the promotion of cytoplasm-to-nuclear recruitment as well as enhancement of transcription, while the episomal replication of the EBV-AC was not essentially involved.

Soluble TRAIL gene and actinomycin D synergistically suppressed multiple metastasis of TRAIL-resistant colon cancer in the liver.

Metastatic liver tumors are highly malignant and refractory to conventional therapies. TRAIL-resistant CT-26 cells underwent apoptosis in vitro in the presence of both recombinant TRAIL (rTRAIL) and a suboptimal dose of actinomycin D (ACD). Co-administration of soluble TRAIL (sTRAIL) gene and ACD suppressed the metastatic liver tumors of CT-26, significantly inducing apoptosis in the tumors, while such effects were not demonstrated in mice that received either the sTRAIL gene or ACD alone. The gene therapy of sTRAIL with a suboptimal dose of an anticancer drug is a new strategy for treatment of multiple liver metastasis.

Sonoporation mediated transduction of pDNA/siRNA into joint synovium in vivo.

The purpose of this study was to investigate the usefulness of sonoporation method on in vivo transduction of plasmid DNA (pDNA) and small interfering RNA (siRNA) into joint tissue. pGEG.GL3 plasmid was mixed with microbubble and injected into knee joints of rats. Ultrasound sonication was performed percutaneously. Three days after injection, GL3 expression of synovial tissue was determined by luciferase assay and RT-PCR. siRNA specific for GL3 (siGL3) or nonspecific siRNA were mixed with pGEG.GL3 plasmid and transduced by sonoporation. siRNA specific for EGFP (siEGFP) was transduced into the knee joints of EGFP transgenic rats, and gene silencing effects for endogenous gene were examined. To determine the localization of transduced siRNA, fluorescently labeled siRNA was transduced into joints. The expression of GL3 in the synovium was significantly enhanced by sonoporation. The gene expression was only seen in the synovium of the knee joint. The expression of GL3 was remarkably suppressed by co-transduction of siGL3, but not suppressed by nonspecific siRNA. siEGFP transduced by sonoporation attenuated green fluorescence on the surface layer of synovium of EGFP transgenic rats. The fluorescently labeled siRNA was seen in the synovium around the patella, femur, and tibia. Sonoporation is examined as a recent, novel, gene transduction method, and the advantage of this technique is minimal invasiveness. In this study, we showed that pDNA/siRNA can be transduced specifically into the joint synovium using sonoporation. The present method may be useful in nucleic acid therapy for joint disorders.

Combination vaccine of dendritic cells (DCs) and T cells effectively suppressed preestablished malignant melanoma in mice.

The study aims at establishing a novel vaccine procedure, using bone marrow-derived DCs that have ingested apoptotic B16 melanoma (DCs(+)), alone or in combination with splenic T lymphocytes from a syngenic donor. Co-immunization with DCs(+) and T cells showed the highest antitumor potential against preestablished B16 tumor in mice, in which CTL and NK cytotoxicities were drastically elevated, while either DCs(+) alone, naive DCs (DCs(-)) alone, or a mixture of DCs(-) and T cells induced less significant therapeutic outcomes. Use of extracellular matrix proteins elevated antitumor activity of DC(-)/T cell vaccine. Compared with the CD8(+) cells, the CD4(+)T cells more remarkably improved the efficacy of DC-based immunotherapy. The present system may be a feasible therapeutic modality to eradicate malignancies including melanoma.

Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy.

Multiple sclerosis (MS) is a T cell-mediated autoimmune disease. Fingolimod, a highly effective disease-modifying drug for MS, retains CCR7+ central memory T cells in which autoaggressive T cells putatively exist, in secondary lymphoid organs, although relapse may still occur in some patients. Here, we analyzed the T cell phenotypes of fingolimod-treated, fingolimod-untreated patients, and healthy subjects. The frequency of CD56+ T cells and granzyme B-, perforin-, and Fas ligand-positive T cells significantly increased during fingolimod treatment. Each T cell subpopulation further increased during relapse. Interestingly, T cells from fingolimod-treated patients exhibited interferon-γ biased production, and more myelin basic protein-reactive cells was noted in CD56+ than in CD56- T cells. It is likely that the altered T cell phenotypes play a role in MS relapse in fingolimod-treated patients. Further clinical studies are necessary to investigate whether altered T cell phenotypes are a biomarker for relapse under fingolimod therapy.