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.

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.

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.

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.

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.

In Situ Raman Analysis of Biofilm Exopolysaccharides Formed in Streptococcus mutans and Streptococcus sanguinis Commensal Cultures.

This study probed in vitro the mechanisms of competition/coexistence between Streptococcus sanguinis (known for being correlated with health in the oral cavity) and Streptococcus mutans (responsible for aciduric oral environment and formation of caries) by means of quantitative Raman spectroscopy and imaging. In situ Raman assessments of live bacterial culture/coculture focusing on biofilm exopolysaccharides supported the hypothesis that both species engaged in antagonistic interactions. Experiments of simultaneous colonization always resulted in coexistence, but they also revealed fundamental alterations of the biofilm with respect to their water-insoluble glucan structure. Raman spectra (collected at fixed time but different bacterial ratios) showed clear changes in chemical bonds in glucans, which pointed to an action by Streptococcus sanguinis to discontinue the impermeability of the biofilm constructed by Streptococcus mutans. The concurrent effects of glycosidic bond cleavage in water-insoluble α - 1,3-glucan and oxidation at various sites in glucans' molecular chains supported the hypothesis that secretion of oxygen radicals was the main "chemical weapon" used by Streptococcus sanguinis in coculture.

Raman Metabolomics of Candida auris Clades: Profiling and Barcode Identification.

This study targets on-site/real-time taxonomic identification and metabolic profiling of seven different Candida auris clades/subclades by means of Raman spectroscopy and imaging. Representative Raman spectra from different Candida auris samples were systematically deconvoluted by means of a customized machine-learning algorithm linked to a Raman database in order to decode structural differences at the molecular scale. Raman analyses of metabolites revealed clear differences in cell walls and membrane structure among clades/subclades. Such differences are key in maintaining the integrity and physical strength of the cell walls in the dynamic response to external stress and drugs. It was found that Candida cells use the glucan structure of the extracellular matrix, the degree of α-chitin crystallinity, and the concentration of hydrogen bonds between its antiparallel chains to tailor cell walls' flexibility. Besides being an effective ploy in survivorship by providing stiff shields in the α-1,3-glucan polymorph, the α-1,3-glycosidic linkages are also water-insoluble, thus forming a rigid and hydrophobic scaffold surrounded by a matrix of pliable and hydrated ß-glucans. Raman analysis revealed a variety of strategies by different clades to balance stiffness, hydrophobicity, and impermeability in their cell walls. The selected strategies lead to differences in resistance toward specific environmental stresses of cationic/osmotic, oxidative, and nitrosative origins. A statistical validation based on principal component analysis was found only partially capable of distinguishing among Raman spectra of clades and subclades. Raman barcoding based on an algorithm converting spectrally deconvoluted Raman sub-bands into barcodes allowed for circumventing any speciation deficiency. Empowered by barcoding bioinformatics, Raman analyses, which are fast and require no sample preparation, allow on-site speciation and real-time selection of appropriate treatments.

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 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.

Raman Study of Pathogenic Candida auris: Imaging Metabolic Machineries in Reaction to Antifungal Drugs.

The multidrug-resistant Candida auris often defies treatments and presently represents a worldwide public health threat. Currently, the ergosterol-targeting Amphotericin B (AmB) and the DNA/RNA-synthesis inhibitor 5-flucytosine (5-FC) are the two main drugs available for first-line defense against life-threatening Candida auris infections. However, important aspects of their mechanisms of action require further clarification, especially regarding metabolic reactions of yeast cells. Here, we applied Raman spectroscopy empowered with specifically tailored machine-learning algorithms to monitor and to image in situ the susceptibility of two Candida auris clades to different antifungal drugs (LSEM 0643 or JCM15448T, belonging to the East Asian Clade II; and, LSEM 3673 belonging to the South African Clade III). Raman characterizations provided new details on the mechanisms of action against Candida auris Clades II and III, while also unfolding differences in their metabolic reactions to different drugs. AmB treatment induced biofilm formation in both clades, but the formed biofilms showed different structures: a dense and continuous biofilm structure in Clade II, and an extra-cellular matrix with a "fluffy" and discontinuous structure in Clade III. Treatment with 5-FC caused no biofilm formation but yeast-to-hyphal or pseudo-hyphal morphogenesis in both clades. Clade III showed a superior capacity in reducing membrane permeability to the drug through chemically tailoring chitin structure with a high degree of acetylation and fatty acids networks with significantly elongated chains. This study shows the suitability of the in situ Raman method in characterizing susceptibility and stress response of different C. auris clades to antifungal drugs, thus opening a path to identifying novel clinical solutions counteracting the spread of these alarming pathogens.

Raman Spectroscopy of Oral Candida Species: Molecular-Scale Analyses, Chemometrics, and Barcode Identification.

Oral candidiasis, a common opportunistic infection of the oral cavity, is mainly caused by the following four Candida species (in decreasing incidence rate): Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei. This study offers in-depth Raman spectroscopy analyses of these species and proposes procedures for an accurate and rapid identification of oral yeast species. We first obtained average spectra for different Candida species and systematically analyzed them in order to decode structural differences among species at the molecular scale. Then, we searched for a statistical validation through a chemometric method based on principal component analysis (PCA). This method was found only partially capable to mechanistically distinguish among Candida species. We thus proposed a new Raman barcoding approach based on an algorithm that converts spectrally deconvoluted Raman sub-bands into barcodes. Barcode-assisted Raman analyses could enable on-site identification in nearly real-time, thus implementing preventive oral control, enabling prompt selection of the most effective drug, and increasing the probability to interrupt disease transmission.

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 Imaging of Pathogenic Candida auris: Visualization of Structural Characteristics and Machine-Learning Identification.

Invasive fungal infections caused by yeasts of the genus Candida carry high morbidity and cause systemic infections with high mortality rate in both immunocompetent and immunosuppressed patients. Resistance rates against antifungal drugs vary among Candida species, the most concerning specie being Candida auris, which exhibits resistance to all major classes of available antifungal drugs. The presently available identification methods for Candida species face a severe trade-off between testing speed and accuracy. Here, we propose and validate a machine-learning approach adapted to Raman spectroscopy as a rapid, precise, and labor-efficient method of clinical microbiology for C. auris identification and drug efficacy assessments. This paper demonstrates that the combination of Raman spectroscopy and machine learning analyses can provide an insightful and flexible mycology diagnostic tool, easily applicable on-site in the clinical environment.

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.

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.

Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.

Antifungal activity of polymethyl methacrylate/Si3N4 composites against Candida albicans.

Previous studies using gram-positive and -negative bacteria demonstrated that hydrolysis of silicon nitride (Si3N4) in aqueous suspensions elutes nitrogen and produces gaseous ammonia while buffering pH. According to immunochemistry assays, fluorescence imaging, and in situ Raman spectroscopy, we demonstrate here that the antipathogenic surface chemistry of Si3N4 can be extended to polymethylmethacrylate (PMMA) by compounding it with a minor fraction (~8 vol.%) of Si3N4 particles without any tangible loss in bulk properties. The hydrolytic products, which were eluted from partly exposed Si3N4 particles at the composite surface, exhibited fungicidal action against Candida albicans. Using a specific nitrative stress sensing dye and highly resolved fluorescence micrographs, we observed in situ congestion of peroxynitrite (ONOO-) radicals in the mitochondria of the Candida cells exposed to the PMMA/Si3N4 composite, while these radicals were absent in the mitochondria of identical cells exposed to monolithic PMMA. These in situ observations suggest that the surface chemistry of Si3N4 mimics the antifungal activity of macrophages, which concurrently produce NO radicals and superoxide anions (O2•-) resulting in the formation of candidacidal ONOO-. The fungicidal properties of PMMA/Si3N4 composites could be used in dental appliances to inhibit the uncontrolled growth of Candida albicans and ensuing candidiasis while being synergic with chemoprophylaxis. STATEMENT OF SIGNIFICANCE: In a follow-up of previous studies of gram-positive and gram-negative bacteria, we demonstrate here that the antipathogenic surface chemistry of Si3N4 could be extended to polymethylmethacrylate (PMMA) containing a minor fraction (~8 vol.%) of Si3N4 particles without tangible loss in bulk properties. Hydrolytic products eluted from Si3N4 particles at the composite surface exhibited fungicidal action against Candida albicans. Highly resolved fluorescence microscopy revealed congestion of peroxynitrite (ONOO-) radicals in the mitochondria of the Candida cells exposed to the PMMA/Si3N4 composite, while radicals were absent in the mitochondria of identical cells exposed to monolithic PMMA. The fungicidal properties of PMMA/Si3N4 composites could be used in dental appliances to inhibit uncontrolled growth of Candida albicans and ensuing candidiasis in synergy with chemoprophylaxis.

Silicon nitride: a potent solid-state bioceramic inactivator of ssRNA viruses.

Surface inactivation of human microbial pathogens has a long history. The Smith Papyrus (2600 ~ 2200 B.C.) described the use of copper surfaces to sterilize chest wounds and drinking water. Brass and bronze on doorknobs can discourage microbial spread in hospitals, and metal-base surface coatings are used in hygiene-sensitive environments, both as inactivators and modulators of cellular immunity. A limitation of these approaches is that the reactive oxygen radicals (ROS) generated at metal surfaces also damage human cells by oxidizing their proteins and lipids. Silicon nitride (Si3N4) is a non-oxide ceramic compound with known surface bacterial resistance. We show here that off-stoichiometric reactions at Si3N4 surfaces are also capable of inactivating different types of single-stranded RNA (ssRNA) viruses independent of whether their structure presents an envelop or not. The antiviral property of Si3N4 derives from a hydrolysis reaction at its surface and the subsequent formation of reactive nitrogen species (RNS) in doses that could be metabolized by mammalian cells but are lethal to pathogens. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of viral RNA and in situ Raman spectroscopy suggested that the products of Si3N4 hydrolysis directly react with viral proteins and RNA. Si3N4 may have a role in controlling human epidemics related to ssRNA mutant viruses.

Surface functionalization of PEEK with silicon nitride.

Surface roughness, bioactivity, and antibacterial properties are desirable in skeletal implants. We hot-pressed a mix of particulate sodium chloride (NaCl) salt and silicon nitride (ß-Si3N4) onto the surface of bulk PEEK. NaCl grains were removed by leaching in water, resulting in a porous PEEK surface embedded with sim15 vol% ß-Si3N4particles. This functionalized surface showed the osteogenic and antibacterial properties previously reported in bulk silicon nitride implants. Surface enhancement of PEEK with ß-Si3N4could improve the performance of spinal fusion cages, by facilitating arthrodesis and resisting bacteria.