G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol.

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy-in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.

Coccomyxa sp.KJ extract affects the fate of T cells stimulated by toxic shock syndrome toxin-1, a superantigen secreted by Staphylococcus aureus.

T cell stimulation by bacterial superantigens induces a cytokine storm. After T cell activation and inflammatory cytokine secretion, regulatory T cells (Treg) are produced to suppress the immune response. Coccomyxa sp.KJ (IPOD FERM BP-22254), a green alga, is reported to regulate immune reactions. Therefore, we examined the effects of Coccomyxa sp.KJ extract (CE) on the superantigen-induced immune response. When human peripheral blood mononuclear cells (PBMCs) were stimulated with toxic shock syndrome-1 (TSST-1) in the presence of CE, the number of activated T cells decreased moderately. Purified T cells stimulated in the presence of CE comprised more non-proliferating cells than those stimulated in the absence of CE, whereas some T cells proliferated more quickly. The levels of activation markers on the stimulated T cells increased in the presence of CE. Most of the inflammatory cytokines did not change but IL-1ß, IL-17, IL-4, and IL-13 secretion increased, whereas that of IL-2, TNF-α, and IL-18 decreased. IL-10 secretion was also decreased by CE treatment, suggesting that the immune response was not suppressed by Treg cells. CE enhanced the expression of stem cell-like memory cell markers in T cells. These results suggest that CE can regulate the fate of T cells and can help to ameliorate superantigen-induced T cell hyperactivation and immune suppression.

Virucidal effect of monogalactosyl diacylglyceride from a green microalga, Coccomyxa sp. KJ, against clinical isolates of SARS-CoV-2 as assessed by a plaque assay.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and is capable of human-to-human transmission and rapid global spread. Thus, the establishment of high-quality viral detection and quantification methods, and the development of anti-SARS-CoV-2 agents are critical. METHODS: Here, we present the rapid detection of infectious SARS-CoV-2 particles using a plaque assay with 0.5% agarose-ME (Medium Electroosmosis) as an overlay medium. RESULTS: The plaques were capable of detecting the virus within 36-40 h post-infection. In addition, we showed that a monogalactosyl diacylglyceride isolated from a microalga (Coccomyxa sp. KJ) could inactivate the clinical isolates of SARS-CoV-2 in a time- and concentration-dependent manner. CONCLUSIONS: These results would allow rapid quantification of the infectious virus titers and help develop more potent virucidal agents against SARS-CoV-2.

Virucidal and Immunostimulating Activities of Monogalactosyl Diacylglyceride from Coccomyxa sp. KJ, a Green Microalga, against Murine Norovirus and Feline Calicivirus.

Human noroviruses are the most common pathogens causing acute gastroenteritis and may lead to more severe illnesses among immunosuppressed people, including elderly and organ transplant recipients. To date, there are no safe and effective vaccines or antiviral agents for norovirus infections. In the present study, we aimed to demonstrate the antiviral activity of monogalactosyl diacylglyceride (MGDG) isolated from a microalga, Coccomyxa sp. KJ, against murine norovirus (MNV) and feline calicivirus (FCV), the surrogates for human norovirus. MGDG showed virucidal activities against these viruses in a dose- and time-dependent manner-MGDG at 100 µg/mL reduced the infectivity of MNV and FCV to approximately 10% after 60 min incubation. In the animal experiments of MNV infection, intraoral administration of MGDG (1 mg/day) exerted a therapeutic effect by suppressing viral shedding in the feces and produced high neutralizing antibody titers in sera and feces. When MGDG was orally administered to immunocompromised mice treated with 5-fluorouracil, the compound exhibited earlier stopping of viral shedding and higher neutralizing antibody titers of sera than those in the control mice administered with distilled water. Thus, MGDG may offer a new therapeutic and prophylactic alternative against norovirus infections.

Immune and allergenic effects of the microalga Coccomyxa sp. strain KJ in healthy humans: A pilot study.

BACKGROUND: The Coccomyxa sp. strain KJ (Coccomyxa KJ), a microalga found in Japan, has a potential function in controlling viral infections. Recently, its dry powder has been marketed as a health food product. OBJECTIVES: This pilot study investigated the effects of Coccomyxa KJ powder tablet intake on allergic reactions and immune functions in healthy participants. MATERIAL AND METHODS: Nine healthy volunteers (4 males and 5 females) who expressed interest in foods containing Coccomyxa KJ, and were willing to undergo blood tests, were recruited. Each individual was asked to take 2 Coccomyxa KJ powder tablets (0.3 g) before breakfast once a day for 4 weeks. The salivary immunoglobulin A (IgA) level and blood parameters (white blood cell (WBC) count, eosinophil and lymphocyte counts and percentages, natural killer (NK) cell activity, interleukin (IL)-6 level, and T helper (Th)1/Th2 cell ratio) were evaluated at baseline and weeks 2 and 4. RESULTS: The 4-week intake of Coccomyxa KJ did not affect salivary IgA levels, WBC count, eosinophil and lymphocyte counts and percentages, or the Th1/Th2 ratio. There were significant differences in the NK cell activity after 4 weeks, with an average increase of 11.78 (95% confidence interval (95% CI): 6.80-16.76). None of the patients experienced adverse reactions during or after the study. CONCLUSIONS: Long-term Coccomyxa KJ intake improved NK cell activity without causing adverse effects on the indicators of local immunity, systemic inflammation and immune response balance. This study suggests that Coccomyxa KJ powder tablets can induce beneficial immune modifications without causing any adverse effects.

Coccomyxa subellipsoidea KJ Components Enhance the Expression of Metallothioneins and Th17 Cytokines during Human T Cell Activation.

Coccomyxa subellipsoidea KJ (C-KJ) is a green alga with unique immunoregulatory characteristics. Here, we investigated the mechanism underlying the modification of T cell function by C-KJ components. The water-soluble extract of C-KJ was fractionated into protein (P) and sugar (S) fractions acidic (AS), basic (BS), and neutral (NS). These fractions were used for the treatment of peripheral blood mononuclear cells stimulated with toxic shock syndrome toxin-1. Transcriptome analysis revealed that both P and AS enhanced the expression of the genes encoding metallothionein (MT) family proteins, inflammatory factors, and T helper (Th) 17 cytokine and suppressed that of those encoding Th2 cytokines in stimulated T cells. The kinetics of MT1 and MT2A gene expression showed a transient increase in MT1 and maintenance of MT2A mRNA after T cell stimulation in the presence of AS. The kinetics of Th17-related cytokine secretion in the early period were comparable to those of MT2A mRNA. Furthermore, our findings revealed that static, a STAT-3 inhibitor, significantly suppressed MT2A gene expression. These findings suggest that the expression of MTs is involved in the immune regulatory function of C-KJ components, which is partially regulated by Th17 responses, and may help develop innovative immunoregulatory drugs or functional foods.

Electrical biosensing system utilizing ion-producing enzymes conjugated with aptamers for the sensing of severe acute respiratory syndrome coronavirus 2.

Viral outbreaks, which include the ongoing coronavirus disease 2019 (COVID-19) pandemic provoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are a major global crisis that enormously threaten human health and social activities worldwide. Consequently, the rapid and repeated treatment and isolation of these viruses to control their spread are crucial to address the COVID-19 pandemic and future epidemics of novel emerging viruses. The application of cost-efficient, rapid, and easy-to-operate detection devices with miniaturized footprints as a substitute for the conventional optic-based polymerase chain reaction (PCR) and immunoassay tests is critical. In this context, semiconductor-based electrical biosensors are attractive sensing platforms for signal readout. Therefore, this study aimed to examine the electrical sensing of patient-derived SARS-CoV-2 samples by harnessing the activity of DNA aptamers directed against spike proteins on viral surfaces. We obtained rapid and sensitive virus detection beyond the Debye length limitation by exploiting aptamers coupled with alkaline phosphatases, which catalytically generate free hydrogen ions which can readily be measured on pH meters or ion-sensitive field-effect transistors. Furthermore, we demonstrated the detection of the viruses of approximately 100 copies/µL in 10 min, surpassing the capability of typical immunochromatographic assays. Therefore, our newly developed technology has great potential for point-of-care testing not only for SARS-CoV-2, but also for other types of pathogens and biomolecules.

Morphology, Ultrastructure, and Molecular Phylogeny of Aphelidium collabens sp. nov. (Aphelida), a Parasitoid of a Green Alga Coccomyxa sp.

Aphelids (Aphelida) are intracellular parasitoids of algae and represent one of the early diverging or sister lineages of the kingdom Fungi. Although Aphelida is a small group, molecular phylogenetic analyses revealed that many environmental sequences belong to Aphelida, suggesting that aphelids are distributed worldwide; however, the extent of their diversity is unclear. Here, we investigated a novel aphelid culture APH2 that parasitizes the green alga Coccomyxa sp. APH2 produced posteriorly uniflagellate zoospores, a defining character of the genus Aphelidium. The residual body of APH2 was spherical in the mature plasmodium, but became amorphous during zoospore formation and collapsed after zoospore discharge, which has not been described for other Aphelidium species. Zoospores of APH2 possessed a striated rhizoplast that extended anteriorly from the kinetosome to the posterior end of the nucleus, and a microtubular root arising from the side of the kinetosome and lying almost parallel to the rhizoplast, both of which are unique among aphelid taxa. A molecular phylogenetic analysis based on the 18S rDNA sequences placed APH2 as sister lineage to all other known aphelid sequences. Based on these results, we describe this aphelid as a new species, Aphelidium collabens.