Serodiagnosis of multiple cancers using an extracellular protein kinase A autoantibody-based lateral flow platform.

Extracellular protein kinase A autoantibody (ECPKA-AutoAb) has been suggested as a universal cancer biomarker due to its higher amounts in serum of several types of cancer patients than that of normal individuals. Herein, we first developed a lateral flow immunoassay (LFIA) tool, using a sandwich format, toward ECPKA-AutoAb in human serum. For this format, 3G2 as a capture antibody was identified using hybridoma technique and a series of screenings where it showed superior capacity to recognize Enzo PKA catalytic subunit alpha (Cα), compared to other PKA antibodies and antigens. Using these components, we performed sandwich ELISA toward a mimic and real sample of ECPKA-AutoAb. As per the results, limit of detection (LOD) was found to be 135 ng/mL and ECPKA-AutoAb levels were higher in various cancer patients than in normal individuals like previous studies. Based on these results, we applied this sandwich format into LFIA tool and found that the LOD of the fabricated LFIA tool showed about 3.8 ng/mL using spiked PKA-Ab, which is significantly improved compared to the LOD of sandwich ELISA. Also, the developed LFIA tool demonstrated a remarkable ability to detect significant differences in ECPKA-AutoAb levels between normal and cancer patients within 15 min, showing a potential for point-of-care (PoC) detection. One interesting point is that our LFIA strip contains an additional conjugation pad II, named because of its position behind the conjugation pad, in which PKA Cα is dried, enabling a sandwich format.

Molecular Analysis of the Avian H7 Influenza Viruses Circulating in South Korea during 2018-2019: Evolutionary Significance and Associated Zoonotic Threats.

Avian influenza virus (AIV) subtypes H5 and H7, possessing the ability to mutate spontaneously from low pathogenic (LP) to highly pathogenic (HP) variants, are major concerns for enormous socio-economic losses in the poultry industry, as well as for fatal human infections. Through antigenic drift and shift, genetic reassortments of the genotypes pose serious threats of increased virulence and pathogenicity leading to potential pandemics. In this study, we isolated the H7-subtype AIVs circulating in the Republic of Korea during 2018-2019, and perform detailed molecular analysis to study their circulation, evolution, and possible emergence as a zoonotic threat. Phylogenetic and nucleotide sequence analyses of these isolates revealed their distribution into two distinct clusters, with the HA gene sharing the highest nucleotide identity with either the A/common teal/Shanghai/CM1216/2017, isolated from wild birds in Shanghai, China, or the A/duck/Shimane/2014, isolated from Japan. Mutations were found in HA (S138A (H3 numbering)), M1 (N30D and T215A), NS1 (P42S), PB2 (L89V), and PA (H266R and F277S) proteins-the mutations had previously been reported to be related to mammalian adaptation and changes in the virulence of AIVs. Taken together, the results firmly put forth the demand for routine surveillance of AIVs in wild birds to prevent possible pandemics arising from reassortant AIVs.

Development and Clinical Evaluation of an Immunochromatography-Based Rapid Antigen Test (GenBody™ COVAG025) for COVID-19 Diagnosis.

Antigen tests for SARS-CoV-2 diagnosis are simpler and faster than their molecular counterparts. Clinical validation of such tests is a prerequisite before their field applications. We developed and clinically evaluated an immunochromatographic immunoassay, GenBody™ COVAG025, for the rapid detection of SARS-CoV-2 nucleocapsid (NP) antigen in two different clinical studies. Retrospectively, 130 residual nasopharyngeal swabs transferred in viral transport medium (VTM), pre-examined for COVID-19 through emergency use authorization (EUA)-approved real-time RT-PCR assay and tested with GenBody™ COVAG025, revealed a sensitivity and specificity of 90.00% (27/30; 95% CI: 73.47% to 97.89%) and 98.00% (98/100; 95% CI: 92.96% to 99.76%), respectively, fulfilling WHO guidelines. Subsequently, the prospective examination of 200 symptomatic and asymptomatic nasopharyngeal swabs, collected on site and tested with GenBody™ COVAG025 and EUA-approved real-time RT-PCR assay simultaneously, revealed a significantly higher sensitivity and specificity of 94.00% (94/100; 95% CI: 87.40% to 97.77%) and 100.00% (100/100; 95% CI: 96.38% to 100.00%), respectively. Clinical sensitivity and specificity were significantly high for samples with Ct values ≤ 30 as well as within 3 days of symptom onset, justifying its dependency on the viral load. Thus, it is assumed this can help with the accurate diagnosis and timely isolation and treatment of patients with COVID-19, contributing to better control of the global pandemic.

Clinical Evaluation of an Immunochromatographic-Based IgM/IgG Antibody Assay (GenBody™ COVI040) for Detection of Antibody Seroconversion in Patients with SARS-CoV-2 Infection.

There is a need for accurate diagnostic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19). This study aimed to evaluate the diagnostic accuracy of an immunochromatography-based immunoglobulin G (IgG)/immunoglobulin M (IgM) antibody assay (GenBody™ COVI040) for detecting SARS-CoV-2 antibody seroconversion in COVID-19 patients. A total of 130 samples, serially collected from patients with confirmed COVID-19, and 100 negative control samples were tested for anti-SARS-CoV-2 IgM and IgG using the GenBody™ COVI040 assay following the South Korean Ministry of Food and Drug Safety guidelines on the review and approval of in vitro diagnostic devices for COVID-19. Reverse-transcription polymerase chain reaction results were used as the comparator. The overall sensitivity of the GenBody™ COVI040 assay was 97.69% (95% confidence interval (CI): 93.40-99.52%). The sensitivity of the assay increased with time post symptom onset (PSO) (sensitivity ≤6 days PSO: 78.57%, 95% CI: 49.20-95.34%; sensitivity 7-13 days PSO: 100%, 95% CI: 87.23-100%; and sensitivity ≥14 days PSO: 100%, 95% CI: 95.94-100%). The specificity of the assay was 100% (95% CI: 96.38-100%). The GenBody™ COVI040 assay showed high sensitivity and specificity, making it a promising diagnostic test to monitor COVID-19.

Evaluation of cell-surface displayed synthetic consensus dengue EDIII cells as a potent oral vaccine candidate.

BACKGROUND: Dengue is a rapidly spreading mosquito borne tropical viral disease affecting hundreds of millions of people across the globe annually. The dengue virus (DENV) includes four genetically distinct serotypes that cause serious life-threatening infections, including dengue hemorrhagic fever/dengue shock syndrome. Dengue vaccine development is complicated by the possibility of vaccine-enhanced severe dengue disease due to antibody-dependent enhancement by pre-existing cross-reactivity, as well as homotypic antibodies. Thus, the development of an efficacious dengue vaccine conferring simultaneous and durable immunity to each of the four DENV serotypes has not yet been developed despite years of research. For mass immunization in deeply affected resource-limited countries, oral vaccination is considered more beneficial than conventional approaches. Therefore, in a continuing effort towards designing economical and potent vaccine candidates, the current study applied yeast surface display technology to develop an oral dengue vaccine candidate using whole recombinant yeast cells displaying the recombinant fusion protein of M cell targeting ligand Co1 fused to the synthetic consensus dengue envelope domain III (scEDIII). Female Balb/c mice were orally fed with recombinant yeast cells and immunogenicity in terms of systemic and mucosal immune responses was monitored. RESULTS: Immunofluorescence microscopy with dengue specific antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG antibody clearly showed that recombinant protein Co1-scEDIII-AGA was localized on the cell surface of the respective clones in comparison with scEDIII-Co1 and Mock cells with no fluorescence. Oral dosage applications of surface displayed Co1-scEDIII-AGA stimulated a systemic humoral immune response in the form of dengue-specific serum IgG, as well as a mucosal immune response in the form of secretory immunoglobulin A (sIgA). Antigen-specific B cell responses in isolated lymphoid cells from the spleen and Peyer's patches further supported an elevated mucosal immune response. In addition, surface displayed Co1-scEDIII-AGA feeding elicited strong immune responses in comparison with scEDIII-Co1 and Mock following intraperitoneal booster with purified scEDIII antigen. CONCLUSIONS: Surface displayed preparations of Co1-scEDIII-AGA induced strong immunogenicity compared with non-displayed scEDIII-Co1. Prior studies have supported the neutralization potential of scEDIII constructs against all four serotypes. Thus, the oral administration of genetically engineered yeast whole cells displaying biologically active Co1-scEDIII fusion protein without any further processing shows prospective as a potent oral vaccine candidate against dengue viral infection.

Global DNA Methylation in the Chestnut Blight Fungus Cryphonectria parasitica and Genome-Wide Changes in DNA Methylation Accompanied with Sectorization.

Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK) of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase), demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

Comparative immunogenicity of preparations of yeast-derived dengue oral vaccine candidate.

BACKGROUND: Dengue is listed as a neglected tropical disease by the Center for Disease Control and Preservation, as there are insufficient integrated surveillance strategies, no effective treatment, and limited licensed vaccines. Consisting of four genetically distinct serotypes, dengue virus (DENV) causes serious life-threatening infections due to its complexity. Antibody-dependent enhancement by pre-existing cross-reactive as well as homotypic antibodies further worsens the clinical symptoms of dengue. Thus, a vaccine conferring simultaneous and durable immunity to each of the four DENV serotypes is essential to restrict its escalation. In deeply affected resource-limited countries, oral vaccination using food-grade organisms is considered to be a beneficial approach in terms of costs, patient comfort, and simple logistics for mass immunization. The current study used a mouse model to explore the immunogenicity of an oral dengue vaccine candidate prepared using whole recombinant yeast cells (WC) and cell-free extracts (CFE) from cells expressing recombinant Escherichia coli heat-labile toxin protein B-subunit (LTB) fused to the consensus dengue envelope domain III (scEDIII). Mice were treated orally with recombinant WC and CFE vaccines in 2-week intervals for 4 weeks and changes in systemic and mucosal immune responses were monitored. RESULTS: Both WC and CFE dosage applications of LTB-scEDIII stimulated a systemic humoral immune response in the form of dengue-specific serum IgG as well as mucosal immune response in the form of secretory sIgA. Antigen-specific B cell responses in isolated lymphoid cells from the spleen and Peyer's patches further indicated an elevated mucosal immune response. Cellular immune response estimated through lymphocyte proliferation assay indicated higher levels in CFE than WC dosage. Furthermore, sera obtained after both oral administrations successfully neutralized DENV-1, whereas CFE formulation only neutralized DENV-2 serotype, two representative serotypes which cause severe dengue infection. Sera from mice that were fed CFE preparations demonstrated markedly higher neutralizing titers compared to those from WC-fed mice. However, WC feeding elicited strong immune responses, which were similar to the levels induced by CFE feeding after intraperitoneal booster with purified scEDIII antigen. CONCLUSIONS: CFE preparations of LTB-scEDIII produced strong immunogenicity with low processing requirements, signifying that this fusion protein shows promise as a potent oral vaccine candidate against dengue viral infection.

Effects of initial moisture content of Korean traditional wheat-based fermentation starter nuruk on microbial abundance and diversity.

The brewing of makgeolli, one of Korea's most popular alcoholic beverages that is gaining popularity globally, is facilitated by nuruk, a traditional Korean cereal starter. The nuruk microbiome greatly influences the fermentation process as well as the nutritional, hygienic, and aromatic qualities of the product. This study is a continuation of our efforts to examine nuruk biodiversity at a depth previously unattainable. In this study, microfloral dynamics in wheat-based nuruk C, composed of traditional ingredients such as barley, green gram, and wheat and fermented under various internal moisture contents of 20% (C20), 26% (C26), and 30% (C30), was evaluated using 454 pyrosequencing during the 30-day fermentation process. Rarefaction analysis and alpha diversity parameters indicated adequate sampling. C20 showed the greatest fungal richness and diversity, C20 and C26 exhibited similar bacterial richness and diversity, while C30 had low fungal and bacterial richness. Fungal taxonomic assignments revealed that the initial moisture content caused selective enrichment of Aspergillus candidus with a decreasing trend during fermentation, whereas Saccharomycetales sp. exhibited increasing relative abundance with increasing moisture content from day 6 of the fermentation process. Depending on initial moisture level, changes in bacterial communities were also observed in the genera Streptomyces, Bacillus, and Staphylococcus, with decreasing trends whereas Saccharopolyspora exhibited a sigmoidal trend with the highest abundance in C26. These findings demonstrate the possible impact of initial moisture content of nuruk on microfloral richness, diversity, and dynamics; this study is thus a step toward our ultimate goal of enhancing the quality of nuruk.

Taxonomic Characterization, Evaluation of Toxigenicity, and Saccharification Capability of Aspergillus Section Flavi Isolates from Korean Traditional Wheat-Based Fermentation Starter Nuruk.

The most economically important species used in a wide range of fermentation industries throughout Asia belong to Aspergillus section Flavi, which are morphologically and phylogenetically indistinguishable, with a few being toxigenic and therefore a major concern. They are frequently isolated from Korean fermentation starters, such as nuruk and meju. The growing popularity of traditional Korean alcoholic beverages has led to a demand for their quality enhancement, therefore requiring selection of efficient non-toxigenic strains to assist effective fermentation. This study was performed to classify the most efficient strains of Aspergillus section Flavi isolated from various types of traditional wheat nuruk, based on a polyphasic approach involving molecular and biochemical evaluation. A total of 69 strains were isolated based on colony morphology and identified as Aspergillusoryzae/flavus based on internal transcribed spacer and calmodulin gene sequencing. Interestingly, none were toxigenic based on PCR amplification of intergenic regions of the aflatoxin cluster genes norB-cypA and the absence of aflatoxin in the culture supernatants by thin-layer chromatography analysis. Saccharification capability of the isolates, assessed through α-amylase and glucoamylase activities, revealed that two isolates, TNA24 and TNA15, showed the highest levels of activity. Although the degrees of variation in α-amylase and glucoamylase activities among the isolates were higher, there were only slight differences in acid protease activity among the isolates with two, TNA28 and TNA36, showing the highest activities. Furthermore, statistical analyses showed that α-amylase activity was positively correlated with glucoamylase activity (p < 0.001), and therefore screening for either was sufficient to predict the saccharifying capacity of the Aspergillus strain.

Metagenomic analysis of fungal diversity in Korean traditional wheat-based fermentation starter nuruk.

Nuruk, a traditional natural starter, is extensively used in the brewing of Makgeolli, one of Korea's most popular alcoholic beverages that has been recently gaining global popularity. Thus, the quality of traditional nuruk needs to be enhanced. The nuruk mycobiome greatly influences both fermentation process as well as palatability enhancement. Limitations of culture-dependent identification restrict an accurate analysis of fungal diversity and distribution in nuruks. 454 pyrosequencing of two traditional wheat-based nuruks, prepared at two representative temperature conditions revealed a total of 153 and 53 OTUs for nuruks A and B, respectively, from a total of 33,157 ITS sequences. Phylogenetic assignments indicated that nuruk A mycobiota was dominated by the genera Aspergillus and Mucorales, whereas nuruk B by Rhizomucor. Species-level identification indicated that Mucorales sp., Aspergillus candidus, and Aspergillus cibarius predominated in nuruk A mycoflora whereas Rhizomucor pusillus, Mucorales sp., and Thermoascus crustaceus in nuruk B. The alpha diversity indices suggest nuruk A mycobiota to be more diverse than that of nuruk B at almost all time points of fermentation. Resemblances of patterns of predominant species composition and succession between culture-dependent and -independent phylogenetic analysis creates the potential to reconstruct the nuruk mycobiome in vitro, which allows the establishment of a standard inoculum for scientific comparison.

Pyrosequencing reveals bacterial diversity in Korean traditional wheat-based nuruk.

The emerging global importance of Korea's alcoholic beverages emphasizes the need for quality enhancement of nuruk, a traditional Korean cereal starter that is used extensively in traditional brewing. Apart from fungi and yeasts, bacteria known to be ubiquitously present are also a part of the nuruk ecosystem and are known to influence fermentation activity by influencing fermentation favorable factors. In the current study, bacterial diversity and temporal variations in the traditional wheat-based nuruk, fermented at two representative temperature conditions for 30 days, along with two commercial wheat-based nuruk samples for comparison analysis were evaluated using libraries of PCR amplicons and 454 pyrosequencing targeting of the hypervariable regions V1 to V3 of the 16S rRNA gene. A total of 90,836 16S reads were analyzed and assigned to a total of 314, 321, and 141 Operational Taxonomic Units (OTUs) for nuruk A, B, and C, respectively. Diversity parameters clearly indicated nuruk B to be more diverse in terms of bacterial composition than nuruk A. Taxonomic assignments indicated that nuruk A was dominated by phylum Cyanobacteria, whereas nuruk B was dominated by phylum Actinobacteria. For both nuruk A and B, members of the phylum Firmicutes mostly converged into the family Bacillaceae; these microorganisms might be present in negligible numbers at the beginning but became significant as the fermentation progressed. The commercial samples were predominated by phylum Firmicutes, which is composed of Lactobacillaceae and Leoconostocaceae. The findings of this study provide new insights into understanding the changes in bacterial community structure during traditional nuruk starter production.

Mycoflora dynamics analysis of Korean traditional wheat-based nuruk.

The growing popularity of traditional Korean alcoholic beverages has led to a demand for quality enhancement of the traditional starter culture nuruk, which consists primarily of wheat. Therefore, this study focused on mycoflora characterization and the temporal variations in traditional wheat-based nuruks fermented at two representative traditional temperature conditions for 30 days. Nuruk A was fermented at a constant temperature of 36°C for 30 days and nuruk B was fermented at a high initial temperature of 45°C for 10 days followed by 35°C for 20 days. The average mycoflora load in the two different nuruk conditions did not vary significantly between the 0 and 30 day cultures, and a maximum load of 8.39 log CFU/g was observed for nuruk A on culture day 3 and 7.87 log CFU/g for nuruk B on culture day 30. Within two samples, pH was negatively correlated with temporal changes in mycoflora load. The pH of nuruk A was significantly lower than that of nuruk B at all of the time points evaluated. Culture-dependent characterization led to the identification of 55 fungal isolates belonging to 9 genera and 15 species, with the most prominent genera comprising Lichtheimia, Penicillium, Trametes, Aspergillus, Rhizomucor, and Mucor. A total of 25 yeast isolates were characterized belonging to 6 genera and 7 species, the most prominent among which were Rhodotorula, Pichia, Debaryomyces, Saccharomycopsis, and Torulospora. Mycofloral community dynamics analysis revealed that both samples A and B varied considerably with respect to the fungal communities over a span of 30 days.

Ylpex5 mutation partially suppresses the defective hyphal growth of a Yarrowia lipolytica ceramide synthase mutant, Yllac1, by recovering lipid raft polarization and vacuole morphogenesis.

Sphingolipids are involved in cell differentiation and morphogenesis in eukaryotic cells. In this study, YlLac1p, a ceramide synthase required for glucosylceramide (GlcCer) synthesis, was found to be essential for hyphal growth in Yarrowia lipolytica. Y. lipolytica GlcCer was shown to be composed of a C16:0 fatty acid, which is hydroxylated at C2, and a C18:2 long chain base, which is unsaturated at both C4 and C8 and methylated at C9. Domain swapping analysis revealed that the entire TRAM/Lag1/CLN8 (TLC) domain, not the Lag1 motif, is crucial for the function of YlLac1p. YlDes1p, the C4 desaturase of the ceramide synthesized by YlLac1p, was also required for Y. lipolytica morphogenesis. Both Yllac1Δ and Yldes1Δ mutants neither polarize lipid rafts nor form normal vacuoles. Interestingly, mutation in YlPEX5, which encode a peroxisomal targeting signal receptor, partially suppressed the defective hyphal growth of Yllac1Δ. The Yllac1ΔYlpex5Δ mutant restored the ability to polarize lipid rafts and to form normal vacuoles, although it could not synthesize GlcCer. Taken together, our results suggest that GlcCer or GlcCer derivatives may be involved in hyphal morphogenesis in Y. lipolytica, at least in part, by affecting polarization of lipid rafts and vacuole morphogenesis.

Distinct roles of two ceramide synthases, CaLag1p and CaLac1p, in the morphogenesis of Candida albicans.

Lag1p and Lac1p catalyse ceramide synthesis in Saccharomyces cerevisiae. This study shows that Lag1 family proteins are generally required for polarized growth in hemiascomycetous yeast. However, in contrast to S. cerevisiae where these proteins are functionally redundant, C. albicans Lag1p (CaLag1p) and Lac1p (CaLac1p) are functionally distinct. Lack of CaLag1p, but not CaLac1p, caused severe defects in the growth and hyphal morphogenesis of C. albicans. Deletion of CaLAG1 decreased expression of the hypha-specific HWP1 and ECE1 genes. Moreover, overexpression of CaLAG1 induced pseudohyphal growth in this organism under non-hypha-inducing conditions, suggesting that CaLag1p is necessary for relaying signals to induce hypha-specific gene expression. Analysis of ceramide and sphingolipid composition revealed that CaLag1p predominantly synthesizes ceramides with C24:0/C26:0 fatty acid moieties, which are involved in generating inositol-containing sphingolipids, whereas CaLac1p produces ceramides with C18:0 fatty acid moieties, which are precursors for glucosylsphingolipids. Thus, our study demonstrates that CaLag1p and CaLac1p have distinct substrate specificities and physiological roles in C. albicans.

A circularly permuted beta-lactamase as a novel reporter for evaluation of protein cyclization efficiency.

Split inteins have been used as a versatile tool in protein engineering to mediate efficient in vivo and in vitro trans-splicing of a protein. The trans-splicing ability of split inteins was also applied to the in vivo cyclization of a protein. However, cyclization efficiency is dependent upon the type of split inteins employed and the conditions under which cyclization occur. In this study, a novel reporter system that easily measures the cyclization efficiency of split inteins was developed. For this purpose TEM-1 beta-lactamase was divided into two fragments (24-215 and 216-286 amino acids) and circularly permuted. The circularly permuted beta-lactamase expressed in Escherichia coli showed little beta-lactamase activity, most likely due to the structural modification of the protein. However, when the circularly permuted beta-lactamase was cyclized by the Synechocystis sp. PCC6803 DnaB split mini-intein, beta-lactamase activity both in vitro and in vivo was recovered. These results suggest that the novel reporter system can be exploited to develop new inteins with high efficiency of in vivo protein cyclization.