Detection limitations of prion seeding activities in blood samples from patients with sporadic prion disease.

BACKGROUND: Human prion diseases (HPDs) are fatal neurodegenerative disorders characterized by abnormal prion proteins (PrPSc). However, the detection of prion seeding activity in patients with high sensitivity remains challenging. Even though real-time quaking-induced conversion (RT-QuIC) assay is suitable for detecting prion seeding activity in a variety of specimens, it shows lower accuracy when whole blood, blood plasma, and blood-contaminated tissue samples are used. In this study, we developed a novel technology for the in vitro amplification of abnormal prion proteins in HPD to the end of enabling their detection with high sensitivity known as the enhanced quaking-induced conversion (eQuIC) assay. METHODS: Three antibodies were used to develop the novel eQUIC method. Thereafter, SD50 seed activity was analyzed using brain tissue samples from patients with prion disease using the conventional RT-QUIC assay and the novel eQUIC assay. In addition, blood samples from six patients with solitary prion disease were analyzed using the novel eQuIC assay. RESULTS: The eQuIC assay, involving the use of three types of human monoclonal antibodies, showed approximately 1000-fold higher sensitivity than the original RT-QuIC assay. However, when this assay was used to analyze blood samples from six patients with sporadic human prion disease, no prion activity was detected. CONCLUSION: The detection of prion seeding activity in blood samples from patients with sporadic prion disease remains challenging. Thus, the development of alternative methods other than RT-QuIC and eQuIC will be necessary for future research.

Chicken Interleukin-5 is Expressed in Splenic Lymphocytes and Affects Antigen-Specific Antibody Production.

Vaccination is important for reducing disease incidence in the poultry industry. To enhance immunity and vaccine efficacy, chicken cytokines associated with antibody production must be identified. In this study, we focused on interleukin-5 (IL-5), involved in antibody production in mice, measuring its expression and effects on antibody production. Concanavalin A-stimulated splenocytes were used for RT-PCR to clone IL5 cDNAs. Recombinant IL-5 was prepared from the clone and administered to chickens with antigen via the ocular-topical route twice every alternate week. IL-5 enhanced antigen-specific IgY and inhibited antigen-specific serum IgA production in serum. Our findings suggest that IL-5 plays an important role in chicken antibody production, with possible unique functions.

Evaluation of expression systems for recombinant protein production in chicken egg bioreactors.

Chicken eggs have gained attention as excellent bioreactors because of their genetic modifications. However, the development of chicken egg bioreactors requires a long time from the construction of the production system to the evaluation of the products. Therefore, in this study, a chicken cell line producing ovalbumin (OVA) was established and constructed a system for the rapid evaluation of the production system. First, the EF1α promoter was knocked in upstream of the OVA locus in chicken DF-1 cells for continuous OVA expression. Furthermore, an ideal position at the OVA locus for the insertion of useful protein genes to maximize recombinant protein yield was analyzed and identified. The knocking in the EF1α promoter upstream of exon1 yielded the maximum production of OVA protein was achieved. In addition, Linking a recombinant hFGF2 cDNA to the 5' side of the OVA was found to increase production efficiency. Therefore, an OVA-expressing cell line and an evaluation system for proteins in chicken egg bioreactors was established. The findings may improve the efficiency of chicken expression systems and expand their applications in protein production.

Wnt signaling blockade is essential for maintaining the pluripotency of chicken embryonic stem cells.

Activation of Wnt/ß-catenin signaling supports the self-renewal of mouse embryonic stem cells. We aimed to understand the effects of Wnt signaling activation or inhibition on chicken embryonic stem cells (chESCs), as these effects are largely unknown. When the glycogen synthase kinase-3 ß inhibitor CHIR99021-which activates Wnt signaling-was added to chESC cultures, the colony shape flattened, and the expression levels of pluripotency-related (NANOG, SOX2, SOX3, OCT4, LIN28A, DNMT3B, and PRDM14) and germ cell (CVH and DAZL) markers showed a decreasing trend, and the growth of chESCs was inhibited after approximately 7 d. By contrast, when the Wnt signaling inhibitor XAV939 was added to the culture, dense and compact multipotent colonies (morphologically similar to mouse embryonic stem cell colonies) showing stable expression of pluripotency-related and germline markers were formed. The addition of XAV939 stabilized the proliferation of chESCs in the early stages of culture and promoted their establishment. Furthermore, these chESCs formed chimeras. In conclusion, functional chESCs can be stably cultured using Wnt signaling inhibitors. These findings suggest the importance of Wnt/ß-catenin signaling in avian stem cells, offering valuable insights for applied research using chESCs.

[A Long-Term Survival Case of Gastric Cancer with Pyloric Stenosis and Peritoneum Dissemination That Received Intravenous and Intraperitoneal Paclitaxel Combined with S-1 Therapy after Bypass Surgery].

Although a 74-year-old man with gastric cancer with pyloric stenosis(cT4aN[+]M0, Stage Ⅲ)had undergone surgery, he was diagnosed with peritoneum dissemination. He received bypass surgery, and an intraperitoneal access port was implanted in his subcutaneous space. Postoperatively, he received 4 courses of SOX therapy. In treatment effect, the primary tumor showed no change, and ascites developed. Therefore, we changed the chemotherapy regimen in intravenous and intraperitoneal paclitaxel combined with S-1 therapy. After starting this regimen, the primary tumor decreased in size, and the pyloric stenosis improved. Currently, the patient is alive without recurrence for 5 years and 8 months after intravenous and intraperitoneal paclitaxel combined with S-1 therapy and receiving this treatment regularly.

Comprehensive analysis of the composition of the major phospholipids during the asexual life cycle of the filamentous fungus Aspergillus nidulans.

Filamentous fungi undergo significant cellular morphological changes during their life cycle. It has recently been reported that deletions of genes that are involved in phospholipid synthesis led to abnormal hyphal morphology and differentiation in filamentous fungi. Although these results suggest the importance of phospholipid balance in their life cycle, comprehensive analyses of cellular phospholipids are limited. Here, we performed lipidomic analysis of A. nidulans during morphological changes in a liquid medium and of colonies on a solid medium. We observed that the phospholipid composition and transcription of the genes involved in phospholipid synthesis changed dynamically during the life cycle. Specifically, the levels of phosphatidylethanolamine, and highly unsaturated phospholipids increased during the establishment of polarity. Furthermore, we demonstrated that the phospholipid composition in the hyphae at colony margins is similar to that during conidial germination. Furthermore, we demonstrated that common and characteristic phospholipid changes occurred during germination in A. nidulans and A. oryzae, and that species-specific changes also occurred. These results suggest that the exquisite regulation of phospholipid composition is crucial for the growth and differentiation of filamentous fungi.

Lipofection with Lipofectamine™ 2000 in a heparin-free growth medium results in high transfection efficiency in chicken primordial germ cells.

Primordial germ cells (PGCs) that can differentiate into gametes are used to produce genome-edited chickens. However, the transfection efficiency into PGCs is low in chickens; therefore, the yield efficiency of PGCs modified via genome editing is problematic. In this study, we improved transfection efficiency and achieved highly efficient genome editing in chicken PGCs. For transfection, we used lipofection, which is convenient for gene transfer. Chicken PGC cultures require adding heparin to support growth; however, heparin significantly reduces lipofection efficiency (p < 0.01). Heparin-induced lipofection efficiency was restored by adding protamine. Based on these results, we optimized gene transfer into chicken PGCs. Lipofectamine 2000 and our PGC medium were the most efficient transfection reagent and medium, respectively. Finally, based on established conditions, we compared the gene knock-out efficiencies of ovomucoid, a major egg allergen, and gene knock-in efficiencies at the ACTB locus. These results indicate that optimized lipofection is useful for CRISPR/Cas9-mediated knock-out and knock-in. Our findings may contribute to the generation of genome-edited chickens and stimulate research in various applications involving them.

The N-terminal disordered region of ChsB regulates its efficient transport to the hyphal apical surface in Aspergillus nidulans.

In fungi, the cell wall plays a crucial role in morphogenesis and response to stress from the external environment. Chitin is one of the main cell wall components in many filamentous fungi. In Aspergillus nidulans, a class III chitin synthase ChsB plays a pivotal role in hyphal extension and morphogenesis. However, little is known about post-translational modifications of ChsB and their functional impacts. In this study, we showed that ChsB is phosphorylated in vivo. We characterized strains that produce ChsB using stepwise truncations of its N-terminal disordered region or deletions of some residues in that region and demonstrated its involvement in ChsB abundance on the hyphal apical surface and in hyphal tip localization. Furthermore, we showed that some deletions in this region affected the phosphorylation states of ChsB, raising the possibility that these states are important for the localization of ChsB to the hyphal surface and the growth of A. nidulans. Our findings indicate that ChsB transport is regulated by its N-terminal disordered region.

srdA mutations suppress the rseA/cpsA deletion mutant conidiation defect in Aspergillus nidulans.

Conidiation is an important reproductive process in Aspergillus. We previously reported, in A. nidulans, that the deletion of a putative glycosyltransferase gene, rseA/cpsA, causes an increase in the production of extracellular hydrolases and a severe reduction in conidiation. The aim of this study was to obtain novel genetic factors involved in the repression of conidiation in the rseA deletion mutant. We isolated mutants in which the rseA deletion mutant conidiation defect is suppressed and performed a comparative genomic analysis of these mutants. A gene encoding a putative transcription factor was identified as the associated candidate causative gene. The candidate gene was designated as srdA (suppressor gene for the conidiation defect of the rseA deletion mutant). The conidiation efficiency of the rseAsrdA double-deletion mutant was increased. Introduction of wild-type srdA into the suppressor mutants caused a conidiation defect similar to that of the rseA deletion mutant. Notably, the conidiation efficiencies of the rseAsrdA double-deletion and srdA single-deletion mutants were higher than that of the wild-type strain. These results indicate that srdA is a novel genetic factor that strongly represses conidiation of the rseA deletion mutant, and a putative transcriptional regulator, SrdA is a negative regulator of conidiation in A. nidulans.

Transcription activator-like effector nuclease-mediated deletion safely eliminates the major egg allergen ovomucoid in chickens.

Among the major egg allergens, ovomucoid (OVM) is very stable against heat and digestive enzymes, making it difficult to remove physiochemically and inactivate allergens. However, recent genome editing technology has made it possible to generate OVM-knockout chicken eggs. To use this OVM-knockout chicken egg as food, it is important to evaluate its safety as food. Therefore, in this study, we examined the presence or absence of mutant protein expression, vector sequence insertion, and off-target effects in chickens knocked out with OVM by platinum TALENs. The eggs laid by homozygous OVM-knockout hens showed no evident abnormalities, and immunoblotting showed that the albumen contained neither the mature OVM nor the OVM truncated variant. Whole genome sequencing (WGS) revealed that the potential TALEN-induced off-target effects in OVM-knockout chickens were localized in the intergenic and intron regions. The WGS information confirmed that plasmid vectors used for genome editing were only transiently present and did not integrate into the genome of edited chickens. These results indicate the importance of safety evaluation and reveal that the eggs laid by this OVM knockout chicken solve the allergy problem in food and vaccines.

Fate Decisions of Chicken Primordial Germ Cells (PGCs): Development, Integrity, Sex Determination, and Self-Renewal Mechanisms.

Primordial germ cells (PGCs) are precursor cells of sperm and eggs. The fate decisions of chicken PGCs in terms of their development, integrity, and sex determination have unique features, thereby providing insights into evolutionary developmental biology. Additionally, fate decisions in the context of a self-renewal mechanism have been applied to establish culture protocols for chicken PGCs, enabling the production of genome-edited chickens and the conservation of genetic resources. Thus, studies on the fate decisions of chicken PGCs have significantly contributed to both academic and industrial development. Furthermore, studies on fate decisions have rapidly advanced owing to the recent development of essential research technologies, such as genome editing and RNA sequencing. Here, we reviewed the status of fate decisions of chicken PGCs and provided insight into other important research issues that require attention.

Screening of Optimal CpG-Oligodeoxynucleotide for Anti-Inflammatory Responses in the Avian Macrophage Cell Line HD11.

CpG-oligodeoxynucleotides (. CpG-ODNs: ) have been shown to possess immunostimulatory features in both mammals and birds. However, compared to their proinflammatory effects, little is known about the anti-inflammatory responses triggered by CpG-ODN in avian cells. Hence, in this study, the anti-inflammatory response in the chicken macrophage cell line HD11 was characterized under stimulation with five types of CpG-ODNs: CpG-A1585, CpG-AD35, CpG-B1555, CpG-BK3, and CpG-C2395. Single-stimulus of CpG-B1555, CpG-BK3, or CpG-C2395 induced interleukin (IL)-10 expression without causing cell injury. The effects of pretreatment with CpG-ODNs before subsequent lipopolysaccharide stimulation were also evaluated. Interestingly, pretreatment with only CpG-C2395 resulted in high expression levels of IL-10 mRNA in the presence of lipopolysaccharide. Finally, gene expression analysis of inflammation-related cytokines and receptors revealed that pre-treatment with CpG-C2395 significantly reduced the mRNA expression of tumor necrosis factor-α, IL-1ß, IL-6, and Toll-like receptor 4. Overall, these results shed light on the anti-inflammatory responses triggered by CpG-C2395 stimulation through a comparative analysis of five types of CpG-ODNs in chicken macrophages. These results also offer insights into the use of CpG-ODNs to suppress the expression of proinflammatory cytokines, which may be valuable in the prevention of avian infectious diseases in the poultry industry.

Prediction of sex-determination mechanisms in avian primordial germ cells using RNA-seq analysis.

In birds, sex is determined through cell-autonomous mechanisms and various factors, such as the dosage of DMRT1. While the sex-determination mechanism in gonads is well known, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism underlying sex determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also exhibited sex-biased expression, and the number of female-biased genes detected was higher than that of male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Retinoic acid stimulation of cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination in avian PGCs involves aspects of both cell-autonomous and somatic-cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method.

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.

Gene manipulation in the Mucorales fungus Rhizopus oryzae using TALENs with exonuclease overexpression.

The Mucorales fungal genus Rhizopus is used for the industrial production of organic acids, enzymes and fermented foods. The metabolic engineering efficiency of Rhizopus could be improved using gene manipulation; however, exogenous DNA rarely integrates into the host genome. Consequently, a genetic tool for Mucorales fungi needs to be developed. Recently, programmable nucleases that generate DNA double-strand breaks (DSBs) at specific genomic loci have been used for genome editing in various organisms. In this study, we examined gene disruption in Rhizopus oryzae using transcription activator-like effector nucleases (TALENs), with and without exonuclease overexpression. TALENs with an overexpressing exonuclease induced DSBs, followed by target site deletions. Although DSBs are repaired mainly by nonhomologous end joining in most organisms, our results suggested that in R. oryzae microhomology-mediated end joining was the major DSB repair system. Our gene manipulation method using TALENs coupled with exonuclease overexpression contributes to basic scientific knowledge and the metabolic engineering of Rhizopus.

Orthologs of Saccharomyces cerevisiae SFH2, genes encoding Sec14 family proteins, implicated in utilization of n-alkanes and filamentous growth in response to n-alkanes in Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica has an ability to assimilate n-alkanes as carbon and energy sources. In this study, the roles of orthologs of Saccharomyces cerevisiae SEC14 family gene SFH2, which we named SFH21, SFH22, SFH23 and SFH24, of Y. lipolytica were investigated. The transcript levels of SFH21, SFH22 and SFH23, determined by RNA-seq analysis, qRT-PCR analysis and northern blot analysis, were found to increase in the presence of n-alkanes. The deletion mutant of SFH21, but not that of SFH22, SFH23 or SFH24, showed defects in growth in the media containing n-alkanes and in filamentous growth on the solid media containing n-alkanes. Additional deletions of SFH22 and SFH23 significantly exaggerated the defect in filamentous growth of the deletion mutant of SFH21, and expression of SFH22 or SFH24 using the SFH21 promoter partially suppressed the growth defect of the deletion mutant of SFH21 on n-alkanes. These results suggest that SFH2 orthologs are involved in the utilization of n-alkanes and filamentous growth in response to n-alkanes in Y. lipolytica.

AP-2 complex contributes to hyphal-tip-localization of a chitin synthase in the filamentous fungus Aspergillus nidulans.

Filamentous fungi maintain hyphal growth to continually internalize membrane proteins related to cell wall synthesis, transporting them to the hyphal tips. Endocytosis mediates protein internalization via target recognition by the adaptor protein 2 complex (AP-2 complex). The AP-2 complex specifically promotes the internalization of proteins important for hyphal growth, and loss of AP-2 complex function results in abnormal hyphal growth. In this study, deletion mutants of the genes encoding the subunits of the AP-2 complex (α, ß2, µ2, or σ2) in the filamentous fungus Aspergillus nidulans resulted in the formation of conidiophores with abnormal morphology, fewer conidia, and activated the cell wall integrity pathway. We also investigated the localization of ChsB, which plays pivotal roles in hyphal growth in A. nidulans, in the Δµ2 strain. Quantitative analysis suggested that the AP-2 complex is involved in ChsB internalization at subapical collar regions. The absence of the AP-2 complex reduced ChsB localization at the hyphal tips. Our findings suggest that the AP-2 complex contributes to cell wall integrity by properly localizing ChsB to the hyphal tips.

Knock-in of the duck retinoic acid-inducible gene I (RIG-I) into the Mx gene in DF-1 cells enables both stable and immune response-dependent RIG-I expression.

Waterfowls, such as ducks, are natural hosts of avian influenza virus (AIV) and can genetically limit the pathogenicity. On the other hand, some AIV strains cause severe pathogenicity in chickens. It is suggested that differences in the pathogenicity of AIV infection between waterfowls and chickens are related to the expression of retinoic acid-inducible gene I (RIG-I), a pattern recognition receptor that chickens evolutionally lack. Here, we knocked-in the duck RIG-I bearing the T2A peptide sequence at the 3' region of the Mx, an interferon-stimulated gene (ISG), in chicken embryo fibroblast cells (DF-1) using the precise integration into target chromosome (PITCh) system to control the duck RIG-I expression in chickens. The expression patterns of the duck RIG-I were then analyzed using qPCR. The knocked-in DF-1 cells expressed RIG-I via the stimulation of IFN-ß and poly(I:C) in a dose-dependent manner. Moreover, poly(I:C) stimulation in the knocked-in DF-1 cells upregulated RIG-I-like receptor (RLR) family signaling pathway-related genes IFN-ß, OASL, and IRF7. The IFN-ß-dependent expression of RIG-I and upregulation of IFN-ß in the poly(I:C) stimulation demonstrated a positive-feedback loop via RIG-I, usually evident in ducks. Overall, this novel strategy established RIG-I-dependent immune response in chickens without overexpression of RIG-I and disruption of the host genes.

Deletion of Aspergillus nidulans cpsA/rseA induces increased extracellular hydrolase production in solid-state culture partly through the high osmolarity glycerol pathway.

Koji molds, such as Aspergillus oryzae and Aspergillus sojae, are used in the food industry in East Asia and have been explored for the large-scale production of extracellular hydrolases. We previously found that the deletion of a gene encoding a putative GT2 glycosyltransferase increased production of extracellular hydrolases in A. sojae. The gene was named rseA (regulator of the secretory enzyme A). We predicted that intracellular signaling pathways were involved in the increased production of hydrolases in the ΔrseA mutant of A. sojae. However, little has been reported on molecular biological knowledge about A. sojae. Hence, Aspergillus nidulans, a typical model organism used in molecular biology, was employed for the functional characterization of rseA in this study. Deletion of the rseA ortholog in A. nidulans induced increased extracellular production of hydrolases under the solid-state cultivation condition, similar to that in A. sojae. The involvement of the cell wall integrity pathway and the high osmolarity glycerol pathway in ΔrseA was further investigated. The results indicated that the HOG pathway played an important role in the increased extracellular production of hydrolases caused by the deletion of the rseA gene. rseA ortholog in A. nidulans was identical to cpsA, which was reported to function as a regulator of mycotoxin production, morphogenesis, and cell wall biosynthesis. However, this is the first study reporting that rseA/cpsA regulates extracellular hydrolase production in A. nidulans.

Acyl-CoA synthetases, Aal4 and Aal7, are involved in the utilization of exogenous fatty acids in Yarrowia lipolytica.

The yeast Yarrowia lipolytica assimilates hydrophobic compounds, such as n-alkanes and fatty acids, as sole carbon and energy sources. It has been shown that the acyl-CoA synthetase (ACS) genes, FAT1 and FAA1, are involved in the activation of fatty acids produced during the metabolism of n-alkanes, but the ACS genes that are involved in the metabolism of fatty acids from the culture medium remains to be identified. In this paper, we have identified the ACS genes involved in the utilization of exogenous fatty acids. RNA-seq analysis and qRT-PCR revealed that the transcript levels of the peroxisomal ACS-like protein-encoding genes AAL4 and AAL7 were increased in the presence of oleic acid. The single deletion mutant of AAL4 or AAL7 and double deletion mutant of AAL4 and AAL7 did not show any defects in the growth on the medium containing glucose, glycerol, n-alkanes, or fatty acids. In contrast, the mutant with deletion of seven genes, FAA1, FAT1-FAT4, AAL4, and AAL7, showed severe growth defects on the medium containing dodecanoic acid or oleic acid. These results suggest that Aal4p and Aal7p play important roles in the metabolism of exogenous fatty acids in collaboration with Faa1p and Fat1p-Fat4p.