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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.

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 rseAdeletion 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.

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.

Hydroxykenopyrochlore, (□,Ce,Ba)2(Nb,Ti)2O6(OH,F), occurs in a weathered Nb-ore from alkaline-carbonatite complexes and pegmatites of the Brazilian shield mined by Compania Brasileira de Metalurgia e Mineração (CBMM), Araxá, Minas Gerais, Brazil. The mineral is a product of alkali metasomatism. It occurs as parts of granular grains up to 0.1 mm in size in association with Ba-bearing hydrokenopyrochlore. Hydroxykenopyrochlore is lemon yellow to yellow in color, non-fluorescent, and brittle. The hardness is 4½ on the Mohs scale. The calculated density is 4.36 g/cm3. It is cubic, Fd–3m, with cell parameters a 10.590(5) Å, V = 1187.6(10) Å3, and Z = 2. The strongest seven lines in the powder XRD pattern [d in Å (I/I0) hkl] are 6.06 (49) 111, 3.18 (27) 311, 3.05 (100) 222, 2.64 (29) 400, 1.870 (56) 440, 1.594 (50) 622, 1.213 (15) 662, and 1.182 (13) 840. The empirical formula derived from electron-microprobe analyses is [□1.117Ce0.532Nd0.035La0.021Pr0.010Sm0.003Y0.002Ba0.101Ca0.030Pb0.004Th0.061U0.007K0.040Na0.036]Σ2(Nb1.368Ti0.325P0.095Fe0.091Al0.082Zr0.039)Σ2[O4.719(OH)1.281]Σ6[(OH)0.846F0.154]. Hydroxykenopyrochlore is a member of the pyrochlore supergroup (class 4.DH.15 of Strunz & Nickel; class 8.2.1. of Dana). It is the vacancy-dominant analogue of hydroxycalciopyrochlore, (Ca,Na,U,□)2(Nb,Ti)2O6(OH), and the Nb-dominant analogue of hydroxykenomicrolite, (□,Na,Sb3+)2Ta2O6(OH), and of hydroxykenoelsmoreite, (□,Pb)2(W,Fe3+,Al)2(O,OH)6(OH).

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.

Introduction Pancreatic trauma is potentially lethal despite recent improvements in surgical techniques and conservative management. However, no guidelines for the management of pancreatic trauma have been established. In this report, we propose an algorithm for the management of pancreatic trauma based on our experience of 9 cases and 1 literature review. Case presentation This study included 9 patients with pancreatic trauma (5 men and 4 women). The patient median age was 40 years (range, 17–75 years). The overall mortality rate was 22.2%, and the postoperative mortality rate was 16.7%. Superficial trauma was present in 2 patients. Deep trauma without injury to the main pancreatic duct was present in 1 patient, and this patient was treated successfully with endoscopic nasopancreatic drainage. Active bleeding was present in 2 patients and controlled by interventional radiology. Deep trauma with injury to the main pancreatic duct was present in 6 patients. Among them, 1 patient died after conservative treatment with endoscopic nasopancreatic drainage. The other 5 patients underwent surgery (pancreatic resection in 4 and necrosectomy in 1). Conclusion The herein-described algorithm recommends interventional radiology for active arterial bleeding, conservative management for trauma without ductal injury, and surgery for trauma with ductal injury. This algorithm may provide a basis for future establishment of guidelines.

CpG-oligodeoxynucleotides (CpG-ODN) has been shown to possess immunostimulatory features in both mammals and birds. However, compared to its pro-inflammatory effects, little is known about the anti-inflammatory responses triggered by CpG-ODN in avian cells. Hence, in this study, we aimed to characterize the anti-inflammatory response in the chick macrophage cell line HD11 under the stimulation of five kinds of CpG-ODN: CpG-A1585, CpG-AD35, CpG-B1555, CpG-BK3, and CpG-C2395. Single-stimulus CpG-B1555, CpG-BK3, and CpG-C2395 induced the interleukin (IL)-10 expression without causing cellular injury. The effects of pretreatment with each CpG-ODN before subsequent lipopolysaccharide stimulation were also evaluated. Interestingly, only CpG-C2395 maintained a high expression level in this situation. Finally, expression analysis of inflammation-related genes, such as the tumor necrosis factor-α, IL-1β, IL-6, and Toll-like receptor 4, was conducted, and pretreatment with CpG-C2395 significantly reduced their expression. Overall, our results shed light on the anti-inflammatory responses triggered by CpG-C2395 stimulation using a comparative analysis of three major classes of CpG-ODN in chick macrophages.

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.

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.

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-β 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-β, OASL, and IRF7. The IFN-β-dependent expression of RIG-I and upregulation of IFN-β 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., Graphical abstract Image 1, Highlights • RIG-I activates the innate immune response-related genes such as type I interferons. • Loss of chicken RIG-I accounts for the pathogenicity of the avian influenza virus. • This strategy controls RIG-I by host gene promoter activation via gene targeting. • The knocked-in DF-1 cells express RIG-I upon IFN-β and poly (I:C) stimulation. • A RIG-I-dependent immune response was observed without overexpression.

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 (

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.

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, β2, 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.

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.

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.

Phosphatidylserine decarboxylases (PSDs) catalyze the production of phosphatidylethanolamine (PE) from phosphatidylserine (PS) and are crucial for the maintenance of PE levels in fungi. The PSDs are classified into two types; the type I PSDs are conserved from bacteria to humans, while the type II PSDs exist only in fungi and plants. In yeasts, the deletion of type I PSD-encoding genes causes severe growth retardation. In contrast, the deletion of type II PSD-encoding genes has little or no effect. In this study, we found four genes encoding type II PSD orthologs in the filamentous fungus Aspergillus nidulans; these included psdB, psdC, psdD, and psdE. Deletion of psdB caused severe growth defects on minimal medium and these defects were partially restored by the addition of ethanolamine, choline, PE, or phosphatidylcholine into the medium. The conidiation efficiency of the psdB deletion mutant was dramatically decreased and its conidiophore structures were aberrant. In the psdB deletion mutant, the PE content decreased while the PS content increased. We further showed that PsdB had a major PSD activity. Our findings suggest that the type II PSDs exert important roles in the phospholipid homeostasis, and in the growth and morphogenesis of filamentous fungi.

At present, the most reliable method for creating genetically modified chickens is the modification of the DNA sequence of primordial germ cells (PGCs). However, during embryogenesis, only a small number of chicken PGCs can be obtained. Therefore, in vitro PGC culturing is necessary to obtain sufficient cells for further genetic engineering. Previously reported PGC culturing methods lack versatility. We report here a new protocol for stable and efficient culturing of chicken PGCs using small-molecule inhibitors. The growth rate of PGCs was investigated following the addition of three small-molecule inhibitors, including blebbistatin, into the culture medium. Chicken PGC survival and proliferation rates increased after the addition of small-molecule inhibitors, compared with the untreated control. Blebbistatin was shown to be the most effective inducer of PGC growth. Long-term culturing of PGCs with blebbistatin maintained the morphology of typical PGCs, and these cells expressed marker proteins such as chicken vasa homolog (CVH) and NANOG. Additionally, PGCs transfected with a fluorescent protein gene were shown to migrate into the gonads of the recipient embryo, and progeny derived from PGCs cultured by this method were efficiently obtained. These results demonstrate that small-molecule inhibitors represent a useful tool for stable and efficient chicken PGC culturing., This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers 19H03107, and 19K22286.

DDL1 encodes a mitochondrial phospholipase A1 involved in acyl chain remodeling of mitochondrial phospholipids and degradation of cardiolipin in Saccharomyces cerevisiae. The deletion of DDL1 leads to respiratory growth defects. To elucidate the physiological role of DDL1, we screened for genes that, when overexpressed, suppress the respiratory growth defect of the DDL1 deletion mutant. Introduction of COQ8, COQ9, or COQ5, which are involved in coenzyme Q (CoQ) synthesis, using a multicopy vector suppressed the respiratory growth defect of the DDL1 deletion mutant. In contrast, introduction of COQ8 using a multicopy vector did not accelerate the growth of the deletion mutants of TAZ1 or CLD1, which encode an acyltransferase or phospholipase A2, respectively, involved in the remodeling of cardiolipin. These results suggest genetic interactions between the mitochondrial phospholipase A1 gene and the genes involved in CoQ synthesis.

We investigated the role of FAD2, which was predicted to encode a fatty acid desaturase of the n-alkane-assimilating yeast Yarrowia lipolytica. Northern blot analysis suggested that FAD2 transcription was upregulated at low temperature or in the presence of n-alkanes or oleic acid. The FAD2 deletion mutant grew as well as the wild-type strain on glucose, n-alkanes, or oleic acid at 30 °C, but grew at a slower rate at 12 °C, when compared to the wild-type strain. The growth of the FAD2 deletion mutant at 12 °C was restored by the addition of 18:2, but not 18:1, fatty acids. The amount of 18:2 fatty acid in the wild-type strain was increased by the incubation at 12 °C and in the presence of n-octadecane. In contrast, 18:2 fatty acid was not detected in the deletion mutant of FAD2, confirming that FAD2 encodes the Δ12-fatty acid desaturase. These results suggest that Δ12-fatty acid desaturase is involved in the growth of Y. lipolytica at low temperature.

Plecoglossus altivelis (ayu) is one of the most important fish species in the Japanese islands and in internal fish hatcheries. Living in open aquatic environments exposes fish to many pathogens. Therefore, they require rapid and strong immune defenses. We investigated in vivo the direct association between the ayu innate immune response, represented by the relative transcription of genes encoding the cathelicidin and hepcidin antimicrobial peptides, and lipopolysaccharide (LPS), a conventional pathogen-associated molecular patterns (PAMPs) of Gram-negative bacteria. Different concentrations of LPS (1, 10 and 100 µg/fish) were injected intraperitoneally into young (sexually immature) and adult (fully sexually mature) ayu. The relative expression of the antimicrobial peptide genes was measured 6 hr, 24 hr and 1 week after stimulation with LPS. We found a direct association between the expression of the antimicrobial peptide genes investigated and LPS stimulation. This relationship was time-, dose- and age-dependent. Further research is required to determine the cell-specific transcriptional regulation and posttranscriptional regulation of these antimicrobial peptides.

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection., Author summary We show here that stimulation of PrPC with anti-PrP mAbs protected mice from lethal infection with IAVs by stimulating macrophage polarization to an anti-inflammatory M2 phenotype through activation of SFKs in infected lungs. IAVs are causative agents for seasonal epidemic outbreaks of influenza, and often cause high morbidity and mortality in infected people, particularly in the young and elderly and those with underlying chronic diseases. It has also been reported that highly pathogenic avian IAVs have the potential to infect humans and cause high morbidity and mortality in infected individuals. The other problem with IAV infections is that new IAV strains, which are resistant to currently available anti-influenza agents such as neuraminidase inhibitors, have been emerging in human populations. Our current results showing that targeting PrPC with anti-PrP mAbs protected against lethal infection with IAVs in mice give rise to the possibility that PrPC-targeting therapeutics might be beneficial in influenza infection. Moreover, since PrPC is a host molecule, PrPC-targeting drugs might not induce drug-resistant IAVs. Thus, PrPC might be a novel target molecule for therapeutic development for IAV infection, although further studies are needed.

Background Birds harbor specific sex determination and differentiation mechanisms. Although the molecular mechanisms associated with sex determination in somatic cells have been elucidated, those for germ cells remain unclear. Results Here, we characterized the chicken forkhead box L3 (foxl3)‐like gene as a sex‐determination factor in sexually indifferent medaka germline stem cells. The foxl3‐like gene was cloned by rapid amplification of cDNA ends, and the nucleotide sequence was analyzed. The deduced amino acid sequence was compared with FOXL3 sequences from other species, revealing low identity and similarity scores. Expression analysis of foxl3‐like mRNA during gonadogenesis showed female left‐gonad‐specific temporal expression in an egg incubated from 10 to 16 days, as well as low general expression in certain hatched female chicken organs. Moreover, the amino acid sequence deduced for the FOXL3‐like protein displayed low identity with medaka FOXL3, with the FOXL3‐like protein specifically localized in the oogonia, whereas medaka FOXL3 was found in sexually indifferent germline stem cells. Furthermore, the timing of expression differed between the foxl3‐like gene and that of medaka foxl3. Conclusions These results suggest that chicken FOXL3‐like protein and medaka FOXL3 differ in terms of their functions as female sex‐determination factors., Key Findings The nucleotide sequence of the chicken foxl3‐like gene was determined by RACE.The expression of chicken foxl3‐like mRNA was virtually undetectable in specific organs, including the ovary, of 2‐week‐old female chickens.Chicken FOXL3‐like protein was detected in the oogonia of an egg incubated for 14 days.Temporal expression of chicken foxl3‐like mRNA was observed only in the oogonia of an egg incubated from 8 to 18 days during gonadogenesis, and the timing of gene expression differed from that of medaka foxl3.

The cell wall integrity signaling (CWIS) pathway is involved in fungal cell wall biogenesis. This pathway is composed of sensor proteins, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway, and it controls the transcription of many cell wall-related genes. PKC plays a pivotal role in this pathway; deficiencies in PkcA in the model filamentous fungus Aspergillus nidulans and in MgPkc1p in the rice blast fungus Magnaporthe grisea are lethal. This suggests that PKC in filamentous fungi is a potential target for antifungal agents. In the present study, to search for MgPkc1p inhibitors, we carried out in silico screening by three-dimensional (3D) structural modeling and performed growth inhibition tests for M. grisea on agar plates. From approximately 800,000 candidate compounds, we selected Z-705 and evaluated its inhibitory activity against chimeric PKC expressed in Saccharomyces cerevisiae cells in which the kinase domain of native S. cerevisiae PKC was replaced with those of PKCs of filamentous fungi. Transcriptional analysis of MLP1, which encodes a downstream factor of PKC in S. cerevisiae, and phosphorylation analysis of the mitogen-activated protein kinase (MAPK) Mpk1p, which is activated downstream of PKC, revealed that Z-705 specifically inhibited PKCs of filamentous fungi. Moreover, the inhibitory activity of Z-705 was similar to that of a well-known PKC inhibitor, staurosporine. Interestingly, Z-705 inhibited melanization induced by cell wall stress in M. grisea. We discuss the relationships between PKC and melanin biosynthesis. IMPORTANCE A candidate inhibitor of filamentous fungal protein kinase C (PKC), Z-705, was identified by in silico screening. A screening system to evaluate the effects of fungal PKC inhibitors was constructed in Saccharomyces cerevisiae. Using this system, we found that Z-705 is highly selective for filamentous fungal PKC in comparison with S. cerevisiae PKC. Analysis of the AGS1 mRNA level, which is regulated by Mps1p mitogen-activated protein kinase (MAPK) via PKC, in the rice blast fungus Magnaporthe grisea revealed that Z-705 had a PKC inhibitory effect comparable to that of staurosporine. Micafungin induced hyphal melanization in M. grisea, and this melanization, which is required for pathogenicity of M. grisea, was inhibited by PKC inhibition by both Z-705 and staurosporine. The mRNA levels of 4HNR, 3HNR, and SCD1, which are essential for melanization in M. grisea, were suppressed by both PKC inhibitors.

Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott–Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination.

Yarrowia lipolytica possesses twelve ALK genes, which encode cytochromes P450 in the CYP52 family. In this study, using a Y. lipolytica strain from which all twelve ALK genes had been deleted, strains individually expressing each of the ALK genes were constructed and their roles and substrate specificities were determined by observing their growth on n-alkanes and analyzing fatty acid metabolism. The results suggested that the twelve Alk proteins can be categorized into four groups based on their substrate specificity: Alk1p, Alk2p, Alk9p, and Alk10p, which have significant activities to hydroxylate n-alkanes; Alk4p, Alk5p, and Alk7p, which have significant activities to hydroxylate the ω-terminal end of dodecanoic acid; Alk3p and Alk6p, which have significant activities to hydroxylate both n-alkanes and dodecanoic acid; and Alk8p, Alk11p, and Alk12p, which showed faint or no activities to oxidize these substrates. The involvement of Alk proteins in the oxidation of fatty alcohols and fatty aldehydes was also analyzed by measuring viability of the mutant deleted for twelve ALK genes in medium containing dodecanol and by observing growth on dodecanal of a mutant strain, in which twelve ALK genes were deleted along with four fatty aldehyde dehydrogenase genes. It was suggested that ALK gene(s) is/are involved in the detoxification of dodecanol and the assimilation of dodecanal. These results imply that genes encoding CYP52-family P450s have undergone multiplication and diversification in Y. lipolytica for assimilation of various hydrophobic compounds.

DDL1 encodes a mitochondrial phospholipase A

To elucidate the mechanism of acyl chain remodeling at the sn-1 position of phosphatidylcholine (PC), we investigated acyl chain introduction using a newly synthesized 1-hydroxy-2-hexadecyl-sn-glycero-3-phosphocholine (HHPC) in Saccharomyces cerevisiae. HHPC is incorporated into yeast cells and converted to a PC species containing acyl residues of 16 or 18 carbons. The efficiency of palmitoleic acid introduction to HHPCin vitro is lower in the reaction with the extract from the deletion mutant of ALE1, which encodes a membrane-bound O-acyltransferase, than in that with extracts from the wild-type strain. In addition, deletion of ALE1 causes reductions in the molecular species containing acyl residues in HHPC. These results reveal that ALE1 is involved in acyl chain transfer to the sn-1 position of HHPC in yeast.

In this study, we investigated the role of OSH6, which encodes a homolog of the oxysterol-binding protein, in the assimilation of n-alkanes in the yeast Yarrowia lipolytica. The deletion mutant of OSH6 showed growth defects on n-alkanes of 10–16 carbons. In the deletion mutant, production of the functional cytochrome P450 was not observed. However, transcription of ALK1, encoding a major P450 belonging to the CYP52 family that plays a critical role in n-alkane hydroxylation, and further translation of its transcript were noted in the deletion mutant as well as in the wild-type strain. The phospholipid composition was altered and, the ratio of phosphatidylserine (PS) was reduced by the deletion of OSH6. Residues involved in the transport of PS and phosphatidylinositol-4-phosphate in Osh6 of Saccharomyces cerevisiae are conserved in Y. lipolytica Osh6p and substitutions of these residues resulted in a defect in the n-alkane assimilation by Y. lipolytica. From these results, we propose a hypothesis that Osh6p provides an ideal endoplasmic reticulum membrane environment for Alk proteins to have a functional conformation via lipid transport activity in Y. lipolytica.

In the n-alkane-assimilating yeast Yarrowia lipolytica, the transcription of ALK1, encoding cytochrome P450, that catalyses n-alkane hydroxylation is activated by a complex composed of Yas1p and Yas2p via a promoter element, ARE1, in response to n-alkanes. An Opi1-family transcription factor, Yas3p, represses the transcription by binding to Yas2p in the nucleus when cultured in glucose-containing medium, but it is localized to the ER, presumably through interaction with acidic phospholipids, phosphatidic acid and/or phospho inositides, when cultured in n-alkane-containing medium. Here, to elucidate the mechanisms regulating the localization of Yas3p, point and deletion mutants of Yas3p were constructed and analysed. The substitution of Trp(360) and Cys(361) by Arg abrogated the localization of Yas3p to the ER and decreased ARE1-mediated transcriptional activation by n-alkane. A Yas3p truncation mutant consisting of residues 259-422 did not bind to acidic phospholipids, but it was localized to the ER in the presence of n-alkane, implying the acidic-phospholipid-independent recruitment of this mutant to the ER in response to n-alkane. The W360R and C361R substitutions in this truncation mutant abolished its localization to the ER. The results suggest that these residues are implicated in the acidic phospholipid-independent interaction of Yas3p to the ER.

OBJECTIVE Several reports showed that neoplastic spindle cells (NSCs) may be strongly involved in the invasion, metastasis, and poor prognosis, as well as in epithelial-mesenchymal transition (EMT). It has not yet been investigated that NSCs relate to the recurrence and prognosis in various cancers. Furthermore, NSCs participate in EMT in pancreatic cancer (PC) too. We clinicopathologically investigated the association between NSCs and the recurrence, prognosis, and EMT in PC. METHODS We studied 68 PC patients. Cancer cells with a spindle or oval shape that do not exhibit luminal structures were defined as NSCs. We graded NSCs regarding to an area of NSCs at hematoxylin and eosin stain (NSC grade) and examined the participation in NSCs and EMT by immunohistostaining of snail antibody and E-cadherin antibody. RESULTS In multivariate analysis, NSC grade was an independent risk factor for disease-free survival and overall survival. This was independent of TNM stage and histological grade. Neoplastic spindle cells were related to EMT pattern in immunohistostaining significantly. CONCLUSIONS Neoplastic spindle cell grade significantly related to the recurrence and prognosis of PC. The NSC grade assessment can be not only performed inexpensively and conveniently, but also used to guide future individualized therapeutic approaches. Furthermore, NSCs were found to relate to EMT profoundly.

A protein kinase C of Aspergillus nidulans, PkcA, is required for cell wall integrity (CWI) and is considered a major component of the regulating pathway. To investigate whether PkcA regulates the transcription of cell wall-related genes, we constructed strains expressing pkcA(R429A) that encodes an activated form of PkcA. The mRNA levels of most chitin synthase genes and an α-glucan synthase gene, agsB, were increased when pkcA(R429A) expression was induced. These mRNA increases were not observed or were only partially observed, in a deletion mutant of rlmA, an ortholog of RLM1 that encodes a transcription factor in the CWI pathway in Saccharomyces cerevisiae. In addition, in a pkcA temperature-sensitive mutant under heat stress, the mRNA levels of some chitin synthase genes and agsB did not increase. These results suggest that PkcA is involved in CWI maintenance through the transcriptional regulation of cell wall-related genes.

Question: A 68year-old man with severe alcoholic liver cirrhosis underwent a left lobe adult-to-adult living donor liver transplantation. The volume of the donor’s liver was relatively low for the recipient; therefore, portacaval shunting was performed between the right branch of the portal vein and the inferior vena

Sterols are present in eukaryotic membranes and significantly affect membrane fluidity, permeability, and microdomain formation. They are synthesized in the endoplasmic reticulum (ER) and transported to other organelles and the plasma membrane. Sterols play important roles in the biogenesis and maintenance of mitochondrial membranes. However, the mechanisms underlying ER-to-mitochondrion sterol transport remain to be identified. Here, using purified yeast membrane fractions enriched in ER and mitochondria, we show that the oxysterol-binding protein homologs encoded by the OSH genes in the yeast Saccharomyces cerevisiae mediate sterol transport from the ER to mitochondria. Combined depletion of all seven Osh proteins impaired sterol transport from the ER to mitochondria in vitro; however, sterol transport was recovered at different levels upon adding one of the Osh proteins. Of note, the sterol content in the mitochondrial fraction was significantly decreased in vivo after Osh4 inactivation in a genetic background in which all the other OSH genes were deleted. We also found that Osh5–Osh7 bind cholesterol in vitro. We propose a model in which Osh proteins share a common function to transport sterols between membranes, with varying contributions by these proteins, depending on the target membranes. In summary, we have developed an in vitro system to examine intracellular sterol transport and provide evidence for involvement of Osh proteins in sterol transport from the ER to mitochondria in yeast.

Vaccination is the most effective method for controlling the infectious diseases that threaten the poultry industry worldwide. The use of adjuvants or immunostimulants is often necessary to improve vaccine efficacy, particularly for vaccines based on recombinant protein or inactivated pathogens. The adjuvant effects of zymosan A on antigen-specific antibody production were investigated in chickens. First, the optimal adjuvant dose of zymosan A was determined. Chicks were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) at a dosage of 2 mg/kg body weight (BW) with or without zymosan A (at a dosage of 0.5 mg/kg BW) co-administration at 4, 5 and 6 weeks of age. Different routes of immunization (oral, intranasal (i.n.), intraocular (i.o.), subcutaneous (s.c.), intramuscular (i.m.) and intraperitoneal (i.p.) were tested. Anti-DNP IgY and IgA concentrations in serum samples from all chicks were measured by an enzyme-linked immunosorbent assay. The results revealed that co-administration of zymosan A with DNP-KLH significantly increased anti-DNP IgY concentrations in chicks immunized by the oral and s.c. routes of administration when compared with control groups. In addition, co-administration of zymosan A with DNP-KLH significantly increased anti-DNP IgA concentrations in chicks immunized by the oral, i.o. and s.c. routes compared with control groups. In conclusion, zymosan A is a useful immune-potentiator adjuvant in chickens, and its co-administration with vaccine antigens enhances humoral immune responses.

T2 (tumor invades perimuscular connective tissue; no extension beyond serosa or into liver) gallbladder cancer has generally been treated by S4aS5 subsegmentectomy (S4aS5 HR). We investigated the therapeutic effect of full-thickness cholecystectomy (FC) and gallbladder bed resection (GBR), in terms of tumor location and resection margin (distance from the tumor). At our department we employ the following protocol to determine the extent of resection needed to achieve R0 status: (1) A tumor located in the gallbladder fundus (Gf) or body (Gb) and only on the free peritoneal side was classified as P-type, for which full-thickness cholecystectomy and regional lymph node dissection were performed. (2) A tumor located in Gf or Gb and in contact with the liver bed was classified as H-type, for which gallbladder bed resection and regional lymph node dissection were performed. (3) A tumor located in the gallbladder neck (Gn) was classified as N-type, for which gallbladder bed resection, bile duct resection, and regional lymph node dissection were performed. Twenty-two patients admitted to our department between January 2000 and December 2014 with pT2gallbladder cancers were included in our study. Surgical procedures performed were compared with those specified in our protocol, and patients in whom the extent of resection was greater than that specified in our strategy were evaluated clinicopathologically and in terms of recurrence and the prognosis. Six (27.2%), 7 (31.8%), and 9 (40.9%) patients underwent limited, standard, and extended surgery, respectively. Ten (66.7%) of 15 patients with tumors close to the liver bed underwent cholecystectomy or extended surgery, 7 (85.7%) of 8 patients with tumors close to the bile duct underwent bile duct resection, and 16 (72.7%) of 22 patients underwent regional lymph node dissection. Recurrence at the bile duct resection margin, para-aortic lymph node metastasis, and hepatic metastasis occurred in 2, 1, and 3 patients, respectively. The 3-year survival rates (for patients including those dying of noncancer causes) were 50, 100, and 75% after limited, standard, and extended surgery, respectively. There was a significant difference in the survival rate of patients who underwent standard or extended surgery (P=0.0273). Favorable results were obtained in T2 gallbladder cancer patients without performing S4aS5 subsegmentectomy. Depending on the tumor location, neither full-thickness cholecystectomy nor gallbladder bed resection appeared to pose problems regarding recurrence or prognosis. In conclusion, surgical treatment based on our protocol, which aims to achieve the condition of R0, may result in a sufficient therapeutic effect.

In the n-alkane assimilating yeast Yarrowia lipolytica, n-alkanes are oxidized to fatty acids via fatty alcohols and fatty aldehydes, after which they are utilized as carbon sources. Here, we show that four genes (HFD1-HFD4) encoding fatty aldehyde dehydrogenases (FALDHs) are involved in the metabolism of n-alkanes in Y. lipolytica. A mutant, in which all of four HFD genes are deleted (Δhfd1-4 strain), could not grow on n-alkanes of 12-18 carbons; however, the expression of one of those HFD genes restored its growth on n-alkanes. Production of Hfd2Ap or Hfd2Bp, translation products of transcript variants generated from HFD2 by the absence or presence of splicing, also supported the growth of the Δhfd1-4 strain on n-alkanes. The FALDH activity in the extract of the wild-type strain was increased when cells were incubated in the presence of n-decane, whereas this elevation in FALDH activity by n-decane was not observed in Δhfd1-4 strain extract. Substantial FALDH activities were detected in the extracts of Escherichia coli cells expressing the HFD genes. Fluorescent microscopic observation suggests that Hfd3p and Hfd2Bp are localized predominantly in the peroxisome, whereas Hfd1p and Hfd2Ap are localized in both the endoplasmic reticulum and the peroxisome. These results suggest that the HFD multigene family is responsible for the oxidation of fatty aldehydes to fatty acids in the metabolism of n-alkanes, and raise the possibility that Hfd proteins have diversified by gene multiplication and RNA splicing to efficiently assimilate or detoxify fatty aldehydes in Y. lipolytica.

CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis–Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not.

Peripheral blood cells from ayu, Plecoglossus altivelis altivelis, were separated using a density gradient. Blood cells were then smeared using Shandon Cytospin and subjected to cytochemical staining. Blood cells were categorized based on morphological and cytochemical characteristics, and the density fractionation range and nucleus area/cell area ratio were observed. Lymphocytes are distinguished from neutrophils by their basophilic cytoplasm and Golgi-like field. The features of chromatin in thrombocytes are different from those of lymphocytes or neutrophils, but some small neutrophils have similar chromatin. Therefore, it is necessary to perform peroxidase staining to distinguish small neutrophils from thrombocytes. Basophils have large basophilic granules in cytoplasm. Based on density fractionation of blood cells, thrombocytes in the low-density area were separated from other blood cells. Identification of peripheral blood cells from ayu was possible with these staining methods. Monocytes/macrophages from spleen are specifically positive for esterase staining by α-naphthyl butyrate. As a result, thrombocytes, lymphocytes, neutrophils, basophils and monocytes/macrophages were identified in smears from peripheral blood or spleen tissue. In this paper, we confirmed that the peripheral blood corpuscles of ayu are able to be identified using the present staining methods.

Interorganelle phospholipid transfer is critical for eukaryotic membrane biogenesis. In the yeast Saccharomyces cerevisiae, phosphatidylserine (PS) synthesized by PS synthase, Pss1, in the endoplasmic reticulum (ER) is decarboxylated to phosphatidylethanolamine (PE) by PS decarboxylase, Psd1, in the ER and mitochondria or by Psd2 in the endosome, Golgi, and/or vacuole, but the mechanism of interorganelle PS transport remains to be elucidated. Here we report that Sfh1, a member of Sec14 family proteins of S. cerevisiae, possesses the ability to enhance PE production by Psd2. Overexpression of SFH1 in the strain defective in Psd1 restored its growth on non-fermentable carbon sources and increased the intracellular and mitochondrial PE levels. Sfh1 was found to bind various phospholipids, including PS, in vivo. Bacterially expressed and purified Sfh1 was suggested to have the ability to transport fluorescently labeled PS between liposomes by fluorescence dequenching assay in vitro. Biochemical subcellular fractionation suggested that a fraction of Sfh1 localizes to the endosome, Golgi, and/or vacuole. We propose a model that Sfh1 promotes PE production by Psd2 by transferring phospholipids between the ER and endosome.

In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane.

We classified resected intraductal papillary mucinous neoplasms (IPMNs) into four subtypes (gastric, intestinal, pancreatobiliary and oncocytic) according to their morphological features and mucin expression, determined their clinicopathological characteristics and investigated the possibility of preoperatively diagnosing these subtypes. Sixty resected tumors, 4 preoperative tumor biopsies and 10 preoperative pancreatic juice cytology specimens were analyzed. The gastric and intestinal types accounted for the majority of IPMNs. Non-gastric type IPMNs were of high-grade malignancy. Many of the pancreatobiliary-type IPMNs were in an advanced stage and were associated with a poor prognosis. The results of mucin immunohistochemical staining of preoperative biopsy and surgically resected specimens were in agreement with each other, and in close agreement with those for pancreatic juice cytology specimens obtained from 10 patients during endoscopic retrograde cholangiopancreatography (ERCP). The immunostaining of preoperative biopsy specimens and ERCP-obtained pancreatic juice cytology specimens may be useful in the differential diagnosis of gastric and intestinal types of IPMN. If such techniques enable the preoperative diagnosis of IPMN subtypes, their use in combination with conventional preoperative imaging modalities may lead to surgical treatment best suited for the biological characteristics of the four subtypes.

In treating liver tumors, preserving hepatic reserve and reducing surgical invasiveness are important for minimizing postoperative complications. Geranylgeranylacetone (GGA) is reported to selectively induce heat-shock protein 70 (HSP70), which initiates a powerful cytoprotective effect. We investigated the function of HSP70 under conditions of radiofrequency ablation (RFA) of the liver.Male Wistar rats were divided into three groups: a control group, a group administered GGA, and a group administered GGA plus quercetin, an HSP70 synthesis inhibitor. Expression of HSP70 and heat-shock factor-1 (HSF1) in the liver was measured at the protein level, and severity of liver damage was investigated using serum and hepatic tissue.The GGA-treated group had higher expression of HSP70 and HSF1 than the other groups. Peak liver damage in all groups occurred 6 h after RFA. The GGA-treated group also had significantly less liver damage and lower serum level of the inflammatory cytokine tumor necrosis factor-α, and a lower rate of apoptosis in tissue around post-ablation necrosis. Expression of HSP70 and HSF1 was suppressed in the group treated with GGA and quercetin, and this group had severe liver damage.Induction of HSP in the liver by GGA may be applicable in future treatments for hepatocellular carcinoma or liver metastasis. The present findings suggest that if preoperative administration of GGA can offer protective effects in the liver, treatment options could be increased and liver failure and other complications might be avoided.

Heat-shock protein 27 (HSP27), a low molecular weight stress protein, is recognized as a molecular chaperone. The expression of HSP27 has been detected in some human tumors and while HSP27 is phosphorylated as a reresponse to stress, the function of phosphorylated HSP27 (p-HSP27) is not known. The aim of this study was to investigate what kind of effect expression of HSP27 and p-HSP27 in HCC has on clinicopathological characteristics and prognosis.An immunohistochemical study for HSP27 and p-HSP27 was performed on 194 resected HCC cases. We analyzed the correlation of HSP27 expression with various parameters statistically.There was no correlation between expression of HSP27 and the clinicopathological characteristics and prognosis from the analysis of 194 cases. From the analysis of the hepatitis C virus (HCV)-positive group of 142 cases, those that were p-HSP27-positive had a larger tumor diameter and the portal vein invasion rate was high.The expression of total HSP27 may serve as a new, clinically useful marker of HCC. In addition, the present study suggests that the expression of phosphorylated HSP27 is useful in the screening and grading of HCC occurring in the setting of HCV.