Production of Ergot Alkaloids by the Japanese Isolate Claviceps purpurea var. agropyri on Rice Medium

Ergot alkaloids (EAs) are secondary metabolites produced by ergot fungi (e.g., Claviceps purpurea), which are parasites of Gramineae grasses. EAs and their analogs are used to treat migraine, postpartum uterine bleeding, and Parkinson's syndrome. Recent studies have reported additional new bioactive activities of EAs and their analogs, making them essential compounds for drug development, drug repositioning, and clinical applications. EAs are produced industrially by field cultivation of ergot or liquid fermentation in the mycelial phase, but there are few published studies of the production of EAs by cereal culture and thus this approach is poorly understood. This study searched for Claviceps strains that produce EAs cultured artificially in the mycelial phase, then the selected strains were cultured on cereal media (white rice, brown rice, and rye) to examine their ability to produce EAs on each medium. C. purpurea var. agropyri produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase. When cultured with white rice, brown rice, or rye, C. purpurea var. agropyri produced 1 - 3 on all cereal media. The total amount of 1 - 3 in each cereal medium (150 g of cereal per Roux flask) was 2220.5 ± 564.1 μg for white rice, 920.0 ± 463.6 μg for brown rice, and 595.4 ± 52.1 μg for rye. The white rice medium supported the highest production of 1 - 3, with the total amount of EAs (150 g of white rice per Roux flask) being about 34 times higher than that in the T25 liquid medium (190 mL per 1 L Erlenmeyer flask) (equivalent amount per flask).

Secalonic Acid and Benzoic Acid Analogues Exhibiting Cyto toxicity against Cancer Cells Isolated from Claviceps yanagawaensis

The genus Claviceps (Clavicipitaceae) is noted for producing ergot alkaloids that cause ergotism. Claviceps yanagawaensis, a parasite of Zoysia japonica (family: Poaceae), has been isolated in Japan. Bioactivity screening showed that a methanol extract from a rice culture of C. yanagawaensis was cytotoxic to cancer cells. In our search for active substances, the new secalonic acid analogues (-)-5-epi-F-7 (1) and ergochrysin C (2) and a new benzoic acid analogue, dimethyl bigutol (3), were isolated along with the known compounds 3,4-dihydroxy-5-(3-methyl-2-buten-1-yl)benzoic acid (4) and methyl veratrate (5). The structures of 1 - 3 were elucidated by NMR, MS, and circular dichroism spectroscopy. MTT assays of 1 - 5 using cancer cell lines (HepG2, HL60, HT29, PANC-1, and T98G) showed that 1 - 4 exhibited cytotoxicity against cancer cells.

Current status, problems, and perspectives of non-alcoholic fatty liver disease research

Non-alcoholic fatty liver disease(NAFLD) is a major chronic liver disease that can lead to liver cirrhosis, liver cancer, and ultimately death. NAFLD is pathologically classified as non-alcoholic fatty liver(NAFL) or non-alcoholic steatohepatitis(NASH) based on the existence of ballooned hepatocytes,although the states have been known to transform into each other. Moreover,since the detection of ballooned hepatocytes may be difficult with limited biopsied specimens, its clinical significance needs reconsideration. Repeated liver biopsy to assess histological NAFLD activity for therapeutic response is also impractical, creating the need for body fluid biomarkers and less invasive imaging modalities. Recent longitudinal observational studies have emphasized the importance of advanced fibrosis as a determinant of NAFLD outcome. Thus,identifying predictors of fibrosis progression and developing better screening methods will enable clinicians to isolate high-risk NAFLD patients requiring early intensive intervention. Despite the considerable heterogeneity of NAFLD with regard to underlying disease, patient age, and fibrosis stage, several clinical trials are underway to develop a first-in-class drug. In this review, we summarize the present status and future direction of NAFLD/NASH research towards solving unmet medical needs.

Mild drinking habit is a risk factor for hepatocarcinogenesis in non-alcoholic fatty liver disease with advanced fibrosis

AIM The impact of mild drinking habit(less than 20 g/d of ethanol) on the clinical course of non-alcoholic fatty liver disease(NAFLD) has not been determined. We examined the influence of a mild drinking habit on liver carcinogenesis from NAFLD. METHODS A total of 301 patients who had been diagnosed as having NAFLD by liver biopsy between 2003 and 2016 [median age: 56 years, 45% male, 56% with nonalcoholic steatohepatitis, 26% with advanced fibrosis(F3-4)] were divided into the mild drinking group withe thanol consumption of less than 20 g/d(mild drinking group, n = 93) and the non-drinking group(n = 208). Clinicopathological features at the time of liver biopsy and factors related to hepatocellular carcinoma(HCC) occurrence were compared between the groups.RESULTS We observed significant differences in male prevalence(P = 0.01), platelet count(P = 0.04), and gammaglutamyl transpeptidase(P = 0.02) between the test groups. Over 6 years of observation, the HCC appearance rate was significantly higher in the mild drinking group(6.5% vs 1.4%, P = 0.02). Multivariate survival analysis using Cox's regression model revealed that hepatic advanced fibrosis(F3-4)(P < 0.01, risk ratio: 11.60), diabetes mellitus(P < 0.01, risk ratio: 89.50), and serum triglyceride(P = 0.04, risk ratio: 0.98) were factors significantly related to HCC in all NAFLD patients, while the effect of a drinking habit was marginal(P = 0.07, risk ratio: 4.43). In patients with advanced fibrosis(F3-4), however, a drinking habit(P = 0.04, risk ratio: 4.83), alpha-fetoprotein(P = 0.01, risk ratio: 1.23), and diabetes mellitus(P = 0.03, risk ratio: 12.00) were identified as significant contributors to HCC occurrence. CONCLUSION A mild drinking habit appears to be a risk factor for hepatocarcinogenesis in NAFLD patients, especially those with advanced fibrosis.

Serum autotaxin levels are correlated with hepatic fibrosis and ballooning in patients with non-alcoholic fatty liver disease

AIM To examine the relationship between serum autotaxin(ATX) concentrations and clinicopathological findings in non-alcoholic fatty liver disease(NAFLD) patients.METHODS One hundred eighty-six NAFLD patients who had undergone liver biopsy between 2008 and 2017 were retrospectively enrolled.Serum samples were collected at the time of biopsy and ATX was measured by enzyme immunoassays.Sera obtained from 160 healthy,nonobese individuals were used as controls.Histological findings were graded according to an NAFLD scoring system and correlations with serum ATX were calculated by Spearman's test.Diagnostic accuracy was evaluated using the area under the receiver operating characteristic curve(AUC).Cut-off values were identified by the Youden index,and the nearest clinically applicable value to the cutoff was considered the optimal threshold for clinical convenience.RESULTS Serum ATX levels were significantly higher in NAFLD patients than in controls(0.86 mg/L vs 0.76 mg/L,P < 0.001) and correlated significantly with ballooning score and fibrosis stage(r = 0.36,P < 0.001 and r = 0.45,P < 0.001,respectively).Such tendencies were stronger in female patients.There were no remarkable relationships between ATX and serum alanine aminotransferase,lipid profiles,or steatosis scores.The AUC values of ATX for predicting the presence of fibrosis(≥ F1),significant fibrosis(≥ F2),severe fibrosis(≥ F3),and cirrhosis(F4),were all more than 0.70 in respective analyses.CONCLUSION Serum ATX levels may at least partially reflect histological severity in NAFLD.

Association between endotoxemia and histological features of nonalcoholic fatty liver disease

AIMTo assess whether surrogate biomarkers of endotoxemia were correlated with the histological features of nonalcoholic fatty liver disease (NAFLD).METHODSOne hundred twenty-six NAFLD patients who had undergone percutaneous liver biopsy were enrolled. Serum lipopolysaccharide (LPS)-binding protein (LBP) and anti-endotoxin core immunoglobulin G (EndoCab IgG) antibody concentrations at the time of liver biopsy were measured using the enzyme-linked immunosorbent assays to examine for relationships between biomarker levels and histological scores.RESULTSSerum LBP concentration was significantly increased in nonalcoholic steatohepatitis (NASH) patients as compared with nonalcoholic fatty liver (NAFL) subjects and was correlated with steatosis (r = 0.38, P &#x0003c; 0.0001) and ballooning scores (r = 0.23, P = 0.01), but not with the severity of lobular inflammation or fibrosis. Multivariate linear regression analysis revealed that LBP was associated with steatosis score and circulating C-reactive protein, aspartate aminotransferase, and fibrinogen levels. Serum EndoCab IgG concentration was comparable between NASH and NAFL patients. No meaningful correlations were detected between EndoCab IgG and histological findings.CONCLUSIONLBP/EndoCab IgG were not correlated with lobular inflammation or fibrosis. More accurate LPS biomarkers are required to stringently assess the contribution of endotoxemia to conventional NASH.

Assessment of KL-6 as a tumor marker in patients with hepatocellular carcinoma

AIM： To investigate the clinical significance of KL-6 as a tumor marker of HCC in two different ethnic groups with chronic liver disease consecutively encountered at outpatient clinics. METHODS： Serum KL-6 was measured by the sandwich enzyme immunoassay method using the KL-6 antibody （Ab） as both the capture and tracer Ab according to the manufacturer＇s instructions （Eisai, Tokyo, Japan）. Assessment of alpha fetoprotein （AFP） and protein induced vitamin K deficiency or absence （PIVKA-II） was performed in both groups using commercially available kits. RESULTS： A significantly higher mean serum KL-6 （556±467 U/L） was found in HCC in comparison with non-HCC groups either with （391±176 U/L; P〈0.001） or without （361±161 U/L; P〈0.001） liver cirrhosis （LC）. Serum KL-6 level did not correlate with either AFP or PIVKA-II serU/Levels. Using rec：eiver operating curve analysis for KL-6 as a predictor for HCC showed that the area under the curve was 0.574 （95%CI = 0.50-0.64） and the KL-6 level that gave the best sensitivity （61%） was found to be 334 U/L but according to the manufacturer＇s instructions; a cut-off point of 500 U/L was used that showed the highest specificity （80%） in comparison with AFP and PIVKA-II （78% vs 72% respectively）. Combining the values of the three markersimproved specificity of AFP for HCC diagnosis from 78% for AFP alone; 93% for AFP plus PIVKA-II to 99% for both plus KL-6 value （P〈0.001）. Mean serum alkaline phosphatase level was significantly higher in KL-6 positive （564＋475） in comparison with KL-6 negative （505＋469） HCC patients （P = 0.021）, but such a difference was not found among non-HCC corresponding groups. CONCLUSION： KL-6 is suggested as a tumor for HCC. Its positivity may reflect HCC-associated cholestasis and/ or local tumor invasion.

Mutations of pre-core and basal core promoter before and after hepatitis B e antigen seroconversion

AIM: To investigate the role of pre-core and basal core promoter(BCP) mutations before and after hepatitis Be antigen(HBe Ag) seroconversion.METHODS: The proportion of pre-core(G1896A) and basal core promoter(A1762T and G1764A) mutant viruses and serum levels of hepatitis B virus(HBV) DNA, hepatitis B surface antigen(HBs Ag), and HB core-related antigen were analyzed in chronic hepatitis B patients before and after HBe Ag seroconversion(n = 25), in those who were persistently HBe Ag positive(n = 18), and in those who were persistently anti-HBe positive(n = 43). All patients were infected with HBV genotype C and were followed for a median of 9 years.RESULTS: Although the pre-core mutant became predominant(24% to 65%, P = 0.022) in the HBe Ag seroconversion group during follow-up, the proportion of the basal core promoter mutation did not change. Median HBV viral markers were significantly higher in patients without the mutations in an HBe Ag positive status(HBV DNA: P = 0.003; HBs Ag: P < 0.001; HB core-related antigen: P = 0.001). In contrast, HBV DNA(P = 0.012) and HBs Ag(P = 0.041) levels were significantly higher in patients with the pre-core mutation in an anti-HBe positive status.CONCLUSION: There is an opposite association of the pre-core mutation with viral load before and after HBe Ag seroconversion in patients with HBV infection.

Longitudinal changes in the height and location of bone bridge from autogenous iliac bone graft in patients with cleft lip and palate

Background: Secondary alveolar bone graft has been widely used for the closure of maxillary bone defects in patients with cleft lip and palate (CLP). However, the log-term stability of grafted bone in detail remains unclear. Purpose: To evaluate the nature of longitudinal changes in the grafted bone from autogenous iliac bone in patients with CLP for more than 2 years after the surgery. Methods and Subjects: The subjects were 124 CLP patients treated with iliac bone graft in Hiroshima University Hospital from 1997 to 2007. The height and location of the bone bridge was evaluated radiographically before and after surgery. Results: 1) Bone graft was performed at a mean age of 12.5 (±4.2) years old. The canines on the cleft side were not erupted at the surgery in 60.0%. All canines erupted except for the congenital missing and impacted ones (11.3%) within 2 years after bone graft;2) The height of the bone bridge was more than 11 mm in 71.8% of all patients 1 month after surgery. In addition, the location of bone bridge in 66.1% of all patients were clinically satisfactory. However, these rates decreased time-dependently owing to bone resorption after the bone graft. Conclusions: In conclusion, it is shown that autogenous bone graft produce a sufficient bone bridge in the jaw cleft area by the long-term evaluation of the changes in height and location of bone bridge, although some amount of time-dependent bone resorption is inevitable.

Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China

Apple ring rot inflicts severe economic losses in the main apple producing areas of East Asia.The causal agent of the disease has been variously identified as Macrophoma kuwatsukai,Physalospora piricola and Botryosphaeria berengeriana f.sp.piricola,although B.dothidea is currently the most widely accepted pathogen name.The taxonomic uncertainty has delayed research that is needed to manage effectively this destructive disease.In the present study,genealogical concordance phylogenetic species recognition(GCPSR)was applied to pathogenic fungal isolates from apple and pear from several locations in China,along with several reference isolates.Phylogenetic results based on sequences of four nuclear loci(ITS,EF-1α,HIS and HSP)revealed the existence of two species within the examined isolates.One includes an ex-epitype isolate of B.dothidea and the other includes an isolate that was previously designated as B.berengeriana f.sp.piricola.Morphologically,the latter taxon presented an appressed mycelial mat on PDA whereas B.dothidea displayed columns of aerial mycelia reaching the lids,and conidia of the latter species were longer than B.dothidea.Botryosphaeria dothidea had a faster growth rate than the latter taxon under relatively high temperatures.Pathogenicity tests showed that on pear stems the latter taxon caused large-scale cankers along with blisters whereas B.dothidea was non-pathogenic,but on apple shoots the two fungi induced large and small wart-like prominences,respectively.Overall,this cryptic species demonstrated sufficient genetic variations and biological differences from B.dothidea.As a result of taxonomic study,we described here the latter taxon in a new combination,Botryosphaeria kuwatsukai and designate an epitype.Both B.kuwatsukai and B.dothidea are considered to be the main causal agents for apple ring rot in China and Japan.

What will happen in patients with advanced nonalcoholic fatty liver disease?

Non-alcoholic fatty liver disease(NAFLD)and advanced form non-alcoholic steatohepatitis(NASH)have been become globally major chronic liver diseases(1,2).NAFLD is one of the leading causes of liver cirrhosis,hepatic failure,and hepatocellular carcinoma(HCC)(3).A large number of retrospective studies reported the importance of hepatic fibrosis with NAFLD patients as the strongest factor of liver-related mortality(4,5).On the other hand,NAFLD patients with advanced hepatic fibrosis are thought to have higer risk of developing vascular events and extrahepatic cancers because of the high prevalence of coexisting cardiometabolic risk factors,such as hypertension,diabetes mellitus,dyslipidemia,and obesity(6).However,since former literatures included only few NAFLD patients with advanced hepatic fibrosis,it is hard to estimate the accurate risk on the whole spectrum of these complications.

腹腔镜下全胃切除术后应用线性吻合器进行功能性食管空肠端端吻合术

限制腹腔镜下全胃切除术（LTG）在临床广泛运用的主要原因是技术上存在腔镜下行Roux—en—Y消化道重建的难度。报道称，诸如吻合口瘘或狭窄等重建相关并发症的发生率较远端胃切除术高。为了克服技术上的瓶颈并建立起标准的重建方法，我们于2006年9月引入使用线性吻合器行体内功能性食管一空肠端端吻合术（FETE）。