Diversity of endophytic bacterial microbiota in grapevine shoot xylems varies depending on wine grape-growing region, cultivar, and shoot growth stage.

Next-generation sequencing technology may clarify microbiota that are as yet poorly understood in the soil, the rhizosphere, and the phyllosphere of vineyards. To provide new information on the interaction between grapevine and microorganisms, we focused on the endophytic microbiota in grapevine. We performed endophytic microbiome analysis of the shoot xylems of four cultivars, Vitis vinifera cvs. Chardonnay, Pinot Noir, Cabernet Sauvignon, and Vitis sp. cv. Koshu, grown in eleven vineyards in Japan. The number of endophytic fungal species was small in the grapevine shoot xylems and could not be analyzed further, whereas a total of 7,019,600 amplicon sequences (46,642-285,003 per shoot xylem) and 1305 bacterial operational taxonomic units were obtained by analysis of the V3-V4 region of the bacterial 16S rRNA gene. Gammaproteobacteria was predominant in the shoot xylems at the shoot elongation stage irrespective of the cultivar, whereas Alphaproteobacteria and Oxyphotobacteria were predominant at véraison. Actinobacteria, Bacteroidia, Bacilli, and Clostridia were also detected in the shoot xylems. The endophytic bacterial microbiota in Koshu and Pinot Noir shoot xylems were similar irrespective of the grapevine-growing region. In contrast, the endophytic bacterial microbiota in Chardonnay and Cabernet Sauvignon showed diversity and complexity among grapevine-growing regions. Alpha diversity analysis revealed that Koshu shoot xylems had a higher diversity of endophytic bacterial microbiota than Pinot Noir, Chardonnay, and Cabernet Sauvignon shoot xylems, and that grapevine shoot xylems at the shoot elongation stage had a higher diversity of endophytic bacterial microbiota than those at véraison. Principal coordinate analysis (PCoA) demonstrated that the profiles of the endophytic bacterial microbiota in grapevine shoot xylems at véraison were relatively uniform compared with those at the shoot elongation stage. Multidimensional scaling analysis showed that the plots of all cultivars were generally apart from each other at the shoot elongation stage and then became close to each other at véraison. The plots of all grapevine-growing regions cultivating Koshu were close to each other, whereas those of grapevine-growing regions cultivating Chardonnay and Cabernet Sauvignon were apart from each other. The findings of this study suggest that the endophytic bacterial microbiota in grapevine shoot xylems varied depending on the cultivar and the grapevine-growing region even for the same cultivars, and that the microbiota fluctuated depending on the shoot growth stage.

Isolation and Characterization of Endophyte Bacillus velezensis KOF112 from Grapevine Shoot Xylem as Biological Control Agent for Fungal Diseases.

As the use of chemical fungicides has raised environmental concerns, biological control agents have attracted interest as an alternative to chemical fungicides for plant-disease control. In this study, we attempted to explore biological control agents for three fungal phytopathogens causing downy mildew, gray mold, and ripe rot in grapevines, which are derived from shoot xylem of grapevines. KOF112, which was isolated from the Japanese indigenous wine grape Vitis sp. cv. Koshu, inhibited mycelial growth of Botrytis cinerea, Colletotrichum gloeosporioides, and Phytophthora infestans. The KOF112-inhibited mycelial tips were swollen or ruptured, suggesting that KOF112 produces antifungal substances. Analysis of the 16S rDNA sequence revealed that KOF112 is a strain of Bacillus velezensis. Comparative genome analysis indicated significant differences in the synthesis of non-ribosomal synthesized antimicrobial peptides and polyketides between KOF112 and the antagonistic B. velezensis FZB42. KOF112 showed biocontrol activities against gray mold caused by B. cinerea, anthracnose by C. gloeosporioides, and downy mildew by Plasmopara viticola. In the KOF112-P. viticola interaction, KOF112 inhibited zoospore release from P. viticola zoosporangia but not zoospore germination. In addition, KOF112 drastically upregulated the expression of genes encoding class IV chitinase and ß-1,3-glucanase in grape leaves, suggesting that KOF112 also works as a biotic elicitor in grapevine. Because it is considered that endophytic KOF112 can colonize well in and/or on grapevine, KOF112 may contribute to pest-management strategies in viticulture and potentially reduce the frequency of chemical fungicide application.

Complete Genome Sequence of Bacillus velezensis KOF112, an Antifungal Endophytic Isolate from Shoot Xylem of the Indigenous Japanese Wine Grape Vitis sp. cv. Koshu.

Bacillus species are the most well-studied biological control agents and produce a large variety of antibiotics that exert antifungal activity against phytopathogens and induce defense responses in plants. Here, we introduce the genome sequence of Bacillus velezensis KOF112, an antifungal endophytic isolate from the shoot xylem of a wine grape.

Transcatheter arterial chemoembolization for hepatocellular carcinoma: Comparison of the therapeutic efficacies between miriplatin and epirubicin.

AIM: The therapeutic efficacy of transcatheter arterial chemoembolization (TACE) using miriplatin was evaluated in comparison with that using epirubicin in patients with hepatocellular carcinoma (HCC). METHODS: Two hundred and eight-nine HCC patients receiving TACE were retrospectively enrolled; none of the patients gave a previous TACE history. The short-term therapeutic efficacy was evaluated by computed tomography (CT) performed 1 month later. In patients showing TE-4, CT and/or magnetic resonance imaging examinations were performed repeatedly and the long-term therapeutic efficacy was assessed based on local tumor recurrence. RESULTS: After exclusion of 68 patients (CT not performed at 1 month), 97 patients treated with epirubicin and 124 treated with miriplatin were analyzed. The percentage of patients showing TE-4 was 46.8% in the miriplatin-TACE group, being significantly higher than that in the epirubicin-TACE group (33.0%). The cumulative local recurrence rates at 18 months were 71.2% in the miriplatin-TACE group and 43.1% in the epirubicin-TACE group; multivariate analysis revealed higher local tumor recurrence rates in the miriplatin-TACE group than in the epirubicin-TACE group. CONCLUSION: For HCC patients, although miriplatin-TACE was superior to epirubicin-TACE in the short term, it proved inferior to the latter in the long term. The merits of TACE using miriplatin should be further investigated, because adverse effects appear to be minimal after miriplatin administration.

Balloon-occluded retrograde transvenous obliteration using a microballoon catheter for intractable gastric fundal varices.

BACKGROUND AND AIM: Balloon-occluded retrograde transvenous obliteration (B-RTO) is recognized as the standard therapy for patients with gastric fundal varices in Japan; however, the procedure is difficult when drainage veins other than the gastrorenal shunt developed. The efficacy and safety of B-RTO using a microballoon catheter for such patients were evaluated. METHODS: The subjects were 99 patients with gastric fundal varices who fulfilled the criteria for receiving endoscopic and/or interventional therapies. Among these, 95 patients underwent B-RTO. Of the 95 patients, 14 were treated with the use of microballoon catheters, including nine in whom the left inferior phrenic vein was found as a secondary drainage vein in addition to the gastrorenal shunt, and five in whom a gastrorenal shunt was absent. The B-RTO procedure performed using a microballoon catheter inserted through the left inferior phrenic vein in 13 patients, and through the pericardiophrenic vein in one patient. RESULTS: The B-RTO procedure using microballoon catheters was successful in 13 of the 14 patients (93%), while in the remaining one patient, multiple drainage veins were visualized on venography. Complete obliteration of the varices was achieved in all the 13 patients by injection of 5% ethanolamine oleate iopamidol at a median volume of 25 mL (range, 11 to 40 mL) through the catheters. None of the patients showed injuries of the drainage veins or any systemic complications. CONCLUSIONS: B-RTO using a microballoon catheter is useful for the treatment of gastric fundal varices in which drainage veins other than the gastrorenal shunt developed.

Therapeutic strategy for patients with bleeding rectal varices complicating liver cirrhosis.

AIM: Although rupture of rectal varices is rarely encountered, it may provoke massive and fatal hemorrhage in patients with liver cirrhosis. We examined the clinical features of patients showing bleeding from rectal varices to establish a suitable therapeutic strategy for the lesions. METHODS: Twelve cirrhotic patients with bleeding rectal varices were enrolled. Surgical suture, endoscopic variceal ligation (EVL) or balloon tamponade was performed to achieve the initial hemostasis. Then, the feeding and drainage vessels of the varices were evaluated by computed tomography, and additional procedures were undertaken: EVL was performed when the sizes of the varices and feeding vessels were small. In contrast, in patients with varices of large sizes, balloon-occluded retrograde transvenous obliteration (B-RTO) was performed when single or two drainage vessels were identified, while endoscopic injection sclerotherapy (EIS) using ethanolamine oleate was carried out for varices with three or more drainage vessels. RESULTS: The Child-Pugh class was grade A in four, B in six and C in two patients. Eleven patients had received previous therapy for esophageal varices. Initial hemostasis was achieved by surgical suture in three patients, EVL in one patient and balloon tamponade in two patients. EVL, EIS and B-RTO were carried out as additional procedures in seven, three and one patient, respectively. Rebleeding from the rectal varices occurred in only one patient who underwent EVL as an additional procedure. CONCLUSION: Bleeding from rectal varices was controlled satisfactorily by the therapeutic strategy of selecting EVL, EIS or B-RTO as an additional therapy according to the size and hemodynamics of the varices.

SNPs in the promoter region of the osteopontin gene as a possible host factor for sex difference in hepatocellular carcinoma development in patients with HCV.

AIMS: Four single nucleotide polymorphisms (SNPs) exist in the promoter region of the osteopontin (OPN) gene, namely, the SNPs at nucleotide (nt) -155, -616, and -1748 showing linkage disequilibrium to each other, and an independent SNP at nt -443. The significance of these SNPs in the risk of hepatocellular carcinoma (HCC) development was examined in patients with hepatitis C virus (HCV). METHODS: The SNPs at nt -155 and nt -443 were analyzed in 120 patients with HCC. The promoter activity was measured in HepG2 cells by the dual-luciferase reporter assay. The electrophoretic mobility shift assay was performed using nuclear extracts from the cells. RESULTS: Peripheral platelet counts at the time of HCC detection were greater in women with homozygous deletion at nt -155 and C/C or C/T at nt -443 than in those showing other allelic combinations, while no such difference was observed in men. The promoter activity was greater in oligonucleotides with deletions at nt -155 and C at nt -443 than in those with other haplotypes. The mobility shift assay showed double and single complexes with oligonucleotides around nt -155 and nt -443, respectively. Binding activities were greater in deletion than in G in the case of the retarded complex in the former assay and in T than in C in the latter assay. The other complex in the former assay included SRY, showing an equivalent binding activity to oligonucleotides with both alleles. CONCLUSION: OPN promoter SNPs may play a role in the sexual difference in the risk of HCC development through the regulation of OPN expression in patients with HCV.

Usefulness of miriplatin as an anticancer agent for transcatheter arterial chemoembolization in patients with unresectable hepatocellular carcinoma.

BACKGROUND: Injection of a suspension of miriplatin into the hepatic artery has been employed for the treatment of hepatocellular carcinoma (HCC). The efficacy and safety of transcatheter arterial chemoembolization (TACE) using miriplatin were evaluated. METHODS: A total of 236 patients with unresectable HCC received miriplatin administration through the hepatic artery, followed by embolization with porous gelatin particles. The efficacy of this treatment modality was evaluated by contrast-enhanced computed tomography performed 1 month later and its safety based on the Common Terminology Criteria for Adverse Events (CTCAE). RESULTS: Miriplatin was used at a median dose of 66 mg. The therapeutic efficacy was evaluated in 130 patients, and the overall and complete response rates were 70.0 and 37.7%, respectively. The efficacies differed depending on the staging and Japan integrated staging (JIS) scores of the HCCs, with the overall and complete response rates being 87.7 and 66.7% for stage I and stage II HCC, and 56.2 and 15.1% for stage III and stage IV HCC, respectively; the corresponding rates were 93.2 and 70.5%, respectively, for HCCs with score 0 and score 1, and 58.1 and 20.9%, respectively, for those with scores 2-4. The stage of HCC was a significant independent factor associated with curative effects of TACE using miriplatin. Grade 3 elevation of serum transaminase levels was found in 23.4% of the patients; however, the values returned to the baseline levels. CONCLUSIONS: Miriplatin is a useful and safe agent for TACE in patients with HCC stage I or II and/or JIS score 0 or 1 only when radiofrequency ablation and liver resection cannot be performed.

A case of liver cirrhosis due to hepatits C virus infection complicating giant anorectal varices treated with balloon-occluded retrograde transvenous obliteration.

A 73-year-old man with liver cirrhosis due to hepatitis C virus infection was admitted to our hospital because of massive bleeding from external varices. Colonoscopic examination revealed that giant anorectal varices had developed between the anus and rectal ampulla, and had ruptured at the perianal site. On three-dimensional computed tomography imaging, the feeding and drainage vessels of the varices were identified as the inferior mesenteric vein and right inferior hemorrhoidal vein, respectively. Endoscopic therapies were not employed for the bleeding varices, because the blood flow volume of the feeding vessel was extremely large. Balloon-occluded retrograde transvenous obliteration (B-RTO) was therefore carried out through the drainage vessels. The variceal blood flow disappeared after B-RTO therapy, and the varices decreased in size with thrombus formation verified by colonoscopy. Bleeding from the external varices also ceased. B-RTO therapy may be an effective approach for giant anorectal varices presenting as a complication in liver cirrhosis patients in whom the main drainage vessels can be determined.

High-dose vitamins E and C supplementation prevents ribavirin-induced hemolytic anemia in patients with chronic hepatitis C.

AIM: In combination therapy using interferon (IFN) and ribavirin for chronic hepatitis C, reduced doses should be used due to ribavirin-induced hemolytic anemia. The present study aimed to elucidate whether high-dose vitamins E and C supplementation attenuated ribavirin-induced hemolytic anemia. METHODS: Twenty-one consecutive patients with chronic hepatitis C were enrolled in this study between July 2003 and December 2004, and received high-dose vitamins E (2000 mg) and C (2000 mg) supplementation, daily, in addition to IFN alfa-2b and ribavirin combination therapy (vitamins E/C group). Twenty-one sex- and age-matched patients who received a standard regimen of IFN alfa-2b and ribavirin for chronic hepatitis C between January 2001 and June 2003 were evaluated as the control group. RESULTS: Decrease in hemoglobin level was significantly prevented in the vitamins E and C group compared to that in the control group (P = 0.029). Three (14.3%) patients in the control group discontinued treatment because of anemia, while no treated patient dropped out of the study due to anemia. Sustained virological response was not significantly different between the two groups. CONCLUSION: High-dose vitamins E and C supplementation prevented ribavirin-induced hemolytic anemia during combination therapy with ribavirin and IFN alfa-2b in patients with chronic hepatitis C.

Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium, Microlunatus phosphovorus.

ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P(i) from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis.