A large deletion within intron 20 sequence of single-copy PolA1 gene as a useful marker for the speciation in Oryza AA-genome species.

Oryza AA-genome complex comprises five wild species, O. rufipogon, O. barthii, O. longistaminata, O. glumaepatula, and O. meridionalis. Evolutionary relationships among these five wild species have remained contentious and inconclusive. We found that intron 20 of PolA1, a single-copy nuclear gene, was short (S-type: 141-142 bp) in O. rufipogon, O. barthii, and O. glumaepatula, while long (L-type: ca. 1.5 kb) introns were apparent in O. longistaminata and O. meridionalis. Because Oryza species containing BB, CC, EE, FF, and GG genome showed L-type introns, the S-type intron was probably derived from the L-type intron by the deletion of a 1.4 kb fragment through intramolecular homologous recombination between two tandem TTTTGC repeats. Excluding the large deletion sequence, intron 20 sequence of O. barthii was identical to that of O. longistaminata. As more than 3,470 accessions of O. rufipogon and O. sativa also contained the same intron 20 sequence with O. longistaminata except for single T-nucleotide deletion, which was shared with O. glumaepatuala, the deletion of the T-nucleotide probably occurred in the L-type intron 20 of O. logistaminata. Deletions of a large 1.4 kb fragment and single T-nucleotide within the intron 20 of PolA1 gene were considered as useful DNA markers to study the evolutionary relationships among Oryza AA-genome species.

Liver stiffness measured by virtual touch quantification predicts the occurrence of posthepatectomy refractory ascites in patients with hepatocellular carcinoma.

PURPOSE: This study assessed the significance of measuring liver stiffness using virtual touch quantification before hepatectomy to predict posthepatectomy refractory ascites. METHODS: A total of 267 patients with hepatocellular carcinoma who underwent hepatectomy were prospectively analyzed. Liver stiffness was defined as the median value of the virtual touch quantification (Vs; m/s) by acoustic radio-force-impulse-based virtual touch. RESULTS: A multivariate analysis showed that Vs and the aspartate aminotransferase-to-platelet ratio index were independent risk factors for postoperative refractory ascites (odds ratio = 3.27 and 3.08, respectively). The cutoff value for Vs was 1.52 m/s (sensitivity: 59.5%, specificity: 88.6%) as determined by the analysis of the receiver-operating characteristic curve, and the area under the receiver-operating characteristic curve was 0.79. The cutoff value for the aspartate aminotransferase-to-platelet ratio was 0.952 (sensitivity: 65.5%, specificity: 82.9%), and the area under the receiver-operating characteristic curve was 0.75. CONCLUSIONS: Vs is an independent risk factor for refractory ascites after hepatectomy. The measurement of liver stiffness by virtual touch quantification before hepatectomy can help estimate the risk of postoperative refractory ascites. Nonsurgical treatments should be considered for the management of patients who are at high risk for refractory ascites.

Risk stratification and prognostic factors in patients with unresectable undifferentiated carcinoma of the pancreas.

BACKGROUND: Undifferentiated carcinoma (UC) of the pancreas has been considered a highly aggressive malignancy. However, only a few studies have systematically described the clinical course of UC patients. The aim of this study was to clarify the prognosis and construct a prognostic model for patients with unresectable UC. METHODS: This study was conducted at 17 institutions in Japan, and a total of 55 patients were analyzed. RESULTS: The median overall survival (OS) of patients with unresectable UC was 3.95 months. In the multivariate Cox proportional hazards (CPH) model, age ≥65 years, Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2, and C-reactive protein (CRP) >10 mg/L were independent prognostic factors for OS (age ≥65 years: hazard ratio [HR], 2.732; 95% confidence interval [CI], 1.353-5.515; ECOG PS ≥ 2: HR, 7.866; 95% CI, 1.981-31.241; CRP >10 mg/L: HR, 1.956; 95% CI, 1.013-3.775). Based on the ß coefficients from the CPH model, the prognostic scores were defined as follows: age ≥65 years (3 points), ECOG PS ≥ 2 (6 points), and CRP >10 ml/L (2 points). The final prognostic model was the sum of the points. The derived prognostic model stratified patients into high-risk (score ≥4) and low-risk (score 0-3) groups, with significant differences in OS (1.45 vs. 8.19 months, respectively; p < 0.001). CONCLUSIONS: The prognostic model stratified patients into high-risk and low-risk groups. These findings suggest that this model can serve as a tool for patient information and decision-making with regard to the therapeutic strategy for UC.

Enhanced patterns on intraoperative contrast-enhanced ultrasonography predict outcomes after curative liver resection in patients with hepatocellular carcinoma.

PURPOSE: This study aimed to clarify what hepatocellular carcinoma (HCC) phenotype, as categorized by intraoperative contrast-enhanced ultrasonography (CEUS), showed a high risk of recurrence after hepatic resection. METHODS: Patients who underwent initial curative hepatectomy with intraoperative CEUS for a single HCC nodule were retrospectively assigned to three patterns of fine (FI), vascular (VA), and irregular (IR) according to the maximum intensity projection pattern based on intraoperative CEUS. Staining was performed for Ki-67, pyruvate kinase type M2 (PKM2), and vascular endothelial growth factor (VEGF) to assess the tumor proliferative activity, tumor glucose metabolism, and angiogenesis, respectively. RESULTS: Of 116 patients, 18, 50, and 48 were assigned to the FI, VA and IR patterns, respectively. IR patients demonstrated a significantly worse prognosis for both the recurrence-free survival (RFS) and overall survival (OS) (P = 0.0002, 0.0262, respectively) than did patients with other patterns. A multivariate analysis revealed an IR pattern in intraoperative CEUS to be an independent predictive factor for a poor RFS, and major hepatectomy and an IR pattern were independent predictive factors for a poor OS. An IR pattern was closely related to the tumor size (≥ 3.3 cm) and poor histological differentiation and showed a high Ki-67 index, low VEGF expression, and high PKM2 expression. CONCLUSION: IR-pattern HCCs as classified by intraoperative CEUS may be associated with a higher risk of recurrence and worse outcomes in HCC patients after hepatic resection than other patterns.

Efficient elimination of Yam mosaic virus (YMV) from white yam (Dioscorea rotundata Poir.) by cryotherapy of axillary buds.

Dioscorea rotundata is an economically important food crop in many tropical countries as many people in this region depend on it for food and livelihood. Viral diseases, especially Yam mosaic virus (YMV), constitute a major constraint in the cultivation of this crop as they perpetuate through generations in the vegetatively propagated planting materials. Getting resistant or at least virus-free planting materials for farmers thus becomes crucial. This study was aimed at eliminating YMV in Dioscorea rotundata by cryotherapy of axillary buds. Enlarged axillary buds of YMV-infected TDr 2269 were frozen in liquid nitrogen for 1 h, re-warmed at 40 °C and cultured to regenerate plantlets. Approximately 76.33% plantlet regeneration and 100% YMV eradication were obtained for cryo-treated buds, against 95% and 0% obtained respectively for non-treated buds. RT-PCR and RT-qPCR analyses did not reveal detectable quantity of YMV in treated plants but did in control plants. Plants from cryo-treated buds showed no mosaic symptoms and produced slightly more tubers, and heavier mini-tubers (20.48±3.11 g) under greenhouse conditions contrary to non-treated plants that showed severe mosaic symptoms with significantly smaller tubers (1.91±0.39 g) (P < 0.05). This is the first report showing the elimination of YMV from infected white yam stock plant by cryotherapy and would be useful for producing clean planting materials.

Restoration of natural killer cell activity by interferon-free direct-acting antiviral combination therapy in chronic hepatitis C patients.

AIM: Interferon-free direct-acting antiviral (DAA) therapy is an effective treatment for chronic hepatitis C (CH(C)) patients. Activity of natural killer (NK) cells was reported to be impaired in patients with hepatitis C virus infection. The aim of this study was to examine whether DAA therapy could restore NK activity in patients with CH(C). METHODS: Direct-acting antiviral therapy was given to 31 CH(C) patients as asunaprevir/daclatasvir (ASV/DCV) (n = 15), ledipasvir/sofosbuvir (n = 7), ombitasvir/paritaprevir/ritonavir (n = 6), or elbasvir/grazoprevir (n = 3). Prior to therapy (0M), at the completion of the therapy (EOT), and at 24 weeks after completion (AFTER), NK activity and the frequency of CD56dim NK and CD56bright NK cells in peripheral blood were estimated by Cr release assay and flow cytometry. Statistical analysis was carried out by anova and the Mann-Whitney U-test. RESULTS: In one of the ASV/DCV-treated patients, treatment was stopped 12 weeks after initiation of therapy because of viral breakthrough. The anova showed that NK activity significantly improved at EOT (vs. 0M, P < 0.01) and at AFTER (vs. 0M, P < 0.001) in 30 patients with sustained virologic response. It also showed that the frequency of CD56dim NK cells was significantly increased at EOT and at AFTER (vs. 0M, P < 0.05). In addition, the NK activity ratio (AFTER/0M) had no significant difference between patient groups with higher and lower Fibrosis-4 scores. CONCLUSION: Direct-acting antiviral therapy in CH(C) patients could improve NK activity by increasing the frequency of CD56dim NK cells. Additionally, our results might imply that DAAs therapy could reduce the risk of hepatocarcinogenesis by restoring innate immune responses.

Impaired Value of 99m Tc-GSA Scintigraphy as an Independent Risk Factor for Posthepatectomy Liver Failure in Patients with Hepatocellular Carcinoma.

BACKGROUND: Posthepatectomy liver failure (PHLF) was recently defined with the corresponding recommendations as follows: grade A, no change in clinical management; grade B, clinical management with noninvasive treatment; and grade C, clinical management with invasive treatment. In this study, we identified the risk factors for grade B and C PHLF in patients with hepatocellular carcinoma (HCC). METHODS: Of 339 HCC patients who underwent curative hepatic resection, 218 were included for analysis. The LHL15 index (uptake ratio of the liver to that of the liver and heart at 15 min) was measured by 99m Tc-GSA (99m technetium-labelled galactosyl human serum albumin); remnant LHL15 was calculated as LHL15 × [1 - (resected liver weight - tumor volume)/whole liver volume without tumor]. RESULTS: A total of 163 patients were classified as having no PHLF, whereas 17, 37, and 1 patient had PHLF grade A, B, and C, respectively. There were significant differences in indocyanine green R15, serum albumin, prothrombin time, Child-Pugh classification, LHL15 and remnant LHL15 between patients with grades B/C PHLF and patients with grade A or no PHLF. Only remnant LHL15 was identified as an independent risk factor for grades B/C PHLF (p = 0.023), with a cut-off value of 0.755. CONCLUSIONS: Remnant LHL15 was an independent risk factor for grades B/C PHLF. Patients with impaired remnant LHL15 value of <0.755 should be carefully monitored for PHLF.

Short- and long-term outcomes of the Frey procedure for chronic pancreatitis: a single-center experience and summary of outcomes in Japan.

PURPOSE: To evaluate the short- and long-term outcomes of the Frey procedure for chronic pancreatitis (CP). METHODS: The subjects of this study were 12 patients who underwent the Frey procedure for CP between January, 2000 and December, 2016. We assessed pain relief, weight gain, and exocrine/endocrine insufficiency during follow-up. RESULTS: The study population comprised 11 men and 1 woman (91.7% vs. 8.3%; mean age, 50.3 ± 6.8 years; range 39-61 years). Pancreatitis was caused by alcohol in 9 (75%) patients and was idiopathic in 3 (25%) patients. The mean follow-up period was 82.5 ± 46.5 months (range 16.9-152.1 months). There was no operative mortality, but three patients (25%) suffered postoperative morbidity. All patients were pain-free at the time of discharge. There was no case of new-onset diabetes mellitus after surgery, although one patient (8.3%) suffered exocrine insufficiency. The body weight and body mass index of all patients improved during follow-up. Only one patient continued to suffer pain in the long term. CONCLUSION: The findings of this long-term follow-up of patients who underwent the Frey procedure suggest that it offers effective pain relief and is a safe technique for the management of CP.

A new marker sequence for systematics of medically important fungi based on amino acid sequence of the largest subunit of RNA polymerase I.

Molecular evolution has dominated taxonomic studies for decades, replacing traditional methods for identification and classification. However, there is a need for better markers to resolve the problems that have limited their usefulness. In this report, we introduce the protein tag (Ptag) sequence, a highly polymorphic amino acid sequence within the C-terminal region of the largest subunit of RNA polymerase I, as a new systematic tag sequence for delineating the evolutionary history of medically important fungi. As Ptag sequences are highly polymorphic between species and low within species, 42 fungal species representing the main taxonomic groups in the phyla Ascomycota and Basidiomycota were tested. The phylogenetic tree inferred from the Ptag sequences showed high consistency with the accepted classification of the Assembling the Fungal Tree of Life (AFTOL) project. Moreover, it could resolve the interspecies phylogenetic relationships of the tested taxa. In contrast, the phylogeny inferred from the nucleotide tag (Ntag) sequence, encoding the Ptag peptide, displayed lesser discriminatory power in resolving the phylogenetic relationships among distantly related taxa. In the case of closely related fungal species, the phylogenetic trees for Ptag and Ntag sequences were consistent with the tree for ITS1 sequences of 11 dermatophytic species. Taken together, the Ptag sequences should contribute to inferring phylogeny among species whereas the Ntag sequences should be useful to analyze variations among closely related species to resolve taxonomic issues in fungi.

Growth increase of Arabidopsis by forced expression of rice 45S rRNA gene.

KEY MESSAGE: Forced expression of rice 45S rRNA gene conferred ca. 2-fold increase of above-ground growth in transgenic Arabidopsis . This growth increase was probably brought by cell proliferation, not by cell enlargement. Recent increase in carbon dioxide emissions is causing global climate change. The use of plant biomass as alternative energy source is one way to reduce these emissions. Therefore, reinforcement of plant biomass production is an urgent key issue to overcome both depletion of fossil energies and emission of carbon dioxide. Here, we created transgenic Arabidopsis with a 2-fold increase in above-ground growth by forced expression of the rice 45S rRNA gene using the maize ubiquitin promoter. Although the size of guard cells and ploidy of leaf-cells were similar between transgenic and control plants, numbers of stomata and pavement cells were much increased in the transgenic leaf. This data suggested that cell number, not cell expansion, was responsible for the growth increase, which might be brought by the forced expression of exogenous and full-length 45S rRNA gene. The expression level of rice 45S rRNA transcripts was very low, possibly triggering unknown machinery to enhance cell proliferation. Although microarray analysis showed enhanced expression of ethylene-responsive transcription factors, these factors might respond to ethylene induced by abiotic/biotic stresses or genomic incompatibility, which might be involved in the expression of species-specific internal transcribed spacer (ITS) sequences within rice 45S rRNA transcripts. Further analysis of the mechanism underlying the growth increase will contribute to understanding the regulation of the cell proliferation and the mechanism of hybrid vigor.

Activation of the transforming growth factor-ß/SMAD transcriptional pathway underlies a novel tumor-promoting role of sulfatase 1 in hepatocellular carcinoma.

UNLABELLED: In vitro studies have proposed a tumor suppressor role for sulfatase 1 (SULF1) in hepatocellular carcinoma (HCC); however, high expression in human HCC has been associated with poor prognosis. The reason underlying this paradoxical observation remains to be explored. Using a transgenic (Tg) mouse model overexpressing Sulf1 (Sulf1-Tg), we assessed the effects of SULF1 on the diethylnitrosamine model of liver carcinogenesis. Sulf1-Tg mice show a higher incidence of large and multifocal tumors with diethylnitrosamine injection compared to wild-type mice. Lung metastases were found in 75% of Sulf1-Tg mice but not in wild-type mice. Immunohistochemistry, immunoblotting, and reporter assays all show a significant activation of the transforming growth factor-ß (TGF-ß)/SMAD transcriptional pathway by SULF1 both in vitro and in vivo. This effect of SULF1 on the TGF-ß/SMAD pathway is functional; overexpression of SULF1 promotes TGF-ß-induced gene expression and epithelial-mesenchymal transition and enhances cell migration/invasiveness. Mechanistic analyses demonstrate that inactivating mutation of the catalytic site of SULF1 impairs the above actions of SULF1 and diminishes the release of TGF-ß from the cell surface. We also show that SULF1 expression decreases the interaction between TGF-ß1 and its heparan sulfate proteoglycan sequestration receptor, TGFßR3. Finally, using gene expression from human HCCs, we show that patients with high SULF1 expression have poorer recurrence-free survival (hazard ratio 4.1, 95% confidence interval 1.9-8.3; P = 0.002) compared to patients with low SULF1. We also found strong correlations of SULF1 expression with TGF-ß expression and with several TGF-ß-related epithelial-mesenchymal transition genes in human HCC. CONCLUSION: Our study proposes a novel role of SULF1 in HCC tumor progression through augmentation of the TGF-ß pathway, thus defining SULF1 as a potential biomarker for tumor progression and a novel target for drug development for HCC.

Branched-chain amino acid-enriched nutrient increases blood platelet count in patients after endoscopic injection sclerotherapy.

AIM: Protein and energy malnutrition is a severe problem for patients with liver cirrhosis (LC) and fasting often induces starvation which is a vitally important outcome. Dietary restriction is essential for endoscopic injection sclerotherapy (EIS) in patients with risky esophageal varices, thereby creating the possible exacerbation of nutritional state and inducing liver dysfunction. Whether EIS induces nutritional deficiency in LC patients and the effects of branched-chain amino acid (BCAA)-enriched nutrient are prospectively investigated. METHODS: A total of 61 LC patients were randomly divided into an EIS monotherapy group (non-BCAA group, n = 31) and an EIS combined with BCAA therapy group (n = 30). Platelet count, blood chemistry and somatometry values were prospectively measured at five time points. RESULTS: The platelet counts before treatment were at the same level in both groups (P = 0.72). Three months after treatment, the counts decreased in the non-BCAA group; however, they increased in the BCAA group (P = 0.019). Body mass index, triceps skin fold thickness and arm muscle circumference significantly decreased in both groups. The BCAA and tyrosine ratio value increased only in the BCAA group (P < 0.01). The skeletal muscle volume measured by InBody720 significantly decreased in the non-BCAA group (P < 0.001). CONCLUSION: EIS induced protein-energy malnutrition, however, skeletal muscle volume was maintained by taking BCAA. Administration of BCAA had some effect in maintaining the nutritional state, and may improve the platelet count. Taking a greater amount of nutrients and shorter dietary restriction period or hospitalization was desirable.

Restoration of natural killer cell activity by pegylated interferon-alpha/ribavirin therapy in chronic hepatitis C patient.

AIM: The combination therapy of pegylated interferon-α and ribavirin (PEG IFN/RBV) is one of the effective treatments for chronic hepatitis C (CHC) patients. Natural killer (NK)-cell activity was reported to be impaired in patients with hepatitis C virus (HCV). The aim of this study was to examine whether PEG IFN/RBV therapy could restore NK activity in CHC patients. METHODS: In 19 CHC patients, PEG IFN/RBV therapy was performed. Just before (0M), at 3 months of the therapy (3M) and at 6 months after completion of the therapy (6M), NK activity and the frequency of NK cells, CD56(dim) NK cells and CD56(bright) NK cells in peripheral blood was estimated by creatinine release assay and flow cytometry. Statistical analysis was performed by anova and Mann-Whitney U-test. RESULTS: anova showed that NK activity significantly improved at 6M (vs 0M, P < 0.05) in the patients studied and in the patients with sustained virological response (SVR). It also showed that frequency of CD56(bright) NK cells was significantly increased at 6M (vs 0M, P < 0.05) in the patients studied and in the SVR group. However, no significant change in NK activity and frequency of CD56(bright) NK cells were detected in non-SVR group. Furthermore, NK activity ratio (6M/0M) in the SVR group was revealed to be higher compared with that in the non-SVR group by analysis using Mann-Whitney U-test (P < 0.05). CONCLUSION: PEG IFN/RBV therapy in CHC patients could improve NK activity by increasing the frequency of CD56(bright) NK cells in SVR patients. Our study also revealed that eradication of HCV could restore NK-cell activity.

Amiprophosmethyl-induced efficient in vitro production of polyploids in raphanobrassica with the aid of aminoethoxyvinylglycine (AVG) in the culture medium.

Optimum conditions for obtaining tetraploid were investigated in raphanobrassica, the intergeneric hybrid between radish (Raphanus sativus) and kale (Brassica oleracea var. acephala) by treating in vitro plants with an anti-mitotic agent, amiprophosmethyl (APM). Initially, no tetraploids but hexaploids and octaploids were induced by the treatments. Although the leaves of these polyploids of raphanobrassica showed chlorosis during subcultures in in vitro conditions, the chlorosis could be successfully prevented by the ethylene inhibitors, both AVG and AgNO3. Based on this result, AVG was added into medium used for the culture after the chromosome doubling treatment, which subsequently resulted in increased survival rates of the treated plant materials as well as increased production rates of polyploids including tetraploid. These polyploid plants showed obviously different characters from the original diploid plant. The tetraploid plant had bigger sizes in shoot, flower and leaf, and more number of leaves than the diploid. On the other hand, the hexaploid and octaploid plants had smaller sizes in shoots and leaves, and less number of leaves than the diploid. Concentration of glucosinolates, functional substances of Brassicaceae crops, did not significantly differ between diploid and tetraploid of raphanobrassica, but reduced in hexaploid and octaploid.

Activation of the transcription factor GLI1 by WNT signaling underlies the role of SULFATASE 2 as a regulator of tissue regeneration.

Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced ß-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and ß-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.

The MET1b gene encoding a maintenance DNA methyltransferase is indispensable for normal development in rice.

While Arabidopsis bears only one MET1 gene encoding the DNA methyltransferase that is mainly responsible for maintaining CG methylation after DNA replication, rice carries two MET1 genes, MET1a and MET1b, expressed in actively replicating and dividing cells, and MET1b is more abundantly expressed than is MET1a. A met1a null mutant displayed no overt phenotypes, implying that MET1b must play a major role in the maintenance DNA methylation. Here, we employed two met1b null mutants, generated by homologous recombination-mediated knock-in targeting and insertion of endogenous retrotransposon Tos17. These MET1a/MET1a met1b/met1b homozygotes exhibited abnormal seed phenotypes, which is associated with either viviparous germination or early embryonic lethality. They also displayed decreased levels of DNA methylation at repetitive CentO sequences and at the FIE1 gene locus in the embryos. In addition, independently isolated knock-in-targeted plants, in which the promoterless GUS reporter gene was fused with the endogenous MET1b promoter, showed the reproducible, dosage-dependent, and spatiotemporal expression patterns of GUS. The genotyping analysis of selfed progeny of heterozygous met1a met1b null mutants indicated that weakly active MET1a seems to serve as a genetic backup mechanism in rice met1b gametophytes, although the stochastic and uncoordinated activation of epigenetic backup mechanisms occurred less efficiently in the met1b homozygotes of rice than in the met1 homozygotes of Arabidopsis. Moreover, passive depletion of CG methylation during the postmeiotic DNA replication in the haploid nuclei of the met1a met1b gametophytes in rice results in early embryonic lethality. This situation somewhat resembles that of the met1 gametophytes in Arabidopsis.

Generation of selectable marker-free transgenic eggplant resistant to Alternaria solani using the R/RS site-specific recombination system.

KEY MESSAGE: Marker-free transgenic eggplants, exhibiting enhanced resistance to Alternaria solani , can be generated on plant growth regulators (PGRs)- and antibiotic-free MS medium employing the multi-auto-transformation (MAT) vector, pMAT21 - wasabi defensin , wherein isopentenyl transferase ( ipt ) gene is used as a positive selection marker. ABSTRACT: Use of the selection marker genes conferring antibiotic or herbicide resistance in transgenic plants has been considered a serious problem for environment and the public. Multi-auto-transformation (MAT) vector system has been one of the tools to excise the selection marker gene and produce marker-free transgenic plants. Ipt gene was used as a selection marker gene. Wasabi defensin gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), was used as a gene of interest. Wasabi defensin gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledon explants of eggplant were cultured on PGRs- and antibiotic-free MS medium. Extreme shooty phenotype/ipt shoots were produced by the explants infected with the pMAT21-wasabi defensin (WD). The same PGRs- and antibiotic-free MS medium was used in subcultures of the ipt shoots. Subsequently, morphologically normal shoots emerged from the Ipt shoots. Molecular analyses of genomic DNA from transgenic plants confirmed the integration of the WD gene and excision of the selection marker (ipt gene). Expression of the WD gene was confirmed by RT-PCR and Northern blot analyses. In vitro whole plant and detached leaf assay of the marker-free transgenic plants exhibited enhanced resistance against Alternaria solani.

Surgical Intervention After Lenvatinib Treatment in Patients With Advanced Hepatocellular Carcinoma.

BACKGROUND/AIM: The survival and prognostic factors in patients with advanced hepatocellular carcinoma (HCC) who underwent surgical intervention after lenvatinib treatment is not well-understood. PATIENTS AND METHODS: Seventy-six patients with advanced HCC who had lenvatinib treatment were retrospectively analyzed. RESULTS: Of 70 patients who were treated with lenvatinib, 14 patients underwent surgical intervention after lenvatinib treatment for 4-28 weeks. Progression-free survival (PFS) was significantly longer in patients who underwent surgical intervention than in patients with non-surgical treatment (median, 8.6 vs. 5.1 months, p=0.019). Non-significantly longer overall survival (OS) was also observed in patients with surgical intervention compared to patients with non-surgical treatment (median, unreached vs. 21.0 months, p=0.206). In patients who underwent surgical intervention, two patients had a partial response, and 12 had stable disease according to RECIST ver. 1.1 criteria. The serum alpha-fetoprotein (AFP) level was significantly lower after lenvatinib treatment than before lenvatinib treatment (median, 19.2 vs. 196.5 ng/ml, p=0.0081). Eleven patients underwent curative surgery with a 14% major postoperative complication (Clavien-Dindo ≥IIIa) rate. Patients who exhibited decreases in AFP levels or maintained AFP levels within the normal range during lenvatinib treatment had significantly longer PFS (median, 8.6 vs. 3.0 months, p=0.0009) and OS (median, unreached vs. 12.4 months, p=0.012) compared to those with persistently elevated AFP levels beyond the normal range. CONCLUSION: Surgical intervention after lenvatinib treatment for advanced HCC was associated with longer PFS. Patients exhibiting decreased AFP levels or maintaining AFP levels within the normal limit may be suitable candidates for surgical intervention after lenvatinib treatment for advanced HCC.

A patient who underwent conversion surgery after atezolizumab plus bevacizumab for hepatocellular carcinoma with portal vein thrombosis and perihepatic lymph node metastases achieved a pathological complete response.

Here, we present a patient with hepatocellular carcinoma complicated by tumor thrombosis into the main portal trunk and perihepatic lymph node metastases who was treated with atezolizumab plus bevacizumab. Shrinkage of the main tumor, portal vein thrombosis, and lymph node metastases were achieved; therefore, hepatectomy with lymphadenectomy could be performed. Final pathology indicated a complete pathological response in the main tumor, portal vein thrombosis, and perihepatic lymph nodes. The patient is currently alive with no evidence of recurrence on radiological assessment at 3 months after surgery.

Transgenic accumulation of a defective cucumber mosaic virus (CMV) replicase derived double stranded RNA modulates plant defence against CMV strains O and Y in potato.

Cucumber mosaic virus is an important plant pathogen with a broad host range encompassing many plant species. This study demonstrates the production of transgenic potato lines exhibiting complete resistance to cucumber mosaic virus strain O and Y by post transcriptional gene silencing. Two constructs were used, one, pEKH2IN2CMVai, contains inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O), while the other, TRV-based VIGS vector (pTRV2CMVai), contains the same fragment of the replicase gene, but without inverted repeat. These constructs were used to produce transgenic potato lines of cultivar 'Danshaku', a susceptible genotype to CMV. Transgenic lines derived from pEKH2IN2CMVai accumulated small interfering RNA (siRNA) before and after virus challenge, whereas those derived from pTRV2CMVai showed siRNA expression after virus challenge. When transgenic lines were challenged with CMV-O or CMV-Y, four lines exhibited complete (100%) resistance to both strains, whereas the other lines had high levels of resistance. Infectivity of CMV-O was lower than that of CMV-Y in the highly resistant plants. There were no significant differences with regard to resistance between plants derived from pEKH2IN2CMVai and those obtained from pTRV2CMVai. The presence of CMV-specific siRNA in the resistant phenotypes indicates that the resistance was acquired through RNA silencing.