Randomized clinical trial of duct-to-mucosa versus invagination pancreaticojejunostomy after pancreatoduodenectomy.

BACKGROUND: The postoperative pancreatic fistula (POPF) rate for duct-to-mucosa and invagination anastomosis after pancreatoduodenectomy is still debated. The aim of this RCT was to investigate the POPF rate for duct-to-mucosa versus invagination pancreaticojejunostomy. METHODS: Patients were stratified by pancreatic texture and diameter of the main pancreatic duct and randomized to the duct-to-mucosa or invagination group. The primary endpoint was the rate of clinically relevant POPF (defined as grade B or C). Secondary endpoints were suture material cost for pancreaticojejunostomy, drain insertion duration and duration of postoperative hospital stay. RESULTS: Some 120 patients undergoing pancreatoduodenectomy were included following consent. Clinically relevant POPF developed in six of 59 patients (10 per cent) in the invagination group and in 14 of 61 patients (23 per cent) in the duct-to-mucosa group (P = 0·077). Duration of drain insertion (6 versus 7 days respectively; P = 0·027) and postoperative hospital stay (19 versus 24 days; P = 0·015) were shorter in the invagination group. Subgroup analysis for 61 patients with a soft pancreas revealed a lower rate of clinically relevant POPF in the invagination group (10 per cent versus 42 per cent in the duct-to-mucosa group; P = 0·010). Among 20 patients with a clinically relevant POPF, the six patients in the invagination group had a shorter duration of drain insertion (38·5 days versus 49 days for 14 patients in the duct-to-mucosa group; P = 0·028) and postoperative hospital stay (42 versus 54·5 days respectively; P = 0·028). CONCLUSION: This study did not demonstrate a superiority of invagination over duct-to-mucosa pancreaticojejunostomy in the risk of POPF. However, in high-risk patients with a soft pancreas, invagination may reduce the risk of clinically relevant POPF compared with duct-to-mucosa. Registration number: UMIN000005890 (http://www.umin.ac.jp).

Contribution of pharmacists with expertise in infectious diseases to appropriate individualized vancomycin dosing.

BACKGROUND/AIM: Dose adjustment of vancomycin (VCM) is important in improving clinical outcomes and avoiding adverse effects such as nephrotoxicity. Although pharmacist-managed VCM therapy has been reported to optimize treatment, there are no studies focused on pharmacist expertise to date. In this study, we compared the contribution of pharmacists trained for infectious diseases and general pharmacists to dose adjustment of VCM. PATIENTS AND METHODS: We retrospectively investigated VCM trough concentration after dose adjustment by both trained (n = 67) and general (without special training for infectious diseases; n = 85) pharmacists. We also compared the incidence of nephrotoxicity during VCM treatment in both groups. RESULTS: The rate of achieving therapeutic VCM trough concentration (10-20 µg/mL) was higher in the trained group than in the control group (80.6 vs. 54.1%, p < 0.001). No significant differences in incidence of nephrotoxicity were observed between the two groups (p = 0.744). Trained pharmacists could contribute more successfully to the achievement of therapeutic VCM concentration ranges without increasing the risk of nephrotoxicity.

Validation of the 8th Edition of the AJCC TNM Staging System for Gastric Cancer using the National Cancer Database.

BACKGROUND: The 8th edition AJCC gastric cancer staging manual was refined using Japanese and Korean data from the International Gastric Cancer Association (IGCA). This study evaluated the eighth edition's validity for U.S. METHODS: National Cancer Database (NCDB) was used to obtain data on gastric cancer patients diagnosed from 2004 to 2008 who underwent surgery and to examine differences in stage grouping and survival between AJCC 7th and 8th editions. Discrimination of models derived from NCDB and IGCA data was compared. RESULTS: Of 12,041 patients, median age was 65, 57.6% were male, median lymph nodes retrieved was 2 (0-76), 30.9% underwent distal/partial gastrectomy, and 49.8% received no adjuvant treatment. The 8th edition differed in that T1-T3 disease was upstaged with N3b, T4aN3a was downstaged from IIIC to IIIB, and T4bN0 and T4aN2 were downstaged from IIIB to IIIA. These changes resulted in increased patients in IIIA (1436 in the 7th edition to 2310 in the 8th) and IIIB (1737-1896) and decreased in IIIC (2100-1067). This also resulted in lower median survival for IIIA (28.7-25.0 months), IIIB (19.6-17.4), IIIC (13.7-11.8). The concordance index for the 8th edition applied to NCDB data was 0.719 [95% confidence interval (CI) 0.703-0.734), which is comparable to that for the 8th edition developed from IGCA data (0.775, 95% CI 0.770-0.780) and the 7th edition applied to NCDB data (0.720, 95% CI 0.704-0.735). CONCLUSIONS: The 8th edition is valid for U.S. populations, showing clear separation of data with preservation of group order.

Role of Prealbumin as a Powerful and Simple Index for Predicting Postoperative Complications After Gastric Cancer Surgery.

BACKGROUND: Preoperative factors, including nutritional status, may have strong correlations with postoperative morbidities. The current study evaluated preoperative prealbumin concentrations as a predictor of postoperative complications after gastric surgery. METHODS: A retrospective study of 1798 patients who underwent gastrectomy for gastric adenocarcinoma was performed. Information was collected on basic patient characteristics, preoperative laboratory findings, and 30 day postoperative complications. The patients were divided into three groups based on prealbumin concentrations (≥22 mg/dL, <22 to ≥15 mg/dL, and <15 mg/dL) for analysis. RESULTS: The overall complication rate was 21.7 %, and the infection rate was 16 %. Subgroup analysis based on prealbumin concentrations showed that complication rates were markedly elevated with decreasing concentrations of prealbumin. Multivariate analysis using a logistic regression model showed that both overall and infectious complications were strongly associated with male gender, elevated C-reactive protein (CRP), and decreased prealbumin levels (p < 0.05). Even in patients with a CRP level higher than 0.1 mg/dL, male gender and low prealbumin concentrations (<15 mg/dL) were significantly correlated with overall and infectious morbidities (p < 0.05). CONCLUSIONS: Preoperative prealbumin concentrations are useful predictors of short-term postoperative outcomes after gastrectomy.

Use of endoscopy to determine the resection margin during laparoscopic gastrectomy for cancer.

BACKGROUND: It can be difficult to determine the transection line during totally laparoscopic surgery for early gastric cancer owing to lack of tactile feedback. This retrospective cohort study aimed to assess the role of intraoperative endoscopy in determining the resection margin in totally laparoscopic gastrectomy. METHODS: Consecutive patients with histologically confirmed gastric cancer who underwent laparoscopic gastrectomy between March 2012 and July 2015 were eligible. Preoperative placement of marking clips and intraoperative endoscopy were performed to determine the resection margin. Frozen-section analyses were also performed to confirm the absence of cancer cells at the surgical margin. Success was defined as the proportion of specimens with all clips present and by the proportion of resections with a negative surgical margin following initial transection. RESULTS: Total laparoscopic gastrectomy with intraoperative endoscopy was performed in 522 patients; a total of 662 surgical margins were analysed. The overall success rate was 99·8 per cent (661 of 662 margins). The success rate of achieving a negative surgical margin during the initial transection was 98·9 per cent (550 of 556 margins). CONCLUSION: Preoperative placement of marking clips and intraoperative endoscopy is helpful in the determination of a safe surgical margin in patients with gastric cancer who undergo laparoscopic gastrectomy.

Prognostic factors for survival in patients with pT1 N+ or T2-3 N0 gastric cancer in Japan.

BACKGROUND: The outcome for pT1 N+ or pT2-3 N0 gastric cancer is favourable, but some patients suffer from recurrent disease. The aim of this study was to identify prognostic factors in patients with pT1 N+ or pT2-3 N0 gastric cancer. METHODS: This was a multicentre, retrospective cohort study. All patients with pT1 N+ or pT2-3 N0 gastric cancer who underwent curative gastrectomy at five high-volume, specialized cancer centres in Japan between 2000 and 2008 were included. Demographic, clinical, surgical and pathological data were collected. Independent prognostic factors were identified using a Cox proportional hazards regression model. RESULTS: Some 1442 patients were included. The 5-year overall survival rate for patients with pT1 N+ or pT2-3 N0 gastric cancer was 92·0 per cent. Multivariable analysis for overall survival identified age (hazard ratio (HR) 2·67, 95 per cent c.i. 2·09 to 3·43), sex (HR 0·57, 0·39 to 0·83) and clinical tumour depth (cT) (HR 1·45, 1·06 to 1·98) as independent prognostic factors. CONCLUSION: Survival of patients with pT1 N+ or pT2-3 N0 gastric cancer is good. Age 65 years or above, male sex and cT2-4 category are associated with worse overall survival.

Molecular diversity among viroids infecting chrysanthemum in India.

Association of Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) with the Chrysanthemum plants exhibiting severe stunting, distinct yellow leaf mottling, and chlorosis was detected in the main chrysanthemum-growing regions of India. Sequence analysis of 90 cDNA clones obtained for CSVd and CChMVd, representing the chrysanthemum-growing regions of India, revealed the high degree of sequence variation throughout the genome under natural conditions. Additionally, all the analyzed CChMVd clones revealed the presence of UUUC in the tetraloop, a signature of symptomatic variants in susceptible cultivars. Phylogenetic analysis revealed that Indian CSVd is closely related to European isolates from ornamentals, whereas CChMVd clustered along with the isolates reported from the East Asian countries.

Epicatechin downregulates adipose tissue CCL19 expression and thereby ameliorates diet-induced obesity and insulin resistance.

BACKGROUND AND AIMS: Epicatechin (EC) intake has been suggested to be beneficial for the prevention of cardiovascular disorders, and it is well known that adipose tissue inflammation is one of the major risk factors for coronary heart diseases. The purpose of the present study was to determine the in vitro and in vivo effects of EC on adipose tissue inflammation and obesity. METHODS AND RESULTS: DNA microarray analysis was performed to evaluate the effects of EC on gene expression in adipocytes co-cultured with bacterial endotoxin-stimulated macrophages. To determine the in vivo effects of the catechin, C57BL/6 mice were fed either a high-fat diet (HFD) or HFD combined with EC, and metabolic changes were observed EC suppressed the expression of many inflammatory genes in the adipocytes co-cultured with endotoxin-stimulated macrophages. Specifically, EC markedly suppressed chemokine (CC motif) ligand 19 (CCL19) expression. The target cell of EC appeared to macrophages. The in vivo study indicated that mice fed the EC-supplemented HFD were protected from diet-induced obesity and insulin resistance. Accordingly, the expression levels of genes associated with inflammation in adipose tissue and in the liver were downregulated in this group of mice. CONCLUSIONS: EC exerts beneficial effects for the prevention of adipose tissue inflammation and insulin resistance. Since we previously reported that mice deficient in the CCL19 receptor were protected from diet-induced obesity and insulin resistance, it can be concluded that the beneficial effects of EC could be mediated, at least in part, by marked suppression of CCL19 expression.

Immunocytochemical analysis of p63 and 34ßE12 in fine needle aspiration cytology specimens for breast lesions: a potentially useful discriminatory marker between intraductal papilloma and ductal carcinoma in situ.

OBJECTIVE: We applied immunocytochemistry to fine needle aspiration (FNA) breast lesion slides in an attempt to enhance their objectivity and specificity. METHODS: We analysed 56 FNA specimens from patients with histologically confirmed breast lesions, using 34ßE12 and p63 antibodies. Immunostained slides were examined in terms of both solitary positive cells (within clusters and non-clusters) and positive clusters within a slide. RESULTS: Positive scores (≥2) for p63(+) cells and percentages of p63(+) clusters differed significantly (P < 0.001) between malignant (3 of 34; 9%) and benign (11 of 22; 50%) cases and varied between benign and malignant groups: intraductal papilloma (IDP) (2 of 8), other benign lesions (9 of 14), ductal carcinoma in situ (DCIS) (1 of 11) and invasive carcinoma (IC) (2 of 23). Similarly, 34ßE12 scores were higher in benign cases (IDP, 8 of 8; other benign, 9 of 14) than in malignant cases (DCIS, 1 of 11; IC, 3 of 23). As well as a significant difference between benign and malignant cases (17 of 22, 77% versus 4 of 34, 12%; P < 0.001), the percentage of 34ßE12(+) clusters was significantly higher in IDP compared with DCIS (P = 0.001). CONCLUSIONS: The immunostaining of FNA breast specimens for p63 and 34ßE12 may help in difficult diagnoses.

Ten-year follow-up results of a randomized clinical trial comparing left thoracoabdominal and abdominal transhiatal approaches to total gastrectomy for adenocarcinoma of the oesophagogastric junction or gastric cardia.

BACKGROUND: The optimal surgical approach for treatment of oesophagogastric junction (OGJ) cancer is controversial. A randomized clinical trial (JCOG9502) comparing transhiatal (TH) and left thoracoabdominal (LTA) approaches was stopped after the first interim analysis owing to limited efficacy for LTA resections. Complete 10-year follow-up data are now available. METHODS: Patients with histologically proven adenocarcinoma of the OGJ or gastric cardia with oesophageal invasion of 3 cm or less were randomized to a TH or LTA approach. Both groups underwent total gastrectomy and splenectomy with D2 nodal dissection plus para-aortic lymphadenectomy above the left renal vein. For LTA, a thorough mediastinal lymphadenectomy below the left inferior pulmonary vein was also mandatory. The primary endpoint was overall survival. RESULTS: A total of 167 patients (82 TH, 85 LTA) were enrolled. The 10-year overall survival rate was 37 (95 per cent c.i. 26 to 47) per cent for the TH approach and 24 (15 to 34) per cent for the LTA technique (P = 0·060). The hazard ratio for death was 1·42 (0·98 to 2·05) for the LTA technique. Subgroup analysis based on the Siewert classification indicated non-significant survival advantages in favour of the TH approach. CONCLUSION: LTA resections should be avoided in the treatment of adenocarcinoma of the OGJ or gastric cardia. REGISTRATION NUMBER: NCT00149266 (https://www.clinicaltrials.gov).

P-ρ-T measurements of H2O up to 260 GPa under laser-driven shock loading.

Pressure, density, and temperature data for H2O were obtained up to 260 GPa by using laser-driven shock compression technique. The shock compression technique combined with the diamond anvil cell was used to assess the equation of state models for the P-ρ-T conditions for both the principal Hugoniot and the off-Hugoniot states. The contrast between the models allowed for a clear assessment of the equation of state models. Our P-ρ-T data totally agree with those of the model based on quantum molecular dynamics calculations. These facts indicate that this model is adopted as the standard for modeling interior structures of Neptune, Uranus, and exoplanets in the liquid phase in the multi-Mbar range.

Detection of human papillomavirus (HPV) in patients with squamous cell carcinoma and the clinical characteristics of HPV-positive cases.

BACKGROUND: The association between human papillomavirus (HPV) and squamous cell carcinoma (SCC) has been reported; however, the prevalence of HPV infection varies, and the clinical characteristics of HPV-positive cases remain unknown. OBJECTIVES: To elucidate the frequency of HPV infection in a series of Japanese patients with SCC and to identify the characteristics of HPV-positive cases. METHODS: We evaluated 38 patients with SCC treated at our department. HPV typing was performed using SCC samples from different body sites. Immunohistochemical staining for HPV proteins and p16(INK) (4a) was performed, in addition to polymerase chain reaction and in situ hybridization. The clinical characteristics of the HPV-positive cases were clarified. RESULTS: Two genital lesions were positive for HPV type 16. Both cases showed basaloid features histopathologically, and were considered to have SCC that had arisen from bowenoid papulosis. p16(INK) (4a) expression was observed in 11 cases, including the two HPV-positive cases. CONCLUSIONS: The present study indicates that the prevalence of HPV is not high and that bowenoid papulosis is an HPV-associated precancerous lesion. Further investigation is necessary to assess the relationship between HPV infection and SCC.

Current status of viroid taxonomy.

Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.

ROCK activation in lung of idiopathic pulmonary fibrosis with oxidative stress.

The Rho-associated coiled-coil containing protein kinase, (Rho-kinase or ROCK) undergoes activation by oxidative stress. ROCK-II, which is an isoform of ROCK, is activated in a murine model of lung fibrosis. The present study evaluated the level of oxidative stress and activation of ROCK-II in patients with idiopathic pulmonary fibrosis (IPF). The ROCK-II level and the phosphorylation of myosin phosphatase subunit-1 (p-MYPT-1), a hallmark of ROCK activation, were examined by immunohistochemistry of lung tissue sections. The 8-iso prostaglandin-F2alpha (8-isoPGF2alpha) level, as a marker of oxidative stress, of exhaled breath condensate was significantly higher in IPF patients than in control patients. In IPF lungs, ROCK-II was predominantly expressed by bronchial epithelial cells, as well as at a lower level by airway smooth muscle cells, vascular smooth muscle cells, and the fibroblasts of fibroblastic foci (FF). In addition, there was moderate p-MYPT-1 expression in these cells of IPF lungs. In control lungs, ROCK-II was expressed by these cells. p-MYPT-1 was weakly expressed by the bronchial epithelial cells. In conclusion, ROCK-II was activated in various lung cells of IPF patients along with oxidative stress detected by 8-isoPGF2alpha elevation. The ROCK pathway may play a role in the development of IPF oxidative stress.

Extraordinary effects of an argon atmosphere on TiO2 photocatalysis.

When used as a photocatalyst under solar light, TiO2 tends to show a low photocatalytic activity. We report here that even TiO2 exhibits a high photocatalytic activity toward the decomposition (mineralization) of aqueous organic contaminants under simulated solar light when used under an Ar atmosphere (Ar flow). The photocatalytic decomposition rate of a cationic dye, crystal violet, and the CO2 evolution rate over TiO2 (P25, mainly composed of anatase and rutile) under Ar flow were substantially higher than those obtained in air and under air, O2, N2 and CO2 flows. Similar positive effects of Ar flow on photocatalytic reactions were observed when another cationic dye, basic violet 4, and TiO2 (anatase) were used, suggesting the versatility of Ar flow for improving photocatalytic activities.

Lymphadenectomy around the left renal vein in Siewert type II adenocarcinoma of the oesophagogastric junction.

BACKGROUND: The extent of lymphadenectomy in patients with Siewert type II adenocarcinoma of the oesophagogastric junction is controversial. The aim of this study was to investigate lymph node involvement around the left renal vein. METHODS: Lymph node involvement and prognosis in patients with Siewert type II cancers treated by R0-1 surgical resection were investigated, with regard to lymphadenectomy around the left renal vein. Based on the incidence of involvement at each node, the node stations were divided into three tiers (first tier, more than 20 per cent involvement; second tier, 10-20 per cent involvement; third tier, less than 10 per cent involvement). RESULTS: Of 150 patients with type II oesophagogastric adenocarcinoma, 94 had left renal vein lymphadenectomy. The first lymph node tier included nodes along the lesser curvature, right cardia, left cardia and left gastric artery, with involvement of 28·0-46·0 per cent and a 5-year survival rate of 42-53 per cent in patients with positive nodes. The nodes around the lower mediastinum, left renal vein, splenic artery and coeliac axis constituted the second tier, with involvement of 12·7-18 per cent and a 5-year survival rate of 11-35 per cent. With regard to the left renal vein, the incidence of involvement was 17 per cent and the 5-year rate survival rate was 19 per cent. Multivariable analysis showed that left renal vein lymphadenectomy was an independent prognostic factor in patients with pathological tumour category pathological T3-4 disease (hazard ratio 0·51, 95 per cent confidence interval 0·26 to 0·99; P = 0·048). CONCLUSION: Left renal vein nodal involvement is similar to that seen along the splenic artery, in the lower mediastinum and coeliac axis, with similar impact on patient survival.

Left hepatic trisectionectomy for advanced perihilar cholangiocarcinoma.

BACKGROUND: Data on outcomes of left hepatic trisectionectomy (LT) for perihilar cholangiocarcinoma are limited. The aim of this study was to clarify short- and long-term outcomes of LT for perihilar cholangiocarcinoma. METHODS: Patients with perihilar cholangiocarcinoma who underwent LT between January 2000 and October 2011 were analysed. Surgical variables, mortality, morbidity (Clavien grade I-V), recurrence sites and survival were compared between subjects who underwent LT, right hemihepatectomy or left hemihepatectomy. RESULTS: A total 214 patients underwent resection for perihilar cholangiocarcinoma, 25 (11·7 per cent) of whom underwent LT, 88 (41·1 per cent) right hemihepatectomy and 94 (43·9 per cent) left hepatectomy. There were no deaths among those who had LT, but 20 patients developed complications. The incidence of grade IIIa complications was significantly higher among patients who underwent LT than in patients who had right or left hemihepatectomy (P = 0·001 and P < 0·001 respectively). Only one patient developed a grade IIIb or IV complication (liver failure) after LT. The overall 5-year survival rate after LT was 39 per cent and median survival was 45 months. There were no significant differences in survival between patients who underwent LT and those who had a right or left hemihepatectomy. CONCLUSION: LT may provide a good outcome for advanced perihilar cholangiocarcinoma.

Proximal margin length with transhiatal gastrectomy for Siewert type II and III adenocarcinomas of the oesophagogastric junction.

BACKGROUND: A gross proximal oesophageal margin greater than 5 cm is considered to be necessary for curative surgery of adenocarcinoma of the oesophagogastric junction. This study investigated whether a shorter proximal margin might suffice in the context of total gastrectomy for Siewert type II and III tumours. METHODS: The gross proximal margin was measured on stretched specimens just after resection. Relationships between gross proximal margin lengths and clinicopathological features were investigated in patients with Siewert type II and III adenocarcinoma of the oesophagogastric junction treated by R0-1 surgical resection. For survival analyses, only patients who had undergone R0 resection for pathological (p) T2-4N0-3M0 tumour via a transhiatal approach were evaluated. RESULTS: Of the 140 patients, 120 had a total gastrectomy. Two patients (1·4 per cent) had histologically positive proximal margins and another two (1·4 per cent) developed anastomotic recurrence. Of 100 patients with pT2-4N0-3M0 tumours who underwent gastrectomy via a transhiatal approach, those with gross proximal margins larger than 20 mm appeared to have better survival than those with shorter margins (P = 0·027). Multivariable analysis demonstrated that a gross proximal margin of 20 mm or less was an independent prognostic factor (hazard ratio (HR) 3·56, 95 per cent confidence interval 1·39 to 9·14; P = 0·008), as was pathological node status (HR 1·76, 1·08 to 2·86; P = 0·024). CONCLUSION: Gross proximal margin lengths of more than 20 mm in resected specimens seem satisfactory for patients with type II and III adenocarcinoma of the oesophagogastric junction treated by transhiatal gastrectomy.

First Report of Coleus blumei viroid Infecting Coleus in India.

Members of the genus Coleviroid (Coleus blumei viroid [CbVd]), family Pospiviroidae have been reported to infect Coleus (Solenostemon sp.). CbVd-1 was first reported from Brazil, CbVd-2, -3, and -4 were first reported from Germany, whereas CbVd-5 and -6 were recently identified in China (2). In India, Coleus is extensively cultivated as an ornamental plant in home gardens. In March to June 2012, Coleus leaf samples with irregular chlorotic spots/patches were collected from home gardens of two different districts of Karnataka (Kodagu and Mysore districts), India, suspecting the presence of Coleus blumei viroids (CbVd 1 to 6). Low molecular weight RNAs were extracted using 2% CTAB buffer containing 1.4 M NaCl, 20 mM EDTA, pH 8.0 and 100 mM Tris-Cl, pH 8.0 (1). Viroid-like RNA was enriched by fractionation 2M LiCl soluble nucleic acids (4). A DNA fragment with the expected size of CbVd-1 was detected in 10 (including both districts) of 14 analyzed (incident rate of 71%) from reverse transcription (RT)-PCR assay using Coleviroid specific primers (forward 5'-TGGATCCAGCGCTGCAACGGAATCCA-3' and reverse 5'-TTGGATCCGCCAGGGAACCCAGGTAAG-3'). RT was performed at 37°C for 60 min in 25 µl reaction mix containing 5 µl RNA extracts, 1 µl reverse primer, 1× first strand buffer, 10 mM dNTPs, and 200U M-MuLV-RT (Invitrogen, USA). For PCR, 2 µl RT was mixed in 25 µl PCR mix containing 0.2 µM each forward and reverse primers and 2U LA Taq (Takara-Bio, Japan) according to the manufacturer's instruction. PCR parameter was one cycle at 94°C for 2 min, 35 cycles at 94°C for 45 s, 53°C for 30 s, and 72°C for 60 s, followed by final extension at 72°C for 10 min (4). Sequence analysis of cloned amplicons detected CbVd-1 in India. To confirm the sequence of the primer regions, an additional set of tail-tail primers were designed, CbVd1-136F (5'-CTTCGTGGAACGGCTCCGCG-3') and CbVd1-136Rev (5'-GAAGAAGCCGAAGCAACTCTC-3') and were used for RT-PCR. Amplified products were cloned, sequenced and compared with previously obtained data. Further, the presences of CbVd-1 in Coleus samples was confirmed by a RNA gel blot assay using digoxigenin-labeled CbVd-1 cRNA probe (3). Alignment of 19 sequences obtained from four representative Coleus samples found the presence of two sequence variants of CbVd-1, namely Ind-1 (GenBank Accession No. AB740017) and Ind-2 (AB740018). Ind-1 was found to differ from Ind-2 by two nucleotide substitutions at position 40 (C to T) and 211 (T to C). BLAST analysis of Ind-1 showed 100% sequence similarity with CbVd-1 isolates from China (DQ178399) and South Korea (EU 410620), whereas Ind-2 was 99% identical to these two Chinese and Koreans isolates. Furthermore, 97% and 96% sequence identity with CbVd 1-RL RNA (Accession no. X95366) was observed for Ind-1 and Ind-2, respectively. Isolates from India were 88% similar with Coleus blumei viroid 1-RG (X95291). To the best of our knowledge, this is the first molecular evidence for the presence of CbVd-1 infecting Coleus in India. Coleus harbors various viroid species and CbVd-1, reported widely, can transmit efficiently through seed and also could infect the other herbaceous plants (3). This report from India will contribute further understanding of a potential risk of Coleus viroids in ornamental species. References: (1) J. J. Doyle and J. L. Doyle. Phytochem. Bull. 19:11, 1987. (2) F. H. Fu et al. Plant Dis. 95:494, 2011. (3) Ishiguro et al. Ann. Phytopathol. Soc. Jpn. 62:84, 1996. (4) S.-F. Li et al., Plant Pathol. 55:565, 2006.

First Report of Grapevine yellow speckle viroid-1 and Hop stunt viroid Infecting Grapevines (Vitis vinifera) in India.

During March through July 2012, 10 to 15% of the Vitis vinifera cultivars Thompson Seedless and Anab-e-Shahi exhibited yellow leaf spots and flecks, shortened internodes, and tiny yellow leaves in vineyards of the Bijapur, Doddaballapur, and Kolar districts of Karnataka State, India. These are the major grapevine cultivation regions in India. Samples were collected from four different plants from each district (12 samples in total) and RNA was extracted using 2X CTAB buffer (1). Presence of Grapevine yellow speckle viroid1 (GYSVd-1, genus Apscaviroid) was tested by reverse transcription (RT)-PCR with primer pair PBCVd100C/194H (4) for the amplification of a 220-bp region of the genome. In agarose gel electrophoresis, five samples showed amplicons of the expected size. These amplicons were cloned and sequenced. BLAST analysis confirmed the presence of GYSVd-1. Based on this data, the full-length genome of GYSVd-1 was amplified by RT-PCR using primer pair 341M (5'-CACTCGCGGGGCGCGTTGGA-3') and 342P (5'-CAATCCCCGGAACCCCCGCT-3') and the amplicons were cloned and sequenced. Sequence analysis revealed two sequence variants namely Kar-1 (GenBank Accession No. AB742222) and Kar-2 (AB742223) with 98% and 99% identity to GYSVd-1 variants IXc (X87913) and II (X87906), respectively. GYSVd-1 variants Kar-1 and Kar-2 clustered in two distinct phylogenetic sub-clades. All 12 samples also tested positive for Hop stunt viroid (HpSVd, genus Hostuviroid) in two separate sets of RT-PCR using HSV-78P (5'-AACCCGGGGCAACTCTTCTC-3') and HSV-83M (5'-AACCCGGGGCTCCTTTCTCA-3'); and HSV-7P (5'-AATTCTCGAGTTGCCGC-3') and HSV-220M (5'-CGAACCGAGAGGTGATGCCA-3'), with the expected size of 303 and 213 bp, respectively (3). Sequence analysis of the amplicons confirmed the presence of HpSVd. Alignment of HpSVd nucleotide sequences obtained from the 12 samples showed the presence of a single type of sequence variant, namely Ind-2 (AB742225). BLAST analysis showed 99% sequence identity of Ind-2 with a HpSVd variant isolated from a 100-year-old grapevine in China. All 12 grapevine samples were also tested for the presence of Australian grapevine viroid (AGVd, genus Apscaviroid), Grapevine yellow speckle viroid 2 (GYSVd 2, genus Apscaviroid), and Citrus exocortis viroid (CEVd, genus Pospiviroid) by RT-PCR as described previously (2). None of the samples showed any positives. Northern blot assay using appropriate digoxigenin-labeled riboprobes performed as described previously (2) further confirmed RT-PCR results. Positive controls for RT-PCR and Northern blot were obtained from viroid-infected grapevines maintained in the greenhouse. GYSVd-1 and HpSVd were detected in symptomatic and symptomless plants. Hence, the symptoms observed in the vineyard cannot be attributed to viroid infection. More work is needed to identify the causal agent(s) of the decline of Thompson Seedless and Anab-e-Shadi cultivars. References: (1) C. R. Adkar-Purushothama et al. Plant Dis. 97:149, 2013. (2) D. Jiang et al. Virus Res, 169:237, 2012. (3) Y. Kawaguchi-Ito et al. PLoS One 4:e8386, 2009. (4) L. I. Ward et al. Plant Dis. 95:617, 2011.