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Three semi-selective media, DTarTA, SPbc, and SPamt, were developed and tested to isolate Pseudomonas syringae pv. maculicola ( Psm ) and P . cannabina pv. alisalensis ( Pca ) from Raphanus sativus seeds. DTarTA contained D-tartaric acid as a carbon source and potassium tellurite, ampicillin sodium, and methyl violet as antibiotics. DTarTA suppressed growth in 19 of the 24 pathovars from the P . syringae complex, whereas Psm and Pca grew and formed gray to black colonies. SPamt contained sucrose and peptone as nutrient sources and was supplemented with bromothymol blue and the same antibiotics present in DTarTA and Psm and Pca formed yellowish to dark brown colonies on the SPamt medium. SPbc contained sucrose and peptone and was supplemented with cephalexin and boric acid as antibiotics and Psm and Pca formed semi-translucent to white colonies on the SPbc medium. SPamt and SPbc suppressed the growth of several plant-associated bacteria (except the P . syringae complex). The growth of saprophytic bacteria in seeds on the different media was compared with that on King’s B medium, using five types of commercially available Raphanus sativus seeds. The suppression rate of DTarTA was 85–99% and was lower for seeds with more saprophytic bacteria. The suppression rates of SPamt and SPbc were 90–99%. In detection tests using 10,000 seed samples mixed with Pca or Psm -contaminated seeds, it was possible to selectively isolate Psm and Pca using SPamt and SPbc, even when the colony numbers of the target bacterium constituted less than 10% of the total colonies. Key points • Bacterial leaf spot and blight pathogens were selectively isolated from seeds . • DTarTA medium distinguishes these pathogens from P. syringae complex pathovars . • SPamp and SPbc media have different selectivity for plant-associated bacteria .

Bacterial leaf spot and bacterial leaf blight are global threats to the cultivation of cruciferous vegetables, and it is necessary to develop methods to easily detect, identify, and distinguish the causative pathogens Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca). Here, we used the sequence specificity of the exchangeable effector loci flanking the hrp gene cluster to design primers that can help detect and discriminate between Psm and Pca. Primers common to both bacteria (hrpK_fw1 and hrpK_fw2) were designed within hrpK at the end of the hrp gene cluster. Psm-specific primers (MAC_rv1 and MAC_rv2) were designed in hopPtoB1 and Pca-specific primers (ALS_rv1 and ALS_rv2) were designed in hopX1 adjacent to hrpK. PCR using hrpK_fw1 and MAC_rv1 or hrpK_fw2 and MAC_rv2 amplified DNA fragments of only Psm, P. syringae pv. tomato (causal agent of tomato bacterial speck), and P. syringae pv. spinaciae (causal agent of spinach bacterial leaf spot), among 76 strains of phytopathogenic bacteria. PCR using hrpK_fw1 and ALS_rv1 or hrpK_2 and ALS_rv2 amplified DNA fragments of only Pca. Multiplex PCR with these primers could easily distinguish Psm and Pca from bacterial colonies isolated on growth media and detect the pathogen in symptomatic leaves. Multiplex nested PCR with the primers detected contamination in one Psm- and/or one Pca-infected seeds in 1000 seeds. These results suggest that these PCR primers could help detect and discriminate Psm and Pca.Key points• We investigated Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis.• Novel primers common to both bacteria were designed following genome comparison.• Multiplex PCR with new primers could discriminate Psm and Pca.

Background The prognostic nutritional index (PNI), which is calculated based on the serum albumin concentration and peripheral blood lymphocyte count, is a useful tool for predicting short-term and long-term postoperative outcome in patients undergoing cancer surgery. However, few studies have investigated PNI in colorectal cancer surgery. We examined the ability of PNI to predict short- and long-term outcomes in patients with colorectal cancer. Methods This retrospective study included 365 patients who underwent resection for colorectal cancer. The prognostic nutritional status was calculated on the basis of admission data as follows: 10 × serum albumin (g/dl) + 0.005 × total lymphocyte count (per mm 3 ). The primary outcomes measured were the impact of PNI on overall survival and postoperative complications. Results Kaplan–Meier analysis and the log rank test revealed that low PNI was significantly associated with poor survival ( P  < 0.0001). In multivariate analysis for survival, preoperative low PNI was an independent prognostic factor for poor survival: odds ratio: 2.25, 95 % confidence interval 1.42–3.59). Moreover, low PNI significantly correlated with the incidence of postoperative complications, especially serious ones. Conclusions Preoperative PNI is a useful predictor of postoperative complications and survival in patients with colorectal cancer.

Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model in vivo and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.

Black rot and bacterial spots threaten the cultivation of cruciferous vegetables worldwide, and the development of a method that can easily detect, identify, and distinguish their respective pathogens Xanthomonas campestris pv. campestris (Xcc) and X. campestris pv. raphani (Xcr) is required. Multiple whole-genome sequences of Xcc and Xcr were aligned to identify specific regions and subsequently design gene markers. A region present in Xcr, but absent in Xcc, was detected, which was approximately 11.5 kbp in length, sandwiched between the serine protease homolog (SPH) and nicotinate phosphoribosyltransferase gene (pncB). It contained putative cellulose synthesis-related genes, whereas Xcc only had a modified cellulose synthase gene. Designed primers were pncB_fw1 and pncB_fw2 (from the pncB gene), Xcc_rv1 and Xcc_rv2 (from the modified cellulose synthesis gene), and Xcr_rv1 and Xcr_rv2 (from the putative first and second open reading frames of the gene cluster). PCR using pncB_fw1 and Xcc_rv1, or pncB_fw2 and Xcc_rv2, amplified DNA fragments only in Xcc and X. campestris pv. incanae (Xci). Xci is the causal agent of black rot of garden stock and closely related to Xcc. PCR using pncB_fw1 and Xcr_rv1, or pncB_2 and Xcr_rv2, amplified DNA fragments only in Xcr. Multiplex PCR analysis easily distinguished Xcc and Xcr from bacterial colonies isolated on growth media and detected the pathogen in symptomatic leaves. Multiplex nested PCR detected the contamination of one seed with Xcc and/or Xcr infection from 1000 seeds. Therefore, the PCR primers designed in this study therefore helped detect and discriminate between Xcc and Xcr.Key points• Xanthomonas campestrispv.campestris(Xcc)andpv.raphani(Xcr)were investigated.• Novel primers were designed following whole-genome comparison analyses.• Multiplex PCR with new primers distinguished Xcc and Xcr simultaneously.

PurposeDespite recent advances in colorectal cancer (CRC) treatment, the prognosis of patients suffering from this malignancy still remains substandard, and metastatic recurrence following curative surgery is the leading cause of mortality. Therefore, it is imperative to identify prognostic markers to predict the clinical outcome of CRC patients. Recent evidence revealed the new role of small nucleolar RNAs (snoRNAs) in oncogenesis. Herein, we systematically evaluated dysregulation of snoRNAs in CRC and clarified their biomarker potential and biological significance in CRC.Experimental designWe analysed expression levels of 4 snoRNAs in 274 colorectal tissues from 3 independent cohorts and 6 colon cancer cell lines. The functional characterisation for the role of SNORA42 in CRC was investigated through a series of in vitro and in vivo experiments.ResultsIn the screening phase, expression levels of all four snoRNAs were significantly elevated in CRC tissues than in corresponding normal mucosa. In the clinical validation cohort, increased SNORA42 expression was an independent prognostic factor for overall survival and disease-free survival, and was a risk factor for distant metastasis. SNORA42 expression negatively correlated with overall survival in an additional independent cohort and identified the patients with high risk for recurrence and poor prognosis in stage II CRC. Furthermore, in vitro and in vivo analyses showed that SNORA42 overexpression resulted in enhanced cell proliferation, migration, invasion, anoikis resistance and tumorigenicity.ConclusionsSNORA42 appears to be a novel oncogene and could serve as a promising predictive biomarker for recurrence and prognosis in patients with CRC.

The beetle horn primordium is a complex and compactly folded epithelial sheet located beneath the larval cuticle. Only by unfolding the primordium can the complete 3D shape of the horn appear, suggesting that the morphology of beetle horns is encoded in the primordial folding pattern. To decipher the folding pattern, we developed a method to manipulate the primordial local folding on a computer and clarified the contribution of the folding of each primordium region to transformation. We found that the three major morphological changes (branching of distal tips, proximodistal elongation, and angular change) were caused by the folding of different regions, and that the folding mechanism also differs according to the region. The computational methods we used are applicable to the morphological study of other exoskeletal animals.

Backgrounds and aim Anastomotic leak (AL) following esophagectomy for esophageal cancer (EC) remains an important cause of prolonged hospitalization and impaired quality of life. Recently, indocyanine green (ICG) fluorescein imaging has been used to evaluate the gastric conduit blood supply during EC surgery. Although several factors have been reported to be associated with AL, no studies have evaluated the relationships between risk factors for AL, including ICG fluorescein imaging. The purpose of this study was to investigate the risk factors associated with AL following esophagectomy and to evaluate the impact of ICG fluorescein imaging of the gastric conduit during EC surgery. Methods One hundred and twenty patients undergoing esophagectomy with esophagogastric anastomosis for EC were enrolled in this retrospective study. Clinicopathological factors, preoperative laboratory variables, and surgical factors, including ICG fluorescence imaging, were analyzed to determine their association with AL. Univariate and multivariate logistic regression analysis was used to evaluate the impact of each of these factors on the incidence of AL. Results Among the 120 patients enrolled in the study, 10 (8.3%) developed AL. Univariate analysis demonstrated an increased risk of AL in patients with a high-neutrophil-to-lymphocyte ratio ( p  = 0.0500) and in patients who did not undergo ICG fluorescein imaging ( p  = 0.0057). Multivariate analysis revealed that the absence of ICG imaging was an independent risk factor for AL ( p  = 0.0098). Conclusions Using ICG fluorescein imaging to evaluate blood flow in the gastric conduit might decrease the incidence of AL following EC surgery.

Background Metastasis is a major cause of death in patients with gastric cancer (GC). MicroRNAs (miRNAs) relating to the epithelial–mesenchymal transition (EMT) control GC progression and metastasis. The aim of this study was to evaluate serum EMT-associated miRNAs for metastatic and prognostic noninvasive biomarkers in GC. Methods In the first step of this study (preliminary experiments), we selected candidate miRNAs associated with metastasis by analyzing the expression of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) and miR-203 in serum samples from stage I ( n  = 12) and stage IV ( n  = 12) GC patients. The second phase involved the independent validation of candidate miRNAs in serum specimens from 130 patients with GC and 22 controls. Results Based on the preliminary experiments, miR-203 was selected as the candidate serum miRNA that was most closely associated with metastasis. Validation analysis revealed that serum miR-203 levels were significantly lower in stage IV than stage I–III GC patients. Serum miR-203 expression was significantly lower in GC patients with a higher T stage, vessel invasion, and lymph node, peritoneal, and distant metastases. Low expression of serum miR-203 was significantly associated with poor disease-free and overall survival. Multivariate analysis revealed that low serum miR-203 expression was an independent predictive marker for lymph node, peritoneal, and distant metastases and a poor prognosis in patients with GC. Conclusions Serum miR-203 has the potential to serve as a noninvasive biomarker for prognosis and to predict metastasis in patients with GC.

Although there is a growing interest in developing circulating microRNA (miRNA) as noninvasive diagnostic biomarkers for the detection of high-risk colorectal adenomas and early-stage CRCs, but the comparative diagnostic significance of serum vs. exosomal miRNAs remains unexplored. Based upon published literature, we performed an initial discovery step by investigating the expression of a miRNA panel in 20 normal colonic mucosa, 27 adenomas, and 19 CRC tissues. We performed subsequent validation by quantifying expression of candidate miRNAs in total serum and in exosomes from 26 adenoma patients and 47 healthy controls, and evaluated their clinical significance and potential diagnostic value in colorectal adenomas. We observed that the expression of four miRNAs, miR-21, miR-29a, miR-92a, and miR-135b, was significantly higher in colorectal adenomas vs. normal colonic mucosa. During validation, expression of miR-21, miR-29a and miR-92a in serum was significantly higher in adenomas vs. healthy controls, significantly correlated with adenoma size and total adenoma number within the colorectum, and significantly discriminated patients with advanced adenomas. In contrast, although exosomal miR-21 and miR-29a levels in adenoma patients were significantly higher than those of healthy volunteers, only exosomal miR-21 significantly correlated with adenoma size and total adenoma number, and could discriminate patients with high-risk adenomas. Compared to exosomal miRNAs, serum levels of miR-21, miR-29a and miR-92a are superior diagnostic biomarkers in patients with high-risk adenomatous polyps.