Tumor budding as a predictor of disease-free survival in patients with cholangiocarcinoma.

Background: Tumor budding is considered a prognostic factor in several solid cancer types. However, we lack comprehensive information on the importance of tumor budding in cholangiocarcinoma. Therefore, we aimed to assess the prognostic value of tumor budding in intrahepatic and extrahepatic cholangiocarcinomas and to evaluate its correlations with other clinicopathological parameters. Methods: We monitored 219 patients who underwent surgery for intrahepatic or extrahepatic cholangiocarcinoma at the Pusan National University Hospital between 2012 and 2021. Tumor budding was evaluated using the International Tumor Budding Consensus Conference scoring system. Tumor budding was classified into low (0-4), intermediate (5-9), and high (≥10). For statistical analysis, tumor budding was divided into two groups based on the cut-off value of 10 (lower: 0-9 vs. higher: ≥10). The correlations between clinicopathological parameters were examined using the chi-square and Fisher's exact test. The prognostic values of the variables were analyzed using the log-rank test and Cox regression analysis. Results: Low, intermediate, and high tumor buddings were identified in 135 (61.6%), 63 (28.8), and 21 (9.6%), patients, respectively. Higher tumor budding was related to the presence of lymphatic invasion (p = 0.017), higher tumor grade (p = 0.001), higher N category (p = 0.034). In the univariable and multivariable analyses, higher tumor budding was associated with shorter disease-free survival in 97 (44.3%) patients who underwent R0 resection (p < 0.001 and p = 0.011). Tumor budding did not significantly correlate with disease-specific survival in entire patients. Conclusion: Tumor budding may serve as a prognostic factor for intrahepatic and extrahepatic cholangiocarcinomas treated with R0 resection.

Lymphocyte-activating gene-3 expression is associated with tumor-infiltrating lymphocyte levels in HER2-positive breast cancers.

ABSTRACT: Lymphocyte-activating gene-3 (LAG-3, CD223) is the third inhibitory receptor targeted for immunotherapy. Several clinical trials investigating the use of interventions targeting LAG-3 are underway. The exact signaling mechanism downstream of LAG-3 is largely unknown, especially in breast cancer. The prognostic significance of tumor-infiltrating lymphocytes (TILs) in breast cancer has been previously determined.Among 167 human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients, 90 and 78 patients were positive and negative for the hormone receptor, respectively. LAG-3 mRNA and protein expression levels in TILs were evaluated by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively, among 12 and 167 HER2-positive breast cancer samples, respectively.High expression of LAG-3 in TILs was significantly correlated with high levels of TILs (P = .003) and an abundance of tertiary lymphoid structures around invasive components (P = .014). In addition, high expression of LAG3 was significantly associated with positivity for programmed death-ligand 1 (PD-L1) in tumor cells, a high immunostaining score of PD-L1 in TILs, and a high total immunostaining score for PD-L1 in tumor cells and TILs (all, P < .001). High expression levels of LAG-3 mRNA were associated with high levels of TILs (P = .091).LAG-3 protein expression was not a prognostic factor in HER2-positive breast cancers, and LAG-3 expression in TILs was significantly associated with the levels of TILs in HER2-positive breast cancer, although it was not a prognostic factor.

Unveiling the Crucial Role of Type IV Secretion System and Motility of Helicobacter pylori in IL-1ß Production via NLRP3 Inflammasome Activation in Neutrophils.

Helicobacter pylori is a gram-negative, microaerophilic, and spiral-shaped bacterium and causes gastrointestinal diseases in human. IL-1ß is a representative cytokine produced in innate immune cells and is considered to be a key factor in the development of gastrointestinal malignancies. However, the mechanism of IL-1ß production by neutrophils during H. pylori infection is still unknown. We designed this study to identify host and bacterial factors involved in regulation of H. pylori-induced IL-1ß production in neutrophils. We found that H. pylori-induced IL-1ß production is abolished in NLRP3-, ASC-, and caspase-1/11-deficient neutrophils, suggesting essential role for NLRP3 inflammasome in IL-1ß response against H. pylori. Host TLR2, but not TLR4 and Nod2, was also required for transcription of NLRP3 and IL-1ß as well as secretion of IL-1ß. H. pylori lacking cagL, a key component of the type IV secretion system (T4SS), induced less IL-1ß production in neutrophils than did its isogenic WT strain, whereas vacA and ureA were dispensable. Moreover, T4SS was involved in caspase-1 activation and IL-1ß maturation in H. pylori-infected neutrophils. We also found that FlaA is essential for H. pylori-mediated IL-1ß production in neutrophils, but not dendritic cells. TLR5 and NLRC4 were not required for H. pylori-induced IL-1ß production in neutrophils. Instead, bacterial motility is essential for the production of IL-1ß in response to H. pylori. In conclusion, our study shows that host TLR2 and NLRP3 inflammasome and bacterial T4SS and motility are essential factors for IL-1ß production by neutrophils in response to H. pylori.

Characterizing antigenic determinants in Helicobacter pylori CagA capable of detecting serum antibodies in children.

Helicobacter pylori can persistently colonize the mucosa of the human stomach, resulting in gastric disorders. Endoscopic biopsy for rapid urease test and histopathologic examination are considered as the most accurate diagnostic methods for H. pylori infection. Serological methods are recommended for children because of invasiveness of the diagnosis mentioned above. Here, the cytotoxin-associated gene A protein (Cag A), as an immunodominant antigen, was subdivided to determine which regions harbor antigenicity for humans. CagA was divided into 17 overlapping fragments of ∼400 bp, which were used for the analysis of antigenic determinants. The partial proteins were subjected to immunoblot analysis using pooled serum samples from children with gastric symptoms. A partial recombinant CagA protein containing epitope regions (683-749 amino acids), which were identified in this study, was produced and used for the detection of anti-CagA antibodies and further investigated its serodiagnostic value for determination of H. pylori infection in children. The serum IgG reactivities from children with gastric symptoms were significantly three times more than that of serum samples from children with non-gastric symptoms (P < 0.005). Moreover, the serum IgG reactivities from children showing strong urease activity of gastric biopsies were significantly higher than those with moderate and weak urease activities (P < 0.05). Hence, the partial CagA is a candidate antigen for diagnosis of H. pylori infection.

Transcriptome analysis of grain-filling caryopses reveals the potential formation mechanism of the rice sugary mutant.

A sugary mutant with low total starch and high sugar contents was compared with its wild type Sindongjin for grain-filling caryopses. In the present study, developing seeds of Sindongjin and sugary mutant from the 11th day after flowering (DAF) were subjected to RNA sequencing (RNA-Seq). A total of 30,385 and 32,243 genes were identified in Sindongjin and sugary mutant. Transcriptomic change analysis showed that 7713 differentially expressed genes (DEGs) (log2 fold change ≥1, false discovery rate (FDR)≤0.001) were identified based on our RNA-Seq data, with 7239 genes up-regulated and 474 down-regulated in the sugary mutant. A large number of DEGs were found related to metabolic, biosynthesis of secondary metabolites, plant-pathogen interaction, plant hormone signal transduction and starch/sugar metabolism. Detailed pathway dissection and quantitative real time PCR (qRT-PCR) demonstrated that most genes involved in sucrose to starch synthesis are up-regulated, whereas the expression of the ADP-glucose pyrophosphorylase small subunit (OsAGPS2b) catalyzing the first committed step of starch biosynthesis was specifically inhibited during the grain-filling stage in sugary mutant. Further analysis suggested that the OsAGPS2b is a considerable candidate gene responsible for phenotype of sugary mutant.

Evaluation of genetic diversity and linkage disequilibrium in Korean-bred rice varieties using SSR markers

Background: In order to evaluate the variation among different rice types, the genetic diversity in a rice collection composed by 59 breeding lines, 23 landraces, 18 weedy rice lines, and 35 introduced lines that collected from countries worldwide was analyzed using 134 simple sequence repeat markers. Results: In total, 1264 alleles were identified (average, 9.43 per locus). Rare alleles made up a large portion (58.4%) of the detected alleles, and 29 unique alleles associated with rice accessions were also discovered. A model-based structural analysis revealed the presence of three subpopulations. The genetic relationships revealed by the neighbour-joining tree method were fairly consistent with the structure-based membership assignments for most of the accessions. A total of 105 accessions (79.5%) showed a clear relationship to each cluster, while the remaining 27 accessions (20.5%) were categorized as admixtures. Linkage disequilibrium (LD) patterns and distributions are of fundamental importance for genome-wide association mapping. The mean r² value for all intrachromosomal loci pairs was 0.1286. The LD between linked markers decreased with the genetic distance between pairs of linked loci. Conclusions: These results will provide an effective aid for future allele mining, association genetics, mapping and cloning gene(s), germplasm conservation, and improvement programs.

Development of cDNA-derived ssr markers and their efficiency in diversity assessment of cymbidium accessions

Cymbidium spp. are popular flowering plants. Assessment of the genetic diversity in cultivated Cymbidium facilitates conservation of germplasm and subsequent cultivar improvement. Thus, it is important to develop more efficient polymorphic DNA markers. Although more motifs (403) were identified and more primers (206) were designed in the genomic library compared to the cDNA library, a larger number of successful primers were obtained from the cDNA library (59.9 percent) than from genomic DNA library (51.1 percent). However, higher PIC and gene diversity were identified in genomic SSRs. The average allele number per locus was also higher in genomic SSRs (7.3) than EST-SSRs (5.2), among the 24 evaluated Cymbidium accessions. AT/TA was comparatively high in EST-SSRs, while this motif was not as common in genomic SSRs. The CTT/AAG/TCT/AGA/TTC/GAA and TGC/GCA/GCT/AGC/CTG/CAG motifs were the most abundant tri-nucleotide sequences in EST-SSRs, while GTT/AAC/TGT/ACA/TTG/CAA was the most frequent in genomic SSRs. The number of repeats ranged from 3 to 12 in EST-SSRs. Currently, 52 novel polymorphic SSR markers have been evaluated, which will be useful for germplasm assessments, core set construction, evaluation of genetic diversity, and marker assisted selection (MAS) based Cymbidium breeding.