Comparative Genome Analysis of 'Candidatus Phytoplasma luffae' Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution.

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of 'Candidatus Phytoplasma luffae' strain NCHU2019 that is associated with witches' broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has a remarkably repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for subfunctionalization or neofunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

Complete Genome Sequence of Xylella taiwanensis and Comparative Analysis of Virulence Gene Content With Xylella fastidiosa.

The bacterial genus Xylella contains plant pathogens that are major threats to agriculture in America and Europe. Although extensive research was conducted to characterize different subspecies of Xylella fastidiosa (Xf), comparative analysis at above-species levels was lacking due to the unavailability of appropriate data sets. Recently, a bacterium that causes pear leaf scorch (PLS) in Taiwan was described as the second Xylella species (i.e., Xylella taiwanensis; Xt). In this work, we report the complete genome sequence of Xt type strain PLS229T. The genome-scale phylogeny provided strong support that Xf subspecies pauca (Xfp) is the basal lineage of this species and Xylella was derived from the paraphyletic genus Xanthomonas. Quantification of genomic divergence indicated that different Xf subspecies share ∼87-95% of their chromosomal segments, while the two Xylella species share only ∼66-70%. Analysis of overall gene content suggested that Xt is most similar to Xf subspecies sandyi (Xfs). Based on the existing knowledge of Xf virulence genes, the homolog distribution among 28 Xylella representatives was examined. Among the 11 functional categories, those involved in secretion and metabolism are the most conserved ones with no copy number variation. In contrast, several genes related to adhesins, hydrolytic enzymes, and toxin-antitoxin systems are highly variable in their copy numbers. Those virulence genes with high levels of conservation or variation may be promising candidates for future studies. In summary, the new genome sequence and analysis reported in this work contributed to the study of several important pathogens in the family Xanthomonadaceae.

Genome-Wide Association Study Reveals the Genetic Architecture of Seed Vigor in Oats.

Seed vigor is crucial for crop early establishment in the field and is particularly important for forage crop production. Oat (Avena sativa L.) is a nutritious food crop and also a valuable forage crop. However, little is known about the genetics of seed vigor in oats. To investigate seed vigor-related traits and their genetic architecture in oats, we developed an easy-to-implement image-based phenotyping pipeline and applied it to 650 elite oat lines from the Collaborative Oat Research Enterprise (CORE). Root number, root surface area, and shoot length were measured in two replicates. Variables such as growth rate were derived. Using a genome-wide association (GWA) approach, we identified 34 and 16 unique loci associated with root traits and shoot traits, respectively, which corresponded to 41 and 16 unique SNPs at a false discovery rate < 0.1. Nine root-associated loci were organized into four sets of homeologous regions, while nine shoot-associated loci were organized into three sets of homeologous regions. The context sequences of five trait-associated markers matched to the sequences of rice, Brachypodium and maize (E-value < 10-10), including three markers matched to known gene models with potential involvement in seed vigor. These were a glucuronosyltransferase, a mitochondrial carrier protein domain containing protein, and an iron-sulfur cluster protein. This study presents the first GWA study on oat seed vigor and data of this study can provide guidelines and foundation for further investigations.

Effect of Peptide Sequences on Supramolecular Interactions of Naphthaleneimide/Tripeptide Conjugates.

In this study, we reported a significant difference in the supramolecular hydrogelation of newly discovered NI-GFF (NI-Gly-l-Phe-l-Phe) and NI-FFG (NI-l-Phe-l-Phe-Gly) on the basis of their phase diagrams. With a small difference in the peptide chain between NI-GFF and NI-FFG, we observed a significant difference in their self-assembly properties; NI-GFF formed a stable gel at neutral pH, whereas NI-FFG did not, under the same conditions. From spectroscopic and computational studies, intermolecular π-π interactions and extended hydrogen bonding interactions might reinforce the intermolecular interactions of NI-GFF, which may facilitate the formation of the self-assembled nanostructures and the hydrogel. In addition, the aggregation-induced emission (AIE)-active NI-GFF reveals relatively good biocompatibility compared with that of NI-FFG for two commonly used cell lines, suggesting that it is a promising candidate for use as a supramolecular material in biomedical applications. Our results highlight the importance of tripeptide sequences in a self-assembling hydrogel system.

Lack of Genotype and Phenotype Correlation in a Rice T-DNA Tagged Line Is Likely Caused by Introgression in the Seed Source.

Rice (Oryza sativa) is one of the most important crops in the world. Several rice insertional mutant libraries are publicly available for systematic analysis of gene functions. However, the tagging efficiency of these mutant resources-the relationship between genotype and phenotype-is very low. We used whole-genome sequencing to analyze a T-DNA-tagged transformant from the Taiwan Rice Insertional Mutants (TRIM) resource. The phenomics records for M0028590, one of the TRIM lines, revealed three phenotypes-wild type, large grains, and tillering dwarf-in the 12 T1 plants. Using the sequencing data for 7 plants from three generations of this specific line, we demonstrate that introgression from an indica rice variety might occur in one generation before the seed was used for callus generation and transformation of this line. In addition, the large-grain trait came from the GS3 gene of the introgressed region and the tillering dwarf phenotype came from a single nucleotide change in the D17 gene that occurred during the callus induction to regeneration of the transformant. As well, another regenerant showed completely heterozygous single-nucleotide polymorphisms across the whole genome. In addition to the known sequence changes such as T-DNA integration, single nucleotide polymorphism, insertion, deletion, chromosome rearrangement and doubling, spontaneous outcrossing occurred in the rice field may also explain some mutated traits in a tagged mutant population. Thus, the co-segregation of an integration event and the phenotype should be checked when using these mutant populations.

IL17a and IL21 combined with surgical status predict the outcome of ovarian cancer patients.

Aside from tumor cells, ovarian cancer-related ascites contains the immune components. The aim of this study was to evaluate whether a combination of clinical and immunological parameters can predict survival in patients with ovarian cancer. Ascites specimens and medical records from 144 ovarian cancer patients at our hospital were used as the derivation group to select target clinical and immunological factors to generate a risk-scoring system to predict patient survival. Eighty-two cases from another hospital were used as the validation group to evaluate this system. The surgical status and expression levels of interleukin 17a (IL17a) and IL21 in ascites were selected for the risk-scoring system in the derivation group. The areas under the receiver operating characteristic (AUROC) curves of the overall score for disease-free survival (DFS) of the ovarian cancer patients were 0.84 in the derivation group, 0.85 in the validation group, and 0.84 for all the patients. The AUROC curves of the overall score for overall survival (OS) of cases were 0.78 in the derivation group, 0.76 in the validation group, and 0.76 for all the studied patients. Good correlations between overall risk score and survival of the ovarian cancer patients were demonstrated by sub-grouping all participants into four groups (P for trend <0.001 for DFS and OS). Therefore, acombination of clinical and immunological parameters can provide a practical scoring system to predict the survival of patients with ovarian carcinoma. IL17a and IL21 can potentially be used as prognostic and therapeutic biomarkers.

Urokinase-type plasminogen activator resulting from endometrial carcinogenesis enhances tumor invasion and correlates with poor outcome of endometrial carcinoma patients.

The purpose of this study was to identify the dysregulated genes involved in the tumorigenesis and progression of endometrial endometrioid adenocarcinoma (EEC), and their possible mechanisms. Endometrial specimens including normal endometrial tissues, atypical endometrial hyperplasia, and EEC were analyzed. The expression profiles were compared using GeneChip Array. The gene expression levels were determined by real-time RT-PCR in the training and testing sets to correlate the clinico-pathological parameters of EEC. Immunoblotting, in vitro cell migration and invasion assays were performed in human endometrial cancer cell lines and their transfectants. In microarray analysis, seven dysregulated genes were identified. Only the levels of urokinase-type plasminogen activator (uPA) were higher in EEC with deep myometrial invasion, positive lympho-vascular space invasion, lymph node metastasis, and advanced stages. After multivariate analysis, uPA was the only independent poor prognostic factor for disease-free survival in the EEC patients (hazard ratio: 4.65, p = 0.03). uPA may enhance the migratory and invasive capabilities of endometrial tumor cells by the phosphorylation of ERK1/2, Akt and p38 molecules. uPA is a dysregulated gene involved in the tumorigenesis, bio-pathological features and outcomes of EEC. uPA may be a potential molecule and target for the detection and treatment of EEC.

Insulin-like growth factors inhibit dendritic cell-mediated anti-tumor immunity through regulating ERK1/2 phosphorylation and p38 dephosphorylation.

Insulin-like growth factors (IGFs) can promote tumorigenesis via inhibiting the apoptosis of cancer cells. The relationship between IGFs and dendritic cell (DC)-mediated immunity were investigated. Advanced-stage ovarian carcinoma patients were first evaluated to show higher IGF-1 and IGF-2 concentrations in their ascites than early-stage patients. IGFs could suppress DCs' maturation, antigen presenting abilities, and the ability to activate antigen-specific CD8(+) T cell. IGF-treated DCs also secreted higher concentrations of IL-10 and TNF-α. IGF-treated DCs showed decreased ERK1/2 phosphorylation and reduced p38 dephosphorylation. The percentages of matured DCs in the ascites were significantly lower in the IGF-1 or IGF-2 highly-expressing WF-3 tumor-bearing mice. The IGF1R inhibitor - NVP-AEW541, could block the effects of IGFs to rescue DCs' maturation and to restore DC-mediated antigen-specific immunity through enhancing ERK1/2 phosphorylation and p38 dephosphorylation. IGFs can inhibit DC-mediated anti-tumor immunity through suppressing maturation and function and the IGF1R inhibitor could restore the DC-mediated anti-tumor immunity. Blockade of IGFs could be a potential strategy for cancer immunotherapy.

Interferon-gamma in ascites could be a predictive biomarker of outcome in ovarian carcinoma.

OBJECTIVE: The ovarian cancer-associated ascites is an ideal material for evaluating the interaction between the host immune system and cancer cells in the tumor micro-environment. The aim of this study was to investigate whether the selected target cytokine expression levels in ascites could serve as an immune biomarker for predicting outcomes in ovarian cancer. METHODS: Eighty-eight specimens of ovarian cancer-associated ascites were evaluated to select the target cytokine by a cytokine profiling kit. The 144 total samples were subsequently analyzed for this target cytokine. The correlation between the target cytokine and clinical characteristics was analyzed. RESULTS: Interferon-gamma (IFN-γ) was identified as the target cytokine. Higher levels of IFN-γ in the ascites of the tumor micro-environment were associated with advanced disease (p=0.012), higher tumor histological grading (p=0.004), and sub-optimal surgical status (p=0.040). By multivariate analysis, the adjusted hazard ratios (HRs) were 2.74 (95% confidence interval (CI) 1.85-4.05, p<0.001) for disease-free survival (DFS) and 1.72 (95% CI 1.01-2.93, p=0.048) for overall survival (OS) for a 10-fold increase in IFN-γ concentration in the ascites. An inverse dose-response relationship between IFN-γ level and survival was also noted (Ptrend<0.001 for DFS and Ptrend<0.042 for OS). CONCLUSIONS: Patients with ovarian cancer and higher IFN-γ expression levels in cancer-associated ascites will have shorter DFS and OS. IFN-γ levels in the ascites may be a prognostic marker and a potential reference for immunotherapy targeting IFN-γ.

A scoring system for predicting results of influenza rapid test in children: a possible model facing overwhelming pandemic infection.

BACKGROUND: The pandemic novel influenza H1N1 (swine) influenza A virus (H1N1v) infection has caused large-scale community infection in Taiwan. Anxiety developed in the general public and physicians faced a huge challenge in many aspects. We conducted this prospective study to develop a scoring system based on the clinical manifestations for predicting the results of influenza rapid testing, as a surrogate of influenza rapid testing, to lower the anxiety and decrease the burden for the test. METHODS: From September 1, 2009 to October 5, 2009, pediatric patients who received influenza rapid tests were enrolled, and questionnaires were recorded and analyzed in the first 2 weeks. A further scoring system was conducted to predict the results of influenza rapid tests and validated in the next 3 weeks. RESULTS: Eight hundred and forty-five children were enrolled in our study. In the first phase, data from 506 patients showed that those with age ≥ 5 years, fever ≥ 38°C, contact history of influenza A infection, myalgia, lethargy, sore throat, cough, and headache had a higher risk of positive results (odds ratio: 1.1-2.53). A scoring system was designed, with ≥5 points indicating acceptable sensitivity (69.5%) and specificity (63.6%). Three hundred and thirty-nine patients in the second phase were enrolled to validate the scoring system and the positive and negative predictive values were 52.0% and 73.8%. CONCLUSION: The emergence of H1N1v infection is not only an important medical issue, but also a socioeconomic problem. Based on easily available clinical information, we develop a scoring system as a preliminary screening tool for the general public and first-line health care providers to evaluate the possibility of influenza virus infection. Although this study was limited by the sensitivity of rapid tests, this type of model may be a surrogate weapon when faced with overwhelming pandemic infection in the future, especially in areas with scarce medical resources.

Vasodilator action of docosahexaenoic acid (DHA) in human coronary arteries in vitro.

Coronary arterial tissues obtained from mammalian hearts are known to develop spontaneous phasic contractions. The aim of the present study was to investigate the vasodilatory effects of docosahexaenoic acid (DHA) on the rhythmic contractions of isolated human coronary arterial (HCA) preparations obtained from the recipient hearts of patients undergoing cardiac transplantation. Results from 8 hearts show that: (i) most HCA tissues displayed spontaneous rhythmic phasic contractions with a cycle length around 10 min in the absence or presence of PGF2alpha or elevated [K+]0 (20 mM); (ii) the rhythmic activity could be suppressed by a free fatty acid DHA (30 microM); (iii) high [K+]0 (20 and 80 mM) could induce sustained tonic contraction in addition to phasic contractions in HCA tissues, the tonic contraction could be antagonized by L-type Ca(2+) channel blockers or by DHA (depending on [K+]0); (iv) a digitalis substance ouabain also could induce tonic contraction and suppress phasic contraction; (v) in isolated HCA vascular smooth muscle cells, DHA increased the magnitude of outward voltage-gated K+ (IKV) currents and the inwardly rectifying IK1 currents. Enhancement of K+ currents could be related to vasorelaxation induced by DHA in HCA preparations. Further studies on the effects of DHA on various ionic currents and intracellular Ca(2+) transient are needed to clarify the Ca(2+)-dependent and the Ca(2+)-independent actions of DHA in HCA.