Chinese cherry CpMYB44-CpSPDS2 module regulates spermidine content and florescence in tobacco.

The flower bud differentiation plays a crucial role in cherry yield and quality. In a preliminary study, we revealed the promotion of spermidine (Spd) in bud differentiation and quality. However, the molecular mechanism underlying Spd regulating cherry bud differentiation remains unclear. To address this research gap, we cloned CpSPDS2, a gene that encodes Spd synthase and is highly expressed in whole flowers and pistils of the Chinese cherry (cv. 'Manaohong'). Furthermore, an overexpression vector with this gene was constructed to transform tobacco plants. The findings demonstrated that transgenic lines exhibited higher Spd content, an earlier flowering time by 6 d, and more lateral buds and flowers than wild-type lines. Additionally, yeast one-hybrid assays and two-luciferase experiments confirmed that the R2R3-MYB transcription factor (CpMYB44) directly binds to and activates the CpSPDS2 promoter transcription. It is indicated that CpMYB44 promotes Spd accumulation via regulating CpSPDS2 expression, thus accelerating the flower growth. This research provides a basis for resolving the molecular mechanism of CpSPDS2 involved in cherry bud differentiation.

The ATP Synthase Subunits FfATPh, FfATP5, and FfATPb Regulate the Development, Pathogenicity, and Fungicide Sensitivity of Fusarium fujikuroi.

ATP synthase catalyzes the synthesis of ATP by consuming the proton electrochemical gradient, which is essential for maintaining the life activity of organisms. The peripheral stalk belongs to ATP synthase and plays an important supporting role in the structure of ATP synthase, but their regulation in filamentous fungi are not yet known. Here, we characterized the subunits of the peripheral stalk, FfATPh, FfATP5, and FfATPb, and explored their functions on development and pathogenicity of Fusarium Fujikuroi. The FfATPh, FfATP5, and FfATPb deletion mutations (∆FfATPh, ∆FfATP5, and ∆FfATPb) presented deficiencies in vegetative growth, sporulation, and pathogenicity. The sensitivity of ∆FfATPh, ∆FfATP5, and ∆FfATPb to fludioxonil, phenamacril, pyraclostrobine, and fluazinam decreased. In addition, ∆FfATPh exhibited decreased sensitivity to ionic stress and osmotic stress, and ∆FfATPb and ∆FfATP5 were more sensitive to oxidative stress. FfATPh, FfATP5, and FfATPb were located on the mitochondria, and ∆FfATPh, ∆FfATPb, and ∆FfATP5 disrupted mitochondrial location. Furthermore, we demonstrated the interaction among FfATPh, FfATP5, and FfATPb by Bimolecular Fluorescent Complimentary (BiFC) analysis. In conclusion, FfATPh, FfATP5, and FfATPb participated in regulating development, pathogenicity, and sensitivity to fungicides and stress factors in F. fujikuroi.

Radiomic signatures based on pretreatment 18F-FDG PET/CT, combined with clinicopathological characteristics, as early prognostic biomarkers among patients with invasive breast cancer.

Purpose: The aim of this study was to investigate the predictive role of fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) in the prognostic risk stratification of patients with invasive breast cancer (IBC). To achieve this, we developed a clinicopathologic-radiomic-based model (C-R model) and established a nomogram that could be utilized in clinical practice. Methods: We retrospectively enrolled a total of 91 patients who underwent preoperative 18F-FDG PET/CT and randomly divided them into training (n=63) and testing cohorts (n=28). Radiomic signatures (RSs) were identified using the least absolute shrinkage and selection operator (LASSO) regression algorithm and used to compute the radiomic score (Rad-score). Patients were assigned to high- and low-risk groups based on the optimal cut-off value of the receiver operating characteristic (ROC) curve analysis for both Rad-score and clinicopathological risk factors. Univariate and multivariate Cox regression analyses were performed to determine the association between these variables and progression-free survival (PFS) or overall survival (OS). We then plotted a nomogram integrating all these factors to validate the predictive performance of survival status. Results: The Rad-score, age, clinical M stage, and minimum standardized uptake value (SUVmin) were identified as independent prognostic factors for predicting PFS, while only Rad-score, age, and clinical M stage were found to be prognostic factors for OS in the training cohort. In the testing cohort, the C-R model showed superior performance compared to single clinical or radiomic models. The concordance index (C-index) values for the C-R model, clinical model, and radiomic model were 0.816, 0.772, and 0.647 for predicting PFS, and 0.882, 0.824, and 0.754 for OS, respectively. Furthermore, decision curve analysis (DCA) and calibration curves demonstrated that the C-R model had a good ability for both clinical net benefit and application. Conclusion: The combination of clinicopathological risks and baseline PET/CT-derived Rad-score could be used to evaluate the prognosis in patients with IBC. The predictive nomogram based on the C-R model further enhanced individualized estimation and allowed for more accurate prediction of patient outcomes.

Exosomal miR-93-5p as an important driver of bladder cancer progression.

Background: Tumor-derived exosomes are involved in the process of tumor metastasis and angiogenesis. MicroRNAs (miRNAs) are the most widely investigated factors in exosomes. Therefore, we hope to find a new therapeutic target in bladder cancer (BLCA), which has high incidence rate and mortality. Methods: Exosomal microRNA(miR)-93-5p expression level, downstream target molecules, and biological functions were examined with bioinformatics technology. Exosomes were extracted by sequential differential centrifugation and verified by transmission electron microscopy. The exosomal miR-93-5p on cell proliferation, invasion, and angiogenesis abilities in 5637 and T24 cells was determined by Cell Counting Kit 8 (CCK-8), colony-forming assay, Transwell assay, and vascular ring formation assay. A mouse xenograft model with intratumor injection was adopted to evaluate the correlation between BLCA-derived exosomes and tumor growth in vivo. Results: The results revealed that exosomes play an important role in the biological progression of BLCA, with miR-93-5p being a particularly important molecule. Compared to normal cells, more malignant cells release more exosomal miR-93-5p, and tumor-derived exosomal miR-93-5p could significantly promote cell proliferation, invasion, and angiogenesis in vitro and in vivo. We identified phosphatase and tensin homolog (PTEN) as the most significant target of miR-93-5p in BLCA and human umbilical vein endothelial cells. Conclusions: Our study successfully revealed the biological role and mechanism of BLCA-derived exosomes in tumor progression. Target at tumor exosomes and exosomal miR-93-5p may be an effective treatment in BLCA.

Copper Amine Oxidase (CuAO)-Mediated Polyamine Catabolism Plays Potential Roles in Sweet Cherry (Prunus avium L.) Fruit Development and Ripening.

Copper amine oxidases (CuAOs) play important roles in PA catabolism, plant growth and development, and abiotic stress response. In order to better understand how PA affects cherry fruit, four potential PavCuAO genes (PavCuAO1-PavCuAO4) that are dispersed over two chromosomes were identified in the sweet cherry genome. Based on phylogenetic analysis, they were classified into three subclasses. RNA-seq analysis showed that the PavCuAO genes were tissue-specific and mostly highly expressed in flowers and young leaves. Many cis-elements associated with phytohormones and stress responses were predicted in the 2 kb upstream region of the promoter. The PavCuAOs transcript levels were increased in response to abscisic acid (ABA) and gibberellin 3 (GA3) treatments, as well as abiotic stresses (NaCl, PEG, and cold). Quantitative fluorescence analysis and high-performance liquid chromatography confirmed that the Put content fell, and the PavCuAO4 mRNA level rose as the sweet cherry fruit ripened. After genetically transforming Arabidopsis with PavCuAO4, the Put content in transgenic plants decreased significantly, and the expression of the ABA synthesis gene NCED was also significantly increased. At the same time, excessive H2O2 was produced in PavCuAO4 transiently expressed tobacco leaves. The above results strongly proved that PavCuAO4 can decompose Put and may promote fruit ripening by increasing the content of ABA and H2O2 while suppressing total free PA levels in the fruit.

Genome-wide characterization of chalcone synthase genes in sweet cherry and functional characterization of CpCHS1 under drought stress.

Cherries are one of the important fruit trees. The growth of cherry is greatly affected by abiotic stresses such as drought, which hinders its development. Chalcone synthase (CHS, EC 2.3.1.74) is a crucial rate-limiting enzyme in the flavonoid biosynthetic pathway that plays an important role in regulating plant growth, development, and abiotic stress tolerance. In the current study, three genes encoding chalcone synthase were identified in the genome of sweet cherry (Prunus avium L.). The three genes contained fewer introns and showed high homology with CHS genes of other Rosaceae members. All members are predicted to localize in the cytoplasm. The conserved catalytic sites may be located at the Cys163, Phe214, His302, and Asn335 residues. These genes were differentially expressed during flower bud dormancy and fruit development. The total flavonoid content of Chinese cherry (Cerasus pseudocerasus Lindl.) was highest in the leaves and slightly higher in the pulp than in the peel. No significant difference in total flavonoid content was detected between aborted kernels and normally developing kernels. Overexpression of Chinese cherry CpCHS1 in tobacco improved the germination frequency of tobacco seeds under drought stress, and the fresh weight of transgenic seedlings under drought stress was higher than that of the wild type, and the contents of SOD, POD, CAT, and Pro in OE lines were significantly increased and higher than WT under drought stress. These results indicate cherry CHS genes are conserved and functionally diverse and will assist in elucidating the functions of flavonoid synthesis pathways in cherry and other Rosaceae species under drought stress.

End-diastolic forward flow in repaired tetralogy of Fallot: Mid-term outcomes from a single center.

Background: Pulmonary arterial end-diastolic forward flow (EDFF) following repaired tetralogy of Fallot (rTOF) is recognized as right ventricular (RV) restrictive physiology, which is closely related to poor prognosis. This study sought to review mid-term experience and investigate the risk factors of EDFF in the rTOF patients. Methods: From September 2016 to January 2019, 100 patients (age < 18 years old) who underwent complete tetralogy of Fallot (TOF) repair were enrolled and were divided into EDFF group (n = 52) and non-EDFF group (n = 48) based on the presence of postoperative EDFF. Elastic net analysis was performed for variable selection. Univariate and multivariate logistic analyses were used to analyze the correlation between risk factors and EDFF. Results: End-diastolic forward flow group had lower systolic blood pressure (P = 0.037), diastolic blood pressure (P = 0.027), and higher vasoactive-inotrope score within 24 h after surgery (P = 0.022) than non-EDFF group. Transannular patch (TAP) was an independent predictor of postoperative EDFF [P = 0.029, OR: 2.585 (1.102∼6.061)]. Patients were followed up for a median of 2.6 years [interquartile range (IQR) 1.6] after the first TOF repair. During follow-up, the prevalence of the EDFF was lower in those with pulmonary valve (PV) reconstructions than that in those undergoing patch enlargement without PV reconstructions in the primary TOF repair (P < 0.001). Conclusion: End-diastolic forward flow was associated with TAP. Patients with EDFF might have a transient hemodynamic instability in the early postoperative period. PV reconstructions in the TOF repair might reduce the incidence of EDFF in the mid-term follow-up.

Comprehensive analysis of CXCL12 expression reveals the significance of inflammatory fibroblasts in bladder cancer carcinogenesis and progression.

BACKGROUND: Bladder cancer (BLCA) is the most common genitourinary tumor but lacks specific diagnostic biomarkers. Recent years have witnessed significant advances in the use and approval of immune checkpoint blockade (ICB) therapy to manage BLCA at advanced stages when platinum-based therapy has failed. The tumor microenvironment (TME) is essential in impacting BLCA patients' prognosis and responsiveness to ICB therapy. CXCL12 is a stromal secreted factor that was essentially involved in regulating the TME among cancers. In this article, we thoroughly investigated the TME regulating roles of CXCL12 in BLCA and revealed its critical involvement in the development of BLCA, which was closely correlated with inflammatory fibroblasts (iCAFs). METHODS: We examined the gene expression profiles in the TCGA and GEO database to reveal the potential association of CXCL12 with the carcinogenesis and prognosis of BLCA. The receiver operating characteristic curve was used to explore the accuracy of CXCL12 along with multiple iCAFs-associated genes in the diagnosis of BLCA. The MCP-COUNTER, ESTIMATE, and TIDE algorithms were applied to estimate the TME components and predict immunotherapy responsiveness. An iCAFs signature was constructed using the ssGSEA algorithm. The "maftool" R package analyzed the oncogenic mutations in BLCA patients. Bioinformatics analysis results were further validated through immunohistochemistry of clinical samples. IMvigor210 cohort was used to validate bioinformatic predictions of therapeutic responsiveness to immune checkpoint inhibitors. RESULTS: This manuscript revealed a significantly reduced expression of CXCL12 in BLCA compared with normal tissue. The expressions of various marker genes for iCAFs were also reduced considerably in BLCA tissues, highlighting the reduction of iCAFs in the pathogenesis of BLCA. Further studies revealed that CXCL12 and iCAFs were associated with pathological features, TME remodeling and aging in BLCA patients. The iCAFs signature further confirmed the intricate immunomodulatory roles of iCAFs in BLCA. Gene mutation analysis revealed the essential relationship between iCAFs and the mutation frequency of oncogenic genes, including TP53 and FGFR3. Meantimes, iCAFs levels also significantly affected BLCA patients' mutations in the TP53 and RTK-RAS pathways. Finally, our results confirmed the significant exclusion of CD8 + T cells by iCAFs, which further influenced the immunotherapy responsiveness in BLCA patients. CONCLUSIONS: This article highlighted the impact of CXCL12 on the pathogenesis and progression of BLCA. The reduced expression levels of iCAFs markers, including CXCL12, were highly accurate in the diagnosis of BLCA, suggesting the reduction of iCAFs accompanied bladder carcinogenesis. However, both CXCL12 and iCAFs significantly impacted the prognosis and immunotherapy responsiveness for BLCA patients by remodeling the TME. Our results critically suggested the dual roles of iCAFs in the carcinogenesis and progression of BLCA. Further exploration of iCAFs might unravel potential diagnostic biomarkers and therapeutic targets for BLCA.

LncRNA CCAT1 promotes prostate cancer cells proliferation, migration, and invasion through regulation of miR-490-3p/FRAT1 axis.

Prostate cancer (PCa) is a prevalent cancer in males, with high incidence and mortality. Recent studies have shown the crucial role of long non-coding RNA (lncRNA) in PCa. Here, we aimed to explore the functional roles and inner mechanisms of lncRNA CCAT1 in PCa cells. qRT-PCR results showed that CCAT1 was upregulated in PCa tissues and cells. Functional assays demonstrated that CCAT1 knockdown suppressed cell proliferation, migration, invasion, yet promoted apoptosis, while CCAT1 promotion showed the opposite results. We also found that CCAT1 negatively regulated miR-490-3p expression and subsequently regulated FRAT1 expression. Inhibition of miR-490-3p or up-regulation of FRAT1 reversed the suppressive effects of CCAT1 knockdown on the PCa cells. In conclusion, CCAT1 regulated FRAT1 expression through miR-490-3p and then promote the PCa cells proliferation, migration, and invasion, which reveals the oncogenic function of CCAT1 in PCa progress.

Value of pulmonary annulus area index in predicting transannular patch placement in tetralogy of Fallot repair.

BACKGROUND: Precisely evaluating the need for transannular patch (TAP) placement is very important in the surgical treatment of tetralogy of Fallot. We hypothesized that the pulmonary annulus area index (PAAI, the pulmonary-to-aortic valve annulus cross-sectional area ratio) could be a useful and accessible predictor for TAP placement. METHODS: The medical records of patients who underwent tetralogy of Fallot repair between 1 January 2016 and 31 December 2017 were reviewed retrospectively. A total of 255 patients were included and categorized into two groups: patients who needed TAP placement and patients who did not. Various candidate predictors for TAP placement (PAAI, pulmonary annulus z-score, and velocity across the pulmonary annulus) were compared using receiver operating characteristic curves. The optimal cutoff for each predictor was assessed. RESULTS: Among the 255 patients included, 156 needed TAP placement (156/237, 65.8%). Both the PAAI (0.28 [0.20/0.34] vs 0.14 [0.09/0.19]; P < .0001) and z-score (-1.5 [-2.9, -0.4] vs -3.6 [-5.3/-2.6]; P < .0001) were smaller in the TAP group. The PAAI is a useful predictor of the pulmonary annulus z-score (AUC 0.830 vs 0.811, P = .19). Combination analysis of the PAAI and velocity across the pulmonary annulus (PV vmax ) showed better predictive value than the PAAI and z-score (AUC 0.860, sensitivity 89.7%, specificity 61.7%, P < .0001). CONCLUSIONS: Our results suggest that the PAAI is a useful and accessible predictor for TAP placement and can be applied readily and simply in clinical practice. A combination with the velocity across the pulmonary annulus could promote the accuracy of prediction.

Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells.

BACKGROUND: The stem cell-based therapies for intervertebral disc degeneration have been widely studied. However, the mechanisms of mesenchymal stem cells interacting with intervertebral disc cells, such as nucleus pulposus cells (NPCs), remain unknown. Exosomes as a vital paracrine mechanism in cell-cell communication have been highly focused on. The purpose of this study was to detect the role of exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) and NPCs in their interaction with corresponding cells. METHODS: The exosomes secreted by BM-MSCs and NPCs were purified by differential centrifugation and identified by transmission electron microscope and immunoblot analysis of exosomal marker proteins. Fluorescence confocal microscopy was used to examine the uptake of exosomes by recipient cells. The effects of NPC exosomes on the migration and differentiation of BM-MSCs were determined by transwell migration assays and quantitative RT-PCR analysis of NPC phenotypic genes. Western blot analysis was performed to examine proteins such as aggrecan, sox-9, collagen II and hif-1α in the induced BM-MSCs. Proliferation and the gene expression profile of NPCs induced by BM-MSC exosomes were measured by Cell Counting Kit-8 and qRT-PCR analysis, respectively. RESULTS: Both the NPCs and BM-MSCs secreted exosomes, and these exosomes underwent uptake by the corresponding cells. NPC-derived exosomes promoted BM-MSC migration and induced BM-MSC differentiation to a nucleus pulposus-like phenotype. BM-MSC-derived exosomes promoted NPC proliferation and healthier extracellular matrix production in the degenerate NPCs. CONCLUSION: Our study indicates that the exosomes act as an important vehicle in information exchange between BM-MSCs and NPCs. Given a variety of functions and multiple advantages, exosomes alone or loaded with specific genes and drugs would be an appropriate option in a cell-free therapy strategy for intervertebral disc degeneration.

Controlled release of Fe3O4 nanoparticles in encapsulated microbubbles to tumor cells via sonoporation and associated cellular bioeffects.

Fe(3)O(4) nanoparticles embedded in the shells of encapsulated microbubbles could be used therapeutically as in situ drug-delivery vehicles. Bioeffects on liver tumor cells SMMC-7721 due to the excitation of Fe(3)O(4) nanoparticles attached to microbubbles generated by ultrasound (US) are studied in an in vitro setting. The corresponding release phenomenon of Fe(3)O(4) nanoparticles from the shells of the microbubbles into the cells via sonoporation and related phenomena, including nanoparticle delivery efficiency, cell trafficking, cell apoptosis, cell cycle, and disturbed flow of intracellular calcium ions during this process, are also studied. Experimental observations show that Fe(3)O(4) nanoparticles embedded in the shells of microbubbles can be delivered into the tumor cells; the delivery rate can be controlled by adjusting the acoustic intensity. The living status or behavior of Fe(3)O(4) -tagged tumor cells can then be noninvasively tracked by magnetic resonance imaging (MRI). It is further demonstrated that the concentration of intracellular Ca(2+) in situ increases as a result of sonoporation. The elevated Ca(2+) is found to respond to the disrupted site in the cell membrane generated by sonoporation for the purpose of cell self-resealing. However, the excessive Ca(2+) accumulation on the membrane results in disruption of cellular Ca(2+) cycling that may be one of the reasons for the death of the cells at the G1 phase. The results also show that the Fe(3)O(4) -nanoparticle-embedded microbubbles have a lower effect on cell bioeffects compared with the non-Fe(3)O(4) -nanoparticle-embedded microbubbles under the same US intensity, which is beneficial for the delivery of nanoparticles and simultaneously maintains the cellular viability.

Brachybacterium zhongshanense sp. nov., a cellulose-decomposing bacterium from sediment along the Qijiang River, Zhongshan City, China.

A cellulose-decomposing bacterium, strain JBT, was isolated from sediments along the Qijiang River, Zhongshan City, China. Results of morphological, biochemical and chemotaxonomic characterization and 16S rRNA gene sequence analysis revealed that strain JBT belonged to the genus Brachybacterium. Insertion sequence-PCR fingerprinting patterns, DNA base ratio analysis and DNA-DNA hybridization data showed that strain JBT differed from recognized species of the genus Brachybacterium. Based on polyphasic analysis, strain JBT represents a novel species of the genus Brachybacterium, for which the name Brachybacterium zhongshanense sp. nov. is proposed. The type strain is JBT (=LMG 23926T=CGMCC 1.6508T=DSM 18832T).