Multiple amino acid transporters as carriers load L-valine-phenazine-1-carboxylic acid conjugate into Ricinus sieve tubes for the phloem translocation.

Some transporters play important roles in the uptake and acropetal xylem translocation of vectorized agrochemicals. However, it is poorly understood the basipetally phloem-loading functions of transporters toward vectorized agrochemicals. Here, L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate) uptake was demonstrated carrier-mediated. RcAAP2, RcANT7, and RcLHT1 showed a similarly up-regulated expression pattern from 62 transporter coding genes in Ricinus at 1 h after L-Val or L-Val-PCA treatment. Subcellular localization revealed that fusion RcAAP2-eGFP, RcANT7-eGFP and RcLHT1-eGFP proteins were expressed in the plasma membrane of mesophyll and phloem cells. Yeast assays found that RcAAP2, RcANT7, and RcLHT1 facilitated L-Val-PCA uptake. To further demonstrate the phloem-loading functions, using vacuum infiltration strategy, an Agrobacterium-mediated RNA interference (RNAi) protocol was constructed in seedlings. HPLC detection indicated that L-Val-PCA phloem sap concentrations were significantly decreased 54.5 %, 27.6 %, and 41.6 % after silencing for 72 h and increased 48.3 %, 52.6 %, and 52.4 % after overexpression, respectively. In conclusion, the plasma membrane-located RcAAP2, RcANT7, and RcLHT1 can loaded L-Val-PCA into Ricinus sieve tubes for the phloem translocation, which may aid in the utilization of transporters and molecular design of phloem-mobile fungicides target root or vascular pathogens.

Amino acid permease RcAAP1 increases the uptake and phloem translocation of an L-valine-phenazine-1-carboxylic acid conjugate.

Amino acid conjugates of pesticides can promote the phloem translocation of parent ingredients, allowing for the reduction of usage, and decreased environmental pollution. Plant transporters play important roles in the uptake and phloem translocation of such amino acid-pesticide conjugates such as L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate). However, the effects of an amino acid permease, RcAAP1, on the uptake and phloem mobility of L-Val-PCA are still unclear. Here, the relative expression levels of RcAAP1 were found to be up-regulated 2.7-fold and 2.2-fold by the qRT-PCR after L-Val-PCA treatments of Ricinus cotyledons for 1 h and 3 h, respectively. Subsequently, expression of RcAAP1 in yeast cells increased the L-Val-PCA uptake (0.36 µmol/107 cells), which was 2.1-fold higher than the control (0.17 µmol/107 cells). Pfam analysis suggested RcAAP1 with its 11 transmembrane domains belongs to the amino acid transporter family. Phylogenetic analysis found RcAAP1 to be strongly similar to AAP3 in nine other species. Subcellular localization showed that fusion RcAAP1-eGFP proteins were observed in the plasma membrane of mesophyll cells and phloem cells. Furthermore, overexpression of RcAAP1 for 72 h significantly increased the phloem mobility of L-Val-PCA in Ricinus seedlings, and phloem sap concentration of the conjugate was 1.8-fold higher than the control. Our study suggested that RcAAP1 as carrier was involved in the uptake and phloem translocation of L-Val-PCA, which could lay foundation for the utilization of amino acids and further development of vectorized agrochemicals.

Metabolism Characteristics of Mycoplasma pneumoniae Infection in Asthmatic Children.

PURPOSE: Studies have shown that Mycoplasma pneumoniae (Mp) infection can aggravate symptoms in asthmatics. However, the mechanism by which Mp infection exacerbates asthma remains unclear. Metabolomics can help identify the mechanism of Mp aggravating asthma in children, thereby providing more a potential target for improving clinical treatment programs. In this article, we analyzed the metabolic level of patients to explain how Mp aggravates asthma in children. METHODS: We divided the subjects into the asthma, Mp infection, asthma combined with Mp infection and healthy groups. Patients' peripheral blood was collected for metabolic and interaction analysis. Cytokine levels were measured via serum and exhaled breath condensate (EBC). RESULTS: A total of 150 participating subjects were divided into four groups after exclusion. We found out that there were different metabolic pathways between the healthy and disease groups. The major pathways of both asthma and asthma combined with Mp infection were valine, leucine and isoleucine biosynthesis; malate-aspartate shuttle was the main differential pathway for Mp infection. Moreover, even though three disease groups involved 81 metabolites at the same time, compared with asthma combined with Mp infection, 2 single disease groups still involved different amino acid pathways (phenylalanine, tyrosine and tryptophan biosynthesis; valine, leucine and isoleucine biosynthesis). Interaction analysis showed that Mp infection in asthmatic patients not only activated cytokines, but also activated Toll-like receptors (TLRs) 2 and 6. Finally, the levels of interleukin (IL)-4, IL-8, IL-13 and tumor necrosis factor-α in EBC with asthma combined with Mp infection were significantly higher than the 2 single disease groups. CONCLUSIONS: Mp infection in asthmatic children can cause changes in the levels of various amino acids in the body, which were enriched in the pathways such as valine, leucine and isoleucine biosynthesis. Palmitic acid can activate TLR2, and iloprost reduces IL-10 levels, ultimately leading to the increased airway inflammation.

Genome-wide characterization and function analysis uncovered roles of wheat LIMs in responding to adverse stresses and TaLIM8-4D function as a susceptible gene.

The Lin-11, Isl-1, and Mec-3 domains (LIM) transcription factors play essential roles in regulating plant biological processes. Despite that, there is a lack of a full understanding of LIMs in wheat (Triticum aestivum L.). In this study, 28 wheat LIM s (TaLIMs) were identified and designated as TaLIM1-1A to TaLIM12-7D. The cis-regulatory element analysis showed that TaLIMs were rich in elements related to biological and abiotic stresses. Expression profiling analysis showed that certain members of TaLIMs were responsive to biotic and abiotic stresses, such as TaLIM1-1A, TaLIM3-2B, TaLIM8-4D, and TaLIM10-5D, were significantly induced by heat, drought, sodium chloride (NaCl), abscisic acid (ABA) and Fusarium graminearum stresses. Furthermore, the biological function of TaLIM8-4D was analyzed and results showed that it was subcellular localization in the nucleus and could induce weak cell death in Nicotiana benthamiana leaves. Additionally, overexpression of TaLIM8-4D could upregulate plant pathogenesis-related (PR) genes, promoting the infection of hemibiotrophic pathogen, implying that TaLIM8-4D could function as susceptible gene in the nucleus by upregulating PR genes and inducing cell death to promote the colonization of hemibiotrophic agent F. graminearum. Overall, the systematic identification, characterization, expression profiling, evolutionary, and function analyses provided the ability to understand TaLIMs and laid a foundation for the further function study of LIM family members in wheat.

An efficient overexpression method for studying genes in Ricinus that transport vectorized agrochemicals.

BACKGROUND: Plant plasma membrane transporters play essential roles during the translocation of vectorized agrochemicals. Therefore, transporters associated with phloem loading of vectorized agrochemicals have drawn increasing attention. As a model system, castor bean (Ricinus communis L.) has been widely used to detect the phloem mobility of agrochemicals. However, there is still a lack of an efficient protocol for the Ricinus seedling model system that can be directly used to investigate the recognition and phloem loading functions of plasmalemma transporters toward vectorized agrochemicals. RESULTS: Here, using vacuum infiltration strategy, we overexpressed the coding gene for enhanced green fluorescent protein (eGFP) in R. communis seedlings by Agrobacterium tumefaciens-mediated transformation system. Strong fluorescence signals were observed in leaves, demonstrating that exogenous genes can be successfully overexpressed in seedlings. Subsequently, gene expression time and vacuum infiltration parameters were optimized. Observation of fluorescence and qRT-PCR analysis showed that eGFP strength and expression level reached a peak at 72 h after overexpression in seedlings. Parameter optimization showed Agrobacterium concentration at OD600 = 1.2, and infiltration for 20 min (0.09 MPa), return to atmospheric pressure, and then infiltration for another 20 min, were the suitable transformation conditions. To test the application of vacuum agroinfiltration in directly examining the loading functions of plasma membrane transporters to vectorized agrochemicals in seedlings, two LHT (lysine/histidine transporter) genes, RcLHT1 and RcLHT7, were overexpressed. Subcellular localization showed the strong fluorescent signals of the fusion proteins RcLHT1-eGFP and RcLHT7-eGFP were observed on the cell membrane of mesophyll cells, and their relative expression levels determined by qRT-PCR were up-regulated 47- and 52-fold, respectively. Furthermore, the concentrations of L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate) in phloem sap collected from seedling sieve tubes were significantly increased 1.9- and 2.3-fold after overexpression of RcLHT1 and RcLHT7, respectively, implying their roles in recognition and phloem loading of L-Val-PCA. CONCLUSIONS: We successfully constructed a transient expression system in Ricinus seedlings and laid the foundation for researchers to directly investigate the loading functions of plasma membrane transporters to vectorized agrochemicals in the Ricinus system.

Systemic fungicidal activity of phenazine-1-carboxylic acid-valine conjugate against tobacco sore shin and its translocation and accumulation in tobacco (Nicotiana tabacum L.).

BACKGROUND: Tobacco sore shin caused by Rhizoctonia solani Kühn is a major soil-borne fungal disease of tobacco, gradually causing infected stems to become thin and dry, leading to great losses to China's tobacco industry. Fungicides with phloem mobility are needed for application to foliage to effectively control root or vascular system pathogens. In this study, phenazine-1-carboxylic acid-valine conjugate (PCA-Val) with strong phloem mobility was tested for control of tobacco sore shin. In vitro fungicidal activity, systemicity, and in vivo efficacy of PCA-Val against R. solani in tobacco seedling were evaluated. RESULTS: In vitro fungicidal activity of PCA-L-Val against R. solani was lower than that of PCA or PCA-D-Val, but the in vivo protective activity and curative activity of PCA-L-Val was the highest among these chemicals tested. The systemicity tests in tobacco seedlings revealed that PCA did not possess phloem mobility, while PCA-L-Val and PCA-D-Val exhibited strong phloem mobility and could be transported and accumulated in the lower part of the seedling as well as throughout the phloem. In addition, we also found that, just like reported hormone amino acid conjugates, PCA-L-Val could be hydrolyzed by tobacco seedlings, to release free PCA. CONCLUSIONS: The current research results indicated that PCA-L-Val possess good phloem transport in tobacco and promising in vivo antifungal activity against R. solani, which can be used as a phloem-mobile fungicide against tobacco sore shin in production practice.

Clinical application of fractional exhaled nitric oxide and nasal nitric oxide levels for the assess eosinophilic inflammation of allergic rhinitis among children.

BACKGROUND: Allergic rhinitis is one of the most common nasal inflammatory diseases among children. Assessment of clinical symptoms, skin prick test and serum immunoglobulin E (IgE) are common methods used to diagnose allergic rhinitis and assess inflammation degree in clinical settings. However, via blood tests assess eosinophils inflammation is invasive, and may cause fear in children. It makes have burden of the diagnosis of allergic rhinitis. Nasal nitric oxide (nNO) and fractional exhaled nitric oxide (FeNO) are noninvasive, inexpensive, and can provide immediate results. These methods may therefore be preferable to assess the inflammation of allergic rhinitis. METHODS: This study was a retrospective analysis. We recruited 61 children with allergic rhinitis from November 2019 to March 2020. The participants were assessed using the FeNO and nNO tests. We also administered questionnaires and carried out traditional allergen and blood tests. We analyzed the relationship between diagnosis results and FeNO and nNO levels before and after the treatment of allergic rhinitis, to investigate the clinical application of FeNO and nNO levels for assess eosinophilic inflammation of allergic rhinitis in children. RESULTS: We observed a significant association both FeNO, nNO level with eosinophils, total IgE. In different levels of eosinophils (EOS), the correlation of detection parameters had obvious change. FeNO and nNO levels were obvious higher compared to pre-treatment. CONCLUSIONS: Using NO concentration can indicates the extent of allergic inflammation and can measure allergy treatment effects combine other influence indexes. The combined use of FeNO and nNO levels may be a useful method for assess the degree of eosinophilic inflammation of allergic rhinitis in children.

MicroRNA-629 promotes the tumorigenesis of non-small-cell lung cancer by targeting FOXO1 and activating PI3K/AKT pathway.

OBJECTIVE: MicroRNA-629 (miR-629) has been found to play an important role in the pathogenesis of human cancers. However, the function of miR-629 is still unknown in non-small-cell lung cancer (NSCLC). The purpose of this study is to preliminarily elucidate the regulatory mechanism of miR-629 in NSCLC. MATERIALS AND METHODS: The mRNA and protein expression was measured by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The function of miR-629 was investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and Transwell assays. The relationship between miR-629 and FOXO1 was confirmed by dual luciferase assay. RESULTS: MiR-629 was upregulated in NSCLC tissues and cells. High expression of miR-629 predicted poor prognosis in patients with NSCLC. Moreover, miR-629 promoted cell proliferation, migration and invasion in NSCLC cells. In addition, FOXO1 was confirmed as a direct target of miR-629 in NSCLC. Furthermore, knockdown of FOXO1 also promoted proliferation, migration and invasion of NSCLC cells. More importantly, overexpression of FOXO1 weakened the carcinogenesis of miR-629 in NSCLC. Besides that, miR-629 promoted EMT and activated the PI3K/AKT pathway in NSCLC. CONCLUSIONS: MiR-629 promotes the progression of NSCLC by targeting FOXO1 and regulating EMT/PI3K/AKT pathway.

The critical role of RasGRP4 in the growth of diffuse large B cell lymphoma.

BACKGROUND: This study aimed to confirm that blocking RasGRP4 can effectively slow down the growth of DLBCL both in vitro and in vivo and ascertain the role of RasGRP4 in the prognosis of DLBCL clinically. METHODS: RasGRP4 expression levels were examined in benign tissues and lymphomas. In order to verify somatic mutation in RasGRP4 gene, cDNA sequencing was performed in DLBCL patients. RasGRP4-dependent cell proliferation, mitochondrial membrane potential, oxidative stress levels and signaling pathway changes were measured by knockdown of RasGRP4. Tumor growth was monitored in xenografted lymphoma model. Clinical data were collected to confirm the role of RasGRP4 in DLBCL. RESULTS: RasGRP4 expression was significantly elevated in DLBCL while no somatic mutations were detected of this gene in DLBCL patients. Decreased RasGRP4 significantly inhibited cell proliferation by simultaneously reducing mitosis and promoting apoptosis and increased the oxidative stress levels. Mechanistically, reduced expression of RasGRP4 decreased ERK while increased JNK expression in SUDHL-4 cells. Knockdown of RasGRP4 also significantly inhibited tumor formation in vivo. Furthermore, RasGRP4 expression levels were significantly higher in patients with larger DLBCL lesions (P = 0.0004), high-risk international prognostic index score groups (P = 0.0042), and its expression was positively correlated with maximum standardized uptake value in DLBCL (P = 0.0004). CONCLUSIONS: These findings indicate the oncogenic role of RasGRP4 in DLBCL, suggesting it as a prognostic biomarker and potential therapeutic target in DLBCL.

Prognostic value of metabolic tumour volume and total lesion glycolysis measured by 18F-fluorodeoxyglucose positron emission tomography/computed tomography in small cell lung cancer: A systematic review and meta-analysis.

The aim of this study was to evaluate the prognostic value of metabolic tumour volume (MTV) and total lesion glycolysis (TLG) for small cell lung cancer (SCLC). MEDLINE, EMBASE and Cochrane Library databases were systematically searched. The pooled hazard ratio (HR) was used to measure the influence of MTV and TLG on survival. The subgroup analysis according to VALSG stage and the measured extent of MTV was performed. Patients with high MTV values experienced a significantly poorer prognosis with a HR of 2.42 (95% CI 1.46-4.03) for overall survival (OS) and a HR of 2.78 (95% CI 1.39-5.53) for progression-free survival (PFS) from the random effect model, and the pooled HR from the fixed effect model was 2.10 (95% CI 1.77-2.50) for OS and 2.27 (95% CI 1.83-2.81) for PFS. Patients with high TLG experienced a poorer prognosis with a HR of 1.61 (95% CI: 1.24-2.07) for OS from the random effect model, and the pooled HR from the fixed effect model was 1.64 (95% CI 1.37-1.96). Heterogeneity among studies was high for MTV in both OS and PFS meta-analyses (I2  = 87% and 88% respectively). After removing one outlier study the heterogeneity was substantially reduced (I2  = 0%) and the pooled HR for the effect of MTV on OS was 1.80 (1.51-2.16, P < 0.00001), and on PFS it was 1.86 (1.49-2.33, P < 0.00001), using either the fixed or random effects model. High MTV is associated with a significantly poorer prognosis OS and PFS, and high TLG is associated with a significantly poorer prognosis regarding OS for SCLC.

MiR-101 inhibits cell proliferation and invasion of pancreatic cancer through targeting STMN1.

MiRNAs regulated most genes expression, which were proved important in various tumors. In this study, we want to investigate miR-101 effect and molecular mechanism on pancreatic cancer (PC), the research about this was blank now. RT-PCR analysis showed that miR-101 expression was declined in PC. MTT assay found that miR-101 mimic suppressed cell viability, while suppressing miR-101 facilitated cell proliferation. Transwell assay showed that miR-101 mimic inhibited cell invasion, but promoted cell invasion by miR-101 inhibitor. With TargetScanHuman's help, we verified STMN1 as a specific target of miR-101 and luciferase reporter assay was carried out to further confirm this discovery. STMN1 expression was reduced by miR-101 mimic and increased by miR-101 inhibitor. We next found that STMN1 was elevated in PC and its expression was negatively correlated with miR-101 expression. Furthermore, STMN1 siRNA curbed cell proliferation and invasion, which was opposite to miR-101 inhibitor effect on PC progression and STMN1 siRNA attenuated miR-101 inhibitor effect on cell proliferation and invasion. In conclusion, miR-101 inhibited PC cell proliferation and invasion via regulating STMN1, which provided a potential therapeutic for PC patients.