Multi-modal optimization to identify personalized biomarkers for disease prediction of individual patients with cancer.

Finding personalized biomarkers for disease prediction of patients with cancer remains a massive challenge in precision medicine. Most methods focus on one subnetwork or module as a network biomarker; however, this ignores the early warning capabilities of other modules with different configurations of biomarkers (i.e. multi-modal personalized biomarkers). Identifying such modules would not only predict disease but also provide effective therapeutic drug target information for individual patients. To solve this problem, we developed a novel model (denoted multi-modal personalized dynamic network biomarkers (MMPDNB)) based on a multi-modal optimization mechanism and personalized dynamic network biomarker (PDNB) theory, which can provide multiple modules of personalized biomarkers and unveil their multi-modal properties. Using the genomics data of patients with breast or lung cancer from The Cancer Genome Atlas database, we validated the effectiveness of the MMPDNB model. The experimental results showed that compared with other advanced methods, MMPDNB can more effectively predict the critical state with the highest early warning signal score during cancer development. Furthermore, MMPDNB more significantly identified PDNBs containing driver and biomarker genes specific to cancer tissues. More importantly, we validated the biological significance of multi-modal PDNBs, which could provide effective drug targets of individual patients as well as markers for predicting early warning signals of the critical disease state. In conclusion, multi-modal optimization is an effective method to identify PDNBs and offers a new perspective for understanding tumor heterogeneity in cancer precision medicine.

Low iron ameliorates the salinity-induced growth cessation of seminal roots in wheat seedlings.

Wheat plants are ubiquitously simultaneously exposed to salinity and limited iron availability caused by soil saline-alkalisation. Through this study, we found that both low Fe and NaCl severely inhibited the growth of seminal roots in wheat seedlings; however, sufficient Fe caused greater growth cessation of seminal roots than low Fe under salt stress. Low Fe improved the root meristematic division activity, not altering the mature cell sizes compared with sufficient Fe under salt stress. Foliar Fe spray and split-root experiments showed that low Fe-alleviating the salinity-induced growth cessation of seminal roots was dependent on local low Fe signals in the roots. Ionomics combined with TEM/X-ray few differences in the root Na+ uptake and vacuolar Na+ sequestration between two Fe levels under salt stress. Phytohormone profiling and metabolomics revealed salinity-induced overaccumulation of ACC/ethylene and tryptophan/auxin in the roots under sufficient Fe than under low Fe. Differential gene expression, pharmacological inhibitor addition and the root growth performance of transgenic wheat plants revealed that the rootward auxin efflux and was responsible for the low Fe-mediated amelioration of the salinity-induced growth cessation of seminal roots. Our findings will provide novel insights into the modulation of crop root growth under salt stress.

Tumor cell-derived exosomes mediating hsa_circ_0001739/lncRNA AC159540.1 facilitate liver metastasis in colorectal cancer.

BACKGROUND: The current study probed into how tumor cell-derived exosomes (Exos) mediated hsa_circ_0001739/lncRNA AC159540.1 to manipulate microRNA (miR)-218-5p/FTO-N6-methyladenosine (m6A)/MYC signal axis in liver metastasis in colorectal cancer (CRC). METHODS: hsa_circ_0001739 and lncRNA AC159540.1 were identified as the upstream regulator of miR-218-5p using ENCORI and LncBase databases. Expression patterns of miR-218-5p, hsa_circ_0001739, lncRNA AC159540.1, FTO, and MYC were detected, accompanied by loss-and-gain-of function assays to examine their effects on CRC cell biological functions. SW480 cells-derived Exos were purified, followed by in vitro studies to uncover the effect of hsa_circ_0001739/lncRNA AC159540. RESULTS: miR-218-5p was downregulated while hsa_circ_0001739/lncRNA AC159540.1 was upregulated in CRC tissues and cells. Silencing of hsa_circ_0001739/lncRNA AC159540.1 restrained the malignant phenotypes of CRC cells. Exos-mediated hsa_circ_0001739/lncRNA AC159540.1 competitively inhibited miR-218-5p to elevate FTO and MYC. The inducing role of Exos-mediated hsa_circ_0001739/lncRNA AC159540.1 in CRC was also validated in vivo. CONCLUSION: Conclusively, Exos-mediated circ_0001739/lncRNA AC159540.1 regulatory network is critical for CRC, offering a theoretical basis for CRC treatment.

Combined morpho-physiological, ionomic and transcriptomic analyses reveal adaptive responses of allohexaploid wheat (Triticum aestivum L.) to iron deficiency.

BACKGROUND: Plants worldwide are often stressed by low Fe availability around the world, especially in aerobic soils. Therefore, the plant growth, seed yield, and quality of crop species are severely inhibited under Fe deficiency. Fe metabolism in plants is controlled by a series of complex transport, storage, and regulatory mechanisms in cells. Allohexaploid wheat (Triticum aestivum L.) is a staple upland crop species that is highly sensitive to low Fe stresses. Although some studies have been previously conducted on the responses of wheat plants to Fe deficiency, the key mechanisms underlying adaptive responses are still unclear in wheat due to its large and complex genome. RESULTS: Transmission electron microscopy showed that the chloroplast structure was severely damaged under Fe deficiency. Paraffin sectioning revealed that the division rates of meristematic cells were reduced, and the sizes of elongated cells were diminished. ICP-MS-assisted ionmics analysis showed that low-Fe stress significantly limited the absorption of nutrients, including N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, and B nutrients. High-throughput transcriptome sequencing identified 378 and 2,619 genome-wide differentially expressed genes (DEGs) were identified in the shoots and roots between high-Fe and low-Fe conditions, respectively. These DEGs were mainly involved in the Fe chelator biosynthesis, ion transport, photosynthesis, amino acid metabolism, and protein synthesis. Gene coexpression network diagrams indicated that TaIRT1b-4A, TaNAS2-6D, TaNAS1a-6A, TaNAS1-6B, and TaNAAT1b-1D might function as key regulators in the adaptive responses of wheat plants to Fe deficiency. CONCLUSIONS: These results might help us fully understand the morpho-physiological and molecular responses of wheat plants to low-Fe stress, and provide elite genetic resources for the genetic modification of efficient Fe use.

Multiomics reveals an essential role of long-distance translocation in regulating plant cadmium resistance and grain accumulation in allohexaploid wheat (Triticum aestivum).

Cadmium (Cd) is a highly toxic heavy metal that readily enters cereals, such as wheat, via the roots and is translocated to the shoots and grains, thereby posing high risks to human health. However, the vast and complex genome of allohexaploid wheat makes it challenging to understand Cd resistance and accumulation. In this study, a Cd-resistant cultivar of wheat, 'ZM1860', and a Cd-sensitive cultivar, 'ZM32', selected from a panel of 442 accessions, exhibited significantly different plant resistance and grain accumulation. We performed an integrated comparative analysis of the morpho-physiological traits, ionomic and phytohormone profiles, genomic variations, transcriptomic landscapes, and gene functionality in order to identify the mechanisms underlying these differences. Under Cd toxicity, 'ZM1860' outperformed 'ZM32', which showed more severe leaf chlorosis, poorer root architecture, higher accumulation of reactive oxygen species, and disordered phytohormone homeostasis. Ionomics showed that 'ZM32' had a higher root-to-shoot translocation coefficient of Cd and accumulated more Cd in the grains than 'ZM1860'. Whole-genome re-sequencing (WGS) and transcriptome sequencing identified numerous DNA variants and differentially expressed genes involved in abiotic stress responses and ion transport between the two genotypes. Combined ionomics, transcriptomics, and functional gene analysis identified the plasma membrane-localized heavy metal ATPase TaHMA2b-7A as a crucial Cd exporter regulating long-distance Cd translocation in wheat. WGS- and PCR-based analysis of sequence polymorphisms revealed a 25-bp InDel site in the promoter region of TaHMA2b-7A, and this was probably responsible for the differential expression. Our multiomics approach thus enabled the identification of a core transporter involved in long-distance Cd translocation in wheat, and it may provide an elite genetic resource for improving plant Cd resistance and reducing grain Cd accumulation in wheat and other cereal crops.

sMicroRNA-28-5p acts as a metastasis suppressor in gastric cancer by targeting Nrf2.

The transcription factor nuclear factor (erythroid-2)-related factor 2 (Nrf2) can principally serve a mode of protection for both the normal cells and cancer cells from cellular stress, and elevates cancer cell survival. microRNA-28 (miR-28) has been involved in the regulation of Nrf2 expression in breast epithelial cells. However, no comprehensive analysis has been conducted regarding the function of miR-28-5p regulating Nrf2 in gastric cancer (GC). In this study, we aimed to evaluate their interaction and biological roles in the migration and invasion of GC cells. The expression of Nrf2 in the cancer tissues harvested from 42 patients with GC was examined by an array of molecular techniques comprising of Immunohistochemical staining, RT-qPCR and Western blot analysis. Kaplan-Meier method was adopted for analysis of the correlation of Nrf2 with the prognosis of GC patients. Interaction between miR-28-5p and Nrf2 was determined using the bioinformatics analysis and dual luciferase reporter gene assay. Gain- and loss-of-function studies of miR-28-5p and Nrf2 were conducted to elucidate their effects on GC cell migration, invasion and metastasis, as well as expression pattern of several epithelial-mesenchymal transition (EMT)-related proteins. Results indicated that the expression pattern of Nrf2 was significantly upregulated in GC tissues and indicative of poor prognosis of GC patients. miR-28-5p was verified to target Nrf2 and downregulate its expression. GC cells with overexpression of miR-28-5p or Nrf2 knockdown exhibited a marked reduction in the migrated and invasive abilities, along with the N-cadherin expression yet an increase of E-cadherin expression. Furthermore, miR-28-5p exerted an inhibitory function on the metastatic and tumorigenicity of GC cells. In conclusion, miR-28-5p is a comprehensive tumor suppressor that inhibits GC cell migration and invasion through repressing the Nrf2 expression. Therefore, miR-28-5p may serve as a potential biomarker for the prognosis of GC and a novel therapeutic target in advanced GC.

Genome-Scale Investigation of GARP Family Genes Reveals Their Pivotal Roles in Nutrient Stress Resistance in Allotetraploid Rapeseed.

The GARP genes are plant-specific transcription factors (TFs) and play key roles in regulating plant development and abiotic stress resistance. However, few systematic analyses of GARPs have been reported in allotetraploid rapeseed (Brassica napus L.) yet. In the present study, a total of 146 BnaGARP members were identified from the rapeseed genome based on the sequence signature. The BnaGARP TFs were divided into five subfamilies: ARR, GLK, NIGT1/HRS1/HHO, KAN, and PHL subfamilies, and the members within the same subfamilies shared similar exon-intron structures and conserved motif configuration. Analyses of the Ka/Ks ratios indicated that the GARP family principally underwent purifying selection. Several cis-acting regulatory elements, essential for plant growth and diverse biotic and abiotic stresses, were identified in the promoter regions of BnaGARPs. Further, 29 putative miRNAs were identified to be targeting BnaGARPs. Differential expression of BnaGARPs under low nitrate, ammonium toxicity, limited phosphate, deficient boron, salt stress, and cadmium toxicity conditions indicated their potential involvement in diverse nutrient stress responses. Notably, BnaA9.HHO1 and BnaA1.HHO5 were simultaneously transcriptionally responsive to these nutrient stresses in both hoots and roots, which indicated that BnaA9.HHO1 and BnaA1.HHO5 might play a core role in regulating rapeseed resistance to nutrient stresses. Therefore, this study would enrich our understanding of molecular characteristics of the rapeseed GARPs and will provide valuable candidate genes for further in-depth study of the GARP-mediated nutrient stress resistance in rapeseed.

Integrated ionomic and transcriptomic dissection reveals the core transporter genes responsive to varying cadmium abundances in allotetraploid rapeseed.

BACKGROUND: Oilseed rape (B. napus L.) has great potential for phytoremediation of cadmium (Cd)-polluted soils due to its large plant biomass production and strong metal accumulation. Soil properties and the presence of other soluble compounds or ions, cause a heterogeneous distribution of Cd. RESULTS: The aim of our study was to reveal the differential responses of B. napus to different Cd abundances. Herein, we found that high Cd (50 µM) severely inhibited the growth of B. napus, which was not repressed by low Cd (0.50 µM) under hydroponic culture system. ICP-MS assays showed that the Cd2+ concentrations in both shoots and roots under 50 µM Cd were over 10 times higher than those under 0.50 µM Cd. Under low Cd, the concentrations of only shoot Ca2+/Mn2+ and root Mn2+ were obviously changed (both reduced); under high Cd, the concentrations of most cations assayed were significantly altered in both shoots and roots except root Ca2+ and Mg2+. High-throughput transcriptomic profiling revealed a total of 18,021 and 1408 differentially expressed genes under high Cd and low Cd conditions, respectively. The biological categories related to the biosynthesis of plant cell wall components and response to external stimulus were over-accumulated under low Cd, whereas the terms involving photosynthesis, nitrogen transport and response, and cellular metal ion homeostasis were highly enriched under high Cd. Differential expression of the transporters responsible for Cd uptake (NRAMPs), transport (IRTs and ZIPs), sequestration (HMAs, ABCs, and CAXs), and detoxification (MTPs, PCR, MTs, and PCSs), and some other essential nutrient transporters were investigated, and gene co-expression network analysis revealed the core members of these Cd transporters. Some Cd transporter genes, especially NRAMPs and IRTs, showed opposite responsive patterns between high Cd and low Cd conditions. CONCLUSIONS: Our findings would enrich our understanding of the interaction between essential nutrients and Cd, and might also provide suitable gene resources and important implications for the genetic improvement of plant Cd accumulation and resistance through molecular engineering of these core genes under varying Cd abundances in soils.

Genome-wide identification of Brassicaceae B-BOX genes and molecular characterization of their transcriptional responses to various nutrient stresses in allotetraploid rapeseed.

BACKGROUND: B-box (BBX) genes play important roles in plant growth regulation and responses to abiotic stresses. The plant growth and yield production of allotetraploid rapeseed is usually hindered by diverse nutrient stresses. However, no systematic analysis of Brassicaceae BBXs and the roles of BBXs in the regulation of nutrient stress responses have not been identified and characterized previously. RESULTS: In this study, a total of 536 BBXs were identified from nine brassicaceae species, including 32 AtBBXs, 66 BnaBBXs, 41 BoBBXs, 43 BrBBXs, 26 CrBBXs, 81 CsBBXs, 52 BnBBXs, 93 BjBBXs, and 102 BcBBXs. Syntenic analysis showed that great differences in the gene number of Brassicaceae BBXs might be caused by genome duplication. The BBXs were respectively divided into five subclasses according to their phylogenetic relationships and conserved domains, indicating their diversified functions. Promoter cis-element analysis showed that BBXs probably participated in diverse stress responses. Protein-protein interactions between BnaBBXs indicated their functions in flower induction. The expression profiles of BnaBBXs were investigated in rapeseed plants under boron deficiency, boron toxicity, nitrate limitation, phosphate shortage, potassium starvation, ammonium excess, cadmium toxicity, and salt stress conditions using RNA-seq data. The results showed that different BnaBBXs showed differential transcriptional responses to nutrient stresses, and some of them were simultaneously responsive to diverse nutrient stresses. CONCLUSIONS: Taken together, the findings investigated in this study provided rich resources for studying Brassicaceae BBX gene family and enriched potential clues in the genetic improvement of crop stress resistance.

Multiomics reveal pivotal roles of sodium translocation and compartmentation in regulating salinity resistance in allotetraploid rapeseed.

The large size and complexity of the allotetraploid rapeseed (Brassica napus) genome present huge challenges for understanding salinity resistance in this important crop. In this study, we identified two rapeseed genotypes with significantly different degrees of salinity resistance and examined the underlying mechanisms using an integrated analysis of phenomics, ionomics, genomics, and transcriptomics. Under salinity, a higher accumulation of osmoregulation substances and better root-system architecture was observed in the resistant genotype, H159, than in the sensitive one, L339. A lower shoot Na+ concentration and a higher root vacuolar Na+ concentration indicated lower root-to-shoot translocation and higher compartmentation in H159 than in L339. Whole-genome re-sequencing (WGRS) and transcriptome sequencing identified numerous DNA variants and differentially expressed genes involved in abiotic stress responses and ion transport. Combining ionomics with transcriptomics identified plasma membrane-localized BnaC2.HKT1;1 and tonoplast-localized BnaC5.NHX2 as the central factors regulating differential root xylem unloading and vacuolar sequestration of Na+ between the two genotypes. Identification of polymorphisms by WGRS and PCR revealed two polymorphic MYB-binding sites in the promoter regions that might determine the differential gene expression of BnaC2.HKT1;1 and BnaC5.NHX2. Our multiomics approach thus identified core transporters involved in Na+ translocation and compartmentation that regulate salinity resistance in rapeseed. Our results may provide elite gene resources for the improvement of salinity resistance in this crop, and our multiomics approach can be applied to other similar studies.

Genomic identification of nitrogen assimilation-related genes and transcriptional characterization of their responses to nitrogen in allotetraploid rapeseed.

BACKGROUND: Nitrogen (N) is an essential macronutrient to maintain plant growth and development. Plants absorb nitrate-N or ammonium-N in the environment and undergo reduction reactions catalyzed by nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS), and glutamine oxoglutarate aminotransferase (GOGAT) within plants. METHODS AND RESULTS: A total of 42 N assimilation-related genes (NAG) members were identified in rapeseed. Darwin's evolutionary pressure analysis showed that rapeseed NAGs underwent purification selection. Cis-element analysis revealed differences in the transcriptional regulation of NAGs between Arabidopsis and rapeseed. Expression analyses revealed that NRs were expressed mainly in old leaves, NIRs were expressed mainly in old leaves and lower stem peels, while the expression situation between different subfamilies of GSs and GOGATs was more complicated. CONCLUSIONS: Differential expression of NAGs suggested that they might be involved in abiotic stresses. The above results greatly enriched our understanding of NAGs' molecular characteristics and provided central gene resources for NAGs-mediated NUE improvement in rapeseed.

Comprehensive dissection into morpho-physiologic responses, ionomic homeostasis, and transcriptomic profiling reveals the systematic resistance of allotetraploid rapeseed to salinity.

BACKGROUND: Salinity severely inhibit crop growth, yield, and quality worldwide. Allotetraploid rapeseed (Brassica napus L.), a major glycophyte oil crop, is susceptible to salinity. Understanding the physiological and molecular strategies of rapeseed salinity resistance is a promising and cost-effective strategy for developing highly resistant cultivars. RESULTS: First, early leaf senescence was identified and root system growth was inhibited in rapeseed plants under severe salinity conditions. Electron microscopic analysis revealed that 200 mM NaCl induced fewer leaf trichomes and stoma, cell plasmolysis, and chloroplast degradation. Primary and secondary metabolite assays showed that salinity led to an obviously increased anthocyanin, osmoregulatory substances, abscisic acid, jasmonic acid, pectin, cellulose, reactive oxygen species, and antioxidant activity, and resulted in markedly decreased photosynthetic pigments, indoleacetic acid, cytokinin, gibberellin, and lignin. ICP-MS assisted ionomics showed that salinity significantly constrained the absorption of essential elements, including the nitrogen, phosphorus, potassium, calcium, magnesium, iron, mangnese, copper, zinc, and boron nutrients, and induced the increase in the sodium/potassium ratio. Genome-wide transcriptomics revealed that the differentially expressed genes were involved mainly in photosynthesis, stimulus response, hormone signal biosynthesis/transduction, and nutrient transport under salinity. CONCLUSIONS: The high-resolution salt-responsive gene expression profiling helped the efficient characterization of central members regulating plant salinity resistance. These findings might enhance integrated comprehensive understanding of the morpho-physiologic and molecular responses to salinity and provide elite genetic resources for the genetic modification of salinity-resistant crop species.

Genome-wide identification of the amino acid permease genes and molecular characterization of their transcriptional responses to various nutrient stresses in allotetraploid rapeseed.

BACKGROUND: Nitrogen (N), referred to as a "life element", is a macronutrient essential for optimal plant growth and yield production. Amino acid (AA) permease (AAP) genes play pivotal roles in root import, long-distance translocation, remobilization of organic amide-N from source organs to sinks, and other environmental stress responses. However, few systematic analyses of AAPs have been reported in Brassica napus so far. RESULTS: In this study, we identified a total of 34 full-length AAP genes representing eight subgroups (AAP1-8) from the allotetraploid rapeseed genome (AnAnCnCn, 2n = 4x = 38). Great differences in the homolog number among the BnaAAP subgroups might indicate their significant differential roles in the growth and development of rapeseed plants. The BnaAAPs were phylogenetically divided into three evolutionary clades, and the members in the same subgroups had similar physiochemical characteristics, gene/protein structures, and conserved AA transport motifs. Darwin's evolutionary analysis suggested that BnaAAPs were subjected to strong purifying selection pressure. Cis-element analysis showed potential differential transcriptional regulation of AAPs between the model Arabidopsis and B. napus. Differential expression of BnaAAPs under nitrate limitation, ammonium excess, phosphate shortage, boron deficiency, cadmium toxicity, and salt stress conditions indicated their potential involvement in diverse nutrient stress responses. CONCLUSIONS: The genome-wide identification of BnaAAPs will provide a comprehensive insight into their family evolution and AAP-mediated AA transport under diverse abiotic stresses. The molecular characterization of core AAPs can provide elite gene resources and contribute to the genetic improvement of crop stress resistance through the modulation of AA transport.

Global Landscapes of the Na+/H+ Antiporter (NHX) Family Members Uncover their Potential Roles in Regulating the Rapeseed Resistance to Salt Stress.

Soil salinity is a main abiotic stress in agriculture worldwide. The Na+/H+ antiporters (NHXs) play pivotal roles in intracellular Na+ excretion and vacuolar Na+ compartmentalization, which are important for plant salt stress resistance (SSR). However, few systematic analyses of NHXs has been reported in allotetraploid rapeseed so far. Here, a total of 18 full-length NHX homologs, representing seven subgroups (NHX1-NHX8 without NHX5), were identified in the rapeseed genome (AnAnCnCn). Number variations of BnaNHXs might indicate their significantly differential roles in the regulation of rapeseed SSR. BnaNHXs were phylogenetically divided into three evolutionary clades, and the members in the same subgroups had similar physiochemical characteristics, gene/protein structures, and conserved Na+ transport motifs. Darwin´s evolutionary pressure analysis suggested that BnaNHXs suffered from strong purifying selection. The cis-element analysis revealed the differential transcriptional regulation of NHXs between the model Arabidopsis and B. napus. Differential expression of BnaNHXs under salt stress, different nitrogen forms (ammonium and nitrate), and low phosphate indicated their potential involvement in the regulation of rapeseed SSR. Global landscapes of BnaNHXs will give an integrated understanding of their family evolution and molecular features, which will provide elite gene resources for the genetic improvement of plant SSR through regulating the NHX-mediated Na+ transport.

Genome-Wide Differential DNA Methylation and miRNA Expression Profiling Reveals Epigenetic Regulatory Mechanisms Underlying Nitrogen-Limitation-Triggered Adaptation and Use Efficiency Enhancement in Allotetraploid Rapeseed.

Improving crop nitrogen (N) limitation adaptation (NLA) is a core approach to enhance N use efficiency (NUE) and reduce N fertilizer application. Rapeseed has a high demand for N nutrients for optimal plant growth and seed production, but it exhibits low NUE. Epigenetic modification, such as DNA methylation and modification from small RNAs, is key to plant adaptive responses to various stresses. However, epigenetic regulatory mechanisms underlying NLA and NUE remain elusive in allotetraploid B. napus. In this study, we identified overaccumulated carbohydrate, and improved primary and lateral roots in rapeseed plants under N limitation, which resulted in decreased plant nitrate concentrations, enhanced root-to-shoot N translocation, and increased NUE. Transcriptomics and RT-qPCR assays revealed that N limitation induced the expression of NRT1.1, NRT1.5, NRT1.7, NRT2.1/NAR2.1, and Gln1;1, and repressed the transcriptional levels of CLCa, NRT1.8, and NIA1. High-resolution whole genome bisulfite sequencing characterized 5094 differentially methylated genes involving ubiquitin-mediated proteolysis, N recycling, and phytohormone metabolism under N limitation. Hypermethylation/hypomethylation in promoter regions or gene bodies of some key N-metabolism genes might be involved in their transcriptional regulation by N limitation. Genome-wide miRNA sequencing identified 224 N limitation-responsive differentially expressed miRNAs regulating leaf development, amino acid metabolism, and plant hormone signal transduction. Furthermore, degradome sequencing and RT-qPCR assays revealed the miR827-NLA pathway regulating limited N-induced leaf senescence as well as the miR171-SCL6 and miR160-ARF17 pathways regulating root growth under N deficiency. Our study provides a comprehensive insight into the epigenetic regulatory mechanisms underlying rapeseed NLA, and it will be helpful for genetic engineering of NUE in crop species through epigenetic modification of some N metabolism-associated genes.

Through an ITIM-independent mechanism the FcγRIIB blocks B cell activation by disrupting the colocalized microclustering of the B cell receptor and CD19.

B cell activation is regulated through the interplay of the BCR with the inhibitory coreceptor FcγRIIB and the activating coreceptor CD19. Recent studies suggest that Ag-driven BCR microclusters are efficiently converted to a signaling active state on colocalization with CD19 microclusters. Using total internal reflection fluorescence microscopy-based, high-resolution, high-speed live-cell and molecule imaging approaches, we show that when co-ligated to the BCR, the FcγRIIB can inhibit B cell activation by blocking the colocalization of BCR and CD19 microclusters within the B cell immunological synapse. Remarkably, this inhibitory function of FcγRIIB is dependent not on its well-characterized ITIM-containing cytoplasmic domain, but its transmembrane domain. Indeed, human primary B cells from systemic lupus erythematosus patients homozygous for gene encoding the loss-of-function transmembrane domain mutant FcγRIIB-I232T fail to block the synaptic colocalization of the BCR with CD19, leading to dysregulated recruitment of downstream signaling molecule p-PI3K to membrane proximal signalosome. This inhibitory function of FcγRIIB in impairing the spatial-temporal colocalization of BCR and CD19 microclusters in the B cell immunological synapse may help explain the hyper-reactive features of systemic lupus erythematosus patient B cells in reported studies. These observations may also provide new targets for therapies for systemic autoimmune disease.

Value of Chloride Clearance Test in Differential Diagnosis of Gitelman Syndrome.

Objective To investigate the value of chloride clearance test in differential diagnosis of Gitelman syndrome (GS). Methods For patients with hypokalemic metabolic alkalosis and highly suspected GS,clinical data were documented and SLC12A3 gene screening was performed as gold standard to diagnose GS. Hydrochlorothiazide (HCT) test and furosemide (FUR) test were performed according to the standard process. Baseline and maximal increasement of chloride excretion fraction (FECl,the net and relative increase measured as εFECl) were compared between patients and controls to evaluated the reaction to the corresponding diuretics. Receiver operating characteristic (ROC) curve was used to evaluate the sensitivity and specificity of HCT test in GS diagnosis. Results Totally 27 patients and 20 health controls received HCT test. Among those patients,23 were diagnosed with GS genetically. When using the net and relative εFECl to diagnose GS,the areas under the ROC curve were 0.987 (95% CI:0.963～1.000,P<0.001) and 0.984 (95%CI:0.950～1.000,P<0.001),respectively. When a reasonable cutoff value for εFECl was selected,the sensitivity and specificity were both higher than 95%. Eight patients received both HCT test and FUR test. Five of them showed decreased reaction to HCT(net εFECl≤2.86% or relative εFECl≤223%),while normal reaction to FUR.SLC12A3 mutations confirmed their GS. Three patients with blunt reaction to FUR showed normal reaction to HCT,finally they were diagnosed as BS clinically because no SLC12A3 gene mutation was detected. Conclusion Comprehensive application of HCT test and FUR test to evaluate the diuretic reaction can effectively differentiate GS and BS.

Genetic variants of microRNA sequences and susceptibility to sepsis in patients with major blunt trauma.

OBJECTIVE: The objective of this study was to conduct a systematic survey of common precursor microRNA (pre-miRNA) single nucleotide polymorphisms (SNPs) and evaluate their clinical relevance in patients with major blunt trauma. BACKGROUND: Recent evidence indicates that small noncoding RNA molecules known as miRNAs can function as important negative gene regulators and are implicated in the pathogenesis of various diseases. METHODS: We conducted a 2-stage study to examine the impact of 9 selected SNPs with potential functional significance on the susceptibility to sepsis of 1268 trauma patients (1 screening cohort, n = 666) and 2 independent validated cohorts (n = 286 and n = 316, respectively) in China. RESULTS: Among the 9 selected SNPs with potential functional significance, only 1 (miR-608 rs4919510) was found to be strongly associated with a higher risk of developing sepsis and multiple organ dysfunction in all 3 independent study cohorts. An even stronger association was observed for the rs4919510 polymorphism when combining these 3 study cohorts together. In addition, the rs4919510 polymorphism showed a significant correlation with a higher production of proinflammatory cytokines and a lower production of anti-inflammatory cytokines. In vitro experiments further indicated that the G→C variant of this polymorphism could significantly increase the expression of mature miR-608. CONCLUSIONS: Our results indicate that the rs4919510G/C SNP in hsa-mir-608 may be a prognostic biomarker for sepsis in patients with major trauma. Further characterization of miRNA SNPs may open new avenues for studying sepsis and developing novel therapeutic approaches.

Characterization of gene mutations and phenotypes of cystic fibrosis in Chinese patients.

BACKGROUND AND OBJECTIVE: Cystic fibrosis (CF) is a relatively common autosomal recessive disorder in Caucasians. CF is considered a very rare disease in Asians, and fewer than 30 Chinese CF patients are reported in the literature. We enrolled seven patients of Chinese Han origin diagnosed with CF at the Peking Union Medical College Hospital, to characterize gene mutations and phenotypes of CF in Chinese patients. METHODS: We analysed the clinical presentation and screened the coding region of the CFTR gene for each patient. RESULTS: Patients were 0-6 years old at onset of symptoms and were 10-28 years old at the time of diagnosis with CF. None of the seven patients had a family history of CF, and only one patient had parents who were consanguineous. Two patients had gastrointestinal symptoms but stool Sudan III results were normal. Four of the seven CF patients also had allergic bronchopulmonary aspergillosis. The concentration of chloride in patients' sweat ranged from 66 mmol/l to 154 mmol/l. In total, we identified 11 different mutations in seven CF patients, including one novel mutation (△E7-E11). Only one of these mutations (R553X) is present in the Caucasian CFTR common mutation-screening panel; and none of the 11 mutations are common in Caucasian CF patients. CONCLUSIONS: CF in China is difficult to diagnose because of a combination of low awareness, atypical clinical symptoms, and a lack of sweat and genetic testing facilities in most hospitals. The mutations identified in Chinese CF patients are different from the common Caucasian gene mutations.