Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.).

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

Time-/dose- series transcriptome data analysis and traditional Chinese medicine treatment of pneumoconiosis.

Pneumoconiosis' pathogenesis is still unclear and specific drugs for its treatment are lacking. Analysis of series transcriptome data often uses a single comparison method, and there are few reports on using such data to predict the treatment of pneumoconiosis with traditional Chinese medicine (TCM). Here, we proposed a new method for analyzing series transcriptomic data, series difference analysis (SDA), and applied it to pneumoconiosis. By comparison with 5 gene sets including existing pneumoconiosis-related genes and gene set functional enrichment analysis, we demonstrated that the new method was not inferior to two existing traditional analysis methods. Furthermore, based on the TCM-drug target interaction network, we predicted the TCM corresponding to the common pneumoconiosis-related genes obtained by multiple methods, and combined them with the high-frequency TCM for its treatment obtained through literature mining to form a new TCM formula for it. After feeding it to pneumoconiosis modeling mice for two months, compared with the untreated group, the coat color, mental state and tissue sections of the mice in the treated group were markedly improved, indicating that the new TCM formula has a certain efficacy. Our study provides new insights into method development for series transcriptomic data analysis and treatment of pneumoconiosis.

Platelet extracellular vesicles: Darkness and light of autoimmune diseases.

Autoimmune diseases are characterized by a breakdown of immune tolerance, leading to inflammation and irreversible end-organ tissue damage. Platelet extracellular vesicles are cellular elements that are important in blood circulation and actively participate in inflammatory and immune responses through intercellular communication and interactions between inflammatory cells, immune cells, and their secreted factors. Therefore, platelet extracellular vesicles are the "accelerator" in the pathological process of autoimmune diseases; however, this robust set of functions of platelet extracellular vesicles has also prompted new advances in therapeutic strategies for autoimmune diseases. In this review, we update fundamental mechanisms based on platelet extracellular vesicles communication function in autoimmune diseases. We also focus on the potential role of platelet extracellular vesicles for the treatment of autoimmune diseases. Some recent studies have found that antiplatelet aggregation drugs, specific biological agents can reduce the release of platelet extracellular vesicles. Platelet extracellular vesicles can also serve as vehicles to deliver drugs to targeted cells. It seems that we can try to silence or inhibit microRNA carried by platelet extracellular vesicles transcription and regulate the target cells to treat autoimmune diseases as platelet extracellular vesicles can transfer microRNA to other cells to regulate immune-inflammatory responses. Hopefully, the information presented here will provide hope for patients with autoimmune diseases.

Autoimmune diseases patients are characterized by autoimmune disorders, whose immune system cannot distinguish between auto- and foreign-antigens. Autoimmune diseases is the third significant disease threatening human health after cardiovascular disease and cancer. However, the exact etiology of autoimmune diseases has yet to be fully elucidated. Several studies have shown that platelet extracellular vesicle content is elevated in multiple autoimmune disorders and positively correlates with disease activity. However, our knowledge about the details of the mechanisms still remains limited and fragmentary. This article updates the communication function of platelet extracellular vesicles in accelerating autoimmune and inflammatory responses. The interesting thing is every coin has two sides. We put forward a new treatment idea for AD based on the particular volume and powerful intercellular communication function of platelet extracellular vesicles. Inhibition of the communication function of platelet extracellular vesicles seems to be considered in the future, or silence or block miRNA of platelet extracellular vesicles involved in the pathogenesis of AD. We can even use it as a drug carrier to deliver the drug to the relevant target cells, thereby enhancing the role of the medicine in regulating immune response and inhibiting inflammation. This paper not only provides a deeper understanding of the pathogenesis of autoimmune diseases but also provides theoretical support for the use of platelet extracellular vesicles to achieve targeted therapy.

Hybrid Attention Network for Epileptic EEG Classification.

Automatic seizure detection from electroencephalography (EEG) based on deep learning has been significantly improved. However, existing works have not adequately excavate the spatial-temporal information between EEG channels. Besides, most works mainly focus on patient-specific scenarios while cross-patient seizure detection is more challenging and meaningful. Regarding the above problems, we propose a hybrid attention network (HAN) for automatic seizure detection. Specifically, the graph attention network (GAT) extracts spatial features at the front end, and Transformer gets time features as the back end. HAN leverages the attention mechanism and fully extracts the spatial-temporal correlation of EEG signals. The focal loss function is introduced to HAN to deal with the imbalance of the dataset accompanied by seizure detection based on EEG. Both patient-specific and patient-independent experiments are carried out on the public CHB-MIT database. Experimental results demonstrate the efficacy of HAN in both experimental settings.

Expression and regulatory characteristics of peripheral blood immune cells in primary Sjögren's syndrome patients using single-cell transcriptomic.

Immune cell subgroups in peripheral blood mononuclear cells (PBMCs) in primary Sjogren's syndrome (pSS) are thought to regulate immune responses, but the nature and functions of these subgroups remain unclear. Here we performed single-cell RNA sequencing (scRNA-seq) of about 68,500 PBMCs from three patients with pSS and three healthy controls (HCs). We found that CD14+ monocytes from pSS patients expressed high levels of the transcription factor CEBPD, and the direct regulation of target genes expression by CEBPD tends to participate in the TNF-α signaling via NF-κB in monocytes. FOLR3 and IL1B were upregulated separately in CD14+ monocyte subsets from different pSS patients. We proposed a system for classifying CD56-CD16+ NK cells based on FCER1G expression. Compared with HCs, pSS patients showed a significantly higher ratio of CD56-CD16+FCER1G+ NK cells to CD56-CD16+FCER1G- NK cells. Our analysis provides a reference dataset and reveals its immune heterogeneity among PBMCs in pSS.

Comparison of the Effects of Different Forms of Nutrition Education on Adolescent Male Soccer Players.

The purpose of this study was to compare the educational effects on nutrition knowledge of two teaching methods targeting adolescent male soccer players through learning online from WeChat account articles (WeChat group) or taking classroom courses (classroom group). The study investigates whether such teaching methods can improve self-efficacy and nutrition knowledge for athletes. A total of 41 U15 (age 15) youth male soccer players, 21 in the classroom group and 20 in the WeChat group, participated in the experiment by receiving the same nutrition education separately for 12 weeks. An athlete nutrition KAP questionnaire and self-efficacy questionnaire were conducted before the intervention, immediately after the intervention, and 6 weeks and 12 weeks after the intervention. As a result, the nutritional knowledge score and the total score of the athlete nutrition KAP questionnaire in the classroom group increased significantly and were notably higher than those in the WeChat group. Self-efficacy scores improved significantly in both groups. In conclusion, the study showed that the level of nutritional knowledge of U15 male soccer players was mediocre, and both forms of nutrition education can significantly improve the level of nutritional knowledge and self-efficacy of the players. In comparison, the educational effect of classroom teaching is significantly greater and more consistent than that of learning from WeChat public articles.

Low-dose interleukin-2 can improve salivary secretion but not lymphocyte infiltration of salivary glands in a murine model of Sjögren's syndrome.

INTRODUCTION/AIM: Effects of low-dose interleukin-2 (IL-2) on the exocrine glandular glands of Sjögren's syndrome are unknown. The aim of this study was to investigate the effects of low-dose IL-2 on salivary gland structure and function in a murine model of Sjögren's syndrome. MATERIALS AND METHODS: Non-obese diabetic/Ltj (NOD) mice were used as the animal model of Sjögren's syndrome, and low-dose IL-2 or phosphate buffered saline was administered subcutaneously from 5 weeks of age, while ICR mice were used as controls. Some mice were sacrificed at 9 weeks of age, while the other mice that continued to receive treatment were sacrificed at 23 weeks. We determined the salivary flow rate of mice every 3 weeks during the intervention. After the mice were sacrificed, one submandibular gland was removed for pathological evaluation, while the other submandibular gland was used to measure the levels of 25 cytokines by Luminex technology. Cervical lymph nodes and spleens were examined by flow cytometry for the proportions of CD8+ T cells and Treg cells. RESULTS: The results showed that the salivary flow rate of NOD mice was slower than that of control-group mice, and there were more pathological changes in the submandibular gland. The levels of many cytokines in the submandibular gland were elevated. The proportion of CD8+ T cells in the cervical lymph nodes and spleens was increased; however, the proportion of Treg cells was decreased. After treatment with IL-2, the exocrine function of the salivary glands of mice was improved. IL-2 also promoted the proliferation of Treg cells in the cervical lymph nodes and spleens, but it did not alter the extent of lymphocyte infiltration in the submandibular gland. The levels of cytokines in the submandibular glands, as well as the proportion of CD8+ T cells in the cervical lymph nodes and spleens, were unchanged significantly after IL-2 treatment. CONCLUSION: Our results demonstrate that treatment with low-dose IL-2 improves the secretory function of the exocrine glands of mice with Sjögren's syndrome, but it does not reverse the structural damage of the exocrine glands.

Targeting EZH2 prevents the occurrence and mitigates the development of Sjögren's syndrome in mice.

OBJECTIVE: We analyzed RNA-SEQ data and found that EZH2 gene expression in salivary glands (SGs) of Sjögren's syndrome (SS) patients was up-regulated and correlated with pathological injury. In this study, we sought to determine if inhibiting EZH2 would ameliorate SS-like disease in NOD/Ltj (NOD) mice. METHODS: We analyzed RNA-SEQ data of SGs of patients with SS from data obtained from the GEO database to explore the correlation between EZH2 gene expression and the progression of SS. Inhibition of EZH2 in the NOD mice was achieved by intraperitoneal administration of GSK343 using both a preventative and a therapeutic model. The effects of GSK343 on SGs secretion and pathological damage, as well as the levels and functions of T cells, B cells, Myeloid-derived suppressor cells (MDSCs), and other immune cells were evaluated. RESULTS: The expression levels of the gene encoding EZH2 in the SGs of SS patients were significantly higher than the non-SS sicca patients, and the expression levels were positively correlated with the severity of the SGs pathological damage. GSK343 treatment significantly increased the salivary flow rate and pathological damage of the SGs in the NOD mice compared to the control mice. In addition, GSK343 significantly inhibited the number and pro-inflammatory-factor secretion of CD4+ and CD8+ T cells and inhibited the increase in the Th1/Th2 cell ratio caused by SS. RNA-SEQ data also showed that EZH2 inhibited several inflammatory pathways during the pathogenesis of SS. CONCLUSIONS: EZH2 expression was up-regulated in the submandibular gland tissue of SS patients.Inhibition of EZH2 alleviated SS-like disease in NOD mice, suggesting that EZH2 might be a potential target for the clinical treatment of SS.

Differential expression involved in starch synthesis pathway genes reveal various starch characteristics of seed and rhizome in lotus (Nelumbo Nucifera).

Lotus (Nelumbo nucifera) is one of the main aquatic vegetables in China. Its seed and rhizome are main edible parts which are rich in starch. Preliminary experiments of starch contents revealed that seed and rhizome expressed great differences in amylose and amylopectin contents. The rhizomes have higher amylopectin content, while the seeds have higher amylose content. In this study, we have estimated 16 varieties of lotus seeds and found that the amylose content of lotus seeds ranged from 30% to 50%, with an average amylose content of 43%, which showed high-amylose content characteristics. Morphological analysis of lotus seed shown that starch rapid accumulated in 20 DAF (day after fertilization) ∼ 26 DAF. Transcriptome of lotus seeds indicated that starch genes played an important role in seed development. Especially in 22 DAF, the genes which controlled amylose synthesis significantly increased, in contrast, the expression of amylopectin-related genes was stable and might limit the synthesis of amylopectin. We further analyzed the expression patterns of 11 key related genes between lotus seeds and rhizomes, and found that the expression of amylose-related genes to were higher in lotus seed, while the expression of genes related to amylopectin synthesis were higher in rhizome. This study provided a comprehensive research of molecular basis for starch in lotus seed and rhizome. Different expression among key genes during starch accumulation might be the principal cause of the differences in starch properties between seed and rhizome. PRACTICAL APPLICATION: Differential expression involved in starch synthesis pathway genes is the main reason for various starch characteristics of seed and rhizome in lotus. High amylose content in lotus seed is a valuable trait for developing functional food.

Genomic variation reveals demographic history and biological adaptation of the ancient relictual, lotus (Nelumbo Adans).

Lotus (Nelumbo Adans.), a relict plant, is the testimony of long-term sustained ecological success, but the underlying genetic changes related to its survival strategy remains unclear. Here, we assembled the high-quality lotus genome, investigated genome variation of lotus mutation accumulation (MA) lines and reconstructed the demographic history of wild Asian lotus, respectively. We identified and validated 43 base substitutions fixed in MA lines, implying a spontaneous mutation rate of 1.4 × 10-9 base/generation in lotus shoot stem cells. The past history of lotus revealed that the ancestors of lotus in eastern and southern Asia could be traced back ~20 million years ago (Mya) and experienced twice significant bottlenecks and population splits. We further identified the selected genes among three lotus groups in different habitats, suggesting that 453 genes between tropical and temperate group and 410 genes between two subgroups from Northeastern China and the Yangtze River - Yellow River Basin might play important roles in natural selection in lotus's adaptation and resilience. Our findings not only improve an understanding of the lotus evolutionary history and the genetic basis of its survival advantages, but also provide valuable data for addressing various questions in evolution and protection for the relict plants.

Danggui Buxue Decoction Ameliorates Inflammatory Bowel Disease by Improving Inflammation and Rebuilding Intestinal Mucosal Barrier.

OBJECTIVE: This study aimed to determine whether Danggui Buxue decoction (DGBX) can improve inflammatory bowel disease (IBD) by regulating immunity and promoting intestinal mucosal repair. METHOD: Dextran sulfate sodium (DSS) was used to induce the IBD model. Drugs (DGBX or saline) were administered to mice, which were randomly divided into three groups (control, model, and experimental groups). Hematoxylin and eosin staining of intestinal tissues was conducted to observe for morphological changes. Changes in cytokines and immune cells in the intestinal tissues were detected by enzyme-linked immunosorbent assay and flow cytometry. Immunofluorescence techniques were used to assess the status of the intestinal mucosal repair. RESULTS: This study found that treatment with DGBX can effectively improve the inflammatory state and pathological structure of the IBD model. DGBX not only can significantly change the composition of intestinal mucosal immune cells and promote the regression of inflammation but also significantly increase the proliferation of intestinal epithelial cells and promote the rapid repair of intestinal mucosal barrier injury compared with the model group (p < 0.05). CONCLUSION: Taking these results, DGBX shows promising protective effects on IBD by regulating immunity and promoting intestinal mucosal repair.

Genetic, transcriptional, and regulatory landscape of monolignol biosynthesis pathway in Miscanthus × giganteus.

BACKGROUND: Miscanthus × giganteus is widely recognized as a promising lignocellulosic biomass crop due to its advantages of high biomass production, low environmental impacts, and the potential to be cultivated on marginal land. However, the high costs of bioethanol production still limit the current commercialization of lignocellulosic bioethanol. The lignin in the cell wall and its by-products released in the pretreatment step is the main component inhibiting the enzymatic reactions in the saccharification and fermentation processes. Hence, genetic modification of the genes involved in lignin biosynthesis could be a feasible strategy to overcome this barrier by manipulating the lignin content and composition of M. × giganteus. For this purpose, the essential knowledge of these genes and understanding the underlying regulatory mechanisms in M. × giganteus is required. RESULTS: In this study, MgPAL1, MgPAL5, Mg4CL1, Mg4CL3, MgHCT1, MgHCT2, MgC3'H1, MgCCoAOMT1, MgCCoAOMT3, MgCCR1, MgCCR2, MgF5H, MgCOMT, and MgCAD were identified as the major monolignol biosynthetic genes in M. × giganteus based on genetic and transcriptional evidence. Among them, 12 genes were cloned and sequenced. By combining transcription factor binding site prediction and expression correlation analysis, MYB46, MYB61, MYB63, WRKY24, WRKY35, WRKY12, ERF021, ERF058, and ERF017 were inferred to regulate the expression of these genes directly. On the basis of these results, an integrated model was summarized to depict the monolignol biosynthesis pathway and the underlying regulatory mechanism in M. × giganteus. CONCLUSIONS: This study provides a list of potential gene targets for genetic improvement of lignocellulosic biomass quality of M. × giganteus, and reveals the genetic, transcriptional, and regulatory landscape of the monolignol biosynthesis pathway in M. × giganteus.

Efficacy of Rebixiao Chinese herbal tablets and Chinese formula granules in acute gout arthritis patients: a randomized, multicenter, double-blind, controlled trial.

OBJECTIVE: To evaluate the clinical efficacy and safety of Rebixiao (RBX) Chinese herbal tablets (CHT) and Chinese formula granules (CFG) in the treatment of acute gout arthritis (AGA). METHODS: This randomized, multicenter, double-blind, controlled trial included 165 AGA patients with the damp-heat symptom pattern who were randomly divided into an RBX CHT group and an RBX CFG group and treated for 7 d at three centers. The total effective rates of the joint symptom score, Traditional Chinese Medicine (TCM) symptoms score, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were used to evaluate the clinical efficacy. Safety assessments were also performed. RESULTS: Of the 165 enrolled patients, 147 completed the clinical observation. There was no difference in baseline between the two groups. The total effective rates of the joint symptom score were 94.36% and 97.36%, and the total effective rates of the TCM symptoms score were 95.77% and 97.36% in the CFG group and CHT group, respectively. No statistical difference was found between the two groups (P > 0.05). Additionally, ESR and CRP were similar in both groups (P > 0.05). Furthermore, treatment efficacy regarding TCM and joint symptoms, the ESR, and CRP were consistent within each center and among the different centers (P > 0.05). In addition, the incidence of adverse events was 4.22% and 2.63% in the CFG group and CHT group, respectively, and no difference was observed between the two groups (P > 0.05). CONCLUSION: RBX CFG and CHT have significant and similar efficacy in the treatment of AGA, and CFG did not increase adverse side effects.

Comparative population genomics reveals genetic divergence and selection in lotus, Nelumbo nucifera.

BACKGROUND: Lotus (Nelumbo nucifera) is an aquatic plant with important agronomic, horticulture, art and religion values. It was the basal eudicot species occupying a critical phylogenetic position in flowering plants. After the domestication for thousands of years, lotus has differentiated into three cultivated types -flower lotus, seed lotus and rhizome lotus. Although the phenotypic and genetic differentiations based on molecular markers have been reported, the variation on whole-genome level among the different lotus types is still ambiguous. RESULTS: In order to reveal the evolution and domestication characteristics of lotus, a total of 69 lotus accessions were selected, including 45 cultivated accessions, 22 wild sacred lotus accessions, and 2 wild American lotus accessions. With Illumina technology, the genomes of these lotus accessions were resequenced to > 13× raw data coverage. On the basis of these genomic data, 25 million single-nucleotide polymorphisms (SNPs) were identified in lotus. Population analysis showed that the rhizome and seed lotus were monophyletic and genetically homogeneous, whereas the flower lotus was biphyletic and genetically heterogeneous. Using population SNP data, we identified 1214 selected regions in seed lotus, 95 in rhizome lotus, and 37 in flower lotus. Some of the genes in these regions contributed to the essential domestication traits of lotus. The selected genes of seed lotus mainly affected lotus seed weight, size and nutritional quality. While the selected genes were responsible for insect resistance, antibacterial immunity and freezing and heat stress resistance in flower lotus, and improved the size of rhizome in rhizome lotus, respectively. CONCLUSIONS: The genome differentiation and a set of domestication genes were identified from three types of cultivated lotus- flower lotus, seed lotus and rhizome lotus, respectively. Among cultivated lotus, flower lotus showed the greatest variation. The domestication genes may show agronomic importance via enhancing insect resistance, improving seed weight and size, or regulating lotus rhizome size. The domestication history of lotus enhances our knowledge of perennial aquatic crop evolution, and the obtained dataset provides a basis for future genomics-enabled breeding.

Genetic diversity, functional properties and expression analysis of NnSBE genes involved in starch synthesis of lotus (Nelumbo nucifera Gaertn.).

BACKGROUND: Starch branching enzyme (SBE) is one of the key enzymes in starch biosynthetic metabolism, determining amylopectin structure. METHODS: Full length coding sequences (CDS) of SBE genes were cloned using reverse transcription PCR (RT-PCR) technology, and neighbor-joining (NJ) tree was used for phylogenetic analysis. Single nucleotide polymorphisms (SNPs) were determined to assess the genetic polymorphisms and variation indexes between individuals and clusters. Quantitative real time PCR (qRT-PCR) was performed to analyze the spatial and temporal expression of NnSBE genes. The effect of NnSBE genes on amylopectin's fine structures was explored using affinity and the enzyme activity analysis of two isoforms in amylopectin and amylose. RESULTS: In this study, two SBE family genes, NnSBEI and NnSBEIII, were identified in lotus (Nelumbo nucifera Gaertn.). Phylogenetic analysis sorted NnSBEI into SBE family B and NnSBEIII into SBE family A. UPGMA phylogenetic tree divided 45 individuals of lotus into three classes. The homozygous haplotype (A G G A G) of NnSBEIII was observed in seed lotus. During the seed embryo development stage, NnSBEIII reached the peak in the middle of the development stage, while NnSBEI increased in the mid-late developmental stage. The different affinity activity of the two isozymes binding amylopectin and amylose assay indicated NnSBEI has higher activity and wider affinity. DISCUSSION: Genetic diversity showed that NnSBE genes received artificial selection during the process of cultivation and domestication in lotus seeds. Furthermore, the expression pattern and affinity activity analysis indicated that NnSBE genes were related to the chain length of amylopectin.

Dangguibuxue decoction abolishes abnormal accumulation of erythroid progenitor cells induced by melanoma.

ETHNOPHARMACOLOGIC RELEVANCE: Dangguibuxue decoction (DGBX), is a well-known traditional Chinese medicine that contains two types of materials used to treat anemia. In this study, we aimed to explore the effect and mechanism of DGBX on abolishing erythroid progenitor cell (Ter119+CD71+) accumulation induced by melanoma. MATERIALS AND METHODS: B16/F10 melanoma cells were used to establish transplanted and metastatic melanoma models. DGBX or normal saline were administered intragastrically daily after the models were established. Tumor sizes and metastatic nodules were observed after tumor cell inoculation. To further test the function of DGBX on erythroid progenitor cell (EPC) accumulation and immunosuppressive abilities, the percentage of EPCs in the blood, and spleen were quantified with flow cytometry. The proportion of CD8+ T cells and related functional mediators, IFN-γ and TNF-α,were also quantified with flow cytometry. To further strengthen our in vivo observations, DGBX serum was prepared from the rats three days after DGBX was administered. Liquid chromatography-mass spectrometry was carried out to control the quality of the experiments. B16/F10 melanomacells were cultured with DGBX serum, and proliferation and apoptosis were observed with the CCK8 assay and AnnexinV/7AAD staining, respectively. EPCs were isolated from B16/F10-bearing mice and cultured under erythroid differentiation conditions. EPCs were treated with DGBX serum, and mature red cell proportions and cell denucleations were tested with flow cytometry and Giemsa staining of the cultured EPCs. Flow cytometry and qPCR were used to analyze the effects of DGBX on the expression of key molecules involved in erythroid development and to explore the mechanism by which DGBX relieves abnormal EPC accumulation. RESULTS: DGBX treatments significantly reduced B16 melanoma tumor sizes and metastatic nodules. Most importantly, our study strongly suggested that DGBX could alleviate anemia, and systematically enhance anti-tumor immune responses by reducing abnormal EPC accumulation. Moreover, DGBX serum treatments had no direct effect on tumor cell proliferation and apoptosis, but could promote EPCs to differentiate into mature red blood cells, in vitro. Mechanistically, at least in part, DGBX relieved abnormal EPC accumulation by altering the "master switch" transcription factors, Pu.1 and Gata-1. CONCLUSIONS: DGBX significantly alleviates abnormal tumor-induced EPC accumulation, inhibits B16 melanoma progression, and enhances anti-tumor immune responses.

Selection of suitable reference genes for quantitive real-time PCR normalization in Miscanthus lutarioriparia.

Miscanthus lutarioriparia, which is found widespread in China, has attracted great attention as a most potential bioenergy plant for years. The quantitative real time PCR (RT-qPCR) has appeared as a sensitive and powerful technique to measure gene expression in living organisms during different development stages. In this study, we evaluated ten candidate genes, including 25S ribosomal RNA gene (25S rRNA), actin1 gene (ACT1), carotenoid-binding protein 20 gene (CBP20), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), Ubiquitin gene (UBQ), eukaryotic elongation factor 1-αgene (eEF-1α), α-tubulin gene (α-TUB), ß-tubulin gene (ß-TUB), eukaryotic translation initiation factor 4α-1 gene (eIF-4α) and NAC domain protein gene(NAC) in a series of 30 M. lutarioriparia samples followed by statistical algorithms geNorm and Normfinder to analyze the gene expression stability. The results indicated that eIF-4αand UBQ were the most stable expressed genes while CBP20 showed as the least stable among all the samples. Based on above research, we recommend that at least two top-ranked reference genes should be employed for expression data normalization. The best genes selected in this study will provide a starting point to select reference genes in the future in other tissues and under other experimental conditions in this energy crop candidate.

Vasoactive Intestinal Peptide Protects Salivary Glands against Structural Injury and Secretory Dysfunction via IL-17A and AQP5 Regulation in a Model of Sjögren Syndrome.

OBJECTIVE: Sjögren syndrome (SS) is an autoimmune disease involving exocrine glands. Currently, drugs that can improve both abnormal immunity and exocrine gland function are needed. The study aimed to investigate the effect and mechanism of vasoactive intestinal peptide (VIP) on the immune response and exocrine gland function in SS. METHODS: We investigated the effects of VIP on the immune response and secretory function of submandibular glands using NOD mice, and analyzed the expression of IL-17A and AQP5 (aquaporin 5). The submandibular gland cells from healthy 8-day-old Sprague-Dawley rats were used to observe the influence of VIP on AQP5 expression. RESULTS: Our study shows that treatment with VIP in an SS mouse model could not only reduce the immune injury to exocrine glands but also improve the secretory function of these glands. Furthermore, VIP was shown to improve the abnormal immune status by downregulating IL-17A expression in the exocrine glands. It also enhanced the secretory function of exocrine glands by upregulating AQP5 expression. CONCLUSIONS: Using a model of SS, we found that VIP could not only modulate the immune response but also affect exocrine gland function, and that these therapeutic effects were associated with IL-17A and AQP5 regulation.