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Theanine, a unique and the most abundant non-proteinogenic amino acid in tea plants, endows tea infusion with the umami taste and anti-stress effects. Its content in tea correlates highly with green tea quality. Theanine content in new shoots of tea plants is high in mid-spring and greatly decreases in late spring. However, how the decrease is regulated is largely unknown. In a genetic screening, we observed that a yeast mutant, glutamate dehydrolase 2 (gdh2), was hypersensitive to 40 mM theanine and accumulated more theanine. This result implied a role of CsGDH2s in theanine accumulation in tea plants. Therefore, we identified the two homologs of GDH2, CsGDH2.1 and CsGDH2.2, in tea plants. Yeast complementation assay showed that the expression of CsGDH2.1 in yeast gdh2 mutant rescued the theanine hypersensitivity and hyperaccumulation of this mutant. Subcellular localization and tissue-specific expression showed CsGDH2.1 localized in the mitochondria and highly expressed in young tissues. Importantly, CsGDH2.1 expression was low in early spring, and increased significantly in late spring, in the new shoots of tea plants. These results all support the idea that CsGDH2.1 regulates theanine accumulation in the new shoots. Moreover, the in vitro enzyme assay showed that CsGDH2.1 had glutamate catabolic activity, and knockdown of CsGDH2.1 expression increased glutamate and theanine accumulation in the new shoots of tea plants. These findings suggested that CsGDH2.1-mediated glutamate catabolism negatively regulates theanine accumulation in the new shoots in late spring, and provides a functional gene for improving late-spring green tea quality.

Zein, a class of alcohol-soluble prolamines in maize endosperm, is mainly composed of α-zein, β-zein, and γ-zein. It has been recognized as a structural protein for various gluten-free systems since it can form gluten-like viscoelastic network. The formation of viscoelastic zein network can make up for the structural defect of gluten-free doughs caused by the lack of gluten. To make the most of structural functionality of zein in gluten-free foods, it is important to clearly elucidate the fundamental properties of zein network. In this article, these properties have been discussed, analyzed and summarized from the relationship between protein network and structural functionality of zein, the feature and formation mechanism of zein network, factors affecting zein network and the applications of zein network in improving the quality of gluten-free food. In addition, this article also looks forward to potential research areas on zein network.

Extensive global glacial retreats are threatening cryosphere ecosystem functioning and the associated biota in glacier-fed water systems. Understanding multi-group biodiversity distributions and compositional variation across diverse but hydrologically linked habitats under varying glacial influences will help explain the mechanisms underlying glacial community organization and ecosystem processes. However, such data are generally lacking due to the difficulty of obtaining biodiversity information across wide taxonomic ranges. Here, we used a multi-marker environmental DNA metabarcoding approach to simultaneously investigate the spatial patterns of community compositions and assembly mechanisms of four taxonomic groups (cyanobacteria, diatoms, invertebrates, and vertebrates) along the flowpaths of a tributary of Lake Nam Co on the Tibetan Plateau-from its glacier headwaters, through its downstream river and wetlands, to its estuary. We detected 869 operational taxonomic units: 119 cyanobacterial, 395 diatom, 269 invertebrate, and 86 vertebrate. Taxonomic richnesses consistently increased from upstream to downstream, and although all groups showed community similarity distance decay patterns, the trend for vertebrates was the weakest. Cyanobacteria, diatom, and invertebrate community compositions were significantly correlated with several environmental factors, while the vertebrate community was only correlated with waterway width. Variation partitioning analysis indicated that varying extents of environmental conditions and spatial factors affected community organizations for different groups. Furthermore, stochastic processes contributed prominently to the microorganisms' community assembly (Sloan's neutral model R

Organic tea is more popular than conventional tea that originates from fertilized plants. Amino acids inorganic soils constitute a substantial pool nitrogen (N) available for plants. However, the amino-acid contents in soils of tea plantations and how tea plants take up these amino acids remain largely unknown. In this study, we show that the amino-acid content in the soil of an organic tea plantation is significantly higher than that of a conventional tea plantation. Glutamate, alanine, valine, and leucine were the most abundant amino acids in the soil of this tea plantation. When 15N-glutamate was fed to tea plants, it was efficiently absorbed and significantly increased the contents of other amino acids in the roots. We cloned seven CsLHT genes encoding amino-acid transporters and found that the expression of CsLHT1, CsLHT2, and CsLHT6 in the roots significantly increased upon glutamate feeding. Moreover, the expression of CsLHT1 or CsLHT6 in a yeast amino-acid uptake-defective mutant, 22∆10α, enabled growth on media with amino acids constituting the sole N source. Amino-acid uptake assays indicated that CsLHT1 and CsLHT6 are H+-dependent high- and low-affinity amino-acid transporters, respectively. We further demonstrated that CsLHT1 and CsLHT6 are highly expressed in the roots and are localized to the plasma membrane. Moreover, overexpression of CsLHT1 and CsLHT6 in Arabidopsis significantly improved the uptake of exogenously supplied 15N-glutamate and 15N-glutamine. Taken together, our findings are consistent with the involvement of CsLHT1 and CsLHT6 in amino-acid uptake from the soil, which is particularly important for tea plants grown inorganic tea plantations.

Theanine, a unique non-proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H+ -dependent. The tissue-specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root-to-bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root-to-shoot delivery in the tea plant.

Free amino acids, including theanine, glutamine and glutamate, contribute greatly to the pleasant taste and multiple health benefits of tea. Amino acids in tea plants are mainly synthesized in roots and transported to new shoots, which are significantly affected by nitrogen (N) level and forms. However, the regulatory amino acid metabolism genes have not been systemically identified in tea plants. Here, we investigated the dynamic changes of free amino acid contents in response to N deficiency and forms in tea plant roots, and systemically identified the genes associated amino acid contents in individual metabolism pathways. Our results showed that glutamate-derived amino acids are the most dynamic in response to various forms of N and N deficiency. We then performed transcriptomic analyses of roots treated with N deficiency and various forms of N, and differentially expressed amino acid metabolic genes in each pathway were identified. The analyses on expression patterns and transcriptional responses of metabolic genes to N treatments provided novel insights for the molecular basis of high accumulation of theanine in tea plant root. These analyses also identified potential regulatory genes in dynamic amino acid metabolism in tea plant root. Furthermore, our findings indicated that the dynamic expression levels of CsGDH, CsAlaDC, CsAspAT, CsSDH, CsPAL, CsSHMT were highly correlated with changes of amino acid contents in their corresponding pathways. Herein, this study provides comprehensive insights into transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root.

Nitrogen in soil directly influences the production and quality of tea. However, high nitrogen application in tea plantation leads to soil acidification and environmental pollution. Studies in model plants showed that plasma membrane localized amino acid transporter can regulate the distribution of amino acids to enhance nitrogen use efficiency. Our recent study identified six CsAAPs as transporters for theanine, a unique and most abundant non-proteinaceous amino acid in tea plant. In this work, we found these theanine transporters can also transport Glutamine, Glutamate, aspartate, alanine and γ-aminobutyric acid. Tissue-specific expression analyses showed that CsAAP1, CsAAP5 and CsAAP6 mainly expressed in leaves, CsAAP8 in root, CsAAP4 and CsAAP2 in stem. Furthermore, the expression of these CsAAPs was induced by nitrogen deficiency in a tissue-specific manner. Subcellular localization analyses showed that CsAAP1, CsAAP2 and CsAAP6 location were in the plasma membrane and endoplasmic reticulum. Taken together, these results suggested theanine transporters are involved in nitrogen deficiency response probably by mediating amino acid transport from roots to new shoots and from source to sink tissues in tea plants.

Zein can form viscoelastic network, similar to gluten network, to support the weak structure of gluten-free dough caused by the lack of gluten, which greatly improves the textural quality of gluten-free foods. The effect of plasticizer and zein subunit on viscoelasticity of zein network was studied. In this study, total zein (comprising α-, β- and γ-subunits) or α-zein (comprising mainly α-subunit) were used to prepare network with two plasticizer, ethanol or acetic acid, at room temperature, and the glass transition temperature, rheological properties, textural properties, protein secondary structure, and microstructure of the networks were analyzed. The glass transition temperature of total zein was higher than that of α-zein due to β- and γ-zein. The zein network with acetic acid could maintain its viscoelasticity consistently, while the zein network with ethanol hardened gradually. The zein network with acetic acid had a larger yield strain, higher cohesive force, and stronger resistance to external stress than the zein network with ethanol. The existence of β- and γ-zein in total zein could increase the yield strain and cohesive force of zein network. After zein transformed from solid particle to viscoelastic network, its ordered secondary structures decreased, and disordered secondary structures increased. The more plasticized zein was, the more uniform and ordered zein network was. It is concluded that rheological and textural properties of zein network were both subunit and plasticizer-dependent, and these properties of total zein network with acetic acid was the closest to those of gluten network.

Addition of raw black rice flour leads to deficient processability on bread making quality. One of the effective methods to modify the functional properties of black rice flour (BRF) composite dough is to extrude black rice flour (EBRF) before incorporation. This study investigated and compared the effect of BRF and EBRF addition level of 10%–50% on the rheology, microstructure of dough and bread quality. The rheological properties of composite dough were recorded by Mixolab, stress relaxation and tensile test. The substitution of EBRF presented higher water absorption but lower development time, protein weakening, starch gelatinization, starch gel stability and starch retrogradation than wheat flour dough. Both the BRF and EBRF dough presented solid‐like behaviour, while the EBRF dough showed more viscous, higher resistance and extensibility than BRF dough. The dough microstructure of dough was observed by SEM, and a more compact structure of EBRF dough could be seen than BRF dough. The incorporation of EBRF in bread quality presented higher specific volume, lower bake loss and firmness than BRF bread. These findings indicated the potential utilisation value of extruded black rice flour in bread making.

Theanine, a unique and abundant non-proteinogenic amino acid in tea, confers to the tea infusion its umami taste and multiple health benefits. Its content in new tea shoots is dynamic in winter and spring. However, its seasonal accumulation pattern and the underlying regulation mechanism of tea plants remain largely unknown. In this study, we measured the theanine contents in the roots and leaf buds of 13 tea cultivars at four time points from winter to spring (Dec. 12, Mar. 1, Mar. 23, and Apr. 13). We found theanine accumulated significantly in the roots to as high as ∼6% dry weight. We found theanine content in the roots was constant or slightly decreased on Mar. 1 compared with Dec.12 but increased consistently on Mar. 23 and then decreased on Apr. 13 in all 13 cultivars. In the leaf buds, theanine content kept increasing from Mar. 1 to Mar. 23 and decreasing from Apr. 13 in most of the 13 cultivars, meaning it was probably both season- and developmental stage-dependent. The expression of theanine biosynthesis and amino acid transporter genes in the roots and buds at the four time points was then examined. The correlation analyses between the gene expression and theanine content suggested the expression of theanine-biosynthesis genes was generally and negatively correlated with theanine content; however, the expression of amino acid transporter genes including CsLHT was generally and positively correlated with theanine contents. Finally, we showed that CsLHT has theanine transport activity. Taken together, this study provided insight into the seasonal regulation of theanine biosynthesis and transport in tea plants during winter and spring.

Background Many studies have shown the effects of SGLT2 inhibitors on type 2 diabetes, but the effects in patients with type 2 diabetes with chronic kidney disease remains unclear. This study aims to evaluate the effects of SGLT2 inhibitors on renal outcomes in patients with type 2 diabetes mellitus with chronic kidney disease. Methods We conducted systematic searches of PubMed, Embase, and Cochrane Central Register of Controlled Trials up to April 30, 2020 and included randomized controlled trials of SGLT2 inhibitors in adult type 2 diabetes mellitus (T2DM) patients with chronic kidney disease (CKD) reporting estimated glomerular filtration rate (eGFR) and/or urine albumin/creatinine ratio (UACR) changes and/or acute kidney injury or failure (AKI). Random effects models were adopted to measure the pooled outcomes. Results Nine studies with 8826 participants were included. SGLT2 inhibitors were not associated with a significant change in eGFR (mean difference (MD), -0.75 ml/minutes per 1.73 m2, 95% CI -1.61 to 0.10, P = .09) in type 2 diabetic patients with CKD. UACR reduction after SGLT2 inhibitors was significant in type 2 diabetic patients with CKD (MD -24.27 mg/g, 95% CI -44.46 to -4.09, P = .02). SGLT2 inhibitors associated with AKI in the patients were significant (OR 0.80, 95% CI [0.66 to 0.98], P = .03). Conclusion SGLT2 inhibitors had no significant effect on kidney function (eGFR measured) in the pooled analysis. And SGLT2 inhibitors effectively reduced UACR in T2DM with CKD. Besides, SGLT2 inhibitors could reduce the incidence of AKI.

Glycerolipid is a main component of membranes in oxygenic photosynthetic organisms. Up to now, the majority of publication in this area has focused on the physiological functions of glycerolipids and lipoprotein complexes in photosynthesis, but the study on the separation and identification of glycerolipids in thylakoid membrane in cyanobacteria is relatively rare. Here we report a new method to separate and identify five photosynthetic glycerolipid classes, including monoglucosyl diacylglycerol, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol, in cyanobacteria Synechococcus sp. PCC 7002 by two-dimensional (normal- and reversed-phase) liquid chromatography online coupled to quadrupole time-of-flight mass spectrometry. Over twice as many lipid species were detected by our method compared to the previously reported methods. Ten new odd-chain fatty acid glycerolipids were discovered for the first time. Moreover, complete separation of isomers of monogalactosyl diacylglycerol and monoglucosyl diacylglycerol was achieved. According to the tandem mass spectrometry results, we found that the head group of monoglucosyl diacylglycerols was not as stable as that of monogalactosyl diacylglycerols, which might explain why the organism chose monogalactosyl diacylglycerols and digalactosyl diacylglycerols instead of monoglucosyl diacylglycerols as the main content of the photosynthetic membranes in the history of evolution. This work will benefit further research on the physiological function of glycerolipids.

A quantitative method for clopidogrel using online-SPE tandem LC–MS/MS was developed and fully validated according to the well-established FDA guidelines. The method achieves adequate sensitivity for pharmacokinetic studies, with lower limit of quantifications (LLOQs) as low as 10 pg/mL. Chromatographic separations were performed on reversed phase columns Kromasil Eternity-2.5-C18-UHPLC for both methods. Positive electrospray ionization in multiple reaction monitoring (MRM) mode was employed for signal detection and a deuterated analogue (clopidogrel-d4) was used as internal standard (IS). Adjustments in sample preparation, including introduction of an online-SPE system proved to be the most effective method to solve the analyte back-conversion in clinical samples. Pooled clinical samples (two levels) were prepared and successfully used as real-sample quality control (QC) in the validation of back-conversion testing under different conditions. The result showed that the real samples were stable in room temperature for 24 h. Linearity, precision, extraction recovery, matrix effect on spiked QC samples and stability tests on both spiked QCs and real sample QCs stored in different conditions met the acceptance criteria. This online-SPE method was successfully applied to a bioequivalence study of 75 mg single dose clopidogrel tablets in 48 healthy male subjects., Graphical abstract A quantitative method for clopidogrel using online-SPE tandem LC–MS/MS was developed and fully validated according to the well-established FDA guidelines. The method achieves adequate sensitivity for pharmacokinetic studies, with lower limit of quantifications (LLOQs) as low as 10 pg/mL. Adjustments in sample preparation, including introduction of an online-SPE system proved to be the most effective method to solve the analyte back-conversion in clinical samples. fx1

This paper studies the application of augmented reality technology in children’s 3D interactive books, which can bring interesting, interactive, immersive and realistic reading experience for children. First, the relevant audio, video and other materials were processed by multimedia software. And the three-dimensional models were created by 3ds Max. Then, the multimedia resources and 3D models were integrated in Unity 3D. Finally, the recognition function was realized with the help of Vuforia SDK, and the interactive functions were accomplished by writing scripts. The result was that an augmented reality mobile application based on android platform was developed as well as a book was designed. By scanning the image of the book with the mobile camera, it can present animals’ three-dimensional models. Users can watch the animation by clicking the button. The application of augmented reality technology in children’s books makes its expression rich and vivid. It also can achieve the purpose of edutainment. Besides, combining this technology with publishing industry can help to promote the transformation and upgrading of the publishing industry.

Toxoplasmosis and anaplasmosis are severe zoonotic diseases, the former caused by Toxoplasma gondii and the latter by Anaplasma spp. In the present study, 332 goat blood samples were randomly collected from Chongqing Municipality, China to screen for T. gondii and Anaplasma spp. We used a polymerase chain reaction (PCR) to detect DNA, and enzyme-linked immunosorbent assay (ELISA) to test for T. gondii antibodies. The prevalence of T. gondii and Anaplasma spp. was 38% and 35% respectively by PCR, and 42% for T. gondii antibodies by ELISA. The co-infection rate by T. gondii and Anaplasma was 13%, where the two predominant pathogens co-infecting were Anaplasma phagocytophilum + A. bovis (10%), followed by T. gondii + A. phagocytophilum (9.64%). While co-infection by three pathogens varied ranging from 1.81% to 5.72%, less than 1% of goats were found to be positive for four pathogens. This is the first investigation of T. gondii and Anaplasma spp. infection in goats from Chongqing.

Channels that cross cell walls and connect the cytoplasm of neighboring cells in multicellular cyanobacteria are pivotal for intercellular communication. We find that the product of the gene all1140 of the filamentous cyanobacterium Anabaena sp. PCC 7120 is required for proper channel formation. All1140 encodes an amidase that hydrolyses purified peptidoglycans. An All1140-GFP fusion protein is located at the Z-ring in the periplasmic space during most of the cell cycle. An all1140-null mutant (M40) was unable to grow diazotrophically, and no mature heterocysts were observed in the absence of combined nitrogen. Expression of two key genes, hetR and patS, was studied in M40 using GFP as a reporter. Upon nitrogen step-down, the patterned distribution of green fluorescent cells in filaments seen in the wild type were not observed in mutant M40. Intercellular communication in M40 was studied by measuring fluorescence recovery after photobleaching (FRAP). Movement of calcein (622 Da) was aborted in M40, suggesting that the channels connecting the cytoplasm of neighboring cells are impaired in the mutant. The channels were examined with electron tomography; their diameters were nearly identical, 12.7 nm for the wild type and 12.4 nm for M40, suggesting that AmiC3 is not required for channel formation. However, when the cell wall sacculi isolated by boiling were examined by EM, the average sizes of the channels of the wild type and M40 were 20 nm and 12 nm, respectively, suggesting that the channel walls of the wild type are expandable and that this expandability requires AmiC3.

This study aimed to develop a web image processing application in order to process the pictures online. First, the front-page was designed and layout by HTML, CSS and BootStrap, then using File API to read the pictures or invoke the local camera to take pictures, and draw them into Canvas. Next the basic image processing algorithm were studied, and through the JavaScript scripting language to achieve pixels processing. The result is that a web image processing application was developed. Users can get the basic adjustment function to the acquired images such as brightness, contrast and basic filter such as mosaic. This study used Canvas technology that provided by HTML5 to develop a high quality native applications without relying on plug-ins. Users do not need to download the client, just a web site that can get the basic adjustment function to the images. It was worth researching.

Lipids are important components of cell membranes and thylakoids in cyanobacteria, and they play vital roles in various biological activities. Real-time tracing of the variation of membrane lipids can provide insights of the physiological status of cyanobacterial cells. In this work, easy ambient sonic-spray ionization mass spectrometry (EASI-MS) was utilized to investigate the changes of acidic lipids in unicellular (Synechocystis 6803, Synechococcus 7002) and filamentous (Anabaena 7120) cyanobacteria during different growth phases. A sqdX mutant with a reduced synthesis of sulfoquinovosyl diacylglycerol (SQDG) was constructed to verify the acquired data of EASI-MS. Principal component analysis (PCA) was performed to compare the acquired data, enabling the discrimination of different species of cyanobacteria in day-to-day analysis. The results showed that the three representative cyanobacteria and their growth status can be easily determined on the basis of the lipids components detected by EASI-MS. Very interestingly, significant decreases of the ratios of SQDG/PG and dramatic changes of the unsaturation level of lipids were observed in different culture times in these cyanobacteria, and these two unique characters can be used describe the aging of cyanobacteria.

Glycerolipid is a main component of membranes in oxygenic photosynthetic organisms. Up to now, the majority of publication in this area has focused on the physiological functions of glycerolipids and lipoprotein complexes in photosynthesis, but the study on the separation and identification of glycerolipids in thylakoid membrane in cyanobacteria is relatively rare. Here we report a new method to separate and identify five photosynthetic glycerolipid classes, including monoglucosyl diacylglycerol, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol, in cyanobacteria Synechococcus sp. PCC 7002 by two-dimensional (normal- and reversed-phase) liquid chromatography online coupled to quadrupole time-of-flight mass spectrometry. Over twice as many lipid species were detected by our method compared to the previously reported methods. Ten new odd-chain fatty acid glycerolipids were discovered for the first time. Moreover, complete separation of isomers of monogalactosyl diacylglycerol and monoglucosyl diacylglycerol was achieved. According to the tandem mass spectrometry results, we found that the head group of monoglucosyl diacylglycerols was not as stable as that of monogalactosyl diacylglycerols, which might explain why the organism chose monogalactosyl diacylglycerols and digalactosyl diacylglycerols instead of monoglucosyl diacylglycerols as the main content of the photosynthetic membranes in the history of evolution. This work will benefit further research on the physiological function of glycerolipids.

Phycobilisomes (PBS) are the major light-harvesting, protein–pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phycobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities.

This study investigated the cytotoxic effect of uvangoletin on HL-60 cells, and the effects of uvangoletin on myelosuppression, leucopenia, gastrointestinal tract disturbances and the possible cytotoxic mechanisms by using CCK-8, flow cytometry, western blot, xenograft, cyclophosphamide-induced leucopenia, copper sulfate-induced emesis and ethanol-induced gastric mucosal lesions assays. The results of CCK-8, flow cytometry and western blot assays indicated that uvangoletin showed the cytotoxic effect on HL-60 cells and induced the apoptosis of HL-60 cells by downregulating the expression levels of anti-apoptotic proteins (Survivin, Bcl-xl and Bcl-2), upregulating the expression levels of pro-apoptotic proteins (Smac, Bax, Bad, c-caspase-3 and c-caspase-9), and promoting the release of cytochrome c from mitochondria to cytoplasm. Further, the results of xenograft assay suggested that uvangoletin inhibited the HL-60-induced tumor growth without adverse effect on body weight of nude mice in vivo by regulating the expression levels of above apoptotic proteins. The results indicated that the reductions of WBCs count and thighbone marrow granulocytes percentage in cyclophosphamide-induced leucopenia assay, the incubation period and number of emesis in copper sulfate-induced emesis assay and the gastric mucosal lesions in ethanol-induced gastric mucosal lesions assay were not exacerbated or reversed by uvangoletin. In conclusion, the research preliminarily indicated that uvangoletin induced apoptosis of HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances, and the pro-apoptotic mechanisms may be related to mitochondria-mediated apoptotic pathway.

Multiple myeloma (MM) is one of the most common causes of mortality from hematological malignancy in China. Recent studies have demonstrated that cancerous inhibitor of protein phosphatase 2A (CIP2A) may exhibit a role in promoting the growth of cancer; however, the function of CIP2A in MM remains unknown. In the present study, the expression and molecular mechanism underlying the effects of CIP2A in patients with MM and in MM cell lines were elucidated. Firstly, the expression of CIP2A was detected in patients with MM and in MM cell lines by reverse transcription‑quantitative polymerase chain reaction. Furthermore, silencing of CIP2A with short hairpin RNA was performed in MM cells, and the impact on the proliferation and apoptosis of RPMI‑8226 cells was analyzed (as endogenous CIP2A is highly expressed in RPMI‑8226 cell lines compared with other cells). CIP2A was significantly elevated in patients with MM and in MM cell lines, and silencing of CIP2A could inhibit the proliferation ability of RPMI‑8226 cells in vitro. In addition, CIP2A knockdown induced apoptosis and led to substantial reduction of c‑Myc protein levels in MM cell lines. This study suggested that CIP2A inhibition may provide a promising therapeutic strategy for patients with MM.

Sulfonation of polyvinylidene difluoride (PVDF) resin was achieved by incubation of the resin with sulfuric acid at a moderately high temperature. The sulfonated PVDF (SPVDF) resin was studied for its ability to extract restriction enzymes from DNA digestion solutions. The SPVDF resin was effective in adsorbing restriction enzymes such as EcoRI and BamHI and the extraction procedure was easy and simple to perform. The adsorption depended upon the amount of the resin added. We found that 1 mg of the SPVDF resin could completely remove all restriction enzyme activity routinely used in DNA digestion within 2 min after its addition. Treatment of a digestion solution with the SPVDF resin did not change the reaction solution and the same digestion buffer could be used for another digestion of the same DNA with other enzymes. We also found that, in comparison with normal PVDF, the SPVDF resin adsorbed less DNA, resulting in less loss of DNA in the extraction step. The potential application of the SPVDF resin in other procedures of molecular cloning and enzyme purification is discussed.

Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro. Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends., DNA double-strand breaks (DSBs), a highly deleterious form of DNA damage, are associated with multiple types of broken ends. Here, the authors identify a XRCC4-like factor that functions in the non-homologous end-joining DNA repair pathway to repair DSBs with complex broken ends.

Pbycobilisomes (PBS) are able to transfer absorbed energy to photosystem I and II, and the distribution of light energy between two photosystems is regulated by state transitions. In this study we show that energy transfer from PBS to photosystem I (PSI) requires ApcD. Cells were unable to perform state transitions in the absence of ApcD. The apcD mutant grows more slowly in light mainly absorbed by PBS, indicating that ApcD-dependent energy transfer to PSI is required for optimal growth under this condition. The apcD mutant showed normal blue-light induced quenching, suggesting that ApcD is not required for this process and state transitions are independent of blue-light induced quenching. Under nitrogen fixing condition, the growth rates of the wild type and the mutant were the same, indicating that energy transfer from PBS to PSI in heterocysts was not required for nitrogen fixation.

Abstract 4923 Objective To explore the safety and clinic therapeutic effects of using tumor activated dendritic cells (DC) accompanied cytokine induced killer (CIK) in order to feinforce opposing anti-tumor function and to eliminate minimal residual disease. Methods 59 patients entered into DCIK plus chemotherapy consolidation teams as achieved the 1st complete remission and after 1 or 2 cycle themotherapy. Collect 56 case's bone marrow monocyte cells at diagnosising to prepare tumor antigen, Collect bone marrow monocyte cells at complete remission to culture DC and CIK, using tumor antigen to activate DCs. After 7 days culturing separately then co-culture together for 1 day, collect these cells as DCs activated CIK, after scrubbing, infusing back to patients. Then these patients accept 10–15 day's immuno-regulation treatment, such as interleukine-2(IL-2) 500000u/d for one week or interferon -α(IFN-α)3,000,000u/d three times one week or thymopentin 1mg/d,2 times every week for two weeks. Those cases (one case AML-M3a,one case ALL-B,one case AML-M4a)only have routine culturing and infusion of DCs and CIK and the DCs are not activated by tumor antigen because of having no conservation leukemic cells. Results 59 cases accept DCIK immuno-therapy for 6 to 16 times respectively, in the 24 to 78 months observation stage, 42 cases OS(overall survival)and 33 cases DFS(disease free survival). No one developed therapeutic-dependent auto-allergic disease, such as fever, swollen, diarrhea, rash et al. Those three cases who accepted routine culturing and infusion of CIK and DCs that had not activated by tumor antigen didn't occure any treatment-dependency side-effects and keep hematologic complete remission, minimal residual disease test outcome persist lower under 103. Conclusion Tumor antigen activated DCs combined with CIK can reinforce anti-tumor immune function and have no obviously side-effects,accompanying with chemotherapy,those patients maybe benefit of this kind consolidation treatment and avoid routine chemo-therapeutic side reaction, temporary curative effect are satisfaction and worth popularized. Disclosures: No relevant conflicts of interest to declare.

Abstract 3226 Objective To evaluate mesenchymal stem cells(MSCs) combination with routine therapy to treat refractory aplastic anemia and bone marrow fibrosis, observe the course of the hematopoitic reconstitution and evaluate the security and curative effect. Methods 12 primary myelofibrosis and 19 chronic aplastic anemia patients who had all accepted routine therapy more than 6 months,because of pathogenetic condition, they needed infusing constituent blood such as at least condensed red blood cells 6 units and platelet 4 units per months, and despite this, whole blood cells counts were very lower. Those patients all had intermittent dizzy, fatigue, bad appetite and purpura. Because of age, complication, and psychological reasons, they refused Allogenic stem cell transplantation. So besides routine therapy, they received umbilical MSCs 1×107, every two weeks one time, those cells were injected directly into medullary cavity of ilium. Results 28 recipients showed hematopoietic reconstitution when PLT was up to 20×109/L and granulocytes was up to 2.0×109/L on day 28 post MSCs infusion. Bone marrow biopsy showed hyperplasia from hypoplasia to evident hyperplasia. On day 28, Th cells ascended from 23.0 to 36.2, Ts cells descended from 24.2 to 14.6, Th.. MTs from 0.95 to 2.47:1, IgG from 6.6 to 13.3, NK cells from 2.40 to 13.5, indicating earlier immune recorrect. At the last follow-up of 12 months, 17 patients showed normal hemoglobin and platelet counts, immune globulin was in the normal level, indicating the hematopoietisis and immune persistent reconstitution. 5 patients still needed infusing constituent blood cells, although, the interval of platelet and red blood cells infusion obviously prolonged, and the times to see doctor decreased and life quality elevated greatly. Conclusion This is a novel clinical research of successful treatment of refractory CAA and MF, it tell us infusion HLA-mismatched allogenic MSCs directly into medullary cavity of ilium is efficiency and security. Disclosures: No relevant conflicts of interest to declare.