Maize Golden2-like transcription factors boost rice chloroplast development, photosynthesis, and grain yield.

Chloroplasts are the sites for photosynthesis, and two Golden2-like factors act as transcriptional activators of chloroplast development in rice (Oryza sativa L.) and maize (Zea mays L.). Rice OsGLK1 and OsGLK2 are orthologous to maize ZmGLK1 (ZmG1) and ZmGLK2 (ZmG2), respectively. However, while rice OsGLK1 and OsGLK2 act redundantly to regulate chloroplast development in mesophyll cells, maize ZmG1 and ZmG2 are functionally specialized and expressed in different cell-specific manners. To boost rice chloroplast development and photosynthesis, we generated transgenic rice plants overexpressing ZmG1 and ZmG2, individually or simultaneously, with constitutive promoters (pZmUbi::ZmG1 and p35S::ZmG2) or maize promoters (pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2). Both ZmG1 and ZmG2 genes were highly expressed in transgenic rice leaves. Moreover, ZmG1 and ZmG2 showed coordinated expression in pZmG1::ZmG1/pZmG2::ZmG2 plants. All Golden2-like (GLK) transgenic plants had higher chlorophyll and protein contents, Rubisco activities and photosynthetic rates per unit leaf area in flag leaves. However, the highest grain yields occurred when maize promoters were used; pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2 transgenic plants showed increases in grain yield by 51%, 47%, and 70%, respectively. In contrast, the pZmUbi::ZmG1 plant produced smaller seeds without yield increases. Transcriptome analysis indicated that maize GLKs act as master regulators promoting the expression of both photosynthesis-related and stress-responsive regulatory genes in both rice shoot and root. Thus, by promoting these important functions under the control of their own promoters, maize GLK1 and GLK2 genes together dramatically improved rice photosynthetic performance and productivity. A similar approach can potentially improve the productivity of many other crops.

YULINK regulates vascular formation in zebrafish and HUVECs.

BACKGROUND: The distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation. METHODS: In this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs). RESULTS: Knockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK. CONCLUSION: This study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs.

Maize ANT1 modulates vascular development, chloroplast development, photosynthesis, and plant growth.

Arabidopsis AINTEGUMENTA (ANT), an AP2 transcription factor, is known to control plant growth and floral organogenesis. In this study, our transcriptome analysis and in situ hybridization assays of maize embryonic leaves suggested that maize ANT1 (ZmANT1) regulates vascular development. To better understand ANT1 functions, we determined the binding motif of ZmANT1 and then showed that ZmANT1 binds the promoters of millet SCR1, GNC, and AN3, which are key regulators of Kranz anatomy, chloroplast development, and plant growth, respectively. We generated a mutant with a single-codon deletion and two frameshift mutants of the ANT1 ortholog in the C4 millet Setaria viridis by the CRISPR/Cas9 technique. The two frameshift mutants displayed reduced photosynthesis efficiency and growth rate, smaller leaves, and lower grain yields than wild-type (WT) plants. Moreover, their leaves sporadically exhibited distorted Kranz anatomy and vein spacing. Conducting transcriptomic analysis of developing leaves in the WT and the three mutants we identified differentially expressed genes (DEGs) in the two frameshift mutant lines and found many down-regulated DEGs enriched in photosynthesis, heme, tetrapyrrole binding, and antioxidant activity. In addition, we predicted many target genes of ZmANT1 and chose 13 of them to confirm binding of ZmANT1 to their promoters. Based on the above observations, we proposed a model for ANT1 regulation of cell proliferation and leaf growth, vascular and vein development, chloroplast development, and photosynthesis through its target genes. Our study revealed biological roles of ANT1 in several developmental processes beyond its known roles in plant growth and floral organogenesis.

Reconfigurable scan lens based on an actively controlled optical phased array.

Integrated photonics provides a path for miniaturization of an optical system to a compact chip scale and offers reconfigurability by the integration of active components. Here we report a chip-scale reconfigurable scan lens based on an optical phased array, consisting of 30 actively controlled elements on the InP integrated photonic platform. By configuring the phase shifters, we show scanning of a nearly diffraction-limited focused spot with a full width at half maximum spot size down to 2.7 µm at the wavelength of 1550 nm. We demonstrate the key functions needed for a laser-scanning microscope, including light focusing, collection, and steering. We also perform confocal measurements to detect reflection at selective depths.

Comparative transcriptomics method to infer gene coexpression networks and its applications to maize and rice leaf transcriptomes.

Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light-dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1, a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C4 enzyme genes and RUBISCO SMALL SUBUNIT2 Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C4 photosynthesis.

Large-scale data analysis for robotic yeast one-hybrid platforms and multi-disciplinary studies using GateMultiplex.

BACKGROUND: Yeast one-hybrid (Y1H) is a common technique for identifying DNA-protein interactions, and robotic platforms have been developed for high-throughput analyses to unravel the gene regulatory networks in many organisms. Use of these high-throughput techniques has led to the generation of increasingly large datasets, and several software packages have been developed to analyze such data. We previously established the currently most efficient Y1H system, meiosis-directed Y1H; however, the available software tools were not designed for processing the additional parameters suggested by meiosis-directed Y1H to avoid false positives and required programming skills for operation. RESULTS: We developed a new tool named GateMultiplex with high computing performance using C++. GateMultiplex incorporated a graphical user interface (GUI), which allows the operation without any programming skills. Flexible parameter options were designed for multiple experimental purposes to enable the application of GateMultiplex even beyond Y1H platforms. We further demonstrated the data analysis from other three fields using GateMultiplex, the identification of lead compounds in preclinical cancer drug discovery, the crop line selection in precision agriculture, and the ocean pollution detection from deep-sea fishery. CONCLUSIONS: The user-friendly GUI, fast C++ computing speed, flexible parameter setting, and applicability of GateMultiplex facilitate the feasibility of large-scale data analysis in life science fields.

Magnetically Modulated Fluorescence of Nitrogen-Vacancy Centers in Nanodiamonds for Ultrasensitive Biomedical Analysis.

The negatively charged nitrogen-vacancy center in fluorescent nanodiamonds (FNDs) is a point defect with unique magneto-optical properties. It emits far-red fluorescence at ∼700 nm, and its intensity can be magnetically modulated with a depth of more than 10% at a field strength of 30 mT. We have closely examined this property and illustrated its practical use in biomedicine by applying a periodic, time-varying magnetic field to FNDs deposited on a surface or dispersed in a solution with a lock-in detection method. We achieved selective and sensitive detection of 100 nm FNDs on a nitrocellulose membrane at a particle density of 0.04 ng/mm2 (or ∼2 × 104 particles/mm2) and in an aqueous solution with a particle concentration of 1 ng/mL (or ∼1 fM) in 10 s as the detection limits. The utility and versatility of the technique were demonstrated with an application to background-free detection of FNDs as reporters for FND-based lateral flow immunoassays as well as selective quantification of FNDs in tissue digests for in vivo studies.

Bioactive Compounds, Antioxidants, and Health Benefits of Sweet Potato Leaves.

Sweet potato (Ipomoea batatas) is one of the most important food crops worldwide and its leaves provide a dietary source of nutrients and various bioactive compounds. These constituents of sweet potato leaves (SPL) vary among varieties and play important roles in treating and preventing various diseases. Recently, more attentions in health-promoting benefits have led to several in vitro and in vivo investigations, as well as the identification and quantification of bioactive compounds in SPL. Among them, many new compounds have been reported as the first identified compounds from SPL with their dominant bioactivities. This review summarizes the current knowledge of the bioactive compositions of SPL and their health benefits. Since SPL serve as a potential source of micronutrients and functional compounds, they can be further developed as a sustainable crop for food and medicinal industries.

Critical Assessment of Metagenome Interpretation-a benchmark of metagenomics software.

Methods for assembly, taxonomic profiling and binning are key to interpreting metagenome data, but a lack of consensus about benchmarking complicates performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on highly complex and realistic data sets, generated from ∼700 newly sequenced microorganisms and ∼600 novel viruses and plasmids and representing common experimental setups. Assembly and genome binning programs performed well for species represented by individual genomes but were substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below family level. Parameter settings markedly affected performance, underscoring their importance for program reproducibility. The CAMI results highlight current challenges but also provide a roadmap for software selection to answer specific research questions.

Hyperexpression of type III secretion system of Salmonella Typhi linked to a higher cytotoxic effect to monocyte-derived macrophages by activating inflammasome.

Inflammasome activation is an important host response to infectious diseases, but the difference in inflammasome activation between typhoid fever and non-typhoidal Salmonella infection has been rarely studied. To determine whether inflammasome activation in macrophages after S. Typhi and S. Typhimurium infection is different, we measured pyroptosis, caspase-1 activation, and IL-1ß secretion in monocyte-derived macrophages infected with S. Typhi or S. Typhimurium both in vitro and ex vivo. The role of Vi capsule and virulence genes in Salmonella pathogenicity island-1 (SPI-1), belonging to type III secretion system, was also examined. S. Typhi caused more pyroptosis, caspase-1 activation, and IL-1ß production than S. Typhimurium did, predominantly within 2 h of infection, in the context of high number of infecting bacteria. Mutagenesis and complementation experiments confirmed that SPI-1 effectors but not Vi were associated with greater inflammasome activation. The expression levels of invA and hilA were significantly higher in S. Typhi than in S. Typhimurium at early log phase in SPI-1 environment. Thus, S. Typhi, relative to its non-typhoidal counterpart, S. Typhimurium, induces greater SPI-1-dependent inflammasome activation in monocyte-derived macrophages. This finding may explain why S. Typhi causes a hyperinflammatory state at bacteremic stage in typhoid fever.

Intracellular Delivery of Luciferase with Fluorescent Nanodiamonds for Dual-Modality Imaging of Human Stem Cells.

Delivering functional proteins (such as enzymes) into cells is important in various biological studies and is often accomplished indirectly by transfection with DNA or mRNA encoding recombinant proteins. However, the transfection efficiency of conventional plasmid methods is low for primary cells, which are crucial sources of cell therapy. Here, we present a new platform based on the use of fluorescent nanodiamond (FND) as a biocompatible nanocarrier to enable rapid, effective, and homogeneous labeling of human mesenchymal stem cells (MSCs) with luciferase for multiplex assays and ultrasensitive detection. More than 100 pg of FND and 100 million copies of firefly luciferase can be delivered into each MSC through endocytosis. Moreover, these endocytic luciferase molecules are catalytically active for hours, allowing the cells to be imaged and tracked in vitro as well as in vivo by both fluorescence and bioluminescence imaging. Our results demonstrate that luciferase-conjugated FNDs are useful as multifunctional labels of human stem cells for diverse theranostic applications.

Transcriptome dynamics of developing maize leaves and genomewide prediction of cis elements and their cognate transcription factors.

Maize is a major crop and a model plant for studying C4 photosynthesis and leaf development. However, a genomewide regulatory network of leaf development is not yet available. This knowledge is useful for developing C3 crops to perform C4 photosynthesis for enhanced yields. Here, using 22 transcriptomes of developing maize leaves from dry seeds to 192 h post imbibition, we studied gene up- and down-regulation and functional transition during leaf development and inferred sets of strongly coexpressed genes. More significantly, we developed a method to predict transcription factor binding sites (TFBSs) and their cognate transcription factors (TFs) using genomic sequence and transcriptomic data. The method requires not only evolutionary conservation of candidate TFBSs and sets of strongly coexpressed genes but also that the genes in a gene set share the same Gene Ontology term so that they are involved in the same biological function. In addition, we developed another method to predict maize TF-TFBS pairs using known TF-TFBS pairs in Arabidopsis or rice. From these efforts, we predicted 1,340 novel TFBSs and 253 new TF-TFBS pairs in the maize genome, far exceeding the 30 TF-TFBS pairs currently known in maize. In most cases studied by both methods, the two methods gave similar predictions. In vitro tests of 12 predicted TF-TFBS interactions showed that our methods perform well. Our study has significantly expanded our knowledge on the regulatory network involved in maize leaf development.

Regulatory Divergence among Beta-Keratin Genes during Bird Evolution.

Feathers, which are mainly composed of α- and ß-keratins, are highly diversified, largely owing to duplication and diversification of ß-keratin genes during bird evolution. However, little is known about the regulatory changes that contributed to the expressional diversification of ß-keratin genes. To address this issue, we studied transcriptomes from five different parts of chicken contour and flight feathers. From these transcriptomes we inferred ß-keratin enriched co-expression modules of genes and predicted transcription factors (TFs) of ß-keratin genes. In total, we predicted 262 TF-target gene relationships in which 56 TFs regulate 91 ß-keratin genes; we validated 14 of them by in vitro tests. A dual criterion of TF enrichment and "TF-target gene" expression correlation identified 26 TFs as the major regulators of ß-keratin genes. According to our predictions, the ancestral scale and claw ß-keratin genes have common and unique regulators, whereas most feather ß-keratin genes show chromosome-wise regulation, distinct from scale and claw ß-keratin genes. Thus, after expansion from the ß-keratin gene on Chr7 to other chromosomes, which still shares a TF with scale and claw ß-keratin genes, most feather ß-keratin genes have recruited distinct or chromosome-specific regulators. Moreover, our data showed correlated gene expression profiles, positive or negative, between predicted TFs and their target genes over the five studied feather regions. Therefore, regulatory divergences among feather ß-keratin genes have contributed to structural differences among different parts of feathers. Our study sheds light on how feather ß-keratin genes have diverged in regulation from scale and claw ß-keratin genes and among themselves.

Prolonged QT interval is linked to all-cause and cardiac mortality in chronic peritoneal dialysis patients.

AIM: Prolonged QT interval is related to changes of electrolytes in haemodialysis (HD) and is associated with all-cause mortality in HD patients. It is unknown if prolonged QT interval is associated with all-cause mortality in peritoneal dialysis (PD) patients as the electrolytes were relatively stable in PD. We therefore investigated the association of prolonged QT interval and all-cause mortality in chronic PD patients. METHODS: The QT intervals were measured in 2003 and all patients were followed to December 2012. A prolonged QT interval was defined as a QT interval > 450 ms. The association of prolonged QT interval with all-cause and cardiac-specific mortality was analyzed using Cox regression and Kaplan-Meier analysis. RESULTS: Of 306 patients, 196 (64%) patients had prolonged QT interval. The incidence density rate was 9.7 per 100 persons-years for all-cause mortality and 5.6 for cardiac specific mortality in patients with prolonged QT interval. Prolonged QT interval was associated with all-cause mortality with a hazard ratio (HR) of 1.59 (95% confidence interval (CI): 1.06-2.39, P = 0.03] and cardiac mortality (HR: 1.66, 95% CI: 1.00-2.78, P = 0.05) with adjustments for age, gender, diabetes, and vintage of dialysis. Longer QT interval (>500 ms, 450-500 ms, and < 450 ms) was significantly associated with a worse overall survival (P = 0.03, log-rank test) and cardiac mortality free survival (P = 0.05, log-rank test). CONCLUSIONS: Prolonged QT interval was associated with all-cause and cardiac mortality in patients on peritoneal dialysis. The association is independent of patient's age and diabetes.

Factors associated with serum fetuin-A concentrations after long-term use of different phosphate binders in hemodialysis patients.

BACKGROUND: Fetuin-A is known as a circulating inhibitor of vascular calcification. Factors associated with serum fetuin-A concentrations after long-term use of different phosphate binders in hemodialysis patients is still uncertain. METHODS: In the post-hoc study, we analyzed serum fetuin-A and biochemical factors (Ca, P, i-PTH, hsCRP, TG, LDL-C) in 50 hemodialysis patients, who completed a 48-week, open-Label, controlled randomized parallel-group study. 23 patients received sevelamer and 27 patients received calcium carbonate. RESULTS: After the 48-week treatment, the sevelamer group had less serum calcium increment, less iPTH decrement, more ALK-P increment, more hsCRP decrement and more LDL-C decrement. There was no significant difference in the serum fetuin-A decrement between two groups. Decreased serum fetuin-A levels were found after 48-week treatment in both groups: from 210.61 (104.73) to 153.85 (38.64) ug/dl, P = 0.003 in sevelamer group, from 203.95 (107.87) to 170.90 (58.02) ug/mL, P =0.002 in calcium group. The decrement in serum fetuin-A (Δfetuin-A) levels was associated with ΔCa (ρ = - 0.230, P = 0.040), ΔiPTH (ρ = 0.306, P = 0.031) and Δalbumin (ρ = 0.408, P = 0.003), not associated with sevelamer use, ΔP and ΔhsCRP. CONCLUSION: After long-term sevelamer or calcium carbonate treatment, both groups of maintenance HD patients had lower serum fetuin-A levels. Serum levels of increased calcium, decreased iPTH and decreased albumin were associated with the serum fetuin-A decrement.

Anatomical and transcriptional dynamics of maize embryonic leaves during seed germination.

Our anatomical analysis revealed that a dry maize seed contains four to five embryonic leaves at different developmental stages. Rudimentary kranz structure (KS) is apparent in the first leaf with a substantial density, but its density decreases toward younger leaves. Upon imbibition, leaf expansion occurs rapidly with new KSs initiated from the palisade-like ground meristem cells in the middle of the leaf. In parallel to the anatomical analysis, we obtained the time course transcriptomes for the embryonic leaves in dry and imbibed seeds every 6 h up to hour 72. Over this time course, the embryonic leaves exhibit transcripts of 30,255 genes at a level that can be regarded as "expressed." In dry seeds, ∼25,500 genes are expressed, showing functional enrichment in transcription, RNA processing, protein synthesis, primary metabolic pathways, and calcium transport. During the 72-h time course, ∼13,900 genes, including 590 transcription factor genes, are differentially expressed. Indeed, by 30 h postimbibition, ∼2,200 genes expressed in dry seeds are already down-regulated, and ∼2,000 are up-regulated. Moreover, the top 1% expressed genes at 54 h or later are very different from those before 30 h, reflecting important developmental and physiological transitions. Interestingly, clusters of genes involved in hormone metabolism, signaling, and responses are differentially expressed at various time points and TF gene expression is also modular and stage specific. Our dataset provides an opportunity for hypothesizing the timing of regulatory actions, particularly in the context of KS development.

Tracking and Finding Slow-Proliferating/Quiescent Cancer Stem Cells with Fluorescent Nanodiamonds.

Quiescent cancer stem cells (CSCs) have long been considered to be a source of tumor initiation. However, identification and isolation of these cells have been hampered by the fact that commonly used fluorescent markers are not sufficiently stable, both chemically and photophysically, to allow tracking over an extended period of time. Here, it is shown that fluorescent nanodiamonds (FNDs) are well suited for this application. Genotoxicity tests of FNDs with comet and micronucleus assays for human fibroblasts and breast cancer cells indicate that the nanoparticles neither cause DNA damage nor impair cell growth. Using AS-B145-1R breast cancer cells as the model cell line for CSC, it is found that the FND labeling outperforms 5-ethynyl-2'-deoxyuridine (EdU) and carboxyfluorescein diacetate succinimidyl ester (CFSE) in regards to its long-term tracking capability (>20 d). Moreover, through a quantification of their stem cell activity by measuring mammosphere-forming efficiencies (MFEs) and self-renewal rates, the FND-positive cells are identified to have an MFE twice as high as that of the FND-negative cells isolated from the same dissociated mammospheres. Thus, the nanoparticle-based labeling technique provides an effective new tool for tracking and finding slow-proliferating/quiescent CSCs in cancer research.

Icodextrin decreases technique failure and improves patient survival in peritoneal dialysis patients.

AIM: It remains unclear whether long-term daily icodextrin use can decrease technique failure and improve survival in peritoneal dialysis (PD) patients. The aim of the present study was to investigate whether icodextrin use, once daily, can decrease technique failure and prolong patient survival in incident PD patients. METHODS: Incident PD patients who survived more than 90 days were recruited from the China Medical University Hospital, Taiwan, between 1 January 2007 and 31 December 2011. All patients were followed until transfer to haemodialysis (HD), renal transplantation, transfer to another centre, death, or 31 December 2011. RESULTS: A total of 306 incident PD patients (89 icodextrin users, 217 icodextrin non-users) were recruited during the study period. Icodextrin users were more likely to have hypertension, diabetes and high or high-average peritoneal transport compared with non-users. During the follow-up period, 43 patients were transferred to HD: seven (7.87%) of the icodextrin group, and 36 (16.59%) of the non-icodextrin group. Thirty-two patients died during the follow-up period: five (5.62%) of the icodextrin group, and 27 (12.44%) of the non-icodextrin group. Icodextrin use was significantly associated with a better prognosis, in terms of technique failure (adjusted HR = 0.32; 95% CI = 0.14-0.72). With regard to patient survival, icodextrin use (adjusted HR = 0.33; 95% CI = 0.12-0.87) was associated with a significantly lower risk of death. CONCLUSION: The use of icodextrin once daily may decrease technique failure and improve survival in incident PD patients.

Carbon monoxide regulates the expression of the wound-inducible gene ipomoelin through antioxidation and MAPK phosphorylation in sweet potato.

Carbon monoxide (CO), one of the haem oxygenase (HO) products, plays important roles in plant development and stress adaptation. However, the function of CO involved in wounding responses is seldom studied. A wound-inducible gene, ipomoelin (IPO), of sweet potato (Ipomoea batatas cv. Tainung 57) was used as a target to study the regulation of CO in wounding responses. After wounding for 1h, the endogenous CO content and IbHO expression level were significantly reduced in leaves. IPO expression upon wounding was prohibited by the HO activator hemin, whereas the HO inhibitor zinc protoporphyrin IX elevated IPO expression. The IPO expression induced by wounding, H2O2, or methyl jasmonate was inhibited by CO. CO also affected the activities of ascorbate peroxidase, catalase, and peroxidase, and largely decreased H2O2 content in leaves. CO inhibited the extracellular signal-regulated kinase (ERK) phosphorylation induced by wounding. IbMAPK, the ERK of sweet potato, was identified by immunoblotting, and the interaction with its upstream activator, IbMEK1, was further confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. Conclusively, wounding in leaves repressed IbHO expression and CO production, induced H2O2 generation and ERK phosphorylation, and then stimulated IPO expression.