Transcriptome and Proteome Analysis Revealed the Influence of High-Molecular-Weight Glutenin Subunits (HMW-GSs) Deficiency on Expression of Storage Substances and the Potential Regulatory Mechanism of HMW-GSs.

The processing quality of wheat is affected by seed storage substances, such as protein and starch. High-molecular-weight glutenin subunits (HMW-GSs) are the major components of wheat seed storage proteins (SSPs); they are also key determinators of wheat end-use quality. However, the effects of HMW-GSs absence on the expression of other storage substances and the regulation mechanism of HMW-GSs are still limited. Previously, a wheat transgenic line LH-11 with complete deletions of HMW-GSs was obtained through introducing an exogenous gene Glu-1Ebx to the wild-type cultivar Bobwhite by transgenic approach. In this study, comparative seed transcriptomics and proteomics of transgenic and non-transgenic lines at different seed developmental stages were carried out to explore the changes in genes and proteins and the underlying regulatory mechanism. Results revealed that a number of genes, including genes related to SSPs, carbohydrates metabolism, amino acids metabolism, transcription, translation, and protein process were differentially enriched. Seed storage proteins displayed differential expression patterns between the transgenic and non-transgenic line, a major rise in the expression levels of gliadins were observed at 21 and 28 days post anthesis (DPA) in the transgenic line. Changes in expressions of low-molecular-weight glutenins (LMW-GSs), avenin-like proteins (ALPs), lipid transfer proteins (LTPs), and protease inhibitors (PIs) were also observed. In addition, genes related to carbohydrate metabolism were differentially expressed, which probably leads to a difference in starch component and deposition. A list of gene categories participating in the accumulation of SSPs was proposed according to the transcriptome and proteome data. Six genes from the MYB and eight genes from the NAC transcription families are likely important regulators of HMW-GSs accumulation. This study will provide data support for understanding the regulatory network of wheat storage substances. The screened candidate genes can lay a foundation for further research on the regulation mechanism of HMW-GSs.

Genome-wide identification and expression analysis of the TaRRA gene family in wheat (Triticum aestivum L.).

Cytokinin is an important endogenous hormone in plants performing a wide spectrum of biological roles. The type-A response regulators (RRAs) are primary cytokinin response genes, which are important components of the cytokinin signaling pathway and are involved in the regulation of plant growth and development. By analysis of the whole genome sequence of wheat, we identified 20 genes encoding RRAs which were clustered into eight homologous groups. The gene structure, conserved motifs, chromosomal location, and cis-acting regulatory elements of the TaRRAs were analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the expression levels of most of the TaRRAs increased rapidly on exogenous cytokinin application. Moreover, the TaRRA family members displayed different expression profiles under the stress treatments of drought, salt, cold, and heat. This study provides valuable insights into the RRA gene family in wheat and promotes the potential application of these genes in wheat genetic improvement.

Genome-wide association study of yield-related traits in common wheat (Triticum aestivum L.) under normal and drought treatment conditions.

The primary goal of modern wheat breeding is to develop new high-yielding and widely adaptable varieties. We analyzed four yield-related agronomic traits in 502 wheat accessions under normal conditions (NC) and drought treatment (DT) conditions over three years. The genome-wide association analysis identified 51 yield-related and nine drought-resistance-related QTL, including 13 for the thousand-grain weight (TGW), 30 for grain length (GL), three for grain width (GW), five for spike length (SL) and nine for stress tolerance index (STI) QTL in wheat. These QTL, containing 72 single nucleotide polymorphisms (SNPs), explained 2.23 - 7.35% of the phenotypic variation across multiple environments. Eight stable SNPs on chromosomes 2A, 2D, 3B, 4A, 5B, 5D, and 7D were associated with phenotypic stability under NC and DT conditions. Two of these stable SNPs had association with TGW and STI. Several novel QTL for TGW, GL and SL were identified on different chromosomes. Three linked SNPs were transformed into kompetitive allele-specific PCR (KASP) markers. These results will facilitate the discovery of promising SNPs for yield-related traits and/or drought stress tolerance and will accelerate the development of new wheat varieties with desirable alleles.

Identification and molecular characterization of mutant line deficiency in three waxy proteins of common wheat (Triticum aestivum L.).

Starch is the main component of wheat (Triticum aestivum L.) grain and a key factor in determining wheat processing quality. The Wx gene is the gene responsible for amylose synthesis. An ethyl methanesulfonate (EMS) mutagenized population was generated using common wheat cv. Gao 8901, a popular and high-quality cultivar in China. A waxy mutant (Wx-null) was isolated by screening M3 seeds with KI-I2 staining of endosperm starch. No obvious waxy proteins in Wx-null line were detected using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). DNA sequencing revealed three SNPs and a 3-bp InDel in the first exon, and a 16-bp InDel at the junction region of the first Wx-A1 intron from the Wx-null line. Six SNPs were identified in Wx-B1 gene of Wx-null line compared to the wild-type Gao 8901, including four missense mutations. One nonsense mutation was found at position 857 in the fourth exon, which resulted in a premature stop codon. Expression levels of Wx genes were dramatically reduced in the Wx-null line. There were no detectable differences in granule size and morphology between Wx-null and wild-type, but the Wx-null line contained more B-type starch granules. The amylose content of the Wx-null line (0.22%) was remarkably lower compared to the wild-type Gao 8901 (24.71%). Total starch is also lower in the Wx-null line. The Wx-null line may provide a potential waxy material with high agronomic performance in wheat breeding programs.

Climatechange vs. Globalwarming: Characterizing Two Competing Climate Discourses on Twitter with Semantic Network and Temporal Analyses#.

Distinct perceptions of the global climate is one of the factors preventing society from achieving consensus or taking collaborative actions on this issue. The public has not even reached an agreement on the naming of the global concern, showing preference for either "climate change" or "global warming", and few previous studies have addressed these two competing discourses resulting from distinct climate concerns by differently linking numerous climate concepts. Based on the 6,662,478 tweets containing #climatechange or #globalwarming generated between 1 January 2009 and 31 December 2018, we constructed the semantic networks of the two discourses and examined their evolution over the decade. The findings indicate that climate change demonstrated a more scientific perspective and showed an attempt to condense climate discussions rather than diffuse the topic by frequently addressing sub-topics simultaneously. Global warming triggered more political responses and showed a greater connection with phenomena. Temporal analysis suggests that traditional political discussions were gradually fading in both discourses but more recently started to revive in the form of discourse alliance in the climate change discourse. The associations between global warming and weather abnormalitiessuddenly strengthened around 2012. Climate change is becoming more dominant than global warming in public discussions. Although two discourses have shown more similarities in the rank order of important climate concepts, apparent disagreements continue about how these concepts are associated. These findings lay the groundwork for researchers and communicators to narrow the discrepancy between diverse climate perceptions.

The role of surface modification for TiO2 nanoparticles in cancer cells.

Titanium dioxide nanoparticles (TiO2 NPs) have a potential in the field of biological application. However, its poor dispersibility in water hampered its applications. In this study, 3-phosphonopropionic acid and 3-aminopropyl-triethoxysilane were respectively used for surface modification on TiO2 NPs with negative and positive surface charges (denoted as TiO2-COOH and TiO2-NH2). Zeta potentials of the prepared samples with high absolute value demonstrate the great improvement in their dispersibility. In terms of viability experiment, both TiO2-COOH and TiO2-NH2 showed low cytotoxicity. The cellular uptake efficiency and the uptake pathways of TiO2-COOH and TiO2-NH2 for cancer cells were studied. The exocytosis of TiO2-NH2 was also observed in the experiment.

Targeting and Photodynamic Killing of Cancer Cell by Nitrogen-Doped Titanium Dioxide Coupled with Folic Acid.

Titanium dioxide (TiO2) has attracted wide attention as a potential photosensitizer (PS) in photodynamic therapy (PDT). However, bare TiO2 can only be excited by ultraviolet illumination, and it lacks specific targeting ligands, which largely impede its application. In our study, we produced nitrogen-doped TiO2 and linked it with an effective cancer cell targeting agent, folic acid (FA), to obtain N-TiO2-FA nanoconjugates. Characterization of N-TiO2-FA included Zeta potential, absorption spectra and thermogravimetric analysis. The results showed that N-TiO2-FA was successfully produced and it possessed better dispersibility in aqueous solution than unmodified TiO2. The N-TiO2-FA was incubated with human nasopharyngeal carcinoma (KB) and human pulmonary adenocarcinoma (A549) cells. The KB cells that overexpress folate receptors (FR) on cell membranes were used as FR-positive cancer cells, while A549 cells were used as FR-negative cells. Laser scanning confocal microscopy results showed that KB cells had a higher uptake efficiency of N-TiO2-FA, which was about twice that of A549 cells. Finally, N-TiO2-FA is of no cytotoxicity, and has a better photokilling effect on KB cells under visible light irradiation. In conclusion, N-TiO2-FA can be as high-value as a PS in cancer targeting PDT.

Enhancement of the photokilling effect of aluminum phthalocyanine in photodynamic therapy by conjugating with nitrogen-doped TiO2 nanoparticles.

As a second-generation photodynamic therapy (PDT) photosensitizer, aluminum phthalocyanine chloride tetrasulfonate (Pc) has gained great attention due to its high absorption at the red light region. Yet, its application in PDT is strongly limited by its low cellular uptake efficiency. In this report, nitrogen-doped TiO2 nanoparticles (N-TiO2) conjugated with Pc are synthesized by a two-step surface modification method. The N-TiO2-Pc products are characterized by Zeta potential, transmission electron microscopy and UV-vis absorption spectroscopy. The cellular uptake, intracellular distribution, cytotoxicity and the photokilling effect of the nanoparticles are studied on different cancer cell lines. Compared with Pc, the absorption spectrum of N-TiO2-Pc expands from red to UV region, resulting in a higher production of reactive oxygen species under visible light irradiation. In addition, the cellular uptake of Pc is largely improved by its carrier N-TiO2. The photokilling efficiency of N-TiO2-Pc is over ten times higher than that of Pc. The results suggest that N-TiO2-Pc is an excellent candidate as a photosensitizer in PDT.

Study of polycation-capped Mn:ZnSe quantum dots as a novel fluorescent probe for living cells.

Transition metal manganese ion (Mn(2+)) doped zinc selenide quantum dots (Mn:ZnSe D-Dots) have been considered as a new material for fluorescent probes in biological labeling. However, this application is limited by the low membrane permeability of D-Dots. In this work, Mn:ZnSe D-Dots were capped with the polycation Sofast to label living cells. For the first time, the efficiency of cellular uptake in living cells is significantly enhanced. Various molar ratios of Sofast to D-Dots were explored and compared to obtain the optimal reaction conditions between Sofast and D-Dots for preparing Sofast/D-Dots nano-compound. A comparison on the fluorescence labeling ability of living cells were made between Sofast/D-Dots and pure D-Dots. Results from laser scanning confocal microscope show that Sofast/D-Dots complexes enter the cells more efficiently than pure D-Dots, even with a lower concentration and shorter incubation time. The cytotoxicities of D-Dots and Sofast/D-Dots were also studied. It was found that Sofast/D-Dots have a much lower cytotoxicity than cadmium-containing quantum dots (i.e. CdTe and CdTe/ZnS). Our results suggest that the non-heavy-metal-containing Sofast/D-Dots complexes have a great potential in the application of biological labeling, especially of long-time bioimaging in living cells.

Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation.

The killing effect of nitrogen-doped titanium dioxide (N-TiO2) nanoparticles on human cervical carcinoma (HeLa) cells by visible light photodynamic therapy (PDT) was higher than that of TiO2 nanoparticles. To study the mechanism of the killing effect, the reactive oxygen species produced by the visible-light-activated N-TiO2 and pure-TiO2 were evaluated and compared. The changes of the cellular parameters, such as the mitochondrial membrane potential (MMP), intracellular Ca2+, and nitrogen monoxide (NO) concentrations after PDT were measured and compared for N-TiO2- and TiO2-treated HeLa cells. The N-TiO2 resulted in more loss of MMP and higher increase of Ca2+ and NO in HeLa cells than pure TiO2. The cell morphology changes with time were also examined by a confocal microscope. The cells incubated with N-TiO2 exhibited serious distortion and membrane breakage at 60 min after the PDT.

Study on the visible-light-induced photokilling effect of nitrogen-doped TiO2 nanoparticles on cancer cells.

Nitrogen-doped TiO2 (N-TiO2) nanoparticles were prepared by calcining the anatase TiO2 nanoparticles under ammonia atmosphere. The N-TiO2 showed higher absorbance in the visible region than the pure TiO2. The cytotoxicity and visible-light-induced phototoxicity of the pure- and N-TiO2 were examined for three types of cancer cell lines. No significant cytotoxicity was detected. However, the visible-light-induced photokilling effects on cells were observed. The survival fraction of the cells decreased with the increased incubation concentration of the nanoparticles. The cancer cells incubated with N-TiO2 were killed more effectively than that with the pure TiO2. The reactive oxygen species was found to play an important role on the photokilling effect for cells. Furthermore, the intracellular distributions of N-TiO2 nanoparticles were examined by laser scanning confocal microscopy. The co-localization of N-TiO2 nanoparticles with nuclei or Golgi complexes was observed. The aberrant nuclear morphologies such as micronuclei were detected after the N-TiO2-treated cells were irradiated by the visible light.

Enhancement of intracellular delivery of anti-cancer drugs by the Tat peptide.

The arginine-rich cationic Tat peptides have been reported to enhance the intracellular delivery of macromolecules, including DNA, RNA, and proteins. In this work an arginine cationic peptide derived from the HIV-1 Tat protein was conjugated with noncovalent bonds to sulfonated aluminum phthalocyanine (AlPcS, a photosensitizer for the light-activated photodynamic cancer therapy), doxorubicin (DOX, a chemotherapeutic agent), or quantum dots (QDs, often used as carriers for the delivery of anticancer drugs). The fluorescence of intracellular conjugates of AlPcS-Tat, DOX-Tat, and QDs-Tat was studied by means of confocal laser scanning microscopy in the human nasopharyngeal carcinoma KB cells and cervical carcinoma Hela cells in vitro. The Tat peptide with noncovalent links can enhance at least a twofold of intracellular delivery of AlPcS, DOX, and QDs via an endocytotic pathway in the two tumor cell lines. This finding may suggest that the Tat peptide-mediated intracellular delivery of anticancer drugs may have the potential for improving efficacy of cancer therapy.

Liposome encapsulation of thiol-capped CdTe quantum dots for enhancing the intracellular delivery.

Although water soluble thiol-capped quantum dots (QDs) have been widely used as photoluminescence (PL) probes in various applications, the negative charges on thiol terminals limit the cell uptake hindering their applications in cell imaging. The commercial liposome complex (Sofast®) was used to encapsulate these QDs forming the liposome vesicles with the loading efficiency as high as about 95%. The cell uptakes of unencapsulated QDs and QD loaded liposome vesicles were comparatively studied by a laser scanning confocal microscope. We found that QD loaded liposome vesicles can effectively enhance the intracellular delivery of QDs in three cell lines (human osteosarcoma cell line (U2OS); human cervical carcinoma cell line (Hela); human embryonic kidney cell line (293 T)). The photobleaching of encapsulated QDs in cells was also reduced comparing with that of unencapsulated QDs, measured by the PL decay of cellular QDs with a continuous laser irradiation in the microscope. The flow cytometric measurements further showed that the enhancing ratios of encapsulated QDs on cell uptake are about 4-8 times in 293 T and Hela cells. These results suggest that the cationic liposome encapsulation is an effective modality to enhance the intracellular delivery of thiol-capped QDs.

Conjugates of folic acids with BSA-coated quantum dots for cancer cell targeting and imaging by single-photon and two-photon excitation.

Bovine serum albumin (BSA)-coated CdTe/ZnS quantum dots (BSA-QDs) were selected to conjugate with folic acid (FA), forming FA-BSA-QDs. This study aims to develop these small FA-BSA-QDs (less than 10 nm) for the diagnosis of cancers in which the FA receptor (FR) is overexpressed. The enhancement of cellular uptake in FR-positive human nasopharyngeal carcinoma cells (KB cells) for FA-BSA-QDs was found by means of confocal fluorescence microscopy under single-photon and two-photon excitation. The uptake enhancement for FA-BSA-QDs was further evaluated by flow-cytometric analysis in 10(4) KB cells, and was about 3 times higher than for BSA-QDs on average. The uptake enhancement was suppressed when KB cells had been pretreated with excess FA, reflecting that the enhancement was mediated by the association of FR at cell membranes with FA-BSA-QDs. When human embryonic kidney cells (293T) (FR-negative cells) and KB cells, respectively, were incubated with FA-BSA-QDs (1 µM) for 40 min, the FA-BSA-QD uptake by 293T cells was much weaker than that by KB cells, demonstrating that FA-BSA-QDs could undergo preferential binding on FR-positive cancer cells. These characteristics suggest that FA-BSA-QDs are potential candidates for cancer diagnosis.

Plasmonic gold nanorods can carry sulfonated aluminum phthalocyanine to improve photodynamic detection and therapy of cancers.

Hexadecyltrimethylammonium bromide-coated gold nanorods (AuNRs) with positive charges were effectively bound to negatively charged sulfonated aluminum phthalocyanine (AlPcS), a photosensitizer for photodynamic detection and therapy, due to the electrostatic force, with a loading content of 10(4) AlPcS molecules per rod. A 5-fold increase in the AlPcS fluorescence of the AlPcS-AuNRs complex was seen. The excitation fluorescence spectrum of the AlPcS-AuNRs with a typical 520 nm band fits well with the resonance band of AuNR surface plasmons, suggesting that such increased AlPcS fluorescence is produced from the strong surface plasmons of AuNRs. The intracellular distribution of AlPcS-AuNRs was studied in the QGY liver cancer cells by respectively imaging the AlPcS fluorescence and AuNRs reflectance with a confocal microscope. Furthermore, the AlPcS-AuNRs-loaded cells were photodynamically damaged after being exposed to red light in a light-dose-dependent manner. In contrast, no phototoxicity of the cells was seen after incubation with the same amount of free AlPcS, indicating that the AlPcS-AuNRs can enhance the AlPcS-mediated photodynamic effect. In addition, the loaded AlPcS can be photothermally released from AuNRs in the cells by the irradiation with an 800 nm femtosecond laser, demonstrating the potential for controlled drug release.

Study on the intracellular fate of Tat peptide-conjugated quantum dots by spectroscopic investigation.

The photoluminescence (PL) spectrum of water-soluble thiol-capped CdTe quantum dots (QDs) conjugated with Tat peptide in solution showed a remarkable redshift as compared to that of unconjugated QDs. After cellular uptake of the Tat-QDs conjugates, the micro-PL spectrum of Tat-QDs in lysosomes showed a spectral blueshift, which was most probably due to the fact that Tat peptide was digested by the enzymes, leaving the Tat-detached QDs in lysosomes. The reasons for the spectral changes have been discussed in detail.

High-photoluminescence-yield gold nanocubes: for cell imaging and photothermal therapy.

Gold nanocubes demonstrate unique optical properties of the high photoluminescence (PL) quantum yield and a remarkably enhanced extinction band at 544 nm. The 4 x 10(-2) PL yield, which is about 200 times higher than that of gold nanorods, allows gold nanocubes to be successfully used in cell imaging of human liver cancer cells (QGY) and human embryo kidney cells (293T) with a common method of single-photon excitation. The high extinction coefficients of gold nanocubes also facilitate them carrying out the photothermal therapy of QGY and 293T cells, showing similar photokilling efficiency as compared to gold nanorods.

Poly (N-isopropylacrylamide)-coated multifunctional nanoparticles for cell tracking.

OBJECTIVE: The objective of this work was to explore a new modality of cell tracking that uses multifunctional nanoparticles. The tracking of transfused cells in vivo is an important step to study the therapeutic course and mechanism of cell therapeutics. BACKGROUND DATA: Several methods of cell tracking have been developed. Our novel method uses multifunctional nanoparticles to track cells via both fluorescence and magnetic resonance. MATERIALS AND METHODS: Poly (N-isopropylacrylamide) (PNIPAM)-coated multifunctional nanoparticles containing both CdTe quantum dots (QDs) and Fe(3)O(4) magnetic particles were used to label Chinese hamster ovary (CHO) cells. The labeled cells were measured by confocal fluorescence microscope in vitro at the photoluminescent band (600 nm) of QDs. When these labeled cells were injected into the mouse, the in vivo images were detected by magnetic resonance imaging (MRI). RESULTS: The nanoparticles easily bound to the plasma membrane of CHO cells after incubation at 37 degrees C. The surface PNIPAM of nanoparticles is a well-known thermoresponsive polymer with a volume phase transition temperature. It is hydrophilic at temperatures below critical solution temperatures (32-34 degrees C) and becomes hydrophobic at higher temperatures. The cellular binding of nanoparticles was stable and nontoxic, and the photoluminescence of nanoparticles could still be seen after 48 h in labeled cells. In addition, the labeled cells can be manipulated by an external magnet. The magnetic resonance images showed that these labeled cells also can be measured in vivo in mice. CONCLUSIONS: PNIPAM-coated multifunctional nanoparticles showed potential for labeling cells and for tracking cells both in vitro and in vivo with the use of fluorescence and magnetic resonance. This new modality of cell tracking has the merits of simplicity and reliability.

Thiol-capped CdTe quantum dots with two-photon excitation for imaging high autofluorescence background living cells.

To effectively image living cells with quantum dots (QDs), particularly for those cells containing high content of native fluorophores, the two-photon excitation (TPE) with a femto-second 800 nm laser was employed and compared with the single-photon excitations (SPE) of 405 nm and 488 nm in BY-2 Tobacco (BY-2-T) and human hepatocellular carcinoma (QGY) cells, respectively. The 405 nm SPE produced the bright photoluminescence (PL) signals of cellular QDs but also induced a strong autofluorescence(AF) from the native fluorophores like flavins in cells. The AF occupied about 30% and 13% of the total signals detected in QD imaging channel in the BY-2-T and QGY cells, respectively. With the excitation of 488 nm SPE, the PL signals were lower than those excited with the 405 nm SPE, although the AF signals were also reduced. The 800 nm TPE generated the best PL images of intracellular QDs with the highest signal ratio of PL to AF, because the two-photon absorption cross section of QDs is much higher than that of the native fluorophores. By means of the TPE, the reliable cellular imaging with QDs, even for the cells having the high AF background, can be achieved.

Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells.

UNLABELLED: Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs) in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11671-009-9307-9) contains supplementary material, which is available to authorized users.