A universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle.

A prevalent side-reaction of succinate dehydrogenase oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a strong inhibitor of succinate dehydrogenase. We purified from cow heart mitochondria an enzyme (OAT1) with OAA tautomerase (OAT) activity that converts enol-OAA to the physiological keto-OAA form, and determined that it belongs to the highly conserved and previously uncharacterized Fumarylacetoacetate_hydrolase_domain-containing protein family. From all three domains of life, heterologously expressed proteins were shown to have strong OAT activity, and ablating the OAT1 homolog caused significant growth defects. In Escherichia coli, expression of succinate dehydrogenase was necessary for OAT1-associated growth defects to occur, and ablating OAT1 caused a significant increase in acetate and other metabolites associated with anaerobic respiration. OAT1 increased the succinate dehydrogenase reaction rate by 35% in in vitro assays with physiological concentrations of both succinate and malate. Our results suggest that OAT1 is a universal metabolite repair enzyme that is required to maximize aerobic respiration efficiency by preventing succinate dehydrogenase inhibition.

A cell wall invertase controls nectar volume and sugar composition.

Nectar volume and sugar composition are key determinants of the strength of plant-pollinator mutualisms. The main nectar sugars are sucrose, glucose and fructose, which can vary widely in ratio and concentration across species. Brassica spp. produce a hexose-dominant nectar (high in the monosaccharides glucose and fructose) with very low levels of the disaccharide sucrose. Cell wall invertases (CWINVs) catalyze the irreversible hydrolysis of sucrose into glucose and fructose in the apoplast. We found that BrCWINV4A is highly expressed in the nectaries of Brassica rapa. Moreover, a brcwinv4a null mutant: (i) has greatly reduced CWINV activity in the nectaries; (ii) produces a sucrose-rich nectar; but (iii) with significantly less volume. These results definitively demonstrate that CWINV activity is not only essential for the production of a hexose-rich nectar, but also support a hypothetical model of nectar secretion in which its hydrolase activity is required for maintaining a high intracellular-to-extracellular sucrose ratio that facilitates the continuous export of sucrose into the nectary apoplast. The extracellular hydrolysis of each sucrose into two hexoses by BrCWINV4A also likely creates the osmotic potential required for nectar droplet formation. These results cumulatively indicate that modulation of CWINV activity can at least partially account for naturally occurring differences in nectar volume and sugar composition. Finally, honeybees prefer nectars with some sucrose, but wild-type B. rapa flowers were much more heavily visited than flowers of brcwinv4a, suggesting that the potentially attractive sucrose-rich nectar of brcwinv4a could not compensate for its low volume.

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis.

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

Recognizing Human Daily Activity Using Social Media Sensors and Deep Learning.

The human daily activity category represents individual lifestyle and pattern, such as sports and shopping, which reflect personal habits, lifestyle, and preferences and are of great value for human health and many other application fields. Currently, compared to questionnaires, social media as a sensor provides low-cost and easy-to-access data sources, providing new opportunities for obtaining human daily activity category data. However, there are still some challenges to accurately recognizing posts because existing studies ignore contextual information or word order in posts and remain unsatisfactory for capturing the activity semantics of words. To address this problem, we propose a general model for recognizing the human activity category based on deep learning. This model not only describes how to extract a sequence of higher-level word phrase representations in posts based on the deep learning sequence model but also how to integrate temporal information and external knowledge to capture the activity semantics in posts. Considering that no benchmark dataset is available in such studies, we built a dataset that was used for training and evaluating the model. The experimental results show that the proposed model significantly improves the accuracy of recognizing the human activity category compared with traditional classification methods.

SHB1 and CCA1 interaction desensitizes light responses and enhances thermomorphogenesis.

Light and temperature are two important environmental signals to plants. After dawn, photo-activated phytochromes translocate into the nucleus and interact with a family of negative basic helix-loop-helix PIF regulators. Subsequent phosphorylation and degradation of PIFs triggers a series of photomorphogenic responses. However, excess light can damage the photosynthetic apparatus and leads to photoinhibition. Plants acclimate to a balanced state of photomorphogenesis to avoid photodamage. Here, we show that upregulation of PIF4 expression by SHB1 and CCA1 under red light represents a desensitization step. After dawn, the highly expressed circadian clock protein CCA1 brings circadian signals to the regulatory region of the PIF4 signaling hub. Recruitment of SHB1 by CCA1 modulates red light-specific induction of PIF4 expression thus integrating circadian and light signals. As noon approaches and light intensity and ambient temperature tend to increase, the SHB1-CCA1 interaction sustains PIF4 expression to trigger thermomorphogenic responses to changing light and temperature conditions.

HRB2 and BBX21 interaction modulates Arabidopsis ABI5 locus and stomatal aperture.

Blue light triggers the opening of stomata in the morning to allow CO2 uptake and water loss through transpiration. During the day, plants may experience periodic drought and accumulate abscisic acid (ABA). ABA antagonizes blue light signalling through phosphatidic acid and reduces stomatal aperture. This study reveals a molecular mechanism by which two light signalling proteins interact to repress ABA signalling in the control of stomatal aperture. A hypersensitive to red and blue 2 (hrb2) mutant has a defective ATP-dependent chromatin-remodelling factor, PKL, in the chromodomain/helicase/DNA binding family. HRB2 enhances the light-induced expression of a B-box transcription factor gene, BBX21. BBX21 binds a T/G box in the ABI5 promoter and recruits HRB2 to modulate the chromatin structure at the ABI5 locus. Mutation in either HRB2 or BBX21 led to reduced water loss and ABA hypersensitivity. This hypersensitivity to ABA was well explained by the enhanced expression of the ABA signalling gene ABI5 in both mutants. Indeed, stomatal aperture was significantly reduced by ABI5 overexpression in the absence or presence of ABA under monochromatic light conditions. Overall, we present a regulatory loop in which two light signalling proteins repress ABA signalling to sustain gas exchange when plants experience periodic drought.

Global analysis of canola genes targeted by SHORT HYPOCOTYL UNDER BLUE 1 during endosperm and embryo development.

Seed development in dicots includes early endosperm proliferation followed by growth of the embryo to replace the endosperm. Endosperm proliferation in dicots not only provides nutrient supplies for subsequent embryo development but also enforces a space limitation, influencing final seed size. Overexpression of Arabidopsis SHORT HYPOCOTYL UNDER BLUE1::uidA (SHB1:uidA) in canola produces large seeds. We performed global analysis of the canola genes that were expressed and influenced by SHB1 during early endosperm proliferation at 8 days after pollination (DAP) and late embryo development at 13 DAP. Overexpression of SHB1 altered the expression of 973 genes at 8 DAP and 1035 genes at 13 DAP. We also surveyed the global SHB1 association sites, and merging of these sites with the RNA sequencing data identified a set of canola genes targeted by SHB1. The 8-DAP list includes positive and negative genes that influence endosperm proliferation and are homologous to Arabidopsis MINI3, IKU2, SHB1, AGL62, FIE and AP2. We revealed a major role for SHB1 in canola endosperm development based on the dynamics of SHB1-altered gene expression, the magnitude of SHB1 chromatin immunoprecipitation enrichment and the over-representation of eight regulatory genes for endosperm development. Our studies focus on an important agronomic trait in a major crop for global agriculture. The datasets on stage-specific and SHB1-induced gene expression and genes targeted by SHB1 also provide a useful resource in the field of endosperm development and seed size engineering. Our practices in an allotetraploid species will impact similar studies in other crop species.

SHORT HYPOCOTYL UNDER BLUE1 or HAIKU2 mixepression alters canola and Arabidopsis seed development.

Canola (Brassica napus) is a widely cultivated species and provides important resources of edible vegetable oil, biodiesel production and animal feed. Seed development in Arabidopsis and canola shares a similar path: an early proliferation of endosperm to form a large seed cavity, followed by a second phase in which the embryo grows to replace the endosperm. In Arabidopsis, the seed reaches almost its final volume before the enlargement of the embryo. SHORT HYPOCOTYL UNDER BLUE1 (SHB1) is a key regulatory gene of seed development with a broad expression beyond endosperm development. By contrast, its two target genes, MINISEED3 (MINI3) and HAIKU2 (IKU2), are narrowly expressed in early developing endosperm and early embryo. We overexpressed SHB1 in canola to explore the possibility of altering seed development. As an alternative strategy, we expressed the canola IKU2 ortholog in Arabidopsis endosperm under the control of a stronger MINI3 promoter. SHB1 targeted canola orthologs of Arabidopsis MINI3 and IKU2 and caused a significantly increased seed mass. Overaccumulation of IKU2 in the early stage of Arabidopsis seed development also significantly increased the final seed mass. Our studies provide a strong case for increasing the final seed mass by manipulating endosperm proliferation at a rather early developmental stage in crops.

The draft genome of the grass carp (Ctenopharyngodon idellus) provides insights into its evolution and vegetarian adaptation.

The grass carp is an important farmed fish, accounting for ∼16% of global freshwater aquaculture, and has a vegetarian diet. Here we report a 0.9-Gb draft genome of a gynogenetic female adult and a 1.07-Gb genome of a wild male adult. Genome annotation identified 27,263 protein-coding gene models in the female genome. A total of 114 scaffolds consisting of 573 Mb are anchored on 24 linkage groups. Divergence between grass carp and zebrafish is estimated to have occurred 49-54 million years ago. We identify a chromosome fusion in grass carp relative to zebrafish and report frequent crossovers between the grass carp X and Y chromosomes. We find that transcriptional activation of the mevalonate pathway and steroid biosynthesis in liver is associated with the grass carp's adaptation from a carnivorous to an herbivorous diet. We believe that the grass carp genome could serve as an initial platform for breeding better-quality fish using a genomic approach.

A WRKY transcription factor recruits the SYG1-like protein SHB1 to activate gene expression and seed cavity enlargement.

Seed development in Arabidopsis and in many dicots involves an early proliferation of the endosperm to form a large embryo sac or seed cavity close to the size of the mature seed, followed by a second phase during which the embryo grows and replaces the endosperm. Short hypocotyl under BLUE1 (SHB1) is a member of the SYG1 protein family in fungi, Caenorhabditis elegans, flies, and mammals. SHB1 gain-of-function enhances endosperm proliferation, increases seed size, and up-regulates the expression of the WRKY transcription factor gene MINISEED3 (MINI3) and the LRR receptor kinase gene HAIKU2 (IKU2). Mutations in either IKU2 or MINI3 retard endosperm proliferation and reduce seed size. However, the molecular mechanisms underlying the establishment of the seed cavity and hence the seed size remain largely unknown. Here, we show that the expression of MINI3 and IKU2 is repressed before fertilization and after 4 days after pollination (DAP), but is activated by SHB1 from 2 to 4 DAP prior to the formation of the seed cavity. SHB1 associates with their promoters but without a recognizable DNA binding motif, and this association is abolished in mini3 mutant. MINI3 binds to W-boxes in, and recruits SHB1 to, its own and IKU2 promoters. Interestingly, SHB1, but not MINI3, activates transcription of pMINI3::GUS or pIKU2::GUS. We reveal a critical developmental switch through the activation of MINI3 expression by SHB1. The recruitment of SHB1 by MINI3 to its own and IKU2 promoters represents a novel two-step amplification to counter the low expression level of IKU2, which is a trigger for endosperm proliferation and seed cavity enlargement.

Hypersensitive to red and blue 1 and its modification by protein phosphatase 7 are implicated in the control of Arabidopsis stomatal aperture.

The stomatal pores are located on the plant leaf epidermis and regulate CO(2) uptake for photosynthesis and the loss of water by transpiration. Their stomatal aperture therefore affects photosynthesis, water use efficiency, and agricultural crop yields. Blue light, one of the environmental signals that regulates the plant stomatal aperture, is perceived by the blue/UV-A light-absorbing cryptochromes and phototropins. The signal transduction cascades that link the perception of light to the stomatal opening response are still largely unknown. Here, we report two new players, Hypersensitive to Red and Blue 1 (HRB1) and Protein Phosphatase 7 (PP7), and their genetic and biochemical interactions in the control of stomatal aperture. Mutations in either HRB1 or PP7 lead to the misregulation of the stomatal aperture and reduce water loss under blue light. Both HRB1 and PP7 are expressed in the guard cells in response to a light-to-dark or dark-to-light transition. HRB1 interacts with PP7 through its N-terminal ZZ-type zinc finger motif and requires a functional PP7 for its stomatal opening response. HRB1 is phosphorylated in vivo, and PP7 can dephosphorylate HRB1. HRB1 is mostly dephosphorylated in a protein complex of 193 kDa in the dark, and blue light increases complex size to 285 kDa. In the pp7 mutant, this size shift is impaired, and HRB1 is predominately phosphorylated. We propose that a modification of HRB1 by PP7 under blue light is essential to acquire a proper conformation or to bring in new components for the assembly of a functional HRB1 protein complex. Guard cells control stomatal opening in response to multiple environmental or biotic stimuli. This study may furnish strategies that allow plants to enjoy the advantages of both constitutive and ABA-induced protection under water-limiting conditions.

Transcriptional and hormonal signaling control of Arabidopsis seed development.

In angiosperms, a double-fertilization event leads to the formation of a diploid embryo and a triploid endosperm. In Arabidopsis and many dicots, seed development undergoes an initial phase of active endosperm proliferation followed by a second phase in which embryo grows to full size and replaces most of the endosperm volume at its maturity. Since the seed coat and endosperm growth in Arabidopsis precedes embryo growth, the major volume of the mature seed is largely attained before the enlargement of the embryo. Therefore, the seed size is coordinately regulated by the growth of the triploid endosperm, the diploid maternal ovule, and the diploid embryo. Recent studies have identified many new pathway components and revealed possible mechanisms that underlie seed development and size regulation in Arabidopsis. In this review, we shall discuss the regulation of endosperm proliferation by a few newly identified pathways involving transcriptional, epigenetic, and imprinting regulators, the regulation of integument or seed coat development by a few transcription factors, and the regulation of embryo proliferation by AP2-like and bHLH proteins and phytohormones.

Proteomic profiling of human bone marrow mesenchymal stem cells under shear stress.

Mesenchymal stem cells (MSCs) are promising seed cells for tissue engineering of blood vessels. As seed cells, MSCs must endure blood fluid shear stress after transplantation. It has been shown that fluid shear stress can regulate the proliferation and differentiation of MSCs. However, the effects of fluid shear stress on MSCs including the types of proteins modulated are still not well understood. In this study, we exposed human mesenchymal stem cells (HMSCs) to 3 dyn/cm(2) shear stress for 6 h and compared them to a control group using proteomic analysis. Thirteen specific proteins were affected by shear stress, 10 of which were up-regulated. Shear stress especially induced sustained increases in the expression of Annexin A2 and GAPDH, which have been specifically shown to affect HMSCs function. We present here the first comparative proteome analysis of effect of shear stress on HMSCs.

Myocardial protective effect of urethane on isolated rat hearts in prolonged hypothermic preservation.

BACKGROUND: One of the most important factors restricting heart transplantation is the limited myocardial ischemia time. This study investigated the effects of urethane on the hypothermic preservation of donor rat hearts. MATERIALS AND METHODS: Hearts isolated from rats were divided into 2 groups (n = 8), a control group with histidine-tryptophan-ketoglutarate (HTK) solution alone and an experimental group with HTK solution plus 30 mM urethane. Hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function; the hearts were then arrested and stored in one of the 2 solutions for 6 hours and 18 hours at 4 degrees C. After preservation, the hearts were reperfused, and cardiac function was evaluated. Lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) content, cardiomyocyte apoptosis, and myocardial ultrastructure were examined. RESULTS: Compared with the control group, the experimental group showed a significantly higher recovery of cardiac function for both 6 hours and 18 hours of preservation and demonstrated a lower rate of cardiomyocyte apoptosis (8.5% + or - 1.2% versus 12.2% + or - 1.8% for 6 hours; 14.1% + or - 2.1% versus 31.4% + or - 2.7% for 18 hours). ATP content was significantly higher in the experimental group than in the control group after 18 hours of preservation (229.4 + or - 29.7 microg/g versus 153.2 + or - 21.1 microg/g). The experimental group also showed lower levels of LDH release after 18 hours of preservation. Electron microscopy studies demonstrated better cardiomyocyte structure in the experimental group for both 6 hours and 18 hours of preservation. CONCLUSIONS: Use of urethane improved cardiac functional recovery and led to significant protective effects on rat hearts placed in a hypothermic preservation solution for a prolonged period.

RGD-modified acellular bovine pericardium as a bioprosthetic scaffold for tissue engineering.

Acellular biological tissues, including bovine pericardia (BP), have been proposed as natural biomaterials for tissue engineering. However, small pore size, low porosity and lack of extra cellular matrix (ECM) after native cell extraction directly restrict the seed cell adhesion, migration and proliferation and which is a vital problem for ABP's application in the tissue engineered heart valve (TEHV). In the present study, we treated acellular BP with acetic acid, which increased the scaffold pore size and porosity and conjugated RGD polypeptides to ABP scaffolds. After 10 days of culture in vitro, the human mesenchymal stem cells (hMSCs) attached the best and proliferated the fastest on RGD-modified acellular scaffolds, and the cell has grown deep into the scaffold. In contrast, a low density of cells attached to the unmodified scaffolds, with few infiltrating into the acellular tissues. These findings support the potential use of modified acellular BP as a scaffold for tissue engineered heart valves.

SHORT HYPOCOTYL UNDER BLUE1 associates with MINISEED3 and HAIKU2 promoters in vivo to regulate Arabidopsis seed development.

Seed development in Arabidopsis thaliana undergoes an initial phase of endosperm proliferation followed by a second phase in which the embryo grows at the expense of the endosperm. As mature seed size is largely attained during the initial phase, seed size is coordinately determined by the growth of the maternal ovule, endosperm, and embryo. Here, we identify SHORT HYPOCOTYL UNDER BLUE1 (SHB1) as a positive regulator of Arabidopsis seed development that affects both cell size and cell number. shb1-D, a gain-of-function overexpression allele, increases seed size, and shb1, a loss-of-function allele, reduces seed size. SHB1 is transmitted zygotically. The increase in shb1-D seed size is associated with endosperm cellurization, chalazal endosperm enlargement, and embryo development. SHB1 is required for the proper expression of two other genes that affect endosperm development, MINISEED3 (MINI3) and HAIKU2 (IKU2), a WRKY transcription factor gene and a leucine-rich repeat receptor kinase gene. SHB1 associates with both MINI3 and IKU2 promoters in vivo. SHB1 may act with other proteins that bind to MINI3 and IKU2 promoters to promote a large seed cavity and endosperm growth in the early phase of seed development. In the second phase, SHB1 enhances embryo cell proliferation and expansion through a yet unknown IKU2-independent pathway.

[Research on application of modified polyethylene glycol hydrogels in the construction of tissue engineered heart valve].

OBJECTIVE: To explore the effect of the polyethylene glycol (PEG)-hydrogels to enhance the seeding-cells adhesion to the biomaterial scaffolds. METHODS: Sixteen porcine aortic valves were decellularized with Triton X-100 and trypsin, then divided into A and B group, eight in each group. Group A: the donor goat's autologous bone marrow mesenchymal stem cells (BMSCs) Selected as the seeding-cells were encapsulated into the modified PEG-hydrogels to complete the process of the cells attaching to the acellular porcine aortic valves. Non-PEG but reservation of BMSCs was modified in Group B. After static culture for 7 d, the mono semilunar tissue engineering heart valve (TEHV) were implanted respectively into each donor goat's abdominal aortas. Gross and histology examination, ultrasonic scanning, electron microscopy observation and biomechanics detection were performed at 16 weeks after operation. The 8 native goat aortic valves from the donor goats were selected at the same time as control group (Group C). RESULTS: There were much more improvements compared Group A to Group B (P < 0.05) in tensile strength [(12.9 +/- 1.3) MPa vs. (8.8 +/- 0.4) MPa], ratio of re-endothelial (84.6% vs. 14.8%) and mural thrombosis (0/8 vs. 8/8). The data illustrated the critical importance of BMSCs differentiation to endothelial and myofibroblast for remodeling into native tissue in microenvironment in vivo. CONCLUSIONS: It is feasible to reconstruct TEHV efficiently by combining modified PEG-hydrogels with acellular biomaterial scaffold and autologous MSCs cells. It can improve the integration of the seeding-cells and scaffold. It can also protect the growth and differentiation of the BMSCs in the systemic circulation effectively.

HYPERSENSITIVE TO RED AND BLUE 1 and its C-terminal regulatory function control FLOWERING LOCUS T expression.

The red and far-red light-absorbing phytochromes and UV-A/blue light-absorbing cryptochromes regulate seedling de-etiolation and flowering responses. The signaling steps that mediate the photoreceptor regulation on key flowering genes remain largely unknown. We report that a previously identified photomorphogenic mutant, hypersensitive to red and blue 1 (hrb1), flowered late and showed attenuated expression of FLOWERING LOCUS T (FT) over both long days and short days. Transgenic plants that overexpress the full-length HRB1, or its C-terminal half, flowered early and accumulated more FT messages under short-day conditions. The transgenic plants also displayed hyposensitive de-etiolation phenotypes, and the expression of these phenotypes requires the action of PIF4. The double mutant of hrb1/cry2 showed a flowering phenotype and an FT expression pattern similar to hrb1 under long-day conditions, suggesting that HRB1 may function downstream of cry2 under long-day conditions. In contrast, hrb1/phyB-9 showed a flowering phenotype and an FT expression pattern similar to phyB-9 over both long days and short days, indicating a modulatory role of HRB1 in the flowering pathway mediated by phyB. Overexpression of HRB1 did not affect the expression of the central clock oscillators, TOC1 and CCA1. HRB1 therefore represents a signaling step that regulates FT expression downstream of red and blue light perception.