Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

iTRAQ-Based Proteomics Reveals that the Tomato ms1035 Gene Causes Male Sterility through Compromising Fat Acid Metabolism.

So far, over 50 spontaneous male sterile mutants of tomato have been described and most of them are categorized as genetic male sterility. To date, the mechanism of tomato genetic male sterility remained unclear. In this study, differential proteomic analysis is performed between genetic male sterile line (2-517), which carries the male sterility (ms1035 ) gene, and its wild-type (VF-11) using isobaric tags for relative and absolute quantification-based strategy. A total of 8272 proteins are quantified in the 2-517 and VF-11 lines at the floral bud and florescence stages. These proteins are involved in different cellular and metabolic processes, which express obvious functional tendencies toward the hydroxylation of the ω-carbon in fatty acids, the tricarboxylic acid cycle, the glycolytic, and pentose phosphate pathways. Based on the results, a protein network explaining the mechanisms of tomato genetic male sterility is proposed, finding the compromising fat acid metabolism may cause the male sterility. These results are confirmed by parallel reaction monitoring, quantitative Real-time PCR (qRT-PCR), and physiological assays. Taken together, these results provide new insights into the metabolic pathway of anther abortion induced by ms1035 and offer useful clues to identify the crucial proteins involved in genetic male sterility in tomato.

Transabdominal ultrasound-guided multifetal pregnancy reduction in 10 cases of monkeys.

Intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) in nonhuman primates, e.g. rhesus and cynomolgus monkeys, has been widely used in researches of reproductive and developmental biology, and the success rate has been improved significantly. However, unwanted multiple pregnancy occurs frequently during the ICSI-ET in monkeys, most of which leads to miscarriages. To improve the birth rate of pregnancies and to safeguard health of host and baby monkeys, multifetal pregnancy reduction (MPR) is necessary. In this study, a total of 10 monkeys with multiple pregnancies received MPR through transabdominal ultrasound-guided potassium chloride injection into beating hearts of selective fetuses. To assess MPR efficiency, 31 monkeys with normal singleton pregnancies and 25 monkeys with twin pregnancies without MPR were used as controls. The aim of the reduction is to keep only one fetus, no matter twin or triplet pregnancy originally. Our results show that six cases of MPR were successful and all of them retained single fetus. Moreover, about 1 month (30.2 ± 1.2 days) of gestation is a better timing for MPR than later stage (50.7 ± 1.9 days). We also found that the remaining fetuses developed normally with full-term gestation and normal birth weight. In conclusion, transabdominal ultrasound-guided potassium chloride injection is a safe and effective MPR method for monkeys with multiple pregnancies.

Effect of pretreatment on pd/Al2O3 catalyst for catalytic oxidation of o-xylene at low temperature.

The effect of pretreatment on Pd/Al2O3 catalysts for the catalytic oxidation of o-xylene at low temperature was studied by changing the pretreatment and testing conditions. The fresh and pretreated Pd/Al2O3 catalysts were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). The results showed that the pretreatment dramatically changed the Pd/PdO ratio and then significantly affected the Pd/Al2O3 activity; while the pretreatment had not much influence on Pd particle size. The Pd/Al2O3 pre-reduced at 300 degrees C/400 degrees C, which has fully reduced Pd species, showed the highest activity; while the fresh Pd/Al2O3, which has fully oxidized Pd species, presented the worst performance, indicating the Pd chemical state plays an important role in the catalytic activity for the o-xylene oxidation. It is concluded that metallic Pd is the active species on the Pd/Al2O3 catalyst for the catalytic oxidation of o-xylene at low temperature.

Super-pangenome analyses highlight genomic diversity and structural variation across wild and cultivated tomato species.

Effective utilization of wild relatives is key to overcoming challenges in genetic improvement of cultivated tomato, which has a narrow genetic basis; however, current efforts to decipher high-quality genomes for tomato wild species are insufficient. Here, we report chromosome-scale tomato genomes from nine wild species and two cultivated accessions, representative of Solanum section Lycopersicon, the tomato clade. Together with two previously released genomes, we elucidate the phylogeny of Lycopersicon and construct a section-wide gene repertoire. We reveal the landscape of structural variants and provide entry to the genomic diversity among tomato wild relatives, enabling the discovery of a wild tomato gene with the potential to increase yields of modern cultivated tomatoes. Construction of a graph-based genome enables structural-variant-based genome-wide association studies, identifying numerous signals associated with tomato flavor-related traits and fruit metabolites. The tomato super-pangenome resources will expedite biological studies and breeding of this globally important crop.

Enhanced removal of ammonia nitrogen from rare earth wastewater by NaCl modified vermiculite: Performance and mechanism.

Wastewater from rare earth mining (WREM) is very harmful to environment and human health due to its high concentration of ammonia nitrogen (NH3-N). It is therefore necessary and urgent to find a low-cost and convenient technique to remove high concentration of NH3-N from WREM. In this study, Natural powdered vermiculite (NV) was modified with seven sodium chloride (NaCl) solutions, and seven kinds of sodium chloride modified vermiculite (Na-V) were obtained. The NH3-N adsorption performance of Na-V is greatly improved compared with NV. Among them, vermiculite modified with 180 g/L NaCl yielded the highest ammonium adsorption capacity (Qm, 11.569 mg/g), which was 63.43% higher than NZ (Qm, 7.079 mg/g). The characterizations of 180-Na-V confirmed the removal mechanism of NH3-N that the improved capacity of modified vermiculite was attributed to its higher mesoporous volume and ion-exchange capacity, which are the result of sodium-ion exchange and Interlayer effect from high concentration of NaCl. The adsorption isotherms and kinetics were respectively best consistent with Langmuir model and the pseudo-second-order (PSO) model. The adsorption capacity (3.808 mg/g) of vermiculite after 5 cycles could still maintain 75.09% of the initial adsorption capacity (5.071 mg/g). A large amount of Na-V had little effect on pH of water, which meet the requirements of practical application. Including pH, dosage, coexisting ions, the effects of other factors on ammonium adsorption were also determined. This study provides a new method for vermiculite to remove high concentration of NH3-N.

iTRAQ and PRM -based proteomics analysis for the identification of differentially abundant proteins related to male sterility in ms-7 mutant tomato (Solanum lycoperscium) plants.

Male sterile mutants can be used in breeding or commercial cultivation in tomato, but there are few research reports on their proteomics. In this study, we analyzed the metabolic pathways and biological functions of differentially abundant proteins (DAPs) involved in two stages of stamen development of the tomato flowers by using a high through-put iTRAQ labeled proteomic approach. There was a total of 1476 DAPs which should associated with the occurrence of pollen abortion in tomato. Moreover, there were more DAPs in the four membrane systems. It shows that membrane systems are very important for tomato pollen development. According to KEGG analysis, these signaling pathways including starch and sucrose metabolism (map00500), tropane, piperidine and pyridine alkaloids biosynthesis (map00960), amino sugar and nucleotide sugar metabolism (map00520) have important effects on pollen development. These results were verified by using mass spectrometry PRM. Finally, two candidate genes (Solyc11g065770 and Solyc11g065530) were found that may be related to pollen development and cause pollen abortion by comparison of protein-protein interaction networks and on the basis of previous studies on ms-7 gene. This data and model will provide a new insight into tomato genetic male sterility 7 and contribute to the improvement of tomato hybrid breeding. Biological significance: Artificial emasculation is still the main method of tomato hybrid breeding at present. Adopting male sterility in tomato cross breeding could greatly improve the production efficiency and seed purity; reduce the cost. Although numerous researches have been conducted to select the genes related to male sterility, the molecular mechanism remains unclear in tomato. In this study, we used the high-through-put iTRAQ labeled proteomic approach, to perform a novel comparison of expression profiles in GMS tomato line and its wildtype line. Based on these results, we proposed the potential regulated protein network involved in pollen development mechanism of tomato GMS and two candidate genes. SIGNIFICANCE: Artificial emasculation is still the main method of tomato hybrid breeding at present. Adopting male sterility in tomato cross breeding could greatly improve the production efficiency and seed purity; reduce the cost. Although numerous researches have been conducted to select the genes related to male sterility, the molecular mechanism remains unclear in tomato. In this study, we used the high-through-put iTRAQ labeled proteomic approach, to perform a novel comparison of expression profiles in GMS tomato line and its wildtype line. Based on these results, we proposed the potential regulated protein network involved in pollen development mechanism of tomato GMS and two candidate genes.

Identification and Characterization of Long Non-coding RNA in Tomato Roots Under Salt Stress.

As one of the most important vegetable crops in the world, the production of tomatoes was restricted by salt stress. Therefore, it is of great interest to analyze the salt stress tolerance genes. As the non-coding RNAs (ncRNAs) with a length of more than 200 nucleotides, long non-coding RNAs (lncRNAs) lack the ability of protein-coding, but they can play crucial roles in plant development and response to abiotic stresses by regulating gene expression. Nevertheless, there are few studies on the roles of salt-induced lncRNAs in tomatoes. Therefore, we selected wild tomato Solanum pennellii (S. pennellii) and cultivated tomato M82 to be materials. By high-throughput sequencing, 1,044 putative lncRNAs were identified here. Among them, 154 and 137 lncRNAs were differentially expressed in M82 and S. pennellii, respectively. Through functional analysis of target genes of differentially expressed lncRNAs (DE-lncRNAs), some genes were found to respond positively to salt stress by participating in abscisic acid (ABA) signaling pathway, brassinosteroid (BR) signaling pathway, ethylene (ETH) signaling pathway, and anti-oxidation process. We also construct a salt-induced lncRNA-mRNA co-expression network to dissect the putative mechanisms of high salt tolerance in S. pennellii. We analyze the function of salt-induced lncRNAs in tomato roots at the genome-wide levels for the first time. These results will contribute to understanding the molecular mechanisms of salt tolerance in tomatoes from the perspective of lncRNAs.

Genome-Wide Identification and Evolutionary Analysis of the SRO Gene Family in Tomato.

SRO (SIMILAR TO RCD ONE) is a family of plant-specific small molecule proteins that play an important role in plant growth and development and environmental responses. However, SROs still lack systematic characterization in tomato. Based on bioinformatics methods, SRO family genes were identified and characterized from cultivated tomatoes and several wild tomatoes. qRT-PCR was used to study the expression of SRO gene in cultivated tomatoes. Phylogenetic and evolutionary analyses showed that SRO genes in angiosperms share a common ancestor and that the number of SRO family members changed as plants diverged and evolved. Cultivated tomato had six SRO members, five of which still shared some degree of identity with the ancestral SRO genes. Genetic structure and physicochemical properties showed that tomato SRO genes were highly conserved with chromosomal distribution. They could be divided into three groups based on exon-intron structure, and cultivated tomato contained only two of these subclades. A number of hormonal, light and abiotic stress-responsive cis-regulatory elements were identified from the promoter of the tomato SRO gene, and they also interacted with a variety of stress-responsive proteins and microRNAs. RNA-seq analysis showed that SRO genes were widely expressed in different tissues and developmental stages of tomato, with significant tissue-specific features. Expression analysis also showed that SRO genes respond significantly to high temperature and salt stress and mediate the tomato hormone regulatory network. These results provide a theoretical basis for further investigation of the functional expression of tomato SRO genes and provide potential genetic resources for tomato resistance breeding.

Identification of the Carbohydrate and Organic Acid Metabolism Genes Responsible for Brix in Tomato Fruit by Transcriptome and Metabolome Analysis.

BACKGROUND: Sugar and organic acids not only contribute to the formation of soluble solids (Brix) but also are an essential factor affecting the overall flavor intensity. However, the possible metabolic targets and molecular synthesis mechanisms remain to be further clarified. METHODS: UHPLC-HRMS (ultrahigh-performance liquid chromatography and high-resolution mass spectrometry) combined with comparative transcriptome analysis were performed in fruits at green ripe (S1), turning-color (S2), and red ripe (S3) stages of two tomato genotypes TM-1 (Solanum galapagense L., LA0436) and TM-38 (S. lycopersicum L. cultivar M82, LA3475) that vary in fruit Brix. RESULTS: The fruit Brix of TM-1 was nearly twice that of TM-38 at S3. Nevertheless, TM-1 accumulated 1.84- and 2.77-fold the L-malic acid and citric acid in red ripe fruit (S3) compared with TM-38, respectively. D-glucose and D-fructose in TM-1 and TM-38 fruits tended to be similar at S3. Concomitantly, the sugar/organic acid ratio of TM-38 fruits were 23. 08-, 4. 38-, and 2.59-fold higher than that of TM-1 fruits at S1, S2, and S3, respectively. Among starch and sucrose (carbohydrate, CHO) metabolism (ko00500) genes, SUS (Solyc07g042550.3) and BAM (Solyc08g077530.3) were positively (r = 0.885-0.931) correlated with the sugar/organic acid ratio. Besides, INV (Solyc09g010080.3 and Solyc09g010090.5.1), AAM (Solyc04g082090.3), 4-α-GTase (Solyc02g020980.2.1), BGL2 (Solyc06g073750.4, Solyc06g073760.3, and Solyc01g081170.3), TPS (Solyc01g005210.2 and Solyc07g006500.3), and TPP (Solyc08g079060.4) were negatively (r = -0.823 to -0.918) correlated with the sugar/organic acid ratio. The organic acid (TCA cycle) metabolism (ko00020) gene ALMT (Solyc01g096140.3) was also negatively (r = -0.905) correlated with the sugar/organic acid ratio. CONCLUSION: Citric acid may play a more dominant role in the sugar/organic acid ratio of the tomato fruit, and the contribution of both L-malic acid and citric acid to the fruit Brix was much greater than that of D-glucose and D-fructose. Genes involved in CHO and TCA metabolism, which have a significant correlation with the sugar/organic acid ratio were considered to be the contributing factors of fruit Brix.

Enhanced soluble sugar content in tomato fruit using CRISPR/Cas9-mediated SlINVINH1 and SlVPE5 gene editing.

Soluble sugar is known to improve the sweetness and increase tomato sauce yield. Studies have focused on improving the content of soluble sugar in tomato fruits, usually by promoting functional genes. We studied two genes (SlINVINH1 and SlVPE5) that inhibited the accumulation of soluble sugar in tomato fruits and obtained two genes' knocked-out lines (CRISPR-invinh1 or CRISPR-vpe5) using CRISPR/Cas9. Aggregated lines with CRISPR-invinh1 and CRISPR-vpe5 were gained by hybridization and self-pollination. Compared to wild-type lines, the glucose, fructose, and total soluble solid (TSS) contents of CRISPR-invinh1 and CRISPR-vpe5 increased significantly. Glucose, fructose, and TSS levels further improved simultaneously with CRISPR-invinh1 and CRISPR-vpe5 than with single gene knock-out lines. This indicates that these genes have a synergistic effect and will increase the soluble sugar content. Thus, the knock-out SlINVINH1 and SlVPE5 may provide a practical basis for improving the sweetness of tomato fruits and their processing quality.

Dendritic superstructures and structure transitions of asymmetric poly(L-lactide-b-ethylene oxide) diblock copolymer thin films.

The evolution of morphologies of isothermally crystallized thin films with different thicknesses of poly(L-lactide-b-ethylene oxide) diblock copolymer was observed by optical microscopy (OM) and atomic force microscopy (AFM). Dendritic superstructures stacked with lamellae were investigated in thin films with approximately 200 nm to approximately 400 nm thickness. The lamellar structure was a lozenge- or truncated-lozenge-shaped single crystal of PLLA confirmed by AFM observations. The contour of the dendritic superstructures is hexagonal, and two types of sectors, [110] and [100], can be classified in terms of the chain-folding and crystal growth directions. These phenomena are due to the interplay of the crystallization of the PLLA block, the microphase separation of the block copolymer, and the effect of the film thickness. The growth process of the superstructure can be classified into three steps: the growth of the main branches, the growth of the secondary side branches along the main branch, and the tertiary side branches. PLLA growth rates decrease in copolymer films thinner than 1 microm. Layer-layer phase structure of the copolymer driven by the crystallization of PLLA and the microphase separation of the copolymer appears to be a key factor explaining the crystallization and morphological behavior of this system.

In situ adsorption-catalysis system for the removal of o-xylene over an activated carbon supported Pd catalyst.

An activated carbon (AC) supported Pd catalyst was used to develop a highly efficient in situ adsorption-catalysis system for the removal of low concentrations of o-xylene. In this study, three kinds of Pd/AC catalysts were prepared and tested to investigate the synergistic efficiency between adsorption and catalysis for o-xylene removal. The Pd/AC catalyst was first used as an adsorbent to concentrate dilute o-xylene at low temperature. After saturated adsorption, the adsorbed o-xylene was oxidized to CO2 and H2O by raising the temperature of the catalyst bed. The results showed that more than 99% of the adsorbed o-xylene was completely oxidized to CO2 over a 5% Pd/AC catalyst at 140 degrees C. Brunauer-Emmett-Teller (BET) analysis, scanning electron microscopy (SEM), temperature-programmed desorption (TPD), and temperature-programmed oxidation (TPO) were applied to investigate the physical properties of o-xylene adsorption-desorption and the in situ adsorption-catalysis activity of the AC support and Pd/AC catalyst. A synergistic relationship between the AC support and the active Pd species for the removal of low concentrations of o-xylene was established.

Hepatitis C virus NS4A inhibits cap-dependent and the viral IRES-mediated translation through interacting with eukaryotic elongation factor 1A.

The genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS3 and NS2-3 proteases. Nonstructural protein 4A (NS4A) is a cofactor of the NS3 serine protease and has been demonstrated to inhibit protein synthesis. In this study, GST pull-down assay was performed to examine potential cellular factors that interact with the NS4A protein and are involved in the pathogenesis of HCV. A trypsin digestion followed by LC-MS/MS analysis revealed that one of the GST-NS4A-interacting proteins to be eukaryotic elongation factor 1A (eEF1A). Both the N-terminal domain of NS4A from amino acid residues 1-20, and the central domain from residues 21-34 interacted with eEF1A, but the central domain was the key player involved in the NS4A-mediated translation inhibition. NS4A(21-34) diminished both cap-dependent and HCV IRES-mediated translation in a dose-dependent manner. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. Taken together, NS4A inhibits host and viral translation through interacting with eEF1A, implying a possible mechanism by which NS4A is involved in the pathogenesis and chronic infection of HCV.

Effect of a C/EBP gene replacement on mitochondrial biogenesis in fat cells.

CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPbeta, are required for fat cell differentiation and maturation. Previous studies showed that replacement of C/EBPalpha with C/EBPbeta, generating the beta/beta alleles in the mouse genome, prevents lipid accumulation in white adipose tissue (WAT). In this study, beta/beta mice lived longer and had higher energy expenditure than their control littermates due to increased WAT energy oxidation. The WAT of beta/beta mice was enriched with metabolically active, thermogenic mitochondria known for energy burning. The beta/beta allele exerted its effect through the elevated expression of the G protein alpha stimulatory subunit (Galphas) in WAT. Galphas, when overexpressed in fat-laden 3T3-L1 cells, stimulated mitochondrial biogenesis similar to that seen in the WAT of beta/beta mice, and effectively diminished the stored lipid pool.