Differential heat-response characteristics of two plastid isoforms of triose phosphate isomerase in tomato.

Heat stress causes dysfunction of the carbon-assimilation metabolism. As a member of Calvin-Benson-Bassham (CBB) cycle, the chloroplast triose phosphate isomerases (TPI) catalyse the interconversion of glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The tomato (Solanum lycopersicum) genome contains two individual SlTPI genes, Solyc10g054870 and Solyc01g111120, which encode the chloroplast-located proteins SlTPI1 and SlTPI2, respectively. The tpi1 and tpi2 single mutants had no visible phenotypes, but the leaves of their double mutant lines tpi1tpi2 had obviously reduced TPI activity and displayed chlorotic variegation, dysplasic chloroplasts and lower carbon-assimilation efficiency. In addition to altering carbon metabolism, proteomic data showed that the loss of both SlTPI1 and SlTPI2 severely affected photosystem proteins, reducing photosynthetic capacity. None of these phenotypes was evident in the tpi1 or tpi2 single mutants, suggesting that SlTPI1 and SlTPI2 are functionally redundant. However, the two proteins differed in their responses to heat stress; the protein encoded by the heat-induced SlTPI2 showed a higher level of thermotolerance than that encoded by the heat-suppressed SlTPI1. Notably, heat-induced transcription factors, SlWRKY21 and SlHSFA2/7, which negatively regulated SlTPI1 expression and positively regulated SlTPI2 expression, respectively. Our findings thus reveal that SlTPI1 and SlTPI2 have different thermostabilities and expression patterns in response to heat stress, which have the potential to be applied in thermotolerance strategies in crops.

Bisphenol B induces developmental toxicity in zebrafish via oxidative stress.

Bisphenol A (BPA) is a typical endocrine disruptor, and the use of bisphenol B (BPB) as a substitute is gradually increasing. Some studies have shown that BPB also has endocrine disrupting effects, but its effects on the early stages of fish growth and development have not been reported. In this paper, zebrafish embryos were exposed to different concentrations of BPB until the 6th day post fertilization (dpf), and the toxic effects of BPB on the early development of zebrafish and the possible molecular mechanisms were investigated. The results showed that BPB exposure at 10, 100, and 1000 µg/L induced developmental toxic effects such as early neurotoxicity and cardiovascular toxicity in zebrafish, and the toxic effects were positively correlated with the degree of oxidative damage. These adverse results were ameliorated by the classical antioxidant N-acetyl-L-cysteine (NAC), suggesting the involvement of oxidative stress in BPB-induced early developmental toxicity. The above data suggest that BPB exposure increases oxidative damage and suppresses the expression of genes critical for early neurological and cardiovascular development, ultimately leading to early developmental toxicity in juvenile zebrafish. This study contributes to broadening our understanding of the toxic effects of BPB and provides a basic theoretical basis for the next management support of bisphenol analogs.

GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells.

BACKGROUND: The pulmonary surfactant that lines the air-liquid surface within alveoli is a protein-lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation. METHOD: GCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling. RESULTS: Knocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS-Erk-Foxo1-Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations. CONCLUSIONS: In summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs.

Highly Efficient OLEDs by Using a Brominated Thermally Activated Delayed Fluorescent Host due to Balanced Carrier Transport and Enhanced Exciton Upconversion.

Thermally activated delayed fluorescent (TADF) materials are naturally bipolar and can potentially serve as hosts. However, triplet excitons in TADF materials are long-lived and prone to unfavorable bimolecular processes. Implementing an efficient reverse system intersection (RISC) process is an effective solution. Moreover, although the general TADF host is bipolar, polarity differences still cause a mobility imbalance. In this work, we designed and synthesized a novel TADF host material, 11-(3-(4-(3-bromophenyl)-6-phenyl-1,3,5-triazin-2-yl)phenyl)-12,12-dimethyl-11,12-dihydroindeno[2,1-a]carbazole (Br-DMIC-TRZ). The upconversion of the TADF host and its doped films is facilitated due to enhanced spin-orbit coupling (SOC) induced by bromine, which exhibits a higher rate of RISC. This progress facilitates the involvement of more triplet excitons in luminescence. Meanwhile, the attachment of bromine to the acceptor fragment of TADF enhances the electron mobility, where hole mobility and electron mobility are more comparable. Enhanced exciton upconversion and balanced carrier transport allow devices formed based on brominated TADF hosts to outperform other hosts. The Br-TADF-based devices with three dopants sensitized achieved improvements of 29.8, 21.4, and 24.4% compared to the DMIC-TRZ-based device. This work provides a feasible molecular design strategy for further developing efficient hosts.

[Genetic analysis of a case with 11ß hydroxylase deficiency caused by CYP11B2/CYP11B1 chimeric gene].

OBJECTIVE: To analyze a child with 11ß hydroxylase deficiency (11ß-OHD) due to CYP11B2/CYP11B1 chimeric gene. METHODS: Clinical data of the child who was admitted to Henan Children's Hospital on August 24, 2020 were retrospectively analyzed. Peripheral blood samples of the child and his parents were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. RT-PCR and Long-PCR were carried out to verify the presence of chimeric gene. RESULTS: The patient, a 5-year-old male, had featured premature development of secondary sex characteristics and accelerated growth, and was diagnosed with 21 hydroxylase deficiency (21-OHD). WES revealed that he has harbored a heterozygous c.1385T>C (p.L462P) variant of the CYP11B1 gene, in addition to a 37.02 kb deletion on 8q24.3. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.1385T>C (p.L462P) was rated as a likely pathogenic variant (PM2_Supporting+PP3_Moderate+PM3+PP4). The results of RT-PCR and Long-PCR suggested that CYP11B1 and CYP11B2 genes have recombined to form a CYP11B2 exon 1~7/CYP11B1 exon 7~9 chimeric gene. The patient was diagnosed as 11ß-OHD and effectively treated with hydrocortisone and triptorelin. A healthy fetus was delivered following genetic counseling and prenatal diagnosis. CONCLUSION: 11ß-OHD may be misdiagnosed as 21-OHD due to the potential CYP11B2/CYP11B1 chimeric gene, which will require multiple methods for the detection.

The role of exercise intensity on fatty liver in rats.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is often caused by obesity. Currently, moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) are two effective treatments for reducing fat mass in patients with obesity and NAFLD. However, the comparative fat-reducing effects and underlying molecular mechanisms of MICT and HIIT remain unclear. This comprehensive study was performed on male Wistar rats treated with standard diet, high-fat diet, MICT, and HIIT to explore their comparative fat-reducing effects and corresponding molecular mechanisms. HIIT had a greater effect on hepatic vacuolation density and lipid content reduction than MICT, and triglyceride and total cholesterol levels in the serum and the liver demonstrated different sensitivities to different exercise training programs. At the molecular level, both MICT and HIIT altered the processes of fatty acid synthesis, fatty acid transport, fatty acid ß-oxidation, and cholesterol synthesis, wherein the transcriptional and translational levels of signaling molecules peroxisome proliferator-activated receptors (PPARs) regulating fatty acid and cholesterol synthesis were strongly changed. Moreover, the metabolic pathways of amino acids, bile acids, and carbohydrates were also affected according to transcriptome analysis, and the changes in the above-mentioned processes in the HIIT group were greater than those in the MICT group. In combination with the search tool for the retrieval of interacting genes/proteins (STRING) analysis and the role of PPARs in lipid metabolism, as well as the expression pattern of PPARs in the MICT and HIIT groups, the MICT-and HIIT-induced fat loss was mediated by the PPAR pathway, causing feedback responses in fatty acid, steroid, amino acid, bile acid, and carbohydrate metabolism, and HIIT had a better fat-reducing effect, which may be initiated by PPAR-α. This study provides a theoretical basis for targeted therapy of patients with obesity and NAFLD.

Global negative effects of nutrient enrichment on arbuscular mycorrhizal fungi, plant diversity and ecosystem multifunctionality.

Despite widespread anthropogenic nutrient enrichment, it remains unclear how nutrient enrichment influences plant-arbuscular mycorrhizal fungi (AMF) symbiosis and ecosystem multifunctionality at the global scale. Here, we conducted a meta-analysis to examine the worldwide effects of nutrient enrichment on AMF and plant diversity and ecosystem multifunctionality using data of field experiments from 136 papers. Our analyses showed that nutrient addition simultaneously decreased AMF diversity and abundance belowground and plant diversity aboveground at the global scale. The decreases in AMF diversity and abundance associated with nutrient addition were more pronounced with increasing experimental duration, mean annual temperature (MAT) and mean annual precipitation (MAP). Nutrient addition-induced changes in soil pH and available phosphorus (P) predominantly regulated the responses of AMF diversity and abundance. Furthermore, AMF diversity correlated with ecosystem multifunctionality under nutrient addition worldwide. Our findings identify the negative effects of nutrient enrichment on AMF and plant diversity and suggest that AMF diversity is closely linked with ecosystem function. This study offers an important advancement in our understanding of plant-AMF interactions and their likely responses to ongoing global change.

Glutathione peroxidase 5 deficiency induces lipid metabolism regulated by reactive oxygen species in Chlamydomonas reinhardtii.

BACKGROUND: Reactive oxygen species (ROS) are generated incidentally during natural metabolism process of aerobic photosynthetic organisms which could be either harmful for cellular components. How ROS regulated lipid metabolism and the transcriptomes of stressed cells respond to ROS in aerobic photosynthetic organisms are unclear. Glutathione peroxidases (GPXs) detoxify hydrogen peroxide or organic hydroperoxides, which are important enzymes of the antioxidant system. So the function of GPXs matters the cellular redox state. How the lipid metabolism respond to the GPXs deficiency remains to be explored. METHODS: In this study, we employed a Chlamydomonas reinhardtii gpx5 knockout mutant to examine the effects of ROS on lipid metabolism. The redox state and lipid content of the parental strain CC4348 and the gpx5 mutant were detected. Besides, the transcriptomes of CC4348 and the gpx5 mutant were sequenced before and after treatment with nitrogen-free medium to obtain genome wide respond. Then we performed the functional annotation, classification and enrichment analysis based on KEGG database for the differentially expressed genes (DEGs) before and after nitrogen deprivation of CC4348 and the gpx5 mutant. RESULTS: In the CC4348 cells, the lipid accumulated accompanying with increasing ROS level after treatment with nitrogen-free media. However, in the gpx5 mutant, the ROS level is much higher than that in the parental strain CC4348, unexpectedly with reduced lipid accumulation. By comparing the transcriptomes of CC4348 and gpx5 mutant, we found that both CC4348 and gpx5 mutant cells displayed upregulation of transcripts related to protein, nucleic acid, carbon metabolism and chlorophyll biosynthesis, but more proportion of genes related to lipid metabolism were up-regulated in CC4348 than that in the gpx5 mutant. CONCLUSION: In CC4348, lipid metabolism was up-regulated with increasing ROS level. But in the gpx5 mutant, Lipid accumulation was less with higher ROS level, which was due to the inhibited lipid biosynthesis. Therefore, ROS provides dual-directional regulation of lipid metabolism induced by GPX5 in Chlamydomonas.

Construction of a high-density genetic map and mapping of growth related QTLs in the grass carp (Ctenopharyngodon idellus).

BACKGROUND: Grass carp (Ctenopharyngodon idellus) are important species in Asian aquaculture. A draft genome for grass carp has already been published in 2015. However, there is still a requirement for a suitable genetic linkage map to arrange scaffolds on chromosomal frameworks. QTL analysis is a powerful tool to detect key locations for quantitative traits, especially in aquaculture. There no growth related QTLs of grass carp have been published yet. Even the growth trait is one of the focuses in grass carp culture. RESULTS: In this study, a pair of distantly related parent grass carps and their 100 six-month-old full-sib offspring were used to construct a high-density genetic map with 6429 single nucleotide polymorphisms (SNPs) by 2b-RAD technology. The total length of the consensus map is 5553.43 cM with the average marker interval of 1.92 cM. The map has a good collinearity with both the grass carp draft genome and the zebrafish genome, and it assembled 89.91% of the draft genome to a chromosomal level. Additionally, according to the growth-related traits of progenies, 30 quantitative trait loci (QTLs), including 7 for body weight, 9 for body length, 5 for body height and 9 for total length, were identified in 16 locations on 5 linkage groups. The phenotypic variance explained for these QTLs varies from 13.4 to 21.6%. Finally, 17 genes located in these regions were considered to be growth-related because they either had functional mutations predicted from the resequencing data of the parents. CONCLUSION: A high density genetic linkage map of grass carp was built and it assembled the draft genome to a chromosomal level. Thirty growth related QTLs were detected. After the cross analysis of Parents resequencing data, 17 candidate genes were obtained for further researches.

Transcriptomic and Physiological Responses to Oxidative Stress in a Chlamydomonas reinhardtii Glutathione Peroxidase Mutant.

Aerobic photosynthetic organisms such as algae produce reactive oxygen species (ROS) as by-products of metabolism. ROS damage biomolecules such as proteins and lipids in cells, but also act as signaling molecules. The mechanisms that maintain the metabolic balance in aerobic photosynthetic organisms and how the cells specifically respond to different levels of ROS are unclear. Glutathione peroxidase (GPX) enzymes detoxify hydrogen peroxide or organic hydroperoxides, and thus are important components of the antioxidant system. In this study, we employed a Chlamydomonas reinhardtii glutathione peroxidase knockout (gpx5) mutant to identify the genetic response to singlet oxygen (1O2) generated by the photosensitizer rose bengal (RB). To this end, we compared the transcriptomes of the parental strain CC4348 and the gpx5 mutant sampled before, and 1 h after, the addition of RB. Functional annotation of differentially expressed genes showed that genes encoding proteins related to ROS detoxification, stress-response-related molecular chaperones, and ubiquitin-proteasome pathway genes were upregulated in CC4338. When GPX5 was mutated, higher oxidative stress specifically induced the TCA cycle and enhanced mitochondrial electron transport. Transcription of selenoproteins and flagellar-associated proteins was depressed in CC4348 and the gpx5 mutant. In addition, we found iron homeostasis played an important role in maintaining redox homeostasis, and we uncovered the relationship between 1O2 stress and iron assimilation, as well as selenoproteins. Based on the observed expression profiles in response to different levels of oxidative stress, we propose a model for dose-dependent responses to different ROS levels in Chlamydomonas.

Cold-regulated protein (SlCOR413IM1) confers chilling stress tolerance in tomato plants.

Chilling stress severely affects the growth, development and productivity of crops. Chloroplast, a photosynthesis site, is extremely sensitive to chilling stress. In this study, the functions of a gene encoding a cold-regulated protein (SlCOR413IM1) under chilling stress were investigated using sense and antisense transgenic tomatoes. Under chilling stress, SlCOR413IM1 expression was rapidly induced and the sense lines exhibited better growth state of seedlings and grown tomato plants. Overexpression of SlCOR413IM1 alleviated chilling-induced damage to the chloroplast membrane and structure, whereas suppression of SlCOR413IM1 aggravated the damage to chloroplast. Moreover, the net photosynthetic rate (Pn), maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm), actual photochemical efficiency of PSII (ΦPSII) and the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and stromal fructose-1, 6-bisphosphatase (sFBPase) were higher in the sense lines than those in the antisense lines. Hence, the inhibition of photosynthetic capacity was less severe in the sense lines but more severe in the antisense lines compared with that in wild-type (WT) plants. Taken together, overexpression of SlCOR413IM1 enhanced the chilling stress tolerance, whereas suppression of this gene increased the chilling sensitivity of tomato plants.

Salvianolic Acid B Enhances Hepatic Differentiation of Human Embryonic Stem Cells Through Upregulation of WNT Pathway and Inhibition of Notch Pathway.

Hepatocytes differentiated from human embryonic stem cells (ESCs) could provide a powerful tool for enabling cell-based therapies, studying the mechanisms underlying human liver development and disease, and testing the efficacy and safety of pharmaceuticals. However, currently most in vitro protocols yield hepatocytes with low levels of liver function. In this study, we investigated the potential of Salvianolic acid B (Sal B), an active pharmaceutical compound present in Salvia miltiorrhiza, which has been shown to have an antifibrotic effect in previous studies, to enhance hepatocyte differentiation from human ESCs. After treatment with Sal B, albumin expression and secretion were consistently increased, indicating that Sal B could promote hepatocyte differentiation process. Expression of a large number of important phase 1 and 2 metabolizing enzymes and phase 3 transporters was also increased in treated cells, indicating an enhanced biotransformation function. Our investigations further revealed the activation of Wnt pathway in treated cells, as determined by upregulation of Wnts, which increased amounts of nuclear ß-catenin. This increased nuclear ß-catenin led in turn to the enhanced expression of T cell factor (TCF) 3 and lymphoid enhancer-binding factor (LEF) 1 which upregulated their downstream targets, cyclin D1 and c-Myc. Notch receptors (Notch1, Notch3), Notch ligand (Jagged2), and Notch receptor targets [hairy and enhancer of split (Hes) 1, 5] were downregulated in treated cells, suggesting that Notch pathway was inhibited. Consistent with the inhibition of Notch pathway, expression of cholangiocyte marker, CK7, was significantly reduced by treatment with Sal B. Numb, a direct transcriptional target of Wnt pathway and a negative regulator of Notch pathway, was upregulated, consistent with activation of Wnt signaling and suppression of Notch signaling. In conclusion, our study demonstrated that Sal B enhanced hepatocyte differentiation from human ESCs through activation of Wnt pathway and inhibition of Notch pathway. Therefore, this study suggests that Sal B can be used as a potential agent to generate more mature hepatocytes for cell-based therapeutics and pharmaceutical studies.

SlCOR413IM1: A novel cold-regulation gene from tomato, enhances drought stress tolerance in tobacco.

Drought stress adversely affects plant growth, development, and productivity. Genes functioning in plant response to drought stress are essential for drought tolerance. In this study, SlCOR413IM1, a cold-regulated gene isolated from Solanum lycopersium, was transferred to Nicotiana tabacum to investigate its function under drought stress. The subcellular localisation of SlCOR413IM1-GFP fusion protein in Arabidopsis protoplasts suggested that SlCOR413IM1 is a chloroplast protein. Expression analyses revealed that SlCOR413IM1 responded to drought and cold stresses. Under drought stress, transgenic plants maintained the high maximum photochemical efficiency, net photosynthetic rate (Pn) and D1 protein content of photosystem II (PSII). Compared with wild-type (WT) plants, transgenic plants showed higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and proline and soluble sugar content, which reduced reactive oxygen species (ROS) generation. However, the high SOD and APX activities in transgenic plants were independent of their transcription levels. Moreover, the transgenic plants exhibited better seed germination, water status and survival, as well as lower malondialdehyde (MDA) content and relative electrical conductivity (REC) than WT plants under drought stress. Taken together, these data demonstrated that overexpression of SlCOR413IM1 enhanced drought stress tolerance in transgenic tobacco.

Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas.

BACKGROUND: The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive. RESULTS: By selecting a suitable parental strain, keeping individual mutants using the agar plate, and designing an insertion cassette-specific primer for library screening, we successfully generated and maintained ~150,000 insertional mutants of Chlamydomonas, which was used for both reverse and forward genetics analysis. We obtained 26 individual mutants corresponding to 20 genes and identified 967 motility-defect mutants including 10 mutants with defective accumulation of intraflagellar transport complex at the basal body. We also obtained 929 mutants defective in oil droplet assembly after nitrogen deprivation. Furthermore, a new insertion cassette with splicing donor sequences at both ends was also constructed, which increased the efficiency of gene interruption. CONCLUSION: In summary, this library provides a multifunctional platform both for obtaining mutants of interested genes and for screening of mutants with specific phenotype.

"Secondary Growth" in Hydrothermal Synthesis of Aligned ZnO Nanostructures and Its Application in Dye-Sensitized Solar Cells.

One-dimensional (1D) aligned ZnO nanostructures were prepared on ZnO film seeded substrates using a low-temperature hydrothermal method, and zinc nitrate and hexamethylenetetramine (HMT) precursors. It was observed that increasing the concentration ratio of Zn2+/HMT from 1 to 100 led to a "secondary growth," and a change in the morphologies of the ZnO nanostructures from arrays of thick nanorods to arrays of thin nanorod-step-thick nanorods. The morphological evolution of ZnO nanostructures with increased growth time at high Zn2+/HMT concentration ratios showed the same transformation. Dye-sensitized solar cells (DSSCs) were fabricated using ZnO nanostructures as the photoanodes, and the electron transport properties were determined by electrochemical impedance spectroscopy (EIS). Although the DSSCs showed low power conversion efficiencies due to the short lengths, the arrays of the thin nanorods demonstrated excellent electron transport with an electron diffusion coefficient (Dn) of 1.57 x 10(-3) cm2/s, and an effective diffusion length (L) of 140 µm.

A stress-associated NAC transcription factor (SlNAC35) from tomato plays a positive role in biotic and abiotic stresses.

The NAC transcription factor family participates in responses to various kinds of environmental stimuli in plants. Responses of NAC genes to abiotic stresses have been widely studied, but their functions in response to biotic stress are little reported in plants, especially in crops. In the present study, we examined the functions of a novel tomato (Solanum lycopersicum) NAC protein (SlNAC35) in abiotic and biotic stress resistance by using transgenic tobacco. Expression analysis found that SlNAC35 expression was induced by drought stress, salt stress, bacterial pathogen, and signaling molecules, suggesting its involvement in plant responses to biotic and abiotic stimuli. Moreover, transgenic lines exhibited a greater number of lateral roots and longer root length compared with Vec lines (empty vector lines) after drought and salt treatment. These results indicate that overexpression of SlNAC35 promoted root growth and development under drought and salt stresses. Higher expressions of NtARF1, NtARF2 and NtARF8 were observed under drought and salt stresses in transgenic lines, suggesting that overexpression of SlNAC35 promoted growth and development of roots in transgenic lines possibly by involving auxin signaling and by regulating NtARF expression. In addition, SlNAC35 overexpression improved resistance to bacterial pathogen in transgenic tobacco, and reactive oxygen species may be in the upstream of salicylic acid (SA) signaling in transgenic tobacco during defense response.

Suppression of tomato SlNAC1 transcription factor delays fruit ripening.

Fruit ripening is a complex process involving many physiological and biochemical changes, including those for ethylene, carotenoid, and cell wall metabolism. Tomato (Solanum lycopersicum) serves as a research model for fruit development and ripening because it possesses numerous favorable genetic features. In this study, SlNAC1 was cloned. An antisense (AS) vector was constructed and transferred to tomato to further explore the function of SlNAC1. The results showed that AS fruits exhibited delayed ripening and a deeper red appearance when these fruits were fully ripened. Fully ripened AS fruits also produced higher total carotenoid and lycopene contents than those of the wild-type (WT) line. Ethylene production of AS fruits was delayed but occurred to a higher extent than that of WT fruits. The softening of AS fruits was slower than that of WT fruits. Endogenous abscisic acid (ABA) level in AS-4 fruits was lower than that in WT fruits. Exogenous ABA accelerated the softening of AS fruits. Furthermore, AS fruits demonstrated up-regulated expression of genes related to lycopene and ethylene biosynthesis but down-regulated expression of genes related to cell wall metabolism and ABA synthesis. Therefore, SlNAC1 is likely implicated in fruit ripening.

Enhancement of hepatocyte differentiation from human embryonic stem cells by Chinese medicine Fuzhenghuayu.

Chinese medicine, Fuzhenghuayu (FZHY), appears to prevent fibrosis progression and improve liver function in humans. Here we found that FZHY enhanced hepatocyte differentiation from human embryonic stem cells (hESC). After treatment with FZHY, albumin expression was consistently increased during differentiation and maturation process, and expression of metabolizing enzymes and transporter were also increased. Importantly, expression of mesenchymal cell and cholangiocyte marker was significantly reduced by treatment with FZHY, indicating that one possible mechanism of FZHY's role is to inhibit the formation of mesenchymal cells and cholangiocytes. Edu-labelled flow cytometric analysis showed that the percentage of the Edu positive cells was increased in the treated cells. These results indicate that the enhanced proliferation involved hepatocytes rather than another cell type. Our investigations further revealed that these enhancements by FZHY are mediated through activation of canonical Wnt and ERK pathways and inhibition of Notch pathway. Thus, FZHY not only promoted hepatocyte differentiation and maturation, but also enhanced hepatocyte proliferation. These results demonstrate that FZHY appears to represent an excellent therapeutic agent for the treatment of liver fibrosis, and that FZHY treatment can enhance our efforts to generate mature hepatocytes with proliferative capacity for cell-based therapeutics and for pharmacological and toxicological studies.

Ethanol negatively regulates hepatic differentiation of hESC by inhibition of the MAPK/ERK signaling pathway in vitro.

BACKGROUND: Alcohol insult triggers complex events in the liver, promoting fibrogenic/inflammatory signals and in more advanced cases, aberrant matrix deposition. It is well accepted that the regenerative capacity of the adult liver is impaired during alcohol injury. The liver progenitor/stem cells have been shown to play an important role in liver regeneration -in response to various chronic injuries; however, the effects of alcohol on stem cell differentiation in the liver are not well understood. METHODS: We employed hepatic progenitor cells derived from hESCs to study the impact of ethanol on hepatocyte differentiation by exposure of these progenitor cells to ethanol during hepatocyte differentiation. RESULTS: We found that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitor cells in a dose-dependent manner. There was also a moderate cell cycle arrest at G1/S checkpoint in the ethanol treated cells, which is associated with a reduced level of cyclin D1 in these cells. Ethanol treatment specifically inhibited the activation of the ERK but not JNK nor the p38 MAP signaling pathway. At the same time, the WNT signaling pathway was also reduced in the cells exposed to ethanol. Upon evaluating the effects of the inhibitors of these two signaling pathways, we determined that the Erk inhibitor replicated the effects of ethanol on the hepatocyte differentiation and attenuated the WNT/ß-catenin signaling, however, inhibitors of WNT only partially replicated the effects of ethanol on the hepatocyte differentiation. CONCLUSION: Our results demonstrated that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitors through inhibiting the MAPK/ERK signaling pathway, and subsequently attenuating the WNT signaling pathway. Thus, our finding provides a novel insight into the mechanism by which alcohol regulates cell fate selection of hESC-derived hepatic progenitor cells, and the identified pathways may provide therapeutic targets aimed at promoting liver repair and regeneration during alcoholic injury.

Highly efficient differentiation of functional hepatocytes from human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) hold great potential for use in regenerative medicine, novel drug development, and disease progression/developmental studies. Here, we report highly efficient differentiation of hiPSCs toward a relatively homogeneous population of functional hepatocytes. hiPSC-derived hepatocytes (hiHs) not only showed a high expression of hepatocyte-specific proteins and liver-specific functions, but they also developed a functional biotransformation system including phase I and II metabolizing enzymes and phase III transporters. Nuclear receptors, which are critical for regulating the expression of metabolizing enzymes, were also expressed in hiHs. hiHs also responded to different compounds/inducers of cytochrome P450 as mature hepatocytes do. To follow up on this observation, we analyzed the drug metabolizing capacity of hiHs in real time using a novel ultra performance liquid chromatography-tandem mass spectrometry. We found that, like freshly isolated primary human hepatocytes, the seven major metabolic pathways of the drug bufuralol were found in hiHs. In addition, transplanted hiHs engrafted, integrated, and proliferated in livers of an immune-deficient mouse model, and secreted human albumin, indicating that hiHs also function in vivo. In conclusion, we have generated a method for the efficient generation of hepatocytes from induced pluripotent stem cells in vitro and in vivo, and it appears that the cells function similarly to primary human hepatocytes, including developing a complete metabolic function. These results represent a significant step toward using patient/disease-specific hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies.