Regulation of depressive-like behaviours by palmitoylation: Role of AKAP150 in the basolateral amygdala.

BACKGROUND AND PURPOSE: Protein palmitoylation is involved in learning and memory, and in emotional disorders. Yet, the underlying mechanisms in these processes remain unclear. Herein, we describe that A-kinase anchoring protein 150 (AKAP150) is essential and sufficient for depressive-like behaviours in mice via a palmitoylation-dependent mechanism. EXPERIMENTAL APPROACH: Depressive-like behaviours in mice were induced by chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS). Palmitoylated proteins in the basolateral amygdala (BLA) were assessed by an acyl-biotin exchange assay. Genetic and pharmacological approaches were used to investigate the role of the DHHC2-mediated AKAP150 palmitoylation signalling pathway in depressive-like behaviours. Electrophysiological recording, western blotting and co-immunoprecipitation were performed to define the mechanistic pathway. KEY RESULTS: Chronic stress successfully induced depressive-like behaviours in mice and enhanced AKAP150 palmitoylation in the BLA, and a palmitoylation inhibitor was enough to reverse these changes. Blocking the AKAP150-PKA interaction with the peptide Ht-31 abolished the CRS-induced AKAP150 palmitoylation signalling pathway. DHHC2 expression and palmitoylation levels were both increased after chronic stress. DHHC2 knockdown prevented CRS-induced depressive-like behaviours, as well as attenuating AKAP150 signalling and synaptic transmission in the BLA in CRS-treated mice. CONCLUSION AND IMPLICATIONS: These results delineate that DHHC2 modulates chronic stress-induced depressive-like behaviours and synaptic transmission in the BLA via the AKAP150 palmitoylation signalling pathway, and this pathway may be considered as a promising novel therapeutic target for major depressive disorder.

DHHC2 regulates fear memory formation, LTP, and AKAP150 signaling in the hippocampus.

Palmitoyl acyltransferases (PATs) have been suggested to be involved in learning and memory. However, the underlying mechanisms have not yet been fully elucidated. Here, we found that the activity of DHHC2 was upregulated in the hippocampus after fear conditioning, and DHHC2 knockdown impaired fear induced memory and long-term potentiation (LTP). Additionally, the activity of DHHC2 and its synaptic expression were increased after high frequency stimulation (HFS) or glycine treatment. Importantly, fear learning selectively augmented the palmitoylation level of AKAP150, not PSD-95, and this effect was abolished by DHHC2 knockdown. Furthermore, 2-bromopalmitic acid (2-BP), a palmitoylation inhibitor, attenuated the increased palmitoylation level of AKAP150 and the interaction between AKAP150 and PSD-95 induced by HFS. Lastly, DHHC2 knockdown reduced the phosphorylation level of GluA1 at Ser845, and also induced an impairment of LTP in the hippocampus. Our results suggest that DHHC2 plays a critical role in regulating fear memory via AKAP150 signaling.

Targeted activation of Nrf2/HO-1 pathway by Corynoline alleviates osteoporosis development.

Oxidative stress is preferentially treated as a risk factor for the development and progression of osteoporosis. Corynoline as a component of Corydalis bungeana Turcz presents antioxidative and anti-inflammatory properties. In the present study, the effects of Corynoline on osteoblasts following hydrogen peroxide (H2O2)-induced injury were evaluated accompanied by the investigation of the molecular mechanisms involved. It was found that Corynoline downregulated the intracellular reactive oxygen species (ROS) generation and restored the osteogenic potential of the disrupted osteoblasts by H2O2 exposure. Furthermore, Corynoline was revealed to activate the Nrf2/HO-1 signaling pathway, while ML385 (an Nrf2 inhibitor) would prevent the Corynoline-mediated positive effects on the disrupted osteoblasts. In terms of the animal experiments, Corynoline treatment contributed to a significantly alleviated bone loss. These findings indicate that Corynoline may significantly attenuate the H2O2-induced oxidative damage of osteoblasts via the Nrf2/HO-1 signaling pathway, providing novel insights to the development of treatments for osteoporosis induced by oxidative injury.

Arginine shortage induces replication stress and confers genotoxic resistance by inhibiting histone H4 translation and promoting PCNA ubiquitination.

The arginine dependency of cancer cells creates metabolic vulnerability. In this study, we examine the impact of arginine availability on DNA replication and genotoxicity resistance. Using DNA combing assays, we find that limiting extracellular arginine results in the arrest of cancer cells at S phase and a slowing or stalling of DNA replication. The translation of new histone H4 is arginine dependent and influences DNA replication. Increased proliferating cell nuclear antigen (PCNA) occupancy and helicase-like transcription factor (HLTF)-catalyzed PCNA K63-linked polyubiquitination protect arginine-starved cells from DNA damage. Arginine-deprived cancer cells display tolerance to genotoxicity in a PCNA K63-linked polyubiquitination-dependent manner. Our findings highlight the crucial role of extracellular arginine in nutrient-regulated DNA replication and provide potential avenues for the development of cancer treatments.

Arginine shortage induces replication stress and confers genotoxic resistance by inhibiting histone H4 translation and promoting PCNA polyubiquitination.

The unique arginine dependencies of cancer cell proliferation and survival creates metabolic vulnerability. Here, we investigate the impact of extracellular arginine availability on DNA replication and genotoxic resistance. Using DNA combing assays, we find that when extracellular arginine is limited, cancer cells are arrested at S-phase and DNA replication forks slow or stall instantly until arginine is re-supplied. The translation of new histone H4 is arginine-dependent and impacts DNA replication and the expression of newly synthesized histone H4 is reduced in the avascular nutrient-poor breast cancer xenograft tumor cores. Furthermore, we demonstrate that increased PCNA occupancy and HLTF-catalyzed PCNA K63-linked polyubiquitination protects arginine-starved cells from hydroxyurea-induced, DNA2-catalyzed nascent strand degradation. Finally, arginine-deprived cancer cells are tolerant to genotoxic insults in a PCNA K63-linked polyubiquitination-dependent manner. Together, these findings reveal that extracellular arginine is the "linchpin" for nutrient-regulated DNA replication. Such information could be leveraged to expand current modalities or design new drug targets against cancer.

A Novel Anti-Osteoporosis Mechanism of VK2: Interfering with Ferroptosis via AMPK/SIRT1 Pathway in Type 2 Diabetic Osteoporosis.

Type 2 diabetic osteoporosis (T2DOP) is a chronic bone metabolic disease. Compared with traditional menopausal osteoporosis, the long-term high glucose (HG) microenvironment increases patients' risk of fracture and osteonecrosis. We were accumulating evidence that implicated ferroptosis as a pivotal mechanism of glucolipotoxicity-mediated death of osteocytes and osteoblast, a novel form of programmed cell death resulting from uncontrolled lipid peroxidation depending on iron. Vitamin K2 (VK2), a fat-soluble vitamin, is clinically applied to prevent osteoporosis and improve coagulation. This study aimed to clarify the role and mechanism of VK2 in HG-mediated ferroptosis. We established the mouse T2DOP model by intraperitoneal injection of streptozotocin solution and a high-fat and high-sugar diet. We also cultured bone marrow mesenchymal stem cells (BMSCs) in HG to simulate the diabetic environment in vitro. Based on our data, VK2 inhibited HG-mediated bone loss and ferroptosis, the latter manifested by decreased levels of mitochondrial reactive oxygen species, lipid peroxidation, and malondialdehyde and increased glutathione in vitro. In addition, VK2 treatment was capable of restoring bone mass and strengthening the expression of SIRT1, GPX4, and osteogenic markers in the distal femurs. As for further mechanism exploration, we found that VK2 could activate AMPK/SIRT1 signaling, and knockdown of SIRT1 by siRNA prevented the VK2-mediated positive effect in HG-cultured BMSCs. Summarily, VK2 could ameliorate T2DOP through the activation of the AMPK/SIRT1 signaling pathway to inhibit ferroptosis.

CORM-3 Attenuates Oxidative Stress-Induced Bone Loss via the Nrf2/HO-1 Pathway.

Bone metabolism occurs in the entire life of an individual and is required for maintaining skeletal homeostasis. The imbalance between osteogenesis and osteoclastogenesis eventually leads to osteoporosis. Oxidative stress is considered a major cause of bone homeostasis disorder, and relieving excessive oxidative stress in bone mesenchymal stem cells (BMSCs) is a potential treatment strategy for osteoporosis. Carbon monoxide releasing molecule-3 (CORM-3), the classical donor of carbon monoxide (CO), possesses antioxidation, antiapoptosis, and anti-inflammatory properties. In our study, we found that CORM-3 could reduce reactive oxygen species (ROS) accumulation and prevent mitochondrial dysfunction thereby restoring the osteogenic potential of the BMSCs disrupted by hydrogen peroxide (H2O2) exposure. The action of CORM-3 was preliminarily considered the consequence of Nrf2/HO-1 axis activation. In addition, CORM-3 inhibited osteoclast formation in mouse primary bone marrow monocytes (BMMs) by inhibiting H2O2-induced polarization of M1 macrophages and endowing macrophages with M2 polarizating ability. Rat models further demonstrated that CORM-3 treatment could restore bone mass and enhance the expression of Nrf2 and osteogenic markers in the distal femurs. In summary, CORM-3 is a potential therapeutic agent for the treatment of osteoporosis.

Therapeutic effect of SIRT3 on glucocorticoid-induced osteonecrosis of the femoral head via intracellular oxidative suppression.

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a serious complication after long-term or excess administration of clinical glucocorticoids intervention, and the pathogenic mechanisms underlying have not been clarified yet. Oxidative stress is considered as a major cause of bone homeostasis disorder. This study is aimed to explore the potential relevance between SIRT3 and GIONFH, as well as the effect of resveratrol, which has been reported for its role in SIRT3 activation, on dexamethasone-induced oxidative stress and mitochondrial compromise in bone marrow stem cells (BMSCs). In this study, our data showed that SIRT3 level was declined in GIONFH rat femoral head, corresponding to a resultant decrease of SIRT3 expression in dexamethasone-treated BMSCs in vitro. We also found that dexamethasone could result in oxidative injury in BMSCs, and resveratrol treatment reduced this deleterious effect via a SIRT3-dependent manner. Moreover, our results demonstrated that rewarding effect of resveratrol on BMSCs osteogenic differentiation was via activation of AMPK/PGC-1α/SIRT3 axis. Meanwhile, resveratrol administration prevented the occurrence of GIONFH, enhanced SIRT3 expression and reduced oxidative level in GIONFH model rats. Therefore, our study provides basic evidence that SIRT3 may be a promising therapeutic target for GIONFH treatment and resveratrol could be an ideal agent for clinical uses.

Effect of the Seasonal Climatic Variations on the Accumulation of Fruit Volatiles in Four Grape Varieties Under the Double Cropping System.

The double cropping system has been widely applied in many subtropical viticultural regions. In the 2-year study of 2014-2015, four grape varieties were selected to analyze their fruit volatile compounds in four consecutive seasons in the Guangxi region of South China, which had a typical subtropical humid monsoon climate. Results showed that berries of winter seasons had higher concentrations of terpenes, norisoprenoids, and C6/C9 compounds in "Riesling," "Victoria," and "Muscat Hamburg" grapes in both of the two vintages. However, in the "Cabernet Sauvignon" grapes, only the berries of the 2014 winter season had higher terpene concentrations, but lower norisoprenoid concentrations than those of the corresponding summer season. The Pearson correlation analysis showed the high temperature was the main climate factor that affected volatile compounds between the summer and winter seasons. Hexanal, γ-terpinene, terpinen-4-ol, cis-furan linalool oxide, and trans-pyran linalool oxide were all negatively correlated with the high-temperature hours in all of the four varieties. Transcriptome analysis showed that the upregulated VviDXSs, VviPSYs, and VviCCDs expressions might contribute to the accumulations of terpenes or norisoprenoids in the winter berries of these varieties. Our results provided insights into how climate parameters affected grape volatiles under the double cropping system, which might improve the understanding of the grape berries in response to the climate changes accompanied by extreme weather conditions in the future.

Effect of the Seasonal Climatic Variations on the Flavonoid Accumulation in Vitis vinifera cvs. 'Muscat Hamburg' and 'Victoria' Grapes under the Double Cropping System.

Under the double cropping system, berries usually showed significant quality variations in the summer and winter seasons. In the two-year/four-consecutive-season study, two table grapes of 'Muscat Hamburg' and 'Victoria' were investigated to determine the phenolic compounds in their berries. Different from those of the summer season, the berries in the winter season suffered no high-temperature stress since veraison to harvest in 2014 and 2015. The variations in the season temperatures led to a higher anthocyanin concentration in the winter season berries of 'Muscat Hamburg' grapes than that in the summer berries, while the summer season berries had higher proportions of acylated and methylated anthocyanins than those in the winter season berries. Similar to the anthocyanins, the winter season berries also had a higher flavonol concentration in both varieties. Transcriptome analysis showed that the upregulated genes involved in the flavonoid pathway in the winter season berries were agreed with the changes found in the metabolites. However, the influence of the growing seasons on the flavanols was not consistent in the two varieties, and the variations in VviLARs between the grapes of 'Muscat Hamburg' and 'Victoria' might be the cause. This research helped us better understand the double cropping system and how the climate factors affected the phenolic compounds in the double cropping system.

Effects of gibberellic acid (GA3 ) application before anthesis on rachis elongation and berry quality and aroma and flavour compounds in Vitis vinifera L. 'Cabernet Franc' and 'Cabernet Sauvignon' grapes.

BACKGROUND: Gibberellic acid (GA3 ), a plant-growth regulator, is often used to obtain enlarged table grape berries and induce seedlessness in them. However, the effects of GA3 on rachis elongation and bunch compactness have seldom been reported in wine-grape production. We assessed the effects of GA3 spraying on wine-grape inflorescences and bunches and their practical implications for viticulture in the Jiaodong Peninsula, China. RESULTS: Various GA3 concentrations were sprayed on field-grown Vitis vinifera L. 'Cabernet Franc' (CF) and 'Cabernet Sauvignon' (CS) grapevines before anthesis in the Jiaodong Peninsula, China, in 2015 and 2016. Inflorescence length during berry development was measured, and flavonoids and aroma compounds in the fruit were detected by high-performance liquid chromatography - mass spectrometry (HPLC-MS) and gas chromatography - mass spectrometry (GC-MS), respectively. For both cultivars, 50 and 100 mg L-1 GA3 caused significant elongation of the rachis, whereas there was no significant effect on inflorescence growth and berry seed number. Anthocyanin, flavonol, and flavan-3-ol levels in mature berries were not significantly influenced by GA3 spraying, whereas C13 -norisoprenoids were modified. CONCLUSION: The application of 50-100 mg L-1 GA3 prior to grapevine anthesis caused elongation of inflorescences and bunches, and eased cluster compactness in CF and CS, and no negative effects were observed on the yield and seed numbers. The concentration and composition of flavonoids and most aroma compounds were not influenced, except that the norisoprenoids were increased by 50 mg L-1 GA3 applications. © 2020 Society of Chemical Industry.

Modulation of volatile compound metabolome and transcriptome in grape berries exposed to sunlight under dry-hot climate.

BACKGROUND: Basal leaf removal is widely practiced to increase grape cluster sunlight exposure that controls berry rot and improves quality. Studies on its influence on volatile compounds in grape berries have been performed mostly in Mediterranean or marine climate regions. It is uncertain whether similar efficiency can be achieved when grape berries are grown under continental climate. This study aimed to dissect the variation in volatile compound production and transcriptome in sunlight-exposed grape berries in a dry-hot climate region and to propose the key genes related to the variation. RESULTS: Four cluster sunlight exposure strategies, including basal leaf removal at pepper-corn size stage, leaf removal at véraison (LR-V), leaf moving at véraison (LM-V), and half-leaf removal at véraison, were implemented at the north foot of the Mt. Tianshan region of northwestern China. Various cluster exposure treatments resulted in a decline in the concentrations of norisoprenoids and monoterpenes in ripening grape berries. Both ß-carotene and lutein, the substrates of norisoprenoid biosynthesis, were reduced by cluster sunlight exposure. K-means cluster analysis showed that some genes involved in biosynthesis such as VviTPS55, VviTPS60, VviTPS66, VviCCD4a and VviCCD4b exhibited lower expression levels in exposed berries at least at one of the tested stages. Two C6-derived esters with fruity attributes, ethyl hexanoate and hexyl acetate, were reduced markedly. In contrast, main C6 alcohol compound levels were elevated in the LR-V- and LM-V-treated grape berries, which corresponded to the up-regulated expression of VviLOXA, VviLOXO and VviADH1 in the oxylipin pathway. Most of the differentially expressed genes in the exposed and control berries were enriched to the "stress response" processes, and this transcriptome difference was accumulated as the berries matured. Besides, LR-V treatment stimulated a significant up-regulation in photosynthesis-related genes in the grape berries, which did not happen with LM-V treatment. CONCLUSIONS: Cluster sunlight exposure in dry-hot climate viticulture resulted in different volatile-targeted transcriptomic and metabolic responses from those obtained in the temperate Mediterranean or marine climate region. Therefore, a modified canopy management should be adopted to improve the aroma of grape berries.

Systematic assessment of complications after robotic-assisted total versus distal gastrectomy for advanced gastric cancer: A retrospective propensity score-matched study using Clavien-Dindo classification.

BACKGROUND: Despite increasing evidence demonstrated robot-assisted distal gastrectomy (RADG) is safe and feasible for the treatment of advanced gastric cancer (AGC), robot-assisted total gastrectomy (RATG) remains a challenging procedure due to its technical difficulties and possible postoperative complications (POCs). This study aimed to systematically evaluate POCs following RATG. METHODS: Between January 2017 and January 2019, 319 AGC patients with pathological stage T2-4aN0-3M0 who underwent RADG or RATG were enrolled. POCs were stratified using the Clavien-Dindo classification. One-to-one propensity score matching was performed to reduce confounding differences. RESULTS: After matching, 266 patients met the criteria for further analysis. Ultimately, 64 patients (24.1%) who developed POCs had 126 clinical manifestation events. Overall the POCs rate was significantly greater after RATG in comparison with RADG (29.3% vs. 18.8%; P = 0.045), and more major POCs (Clavien-Dindo grade ≥ IIIa) were observed in the RATG group (14.3% vs. 5.3%; P = 0.013). The POCs were then classified into local and systemic POCs. The rates of local POCs (35.3% vs. 19.5%; P = 0.004) and systemic POCs (24.8% vs. 15.0%; P = 0.046) were significantly higher in the RATG group than the RADG group. Subgroup analysis showed that the anastomotic leakage rate was higher after RATG (5.3% vs. 0.8%; P = 0.031), whereas the remaining POCs were similar between the two groups. Patients with higher POCs significantly had longer postoperative length of stay (R = 0.895, P = 0.003). Multivariate analysis confirmed age, extent of resection, and TNM stage were risk factors for all POCs. CONCLUSIONS: These findings demonstrated that RATG is technically feasible and safe for treatment of AGC with acceptable morbidity and mortality rates. The POCs rate of RATG was higher than RADG, especially for anastomotic leakage. More effective anastomotic techniques are needed in RATG to prevent leakage.

Rootstock-Mediated Effects on Cabernet Sauvignon Performance: Vine Growth, Berry Ripening, Flavonoids, and Aromatic Profiles.

Rootstocks are widely used in viticulture due to their resistance to biotic and abiotic stress. Additionally, rootstocks can affect vine growth and berry quality. This study evaluated the effects of eight rootstocks (101-14, 110R, 5A, 5BB, Ganzin 1, Harmony, Riparia Gloire, and SO4) on the vine growth, berry ripening, and flavonoids and aromatic profiles of Cabernet Sauvignon in two consecutive seasons (2015⁻2016). With few exceptions, minor differences were observed among grafted and own-rooted vines. Own-rooted vines produced the least pruning weight but the highest yield. 101-14, 5BB, and SO4 slightly reduced total soluble solids, but increased acidity, showing tendencies for retarding maturation. Ganzin 1 inhibited the accumulation of flavan-3-ols in berry skins. Furthermore, concentrations and proportions of epicatechin-3-O-galate were decreased by rootstocks, except for 110R. 5A, Harmony, and Riparia Gloire enhanced flavonol concentrations. SO4 slightly decreased most of the individual anthocyanin concentrations. With respect to volatile compounds, 110R, Riparia Gloire, and SO4 induced reductions in concentrations of total esters, whilst 101-14, Ganzin 1, 110R, and 5BB led to increases in the concentrations of C13-norisoprenoids. Therefore, with respect to the negative effects of SO4 on berry ripening and the accumulation of anthocyanin and volatile esters, SO4 is not recommended in practice.

Identification of key genes and associated pathways in KIT/PDGFRA wild­type gastrointestinal stromal tumors through bioinformatics analysis.

Gastrointestinal stromal tumors (GISTs) are the most common type of mesenchymal tumor in the gastrointestinal tract. The present study aimed to identify the potential candidate biomarkers that may be involved in the pathogenesis and progression of v­kit Hardy­Zuckerman 4 feline sarcoma viral oncogene homolog (KIT)/platelet­derived growth factor receptor α (PDGFRA) wild­type GISTs. A joint bioinformatics analysis was performed to identify the differentially expressed genes (DEGs) in wild­type GIST samples compared with KIT/PDGFRA mutant GIST samples. Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs was conducted using Database for Annotation, Visualization and Integrated Discovery and KEGG Orthology­Based Annotation System (KOBAS) online tools, respectively. Protein­protein interaction (PPI) networks of the DEGs were constructed using Search Tool for the Retrieval of Interacting Genes online tool and Cytoscape, and divided into sub­networks using the Molecular Complex Detection (MCODE) plug­in. Furthermore, enrichment analysis of DEGs in the modules was analyzed with KOBAS. In total, 546 DEGs were identified, including 238 upregulated genes primarily enriched in 'cell adhesion', 'biological adhesion', 'cell­cell signaling', 'PI3K­Akt signaling pathway' and 'ECM­receptor interaction', while the 308 downregulated genes were predominantly involved in 'inflammatory response', 'sterol metabolic process' and 'fatty acid metabolic process', 'small GTPase mediated signal transduction', 'cAMP signaling pathway' and 'proteoglycans in cancer'. A total of 25 hub genes were obtained and four modules were mined from the PPI network, and sub­networks also revealed these genes were primarily involved in significant pathways, including 'PI3K­Akt signaling pathway', 'proteoglycans in cancer', 'pathways in cancer', 'Rap1 signaling pathway', 'ECM­receptor interaction', 'phospholipase D signaling pathway', 'ras signaling pathway' and 'cGMP­PKG signaling pathway'. These results suggested that several key hub DEGs may serve as potential candidate biomarkers for wild­type GISTs, including phosphatidylinositol­4,5­bisphosphate 3­kinase, catalytic subunit γ, insulin like growth factor 1 receptor, hepatocyte growth factor, thrombospondin 1, Erb­B2 receptor tyrosine kinase 2 and matrix metallopeptidase 2. However, further experiments are required to confirm these results.

Effects of cluster thinning on vine photosynthesis, berry ripeness and flavonoid composition of Cabernet Sauvignon.

Cluster thinning is a common practice for regulating vine yield and grape quality. The effects of cluster thinning on vine photosynthesis, berry ripeness and flavonoid composition of V. vinifera L. Cabernet Sauvignon were evaluated during two seasons. Half of the clusters were removed at pea-size and veraison relative to two controls, respectively. Both cluster thinning treatments significantly increased pruning weight and decreased yield. No effects of cluster thinning on berry growth, ripeness and flavonol composition were observed. Early cluster thinning decreased the photosynthetic rate at pea-size, but the effect diminished at post-veraison. Early cluster thinning significantly promoted the biosynthesis of anthocyanins but decreased the proportion of 3'5'-hydroxylated and acylated anthocyanins at veraison. Late cluster thinning decreased the proportions of 3'5'-hydroxylated and acylated anthocyanins. Additionally, Cluster thinning showed inconsistent effects on flavan-3-ol composition over the two seasons.

Dissecting the Variations of Ripening Progression and Flavonoid Metabolism in Grape Berries Grown under Double Cropping System.

A double cropping system has been commercially adopted in southern China, where there is abundant sunshine and heat resources. In this viticulture system, the first growing season normally starts as a summer cropping cycle; then, the vine is pruned and forced, resulting in a second crop in winter. Due to climate differences between the summer and winter growing seasons, grape ripening progression and flavonoid metabolism vary greatly. Here, the metabolites and transcriptome of flavonoid pathways were analyzed in grapes grown under two growing seasons at different stages. Notably, the winter cropping cycle strongly increased flavonoid levels by several times in comparison to summer grapes, while the summer season took a major toll on anthocyanin and flavonol accumulation, since the winter cropping greatly triggered the expression of upstream genes in the flavonoid pathway in a coordinated expression pattern. Moreover, the ratio of VviF3'5'Hs (flavonoid 3'5'-hydroxylase) to VviF3'Hs (flavonoid 3'-hydroxylase) transcript levels correlated remarkably well with the ratio of 3'5'-substituted to 3'-substituted flavonoids, which was presumed to control the flux of intermediates into different flavonoid branches. On the other hand, the phenological phase also varied greatly in the two crops. Compared to summer cropping, winter growing season accelerated the duration from budburst to veraison, therefore advancing the onset of ripening, but also prolonging the duration of ripening progression due to the purposes to harvest high-quality grapes. The differential expression pattern of hormone-related genes between the two cropping cycles might explain this phenomenon.

Comparison of transcriptional expression patterns of carotenoid metabolism in 'Cabernet Sauvignon' grapes from two regions with distinct climate.

The downstream flux of carotenoid metabolism in grape berries includes the biosynthesis of norisoprenoids, a group of important aroma compounds, and the production of ABA, a well-known plant hormone. This study focused on the transcriptional profiling comparison of genes participating in the biosynthesis of carotenoids, norisoprenoids, and ABA in Vitis vinifera 'Cabernet Sauvignon' grapes at pea size, veraison, and ripening stages. The grapes were obtained from Changli (CL, eastern China) and Gaotai (GT, western China) regions and analyzed using RNA-sequencing technology. The transcripts required for the carotenoid biosynthesis pathway showed a coordinated expression pattern, mainly expressing at green stage for CL and at veraison for GT, respectively. However, the carotenoid content evolution was not coincident with the timing and pattern of related gene expressions, since more carotenoids were accumulated at veraison in CL relative to two weeks before veraison in GT. Interestingly, norisoprenoid content was higher in GT than in CL, particularly at veraison and ripening, while the key gene encoding carotenoid cleavage dioxygenases, VvCCD1, showed an inverse relationship within the two regions. Higher flux was expected through the carotenoid pathway into ABA production in GT, based on the higher expression level of 9-cis-epoxycarotenoid dioxygenase and drought growing conditions. Most components involved in ABA and ethylene signaling showed distinct expression profiles in the two regions. These results revealed that downstream flux of carotenoid metabolism in grape berries showed regional differences. This study lays a foundation for future research to explore the molecular basis of climatic influences on carotenoid, norisoprenoid, and ABA biosynthesis.

Minimally invasive surgical technique: Percutaneous external fixation combined with titanium elastic nails for selective treatment of tibial fractures.

INTRODUCTION: Several techniques have been described to treat tibial fractures, which respectively remains defects. This article presents a novel intra- and extramedullary fixation technique: percutaneous external fixator combined with titanium elastic nails (EF-TENs system). The purpose of this study is to introduce this new minimally invasive surgical technique and selective treatment of tibial fractures, particularly in segmental fractures, diaphysis fractures accompanied with distal or proximal bone subfissure, or fractures with poor soft-tissue problems. METHODS: Following ethical approval, thirty-two patients with tibial fractures were treated by the EF-TENs system between January 2010 and December 2012. The follow-up studies included clinical and radiographic examinations. All relevant outcomes were recorded during follow-up. RESULTS: All thirty-two patients were achieved follow-ups. According to the AO classification, 3 Type A, 9 Type B and 20 Type C fractures were included respectively. According to the Anderson-Gustilo classification, there were 5 Type Grade II, 3 Type Grade IIIA and 2 Type Grade IIIB. Among 32 patients, 8 of them were segmental fractures. 12 fractures accompanied with bone subfissure. Results showed no nonunion case, with an average time of 23.7 weeks (range, 14-32 weeks). Among them, there were 3/32 delayed union patients and 0/32 malunion case. 4/32 patients developed a pin track infection and no patient suffered deep infection. The external fixator was removed with a mean time of 16.7 weeks (range, 10-26 weeks). Moreover, only 1/32 patient suffered with the restricted ROM of ankle, none with the restricted ROM of knee. CONCLUSION: This preliminary study indicated that the EF-TENs system, as a novel intra- and extramedullary fixation technique, had substantial effects on selective treatment of tibial fractures.

Molecular and biochemical characterization of the UDP-glucose: Anthocyanin 5-O-glucosyltransferase from Vitis amurensis.

Generally, red Vitis vinifera grapes only contain monoglucosidic anthocyanins, whereas most non-vinifera red grapes of the Vitis genus have both monoglucosidic and bis-glucosidic anthocyanins, the latter of which are believed to be more hydrophilic and more stable. Although previous studies have established the biosynthetic mechanism for formation of monoglucosidic anthocyanins, less attention has been paid to that of bis-glucosidic anthocyanins. In the present research, the full-length cDNA of UDP-glucose: anthocyanin 5-O-glucosyltransferase from Vitis amurensis Rupr. cv. 'Zuoshanyi' grape (Va5GT) was cloned. After acquisition and purification of recombinant Va5GT, its enzymatic parameters were systematically analyzed in vitro. Recombinant Va5GT used malvidin-3-O-glucoside as its optimum glycosidic acceptor when UDP-glucose was used as the glycosidic donor. Va5GT-GFP was found to be located in the cytoplasm by analyzing its subcellular localization with a laser-scanning confocal fluorescence microscope, and this result was coincident with its metabolic function of modifying anthocyanins in grape cells. Furthermore, the relationship between the transcriptional expression of Va5GT and the accumulation of anthocyanidin bis-glucosides during berry development suggested that Va5GT is a key enzyme in the biosynthesis of bis-glucosidic anthocyanins in V. amurensis grape berries.