Biotransformation-guided purification of a novel glycoside derived from the extracts of Chinese herb Baizhi.

Our pursuit of new compounds with enhanced bioavailability and bioactivity prompted us to employ the biotransformation-guided purification (BGP) approach which leverages proficient in vitro biotransformation techniques. Angelica dahurica roots, also called Baizhi in Chinese traditional medicine, are famous for their anti-inflammatory and analgesic properties. Herein, we applied the BGP methodology to Baizhi extracts, employing Deinococcus geothermalis amylosucrase (DgAS), an enzyme demonstrating catalytic competence across diverse substrates, for biotransformation. Initiating with a 70 % methanol extraction, we obtained the crude extract of commercial Baizhi powder, followed by an additional extraction using ethyl acetate. Notably, reactions performed on this extract yielded limited quantities of novel compounds. Subsequently, the extract underwent partitioning into four fractions based on HPLC profiling, leading to the successful isolation of a compound with significant yield from fraction 2 mixtures upon reaction with DgAS. Structural elucidation confirmed the compound as byakangelicin-7″-O-α-glucopyranoside (BG-G), a new alpha glycoside derivative of byakangelicin. Furthermore, validation experiments verified the capacity of DgAS to glycosylate pure byakangelicin, yielding BG-G. Remarkably, the aqueous solubility of BG-G exceeded that of byakangelicin by over 29,000-fold. In conclusion, BGP emerges as a potent strategy combining traditional medicinal insights with robust enzymatic tools for generating new compounds.

Molecular cloning and functional characterization of an isoflavone glucosyltransferase from Pueraria thomsonii.

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.

Salicylic acid carboxyl glucosyltransferase UGT87E7 regulates disease resistance in Camellia sinensis.

Plant immune response following pathogenic infection is regulated by plant hormones, and salicylic acid (SA) and its sugar conjugates play important roles in establishing basal resistance. Here, the important pathogen Pseudopestalotiopsis camelliae-sinensis (Pcs) was isolated from tea gray blight, one of the most destructive diseases in tea plantations. Transcriptomic analysis led to the discovery of the putative Camellia sinensis UDP-glucosyltransferase CsUGT87E7 whose expression was significantly induced by SA application and Pcs infection. Recombinant CsUGT87E7 glucosylates SA with a Km value of 12 µM to form SA glucose ester (SGE). Downregulation reduced the accumulation of SGE, and CsUGT87E7-silenced tea plants exhibited greater susceptibility to pathogen infection than control plants. Similarly, CsUGT87E7-silenced tea leaves accumulated significantly less SA after infection and showed reduced expression of pathogenesis-related genes. These results suggest that CsUGT87E7 is an SA carboxyl glucosyltransferase that plays a positive role in plant disease resistance by modulating SA homeostasis through a mechanism distinct from that described in Arabidopsis (Arabidopsis thaliana). This study provides insight into the mechanisms of SA metabolism and highlights the role of SGE in the modulation of plant disease resistance.

Molecular cloning and functional characterization of an isoflavone glucosyltransferase from Pueraria thomsonii / 中国天然药物

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.

Enhanced Accumulation of Betulinic Acid in Transgenic Hairy Roots of Senna obtusifolia Growing in the Sprinkle Bioreactor and Evaluation of Their Biological Properties in Various Biological Models.

Betulinic acid, which is found in transgenic roots of Senna obtusifolia (L.) H.S.Irwin & Barneby, is a pentacyclic triterpene with distinctive pharmacological activities. In this study, we report the differences in the content of betulinic acid and selected anthraquinones in transgenic S. obtusifolia hairy roots with overexpression of the PgSS1 gene (SOPSS2 line) and in transformed hairy roots without this genetic construct (SOA41 line). Both hairy root lines grew in 10 L sprinkle bioreactor. Additionally, the extracts obtained from this plant material were used for biological tests. Our results demonstrated that the SOPSS2 hairy root cultures from the bioreactor showed an increase in the content of betulinic acid (38.125 mg/g DW), compared to the SOA41 hairy root line (4.213 mg/g DW). Biological studies have shown a cytotoxic and antiproliferative effect on U-87MG glioblastoma cells, and altering the level of apoptotic proteins (Bax, p53, Puma and Noxa). Antimicrobial properties were demonstrated for both tested extracts, with a stronger effect of SOPSS2 extract. Moreover, both extracts showed moderate antiviral properties on norovirus surrogates.

Natural products targeting the synthesis of ß(1,3)-D-glucan and chitin of the fungal cell wall. Existing drugs and recent findings.

BACKGROUND: During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viability of fungi, the inhibition of cell wall synthesizing machinery, such as ß(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of ß(1-3)-D-glucan and chitin, respectively, represent an ideal mode of action of antifungal agents. Although the echinocandins anidulafungin, caspofungin and micafungin are clinically well-established GS inhibitors for the treatment of invasive fungal infections, much effort must still be made to identify inhibitors of other enzymes and processes involved in the synthesis of the fungal cell wall. PURPOSE: Since natural products (NPs) have been the source of several antifungals in clinical use and also have provided important scaffolds for the development of semisynthetic analogues, this review was devoted to investigate the advances made to date in the discovery of NPs from plants that showed capacity of inhibiting cell wall synthesis targets. The chemical characterization, specific target, discovery process, along with the stage of development are provided here. METHODS: An extensive systematic search for NPs against the cell wall was performed considering all the articles published until the end of 2020 through the following scientific databases: NCBI PubMed, Scopus and Google Scholar and using the combination of the terms "natural antifungals" and "plant extracts" with "fungal cell wall". RESULTS: The first part of this review introduces the state of the art of the structure and biosynthesis of the fungal cell wall and considers exclusively those naturally produced GS antifungals that have given rise to both existing semisynthetic approved drugs and those derivatives currently in clinical trials. According to their chemical structure, natural GS inhibitors can be classified as 1) cyclic lipopeptides, 2) glycolipids and 3) acidic terpenoids. We also included nikkomycins and polyoxins, NPs that inhibit the CS, which have traditionally been considered good candidates for antifungal drug development but have finally been discarded after enduring unsuccessful clinical trials. Finally, the review focuses in the most recent findings about the growing field of plant-derived molecules and extracts that exhibit activity against the fungal cell wall. Thus, this search yielded sixteen articles, nine of which deal with pure compounds and seven with plant extracts or fractions with proven activity against the fungal cell wall. Regarding the mechanism of action, seven (44%) produced GS inhibition while five (31%) inhibited CS. Some of them (56%) interfered with other components of the cell wall. Most of the analyzed articles refer to tests carried out in vitro and therefore are in early stages of development. CONCLUSION: This report delivers an overview about both existing natural antifungals targeting GS and CS activities and their mechanisms of action. It also presents recent discoveries on natural products that may be used as starting points for the development of potential selective and non-toxic antifungal drugs.

Potato starch modified by Streptococcus thermophilus GtfB enzyme has low viscoelastic and slowly digestible properties.

Potato starch with high viscosity and digestibility cannot be added into some foods. To address this issue, a novel starch-acting enzyme 4,6-α-glucosyltransferase from Streptococcus thermophilus (StGtfB) was used. StGtfB decreased the iodine affinity and the molecular weight, but increased the degree of branching of starch at a mode quite different from glycogen 1,4-α-glucan branching enzyme (GBE). StGtfB at 5 U/g substrate mainly introduced DP 1-7 into amylose (AMY) or DP 1-12 branches into amylopectin (AMP), and increased the ratio of short- to long-branches from 0.32 to 2.22 or from 0.41 to 2.50. The DP 3 branch chain was the most abundant in both StGtfB-modified AMY and StGtfB-modified AMP. The DP < 6 branch chain contents in StGtfB-modified AMY were 42.68%, much higher than those of GBE-modified AMY. StGtfB significantly decreased viscoelasticity but still kept pseudoplasticity of starch. The modifications also slowed down the glucose generation rate of products at the mammalian mucosal α-glucosidase level. The slowly digestible fraction in potato starch increased from 34.29% to 53.22% using StGtfB of 5 U/g starch. This low viscoelastic and slowly digestible potato starch had great potential with respect to low and stable postprandial blood glucose.

Substrate reduction therapy with Miglustat in pediatric patients with GM1 type 2 gangliosidosis delays neurological involvement: A multicenter experience.

BACKGROUND: In GM1 gangliosidosis the lack of function of ß-galactosidase results in an accumulation of GM1 ganglioside and related glycoconjugates in visceral organs, and particularly in the central nervous system, leading to severe disability and premature death. In the type 2 form of the disease, early intervention would be important to avoid precocious complications. To date, there are no effective therapeutic options in preventing progressive neurological deterioration. Substrate reduction therapy with Miglustat, a N-alkylated sugar that inhibits the enzyme glucosylceramide synthase, has been proposed for the treatment of several lysosomal storage disorders such as Gaucher type 1 and Niemann Pick Type C diseases. However, data on Miglustat therapy in patients with GM1 gangliosidosis are still scarce. METHODS: We report here the results of Miglustat administration in four Italian children (average age: 55 months, range 20-125) affected by GM1 gangliosidosis type 2 treated in three different Italian pediatric hospitals specialized in metabolic diseases. CONCLUSION: This treatment was safe and relatively well tolerated by all patients, with stabilization and/or slowing down of the neurological progression in three subjects.

The temporal regulation of TEK contributes to pollen wall exine patterning.

Pollen wall consists of several complex layers which form elaborate species-specific patterns. In Arabidopsis, the transcription factor ABORTED MICROSPORE (AMS) is a master regulator of exine formation, and another transcription factor, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), specifies formation of the nexine layer. However, knowledge regarding the temporal regulatory roles of TEK in pollen wall development is limited. Here, TEK-GFP driven by the AMS promoter was prematurely expressed in the tapetal nuclei, leading to complete male sterility in the pAMS:TEK-GFP (pat) transgenic lines with the wild-type background. Cytological observations in the pat anthers showed impaired callose synthesis and aberrant exine patterning. CALLOSE SYNTHASE5 (CalS5) is required for callose synthesis, and expression of CalS5 in pat plants was significantly reduced. We demonstrated that TEK negatively regulates CalS5 expression after the tetrad stage in wild-type anthers and further discovered that premature TEK-GFP in pat directly represses CalS5 expression through histone modification. Our findings show that TEK flexibly mediates its different functions via different temporal regulation, revealing that the temporal regulation of TEK is essential for exine patterning. Moreover, the result that the repression of CalS5 by TEK after the tetrad stage coincides with the timing of callose wall dissolution suggests that tapetum utilizes temporal regulation of genes to stop callose wall synthesis, which, together with the activation of callase activity, achieves microspore release and pollen wall patterning.

Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

Seed-specific down-regulation of Arabidopsis CELLULOSE SYNTHASE 1 or 9 reduces seed cellulose content and differentially affects carbon partitioning.

KEY MESSAGE: Seed-specific down-regulation of AtCESA1 and AtCESA9, which encode cellulose synthase subunits, differentially affects seed storage compound accumulation in Arabidopsis. High amounts of cellulose can negatively affect crop seed quality, and, therefore, diverting carbon partitioning from cellulose to oil, protein and/or starch via molecular breeding may improve seed quality. To determine the effect of seed cellulose content reduction on levels of storage compounds, Arabidopsis thaliana CELLULOSE SYNTHASE1 (AtCESA1) and AtCESA9 genes, which both encode cellulose synthase subunits, were individually down-regulated using seed-specific intron-spliced hairpin RNA (hpRNAi) constructs. The selected seed-specific AtCESA1 and AtCESA9 Arabidopsis RNAi lines displayed reduced cellulose contents in seeds, and exhibited no obvious visual phenotypic growth defects with the exception of a minor effect on early root development in AtCESA1 RNAi seedlings and early hypocotyl elongation in the dark in both types of RNAi line. The seed-specific down-regulation of AtCESA9 resulted in a reduction in seed weight compared to empty vector controls, which was not observed in AtCESA1 RNAi lines. In terms of effects on carbon partitioning, AtCESA1 and AtCESA9 RNAi lines exhibited distinct effects. The down-regulation of AtCESA1 led to a ~ 3% relative increase in seed protein content (P = 0.04) and a ~ 3% relative decrease in oil content (P = 0.02), but caused no alteration in soluble glucose levels. On the contrary, AtCESA9 RNAi lines did not display a significant reduction in seed oil, protein or soluble glucose content. Taken together, our results indicate that the seed-specific down-regulation of AtCESA1 causes alterations in seed storage compound accumulation, while the effect of AtCESA9 on carbon partitioning is absent or minor in Arabidopsis.

Genome-wide analyses reveals a glucosyltransferase involved in rutin and emodin glucoside biosynthesis in tartary buckwheat.

With people's increasing needs for health concern, rutin and emodin in tartary buckwheat have attracted much attention for their antioxidant, anti-diabetic and reducing weight function. However, the biosynthesis of rutin and emodin in tartary buckwheat is still unclear; especially their later glycosylation contributing to make them more stable and soluble is uncovered. Based on tartary buckwheat' genome, the gene structures of 106 UGTs were analyzed; 21 candidate FtUGTs were selected to enzymatic test by comparing their transcript patterns. Among them, FtUGT73BE5 and other 4 FtUGTs were identified to glucosylate flavonol or emodin in vitro; especially rFtUGT73BE5 could catalyze the glucosylation of all tested flavonoids and emodin. Furthermore, the identical in vivo functions of FtUGT73BE5 were demonstrated in tartary buckwheat hairy roots. The transcript profile of FtUGT73BE5 was consistent with the accumulation trend of rutin in plant; this gene may relate to anti-adversity for its transcripts were up-regulated by MeJA, and repressed by ABA.

Red versus green leaves: transcriptomic comparison of foliar senescence between two Prunus cerasifera genotypes.

The final stage of leaf ontogenesis is represented by senescence, a highly regulated process driven by a sequential cellular breakdown involving, as the first step, chloroplast dismantling with consequent reduction of photosynthetic efficiency. Different processes, such as pigment accumulation, could protect the vulnerable photosynthetic apparatus of senescent leaves. Although several studies have produced transcriptomic data on foliar senescence, just few works have attempted to explain differences in red and green leaves throughout ontogenesis. In this work, a transcriptomic approach was used on green and red leaves of Prunus cerasifera to unveil molecular differences from leaf maturity to senescence. Our analysis revealed a higher gene regulation in red leaves compared to green ones, during leaf transition. Most of the observed DEGs were shared and involved in transcription factor activities, senescing processes and cell wall remodelling. Significant differences were detected in cellular functions: genes related to photosystem I and II were highly down-regulated in the green genotype, whereas transcripts involved in flavonoid biosynthesis, such as UDP glucose-flavonoid-3-O-glucosyltransferase (UFGT) were exclusively up-regulated in red leaves. In addition, cellular functions involved in stress response (glutathione-S-transferase, Pathogen-Related) and sugar metabolism, such as three threalose-6-phosphate synthases, were activated in senescent red leaves. In conclusion, data suggests that P. cerasifera red genotypes can regulate a set of genes and molecular mechanisms that cope with senescence, promoting more advantages during leaf ontogenesis than compared to the green ones.

Acid invertase confers heat tolerance in rice plants by maintaining energy homoeostasis of spikelets.

Heat stress impairs both pollen germination and pollen tube elongation, resulting in pollination failure caused by energy imbalance. Invertase plays a critical role in the maintenance of energy homoeostasis; however, few studies investigated this during heat stress. Two rice cultivars with different heat tolerance, namely, TLY83 (heat tolerant) and LLY722 (heat susceptible), were subjected to heat stress. At anthesis, heat stress significantly decreased spikelet fertility, accompanied by notable reductions in pollen germination on stigma and pollen tube elongation in ovule, especially in LLY722. Acid invertase (INV), rather than sucrose synthase, contributed to sucrose metabolism, which explains the different tolerances of both cultivars. Under heat stress, larger enhancements in NAD(H), ATP, and antioxidant capacity were found in TLY83 compared with LLY722, whereas a sharp reduction in poly(ADP-ribose) polymerase (PARP) activity was found in the former compared with the latter. Importantly, exogenous INV, 3-aminobenzamide (a PARP inhibitor), sucrose, glucose, and fructose significantly increased spikelet fertility under heat stress, where INV activity was enhanced and PARP activity was inhibited. Therefore, INV can balance the energy production and consumption to provide sufficient energy for pollen germination and pollen tube growth under heat stress.

Biocatalytic synthesis of ginsenoside Rh2 using Arabidopsis thaliana glucosyltransferase-catalyzed coupled reactions.

Ginsenoside Rh2, a rare protopanaxadiol (PPD)-type triterpene saponin isolated from Panax ginseng, exhibits notable anticancer and immune-system-enhancing activities. Glycosylation catalyzed by uridine diphosphate-dependent glucosyltransferase (UGT) is the final biosynthetic step of ginsenoside Rh2. In this study, UGT73C5 isolated from Arabidopsis thaliana was demonstrated to selectively transfer a glucosyl moiety to the C3 hydroxyl group of PPD to synthesize ginsenoside Rh2. UGT73C5 was coupled with sucrose synthase (SuSy) from A. thaliana to regenerate costly uridine diphosphate glucose (UDPG) from cheap sucrose and catalytic amounts of uridine diphosphate (UDP). The UGT73C5/SuSy ratio, temperature, pH, cofactor UDP, and PPD concentrations for UGT73C5-SuSy coupled reactions were optimized. Through the stepwise addition of PPD, the maximal ginsenoside Rh2 production was 3.2 mg mL-1, which was the highest yield reported to date. These promising results provided an efficient and cost-effective approach to semisynthesize the highly valuable ginsenoside Rh2.

Enrichment of Polyglucosylated Isoflavones from Soybean Isoflavone Aglycones Using Optimized Amylosucrase Transglycosylation.

Isoflavones in soybeans are well-known phytoestrogens. Soy isoflavones present in conjugated forms are converted to aglycone forms during processing and storage. Isoflavone aglycones (IFAs) of soybeans in human diets have poor solubility in water, resulting in low bioavailability and bioactivity. Enzyme-mediated glycosylation is an efficient and environmentally friendly way to modify the physicochemical properties of soy IFAs. In this study, we determined the optimal reaction conditions for Deinococcus geothermalis amylosucrase-mediated α-1,4 glycosylation of IFA-rich soybean extract to improve the bioaccessibility of IFAs. The conversion yields of soy IFAs were in decreasing order as follows: genistein > daidzein > glycitein. An enzyme quantity of 5 U and donor:acceptor ratios of 1000:1 (glycitein) and 400:1 (daidzein and genistein) resulted in high conversion yield (average 95.7%). These optimal reaction conditions for transglycosylation can be used to obtain transglycosylated IFA-rich functional ingredients from soybeans.

Small Molecule Inhibitor Screen Reveals Calcium Channel Signaling as a Mechanistic Mediator of Clostridium difficile TcdB-Induced Necrosis.

Clostridioides difficile is the leading cause of nosocomial diarrhea in the United States. The primary virulence factors are two homologous glucosyltransferase toxins, TcdA and TcdB, that inactivate host Rho-family GTPases. The glucosyltransferase activity has been linked to a "cytopathic" disruption of the actin cytoskeleton and contributes to the disruption of tight junctions and the production of pro-inflammatory cytokines. TcdB is also a potent cytotoxin that causes epithelium necrotic damage through an NADPH oxidase (NOX)-dependent mechanism. We conducted a small molecule screen to identify compounds that confer protection against TcdB-induced necrosis. We identified an enrichment of "hit compounds" with a dihydropyridine (DHP) core which led to the discovery of a key early stage calcium signal that serves as a mechanistic link between TcdB-induced NOX activation and reactive oxygen species (ROS) production. Disruption of TcdB-induced calcium signaling (with both DHP and non-DHP molecules) is sufficient to ablate ROS production and prevent subsequent necrosis in cells and in a mouse model of intoxication.

Ganglioside deficiency in hypothalamic POMC neurons promotes body weight gain.

BACKGROUND: Glucosylceramide synthase (GCS; gene: UDP-glucose:ceramide glucosyltransferase (Ugcg))-derived gangliosides comprise a specific class of lipids in the plasma membrane that modulate the activity of transmembrane receptors. GCS deletion in hypothalamic arcuate nucleus (Arc) neurons leads to prominent obesity. However, it has not yet been studied how ganglioside depletion affects individual Arc neuronal subpopulations. The current study investigates the effects of GCS deletion specifically in anorexigenic pro-opiomelanocortin (POMC) neurons. Additionally, we investigate insulin receptor (IR) signaling and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) binding to ATP-dependent K+ (KATP) channels of GCS-deficient POMC neurons. MATERIALS AND METHODS: We generated Ugcgf/f-Pomc-Cre mice with ganglioside deficiency in POMC neurons. Moreover, the CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas9 technology was used to inhibit GCS-dependent ganglioside biosynthesis in cultured mouse POMC neurons, yielding UgcgΔ-mHypoA-POMC cells that were used to study mechanistic aspects in further detail. Proximity ligation assays (PLAs) visualized interactions between gangliosides, IR, and KATP channel subunit sulfonylurea receptor-1 (SUR-1), as well as intracellular IR substrate 2 (IRS-2) phosphorylation and PIP3. RESULTS: Chow-fed Ugcgf/f-Pomc-Cre mice showed a moderate but significant increase in body weight gain and they failed to display an increase of anorexigenic neuropeptide expression during the fasting-to-re-feeding transition. IR, IRS-2, p85, and overall insulin-evoked IR and IRS-2 phosphorylation were elevated in ganglioside-depleted UgcgΔ-mHypoA-POMC neurons. A PLA demonstrated that more insulin-evoked complex formation occurred between PIP3 and SUR-1 in ganglioside-deficient POMC neurons in vitro and in vivo. CONCLUSION: Our work suggests that GCS deletion in POMC neurons promotes body weight gain. Gangliosides are required for an appropriate adaptation of anorexigenic neuropeptide expression in the Arc during the fasting-to-re-feeding transition. Moreover, gangliosides might modulate KATP channel activity by restraining PIP3 binding to the KATP channel subunit SUR-1. Increased PIP3/SUR-1 interactions in ganglioside-deficient neurons could in turn potentially lead to electrical silencing. This work highlights that gangliosides in POMC neurons of the hypothalamic Arc are important regulators of body weight.

Ethylene plays an important role in the softening and sucrose metabolism of blueberries postharvest.

We studied the effects of ethylene on softening and sucrose metabolism in postharvest blueberry fruit by examining the responses of fruit firmness, cell wall polysaccharides, cell wall enzymes, four key genes of cell wall degradation and metabolism, enzyme activities, and five key genes of sucrose metabolism to exogenous ethylene treatments. Ethylene was found to accelerate blueberry softening, as it promoted the degradation of pectin and expression of pectinesterase (PE) and polygalacturonase (PG). Sucrose catabolism was accelerated with fruit softening, while sucrose content, sucrose phosphate synthase (SPS) activity were positively correlated with the loss of fruit firmness. Exogenous ethylene treatments promoted sucrose metabolism by inhibiting the expression of VcSPS1 and VcNIN2 and stimulating the expression of VcSS1 and VcCWINV1. These results indicate that ethylene plays an important role in fruit softening and sucrose metabolism of blueberry at 20 °C, and there may be a link between sucrose metabolism and fruit softening.