Functional significance of O-linked N-acetylglucosamine protein modification in regulating autophagy.

Autophagy is a core molecular pathway that preserves cellular and organismal homeostasis. Being susceptible to nutrient availability and stress, eukaryotic cells recycle or degrade internal components via membrane transport pathways to provide sustainable biological molecules and energy sources. The dysregulation of this highly conserved physiological process has been strongly linked to human disease. Post-translational modification, a mechanism that regulates protein function, plays a crucial role in autophagy regulation. O-linked N-acetylglucosamine protein modification (O-GlcNAcylation), a monosaccharide post-translational modification of intracellular proteins, is essential in nutritional and stress regulatory mechanisms. O-GlcNAcylation has emerged as an essential regulatory mechanism of autophagy. It regulates autophagy throughout its lifetime by targeting the core components of the autophagy regulatory network. This review provides an overview of the O-GlcNAcylation of autophagy-associated proteins and their regulation and function in the autophagy pathway. Therefore, this article may contribute to further understanding of the role of O-GlcNAc-regulated autophagy and provide new perspectives for the treatment of human diseases.

Connexin32 ameliorates epithelial-to-mesenchymal-transition in diabetic renal tubular via inhibiting NOX4.

Renal tubulointerstitial fibrosis (RIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs), is the main cause of diabetic renal fibrosis. Oxidative stress plays a pivotal role in the development of diabetic RIF. Connexin32 (Cx32), prominently expressed in renal TECs, has emerged as an important player in the regulation of oxidative stress. However, the role of Cx32 in diabetic RIF has not been explored yet. Here, we showed that adenovirus-mediated Cx32 overexpression suppressed EMT to ameliorate RIF and renal function in STZ-induced diabetic mice, while knockout (KO) of Cx32 exacerbated RIF in diabetic mice. Moreover, overexpression of Cx32 inhibited EMT and the production of extra cellular matrix (ECM) in high glucose (HG) induced NRK-52E cells, whereas knockdown of Cx32 showed the opposite effects. Furthermore, we showed that NOX4, the main source of ROS in renal tubular, was down-regulated by Cx32. Mechanistically, Cx32 down-regulated the expression of PKC alpha in a carboxyl-terminal-dependent manner, thereby inhibiting the phosphorylation at Thr147 of p22phox triggered by PKC alpha, which ultimately repressed the formation of the p22phox-NOX4 complex to reduce the protein level of NOX4. Thus, we establish Cx32 as a novel target and confirm the protection mechanism in RIF.

External modulation of Rotimer exudate secretion in monogonant rotifers.

The Rotimer, a rotifer-specific biopolymer, is an exogenic bioactive exudate secreted by different monogonant species (e.g. Euchlanis dilatata or Lecane bulla). The production of this viscoelastic biomolecule is induced by different micro-particles, thereby forming a special Rotimer-Inductor Conglomerate (RIC) in a web format. In this case, the water insoluble Carmine crystals, filtered to size (max. diameter was 50 µm), functioned as an inductor. The RIC production is an adequate empirical indicator to follow up this filamentous biopolymer secretion experientially; moreover, this procedure is very sensitive to the environmental factors (temperature, pH, metals and possible natural pollutant agents). The above mentioned species show completely different reactions to these factors, except to the presence of calcium and to the modulating effects of different drugs. One of the novelties of this work is that the Rotimer secretion and consequently, the RIC-formation is a mutually obligatory and evolutionary calcium-dependent process in the concerned monogonants. This in vivo procedure needs calcium, both for the physiology of animals and for fiber formation, particularly in the latter case. The conglomerate covered area (%) and the detection of the longest filament (mm) of the given RIC were the generally and simultaneously applied methods in the current modulating experiments. Exploring the regulatory (e.g. calcium-dependency) and stimulating (e.g. Lucidril effect) possibilities of biopolymer secretion are the basis for optimizing the RIC-production capacities of these micro-metazoans.

Elafibranor restricts lipogenic and inflammatory responses in a human skin stem cell-derived model of NASH.

Non-alcoholic steatohepatitis (NASH) is characterized by hepatocellular steatosis with concomitant hepatic inflammation. Despite its pandemic proportions, no anti-NASH drugs have been approved yet. This is partially because drug development is decelerated due to the lack of adequate tools to assess the efficacy of potential new drug candidates. The present study describes the development and application of a new preclinical model for NASH using hepatic cells generated from human skin-derived precursors. Exposure of these cells to lipogenic (insulin, glucose, fatty acids) and pro-inflammatory factors (IL-1ß, TNF-α, TGF-ß) resulted in a characteristic NASH response, as indicated by intracellular lipid accumulation, modulation of NASH-specific gene expression, increased caspase-3/7 activity and the expression and/or secretion of inflammatory markers, including CCL2, CCL5, CCL7, CCL8, CXCL5, CXCL8, IL1a, IL6 and IL11. The human relevance of the proposed NASH model was verified by transcriptomics analyses that revealed commonly modulated genes and the identification of the same gene classes between the in vitro system and patients suffering from NASH. The application potential of this in vitro model was demonstrated by testing elafibranor, a promising anti-NASH compound currently under clinical phase III trial evaluation. Elafibranor attenuated in vitro key features of NASH, and dramatically lowered lipid load as well as the expression and secretion of inflammatory chemokines, which in vivo are responsible for the recruitment of immune cells. This reduction in inflammatory response was NFκB-mediated. In summary, this human-relevant, in vitro system proved to be a sensitive testing tool for the investigation of novel anti-NASH compounds.

Probing the metabolic phenotype of breast cancer cells by multiple tracer stable isotope resolved metabolomics.

Breast cancers vary by their origin and specific set of genetic lesions, which gives rise to distinct phenotypes and differential response to targeted and untargeted chemotherapies. To explore the functional differences of different breast cell types, we performed Stable Isotope Resolved Metabolomics (SIRM) studies of one primary breast (HMEC) and three breast cancer cells (MCF-7, MDAMB-231, and ZR75-1) having distinct genotypes and growth characteristics, using 13C6-glucose, 13C-1+2-glucose, 13C5,15N2-Gln, 13C3-glycerol, and 13C8-octanoate as tracers. These tracers were designed to probe the central energy producing and anabolic pathways (glycolysis, pentose phosphate pathway, Krebs Cycle, glutaminolysis, nucleotide synthesis and lipid turnover). We found that glycolysis was not associated with the rate of breast cancer cell proliferation, glutaminolysis did not support lipid synthesis in primary breast or breast cancer cells, but was a major contributor to pyrimidine ring synthesis in all cell types; anaplerotic pyruvate carboxylation was activated in breast cancer versus primary cells. We also found that glucose metabolism in individual breast cancer cell lines differed between in vitro cultures and tumor xenografts, but not the metabolic distinctions between cell lines, which may reflect the influence of tumor architecture/microenvironment.

Roles of the NLRP3 inflammasome in the pathogenesis of diabetic nephropathy.

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus, and persistent inflammation in circulatory and renal tissues is an important pathophysiological basis for DN. The essence of the microinflammatory state is the innate immune response, which is central to the occurrence and development of DN. Members of the inflammasome family, including both "receptors" and "regulators", are key to the inflammatory immune response. Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) and other inflammasome components are able to detect endogenous danger signals, resulting in activation of caspase-1 as well as interleukin (IL)-1ß, IL-18 and other cytokines; these events stimulate the inflammatory cascade reaction, which is crucial for DN. Hyperglycaemia, hyperlipidaemia and hyperuricaemia can activate the NLRP3 inflammasome, which then mediates the occurrence and development of DN through the K+ channel model, the lysosomal damage model and the active oxygen cluster model. In this review, we survey the involvement of the NLRP3 inflammasome in various signalling pathways and highlight different aspects of their influence on DN. We also explore the important effects of the NLRP3 inflammasome on kidney function and structural changes that occur during DN development and progression. It is becoming more evident that NLRP3 inflammasome targeting has therapeutic potential for the treatment of DN.

Exploring formation of turanose in honey via stable isotope labelling and high-resolution mass spectrometry analysis.

Turanose, an isomer of sucrose, naturally exists in honey. Previous study indicated that turanose content increased gradually in acacia honey as honeybees brewed honey in the hive. However, it is unclear how turanose is generated in honey. We hypothesised that turanose was produced by enzymes from honeybees and performed a series of simulation experiments to prove this hypothesis. We found turanose in honey was produced by honeybees processing sucrose. Furthermore, we determined that sugar composition of simulated nectar influenced the turanose concentration in honey: when sucrose concentration was below 5%, turanose was difficult to form, whereas high concentration of fructose and limited glucose were beneficial in producing turanose. Using 13C-labelled sucrose tests combined with proteomics analysis, we identified that α-glucosidase converted sucrose to turanose through an intermolecular isomerisation process. This study reveals the formation mechanism of turanose in honey and assists in the scientific control and improvement of honey quality.

Sweet-bitter taste interactions in binary mixtures of sweeteners: Relationship between taste receptor activities and sensory perception.

This study investigated the effects of various binary sweetener mixtures on sweetness enhancement and their interactions with sweet or bitter taste receptors, focusing on sensory perception and receptor activity. Acesulfame K or saccharin was mixed with allulose, aspartame, erythritol, fructose, glucose, or sucrose to match a target sucrose sweetness. The effects of the mixtures on sweet and bitter taste receptors (in the human embryonic kidney -293 cells) and sensory taste intensities were evaluated. Sweetness enhancement at the sweet taste receptor level was observed in some cases, with several monosaccharides reducing the acesulfame K- or saccharin-induced bitter taste receptor activity. Combining acesulfame K or saccharin with any of the six sweeteners perceptually enhanced sweetness (60% âˆ¼ 100% in 50:50 ratio), correlating with a reduction in inherent bitterness (-35% âˆ¼ -63% in 50:50 ratio). This finding suggests that sweetness perception likely increased because the monosaccharides mitigate the activation of bitter receptors caused by high-potency sweeteners.

Soluble sugar, organic acid and phenolic composition and flavor evaluation of plum fruits.

Plums (Prunus salicina and Prunus domestica) are prevalent in southwestern China, and have attracted interest owing to their delectable taste and exceptional nutritional properties. Therefore, this study aimed to investigate the nutritional and flavor properties of plum to improve its nutritional utilization. Specifically, we determined the soluble sugars, organic acids, and phenolic components in 86 accessions using high-performance liquid chromatography. Notably, glucose, fructose, malic, and quinic acids were the predominant sweetness and acidity in plums, with sucrose contributing more to the sweetness of the flesh than the peel. Moreover, The peel contains 5.5 fold more phenolics than flesh, epicatechin, gallic acid, and proanthocyanidins C1 and B2 were the primary sources of astringency. Correlation and principal component analyses showed eight core factors for plum flavor rating, and a specific rating criterion was established. Conclusively, these findings provide information on the integrated flavor evaluation criteria and for enhancing optimal breeding of plums.

Raloxifene-driven benzothiophene derivatives: Discovery, structural refinement, and biological evaluation as potent PPARγ modulators based on drug repurposing.

By virtue of the drug repurposing strategy, the anti-osteoporosis drug raloxifene was identified as a novel PPARγ ligand through structure-based virtual high throughput screening (SB-VHTS) of FDA-approved drugs and TR-FRET competitive binding assay. Subsequent structural refinement of raloxifene led to the synthesis of a benzothiophene derivative, YGL-12. This compound exhibited potent PPARγ modulation with partial agonism, uniquely promoting adiponectin expression and inhibiting PPARγ Ser273 phosphorylation by CDK5 without inducing the expression of adipongenesis associated genes, including PPARγ, aP2, CD36, FASN and C/EBPα. This specific activity profile resulted in effective hypoglycemic properties, avoiding major TZD-related adverse effects like weight gain and hepatomegaly, which were demonstrated in db/db mice. Molecular docking studies showed that YGL-12 established additional hydrogen bonds with Ile281 and enhanced hydrogen-bond interaction with Ser289 as well as PPARγ Ser273 phosphorylation-related residues Ser342 and Glu343. These findings suggested YGL-12 as a promising T2DM therapeutic candidate, thereby providing a molecular framework for the development of novel PPARγ modulators with an enhanced therapeutic index.

Gelation behavior and mechanism of Nicandra physalodes (Linn.) Gaertn. seeds pectin induced by Glucono-delta-lactone.

In this study, the physicochemical properties of pectin from Nicandra physalodes (Linn.) Gaertn. seeds (NPGSP) were analysed firstly, and the rheological behavior, microstructure and gelation mechanism of NPGSP gels induced by Glucono-delta-lactone (GDL) were investigated. The hardness of NPGSP gels was increased from 26.27 g to 226.77 g when increasing GDL concentration from 0 % (pH = 4.0) to 1.35 % (pH = 3.0), and the thermal stability was improved. The peak around 1617 cm-1 was decreased as the adsorption peak of the free carboxyl groups was attenuated with addition of GDL. GDL increased the crystalline degree of NPGSP gels, and its microstructure exhibited more smaller spores. Molecular dynamics was performed on systems of pectin and gluconic acid (GDL hydrolysis product), indicating that inter-molecular hydrogen bonds and van der Waals forces were the main interactions to promote gels formation. Overall, NPGSP has the potential commercial value for developing as a thickener in food processing.

Soluble sugar and organic acid composition and flavor evaluation of Chinese cherry fruits.

Chinese cherry is an economically important fruit crop native to China. Flavor quality is greatly influenced by compositions of soluble sugars and organic acids. To better understand the flavor quality of Chinese cherry, we determined sugar and acid components in thirty-eight landrace and cultivar collections, and two wild resources using the HPLC method. Glucose and fructose were the main components, accounting for 85.91% of soluble sugars. Malic acid was the predominant organic acid, with an average proportion of 65.73% of total acids. Correlation and PCA analysis revealed seven key indicators for evaluating fruit flavor. Compared with wild Chinese cherry, the cultivated collections exhibited higher levels of soluble sugars, especially fructose, and lower levels of organic acid, particularly malic acid in fruits. Finally, we have established grading criteria for seven flavor indicators in Chinese cherry. Our study provides valuable references for identifying flavor compounds and improving flavor quality of Chinese cherry.

A regular Chlorella mannogalactan and its sulfated derivative as a promising anticoagulant: Structural characterization and anticoagulant activity.

Chlorella is one of the most widely cultivated species of microalgae and has been consumed as a "green healthy food". In this study, a novel polysaccharide (CPP-1) was isolated from Chlorella pyrenoidosa, structurally analyzed, and sulfated as a promising anticoagulant. Structural analyses by chemical and instrumental methods such as monosaccharide composition, methylation-GC-MS and 1D/2D NMR spectroscopy analysis revealed that CPP-1 had a molecular weight of ~13.6 kDa, and mainly consisted of d-mannopyranose (d-Manp), 3-O-methylated d-Manp (3-O-Me-d-Manp), and d-galactopyranose (d-Galp). The molar ratio of d-Manp and d-Galp was 1.0:2.3. CPP-1 consisted of a (1→6)-linked ß-d-Galp backbone substituted at C-3 by the d-Manp and 3-O-Me-d-Manp residues in a molar ratio of 1:1, which was a regular mannogalactan. The sulfated Chlorella mannogalactan (SCM) with sulfated group content of 40.2 % equivalent to that of unfractionated heparin was prepared and analyzed. NMR analysis confirmed its structure, indicating that most free hydroxyl groups in the side chains and partial hydroxyl groups in the backbone were sulfated. Anticoagulant activity assays indicated that SCM exhibited strong anticoagulant activity by inhibiting intrinsic tenase (FXase) with IC50 of 13.65 ng/mL, which may be a safer anticoagulant as an alternative to heparin-like drugs.

A new non-enzymatic biosensor for the determination of bisphenol-A.

In this study, a new non-enzymatic carbon paste biosensor was developed for the determination of Bisphenol-A (BPA) based on Multiwalled Carbon Nanotube (MWCNT) modified Myoglobin (Mb). The measurement principle of the biosensor was developed based on the inhibition effect of BPA on the heme group of myoglobin in the presence of hydrogen peroxide. With the designed biosensor, measurements were taken in the potential range of (-0.15 V & +0.65 V) using the differential pulse voltammetry (DPV) method in the medium containing K4[Fe(CN)6]. The linear range for BPA was determined to be 100-1000 µM. Response time was calculated as 16 s. The limit of detection was set at 89 µM. As a result, it has been proven that MWCNT modified myoglobin based biosensor is an alternative method that can be used for BPA determination, giving very sensitive and fast results.

The structures of two glucomannans from Bletilla formosana and their protective effect on inflammation via inhibiting NF-κB pathway.

Bletilla formosana is a traditional Chinese herbal medicine and is widely consumed as foods and medicines in China. However, the chemical structure and bioactivity of its polysaccharides remain unknown. Herein, two new polysaccharides, BFP60 and BFP80, with molecular weights of 3.99 kDa and 10.07 kDa, respectively, were isolated and purified from dried tuber of B. formosana. Structural analysis suggested that BFP60 and BFP80 may have backbone consisted of →4)-ß-d-Man-(1→,→4)-ß-d-Glc-(1→,→4)-2-O-acetyl-ß-d-Man-(1→, and →4)-3-O-acetyl-ß-d-Man-(1→. Inflammation assay in LPS-induced RAW264.7 cells showed that the productions of NO and pro-inflammatory cytokines including IL-6, IL-1ß, TNF-α, and IFN-γ were significantly reduced, and the expression of iNOS, COX-2, and target proteins in the NF-κB pathway were suppressed after BFP60 and BFP80 pretreatment. These findings indicated that this novel polysaccharide had significant inflammatory protective effects in vitro.

Preparation and structural properties of selenium modified heteropolysaccharide from the fruits of Akebia quinata and in vitro and in vivo antitumor activity.

Cancer is a complex disease, and blocking tumor angiogenesis has become one of the most promising approaches in cancer therapy. Here, an exopoly heteropolysaccharide (AQP70-2B) was firstly isolated from Akebia quinata. Monosaccharide composition indicated that the AQP70-2B was composed of rhamnose, glucose, galactose, and arabinose. The backbone of AQP70-2B consisted of →1)-l-Araf, →3)-l-Araf-(1→, →5)-l-Araf-(1→, →3,5)-l-Araf-(1→, →2,5)-l-Araf-(1→, →4)-d-Glcp-(1→, →6)-d-Galp-(1→, and →1)-d-Rhap residues. Based on the close relationship between selenium and anti-tumor activity, AQP70-2B was modified with selenium to obtain selenized polysaccharide Se-AQP70-2B. Then, a series of methods for analysis and characterization, especially scanning electron microscopy coupled with energy dispersive spectrometry (SEM-EDS), indicated that Se-AQP70-2B was successfully synthesized. Furthermore, zebrafish xenografts and anti-angiogenesis experiments indicated that selenization could improve the antitumor activity by inhibiting tumor cell proliferation and migration and blocking angiogenesis.

Identification of a novel PPARγ modulator with good anti-diabetic therapeutic index via structure-based screening, optimization and biological validation.

PPARγ is well-known as the target receptor of TZD anti-diabetic drugs. However, recently the therapeutic benefits of these TZD drugs have been compromised by many severe side effects because of their full PPARγ agonistic action to lock the AF-2 helix. Herein, we conducted a virtual screening in the combination with structure-based design, synthesis and biological evaluation both in vitro and in vivo, leading to the identification of a potent candidate YG-C-20 as the SPPARγM with improved and safer anti-diabetic therapeutics. Mechanistically, this compound presented such desired pharmacological profiles (e.g., preferable anti-diabetic efficiencies and minimized side effects) mainly via selectively inhibiting the CDK5-mediated phosphorylation of PPARγ-Ser273 and up-regulating the expression of insulin-sensitive genes Adiponectin and Glut4, yet lacking the classical full agonism to induce the adipogenesis and the expression of key adipogenic genes including PPARγ, aP2, CD36, LPL, C/EBPα and FASN. Further validation led to the final recognition of its (R)-configured isomer as the potential conformational form. Subsequent molecular docking studies revealed a unique hydrogen-bonding network of (R)-YG-C-20 with three full PPARγ agonism-unrelated residues, especially with PPARγ-Ser273 phosphorylation-associated site Ser342, which not only gives a clear verification for our structure-based design but also provides a proof of concept for the abovementioned molecular mechanism.

Simulated digestion, dynamic changes during fecal fermentation and effects on gut microbiota of Avicennia marina (Forssk.) Vierh. fruit non-starch polysaccharides.

Grey mangrove (Avicennia marina (Forssk.) Vierh.) fruit is a traditional folk medicine and health food consumed in many countries. In this study, its polysaccharides (AMFPs) were obtained and analyzed by chemical and instrumental methods, with the results indicating that AMFPs consisted of galactose, galacturonic acid, arabinose, and rhamnose in a molar ratio of 4.99:3.15:5.38:1.15. The dynamic changes in AMFPs during the digestion and fecal fermentation processes were then investigated. The results confirmed that AMFPs were not depolymerized by gastric acid and various digestive enzymes. During fermentation, 56.05 % of the AMFPs were utilized by gut microbiota. Galacturonic acid, galactose, and arabinose from AMFPs, were mostly consumed by gut microbiota. AMFPs obviously decreased harmful bacteria and increased some beneficial microbiota, including Megasphaera, Mistuokella, Prevotella, and Megamonas. Furthermore, AMFPs obviously increased the levels of various short-chain fatty acids. These findings suggest that AMFPs have potential prebiotic applications for improving gut health.

Effect of molecular weight of chitosan on the formation and properties of zein-nisin-chitosan nanocomplexes.

This study evaluated the effect of molecular weight of chitosan (3 kDa,150 kDa, 400 kDa, and 600 kDa) on zein-nisin-chitosan nanocomplexes. The formation mechanism, physicochemical and antibacterial properties of the nanocomplexes (ZNC0.3, ZNC15, ZNC40, and ZNC60) were assessed. The nanocomplexes were characterized by DLS, ζ-potential, atomic force microscopy, scanning electron microscopy, circular dichroism, fourier transform infrared and UV-Vis spectroscopy. The results showed that the lowest molecular weight chitosan (LMWC, 3 kDa) formed nanocomplexes with nisin and zein structurally differed from the higher molecular weights chitosan (HMWC, >3 kDa). LMWC was doped on the surface of the nanocomplexes. HMWC linked and formed a network to adsorb zein and nisin. The antibacterial activity against Staphylococcus aureus showed that the minimum inhibitory concentration of ZNC0.3, ZNC15, ZNC40, and ZNC60 was 7.0625, 14.125, 14.125, and 28.25 µg/mL. ZNC0.3 could be a suitable nisin delivery system for its high encapsulation efficiency (85.38%) and antibacterial properties.

Comparison of pyrazines formation in methionine/glucose and corresponding Amadori rearrangement product model.

The generation of pyrazines in a binary methionine/glucose (Met/Glc) mixture and corresponding methionine/glucose-derived Amadori rearrangement product (MG-ARP) was studied. Quantitative analyses of pyrazines and methional revealed that MG-ARP generated more methional compared to Met/Glc, whereas lower content and fewer species of pyrazines were observed in the MG-ARP model. Comparing the availability of α-dicarbonyl compounds generated from the Met/Glc model, methylglyoxal (MGO) was a considerably effective α-dicarbonyl compound for the formation of pyrazines during MG-ARP degradation, but glyoxal (GO) produced from MG-ARP did not effectively participate in the corresponding formation of pyrazines due to the asynchrony on the formation of GO and recovered Met. Diacetyl (DA) content was not high enough to form corresponding pyrazines in the MG-ARP model. The insufficient interaction of precursors and rapid drops in pH limited the formation of pyrazines during MG-ARP degradation. Increasing reaction temperature could reduce the negative inhibitory effect by promoting the content of precursors.