Engineered dityrosine-bonding of the RSV prefusion F protein imparts stability and potency advantages.

Viral fusion proteins facilitate cellular infection by fusing viral and cellular membranes, which involves dramatic transitions from their pre- to postfusion conformations. These proteins are among the most protective viral immunogens, but they are metastable which often makes them intractable as subunit vaccine targets. Adapting a natural enzymatic reaction, we harness the structural rigidity that targeted dityrosine crosslinks impart to covalently stabilize fusion proteins in their native conformations. We show that the prefusion conformation of respiratory syncytial virus fusion protein can be stabilized with two engineered dityrosine crosslinks (DT-preF), markedly improving its stability and shelf-life. Furthermore, it has 11X greater potency as compared with the DS-Cav1 stabilized prefusion F protein in immunogenicity studies and overcomes immunosenescence in mice with simply a high-dose formulation on alum.

TTLL12 has a potential oncogenic activity, suppression of ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin, that can be overcome by molecules identified by screening a compound library.

Tubulin tyrosine ligase 12 (TTLL12) is a promising target for therapeutic intervention since it has been implicated in tumour progression, the innate immune response to viral infection, ciliogenesis and abnormal cell division. It is the most mysterious of a fourteen-member TTL/TTLL family, since, although it is the topmost conserved in evolution, it does not have predicted enzymatic activities. TTLL12 seems to act as a pseudo-enzyme that modulates various processes indirectly. Given the need to target its functions, we initially set out to identify a property of TTLL12 that could be used to develop a reliable high-throughput screening assay. We discovered that TTLL12 suppresses the cell toxicity of nitrotyrosine (3-nitrotyrosine) and its ligation to the C-terminus of detyrosinated α-tubulin (abbreviated to ligated-nitrotyrosine). Nitrotyrosine is produced by oxidative stress and is associated with cancer progression. Ligation of nitrotyrosine has been postulated to be a check-point induced by excessive cell stress. We found that the cytotoxicities of nitrotyrosine and tubulin poisons are independent of one another, suggesting that drugs that increase nitrotyrosination could be complementary to current tubulin-directed therapeutics. TTLL12 suppression of nitrotyrosination of α-tubulin was used to develop a robust cell-based ELISA assay that detects increased nitrotyrosination in cells that overexpress TTLL12 We adapted it to a high throughput format and used it to screen a 10,000 molecule World Biological Diversity SETTM collection of low-molecular weight molecules. Two molecules were identified that robustly activate nitrotyrosine ligation at 1 µM concentration. This is the pioneer screen for molecules that modulate nitrotyrosination of α-tubulin. The molecules from the screen will be useful for the study of TTLL12, as well as leads for the development of drugs to treat cancer and other pathologies that involve nitrotyrosination.

Lycopene Regulates Dietary Dityrosine-Induced Mitochondrial-Lipid Homeostasis by Increasing Mitochondrial Complex Activity.

SCOPE: Dityrosine (DT), a marker of protein oxidation, is widely found in many high-protein foods. Dietary intake of DT induces myocardial oxidative stress injury and impairs energy metabolism. Lycopene is a common dietary supplement with antioxidant and mitochondrial-lipid homeostasis modulating abilities. This study aimed to examine the effects of lycopene on DT-induced disturbances in myocardial function and energy metabolism. METHODS AND RESULTS: Four-week-old C57BL/6J mice received intragastric administration of either tyrosine (420 µg kg-1 BW), DT (420 µg kg-1 BW), or lycopene at high (10 mg kg-1 BW) and low (5 mg kg-1 BW) doses for 35 days. Lycopene administration effectively reduced oxidative stress, cardiac fatty acid accumulation, and cardiac hypertrophy and improved mitochondrial performance in DT-induced mice. In vitro experiments in H9c2 cells showed that DT directly inhibited the activity of the respiratory chain complex, whereas oxidative phosphorylation and ß-oxidation gene expression is upregulated. Lycopene enhanced the activity of the complexes and inhibited ROS production caused by compensatory regulation. CONCLUSION: Lycopene improves DT-mediated myocardial energy homeostasis disorder by promoting the activity of respiratory chain complexes I and IV and alleviates the accumulation of cardiac fatty acids and myocardial hypertrophy.

Effect of catechin and commercial preparation of green tea essence on the pharmacokinetics of l-dopa in rabbits.

The aim of this study was to investigate drug interactions of L-dopa/carbidopa with catechin and green tea essence in rabbits following the simultaneous administration via an intramuscular injection of catechin or via an intragastric route for green tea essence with L-dopa/carbidopa. The results indicated that catechin at doses of 10, 20 and 50 mg/kg increased the area under the plasma concentration-time curve from time zero to the time of the last quantifiable concentration (AUC0-t ) of L-dopa by about 69, 78 and 42%, respectively. The metabolic ratios of the AUC0-t for 3-O-methyldopa (3-OMD)/L-dopa significantly decreased by about 56, 68 and 76% (P < 0.05), respectively. In addition, a single dose of 5/1.25 mg/kg L-dopa/carbidopa was co-administrated with 150 mg/kg green tea essence via an intragastric route with an oral-gastric tube. Comparing the related pharmacokinetic parameters of L-dopa, the clearance and metabolic ratio of L-dopa decreased by 20 and 19% (P < 0.05), respectively. In conclusion, catechin and green tea essence can significantly affect the metabolism of L-dopa by the catechol-O-methyltransferase (COMT) metabolic pathway. Catechin can enhance L-dopa bioavailability, and both catechin and green tea essence decreased 3-OMD formation. Therefore, catechin and green tea essence may increase L-dopa efficacy for Parkinson's disease treatment.

Silybin alleviates hepatic lipid accumulation in methionine-choline deficient diet-induced nonalcoholic fatty liver disease in mice via peroxisome proliferator-activated receptor α.

Nonalcoholic fatty liver disease (NAFLD) is regarded as the most common liver disease with no approved therapeutic drug currently. Silymarin, an extract from the seeds of Silybum marianum, has been used for centuries for the treatment of various liver diseases. Although the hepatoprotective effect of silybin against NAFLD is widely accepted, the underlying mechanism and therapeutic target remain unclear. In this study, NAFLD mice caused by methionine-choline deficient (MCD) diet were orally administrated with silybin to explore the possible mechanism and target. To clarify the contribution of peroxisome proliferator-activated receptor α (PPARα), PPARα antagonist GW6471 was co-administrated with silybin to NAFLD mice. Since silybin was proven as a PPARα partial agonist, the combined effect of silybin with PPARα agonist, fenofibrate, was then evaluated in NAFLD mice. Serum and liver samples were collected to analyze the pharmacological efficacy and expression of PPARα and its targets. As expected, silybin significantly protected mice from MCD-induced NAFLD. Furthermore, silybin reduced lipid accumulation via activating PPARα, inducing the expression of liver cytosolic fatty acid-binding protein, carnitine palmitoyltransferase (Cpt)-1a, Cpt-2, medium chain acyl-CoA dehydrogenase and stearoyl-CoA desaturase-1, and suppressing fatty acid synthase and acetyl-CoA carboxylase α. GW6471 abolished the effect of silybin on PPARα signal and hepatoprotective effect against NAFLD. Moreover, as a partial agonist for PPARα, silybin impaired the powerful lipid-lowering effect of fenofibrate when used together. Taken together, silybin protected mice against NAFLD via activating PPARα to diminish lipid accumulation and it is not suggested to simultaneously take silybin and classical PPARα agonists for NAFLD therapy.

The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation.

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Omega-7 oil increases telomerase activity and accelerates healing of grafted burn and donor site wounds.

This study investigated the efficacy of Omega-7 isolated from the sea buckthorn oil (Polyvit Co., Ltd, Gangar Holding, Ulaanbaatar, Mongolia) in ovine burn wound healing models. In vitro, proliferation (colony-forming rate) and migration (scratch) assays using cultured primary ovine keratinocytes were performed with or without 0.025% and 0.08% Omega-7, respectively. The colony-forming rate of keratinocytes in the Omega-7 group at 72 and 96 h were significantly higher than in the control (P < 0.05). The percentage of closure in scratch assay in the Omega-7 group was significantly higher than in the control at 17 h (P < 0.05). In vivo, efficacy of 4% Omega-7 isolated from buckthorn oil was assessed at 7 and 14 days in grafted ovine burn and donor site wounds. Telomerase activity, keratinocyte growth factor, and wound nitrotyrosine levels were measured at day 14. Grafted sites: Un-epithelialized raw surface area was significantly lower and blood flow was significantly higher in the Omega-7-treated sites than in control sites at 7 and 14 days (P < 0.05). Telomerase activity and levels of keratinocyte growth factors were significantly higher in the Omega-7-treated sites after 14 days compared to those of control (P < 0.05). The wound 3-nitrotyrosine levels were significantly reduced by Omega-7. Donor sites: the complete epithelialization time was significantly shorter and blood flow at day 7 was significantly higher in the Omega-7-treated sites compared to control sites (P < 0.05). In summary, topical application of Omega-7 accelerates healing of both grafted burn and donor site wounds. Omega-7 should be considered as a cost-efficient and effective supplement therapy for burn wound healing.

Effects of clovamide and its related compounds on the aggregations of amyloid polypeptides.

Alzheimer's disease (AD) and type 2 diabetes (T2D) are common diseases in the elderly, and the increasing number of patients with these diseases has become a serious health problem worldwide. The aggregation and development of plaque of amyloid polypeptides (amyloid ß; Aß and human islet amyloid polypeptide; hIAPP, amylin) are found in the brains of patients with AD and the pancreas of patients with T2D and are considered to be, in part, the causes of both diseases, respectively. Therefore, preventing amyloid aggregation may be a promising therapeutic strategy for preventing AD and T2D. In addition, the disaggregation of the already aggregated amyloid polypeptides is expected to contribute to the prevention and treatment of both diseases as amyloid polypeptide aggregations begin several decades before the onset of disease. Therefore, in this study, we investigated the hIAPP aggregation inhibitory activity and Aß42/hIAPP disaggregation activity of clovamide which had shown inhibitory activity against Aß42 aggregation in our previous studies. In addition, active sites were identified (structure-activity relationship analysis) using clovamide-related compounds in which hydroxyl groups of these compounds were either eliminated or methylated. Our results showed that the compounds with one or two catechol moieties showed strong hIAPP aggregation inhibitory activity and Aß42/hIAPP disaggregation activity; and the non-catechol type compounds showed little or no activity. This suggests that the catechol moiety is important in amyloid polypeptide aggregation inhibition and disaggregation. Thus, clovamide and its related compounds may be promising therapeutic strategies for inhibiting amyloid polypeptide-related pathology in AD and T2D.

Isolation and Characterization of Compounds from Glycyrrhiza uralensis as Therapeutic Agents for the Muscle Disorders.

Skeletal muscle is the most abundant tissue and constitutes about 40% of total body mass. Herein, we report that crude water extract (CWE) of G. uralensis enhanced myoblast proliferation and differentiation. Pretreatment of mice with the CWE of G. uralensis prior to cardiotoxin-induced muscle injury was found to enhance muscle regeneration by inducing myogenic gene expression and downregulating myostatin expression. Furthermore, this extract reduced nitrotyrosine protein levels and atrophy-related gene expression. Of the five different fractions of the CWE of G. uralensis obtained, the ethyl acetate (EtOAc) fraction more significantly enhanced myoblast proliferation and differentiation than the other fractions. Ten bioactive compounds were isolated from the EtOAc fraction and characterized by GC-MS and NMR. Of these compounds (4-hydroxybenzoic acid, liquiritigenin, (R)-(-)-vestitol, isoliquiritigenin, medicarpin, tetrahydroxymethoxychalcone, licochalcone B, liquiritin, liquiritinapioside, and ononin), liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were found to enhance myoblast proliferation and differentiation, and myofiber diameters in injured muscles were wider with the liquiritigenin than the non-treated one. Computational analysis showed these compounds are non-toxic and possess good drug-likeness properties. These findings suggest that G. uralensis-extracted components might be useful therapeutic agents for the management of muscle-associated diseases.

Co-option of PPARα in the regulation of lipogenesis and fatty acid oxidation in CLA-induced hepatic steatosis.

Nonalcoholic-fatty-liver-disease (NAFLD) is the result of imbalances in hepatic lipid partitioning and is linked to dietary factors. We demonstrate that conjugated linoleic acid (CLA) when given to mice as a dietary supplement, induced an enlarged liver, hepatic steatosis, and increased plasma levels of fatty acid (FA), alanine transaminase, and triglycerides. The progression of NAFLD and insulin resistance was reversed by GW6471 a small-molecule antagonist of peroxisome proliferator-activated receptor α (PPARα). Transcriptional profiling of livers revealed that the genes involved in FA oxidation and lipogenesis as two core gene programs controlled by PPARα in response to CLA and GW6471 including Acaca and Acads. Bioinformatic analysis of PPARα ChIP-seq data set and ChIP-qPCR showed that GW6471 blocks PPARα binding to Acaca and Acads and abolishes the PPARα-mediated local histone modifications of H3K27ac and H3K4me1 in CLA-treated hepatocytes. Thus, our findings reveal a dual role of PPARα in the regulation of lipid homeostasis and highlight its druggable nature in NAFLD.

Altered Bioavailability of Nitric Oxide and L-Arginine Is a Key Determinant of Endothelial Dysfunction in Preeclampsia.

BACKGROUND: Preeclampsia is a major cause of maternal and neonatal morbidity and mortality in sub-Saharan Africa. Evidence indicates that endothelial dysfunction is central to the pathogenesis of preeclampsia. This study assessed the level of the components of the arginine-nitric oxide pathway to evaluate endothelial dysfunction in normotensive pregnancies and pregnancies complicated with preeclampsia. METHODS: This case-control study was conducted among pregnant women who visited Comboni Hospital from January 2017 to May 2018. A total of 180 pregnant women comprising 88 preeclamptic women (PE) and 92 healthy normotensive pregnant women (NP) were recruited. Sociodemographic, clinical, and obstetric data were obtained using validated questionnaires. Blood pressure and anthropometrics were measured, and blood samples were collected for the estimation of nitric oxide (NO∙), L-arginine, asymmetric dimethylarginine (ADMA), and 3-nitrotyrosine using an enzyme-linked immunosorbent assay technique. RESULTS: The mean NO∙ (p = 0.010) and L-arginine/ADMA ratio (p < 0.0001) was significantly lower in PE compared to NP while mean L-arginine (p = 0.034), ADMA (p < 0.0001), and 3-nitrotyrosine (p < 0.0001) were significantly higher in PE than NP. ADMA showed a significant positive association with systolic blood pressure (ß = 0.454, p = 0.036) in severe PE. Women with PE had significant intrauterine growth restriction (p < 0.0001) and low birth weight infants (p < 0.0001) when compared to NP. CONCLUSION: Preeclampsia is associated with reduced NO∙ bioavailability, L-arginine/ADMA ratio, and elevated levels of ADMA and 3-nitrotyrosine. Measurements of the levels of these parameters can help in the early prediction of endothelial dysfunction in preeclampsia. Exogenous therapeutic supplementation with L-arginine during pregnancy to increase the L-arginine/ADMA ratio should be considered to improve endothelial function in preeclampsia and pregnant women at risk of developing preeclampsia.

MG53 Protects against Sepsis-Induced Myocardial Dysfunction by Upregulating Peroxisome Proliferator-Activated Receptor-α.

BACKGROUND: The heart is one of the most commonly affected organs during sepsis. Mitsugumin-53 (MG53) has attracted attention in research due to its cardioprotective function. However, the role of MG53 in sepsis-induced myocardial dysfunction (SIMD) remains unknown. The purpose of this study was to explore the underlying mechanism of MG53 in SIMD and investigate its potential relationship with peroxisome proliferator-activated receptor-α (PPARα). METHODS: The cecal ligation and puncture (CLP) model was created to induce SIMD in rats. Protein levels of MG53 and PPARα, cardiac function, cardiomyocyte injury, myocardial oxidative stress and inflammatory indicators, and cardiomyocyte apoptosis were measured at 18 h after CLP. The effects of MG53 on PPARα in SIMD were investigated via preconditioning recombinant human MG53 (rhMG53) and PPARα antagonist GW6471. RESULTS: The expression of MG53 and PPARα sharply decreased in the myocardium at 18 h after CLP. Compared with the sham group, cardiac function was significantly depressed, which was associated with the destructed myocardium, upregulated oxidative stress indicators and proinflammatory cytokines, and excessive cardiomyocyte apoptosis in the CLP group. Supplementation with rhMG53 enhanced myocardial MG53, increased the survival rate with improved cardiac function, and reduced oxidative stress, inflammation, and myocardial apoptosis, which were associated with PPARα upregulation. Pretreatment with GW6471 abolished the abovementioned protective effects induced by MG53. CONCLUSIONS: Both MG53 and PPARα were downregulated after sepsis shock. MG53 supplement protects the heart against SIMD by upregulating PPARα expression. Our results provide a new treatment strategy for SIMD.

Epigallocatechin-3-Gallate (EGCG), an Active Compound of Green Tea Attenuates Acute Lung Injury Regulating Macrophage Polarization and Krüpple-Like-Factor 4 (KLF4) Expression.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are serious clinical complications with a high frequency of morbidity and mortality. The initiation and amplification of inflammation is a well-known aspect in the pathogenesis of ALI and related disorders. Therefore, inhibition of the inflammatory mediators could be an ideal approach to prevent ALI. Epigallocatechin-3-gallate (EGCG), a major constituent of green tea, has been shown to have protective effects on oxidative damage and anti-inflammation. The goal of the present study was to determine whether EGCG improves phenotype and macrophage polarisation in LPS-induced ALI. C57BL/6 mice were given two doses of EGCG (15 mg/kg) intraperitoneally (IP) 1 h before and 3 h after LPS instillation (2 mg/kg). EGCG treatment improved histopathological lesions, Total Leucocyte count (TLC), neutrophils infiltration, wet/dry ratio, total proteins and myeloperoxidase (MPO) activity in LPS-induced lung injury. The results displayed that EGCG reduced LPS-induced ALI as it modulates macrophage polarisation towards M2 status. Furthermore, EGCG also reduced the expression of proinflammatory M1 mediators iNOS TNF-α, IL-1ß and IL-6 in the LPS administered lung microenvironment. In addition, it increased the expression of KLF4, Arg1 and ym1, known to augment the M2 phenotype of macrophages. EGCG also alleviated the expression of 8-OHdG, nitrotyrosine, showing its ability to inhibit oxidative damage. TREM1 in the lung tissue and improved lung regenerative capacity by enhancing Ki67, PCNA and Ang-1 protein expression. Together, these results proposed the protective properties of EGCG against LPS-induced ALI in may be attributed to the suppression of M1/M2 macrophages subtype ratio, KLF4 augmentation, lung cell regeneration and regulating oxidative damage in the LPS-induced murine ALI.

Trimethylamine-N-Oxide Promotes Age-Related Vascular Oxidative Stress and Endothelial Dysfunction in Mice and Healthy Humans.

Age-related vascular endothelial dysfunction is a major antecedent to cardiovascular diseases. We investigated whether increased circulating levels of the gut microbiome-generated metabolite trimethylamine-N-oxide induces endothelial dysfunction with aging. In healthy humans, plasma trimethylamine-N-oxide was higher in middle-aged/older (64±7 years) versus young (22±2 years) adults (6.5±0.7 versus 1.6±0.2 µmol/L) and inversely related to brachial artery flow-mediated dilation (r2=0.29, P<0.00001). In young mice, 6 months of dietary supplementation with trimethylamine-N-oxide induced an aging-like impairment in carotid artery endothelium-dependent dilation to acetylcholine versus control feeding (peak dilation: 79±3% versus 95±3%, P<0.01). This impairment was accompanied by increased vascular nitrotyrosine, a marker of oxidative stress, and reversed by the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. Trimethylamine-N-oxide supplementation also reduced activation of endothelial nitric oxide synthase and impaired nitric oxide-mediated dilation, as assessed with the nitric oxide synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester). Acute incubation of carotid arteries with trimethylamine-N-oxide recapitulated these events. Next, treatment with 3,3-dimethyl-1-butanol for 8 to 10 weeks to suppress trimethylamine-N-oxide selectively improved endothelium-dependent dilation in old mice to young levels (peak: 90±2%) by normalizing vascular superoxide production, restoring nitric oxide-mediated dilation, and ameliorating superoxide-related suppression of endothelium-dependent dilation. Lastly, among healthy middle-aged/older adults, higher plasma trimethylamine-N-oxide was associated with greater nitrotyrosine abundance in biopsied endothelial cells, and infusion of the antioxidant ascorbic acid restored flow-mediated dilation to young levels, indicating tonic oxidative stress-related suppression of endothelial function with higher circulating trimethylamine-N-oxide. Using multiple experimental approaches in mice and humans, we demonstrate a clear role of trimethylamine-N-oxide in promoting age-related endothelial dysfunction via oxidative stress, which may have implications for prevention of cardiovascular diseases.

Peroxisome proliferator-activated receptor-γ mediates the antihypertensive effects of acupuncture in spontaneously hypertensive rats.

We investigated a central antihypertensive effect of acupuncture in rostral ventrolateral medulla (RVLM) in spontaneously hypertensive rats (SHRs). In total, 56 rats were randomly divided into seven groups as follows: the SHR group, SHR+acupuncture (SHR+Acu) group, SHR+nonacupuncture (SHR+Non-acu) group, GW9662+acupuncture (GW9662+Acu) group, GW9662+GW1929 group, GW9662 group, and 2% DMSO group (n = 8 per group). The whole eight Wistar-Kyoto rats were assigned to the WKY group. The acupuncture treatment lasting for 14 days was performed at the Taichong acupoint (LR3) or at a nonacupoint (non-acu) once daily. The peroxisome proliferator-activated receptor (PPAR)-γ agonist GW1929 and the PPAR-γ inhibitor GW9662 were microinjected by the brain stereotactic technique. Blood pressure was measured by the tail-cuff method. Sympathetic vasomotor activity was determined by implanting in a telemetry electrocardiogram radio transmitter. The expression of PPARs in the RVLM of the rats was detected using Western blot. We demonstrated that acupuncture attenuated blood pressure, heart rate, and sympathetic vasomotor activity in SHRs. The protein expression of PPAR-γ was significantly increased in SHRs treated with acupuncture. The antihypertensive effects of acupuncture in SHRs were abrogated by microinjection bilaterally into RVLM of GW9662. Microinjection of GW1929 mimicked the antihypertensive effect of acupuncture. PPAR-γ expression was negatively correlated with blood pressure and sympathetic vasomotor activity in SHRs treated with acupuncture. These results suggested that acupuncture promoted a central antihypertensive effect by increasing the expression of PPAR-γ in RVLM of SHRs.

Consuming decaffeinated coffee with milk and sugar added before a high-glycaemic index meal improves postprandial glycaemic and insulinaemic responses in healthy adults.

The present study aimed to compare the effects of drinking different types of coffee before a high-glycaemic index (GI) meal on postprandial glucose metabolism and to assess the effects of adding milk and sugar into coffee. In this randomised, crossover, acute feeding study, apparently healthy adults (n 21) consumed the test drink followed by a high-GI meal in each session. Different types of coffee (espresso, instant, boiled and decaffeinated, all with milk and sugar) and plain water were tested in separate sessions, while a subset of the participants (n 10) completed extra sessions using black coffees. Postprandial levels of glucose, insulin, active glucagon-like peptide 1 (GLP-1) and nitrotyrosine between different test drinks were compared using linear mixed models. Results showed that only preloading decaffeinated coffee with milk and sugar led to significantly lower glucose incremental AUC (iAUC; 14 % lower, P = 0·001) than water. Preloading black coffees led to greater postprandial glucose iAUC than preloading coffees with milk and sugar added (12-35 % smaller, P < 0·05 for all coffee types). Active GLP-1 and nitrotyrosine levels were not significantly different between test drinks. To conclude, preloading decaffeinated coffee with milk and sugar led to a blunted postprandial glycaemic response after a subsequent high-GI meal, while adding milk and sugar into coffee could mitigate the impairment effect of black coffee towards postprandial glucose responses. These findings may partly explain the positive effects of coffee consumption on glucose metabolism.

Grape Polyphenols Ameliorate Muscle Decline Reducing Oxidative Stress and Oxidative Damage in Aged Rats.

A large number of studies have demonstrated the implication of oxidative stress (OxS) in the pathogenesis of ageing-related muscle decline and atrophy. The key mechanism is related to the OxS-induced production of free radicals, with the consequent increase in oxidative damage, resulting in affected muscle quality and strength. The present study aimed to evaluate the efficacy of a grape polyphenol-based nutraceutical formulation (Taurisolo®) in reducing the OxS in muscle of aged rats. A group of 16 aged (20 months) rats were orally administered with Taurisolo® (n = 8; 100 mg/kg Taurisolo®) or placebo (n = 8; 50 mg/kg maltodextrin); an additional group of eight young (three months) rats were also treated with placebo. All the treatments were orally administered for 30 days. The activities of antioxidant enzymes, the levels of malondialdehyde (MDA) and nitrotyrosine (N-Tyr) and the expression of OxS- and inflammation-related genes were evaluated on the gastrocnemius muscle. In muscle samples of the treated-group, increased activity of antioxidant enzymes, reduced MDA and N-Tyr levels and increased expression of antioxidant and anti-inflammatory genes were observed in respect to the placebo. Data herein presented suggest that the chronic treatment with Taurisolo® significantly reduces oxidative damage and improves muscle performance in aged rats.

Probing the Mechanism of Structure-Switching Aptamer Assembly by Super-Resolution Localization of Individual DNA Molecules.

Oligonucleotide aptamers can be converted into structure-switching biosensors by incorporating a short, typically labeled oligonucleotide that is complementary to the analyte-binding region. Binding of a target analyte can disrupt the hybridization equilibrium between the aptamer and the labeled-complementary oligo producing a concentration-dependent signal for target-analyte sensing. Despite its importance in the performance of a biosensor, the mechanism of analyte-response of most structure-switching aptamers is not well understood. In this work, we employ single-molecule fluorescence imaging to investigate the competitive kinetics of association of a labeled complementary oligonucleotide and a target analyte, l-tyrosinamide (L-Tym), interacting with an L-Tym-binding aptamer. The complementary readout strand is fluorescently labeled, allowing us to measure its hybridization kinetics with individual aptamers immobilized on a surface and located with super-resolution techniques; the small-molecule L-Tym analyte is not labeled in order to avoid having an attached dye molecule impact its interactions with the aptamer. We measure the association kinetics of unlabeled L-Tym by detecting its influence on the hybridization of the labeled complementary strand. We find that L-Tym slows the association rate of the complementary strand with the aptamer but does not impact its dissociation rate, suggesting an SN1-like mechanism where the complementary strand must dissociate before L-Tym can bind. The competitive model revealed a slow association rate between L-Tym and the aptamer, producing a long-lived L-Tym-aptamer complex that blocks hybridization with the labeled complementary strand. These results provide insight about the kinetics and mechanism of analyte recognition in this structure-switching aptamer, and the methodology provides a general means of measuring the rates of unlabeled-analyte binding kinetics in aptamer-based biosensors.

Pine (Pinus sylvestris L.) bark proanthocyanidins affords prevention of peroxynitrite-induced l-tyrosine nitration, DNA damage and hydroxyl radical formation.

Peroxynitrite is known as a strong deleterious species that may readily trigger several geriatric diseases via injuring cellular constituents. Proanthocyanidins, a biological flavonoids constituent of Pinus sylvestris L. bark, has been attributed a large variety of pharmacological functions to its antioxidant potential. The results revealed that peroxynitrite could cause the generation of hydroxyl radical, the breakage of φX-174 plasmid DNA brand as well as the nitration of L-tyrosine. However, pine (Pinus sylvestris L.) bark proanthocyanidins extracts at low concentration range markedly inhibited the peroxynitrite -induced the formation of open circular DNA form (IC50 = 5.03±0.39 mg/mL). The 3-Nitro-L-tyrosine generated by the reaction of peroxynitrite with L-tyrosine was reduced by PBP (IC50 = 1.01±0.01 mg/mL). Besides, electron spin resonance spectroscopy data indicates that the intensive signal of dimethyl pyridine N-oxide hydroxyl radical adduct from peroxynitrite was reversed by pine bark proanthocyanidins extracts (IC50 =1.02±0.04 mg/mL). Moreover, the obtained data shows that PBP provides more efficient protection against peroxynitrite than that of ascorbic acid. Together, the present study suggests that pine bark proanthocyanidins could exert potent preventive activity against peroxynitrite -elicited cytotoxicity on the biomacromolecules, a study-worthy finding with pharmacological importance.

Approaches to develop therapeutics to treat frontotemporal dementia.

Frontotemporal degeneration (FTD) is a complex disease presenting as a spectrum of clinical disorders with progressive degeneration of frontal and temporal brain cortices and extensive neuroinflammation that result in personality and behavior changes, and eventually, death. There are currently no effective therapies for FTD. While 60-70% of FTD patients are sporadic cases, the other 30-40% are heritable (familial) cases linked to mutations in several known genes. We focus here on FTD caused by mutations in the GRN gene, which encodes a secreted protein, progranulin (PGRN), that has diverse roles in regulating cell survival, immune responses, and autophagy and lysosome function in the brain. FTD-linked mutations in GRN reduce brain PGRN levels that lead to autophagy and lysosome dysfunction, TDP43 accumulation, excessive microglial activation, astrogliosis, and neuron death through still poorly understood mechanisms. PGRN insufficiency has also been linked to Alzheimer's disease (AD), and so the development of therapeutics for GRN-linked FTD that restore PGRN levels and function may have broader application for other neurodegenerative diseases. This review focuses on a strategy to increase PGRN to functional, healthy levels in the brain by identifying novel genetic and chemical modulators of neuronal PGRN levels. This article is part of the special issue entitled 'The Quest for Disease-Modifying Therapies for Neurodegenerative Disorders'.