Recombinant H77C gpE1/gpE2 heterodimer elicits superior HCV cross-neutralisation than H77C gpE2 alone.

BACKGROUND & AIMS: An optimal HCV vaccine requires the induction of antibodies that neutralise the infectivity of many heterogenous viral isolates. In this study, we have focused on determining the optimal recombinant envelope glycoprotein component to elicit cross-neutralising antibodies against global HCV genotypes. We compared the immunoreactivity and antigenicity of the recombinant HCV genotype 1a strain H77C envelope glycoprotein heterodimer gpE1/gpE2 with that of recombinant gpE2 alone derived from an infectious molecular clone (H77C). METHODS: Characterization of the envelope glycoproteins was accomplished by determining their ability to bind to a panel of broadly cross-neutralising monoclonal antibodies (bNAbs). Immunogenicity was determined by testing the ability of vaccine antisera to neutralise the infectivity in vitro of a panel of pseudotyped HCV particles in which gpE1/gpE2 derived from representative isolates of the major global HCV genotypes were displayed. RESULTS: gpE1/gpE2 binds to more diverse bNabs than gpE2 alone and elicits a broader profile of cross-neutralising antibodies in animals, especially against more heterologous, non-1a genotypes. While not all heterologous HCV strains can be potently inhibited in vitro by gpE1/gpE2 antisera derived from a single HCV strain, the breadth of heterologous cross-neutralisation is shown to be substantial. CONCLUSIONS: Our work supports the inclusion of gpE1/gpE2 in an HCV vaccine in order to maximise the cross-neutralisation of heterogenous HCV isolates. Our data also offers future directions in formulating a cocktail of gpE1/gpE2 antigens from a small selection of HCV genotypes to further enhance cross-neutralisation of global HCV strains and hopefully, achieving global protection. IMPACT AND IMPLICATIONS: An HCV vaccine is urgently required to prevent the high global incidence of HCV infection and disease. Since HCV is a highly heterogeneous virus, it is desirable for a vaccine to elicit antibodies that neutralise the infectivity of most global strains. To this end, we have compared the immunoreactivity and antigenicity of recombinant H77C E1E2 heterodimer with that of H77C E2 alone and show that the former exhibits more cross-neutralising epitopes and demonstrates a broader cross-neutralisation profile in vitro. In addition, our data suggests a way to further broaden cross-neutralisation using a combination of E1E2 antigens derived from a few different HCV clades. Our work provides encouragement for the development of an effective global HCV vaccine.

Maternal selenium dietary supplementation alters sociability and reinforcement learning deficits induced by in utero exposure to maternal immune activation in mice.

Maternal immune activation (MIA) during pregnancy increases the risk for the unborn foetus to develop neurodevelopmental conditions such as autism spectrum disorder and schizophrenia later in life. MIA mouse models recapitulate behavioural and biological phenotypes relevant to both conditions, and are valuable models to test novel treatment approaches. Selenium (Se) has potent anti-inflammatory properties suggesting it may be an effective prophylactic treatment against MIA. The aim of this study was to determine if Se supplementation during pregnancy can prevent adverse effects of MIA on offspring brain and behaviour in a mouse model. Selenium was administered via drinking water (1.5 ppm) to pregnant dams from gestational day (GD) 9 to birth, and MIA was induced at GD17 using polyinosinic:polycytidylic acid (poly-I:C, 20 mg/kg via intraperitoneal injection). Foetal placenta and brain cytokine levels were assessed using a Luminex assay and brain elemental nutrients assessed using inductively coupled plasma- mass spectrometry. Adult offspring were behaviourally assessed using a reinforcement learning paradigm, the three-chamber sociability test and the open field test. MIA elevated placental IL-1ß and IL-17, and Se supplementation successfully prevented this elevation. MIA caused an increase in foetal brain calcium, which was prevented by Se supplement. MIA caused in offspring a female-specific reduction in sociability, which was recovered by Se, and a male-specific reduction in social memory, which was not recovered by Se. Exposure to poly-I:C or selenium, but not both, reduced performance in the reinforcement learning task. Computational modelling indicated that this was predominantly due to increased exploratory behaviour, rather than reduced rate of learning the location of the food reward. This study demonstrates that while Se may be beneficial in ameliorating sociability deficits caused by MIA, it may have negative effects in other behavioural domains. Caution in the use of Se supplementation during pregnancy is therefore warranted.

Flavonoids and phenolic acids from sugarcane: Distribution in the plant, changes during processing, and potential benefits to industry and health.

Sugarcane (Saccharum sp.) plants are grown in warmer climates throughout the world and processed to produce sugar as well as other useful byproducts such as molasses and bagasse. Sugarcane is rich in (poly)phenols, but there has been no attempt to critically evaluate the published information based on the use of suitable methodologies. The objective of this review is to evaluate the quantitative and qualitative (poly)phenolic profiles of individual parts of the sugarcane plant and its multiple industrial products, which will help develop new processes and uses for sugarcane (poly)phenols. The quantitative analysis involves the examination of extraction, concentration, and analytical techniques used in each study for each plant part and product. The qualitative analysis indicates the identification of various (poly)phenols throughout the sugarcane processing chain, using only compounds elucidated through robust analytical methodologies such as mass spectrometry or nuclear magnetic resonance. In conclusion, sugarcane (poly)phenols are predominantly flavonoids and phenolic acids. The main flavonoids, derivatives of apigenin, luteolin, and tricin, with a substantial proportion of C-glycosides, are consistently found across all phases of sugarcane processing. The principal phenolic acids reported throughout the process include chlorogenic acids, as well as ferulic and caffeic acids mostly observed after hydrolysis. The derivation of precise quantitative information across publications is impeded by inconsistencies in analytical methodologies. The presence of multiple (poly)phenols with potential benefits for industrial applications and for health suggests sugarcane could be a useful provider of valuable compounds for future use in research and industrial processes.

Structure-function relationships in (poly)phenol-enzyme binding: Direct inhibition of human salivary and pancreatic α-amylases.

(Poly)phenols inhibit α-amylase by directly binding to the enzyme and/or by forming starch-polyphenol complexes. Conventional methods using starch as the substrate measure inhibition from both mechanisms, whereas the use of shorter oligosaccharides as substrates exclusively measures the direct interaction of (poly)phenols with the enzyme. In this study, using a chromatography-based method and a short oligosaccharide as the substrate, we investigated the detailed structural prerequisites for the direct inhibition of human salivary and pancreatic α-amylases by over 50 (poly)phenols from the (poly)phenol groups: flavonols, flavones, flavanones, flavan-3-ols, polymethoxyflavones, isoflavones, anthocyanidins and phenolic acids. Despite being structurally very similar (97% sequence homology), human salivary and pancreatic α-amylases were inhibited to different extents by the tested (poly)phenols. The most potent human salivary α-amylase inhibitors were luteolin and pelargonidin, while the methoxylated anthocyanidins, peonidin and petunidin, significantly blocked pancreatic enzyme activity. B-ring methoxylation of anthocyanidins increased inhibition against both human α-amylases while hydroxyl groups at C3 and B3' acted antagonistically in human salivary inhibition. C4 carbonyl reduction, or the positive charge on the flavonoid structure, was the key structural feature for human pancreatic inhibition. B-ring glycosylation did not affect salivary enzyme inhibition, but increased pancreatic enzyme inhibition when compared to its corresponding aglycone. Overall, our findings indicate that the efficacy of interaction with human α-amylase is mainly influenced by the type and placement of functional groups rather than the number of hydroxyl groups and molecular weight.

Inhibitory effect of extracts from edible parts of nuts on α-amylase activity: a systematic review.

Elevated blood glucose concentration is a risk factor for developing metabolic dysfunction and insulin resistance, leading to type 2 diabetes and cardiovascular diseases. Nuts have the potential to inhibit α-amylase activity, and so lower postprandial glucose, due to their content of polyphenols and other bioactive compounds. We conducted a systematic literature review to assess the ability of extracts from commonly consumed edible parts of nuts to inhibit α-amylase. Among the 31 included papers, only four utilised human α-amylases. These papers indicated that polyphenol-rich chestnut skin extracts exhibited strong inhibition of both human salivary and pancreatic α-amylases, and that a polyphenol-rich almond skin extract was a potent inhibitor of human salivary α-amylase. The majority of the reviewed studies utilised porcine pancreatic α-amylase, which has ∼86% sequence homology with the corresponding human enzyme but with some key amino acid variations located within the active site. Polyphenol-rich extracts from chestnut, almond, kola nut, pecan and walnut, and peptides isolated from cashew, inhibited porcine pancreatic α-amylase. Some studies used α-amylases sourced from fungi or bacteria, outcomes from which are entirely irrelevant to human health, as they have no sequence homology with the human enzyme. Given the limited research involving human α-amylases, and the differences in inhibition compared to porcine enzymes and especially enzymes from microorganisms, it is recommended that future in vitro experiments place greater emphasis on utilising enzymes sourced from humans to facilitate a reliable prediction of effects in intervention studies.

Effects of (Poly)phenols on Circadian Clock Gene-Mediated Metabolic Homeostasis in Cultured Mammalian Cells: A Scoping Review.

Circadian clocks regulate metabolic homeostasis. Disruption to our circadian clocks, by lifestyle behaviors such as timing of eating and sleeping, has been linked to increased rates of metabolic disorders. There is now considerable evidence that selected dietary (poly)phenols, including flavonoids, phenolic acids and tannins, may modulate metabolic and circadian processes. This review evaluates the effects of (poly)phenols on circadian clock genes and linked metabolic homeostasis in vitro, and potential mechanisms of action, by critically evaluating the literature on mammalian cells. A systematic search was conducted to ensure full coverage of the literature and identified 43 relevant studies addressing the effects of (poly)phenols on cellular circadian processes. Nobiletin and tangeretin, found in citrus, (-)-epigallocatechin-3-gallate from green tea, urolithin A, a gut microbial metabolite from ellagitannins in fruit, curcumin, bavachalcone, cinnamic acid, and resveratrol at low micromolar concentrations all affect circadian molecular processes in multiple types of synchronized cells. Nobiletin emerges as a putative retinoic acid-related orphan receptor (RORα/γ) agonist, leading to induction of the circadian regulator brain and muscle ARNT-like 1 (BMAL1), and increased period circadian regulator 2 (PER2) amplitude and period. These effects are clear despite substantial variations in the protocols employed, and this review suggests a methodological framework to help future study design in this emerging area of research.

Inhibition of human starch digesting enzymes and intestinal glucose transport by walnut polyphenols.

One approach to controlling type 2 diabetes (T2D) is to lower postprandialglucose spikesby slowing down the digestion of carbohydrates and the absorption of glucose in the small intestine. The consumption of walnuts is associated with a reduced risk of chronic diseases such as T2D, suggested to be partly due to the high content of (poly)phenols. This study evaluated, for the first time, the inhibitory effect of a (poly)phenol-rich walnut extract on human carbohydrate digesting enzymes (salivary and pancreatic α-amylases, brush border sucrase-isomaltase) and on glucose transport across fully differentiated human intestinal Caco-2/TC7 monolayers. The walnut extract was rich in multiple (poly)phenols (70 % w/w) as analysed by Folin-Ciocalteau and by LCMS. It exhibited potent inhibition of both human salivary (IC50: 32.2 ± 2.5 µg walnut (poly)phenols (WP)/mL) and pancreatic (IC50: 56.7 ± 1.7 µg WP/mL) α-amylases, with weaker effects on human sucrase (IC50: 990 ± 20 µg WP/mL), maltase (IC50: 1300 ± 80 µg WP/mL), and isomaltase (IC25: 830 ± 60 µg WP/mL) activities. Selected individual walnut (poly)phenols inhibited human salivary α-amylase in the order: 1,3,4,6-tetragalloylglucose > ellagic acid pentoside > 1,2,6-tri-O-galloyl-ß-D-glucopyranose, with no inhibition by ellagic acid, gallic acid and 4-O-methylgallic acid. The (poly)phenol-rich walnut extract also attenuated (up to 59 %) the transfer of 2-deoxy-D-glucose across differentiated Caco-2/TC7 cell monolayers. This is the first report on the effect of (poly)phenol-rich extracts from any commonly-consumed nut kernel on any human starch-digesting enzyme, and suggests a mechanism through which walnut consumption may lower postprandial glucose spikes and contribute to their proposed health benefits.

The flavonoid quercetin decreases ACE2 and TMPRSS2 expression but not SARS-CoV-2 infection in cultured human lung cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) on host cells, via its spike protein, and transmembrane protease, serine 2 (TMPRSS2) cleaves the spike-ACE2 complex to facilitate virus entry. As rate-limiting steps for virus entry, modulation of ACE2 and/or TMPRSS2 may decrease SARS-CoV-2 infectivity and COVID-19 severity. In silico modeling suggested the natural bioactive flavonoid quercetin can bind to ACE2 and a recent randomized clinical trial demonstrated that oral supplementation with quercetin increased COVID-19 recovery. A range of cultured human cells were assessed for co-expression of ACE2 and TMPRSS2. Immortalized Calu-3 lung cells, cultured and matured at an air-liquid interface (Calu-3-ALIs), were established as the most appropriate. Primary bronchial epithelial cells (PBECs) were obtained from healthy adult males (N = 6) and cultured under submerged conditions to corroborate the outcomes. Upon maturation or reaching 80% confluence, respectively, the Calu-3-ALIs and PBECs were treated with quercetin, and mRNA and protein expression were assessed by droplet digital PCR and ELISA, respectively. SARS-CoV-2 infectivity, and the effects of pre- and co-treatment with quercetin, was assessed by median tissue culture infectious dose assay. Quercetin dose-dependently decreased ACE2 and TMPRSS2 mRNA and protein in both Calu-3-ALIs and PBECs after 4 h, while TMPRSS2 remained suppressed in response to prolonged treatment with lower doses (twice daily for 3 days). Quercetin also acutely decreased ADAM17 mRNA, but not ACE, in Calu-3-ALIs, and this warrants further investigation. Calu-3-ALIs, but not PBECs, were successfully infected with SARS-CoV-2; however, quercetin had no antiviral effect, neither directly nor indirectly through downregulation of ACE2 and TMPRSS2. Calu-3-ALIs were reaffirmed to be an optimal cell model for research into the regulation of ACE2 and TMPRSS2, without the need for prior genetic modification, and will prove valuable in future coronavirus and respiratory infectious disease work. However, our data demonstrate that a significant decrease in the expression of ACE2 and TMPRSS2 by a promising prophylactic candidate may not translate to infection prevention.

Development of a low-fructose carbohydrate gel for exercise application.

This study aimed to develop a low-fructose (<3 g/serve) carbohydrate (CHO) gel for athletes. Various prototypes with 30 g CHO/serve and differing water content (12 %, 21 %, 32 %, 39 % w/v) were created and evaluated for sensory attributes. The final gel contained 62.1 ± 0.2 g CHO/100 g with 0.17 % w/w fructose. Endurance athletes (n = 20) underwent a feeding-challenge protocol, ingesting 30 g gel every 20 min during 2 h of running (60 % V ˙ O2max), followed by a 1 h self-paced distance test. Blood glucose increased significantly from baseline (4.0 ± 0.9 vs. 6.6 ± 0.6 mmol/L, p < 0.001) and remained elevated after the distance test (4.9 ± 0.7 mmol/L, p < 0.05). Breath hydrogen levels increased (5 ± 4 ppm, p < 0.05) without substantial CHO malabsorption detected. Gastrointestinal symptoms (GIS) increased during exercise but were mild. The low-fructose CHO gel demonstrated good tolerance, promoting glucose availability without severe GIS or CHO malabsorption.

Small Molecules Targeting GRP78 Mitigate Anti- GRP78 Autoantibody-Mediated Tissue Factor Procoagulant Activity in Cultured Endothelial Cells.

BACKGROUND: The 78-kDa glucose-regulated protein (GRP78) expressed on the cell surface (csGRP78) has been reported to regulate tissue factor procoagulant activity (TF PCA) in lesion-resident endothelial cells (ECs), which is further enhanced by circulating anti-GRP78 autoantibodies that bind to the Leu98-Leu115 epitope in GRP78. OBJECTIVES: Determine the effects of the engagement of the anti-GRP78 autoantibody to cell surface GRP78 on endothelial cells and the underlying mechanisms that impact TF PCA. METHODS: Immunofluorescent staining was used to determine the presence of csGRP78 in TNFα-treated endothelial cells. An established TF PCA assay was used to evaluate human ECs following treatment with anti-GRP78 autoantibodies. The Fura 2-AM assay was used to quantify changes in intracellular Ca2+ levels. Small molecules predicted to bind csGRP78 were identified using artificial intelligence. ELISAs were used to assess the ability of these GRP78 binders to mitigate TF activity and interfere with the autoantibody/csGRP78 complex. RESULTS: In TNFα-treated ECs, anti-GRP78 autoantibodies increased TF PCA. This observation was further enhanced by ER-stress-induced elevation of csGRP78 levels. Anti-GRP78 autoantibody treatment increased intracellular Ca2+ levels. Sequestering the anti-GRP78 autoantibody with a conformational peptide or blocking with heparin molecules attenuated anti-GRP78 autoantibody-induced TF PCA. We identified B07*, a GRP78 binder that diminished anti-GRP78 autoantibody-induced TF PCA on ECs. CONCLUSIONS: These findings show how anti-GRP78 autoantibodies enhance TF PCA that contributes to thrombosis and identify novel GRP78 binders that represent a potential novel therapeutic strategy for treating and managing atherothrombotic disease.