Fibrinogen αC‐regions are not directly involved in fibrin polymerization as evidenced by a "Double‐Detroit" recombinant fibrinogen mutant and knobs‐mimic peptides

Background: Fibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α‐ and β‐chains N‐termini (GPR, GHR; A‐, B‐knobs, respectively) engaging pre‐existent a and b pockets in other fibrin(ogen) molecules' γ‐ and (B)β‐chains C‐terminal regions. A role for mostly disordered (A)α‐chains C‐terminal regions "bridging" between fibrin molecules/fibrils has been proposed. Objectives: Fibrinogen Detroit is a clinically observed mutation (AαR19→S) with non‐engaging GPS A‐knobs. By analogy, a similar Bβ‐chain mutation, BβR17→S, should produce non‐engaging GHS B‐knobs. A homozygous “Double‐Detroit” mutant (AαR19→S, BβR17→S; DD‐FG) was developed: with A‐a and B‐b engagements endogenously blocked, other interactions would become apparent. Methods: DD‐FG, wild‐type recombinant (WT‐FG), and human plasma (hp‐FG) fibrinogen self‐association was studied by turbidimetry coupled with fibrinopeptides release HPLC/mass spectrometry analyses, and by light‐scattering following size‐exclusion chromatography (SE‐HPLC). Results: In contrast to WT‐FG and hp‐FG, DD‐FG produced no turbidity increase, irrespective of thrombin concentration. The SE‐HPLC profile of concentrated DD‐FG was unaffected by thrombin treatment, and light‐scattering, at lower concentration, showed no intensity and hydrodynamic radius changes. Compared with hp‐FG, both WT‐FG and DD‐FG showed no FpA cleavage difference, while ~50% FpB was not recovered. Correspondingly, SDS‐PAGE/Western‐blots revealed partial Bβ‐chain N‐terminal and Aα‐chain C‐terminal degradation. Nevertheless, ~70% DD‐FG molecules bearing (A)αC‐regions potentially able to associate were available. Higher‐concentration, nearly‐intact hp‐FG with 500‐fold molar excess GPRP‐NH2/GHRP‐NH2 knobs‐mimics experiments confirmed these no‐associations findings. Conclusions: (A)αC‐regions interactions appear too weak to assist native fibrin polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered

Pomegranate juice, but not an extract, confers a lower glycemic response on a high–glycemic index food: randomized, crossover, controlled trials in healthy subjects

Background: Low–glycemic index diets have demonstrated health benefits associated with a reduced risk of developing type 2 diabetes. Objectives: We tested whether pomegranate polyphenols could lower the glycemic response of a high–glycemic index food when consumed together and the mechanism by which this might occur. Design: We compared the acute effect of a pomegranate juice and a polyphenol-rich extract from pomegranate (supplement) on the bread-derived postprandial blood glucose concentration in 2 randomized, crossover, controlled studies (double-blinded for the supplements), each on 16 healthy volunteers. An additional randomized, crossover, controlled study on 16 volunteers consuming constituent fruit acids in a pH-balanced solution (same pH as pomegranate) and bread was conducted to determine any contributions to postprandial responses caused by acidic beverages. Results: As primary outcome, the incremental area under the curve for bread-derived blood glucose (−33.1% ± 18.1%, P = 0.000005) and peak blood glucose (25.4% ± 19.3%, P = 0.0004) were attenuated by pomegranate juice, compared with a control solution containing the equivalent amount of sugars. In contrast, the pomegranate supplement, or a solution containing the malic and citric acid components of the juice, was ineffective. The pomegranate polyphenol punicalagin was a very effective inhibitor of human α-amylase in vitro, comparable to the drug acarbose. Neither the pomegranate extract nor the individual component polyphenols inhibited 14C-D-glucose transport across differentiated Caco-2/TC7 cell monolayers, but they inhibited uptake of 14C-glucose into Xenopus oocytes expressing the human glucose transporter type 2. Further, some of the predicted pomegranate gut microbiota metabolites modulated 14C-D-glucose and 14C-deoxy-D-glucose uptake into hepatic HepG2 cells. Conclusions: These data indicate that pomegranate polyphenols, when present in a beverage but not in a supplement, can reduce the postprandial glycemic response of bread, whereas microbial metabolites from pomegranate polyphenols exhibit the potential to further modulate sugar metabolism much later in the postprandial period. This trial was registered at clinicaltrials.gov as NCT02486978, NCT02624609, and NCT03242876

Nutritional implications of olives and sugar: attenuation of post-prandial glucose spikes in healthy volunteers by inhibition of sucrose hydrolysis and glucose transport by oleuropein

Purpose: The secoiridoid oleuropein, as found in olives and olive leaves, modulates some biomarkers of diabetes risk in vivo. A possible mechanism may be to attenuate sugar digestion and absorption. Methods: We explored the potential of oleuropein, prepared from olive leaves in a water soluble form (OLE), to inhibit digestive enzymes (α-amylase, maltase, sucrase), and lower [¹⁴C(U)]-glucose uptake in Xenopus oocytes expressing human GLUT2 and [¹⁴C(U)]-glucose transport across differentiated Caco-2 cell monolayers. We conducted 7 separate crossover, controlled, randomised intervention studies on healthy volunteers (double-blinded and placebo-controlled for the OLE supplement) to assess the effect of OLE on post-prandial blood glucose after consumption of bread, glucose or sucrose. Results: OLE inhibited intestinal maltase, human sucrase, glucose transport across Caco-2 monolayers, and uptake of glucose by GLUT2 in Xenopus oocytes, but was a weak inhibitor of human α-amylase. OLE, in capsules, in solution or as naturally present in olives, did not affect post-prandial glucose derived from bread, while OLE in solution attenuated post-prandial blood glucose after consumption of 25 g sucrose, but had no effect when consumed with 50 g of sucrose or glucose. Conclusion: The combined inhibition of sucrase activity and of glucose transport observed in vitro was sufficient to modify digestion of low doses of sucrose in healthy volunteers. In comparison, the weak inhibition of α-amylase by OLE was not enough to modify blood sugar when consumed with a starch-rich food, suggesting that a threshold potency is required for inhibition of digestive enzymes in order to translate into in vivo effects

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region

Objective: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. Conclusions: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth

Tangential beam IMRT versus tangential beam 3D-CRT of the chest wall in postmastectomy breast cancer patients: A dosimetric comparison

<p>Abstract</p> <p>Background</p> <p>This study evaluates the dose distribution of reversed planned tangential beam intensity modulated radiotherapy (IMRT) compared to standard wedged tangential beam three-dimensionally planned conformal radiotherapy (3D-CRT) of the chest wall in unselected postmastectomy breast cancer patients</p> <p>Methods</p> <p>For 20 unselected subsequent postmastectomy breast cancer patients tangential beam IMRT and tangential beam 3D-CRT plans were generated for the radiotherapy of the chest wall. The prescribed dose was 50 Gy in 25 fractions. Dose-volume histograms were evaluated for the PTV and organs at risk. Parameters of the dose distribution were compared using the Wilcoxon matched pairs test.</p> <p>Results</p> <p>Tangential beam IMRT statistically significantly reduced the ipsilateral mean lung dose by an average of 21% (1129 cGy versus 1437 cGy). In all patients treated on the left side, the heart volume encompassed by the 70% isodose line (V70%; 35 Gy) was reduced by an average of 43% (5.7% versus 10.6%), and the mean heart dose by an average of 20% (704 cGy versus 877 cGy). The PTV showed a significantly better conformity index with IMRT; the homogeneity index was not significantly different.</p> <p>Conclusions</p> <p>Tangential beam IMRT significantly reduced the dose-volume of the ipsilateral lung and heart in unselected postmastectomy breast cancer patients.</p

Differential patterns of inhibition of the sugar transporters GLUT2, GLUT5 and GLUT7 by flavonoids

Only limited data are available on the inhibition of the sugar transporter GLUT5 by flavonoids or other classes of bioactives. Intestinal GLUT7 is poorly characterised and no information exists concerning its inhibition. We aimed to study the expression of GLUT7 in Caco-2/TC7 intestinal cells, and evaluate inhibition of glucose transport by GLUT2 and GLUT7, and of fructose transport by GLUT2, GLUT5 and GLUT7, by flavonoids. Differentiated Caco-2/TC7 cell monolayers were used to investigate GLUT7 expression, as well as biotinylation and immunofluorescence to assess GLUT7 location. For mechanistic sugar transport studies, X. laevis oocytes were injected with individual mRNA, and GLUT protein expression on oocyte membranes was confirmed. Oocytes were incubated with D-[14C(U)]-glucose or D-[14C(U)]-fructose in the presence of flavonoids, and uptake was estimated by liquid scintilation counting. In differentiated Caco-2/TC7 cell monolayers, GLUT7 was mostly expressed apically. When applied apically, or to both compartments, sorbitol, galactose, L-glucose or sucrose did not affect GLUT7 mRNA expression. Fructose applied to both sides increased GLUT7 mRNA (13%, p ≤ 0.001) and total GLUT7 protein (2.7-fold, p ≤ 0.05), while the ratio between apical, basolateral and total GLUT7 protein was unchanged. In the X. laevis oocyte model, GLUT2-mediated glucose and fructose transport were inhibited by quercetin, (−)-epigallocatechin gallate (EGCG) and apigenin, GLUT5-mediated fructose transport was inhibited by apigenin and EGCG, but not by quercetin, and GLUT7-mediated uptake of both glucose and fructose was inhibited by apigenin, but not by quercetin nor EGCG. Expression of GLUT7 was increased by fructose, but only when applied to Caco-2/TC7 cells both apically and basolaterally. Since GLUT2, GLUT5 and GLUT7 show different patterns of inhibition by the tested flavonoids, we suggest that they have the potential to be used as investigational tools to distinguish sugar transporter activity in different biological settings

Green and Chamomile Teas, but not Acarbose, Attenuate Glucose and Fructose Transport via Inhibition of GLUT2 and GLUT5

1 Scope: High glycaemic sugars result in blood‐glucose spikes, while large doses of post‐prandial fructose inundate the liver, causing an imbalance in energy metabolism, both leading to increased risk of metabolic malfunction and type 2 diabetes. Acarbose, used for diabetes management, reduces post‐prandial hyperglycaemia by delaying carbohydrate digestion. 2 Methods and results: Chamomile and green teas both inhibited digestive enzymes (α‐amylase and maltase) related to intestinal sugar release, as already established for acarbose. However, acarbose had no effect on uptake of sugars using both differentiated human Caco‐2 cell monolayers and Xenopus oocytes expressing human glucose transporter‐2 (GLUT2) and GLUT5. Both teas effectively inhibited transport of fructose and glucose through GLUT2 inhibition, while chamomile tea also inhibited GLUT5. Long term incubation of Caco‐2/TC7 cells with chamomile tea for 16 h or 4 days did not enhance the observed effects, indicating that inhibition is acute. Sucrase activity was directly inhibited by green tea and acarbose, but not chamomile. 3 Conclusion: These findings show that chamomile and green teas are potential tools to manage absorption and metabolism of sugars with efficacy against high sugar bolus stress inflicted, for example, by high fructose syrups, where the drug acarbose would be ineffective

Does fibrin(ogen) bind to monomeric or dimeric GPVI, or not at all?

GPVI is the major signalling receptor for collagen on platelets. Dimerization of GPVI is required for collagen binding and initiation of signalling through the associated FcR-γ chain. Recently, fibrin and fibrinogen have been identified as ligands for GPVI and shown to induce signalling in support of thrombus formation and stabilization. Contrasting observations have been reported on whether fibrin binds to monomeric or dimeric GPVI, or to neither form. In this article, we discuss reasons for the contradictory results and how to reconcile these. We conclude that a lack of structural knowledge regarding the GPVI constructs that are being used, along with the use of non-standardized reagents, might be the main cause of the discrepant results. This article aims to highlight some of the key areas that need to be addressed