Increase In Lag Time Research Articles

Antimicrobial Activities of Ethanol Extracts of Rhus javanica and Terminalia chebula Against Shigella Species

This study investigated the antimicrobial activities of Oriental medicinal plant extracts against four Shigella species, i.e., Shigella sonnei, Shigella flexneri, Shigella boydii and Shigella dysenteriae. Twelve Oriental medicinal plants were extracted with 80% ethanol. When the Rhus javanica extract was used at 0.01% concentration, 98.09% of the S. sonnei cells were injured. And S. boydii showed significantly different percentages of injured cells at 0.01% and 0.5% of R. javanica extracts, and S. dysenteriae at 0.01%, 0.1% and 0.5% concentrations of the extracts (P < 0.05). Among the tested Shigella species, S. sonnei was the most easily injured, showing a higher percentage of injured cells at low R. javanica extract concentrations. The growth inhibitory effects of the R. javanica and Terminalia chebula extracts on the Shigella species at different concentrations (i.e., 1, 10 and 100 ppm) were calculated using the modified Gompertz equation. R. javanica extracts used at 100 ppm inhibited the growth of S. flexneri and S. dysenteriae for the entire cultivation period (>48 h). However, the T. chebula extract used at the same concentration did not completely inhibit the growth and caused an increased lag time corresponding to 227.0% and 174.4% for S. flexneri and S. dysenteriae, respectively.Practical ApplicationsShigellosis results in at least 600,000 deaths worldwide each year. And in the U.S.A., it is a major bacteria involved in food poisoning, reported in 10,000–20,000 patients (Centers for Disease Control and Prevention). In the present study, ethanol extracts of oriental medicinal plants were used as basic materials for controlling Shigella species, and there are potentials for medicinal plants, as a natural antimicrobial agent, to be used in the development of eco‐friendly reduction technology on Shigella species.

Soil moisture modifies the response of soil respiration to temperature in a desert shrub ecosystem

Abstract. The current understanding of the responses of soil respiration (Rs) to soil temperature (Ts) and soil moisture is limited for desert ecosystems. Soil CO2 efflux from a desert shrub ecosystem was measured continuously with automated chambers in Ningxia, northwest China, from June to October 2012. The diurnal responses of Rs to Ts were affected by soil moisture. The diel variation in Rs was strongly related to Ts at 10 cm depth under moderate and high volumetric soil water content (VWC), unlike under low VWC. Ts typically lagged Rs by 3–4 h. However, the lag time varied in relation to VWC, showing increased lag times under low VWC. Over the seasonal cycle, daily mean Rs was correlated positively with Ts, if VWC was higher than 0.08 m3 m−3. Under lower VWC, it became decoupled from Ts. The annual temperature sensitivity of Rs (Q10) was 1.5. The short-term sensitivity of Rs to Ts varied significantly over the seasonal cycle, and correlated negatively with Ts and positively with VWC. Our results highlight the biological causes of diel hysteresis between Rs and Ts, and that the response of Rs to soil moisture may result in negative feedback to climate warming in desert ecosystems. Thus, global carbon cycle models should account the interactive effects of Ts and VWC on Rs in desert ecosystems.

Inhibitory effect on in vitro LDL oxidation and HMG Co-A reductase activity of the liquid-liquid partitioned fractions of Hericium erinaceus (Bull.) Persoon (lion's mane mushroom).

Oxidation of low-density lipoprotein (LDL) has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A) reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ). The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins) for the formation of conjugated diene (CD) at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL) of thiobarbituric acid reactive substances (TBARS) at 1 mg/mL. It also mostly inhibited (59.91%) the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases.

Evaluating tyrosinase (monophenolase) inhibitory activity from fragrant roots of Hemidesmus indicus for potent use in herbal products

Root organs of Hemidesmus indicus are traditionally known for multipurpose medicinal applications. Root extracts of H. indicus contained 2-hydroxy-4-methoxybenzaldehyde (MBALD) as principal fragrant phenolic compound. This methoxybenzaldehyde was shown to have inhibitory potential against diphenolase activity of the tyrosinase. The present work explored the effects of this MBALD (both in crude extracts and pure forms) on the monophenolase activity. The lag time of reaction and the activity of monophenolase were affected upon supplementation of methoxybenzaldehyde. The increase in lag time indicated an inhibition of monophenolase activity which subsequently led to the reduction in total tyrosinase activity. MBALD was shown to be more potent than vanillin in inhibiting monophenolase activity. Inhibition kinetic analysis confirmed the same observation, with a higher Km value (0.395mM) was determined for monophenolase reaction in presence of MBALD as compared to Km value of tyrosinase without any inhibition. As compared to pure MBALD, the crude root extract of H. indicus showed greater inhibition against monophenolase activity, which was also evidenced from the extension in lag time. This indicates its potential application in herbal products.

Reversal of anticoagulant effects of apixaban with non-specific prohaemostatic agents: an in vitro study

Background: As any potent anticoagulant, apixaban, an oral factor Xa inhibitor, exposes to a risk of haemorrhagic complications. Thus in the absence of specific antidote, bleeding management is challenging. Aim: To investigate the efficacy of recombinant activated factor VII (rFVIIa), prothrombin complex concentrate (PCC), and partially activated PCC (aPCC) to compensate, in vitro, the anticoagulant effects of apixaban. Methods: Whole blood (WB) from healthy volunteers was spiked with therapeutic (200 ng.ml-1) or overdose (680 ng.ml-1) amount of apixaban and either rFVIIa (equivalent to 90 and 120 μg.kg-1), PCC (25 and 50 UI.kg-1), or aPCC (80 and 160 UI.kg-1). Reversal was assessed on WB with thromboelastography, and on platelet poor plasma using standard laboratory assays (PT, aPTT) as well as thrombin generation test (TGT). Increase of clot turbidity was also recorded to explore the dynamics of fibrin polymerization. Results: Apixaban concentration dependently lengthened PT, aPTT, and the clotting time (CT) in ROTEM®. It also increased lag time (LT) and decreased endogenous thrombin potential (ETP) as well as peak height in the TGT. Apixaban delayed and slowed down fibrin formation and polymerization with subsequent alteration of the resulting clot. rFVIIa had predominant effects on kinetics parameters. Addition of rFVIIa overcorrected PT, aPTT, and CT at apixaban therapeutic concentration and partly restored them in overdose situation. rFVIIa also decreased LT, and accelerated fibrin polymerization at therapeutic dose. Addition of PCC shortened PT and CT, increased peak height and ETP, but paradoxically lengthened aPTT and little modified the turbidity profile. Addition of aPCC shortened PT, aPTT, and LT in the TGT, whereas it did not affected CT in ROTEM®. aPCC also increased peak high, overcorrected ETP, and induced earlier and faster fibrin polymerization. Conclusions: These non-specific prohaemostatic agents corrected partially or completely several laboratory parameters. In particular aPCC, improving most of them was the most seducing. Further investigation is nevertheless necessary: reversal of aPCC was not confirmed in WB, increasing concentration of the prohaemostatic agents had no additive effect, and putative efficacy was strongly dependent on the observed parameter (rFVIIa was best on CT; PCC on ETP). Overall our observations underline the need for robust endpoints and clinical trials.

In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel

IntroductionPrasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. MethodsThe effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. ResultsPAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. ConclusionsRecombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings.

Nutritional evaluation and health promoting activities of nuts and seeds cultivated in Greece

Available data suggest that genetic as well as environmental factors may influence nuts and seeds nutrients content. In this context nuts and seeds cultivated in Greece were studied. Macronutrients content was in agreement with that from other areas. Total phenolics content was in the range of 43.0 ± 2.1–1512.7 ± 60.7 mg GAE/100 g for chestnut and walnut, respectively. Thirteen to 22 individual phenolics were identified in the studied species. Oleanolic acid was in the range of 0.10–9.03 mg/100 g. Pumpkin seeds contained the higher squalene content (71.6 mg/100 g). β-Sitosterol predominated in all samples except pumpkin seeds. Tocopherols ranged from 8.9 mg/100 g (chestnut) to 29.3 mg/100 g (almond). Nuts and seeds hydrophilic extracts at quantities corresponding to the estimated daily consumption by the Greeks succeeded in inhibiting LDL oxidation in vitro by increasing lag time 1.1–14.1 times. One serving of nuts or seeds may cover a significant fraction of health promoting microconstituents daily intake.

The effect of heavy metals and temperature on microbial growth and lag

A mathematical model to simulate microbial growth and lag time is proposed in the present work in accordance with Sengor et al. (2009). The model is verified experimentally for a mixed microbial culture growing at metal free and at Cr(III) or Cr(VI) contaminated environments. The model is further verified using a monoculture of Pseudomonas sp. growing at metal free and Zn(II) contaminated environment. The effect of temperature on microbial growth and lag is also simulated by the model. The simulation results show for the mixed culture that growth rate decrease with parallel increase in lag time, with the rise of either Cr(III) or Cr(VI) concentration. For Pseudomonas sp., the simulation indicated that there is almost nil lag time for growth at 35 ºC, while approximately 10 h lag time is calculated for growth at ambient temperature (20 ºC). The lag time increases by approximately 6 more hours when 0.01 mM Zn(II) is added in the growth medium (at ambient temperature). The increase of lag time with temperature reduction is explained by the fact that the biochemical reactions are slowed down at lower temperatures. A good correlation between model predictions and simulation results is observed.

Effects of Omega-3 Polyunsaturated Fatty Acids on Platelet Function in Healthy Subjects and Subjects with Cardiovascular Disease

Hyperactivation and aggregation of platelets play a major role in thrombosis and hemostasis. The aims of this study were to investigate the effects of omega-3 polyunsaturated fatty acids (PUFAs) on platelet function. Light transmission aggregometry and flow cytometric analyses of platelet activation and platelet-leukocyte aggregates were determined at baseline and after 4 weeks of omega-3 (docosahexaenoic acid 520 mg and eicosapentaenoic acid 120 mg) supplementation. In total, 40 healthy subjects and 16 patients with a history of cardiovascular disease (CVD) completed the study. In healthy subjects, omega-3 PUFA significantly reduced adenosine diphosphate (ADP)-induced (maximum amplitude, 77.0% ± 3.2% vs. 71.6% ± 3.4%, p = 0.036; maximum slope, 86.3 ± 1.8 vs. 80.7 ± 2.1, p = 0.014) and adrenaline-induced platelet aggregation (maximum slope, 42.8 ± 2.7 vs. 37.4 ± 3.0, p = 0.013; lag time, 00:21 ± 00:02 vs. 00:31 ± 00:03 s, p = 0.002). Omega-3 PUFA also reduced P-selectin expression (40.5% ± 2.9% vs. 34.4% ± 2.4%, p = 0.049) on platelets and platelet-monocyte aggregates (38.5% ± 2.6% vs. 31.4% ± 2.5%, p = 0.022) after activation with ADP 0.5 µM. There were fewer changes in platelet aggregation and activation found in subjects with CVD. Nevertheless, there was a reduction in the slope of arachidonic acid-induced platelet aggregation (13.21 ± 6.41 vs. 4.88 ± 3.01, p = 0.009) and increased lag time for U46619 (00:16 ± 00:00 vs. 00:29 ± 00:07 s, p = 0.018) induced platelet aggregation. Thus, 4-week supplementation of 640 mg omega-3 PUFA reduced measures of platelet aggregation and activation in healthy subjects but effects were less evident in patients with existing CVD. Our findings support the recommendation that the omega-3 PUFA dose be higher in CVD than among healthy subjects.

Antioxidant effects of vitamins C and e on the low-density lipoprotein oxidation mediated by myeloperoxidase.

Oxidative modification of low-density lipoprotein (LDL) appears to be an early step in the pathogenesis of atherosclerosis. Meanwhile, myeloperoxidase (MPO)-catalyzed reaction is one of the potent pathway for LDL oxidation in vivo. The aim of this study was to evaluate in vitro antioxidant effects of vitamins C and E on LDL oxidation mediated by MPO. MPO was isolated from fresh plasma by sequential centrifugation using density ultracentrifugation. It was incubated with LDL and the LDL oxidation level was determined spectrophotometrically by measuring conjugated diene absorbance at 234 nm. Furthermore, vitamin C (50-200 mM) and vitamin E (10-40 mM) were added and the LDL oxidation level was determined. The purity index of MPO and its enzymatic activity were 0.69 and 1127 U/mg protein, respectively. It was demonstrated that vitamin C in vitro inhibited LDL oxidation mediated by MPO; however, vitamin E was unable to act in the same way. The protection by vitamin C was concentration dependent and maximum protective effect of vitamin C was observed at 150 mM, where about 64% of the LDL oxidation was inhibited. Vitamin C increased lag time of LDL oxidation mediated by MPO up to 2.4 times. It can be concluded from our results that vitamin C is able to improve LDL resistance to oxidative modification in vitro. In addition, vitamin C might be effective in LDL oxidation mediated by MPO in vivo, resulting in reduction of atherosclerosis process rate.

Preventive Effects of Multi-Lamellar Emulsion on Low Potency Topical Steroid Induced Local Adverse Effect

BackgroundTopical steroid treatment induces diverse local Wand systemic adverse effects. Several approaches have been tried to reduce the steroid-induced adverse effects. Simultaneous application of physiological lipid mixture is also suggested.ObjectiveNovel vehicles for topical glucocorticoids formulation were evaluated for the efficacy of reducing side-effects and the drug delivery properties of desonide, a low potency topical steroid.MethodsTranscutaneous permeation and skin residual amount of desonide were measured using Franz diffusion cells. The in vivo anti-inflammatory activity was evaluated using murine model.ResultsTopical steroids formulation containing desonide, in either cream or lotion form, were prepared using multi-lamellar emulsion (MLE), and conventional desonide formulations were employed for comparison. MLE formulations did not affect the anti-inflammatory activity of the desonide in phobol ester-induced skin inflammation model, compared with conventional formulations. While the penetrated amounts of desonide were similar for all the tested formulations at 24 hours after application, the increased lag time was observed for the MLE formulations. Interestingly, residual amount of desonide in epidermis was significantly higher in lotion type MLE formulation. Steroid-induced adverse effects, including permeability barrier function impairment, were partially prevented by MLE formulation.ConclusionTopical desonide formulation using MLE as a vehicle showed a better drug delivery with increased epidermal retention. MLE also partially prevented the steroid-induced side effects, such as skin barrier impairment.

Formulation and Evaluation of Pulsatile Drug Delivery System of Metoprolol Tartarate Using Core in Cup Tablet

The purpose of this work is to formulate a pulsatile drug delivery system using metoprolol tartarate as model drug. A core in cup (three component tablet) is prepared where in core tablet, an impermeable material surrounding the tablet except the top and soluble hydrophilic polymer layer at the top is designed. The core tablet contains metoprolol tartarate, cellulose acetate propionate is used as impermeable membrane and sodium alginate 500 cps and sodium alginate 2000 cps used as soluble hydrophilic polymer layer .The top cover layer is prepared using 3<SUP>2</SUP> factorial design. Quantification of water uptake, top layer expansion, in-vitro dissolution studies, radial and axial expansion, stereomicroscopic image, and short term stability studies are performed. The concentration of top layer of hydrophilic polymer is a critical factor governing the release pattern, increase in the concentration increased lag time and delay the release .The T70% and T90 % values are also influenced by the polymer concentration with less polymer concentration of hydrophilic polymer lesser T70% and T90 % values are obtained and as the concentration increased higher values are obtained. It can be concluded from the study that pulsatile drug delivery system useful in chronotherapy of hypertension can be prepared by this technique.

Thrombin Generation Assay Is Superior to Traditional Coagulometric and Chromogenic Assays to Monitor New Oral Anticoagulants

AbstractAbstract 1169 INTRODUCTION:New oral anticoagulants (NOACs) have been recently approved by the European Medicine Agency (EMA) and the Food and Drug Administration (FDA) for several indications. New oral anticoagulants include anti-IIa agent (dabigatran etexilate) and anti-Xa agents (rivaroxaban, apixaban and edoxaban). NOACs do not require monitoring nor frequent dose adjustment. However, searching for the optimal dose in the individual patient may be useful in some situations. Recent studies have shown that aPTT, HTI and ECT could be used to monitor dabigatran whereas PT and anti-Xa chromogenic assays could be used to monitor anti-Xa agents, while standardizing the time between the last intake of rivaroxaban and the sampling is mandatory. These tests only measure the initiation phase of the coagulation cascade. Thrombin generation assay (TGA) which measures the entire thrombin generation process could be used to better discriminate the inhibitory profile of the NOACs in patients. OBJECTIVES:To study the impact of dabigatran and rivaroxaban in patients by thrombin generation assay compared to other traditional coagulometric and chromogenic assays. MATERIALS AND METHODS:Five patients under dabigatran and 5 patients under rivaroxaban for atrial fibrillation were included in this study. Blood samples were taken at different intervals: at Ctrough, 2h and 3h after drug administration. The following tests were performed at each timepoint Rivaroxaban:Prothrombin Time (PT) using the following reagents: Triniclot PT Excel S.. and Innovin.., Thrombin Generation Assay (TGA) using PPP-Reagent and PPP-Reagent High, Biophen Direct Factor-Xa Inhibitor.. (DiXaI). Dabigatran: Activated Partial Thromboplastin Time (aPTT) using CK-Prest.. and Synthasil.., Hemoclot Thrombin Inhibitor.. (HTI) and Thrombin Generation Assay (TGA) using PPP-Reagent and PPP-Reagent Low. All the tests were calibrated by spiking rivaroxaban or dabigatran at increasing concentrations in pooled citrated normal human platelet poor plasma (PPP).RESULTS AND DISCUSSIONS: In vitroRivaroxabanThe Peak and mVRI were the most sensitive CAT parameters with a high sensitivity (Peak IC50 was 3ng/mL with PPP-Reagent Low and PPP-Reagent and 14ng/mL with PPP-Reagent High; mVRI IC50 was 1ng/mL with PPP-Reagent Low and PPP-Reagent and 3ng/mL with PPP-Reagent High) and both reagent showed a low variability (CV<1.0%).The PPP-reagent Low does not allow resolving TGA profiles with different rivaroxaban concentrations. Consequently, PPP-Reagent and PPP-Reagent High were only tested ex vivo.The low sensitivity of the lag time explains the lack of sensitivity of PT, even with the more sensitive reagent (i.e. Triniclot PT Excel S..). DabigatranDabigatran mainly delayed the initiation phase with a strong dose-dependent increase of lag time and Tmax and a slight dose-dependent decrease of Cmax and ETP.The concentration in dabigatran needed to double the lag time was 66 ng/mL and 70ng/mL with the PPP-Reagent and the PPP-Reagent Low respectively. The PPP-reagent High is less sensitive in comparison to PPP-Reagent and PPP-Reagent Low. Ex-VivoSimilar concentrations of rivarovaban or dabigatran in different patients provided with Biophen DiXaI.. and HTI are associated with differences in TGA profile (Figure 1 & 2 showing the dispersion of the different measurement). Therefore, anti-Xa tests and dilute thrombin time are not the most accurate tests to use to propose cut-off associated with a bleeding or thrombosis risk. This illustrates the interest to have tests able to evaluate the entire thrombin generation process to propose cut-off in one or more parameters associated with a bleeding or thrombosis risk. CONCLUSIONThrombin generation assay could be superior to traditional coagulometric and chromogenic assays to monitor new oral anticoagulants in terms of prediction of bleeding or thrombosis risk. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Effects on Thrombin Generation Following Resuscitation with Lactated Ringers and Fresh Frozen Plasma

Following trauma, patients are infused with fluids such as crystalloids or fresh frozen plasma. Observing thrombin generation is key to demonstrating changes in hemostatic potential due to the infusions. Our objective was to explore the effects of resuscitative fluids on coagulation as measured by Calibrated Automated Thrombography (CAT). Male rats underwent laparotomy with gut externalization and hemorrhagic shock to MAP of 35mmHg until decompensation (inability to auto‐resuscitate). Rats were randomized based on fluids received: Lactated Ringers (LR; n=7) and Fresh Frozen Plasma Day 0 (PL‐0; n=8). PL‐0 was obtained by pooling citrated rat plasma from non‐experimental donor rats and PL‐0 coagulation factors were not significantly different from baseline. Prior to resuscitation, there were no significant differences in coagulation factors between LR and PL‐0. Resuscitation with LR resulted in a significant decrease in lagtime but maintenance of high endogenous thrombin potential (ETP) and peak levels. Resuscitation with PL‐0 resulted in a significant increase in lagtime, ETP, and Peak. Following trauma and hemorrhagic shock, resuscitation with LR produced a prothrombotic state while resuscitation with PL‐0 produced a coagulopathic state. Hemostatic potential is important in determining which fluid may be required during trauma management. (UT Health Science Center–Houston CeTIR Dept Grant).

Comparison between Ethylcellulose Aqueous Colloidal Dispersion and Ethylcellulose Ethanolic Solution as Rupturable Coating Materials for Pulsatile Release Tablets

Pulsatile release dosage forms which time of drug release can be determined have shown advantages for various drugs such as chronopharmacological drugs and drugs with high first pass metabolism. The pulsatile release tablets consisting of drug-containing cores coated with inner swelling layer containing a superdisintegrant (croscarmellose sodium (Ac-Di-Sol®)) and outer rupturable layer (ethylcellulose (EC)) were previously developed. In this study, two different types of EC coating membrane, EC ethanolic solution (ECS) versus EC aqueous colloidal dispersion (ECD), were compared. Propranolol HCl was used as a model drug. Effect of rupturable coating level on lag time and drug release was investigated. The results demonstrated that using ECS could provide the pulsatile release tablets with obviously longer lag time than ECD. With ECS, increasing of rupturable coating level increased lag time of the pulsatile release tablets because of the lowered water uptake. In addition, rapid drug release after the predetermined lag time was achieved in the pulsatile release tablets using ECS. However, the pulsatile tablets with ECD ruptured within a few minutes even high levels of rupturable coating were used. It was suggested that the different performance of the pulsatile release tablets with different types of EC might be related to the discrepancy in physical properties of the obtained EC membrane.

Discriminating rapid exhumation from syndepositional volcanism using detrital zircon double dating: Implications for the tectonic history of the Eastern Cordillera, Colombia

Lag time is the difference between the closure age of a thermochronologic system and the depositional age of host strata. Lag-time analysis of sedimentary basin fi ll provides insight into the exhumation history of adjacent eroded orogens. In a case study of the Paleogene Floresta basin in the Eastern Cordillera fold-thrust belt of Colombia, variations in lag time refl ect changes in both sediment source areas and exhumation patterns. However, near-zero lag times can be produced by either syndepositional volcanism or rapid exhumation. We applied U-Pb geochronology and (U-Th)/He (ZHe) thermochronology to individual zircon grains and identifi ed zircons of volcanic origin as those for which the U-Pb age is within the 2σ uncertainty of their ZHe age. Consistent discrimination of young ZHe ages as the products of either rapid exhumation or volcanism reveals three stages in the history of the northern Andean hinterland. (1) Early to late Paleocene: The appearance of syndepositional and Mesozoic volcanic zircons marks the initial infl ux of magmatic arc detritus. (2) Middle to late Eocene: Near-zero lag times point to rapid, regionally extensive exhumation attributable to thrust-induced uplift of the Magdalena Valley basement. (3) Late Eocene to late Oligocene: Increased lag time is interpreted as recycling of shallowly buried foreland-basin strata possibly due to movement on basinbounding thrust systems. The presence of volcanic zircons with ZHe ages younger than or indistinguishable from the youngest exhumationally cooled zircons underscores the need for double dating to reliably identify volcanic infl uence in detrital thermochronology datasets. These data highlight the utility of double-dated ZHe results for extracting tectonic histories and reliably excluding volcanic zircons from lag-time analysis.

Monitoring Insulin Aggregation via Capillary Electrophoresis

Early stages of insulin aggregation, which involve the transient formation of oligomeric aggregates, are an important aspect in the progression of Type II diabetes and in the quality control of pharmaceutical insulin production. This study is the first to utilize capillary electrophoresis (CE) with ultraviolet (UV) detection to monitor insulin oligomer formation at pH 8.0 and physiological ionic strength. The lag time to formation of the first detected species in the aggregation process was evaluated by UV-CE and thioflavin T (ThT) binding for salt concentrations from 100 mM to 250 mM. UV-CE had a significantly shorter (5–8 h) lag time than ThT binding (15–19 h). In addition, the lag time to detection of the first aggregated species via UV-CE was unaffected by salt concentration, while a trend toward an increased lag time with increased salt concentration was observed with ThT binding. This result indicates that solution ionic strength impacts early stages of aggregation and β-sheet aggregate formation differently. To observe whether CE may be applied for the analysis of biological samples containing low insulin concentrations, the limit of detection using UV and laser induced fluorescence (LIF) detection modes was determined. The limit of detection using LIF-CE, 48.4 pM, was lower than the physiological insulin concentration, verifying the utility of this technique for monitoring biological samples. LIF-CE was subsequently used to analyze the time course for fluorescein isothiocyanate (FITC)-labeled insulin oligomer formation. This study is the first to report that the FITC label prevented incorporation of insulin into oligomers, cautioning against the use of this fluorescent label as a tag for following early stages of insulin aggregation.

Thermo‐tectonic evolution of the south‐central Pyrenees from rifting to orogeny: insights from detrital zircon U/Pb and (U‐Th)/He thermochronometry

AbstractConstraining the thermal and denudational evolution of continental margins from extensional episodes to early orogenic stages is critical in the objective to better understand the sediment routing during the growth of orogenic topography. Here, we report 160 detrital zircon U/Pb ages and 73 (U‐Th)/He ages from Albian, Upper Cretaceous and Eocene sandstones from the south‐central Pyrenees. All samples show dominant zircon U/Pb age peaks at 310–320 Ma, indicating a primary contribution from Variscan granites of the central Pyrenean Axial Zone. A secondary population at 450–600 Ma documents zircon grains sourced from the eastern Pyrenees. Zircon (U‐Th)/He ages recovered from older samples document, a Triassic age peak at ca. 241 Ma, corresponding to denudation coeval with the initiation of Atlantic rifting. An Early Cretaceous cooling event at ca. 133 Ma appears consistent with rift‐related exhumation and thermal overprint on the Iberian margin. The (U‐Th)/He age peaks from ca. 80 Ma to ca. 68 Ma with decreasing depositional ages are interpreted to reflect the southward‐migrating thrust‐related exhumation on the pro‐wedge side of the Pyrenean orogen. The increase in lag times, from ca. 15 Ma in the Tremp Formation (ca. 65 Ma) to 28 Ma in the Escanilla Formation (ca. 40 Ma), suggests decreasing exhumation rates from 0.4 km Myr–1 to 0.2 km Myr–1. The apparent inconsistency with convergence rates is used to infer that rocks cooled at 68 Ma may have resided in the crust before final exhumation to the surface. Finally, the cooling event observed at 68 Ma provides support to the inferred acceleration of convergence, shortening and exhumation during Late Cretaceous times.

Effects of copolypeptides on amyloid fibrillation of hen egg‐white lysozyme

The fibrillation of hen egg-white lysozyme (HEWL) in the absence and presence of simple, unstructured D,L-lysine-co-glycine (D,L-Lys-co-gly) and D,L-lysine-co-L-phenylalanine (D,L-Lys-co-Phe) copolypeptides was studied by using a variety of analytical techniques. The attenuating and decelerating effects on fibrillation are significantly dependent on the polypeptide concentration and the composition ratios in the polypeptide chain. Interestingly, D,L-Lys-co-gly and D,L-Lys-co-Phe copolypeptides with the same composition ratio have comparable attenuating effects on fibrillation. The copolypeptide with highest molar fraction of glycine residue exhibits the strongest suppression of HEWL fibrillation. The copolypeptide has the highest hydrophobic interacting capacity due to the more molar ratio of apolar monomer in the polymer backbone. The major driving forces for the association of HEWL and copolypeptides are likely to be hydrogen bonding and hydrophobic interactions, and these interactions reduce the concentration of free protein in solution available to proceed to fibrillation, leading to the increase of lag time and attenuation of fibrillation. The results of this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the unstructured polypeptides and the amyloid proteins.

Secondary Nucleating Sequences Affect Kinetics and Thermodynamics of Tau Aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)(n)Z, Z(X)(n)Z (n ≥ 2), or (XZ)(n) (n ≥ 2), where X is a hydrophobic residue and Z is a charged or polar residue. N-Acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed seven peptides, in addition to well-known primary nucleating sequences Ac(275)VQIINK (AcPHF6*) and Ac(306)VQIVYK (AcPHF6), formed fibers, tubes, ribbons, or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac(10)VME, AcPHF6*, Ac(375)KLTFR, and Ac(393)VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac(375)KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild-type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics.