Sample Agitation Research Articles

Quantification and Stability Determination of Surface Amine Groups on Silica Nanoparticles Using Solution NMR.

Surface chemistry is a critical factor for determining the behavior of a nanomaterial after incorporation in composites, devices, and biomedical products, and is also important for nanotoxicology studies. We have developed an optimized protocol for dissolution of aminated silicas and determination of functional-group contents by quantitative 1H NMR (qNMR) analysis of the released amines. A number of variables were optimized for the dissolution protocol, including the base concentration, mass of silica, time, temperature, and method of sample agitation, in order to achieve adequate NMR signals for quantification. The protocol was tested using nanoparticles from a single commercial supplier with sizes ranging from 20 to 120 nm that were functionalized with 3-aminopropyl groups. Interestingly the batch-to-batch variability for some sizes of these aminated silicas was as high as 50%. Amine contents measured by a ninhydrin colorimetric assay were typically ∼20% lower than those measured by qNMR, consistent with measurement of only ninhydrin-reagent accessible amines. The dissolution-qNMR protocol was compatible with aminated silicas from other commercial suppliers, and in these cases, an even larger variability in surface coverage was observed. Silica nanoparticles with longer-chain amines and variable amine loadings were synthesized to demonstrate the ability to quantify amines with more complex structures and to assess the limit of quantification for the dissolution-qNMR method. Finally, the stability of the aminated nanoparticles was examined. Loss of 3-aminopropyl groups occurred in water at room temperature and was significantly more rapid at higher temperatures. Amine loss increased with increasing surface coverage and was slower for long-chain amines, consistent with studies of amine stability on planar silica. Overall, this work highlights the importance of developing methods for quantifying surface functionalization, particularly given the variability in surface coverage for commercial samples, and for ensuring that the amine group is stable under its usage conditions.

Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

Normal reference intervals and the effects of sample handling on dynamic viscoelastic coagulometry (Sonoclot) in healthy adult horses.

To determine reference intervals and the effect of sample agitation and rest time on Sonoclot analysis in healthy adult horses. Original prospective study. University veterinary medical teaching hospital. Sixty healthy adult horses. Blood was collected for assessment of complete blood count, serum biochemical analysis, and Sonoclot analysis. Horses were determined to be healthy based upon physical examination, CBC, and serum biochemistry analysis. Blood was analyzed in a glass bead-containing cuvette using the Sienco Sonoclot analyzer following 2 rest periods (30 mins and 240 min) and with 2 sample handling interventions (agitated and nonagitated), to obtain values for clot rate, time-to-peak, activated clotting time, and platelet function. This study failed to detect a significant difference when a rest time of 30 minutes was compared with 240 minutes, but based on wide limits of agreement the 2 rest times were not considered interchangeable. Agitation at both rest times significantly affected all Sonoclot analyses leading to changes indicative of hypercoagulability. Sample agitation and rest time should be taken into consideration when developing preanalytical guidelines for Sonoclot analysis in horses. Calculated reference intervals were relatively wide. Further research is needed to evaluate the clinical utility of Sonoclot analysis in horses.

Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry

Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome.Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells.

Research of Anti-Cancer Components in Traditional Chinese Medicine on Hollow Fibre Cell Fishing and Hollow Fibre Liquid Phase Microextraction

Hollow fibre cell fishing with HPLC (HFCF-HPLC) based on the human ovarian cancer cell line SKOV-3, human renal tubular cell line ACHN or hepatoma cell line HepG-2 was employed to screen active groups of coumarin and volatile oil in Radix angelicae sinensis, Radix angelicae dahuricae and Fructus citri sarcodactylis. Simultaneously, hollow fibre liquid phase microextraction with HPLC (HFLPME-HPLC) was conducted to enrich and determine the contents of active components in the same sample solution. Before application, for HFCF-HPLC, cells growth states and survival rates on the fibre, effect of ethanol concentration in the extract of samples on cell survival rates, non-specific binding between fibre active centres and the target components, positive and negative controls and repeatabilities were validated; for HFLPME, extraction solvent, sample phase pH, agitation speed, extraction time and sample phase volume were investigated. Many active components were screened from three medicines. Some of them, such as scoparone, psoralen, bergapten, oxypeucedanin, imperatorin, ligustilide, were identified by MS. The target fishing factors of active components and the cell apoptosis rates of three cells under the medicines effect were researched. The binding sites of active groups on HepG-2 cells were preliminarily determined. The results demonstrated that HFCF-HPLC, coupled with HFLPME-HPLC, is a simple and universal approach to find bioactive components at the cellular level, determine their content and research traditional Chinese medicines (TCMs) entirety effect of multi-component and multi-target. The approach may provide us a new and good solution to clarify the material basis of anti-cancer effect and conduct personalized quality control for the components associated with efficacy in TCMs.

Numerical modeling of solid-phase microextraction: binding matrix effect on equilibrium time.

Solid-phase microextraction (SPME) is a well-known sampling and sample preparation technique used for a wide variety of analytical applications. As there are various complex processes taking place at the time of extraction that influence the parameters of optimum extraction, a mathematical model and computational simulation describing the SPME process is required for experimentalists to understand and implement the technique without performing multiple costly and time-consuming experiments in the laboratory. In this study, a mechanistic mathematical model for the processes occurring in SPME extraction of analyte(s) from an aqueous sample medium is presented. The proposed mechanistic model was validated with previously reported experimental data from three different sources. Several key factors that affect the extraction kinetics, such as sample agitation, fiber coating thickness, and presence of a binding matrix component, are discussed. More interestingly, for the first time, shorter or longer equilibrium times in the presence of a binding matrix component were explained with the help of an asymptotic analysis. Parameters that contribute to the variation of the equilibrium times are discussed, with the assumption that one binding matrix component is present in a static sample. Numerical simulation results show that the proposed model captures the phenomena occurring in SPME, leading to a clearer understanding of this process. Therefore, the currently presented model can be used to identify optimum experimental parameters without the need to perform a large number of experiments in the laboratory.

Dispersive Micro-Solid-Phase Extraction Using Mesoporous Hybrid Materials for Simultaneous Determination of Semivolatile Compounds from Plant Tea by Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry

This report described the use of mesoporous hybrid materials (MHM) in a dispersive micro-solid-phase extraction procedure to extract semivolatile compounds from plant tea that were then analyzed by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Dihydrotanshinone I, tanshinone I, cryptotanshinone, and tanshinone IIA were selected as the model compounds, and the extraction parameters, including mesoporous concentration, extraction time, sample agitation and desorption solvents, were optimized. The interaction with the analytes and the large surface area of the MHM facilitated the adsorption of analytes. The method showed good linearity, with correlation coefficients >0.9980 in the range 0.25-100 ng/mL, and low limits of detection (0.012-0.046 pg). Finally, the recovery values were 91-103% for Danshen tea, 89-102% for Danshen, and 88-96% for tanshinone capsules. The results showed that the proposed method was suitable for the extraction and determination of tanshinones in complex samples.

Dispersive Micro Solid-Phase Extraction with Graphene Oxide for the Determination of Phenolic Compounds in Dietary Supplements by Ultra High Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry

This report described the use of graphite oxide (GO) as sorbent in dispersive micro solid-phase extraction (DMSPE), together with ultra high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS), for the determination of phenolic compounds (protocatechuic aldehyde, caffeic acid, and rosmarinic acid) in dietary supplements. The extraction conditions such as sample agitation, agitation time, sorbent amount, and type and volume of the eluent were optimized. The results showed that GO-assisted DMSPE exhibited higher enrichment factors for tested solutes as compared to the normal extraction. Under the most favorable conditions, good limits of detection (0.07–0.21 ng/mL) and repeatability of extraction (RSDs below 5.6 %, n = 5) were obtained. The developed method was applied to determine phenolic analytes with satisfactory recoveries, which were 90.1–96.4 % for Danshen and 85.5–97.6 % for Danshen injection, respectively.

Preparation of magnetic nanocomposite beads and optimizing the conditions for effective removal of U(VI) from aqueous solutions

Magnetic ion-imprinted polymers (IIPs) were prepared by precipitate polymerization and leached with HCl to remove uranium. Their ability to remove hexavalent uranium from wastewater effluents was studied. Batch adsorption studies to determine the optimum conditions of U(VI) removal were conducted at different levels of sample pH, sorbent amount, agitation time, and initial uranium concentration. It was observed that, under optimum conditions (i.e. pH 4, adsorbent amount of 50 mg, 45 min agitation time, and initial U(VI) concentration of 2 mg L−1), the maximum removal of U(VI) cations was >98% and 80% for the magnetic IIP and the corresponding magnetic non-imprinted polymers (NIP), respectively. Langmuir and Freundlich isotherms were used to describe the adsorption of U(VI) onto magnetic IIP and NIP. The adsorption capacity of U(VI) was determined to be 1.06 and 0.85 mg g−1 for the two isotherms, respectively. The order of selectivity was found to be U(VI) > Fe(III) > Pb(II). For six cycles of regeneration and reuse, the magnetic polymers maintained their stabilities with only a 4% loss in the extraction efficiency. The average extraction efficiencies of the magnetic polymers for the spiked acid mine drainage and sewage wastewater effluents were 71% and 58% for the magnetic IIP and NIP, respectively. From powder X-ray diffraction analysis, application of the Scherrer equation yielded magnetic nanoparticles of an average mean diameter of 11.9 nm. Thermo-gravimetric analysis revealed that the HCl-leached magnetic polymers had a magnetite residual weight of 5%.

Determination of Organochloride and Triazine Pesticides in Natural Waters by Solvent Bar Microextraction

A new analytical methodology based on solvent bar microextraction has been developed to preconcentrate two triazine and two organochloride pesticides in natural waters prior to analysis by gas chromatography. The method was optimized for the organic solvent, sample pH, sample agitation, length of the fiber, extraction time, and salt concentration. The optimized methodology provides high enrichment factors, ranging between 40.2 (aldrin) to 107.0 (lindane). Since no salinity effects were observed, this method may be used to analyze fresh and salt water. Thus, three real samples with different salinities were analyzed with pesticide recoveries higher than 80%. As this methodology agreed with the principles of green analytical chemistry, it offers low solvent and sample consumption, simplicity, and compatibility with current analytical techniques.

Inactivation of Escherichia coli and Staphylococcus aureus by Pulsed Electric Fields Increases with Higher Bacterial Population and with Agitation of Liquid Medium

Inactivation of Escherichia coli, E. coli O157:H7, and Staphylococcus aureus in liquid media by pulsed electric fields (PEF) was conducted at varying bacterial populations with and without sample agitation. A laboratory-scale PEF batch unit with a rectangular electric pulse was used, operating under the following conditions: 25 kV/cm (E. coli, E. coli O157:H7) and 30 kV/cm (S. aureus) electric field strengths, 1-μs pulse width, 1-Hz pulse repetition rate, and 20 to 350 pulses for all samples. Not surprisingly, bacterial inactivation (for all three strains) increased with increasing pulse number, achieving the highest reduction at 350 pulses. Log CFU per milliliter microbial inactivation increased commensurately with increasing bacterial population (P < 0.05) but only when samples were treated with more than 200 pulses. For example, when E. coli was treated with 200 pulses at 10(5) CFU/ml, inactivation was only 3.0 Log versus 4.8 Log at the 10(10) inoculation level. When E. coli O157:H7 was treated with 200 pulses at 10(5) CFU/ml, inactivation was only 2.5 Log versus 4.6 Log at the 10(10) inoculation level. When S. aureus was treated with 200 pulses at 10(6) CFU/ml, inactivation was only 2.6 Log versus 4.8 Log at the 10(10) inoculation level. Inactivation of populations was also found to be statistically greater (P < 0.05) when liquid samples were agitated, in comparison to nonagitated samples. Because PEF inactivation activity is influenced by bacterial population and sample agitation, future studies should carefully consider these factors in experimental designs and/or scaled-up industry application.

A simple cost-effective manometric respirometer: design and application in wastewater biomonitoring

Application of respirometric tools in wastewater engineering fields is still not getting familiarity and acceptance by academy or industry in developing countries as compared to the use of conventional biochemical oxygen demand (BOD) approach. To justify the applicability of respirometry, a low-cost respirometric device suitable for monitoring biodegradation process in wastewater has been developed. This device contains six independently operating reactors placed in a temperature control unit for the bioassay of five wastewater samples simultaneously (along with one blank). Each reactor is equipped with a magnetic stirrer for the continuous agitation of the test sample. Six manometers, linked with the individual reactors, measure the pressure and volume changes in the headspace gas phase of the reactor. Working formulae have been derived to convert the ‘volume-change in gas phase’ data to ‘the oxygen depletion in the whole liquid–gas system’ data. The performance of the device has been tested with glucose–glutamic acid standard solution and found satisfactory. Conventional BOD test and the respirometric measurements were performed simultaneously and it is found that in addition to measuring the BOD of the sample, this device gives oxygen uptake profile for further analysis to determine the biokinetic coefficients. Additionally, in some cases, following a specific test protocol, the respirometer can indirectly estimate the carbon dioxide evolved during biodegradation process for calculating respiratory activity parameter such as respiratory quotient. It is concluded that the device can be used in the laboratories associated with the activated sludge plants and also for teaching and research purposes in developing countries.

Salt saturated single drop microextraction of gold from water samples and its determination by graphite furnace atomic absorption spectrometry

A new microextraction procedure, termed salt saturated single drop microextraction, combined with graphite furnace atomic absorption spectrometry (GFAAS) was introduced and developed for the determination of gold in water samples. This is the first report of the extraction of a cationic species by direct immersion SDME utilizing a polar solvent. A mixture of benzyl alcohol–isopropyl alcohol was used as the extracting solvent from the brine sample, which can be considered as a form of salting-out solvent extraction. The factors influencing the extraction efficiency, such as type and volume of extracting solvent, extraction time, pH, agitation of sample, temperature and volume of sample were investigated and optimized. The developed protocol was found to yield a linear calibration curve in the concentration range of 25 and 550 μg L−1 with a limit of detection of 7.4 μg L−1. The total analysis time including microextraction was less than 10 min. The method has been successfully applied for the analysis of gold in three spiked tap, lake and seawater samples.

Comparative Study of the Methodology Used in the Extraction of Isoflavones from Legumes Applying a Modified QuEChERS Approach

Isoflavones are phytochemicals of great interest because of their association with a large variety of positive effects on human health. The major sources of isoflavones in the diet are plants of the Leguminosae family, especially soybeans, although many other legumes more widely consumed in the Mediterranean diet have also been reported to contain these compounds. In previous work we extracted isoflavones from samples using a modified QuEChERS (Quick Easy Cheap Effective Rugged Safe) methodology. To compare different methods for placing the sample and the solvent in contact to optimise the extraction of isoflavones from legumes (chickpeas, lentils and white beans) using a modified QuEChERS methodology. Five different approaches to sample agitation were tested: vortex agitation, thermostatted stirring agitation and thermostatted tray shaking, and a thermostatted ultrasound bath and an ultrasound probe. To evaluate the different methodologies a modified QuEChERS approach was used as the extraction method. The separation and quantification of isoflavones was carried out using liquid chromatography-triple quadrupole/mass spectrometry (LC-MS/MS). The best methods were found upon using a thermostatted shaking tray for the extraction of chickpeas and white beans and the ultrasound probe for lentil samples. These methods were chosen based on the highest amount of analytes obtained as well as the best recovery values. Determination of isoflavones in foods may be affected by the different methods used to place the sample and the solvent in contact in the extraction step. The main advantages of the proposed extraction procedures are their simplicity, speed, reliability and low cost.

A rapid and selective method for simultaneous determination of six toxic phenolic compounds in mainstream cigarette smoke using single-drop microextraction followed by liquid chromatography–tandem mass spectrometry

Cigarette smoke contains several toxic phenolic compounds, measurements of which are essential from a public health standpoint. This article describes a simple and selective analytical method for quantitative determination of six toxic phenolic compounds (phenol, catechol, resorcinol, hydroquinone, o-cresol, and p-cresol) from mainstream cigarette smoke using single-drop microextraction in combination with liquid chromatography-tandem mass spectrometry. Single-drop microextraction was applied prior to analysis by liquid chromatography-tandem mass spectrometry for the extraction and preconcentration of target phenolic compounds from raw cigarette smoke extract. The effects of the extraction solvent, sampling time, solution pH, salt addition, sample agitation rate, and temperature on the extraction efficiency were examined and optimized. The identification of each analyte was established by chromatographic retention times, analyte-specific fragmentation patterns, and relative peak area ratios of two product/precursor ion pairs. Analytical parameters such as the detection limit, relative recovery, reproducibility, linearity, and enrichment factor were evaluated under the optimized experimental conditions. 1-Decanol was selected as the extraction solvent and the limits of detection were found to be in the range of 0.05-0.3 ng mL(-1) using an extraction time of 12 min. Gradient chromatographic conditions were optimized for the separation of the six phenolic compounds in a run time of 10 min including reequilibration of the column. The present method for determination of phenolic compounds from mainstream cigarette smoke is simple and specific and shows good reproducibility, with relative standard deviations less than 10% for all targeted phenolics.

Detection of TNT-Contamination in spiked soil samples using SPME and GC/MS

Soil-contamination from toxic explosive residues at military bases throughout the world, is a major environmental concern. A strong candidate for TNT soil-contamination within Greece, is the military base/airport of Maleme in Crete. While soil sampling was in progress new routes of trace analysis for explosives in soil were explored. Solid-phase micro-extraction (SPME) was used to selectively preconcentrate analytes prior to GC/MS. In the preliminary experiments (effectuated on water-spiked samples) optimization of the SPME technique was achieved by controlling parameters such as sampling method, salt content and sample agitation. As immersion of the SPME fiber was found to be more efficient for extracting most analytes of interest, a pretreatment step was introduced for the soil samples which simply converted them to water samples. The developed SPME protocol was able to screen explosives in spiked-soils, in concentrations well below the certified reporting and detection limits.

Hollow-Fiber Liquid-Phase Microextraction and Chiral LC–MS/MS Analysis of Venlafaxine and its Metabolites in Plasma

An enantioselective analytical method was developed and validated for determination of venlafaxine and its metabolites O-desmethylvenlafaxine and N-desmethylvenlafaxine in plasma samples. The method employed LC-MS/MS analysis and hollow-fiber liquid-phase microextraction (HF LPME) for sample preparation. After HF LPME optimization the following condition was established: sample volume of 4 ml, sample agitation at 1750 rpm, 20 min of extraction, 0.1 mol/l acetic acid as acceptor phase, 1-octanol as organic phase and donor phase pH adjustment to 10. Under these conditions, the method was linear over the concentration range of 5-500 ng/ml with quantification limits of 5 ng/ml. The use of HF LPME for sample preparation provided suitable recoveries, efficient clean-up and low consumption of organic solvent.

Combination of hollow‐fiber liquid‐phase microextraction and capillary electrophoresis for pioglitazone and its main metabolites determination in rat liver microsomal fraction

Pioglitazone (PGZ), a thiazolidinedione antidiabetic agent, is reported as a potent and selective activator of peroxisome proliferator-activated receptor γ (PPAR γ). This drug has been widely prescribed for the treatment of Type 2 diabetes mellitus. In this regard, this manuscript presents, for the first time, an alternative electrophoretic method for PGZ and its main metabolites determination in rat liver microsomal fraction. The electrophoretic analyses were performed using an uncoated fused-silica capillary of 50 μm id, 48 cm in total length and 40 cm in effective length, and 50 mmol/L sodium phosphate buffer solution (pH 2.5). All experiments were carried out under the normal mode. The capillary temperature was set at 35°C and a constant voltage of +30 kV was applied during the analyses. Samples were introduced into the capillary by hydrodynamic injection (50 mbar, 15 s) and detection was performed at 190 nm. The sample preparation procedure, based on hollow-fiber liquid-phase microextraction, was optimized using multifactorial experiments. Next, the following optimal condition was established: sample agitation at 1500 rpm, extraction for 15 min, 0.01 mol/L hydrochloric acid as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 6.0. The method demonstrated LOQs of 200 ng/mL. Additionally, it was linear over the concentration range of 200-25,000 ng/mL for PGZ and 200-2000 ng/mL for the metabolites. Finally, the validated method was employed to study the in vitro metabolism of PGZ using rat liver microsomal fraction.

Headspace solid‐phase microextraction combined with mass spectrometry as a powerful analytical tool for profiling the terpenoid metabolomic pattern of hop‐essential oil derived from Saaz variety

Hop (Humulus lupulus L., Cannabaceae family) is prized for its essential oil contents, used in beer production and, more recently, in biological and pharmacological applications. In this work, a method involving headspace solid-phase microextraction and gas chromatography-mass spectrometry was developed and optimized to establish the terpenoid (monoterpenes and sesquiterpenes) metabolomic pattern of hop-essential oil derived from Saaz variety as a mean to explore this matrix as a powerful biological source for newer, more selective, biodegradable and naturally produced antimicrobial and antioxidant compounds. Different parameters affecting terpenoid metabolites extraction by headspace solid-phase microextraction were considered and optimized: type of fiber coatings, extraction temperature, extraction time, ionic strength, and sample agitation. In the optimized method, analytes were extracted for 30 min at 40°C in the sample headspace with a 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane coating fiber. The methodology allowed the identification of a total of 27 terpenoid metabolites, representing 92.5% of the total Saaz hop-essential oil volatile terpenoid composition. The headspace composition was dominated by monoterpenes (56.1%, 13 compounds), sesquiterpenes (34.9%, 10), oxygenated monoterpenes (1.41%, 3), and hemiterpenes (0.04%, 1) some of which can probably contribute to the hop of Saaz variety aroma. Mass spectrometry analysis revealed that the main metabolites are the monoterpene β-myrcene (53.0 ± 1.1% of the total volatile fraction), and the cyclic sesquiterpenes, α-humulene (16.6 ± 0.8%), and β-caryophyllene (14.7 ± 0.4%), which together represent about 80% of the total volatile fraction from the hop-essential oil. These findings suggest that this matrix can be explored as a powerful biosource of terpenoid metabolites.

Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂logτ(lag)/∂[urea] and ∂logν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.