Loading Solution Research Articles

Surfactant solutions behavior under electric explosion wire loading

In continuation of work on the study of the behavior of a continuous medium under dynamic, quasi-static and shock-wave loading, in this paper experiments on loading solutions of surfactants (surfactants) or proppant carriers (liquids for fracturing oil and gas reservoirs) based on water (Guar et al. Surfogel) under the influence of an electric explosion of a conductor were carried out. The velocity profiles of the free surface of surfactant solutions were obtained and analyzed. The velocity profiles were used to estimate the amplitude of the compression pulse, the spall strength of liquids, the rate of deformation at the fronts of the compression and rarefaction waves, and the shear viscosity of liquids. Distinctive features of liquids have been found.

Loading Mg<sup>2+</sup> into Erythrocytes with a Proposed Single Syringe Sterile Loading Process

Purpose: Primary hypomagnesemia with secondary hypocalcemia (PHSH) is a genetically defined disease in which there is a malabsorption of magnesium from the intestine. The aim of this research was to encapsulate magnesium into autologous erythrocytes to be used in PHSH patients. A scheme to use a single syringe closed sterile system for the process is proposed. Controlled lysis of erythrocytes and the ability to reseal the loaded erythrocytes were investigated. Method: Suitable concentrations of salt solutions required for making reversible pores and resealing of erythrocytes were identified. For making reversible pores, 0.7% NaCl and 2.3% MgSO4 were used separately. A solution of 3.5% MgSO4 was used as the drug loading solution. After the loading step, the isotonicity for the erythrocytes was restored with 0.9% NaCl and 3.0% MgSO4 solutions separately. The ability to load and reseal erythrocytes were assessed by determining the resulting Mg2+ concentration through Microwave Plasma Atomic Emission Spectrophotometric (MP-AES) analysis. Results: Analysis of results revealed that Mg2+ ions can be successfully loaded into erythrocytes through the formation of reversible pores. Resealing failed with 3.0% MgSO4, but the erythrocytes were successfully resealed with 0.9% NaCl solution. Conclusion: The need for strictly controlled salt solution concentrations for lysis and resealing were identified. In theoretical concentration determinations, the osmotic coefficient had to be taken into account. The study showed the possibility of loading Mg2+ into erythrocytes as a successful drug delivery system.

Polyethyleneimine-modified magnetic carbon nanotubes as solid-phase extraction adsorbent for the analysis of multi-class mycotoxins in milk via liquid chromatography-tandem mass spectrometry.

Polyethyleneimine-modified magnetic multi-walled carbon nanotubes were developed to extract 10 mycotoxins. Simple modification of polyethyleneimine was achieved on the magnetic substrate by using an epoxy-containing silane agent as a linker. The resultant magnetic adsorbent was integrated with reverse phase and anion exchange interaction sites. Under optimal extraction conditions, only 20.0mg adsorbent was used to extract the mycotoxins from 50.0mL loading solution. The maximum adsorption capacities of the adsorbent toward the mycotoxins ranged from 4.9 to 10.2mg/g. Adsorption and desorption were completed within 3.0 and 2.0min, respectively. The adsorbent could be used for six repeated runs without evident change in extraction performance. The adsorbent combined with liquid chromatography-tandem mass spectrometry was applied further to analyze the mycotoxins in milk. The absolute recoveries of the 10 mycotoxins ranged from 88.3 to 103.5% with relative standard deviations that ranged from 2.4 to 6.5%, and their limits of detection were 0.003 to 0.442μg/kg. The proposed adsorbent has great potential in the routine analysis of mycotoxins in ordinary analytical chemistry laboratory.

Coordination Polymers Integrating Metalloimmunology with Immune Modulation to Elicit Robust Cancer Chemoimmunotherapy

Nanoscale coordination polymers (NCPs) constructed by metal ions and organic ligands via metalligand bonds have attracted great attention for their biomedical application. Herein, a new type of NCP...

Efficient activation of oxone by pyrite for the degradation of propanil: Kinetics and degradation pathway

Pyrite (FeS2) is an abundant sulfide-associated iron mineral that exists in the earth. In this study, the pyrite/oxone process was demonstrated to be an effective approach for the catalytic degradation of propanil, where more than 90% decay ([propanil]0 = 0.01 mM) was achieved within 15 min. Typically, the effects of various experimental parameters, including catalyst loading, oxone dosage, propanil concentration, and initial solution pH, were examined. Two optimal reaction pH values were observed at pH 9.1 and pH 2.9. The generated SO4－ and OH were verified to be the dominant reactive radicals and primarily responsible for the propanil degradation. Both Fe(II) regeneration and sulfur conversion play an important role in oxone activation mechanism and effectively aid the catalytic activity of pyrite. Different co-existing natural water constituents exert dissimilar effects on the pyrite/oxone process. Additionally, the reusability test of pyrite exhibited a reasonable catalytic activity. The pyrite/oxone process was proven efficient in terms of propanil mineralization. A series of reaction intermediates was detected via four major degradation pathways. Overall, the pyrite/oxone process could be a promising approach for the removal of organic compounds in water.

Vitrification and proteomic analysis of embryogenic callus of Panax ginseng C. A. Meyer

Panax ginseng C. A. Meyer is a desirable medicinal plant due to its health benefits. With the growing interest in ginseng products, the safe preservation of genetically engineered material with unique attributes is increasingly important. In the present study, an effective protocol for cryopreservation of the ginseng embryogenic callus by vitrification is reported. After 2-wk subculture, the calluses were cold-acclimated at 4°C for 3 wk, then precultured for 2 d with a preculture medium containing 0.3 M sucrose. The precultured embryonic callus was then treated with a loading solution, followed by exposure to plant vitrification solution 2 at 0°C for 90 min. The dehydrated embryogenic calluses were then transferred into fresh 1.5 mL plant vitrification solution 2 and directly immersed in liquid nitrogen. With this protocol, an average of 68.3% regrowth rate for cryopreserved material was obtained, and the number of somatic embryos differentiated from embryogenic callus was significantly increased. Proteomic analysis showed that proteins related to carbohydrate metabolism, the stress response, and oxidative metabolism all significantly up-regulated after cryopreservation. Our results indicate that cold acclimation and preculture may be linked to enhanced cryopreservation tolerance. The up-regulated proteins play an important role in ginseng stress responses and may contribute to cryopreservation tolerance. Carbohydrate metabolism–related protein may be associated with the recovery growth of the cryopreserved embryogenic callus. The increased expression of HSP and 14-3-3-like protein play important roles in the response to stress and could be related with the improvement of somatic embryo production after cryopreservation.

Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources

Sweet potato (Ipomoea batatas) is one of the ten most important staple crops and provides a livelihood for many people around the globe. To adapt to ever-changing circumstances farmers and breeders need to have access to a broad diversity of germplasm. This study focuses on the development of a cryopreservation protocol that allows the long term storage of different sweet potato cultivars. For this, a droplet vitrification protocol was optimized, comparing several parameters; preculture method (0.3 M sucrose vs no preculture); meristem position (axillary vs apical); plant age (3 to 9 weeks); regeneration medium (MS + 2.22 µM BA, Hirai and MS); and length of loading solution treatment (20 to 360 min). Two months after cryopreservation, the regeneration rates of the meristems were compared, which resulted in significant differences for the preculture method, meristem position and loading solution. With these new insights an optimized droplet vitrification protocol was developed with the following parameters: use of 3–9 week old axillary meristems, no preculture phase, 20 min LS treatment, 30 min PVS2 treatment, exposure to liquid nitrogen by droplet vitrification, warming treatment in RS for 15 min, 1 day 0.3 M sucrose recuperation culture, 1 month MS + 2.22 µM BA followed by 1 month of MS cultures. This protocol was subsequently tested on 10 representative accessions resulting in a post cryopreservation regeneration rate of more than 40% for 70% of the tested cultivars, showing that this protocol could be implemented for a large portion of existing sweet potato collections.

Membrane-Based Affinity Purification to Identify Target Proteins of a Small-Molecule Drug

Identifying the target proteins of small-molecule drug candidates is important for determining their molecular mechanisms of action. Porous membranes derivatized with such small molecules may provide an attractive target-identification platform due to a high protein-capture efficiency during flow through membrane pores. This work employs carbonic anhydrase II (CAII) binding to immobilized 4-(2-aminoethyl)benzenesulfonamide (AEBSA) to examine the efficiency and selectivity of affinity capture in modified membranes. Selective elution of captured protein, tryptic digestion, tandem mass spectrometry analysis, and label-free quantification (LFQ) identify CAII as the dominant AEBSA target in diluted serum or cell lysate. CAII identification relies on determining the ratio of protein LFQ intensities in sample and control experiments, where free AEBSA added to the control loading solution limits CAII capture. Global proteomics shows that the spiked CAII is the only protein with a log2 ratio consistently >2, and the detection limit for CAII identification is 0.004 wt % of the total protein in 1:4 diluted human serum or 0.024 wt % of the total protein from breast cancer cell lysates. The same approach also identifies native CAII in human kidney cell lysate as an AEBSA target. Comparison of affinity capture using membranes, Affi-Gel 10 resin or M-270 Dynabeads derivatized with AEBSA suggests that only membranes allow identification of low-abundance CAII as a target.

Molecularly imprinted polymers for selective extraction of rosmarinic acid from Rosmarinus officinalis L.

This study provides a robust and reproducible approach for selective extraction of rosmarinic acid (RA) using molecularly imprinted polymers (MIPs). Computational modeling and UV spectroscopic analysis were performed to optimize MIP synthesis. Consequently, six different bulk and surface imprinted polymers were generated using RA as the template. Binding performance of the imprinted polymers was evaluated using static equilibrium and complementary dynamic rebinding experiments. Despite the high selectivity of thus generated surface imprinted polymers, the corresponding bulk polymers exhibited better binding performance when serving as sorbents during solid phase extraction (SPE). An optimized molecularly imprinted solid phase extraction (MISPE) protocol was developed in respect to loaded amount of RA, composition of the loading solution, washing solvent, and elution volume. Thereby, a remarkably selective extraction of RA from real-world Rosmarinus officinalis L. extract with a recovery rate and purity of 81.96 ± 6.33% and 80.59 ± 0.30%, respectively, was achieved.

Microporous polymer microspheres with amphoteric character for the solid-phase extraction of acidic and basic analytes.

Solid-phase extraction (SPE) is a widely-used and very well-established sample preparation technique for liquid samples. An area of on-going focus for innovation in this field concerns the development of new and improved SPE sorbents that can enhance the sensitivity and/or the selectivity of SPE processes. In this context, mixed-mode ion-exchange sorbents have been developed and commercialised, thereby allowing enhanced capacity and selectivity to be offered by one single material. The ion-selectivity of these materials is such that either anion-exchange or cation-exchange is possible, however one limitation to their use is that more than one sorbent type is required to capture both anions and cations. In this paper, we disclose the design, synthesis and exploitation of a novel SPE sorbent based on microporous polymer microspheres with amphoteric character. We show that it is possible to switch the ion-exchange retention mechanism of the sorbent simply by changing the pH of the loading solution; anion-exchange dominates at low pH, cation-exchange dominates at high pH, and both mechanisms can contribute to retention when the polymer-bound amphoteric species, which are based on the α-amino acid sarcosine (N-methylglycine), are in a zwitterionic state. This is an interesting and useful feature, since it allows distinctly different groups of analytes (acids and bases) to be fractionated using one single amphoteric sorbent with dual-functionality. The sarcosine-based sorbent was applied to the SPE of acidic, basic and amphoteric analytes from ultrapure water, river water and effluent wastewater samples. Under optimised conditions (loading 100mL of sample at pH 6, washing with 1mL of MeOH and eluting with an acidic or basic additive in MeOH) the recoveries for most of the compounds were from 57% to 87% for river water and from 61% to 88% for effluent wastewater. We anticipate that these results will lay the basis for the development of a new family of multifunctional sorbents, where two or more separation mechanisms can be embedded within one single, bespoke material optimised for application to challenging chemical separations to give significant selectivity advantages over essentially all other state-of-the-art SPE sorbents.

Preparation of reference standard extract of Lonicera hypoglauca Miq. with macroporous resin following a scale-up separation

ABSTRACT Bench-scale and pilot-scale methods were developed for the separation of extracted caffeoylquinic acids (CQAs) also called reference standard extracts (RSE) from defined source Lonicerae species using macroporous resin. Various separation parameters of the process were optimized such as Lonicera hypoglauca Miq. batch No. 4, 75% ethanol as extraction solvent, SD3000 macroporous resin, 3 mg/mL of total CQA loading solutions, 2 BV/h of adsorption and desorption flow rates and ethanol-water (30:70, 80:20, v/v) as eluent in gradient mode. The pilot-scale experiments were evaluated in six cycles. The quantitative analysis of CQA using RSE and mixed external standards were compared.

Large-sized nano-TiO2/SiO2 mesoporous nanofilmconstructed macroporous photocatalysts with excellent photocatalytic performance

Novel large-sized mesoporous nanofilm-constructed macroporous SiO2 (LMNCMS) with two sets of well-defined 3D continuous pass-through macropores (pore size of 0.5–1.0 μm, wall thickness of 40–50 nm) was prepared through a dual-templating approach, and used as an advanced support for TiO2 nanocrystalline photocatalyst. The structural and optical properties of the as-prepared materials were investigated by various characterization techniques in order to explore the connections between catalysts’ features and catalytic performance. The photocatalytic activities were evaluated by degradations of methylene blue (MB) and phenol under the simulated sunlight irradiation. To gain insight into the impact of preparation and operation conditions on photocatalytic degradation processes, experiments were conducted at wide ranges of the TiO2 loading content, calcination temperature, solution pH, and photocatalyst dosage. Nano-TiO2/LMNCMS exhibited high photocatalytic activity and stability. Rapid matter transport, good accessibility of pollutants to TiO2 and high light harvesting could mainly account for the superior photocatalytic performance. The trapping experiments were performed to identify the main reactive species in the catalytic reactions.

Enzyme Loading in Internally-Coated Capillary Tubes Via Kinetic Doping

Development of capillary tubes internally doped with enzymes is of great interest for microfluidic reactions, and kinetic doping could provide a facile, inexpensive method for their manufacture. Kinetic doping has previously been demonstrated to have a high loading capacity with thin films coated on flat-surface coverslips. Dip coating of these surfaces was developed with the eventual intention to coat different shapes and sizes of substrates. In this study, we expanded the use of kinetic doping to internally-coated capillary tubes. Parameters for internally doping capillary tubes were developed with rhodamine 6G, which produced internally-coated thin films with a 90 nm thickness. Horseradish peroxidase (HRP) was then loaded into the capillary tubes, with a 47,000× increase in concentration over the loading solution. After excluding surface-adsorbed protein, the increase in HRP concentration in the thin films over the loading solution was determined to be 9850×. The activity of the loaded HRP was determined to be 0.019 ± 0.003 U/mg and shown to have a stronger resistance to denaturation by methanol.

Antibacterial activity of tea saponin from Camellia oleifera shell by novel extraction method

Natural preservatives are substances that are secreted by organisms or present in the body and have bacteriostatic effects, so they are not toxic to humans. Natural preservatives have become a research focus because of their high safety and strong antibacterial effects. Some commodities in the market are added with tea saponins, such as a brand of foaming cleanser, shampoo, and toothpaste rich in tea saponins. In this study, A high concentration of tea saponin was obtained through extraction and purification from Camellia oleifera shells, and its antibacterial activity for the two tested bacteria was determined in vitro. The results revealed that AB-8 macroporous adsorption resin was used in combination with Sephadex G-15 to obtain tea saponin with 82.5 % purity. The optimal purification process conditions were: the loading quantity of sample is 1 bed volume (BV), the loading solution is 25 mg/mL, the loading solution PH is 5, the loading flow rate is 3 BV/h, the elution volume is 4 BV, the elution flow rate is 3 BV/h, the eluent ethanol concentration is 95 %, and the eluent pH is 9. Tea saponin had good bacteriostatic effect on Escherichia coli and Staphylococcus aureus with its minimum inhibitory concentration of 1 and 0.5 mg/mL, respectively. In addition, its minimum bactericidal concentration was 4 mg/mL. Tea saponin could change membrane permeability, destroy cell membrane structure, and inhibit bacterial growth. This study provides basic guidance for the application of tea saponin in the development of natural antibacterial agents, is conducive to the high-value utilization of tea saponin, provides a reference for the economical of camellia tea waste, and plays a benefit in increasing agricultural income.

Synthesis of Carbon Onion and Its Application as a Porous Carrier for Amorphous Drug Delivery

Given the great potential of porous carrier-based drug delivery for stabilising the amorphous form of drugs and enhancing dissolution profiles, this work is focussed on the synthesis and application of carbon onion or onion-like carbon (OLC) as a porous carrier for oral amorphous drug delivery, using paracetamol (PA) and ibuprofen (IBU) as model drugs. Annealing of nanodiamonds at 1100 °C produced OLC with a diamond core that exhibited low cytotoxicity on Caco-2 cells. Solution adsorption followed by centrifugation was used for drug loading and results indicated that the initial concentration of drug in the loading solution needs to be kept below 11.5% PA and 20.7% IBU to achieve complete amorphous loading. Also, no chemical interactions between the drug and OLC could be detected, indicating the safety of loading into OLC without changing the chemical nature of the drug. Drug release was complete in the presence of sodium dodecyl sulphate (SDS) and was faster compared to the pure crystalline drug, indicating the potential of OLC as an amorphous drug carrier.

Standardization of vitrification treatments for safe cryo-conservation of banana (Musa acuminata Colla) cultivars using differential scanning calorimetry

Cryo-conservation is the most promising approach and reliable method for long term conservation of banana and plantain genetic resources. Prior to the development of a safe cryo-conservation procedure for highly hydrated tissues of any vegetatively propagated crop species such as banana, suitable vitrification treatments involving loading solution in combination with Plant Vitrification Solution 2 (PVS2) solution have to be standardized. In the present study, Differential Scanning Calorimetry (DSC) technique was attempted to identify the best vitrification treatment using shoot apices as storage explants in two banana cultivars namely Grand Naine and Red banana. The results showed that DSC thermogram curves detected the formation of ice nucleation at a temperature of -18.67°C for Grand Naine and -19.16°C for Red banana in the control shoot apices. Among the sixteen vitrification treatment combinations tested, the treatments, LP6, LP7, LP10, LP11, LP14 and LP15 were judged as best treatments for cryo-conservation of both banana cultivars as revealed by the formation of glass transition events in the vitrified shoot apices. This information obtained through DSC experiments would further provide scope for designing safe cryo-conservation system for banana. This is the first report to demonstrate the application of DSC to optimize the vitrification treatments that would be adopted for developing cryo-conservation protocol for Musa germplasm.

Amino-modified Scholl-coupling mesoporous polymer for online solid-phase extraction of plant growth regulators from bean sprouts

A new amino-modified Scholl-coupling mesoporous polymer (NH2@SMPA)-online solid-phase extraction method, coupled with high-performance liquid chromatography (online SPE-HPLC) was established for the analysis of six plant growth regulators (PGRs) in bean sprouts. NH2@SMPA was synthesized by acid-catalyzed deacetylation of acetylamino-Scholl-coupling mesoporous polymer (SMPA). The diversity of functional groups, such as aromatic, acetylamino, and NH2, was conducive to multiple binding interactions between NH2@SMPA and PGRs. NH2@SMPA exhibited superior extraction capability for PGRs, compared with SMPA and commercial adsorbents. The extraction conditions, including loading solvent, pH of loading solution, eluting solvent, and flow rates of loading and elution, were optimized. Under the optimized conditions, wide linear ranges (0.01–500 μg kg−1) and low detection limits (2.34–20.2 ng kg−1) were obtained. The recoveries were satisfactory, i.e., 86.0% to 109% with relative standard deviations ≤9.8% (n = 3). Finally, the online SPE-HPLC method was successfully used for determination of PGRs in bean sprouts.

Long-term conservation of Tarenaya rosea (Cleomaceae) root cultures: histological and histochemical analyses during cryopreservation using the encapsulation-vitrification technique.

Adventitious root cultures of Tarenaya rosea were successfully cryopreserved using the encapsulation-vitrification technique. Histological analysis revealed useful information on the successive steps of cryopreservation. Coupled with complementary histochemical approaches, these studies provided cellular and tissue descriptions of T. rosea root cultures during cryopreservation and contributed to an understanding of cellular stress responses, as well as characterization of the anatomical pattern of root regeneration. The effects of exposure duration to PVS3 solution (0-120min), unloading treatment (direct and gradual), and recovery medium (liquid and solid) on recovery of cryopreserved roots were investigated. The highest recovery (91%) after cooling in liquid nitrogen (LN) was reached with PVS3 treatment for 90min, gradual rehydration in unloading solution, and recovery on solid MS medium. The cryopreserved roots showed high multiplication capacity, which was maintained for up to four subcultures. The effect of cryopreservation on root structure was investigated by histological and histochemical studies. Plasmolysis intensified during exposure to loading and PVS3 solutions, but decreased after unloading treatment. The proportion of intercellular spaces increased progressively throughout the cryopreservation protocol, culminating in root cortex disruption. Histochemical analyses revealed polysaccharides, proteins, and both lipidic and pectic substances in intercellular spaces. The vascular cylinder remained intact, ensuring the formation of new roots from the pericycle, showing that proliferative capacity of cryopreserved roots had not diminished.

Detection of trace metal ions in high-purity boric acid by online two-dimensional valve switching coupled with ion chromatography-inductively coupled plasma mass spectrometry

In this paper, a two-dimensional valve switching technique was established to detect 10 trace metal ions in high-purity boric acid by ion chromatography-inductively coupled plasma mass spectrometry (IC-ICPMS). The system consisted of a quantitative loop, a matrix capture column, an iminodiacetic acid cationic chelating column, an ion chromatography column, two sample valves and an inductively coupled plasma mass spectrometer. The iminodiacetic acid cationic chelating column coupled the metal ions in the sample with its carboxyl functional groups to separate them from the matrix boric acid solution, and then eluted the chelated metal ions into the ion chromatographic column and inductively coupled plasma mass spectrometer. The proposed method feasibly achieved an online, rapid and efficient detection of 10 trace ions in high-purity boric acid with the optimized experimental conditions (1.0 mol/L HNO3), such as loading solution, loading flow rate, type of eluent, concentration of the eluent, sample matrix, etc. The experimental data showed that this method had good linearity in the range of 0.01–2.0 μg/L for 10 metal ions, such as Cr, Fe, Ni, Co, V, Mn, Cu, Zn, Cd and Pb in high-purity boric acid samples, with a correlation coefficient greater than 0.998. The limit of detection (LOD, S/N = 3) and the limit of quantitation (LOQ, S/N = 10) were 0.001–0.023 μg/L and 0.003–0.077 μg/L, respectively. The relative standard deviation (RSD) of each element was less than 3.0% (n = 6) and the spiked recoveries ranged between 83% and 110%. This method could be proposed to be suitable for determination of trace metal ions in real samples.

Modulation of Pharmaceutical and Pharmacokinetic Attributes of Curcumin Encapsulated in Chitosan Using Spray Drying Technique

The present study is designed to formulate and characterize Curcumin (CUR) loaded Chitosan nanoparticles by ionic cross linking using TPP by employing spray drying technique to enhance the aqueous solubility, hence oral bio availability of Curcumin. Curcumin is very sparingly aqueous soluble, unstable in alkaline conditions and gets eliminated faster, resulting in poor bio availability. Curcumin in alcohol was used as drug loading solution. Tween 80 served as nano emulsifier and stabilizer. EE of optimized formulation F6 was found to be 98.5 ± 0.16 %. This formulation has an average particle size of 25 ± 0.1 nm. The Zeta potential of the formulation is -200 mV. FTIR results revealed the absence of characteristic CUR peak in Curcumin Chitosan nanoparticles implying the successful incorporation of CUR into the Chitosan matrix. DSC thermograms witnessed that the crystalline form of CUR is transformed into amorphous form, having better dissolution and stability profile. SEM imaging of the nanosuspension formulation showed the topological features of the prepared nanoparticles. The nanoparticles were more spherical, discrete and homogeneous. % cumulative drug release studies exhibited very less drug release in Simulated Gastric Fluid (SGF- 3.97 %) and Simulated Intestinal Fluid (SIF - 4.2 %) exhibiting sustained release. Pure Curcumin showed burst release of approximately 95 % in 2 h in SGF with a decline in the concentration after 2 h in SIF due to significantly increased degradation in the alkaline conditions. The oral bioavailability (AUC) of Curcumin in female wistar rats increased 11-times following ingestion of Curcumin nanosuspension in comparison with conventional Curcumin suspension. The maximum concentration Cmax of 1034 ± 96 ng / ml for nanosuspension and 94 ± 9.4 ng / ml for Curcumin suspension and was observed at tmax of 2 h for both nanosuspension and suspension.