Alternative Purification Method Research Articles

Characterization and engineering of the type 3 secretion system needle monomer from Salmonella through the construction and screening of a comprehensive mutagenesis library.

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needle's assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle's mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS's assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

Validation of a size exclusion method for concomitant purification and formulation of peptide radiopharmaceuticals

BackgroundBoth in clinical routine and in preclinical research, the established standard procedure for the final purification of radiometal-labeled peptide radiopharmaceuticals is cartridge-based reversed-phase (RP) solid phase extraction (SPE). It allows the rapid and quantitative separation of the radiolabeled peptide from hydrophilic impurities and easy integration into automated synthesis procedures. However, product elution from RP cartridges necessitates the use of organic solvents and product recovery is sometimes limited. Thus, an alternative purification method based on commercially available size exclusion cartridges was investigated.ResultsSince most peptide radiopharmaceuticals have a molecular weight > 1 kDa, Sephadex G10 cartridges with a molecular size cut-off of 700 Da were used for the final purification of a broad palette of 68Ga-, 64Cu- and 99mTc-labeled experimental peptide radiotracers as well as the clinically relevant ligand PSMA-617. Results (radiochemical purity (RCP, determined by ITLC), recovery from the solid support) were compared to the respective standard RP-SPE method. Generally, retention of unreacted 68Ga, 64Cu and 99mTc salts on the G10 cartridges was quantitative up to the specified elution volume (1.2 mL) for 68Ga and 99mTc and 99.6% for 64Cu. Even at increased elution volumes of 1.5-2 mL, RCPs of the eluted 68Ga- and 99mTc -radiopeptides were > 99%. For all peptides with a molecular weight ≥ 2 kDa, product recovery from the G10 cartridges was consistently > 85% upon respective adjustment of the elution volume. Product recovery was lowest for [68Ga]Ga-PSMA-617 (67%, 1.2 mL to 84%, 2 mL). The pH of the final product solution was found to be volume-dependent (1.2 mL: pH 6.3; 1.5 mL: pH 5.9; 2 mL: pH 5.5). Notably, the G10 cartridges were reused up to 20 times without compromising performance, and implementation of the method in an automated radiosynthesis procedure was successful.ConclusionsOverall, size exclusion purification yielded all peptide radiopharmaceuticals in excellent radiochemical purities (> 99%) in saline within 10–12 min. Although product recovery is marginally inferior to classical SPE purifications, this method has the advantage of completely avoiding organic solvents and representing a cost-effective, easy-to-implement purification approach for automated radiotracer synthesis.

Enhanced invertase binding from baker's yeast via cryogels included boronic acids.

Invertase, an industrially significant glycoenzyme, was purified from baker's yeast using poly (2-Hydroxyethyl methacrylate) [PHema-Pba] cryogels functionalized with boronic acid. At subzero temperatures, PHema-Pba cryogels were synthesized and characterized using swelling tests, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The surface area of the PHema-Pba cryogels was 14 m2/g with a swelling ratio of 88.3% and macroporosity of 72%. The interconnected macropores of PHema-Pba cryogels were shown via scanning electron microscopy. Invertase binding capacity of PHema-Pba cryogel was evaluated by binding studies in different pH, temperature, and interaction time conditions and the maximum Invertase binding of PHema-Pba cryogel was found as 15.2mg/g. and 23.7 fold Invertase purification was achieved from baker's yeast using PHema-Pba cryogels. The results show that PHema-Pba cryogels have high Invertase binding capacity and may be used as an alternative method for enzyme purification via boronate affinity systems.

Proposal and application of a novel extraction and purification technology for juglone from Juglans mandshurica waste branches: Reverse micellar microemulsion combined with magnetic solid phase extraction

A reverse micellar microemulsion combined with magnetic solid phase extraction (RMM-MSPE) technology was developed for the extraction and purification of juglone from Juglans mandshurica. Reverse micellar microemulsion (RMM) (Tween 80: n-propanol: n-hexane: 0.05 mol/L KCl aqueous solution = 27:13.5:4.5:55; w/w/w/w) was used to extract juglone based on microwave. The optimum conditions were a pH of 5.6, a liquid-solid ratio of 20:1 (mL/g), temperature of 40 °C, time of 63 s, and power of 400 W. Graphene oxide/nano diamonds/ Fe3O4 (GO/ND/Fe3O4) was prepared as adsorbent and applied to purify juglone by magnetic solid-phase extraction (MSPE). The optimum adsorption conditions were time of 14 h, solid to liquid ratio of 30 mg/mL, pH of 5.5 and shaker speed of 200 r/min. Anhydrous methanol provided the best desorption of juglone and the optimum conditions were a desorption time of 16 h, pH of 8, and the solid-liquid ratio of 40 mg/mL. The purity of juglone was 69.08% and the recovery yield was 78.67%. RMM and GO/ND/Fe3O4 could be reused three times and eight times after the first use, respectively. The proposed RMM-MSPE is a green and efficient alternative method for extraction and purification of juglone.

Removal of photoredox catalysts from polymers synthesized by organocatalyzed atom transfer radical polymerization.

Organocatalyzed atom transfer radical polymerization (O-ATRP) is a method of producing polymers with precise structures under mild conditions using organic photoredox catalysts (PCs). Due to the unknown toxicity of PCs and their propensity to introduce color in polymers synthesized by this method, removal of the PC from the polymer product can be important for certain applications of polymers produced using O-ATRP. Current purification methods largely rely on precipitation to remove the PC from the polymer, but a more effective and efficient purification method is needed. In this work, an alternative purification method relying on oxidation of the PC to PC · + followed by filtration through a plug to remove PC · + from the polymer and removal of the volatiles was developed. A range of chemical oxidants and stationary phases were tested for their ability to remove PCs from polymers, revealing chemical oxidation by N-bromosuccinimide followed by a filtration through a silica plug can remove up to 99% of the PC from poly(methyl methacrylate). Characterization of the polymer before and after purification demonstrated that polymer molecular weight, dispersity, and chain-end fidelity are not signficantly impacted by this purification method. Finally, this purification method was tested on a range of dihydrophenazine, phenoxazine, dihydroacridines, and phenothiazine PCs, revealing the strength of the chemical oxidant must match the oxidation potential of the PC for effective purification.

Bactericidal efficacy of non-thermal plasma activation against Aeromonas hydrophila and immunological responses of koi (Cyprinus carpio haematopterus)

In the aquaculture industry, an efficient and safe water purification system is important to prevent mass mortality by virulent pathogens. As extensive use of traditional methods (e.g.: povidone-iodine, ozone, ultraviolet irradiation, formalin, and chlorine dioxide) have adverse effects on cultured fish, an appropriate and alternative water purification method is vital for the sustainability of the industry. Non-thermal plasma technology has been successfully used for various biomedical purposes (e.g: food sterilization, medical device disinfection, wound healing, cancer therapy, etc.) and has great potential to be used as a sterilizing system. However, few studies have been conducted on its usefulness in the aquaculture industry. In this study, we investigated the bactericidal efficacy of plasma-activated water induced by non-thermal plasma and its histopathological as well as immunological adverse effects on koi. A highly virulent Aeromonas hydrophila SNU HS7, which caused massive mortality of koi, was used for this study. Non-thermal plasma was applied for 10 min to the fish tanks with 1.2 × 109 CFU/mL SNU HS7 using PLMB-20 system to confirm the sterilization efficacy and to observe the survival and immunological reaction of koi for 14 days. As a result, gross pathological, histopathological, and immunological investigations did not reveal any significant adverse effects in fish as compared to the control groups. To the best of our knowledge, this is the first study showing that non-thermal plasma can be used for sterilization of rearing water without giving significant physiological damage to the fish, even under the assumption of extreme situations. As plasma can effectively sterilize not only bacteria but also other unknown pathogens, the results of this study are showing a promising future in purifying water in aquaculture practice.

Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology

Adenoviruses (AdVs) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney 239 (HEK293) or PER.C6 virus producer cells with AdV plasmid vectors or infecting these cells withcell lysates containing replication-defective AdV. Cell lysates can be purified further by caesium chloride or chromatographic protocols to research virus seed stocks (RVSS) for characterisation to high quality master virus seed stocks (MVSS) and working virus seed stocks (WVSS) before downstream production of pure, high titre AdV. Lysates are poorly infectious, block filtration columns and have limited storage capability. Aqueous two-phase systems (ATPS) are an alternative method for AdV purification that rapidly generates cleaner RVSS for characterisation to MVSS. After testing multiple ATPS formulations, an aqueous mixture of 20 % PEG 600 and 20 % (NH4)2SO4 (w/w) was found most effective for AdV partitioning, producing up to 97+3% yield of high-titre virus that was devoid of aggregates both effective in vitro and in vivo with no observable cytotoxicity. Importantly, AdV preparations stored at −20 °C or 4 °C show negligible loss of titre and are suitable for downstream processing to clinical grade to support the need for AdV vaccines.

Pristine graphite oxide retains its C-axis registry in methanol. The way to alternative purification method

In this work, we demonstrate that pristine graphite oxide (PGO), formed by the oxidation of graphite by the modified Hummers’ method, when still in synthetic mixture, can be re-intercalated by methanol, while keeping its c-axis registry. The as-formed structure contains two molecular layers of methanol, sandwiched between the neighboring graphene oxide (GO) sheets. The typical water-washed and dried GO intercalates only by one molecular layer of methanol. This finding suggests the route for preparing alcohol-intercalated GO straight from PGO, while bypassing water-purification, drying, and intercalating steps. The methanol-purified GO has significantly higher content of organic sulfates, compared to the typical water-purified sample. We stress one more time that the 1620 cm−1 and 1223 cm−1 absorption bands in the FTIR spectra of GO are originated by water molecules, and organic sulfates, respectively. The GO purification with methanol has some advantages over the currently used water-purification, and might become an attractive alternative to the latter.

Design and optimization of an alternative chymotrypsin purification method by adsorption onto non-soluble alginate–carrageenan bed

In the present work, we have developed a new protocol for the purification of chymotrypsin using adsorption onto non-soluble alginate–carrageenan bed. In the first instance, an experimental statistical design was used to screen which parameters are significant in the adsorption process. The variables tested were pH, temperature, and medium’s ionic strength. Significant statistical differences (p < 0.05) were obtained for pH and the medium’s ionic strength, whereas temperature had no influence on the adsorption efficiency in the studied range. In a second step, response surface methodology was used to find the conditions that produced the highest adsorption of chymotrypsin. The optimal working conditions found were pH 6.5 without the addition of salts, which is consistent with an electrostatic interaction. The adsorption kinetics were studied, showing that the adsorption of the enzyme followed a pseudo-first-order rate expression. Different adsorption isotherm models were applied to describe the equilibrium isotherms. The Freundlich model was found to be more suitable for our data. At optimal conditions, 1 g of hydrated matrix adsorbed 11 mg of chymotrypsin. In the desorption process, 93.1% of the biological activity of chymotrypsin was recovered in only 20 min. Additionally, experimental breakthrough profiles were acquired. The optimized conditions were applied in the design of both batch and packed bed adsorption systems for the purification of chymotrypsin from fresh bovine pancreas, reaching a purification factor of 3.8 and 6.9, respectively.

Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method.

Background:Beta-cardiotoxin (β-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses β-blocker activity as indicated by its negative chronotropy and its binding property to both β-1 and β-2 adrenergic receptors and has been proposed as a novel β-blocker candidate. Previously, β-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein.Methods:β-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of β-CTX concentration.Results:Purified β-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. β-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in β-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis.Conclusion:We present an alternative purification method for β-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from β-CTX.

A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome

For mass spectrometry (MS)-based N-glycoproteomics, selective enrichment of N-glycopeptides prior to MS analysis is a crucial step to reduce sample complexity. Enrichment based on covalent coupling is as an increasingly attractive strategy due to the unbiased and highly specific features. However, most of current covalent coupling reactions for N-glycopeptides enrichment are still limited by long coupling time and harsh coupling conditions. Herein, we developed a thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome. With the use of facile synthesis of Cys-terminated magnetic nanoparticles, the oxidized glycan moieties on glycopeptides could be selectively captured by the β-amino thiols groups on the surface of magnetic nanoparticles through thiazolidine formation. The coupling could be achieved within 30 min under mild condition, eliminating the addition of toxic catalyst or sample-destroying reducing agent. Also, the great enrichment performance for N-glycopeptides were obtained in terms of sensitivity (low fmol levels), selectivity (extracting N-glycopeptides from the mixture of glycopeptides and non-glycopeptides at a 1:100 molar ratio) and reproducibility (CVs<26%). Finally, this proposed method was successfully demonstrated by analyzing the N-glycoproteome from 2 μL human serum, which offers an alternative purification method for analysis of N-glycoproteome from complex biological samples.

A simple and effective method using macroporous resins for the simultaneous decoloration and deproteinisation of Cordyceps militaris polysaccharides

SummaryThe aim of this study was to develop an effective one‐step decoloration and deproteinisation method using macroporous resin for Cordyceps militaris polysaccharide (CMP) extraction. Performances of different resins were compared, and the structures and bioactivities of CMP recovered using this one‐step method were evaluated and compared with traditional methods. The results showed that NKA‐9 resin exhibited the highest adsorption capacity for pigment and free protein than other tested resins. Under the optimised condition, NKA‐9 resin adsorption exhibited high decoloration ratio (80.69%), deproteinisation ratio (62.51%) and polysaccharide recovery ratio (78.08%). Furthermore spectroscopic analysis revealed that the structure of CMP remained unchanged after adsorption, but some reagent residues can be detected using traditional methods. Compared with traditional methods, the antioxidant activity of CMP extracted using a one‐step method was significantly higher, without negative impact on cell viability. Thus, this novel technique is a promising alternative method for CMP separation and purification.

Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography

The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400 SEC, we obtained highly pure, i.e., > 90%, rHBsAg virus-like particles (VLP).

Environmentally Benign and Recyclable Aqueous Two-Phase System Composed of Distillable CO2-Based Alkyl Carbamate Ionic Liquids

Ionic liquid-based aqueous two-phase systems (IL-ATPSs) have been studied comprehensively as a potential alternative method for protein purification. One of the distinct features of IL-ATPS is that the polarity of the IL can be adjusted by different combinations of cation and anion, providing flexibility in governing the partition of proteins between phases. However, the conventional IL-ATPS usually has poor environmental footprint and low recyclability, thereby hampering the systems for practical use in protein separation. To address these shortcomings, here we explored the distillable CO2-based alkyl carbamate ILs as phase-forming components. N,N-dimethylammonium N′,N′-dimethylcarbamate (DIMCARB) was able to form ATPS with polypropylene glycol (PPG). The liquid–liquid equilibrium data of ATPSs composed of PPG with different molecular mass and DIMCARB were determined. As both phase-forming components are thermosensitive, the effect of temperature on the phase diagram of the PPG 400 + DIMCARB system was also evaluated. The partition behavior of model proteins (i.e., bovine serum albumin and lysozyme) in the PPG + DIMCARB systems were assessed. Both model proteins tend to migrate to the more hydrophilic IL-rich phase, when a higher temperature or a higher molecular mass of PPG was used. The DIMCARB was recovered via rotary evaporation, while the PPG 400 was recovered from a secondary two-phase system thermally induced from the primary ATPS. The recovered components were subsequently used to prepare a new batch of ATPS. The phase composition of the new system as well as the partition coefficients of both model proteins in the recycled system were maintained, thereby proving the feasibility of reusing both phase-forming components. Overall, the recyclable and biodegradable phase-forming components used in the newly developed PPG + DIMCARB system make the ATPS a sustainable and eco-friendly system for protein purification.

Single step purification via magnetic nanoparticles of new broad pH active protease from Penicillium aurantiogriseum

A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60–80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1 M NaCl solution) and eluted 3 times (1 M NaCl solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40 kDa on SDS-PAGE and optimum activity at 45 °C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55 °C, did not lose any activity over 48 h at 25 °C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative purification method and novel broad pH tolerant protease.

Purification of Saccharomyces cerevisiae Homologous Recombination Proteins Dmc1 and Rdh54/Tid1 and a Fluorescent D-Loop Assay.

Budding yeast Dmc1 is a member of the RecA family of strand exchange proteins essential for homologous recombination (HR) during meiosis. Dmc1 mediates the steps of homology search and DNA strand exchange reactions that are central to HR. To achieve optimum activity, Dmc1 requires a number of accessory factors. Although methods for purification of Dmc1 and many of its associated factors have been described (Binz, Dickson, Haring, & Wold, 2006; Busygina et al., 2013; Chan, Brown, Qin, Handa, & Bishop, 2014; Chi et al., 2006; Cloud, Chan, Grubb, Budke, & Bishop, 2012; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Van Komen, Macris, Sehorn, & Sung, 2006), Dmc1 has been particularly difficult to purify because of its tendency to aggregate. Here, we provide an alternative and simple high-yield purification method for recombinant Dmc1 that is active and responsive to stimulation by accessory factors. The same method may be used for purification of recombinant Rdh54 (a.k.a. Tid1) and other HR proteins with minor adjustments. We also describe an economical and sensitive D-loop assay for strand exchange proteins that uses fluorescent dye-tagged, rather than radioactive, ssDNA substrates.

Biophysical analysis of BMV virions purified using a novel method.

Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2mg of virus from 1g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.

An alternative purification method for human serum paraoxonase 1 and its interaction with methidathion

In this study, an alternative purification method for human Paraoxonase 1 (hPON1) enzyme was developed using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-L-tyrosine-1-aminoanthracene hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. The enzyme was purified 674-fold with a yield of 16%. Furthermore, we examined the in vitro effect of methidathion on the enzyme activity to understand the better inhibitory properties of the compound. Methidathion is a highly toxic insecticide used to control a broad spectrum of agricultural insect and mite pests. IC50 value was found to be 0.130 mM for the pesticide. Methidathion showed a competitive inhibition with Ki of 0.119 mM for paraoxon.

Asymmetric flow field flow fractionation methods for virus purification

Detailed biochemical and biophysical characterization of viruses requires viral preparations of high quantity and purity. The optimization of virus production and purification is an essential, but laborious and time-consuming process. Asymmetric flow field flow fractionation (AF4) is an attractive alternative method for virus purification because it is a rapid and gentle separation method that should preserve viral infectivity. Here we optimized the AF4 conditions to be used for purification of a model virus, bacteriophage PRD1, from various types of starting materials. Our results show that AF4 is well suited for PRD1 purification as monitored by virus recovery and specific infectivity. Short analysis time and high sample loads enabled us to use AF4 for preparative scale purification of PRD1. Furthermore, we show that AF4 enables the rapid real-time analysis of progeny virus production in infected cells.

Hypolipidemic activities of xanthorrhizol purified from centrifugal TLC

Hyperlipidemia is defined as the presence of either hypertriglyceridemia or hypercholesterolemia, which could cause atherosclerosis. Although hyperlipidemia can be treated by hypolipidemic drugs, they are limited due to lack of effectiveness and safety. Previous studies demonstrated that xanthorrhizol (XNT) isolated from Curcuma xanthorrhizza Roxb. reduced the levels of free fatty acid and triglyceride in vivo. However, its ability to inhibit cholesterol uptake in HT29 colon cells and adipogenesis in 3T3-L1 cells are yet to be reported. In this study, XNT purified from centrifugal TLC demonstrated 98.3% purity, indicating it could be an alternative purification method. The IC50 values of XNT were 30.81 ± 0.78 μg/mL in HT29 cells and 35.07 ± 0.24 μg/mL in 3T3-L1 adipocytes, respectively. Cholesterol uptake inhibition study using HT29 colon cells showed that XNT (15 μg/mL) significantly inhibited the fluorescent cholesterol analogue NBD uptake by up to 27 ± 3.1% relative to control. On the other hand, higher concentration of XNT (50 μg/mL) significantly suppressed the growth of 3T3-L1 adipocytes (5.9 ± 0.58%) compared to 3T3-L1 preadipocytes (81.31 ± 0.55%). XNT was found to impede adipogenesis of 3T3-L1 adipocytes in a dose-dependent manner from 3.125 to 12.5 μg/mL, where 12.5 μg/mL significantly suppressed 36.13 ± 2.1% of lipid accumulation. We postulate that inhibition of cholesterol uptake, adipogenesis, preadipocyte and adipocyte number may be utilized as treatment modalities to reduce the prevalence of lipidemia. To conclude, XNT could be a potential hypolipidemic agent to improve cardiovascular health in the future.