Engineered AAV8 capsid acquires heparin and AVB sepharose binding capacity but has altered in vivo transduction efficiency.

Naturally occurring adeno-associated virus (AAV) serotypes that bind to ligands such as AVB sepharose or heparin can be purified by affinity chromatography, which is a more efficient and scalable method than gradient ultracentrifugation. Wild-type AAV8 does not bind effectively to either of these molecules, which constitutes a barrier to using this vector when a high throughput design is required. Previously, AAV8 was engineered to contain a SPAKFA amino acid sequence to facilitate purification using AVB sepharose resin; however, in vivo studies were not conducted to examine whether these capsid mutations altered the transduction profile. To address this gap in knowledge, a mutant AAV8 capsid was engineered to bind to AVB sepharose and heparan sulfate (AAV8-AVB-HS), which efficiently bound to both affinity columns, resulting in elution yields of >80% of the total vector loaded compared to <5% for wild-type AAV8. However, in vivo comparison by intramuscular, intravenous, and intraperitoneal vector administration demonstrated a significant decrease in AAV8-AVB-HS transduction efficiency without alteration of the transduction profile. Therefore, although it is possible to engineer AAV capsids to bind various affinity ligands, the consequences associated with mutating surface exposed residues have the potential to negatively impact other vector characteristics including in vivo potency and production yield. This study demonstrates the importance of evaluating all aspects of vector performance when engineering AAV capsids.

Biophysical properties of hydrogels for mimicking tumor extracellular matrix.

The extracellular matrix (ECM) is an essential component of the tumor microenvironment. It plays a critical role in regulating cell-cell and cell-matrix interactions. However, there is lack of systematic and comparative studies on different widely-used ECM mimicking hydrogels and their properties, making the selection of suitable hydrogels for mimicking different in vivo conditions quite random. This study systematically evaluates the biophysical attributes of three widely used natural hydrogels (Matrigel, collagen gel and agarose gel) including complex modulus, loss tangent, diffusive permeability and pore size. A new and facile method was developed combining Critical Point Drying, Scanning Electron Microscopy imaging and a MATLAB image processing program (CSM method) for the characterization of hydrogel microstructures. This CSM method allows accurate measurement of the hydrogel pore size down to nanometer resolution. Furthermore, a microfluidic device was implemented to measure the hydrogel permeability (Pd) as a function of particle size and gel concentration. Among the three gels, collagen gel has the lowest complex modulus, medium pore size, and the highest loss tangent. Agarose gel exhibits the highest complex modulus, the lowest loss tangent and the smallest pore size. Collagen gel and Matrigel produced complex moduli close to that estimated for cancer ECM. The Pd of these hydrogels decreases significantly with the increase of particle size. By assessing different hydrogels' biophysical characteristics, this study provides valuable insights for tailoring their properties for various three-dimensional cancer models.

Diffusion coefficients of polar organic compounds in agarose hydrogel and water and their use for estimating uptake in passive samplers.

Diffusion coefficient (D) is an important parameter for prediction of micropollutant uptake kinetics in passive samplers. Passive samplers are nowadays commonly used for monitoring trace organic pollutants in different environmental matrices. Samplers utilising a hydrogel layer to control compound diffusion are gaining popularity. In this work we investigated diffusion of several perfluoroalkyl substances, currently used pesticides, pharmaceuticals and personal care products in 1.5% agarose hydrogel by measuring diffusion coefficients using two methods: a diffusion cell and a sheet stacking technique. Further, diffusion coefficients in water were measured using Taylor dispersion method. The sheet stacking method was used to measure D at 5, 12, 24, and 33 °C in order to investigate temperature effect on diffusion. Median D values ranged from 2.0 to 8.6 × 10-6 cm2 s-1 and from 2.1 to 8.5 × 10-6 cm2 s-1 for the diffusion cell and sheet stack methods respectively. For most compounds, the variability between replicates was higher than the difference between values obtained by the two methods. Rising temperature from 10 to 20 °C increases the diffusion rate by the factor of 1.41 ± 0.10 in average. In water, average D values ranged from 3.03 to 10.0 × 10-6 cm2 s-1 and were comparable to values in hydrogel, but some compounds including perfluoroalkyl substances with a long aliphatic chain could not be evaluated properly due to sorptive interactions with capillary walls in the Taylor dispersion method. Sampling rates estimated using the measured D values were systematically higher than values estimated from laboratory sampler calibration in our previously published study, by the factor of 2.2 ± 1.0 in average.

Perfluorooctyl bromide traces self-assembled with polymeric nanovesicles for blood pool ultrasound imaging.

A novel perfluorooctyl bromide (PFOB)-loaded nanovesicle with a size of about 500 nm was prepared by self-assembly of an amphiphilic block copolymer, poly(ethylene oxide)-b-poly(d,l-lactic acid) (PEG-PDLLA), for blood pool ultrasound imaging. The excellent compatibility of PFOB with the hydrophobic PDLLA block makes PFOB uniformly distribute and integrate well within the nanovesicle shell. In theory, both the compressibility and shell density of the nanovesicle as ultrasound scatterers are enhanced, resulting in much higher echo intensity compared to the other PFOB nanoparticles. In vitro and in vivo imaging results illustrate that these polymeric nanovesicles with extremely low content of PFOB show quite a good contrast-enhancing effect even if highly diluted in blood. Therefore this PFOB-loaded polymeric nanovesicle is anticipated to be applicable as an ultrasound contrast agent for normal angiography and specific imaging of capillary-abundant organs or tissues (e.g. tumors).

Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp.

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and 42.5 degrees C, and the enzyme was stable under 40 degrees C. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a beta-agarase.

Evaluation of HPSEC-ELSD method for precise measurement of ß-agarase activity.

ß-agarase activity was monitored by traditional reducing sugar content methods: Somogyi-Nelson's arsenomolybdate, Miller's dinitrosalicylic acid and Kidby and Davidson's ferricyanide methods, as well as by high-performance size exclusion chromatography coupled with a refractive index detector and an evaporative light scattering detector (ELSD). Calibration curves were established separately for each method to measure the amounts of the neoagaro-oligosaccharides (NAOS) in the reaction mixtures, which are the products from 1-10 units (U) of ß-agarase cleavage activity on agarose. Product quantities from each monitoring method were compared with the isolated NAOS products. The graphs plotted by agarase activity unit and product concentration clearly displayed that the ELSD method closely followed the results of the isolated products. The percentage deviation of results measured by the five methods away from those of the isolated NAOS product mixture amounted to -13.1-35.1, -21.1-25.5, -27.1-23.81, 6.1-24.3 and 16.2-22.8%, respectively. When the loss during product isolation, about 15-17%, was taken into account, the high precision of the ELSD method was confirmed. HPSEC-ELSD methods also accurately measured the enzyme kinetics as well as enabling partial identification of oligosaccharides assembled in the NAOS product mixture. This study established the HPSEC-ELSD system as an alternative method for monitoring agarase activity.

Direct electrochemistry and electrocatalysis of hemoglobin on carbon ionic liquid electrode.

Hemoglobin in agarose was successfully immobilized on a carbon ionic liquid electrode and the direct electrochemical behavior of hemoglobin was investigated. Room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate was used as the modifier. Ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy and cyclic voltammetry were used to characterize the hemoglobin on the modified electrode. The results showed that the immobilized hemoglobin retained its bioelectrocatalytic activity. The electrochemistry of hemoglobin provided an opportunity to manufacture a third generation of biosensors. Experimental conditions influencing the biosensor performances such as pH, and potential were optimized and assessed. Under the optimal conditions, hydrogen peroxide was detected in the concentration range from 2x10(-6) to 1.2x10(-3)M with a detection limit of 0.2 microM at S/N=3. The apparent Michaelis-Menten constant was 1.495 mM. The biosensor exhibited some advantages, such as short time respond, high sensitivity, good reproducibility and long-term stability.

Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose.

We describe a novel technique of phosphate-affinity SDS-PAGE using Phos-tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were clearly separated as up-shifted migration bands in a 3% w/v polyacrylamide gel containing 20 microM Phos-tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high-molecular-mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa).

Biodegradable inflatable balloon for reducing radiation adverse effects in prostate cancer.

Carcinoma of the prostate is one of the most abundant killers for men in the western world, and it is frequently treated via Radiation therapy. Unfortunately, radiotherapy side effects include rectal irritation and bleeding, erectile dysfunction and urinary frequency. Because radiation intensity decays rapidly as a function of distance, displacing irradiated prostate away from normal tissues would reduce damage and therefore side effects. The objective of this study is to develop an inflatable balloon that is implanted via a minimal invasive procedure. The balloon is made of a biodegradable polymer called poly(lactide-co-epsilon-caprolactone). The implant is inserted rolled throughout the perineum; inflated in situ with a physiological saline; sealed and placed between the rectum wall, and the prostate gland. Balloon's mechanical and chemical properties were extensively characterized both in vitro and in vivo. The balloon's preparation ensures no bonding across surfaces as these may endanger the implant mechanical stability. Moreover, the coating method does not alter the polymer's molecular weight and therefore preserve its mechanical properties. Balloon's sterilization was carried out using ethylene oxide which, as our results show and in comparison with gamma-irradiation, doesn't damage the mechanical stability of the implant. The proper functionality of the insertion-mounting device as well as the balloon capability to retain its inflated form during patients' radiation session was demonstrated both in vitro and in vivo.

Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9 / Purificação e caracterização de uma lipase extracelular produzida por uma nova cepa: Pseudomonas aeruginosa SRT9

An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/minrespectively.

Uma lipase extracelular foi isolada e purificada a partir de um caldo de cultura de Pseudomonas aeruginosa SRT9 até homogeneidade visível empregando-se precipitação com sulfato de amônia, seguida de técnicas cromatográficas em colunas de fenil sefarose CL-4B e Mono Q HR 5/5, obtendo-se um fator de purificação de 98 vezes, e atividade especifica de 12307,8 U/mg. Por SDS_PAGE, estimou-se que o peso molecular da lipase purificada é 29kDa, com um ponto isoelétrico de 4,5. A lipase apresentou atividade máxima em uma ampla faixa de temperatura e pH, com ótimos a 55ºC e pH 6,9. A lípase foi mais ativa sobre triacilglicerois de cadeia longa (C14-C16). A lipase foi fortemente inibida por EDTA, o que sugere que a enzima pode ser uma metaloproteína. SDS e íons metálicos, como Hg2+, Zn2+,Cu2+, Ag2+ e Fe2+, diminuíram marcadamente a atividade da lipase. Sua grande estabilidade e atividade em solventes organicos sugerem que esta lípase pode ser uma excelente ferramenta tecnológica com várias aplicações como reações organosintéticas e preparação de produtos farmacêuticos enantiomericamente puros. Os valores de Km e Vmax para a enzima purificada na hidrólise de trioleina foram 1,11 mmol/L e 0,05 mmol/L/min, respectivamente.

An improved protocol for the preparation and restriction enzyme digestion of pulsed-field gel electrophoresis agarose plugs for the analysis of Legionella isolates.

Pulsed-field gel electrophoresis (PFGE), which determines the genomic relatedness of isolates, is currently used for the epidemiological investigation of infectious agents such as bacteria. In particular, this method has been used for the epidemiological investigation of Legionella outbreaks. However, it takes 4 days to complete a Legionella-PFGE analysis. Due to partial digestion and DNA damage, the reproducibility of the obtained fragment digestion patterns is poor for this pathogen. In this study, we report an improved protocol that takes only 2 days to complete and that allows clear discrimination of the restriction profile with higher reproducibility than that previously achieved.

Interactions of trace metals with hydrogels and filter membranes used in DET and DGT techniques.

Equilibrium partitioning of trace metals between bulk solution and hydrogels/filter was studied. Under some conditions, trace metal concentrations were higher in the hydrogels or filter membranes compared to bulk solution (enrichment). In synthetic soft water, enrichment of cationic trace metals in polyacrylamide hydrogels decreased with increasing trace metal concentration. Enrichment was little affected by Ca and Mg in the concentration range typically encountered in natural freshwaters, indicating high affinity but low capacity binding of trace metals to solid structure in polyacrylamide gels. The apparent binding strength decreased in the sequence: Cu > Pb > Ni approximately to Cd approximately to Co and a low concentration of cationic Cu eliminated enrichment of weakly binding trace metal cations. The polyacrylamide gels also had an affinity for fulvic acid and/or its trace metal complexes. Enrichment of cationic Cd in agarose gel and hydrophilic polyethersulfone filter was independent of concentration (10 nM to 5 microM) but decreased with increasing Ca/ Mg concentration and ionic strength, suggesting that it is mainly due to electrostatic interactions. However, Cu and Pb were enriched even after equilibration in seawater, indicating that these metals additionally bind to sites within the agarose gel and filter. Compared to the polyacrylamide gels, agarose gel had a lower affinity for metal-fulvic complexes. Potential biases in measurements made with the diffusive equilibration in thin-films (DET) technique, identified by this work, are discussed.

A novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization.

This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase.

Interaction of porphyran with a hydrophobic surface and stabilization of liposomes.

Three porphyran preparations with high emulsifying ability and varying molecular mass, 3,6-anhydrogalactose content, and sulfate content without any protenaceous component were prepared from dried nori processed from Porphyra yezoensis, a red alga. Each of these preparations was applied to demonstrate adsorption or binding to the surface of oil droplets. The decrease in porphyran concentration of the aqueous phase of O/W emulsions prepared with porphyran and with toluidine blue (TB)-porphyran complex formed by adding TB to the O/W emulsions indicated ready adsorption to the surface of oil droplets. The decrease in zeta-potential of the O/W emulsions suggested that the sulfate groups of the adsorbed porphyran were oriented toward the external aqueous phase. A biomolecular interaction analysis exhibited rapid binding of porphyran to C16-alkane, probably through 3,6-anhydrogalactose. Porphyran-coated liposomes were tolerant to digestion with phospholipase D. The increased molecular weight of the porphyran preparations had an increased effect on these characteristics. The results of this study demonstrate that the emulsifying ability of porphyran is derived from the adequate adsorption to the surface of oil droplets and that porphyran could be effectively applied to stabilize liposomes.

Multiresidue analysis of avermectins in bovine liver by immunoaffinity column cleanup procedure and liquid chromatography with fluorescence detector.

A multiresidue liquid chromatography (LC) method was developed to detect avermectin, ivermectin, doramectin, and eprinomectin simultaneously in bovine liver. The extracted samples were cleaned up by an immunoaffinity column, which was prepared by coupling anti-avermectin polyclonal antibody with CNBr-activated Sepharose 4B. The dynamic column capacities of avermectin, ivermectin, doramectin, and eprinomectin were 3531, 3542, 3543, and 3284 nglmL gel, respectively. The eluate was evaporated to dryness, and residues were derivatized and determined by LC with fluorescence detector set at 365 nm excitation and 465 nm emission wavelengths. Recoveries ranged from 79.3 to 115.9% with coefficients of variation of 1.1-19.4% when avermectin, ivermectin, doramectin, and eprinomectin were spiked at levels of 5-100 ng/g. The limit of quantitation of the method was 2 ng/g for each drug.

Development of mammalian cell-enclosing subsieve-size agarose capsules (<100 microm) for cell therapy.

Agarose capsules were prepared using a droplet breakup method in a coflowing stream. Subsieve-size capsules 76+/-9 microm in diameter were obtained by extruding 4 wt% agarose solution from a needle (300 microm inner diameter) at a velocity of 1.2 cm/s into an ambient liquid paraffin flow of 20.8 cm/s. Increasing the flow rate of the liquid paraffin and decreasing that of the agarose solution resulted in a decreased resultant capsule diameter. Reduction in diameter from several hundred micrometers to subsieve-size (<100 microm) enhanced molecular exchange and mechanical stability. Measurements based on the percentage of intact mitochondria in the cells demonstrated that the viability of the enclosed cells was independent of capsule diameter. No significant difference was observed between the viabilities of cells enclosed in capsules with diameters of 79+/-8 and 351+/-41 microm (p=0.43). Compared with cells seeded in a tissue culture dish, the cells enclosed in the subsieve-size capsules showed 89.2% viability.

A simple, efficient, and economical method for recovering DNA from agarose gel.

A simple method of recovering DNA from agarose gel that is fast, inexpensive, and friendly both to operators and environment is described. Two rows of wells are made in an agarose gel, and a DNA sample is loaded into the well nearest to the negative pole for separation by electrophoresis. Recovery is accomplished by pipetting the DNA-containing TAE buffer from the well near the positive pole after target DNA fragments have migrated into the well. A recovery rate of up to 94 +/- 2.3% was observed with this method.

Residue analysis for halofuginone in sturgeon muscle by immunoaffinity cleanup and liquid chromatography.

A high-performance liquid chromatography (LC) method was developed for the determination of halofuginone (HFG) in sturgeon muscle. The extracted samples were cleaned up by an immunoaffinity chromatography column that was prepared by covalently coupling polyclonal antibodies against HFG to cyanogen bromide (CNBr) activated Sepharose 4B. The eluate was evaporated to dryness, and residues were determined by LC with absorbance detection at 243 nm. Recoveries of HFG from samples fortified at 20-200 microg/kg levels ranged 74.6-81.1%, with coefficients of variation of 0.7-8.6%. The detection limit was estimated to be 10 microg/kg in a 2 g sample.

Dynamic mechanical properties of agarose gels modeled by a fractional derivative model.

The complex modulus (E*) and elastic modulus (E') of agarose gels (2% to 4%) are measured with a dynamic mechanical analyzer in frequency sweep shear sandwich mode between 0.1 and 20 Hz. The data showed that E* and E' increase with frequency according to a power law which can be described by a fractional derivative model to characterize the dynamic viscoelasticity of the gel. The functions between the model parameters including storage modulus coefficient (H) and the power law exponent (beta) and the agarose concentration are established. A molecular basis for the application of the fractional derivative model to gel polymers is also discussed. Such an approach can be useful in tissue culture studies employing dynamic pressurization or for validation of magnetic resonance elastography.

Fingerprinting polysaccharides with single-molecule atomic force microscopy.

We report the use of an atomic force microscopy (AFM)-based force spectroscopy technique to identify, at the single-molecule level, the components of mixtures of polysaccharides. Previously, we showed that the elasticity of certain types of polysaccharides is governed by force-induced conformational transitions of the pyranose ring. These transitions produce atomic fingerprints in the force-extension spectrum that are characteristic of the ground-energy conformation of the pyranose ring and the type of glycosidic linkages. Using this approach we find that commercially available agarose and lambda-carrageenan contain molecules that, when stretched in an atomic force microscope, produce a force spectrum characteristic of alpha-(1-->4) d-glucans. We have identified these molecules as amylopectin or floridean starch, a storage polysaccharide in algae. Our methodology can identify individual polysaccharide molecules in solution, which is not possible by any other spectroscopic technique, and therefore is an important addition to the arsenal of analytical techniques used in carbohydrate research.