Agarose-based Gel Research Articles

Dextrin-assisted gel electromembrane extraction of chiral drugs: Improving the extraction efficiency and investigation of enantioselectivity of extraction

The present study investigates the use of dextrins (maltodextrin, β-cyclodextrin, and hydroxypropyl-β-cyclodextrin) to improve the efficiency of the agarose-based gel electromembrane extraction technique for extracting chiral basic drugs (citalopram, hydroxyzine, and cetirizine). Additionally, it examines the enantioselectivity of the extraction process for these drugs. To achieve these, dextrins were incorporated into either the sample solution, the membrane, or the acceptor solution, and then the extraction procedure was performed. Enantiomers were separated and analyzed using a capillary electrophoresis device equipped with a UV detector. The results obtained under the optimal extraction conditions (sample solution pH: 4.0, acceptor solution pH: 2.0, gel membrane pH: 3.0, agarose concentration: 3 % w/v, stirring rate: 1000 rpm, gel thickness: 4.4 mm, extraction voltage: 62.3 V, and extraction time: 32.1 min) indicated that incorporating dextrins into either the sample solution, membrane or the acceptor solution enhances extraction efficiency by 17.3–23.1 %. The most significant increase was observed when hydroxypropyl-β-cyclodextrin was added to the acceptor solution. The findings indicated that the inclusion of hydroxypropyl-β-cyclodextrin in the sample solution resulted in an enantioselective extraction, yielding an enantiomeric excess of 6.42–7.14 %. The proposed method showed a linear range of 5.0–2000 ng/mL for enantiomers of model drugs. The limit of detection and limit of quantification for all enantiomers were found to be < 4.5 ng/mL and <15.0 ng/mL, respectively. Intra- and inter-day RSDs (n = 4) were less than 10.8 %, and the relative errors were less than 3.2 % for all the enantiomers. Finally, the developed method was successfully applied to determine concentrations of enantiomers in a urine sample with relative recoveries of 96.8–99.2 %, indicating good reliability of the developed method.

Quality Control Method and Device for Producing Agarose Micropads.

In brightfield and fluorescence microscopy, capturing images that show well-focused and immobile microorganisms can be challenging. An agarose-based gel pad reduces the variability of results, especially in conditions like uneven specimen staging, variable fluid dynamics, and Brownian motion that plague conventional wet mount setups. To correct these discrepancies during image acquisition, we analyzed three micropad preparation setups. We tested the quality and consistency of pads and images resulting from each setup. Our examination reveals that improved gel pad flatness is associated with better image quality. Moreover, we observe increased consistency in gel pad construction connected to the use of a 3D-printed setup. These findings highlight the technical benefits arising from incorporating micropad-generating platforms that increase the consistency of results in imaging pipelines. Additionally, our use of a quantitative approach to examine pad flatness suggests its inclusion in quality control pipelines to reduce variation in gel pad construction and image quality over time and between investigators. Finally, our use of a 3D-printed setup coupled with a quantitative downstream routine suggests their application in microscopy experiments that involve model organisms relevant to human health and disease.

Agarose-based Gel Electrolytes for Sustainable Primary and Secondary Zinc-Air Batteries

Present Zn-air batteries (ZABs) are based on concentrated alkaline liquid electrolytes, with high ionic conductivity, but suffer from leakage, evaporation, and carbonate precipitation due to the semi-open characteristic of these systems. To overcome these issues, gel polymer electrolytes (GPEs), based on naturally occurring biopolymers, arise as a green option to overcome the above-mentioned limitations. In this work, a novel GPE based on pure agarose from seaweed is presented as a smart alternative to liquid (adsorbed on a separator) and gel electrolytes (based on synthetic polymers). The innovative synthesis method described can directly encapsulate concentrated KOH liquid electrolytes into an agarose matrix in one-pot; the process requiring approx. 10 min. The unique gel developed in this work, with 2 wt% agarose and 8 M KOH electrolyte, presents the best compromise between physicochemical and electrochemical properties, at lab scale. The characterization results revealed an outstanding ionic conductivity of 0.45 ± 0.05 S cm−1, ≈100% water retention up to 200–250 h, retarded Zn self-corrosion up to 30 days (symmetric cell under open circuit), average Zn utilization > 70–80% in primary ZABs in the range 1–20 mA cm−2 with peaks of ≈96%. In secondary ZABs the gel electrolyte presents high round-trip efficiency and improved cyclability at high areal capacities, under soft and severe cycling conditions, never tested before. This agarose gel represents a potential benchmark for future development of GPE-based ZABs for stationary applications.

Different Drying Techniques Can Affect the Adsorption Properties of Agarose-Based Gels for Crystal Violet Removal

Agarose-based gels were produced either by freeze-drying or by supercritical drying for crystal violet (CV) removal from aqueous solutions. The microporosity features of these structures highly affected the final adsorption properties. In particular, agarose cryogels were characterized by a macroporous and irregular morphology, with a low value of specific surface area (11 ± 6 m2/g) with respect to the nanoporous agarose aerogels (154 ± 12 m2/g). To test the efficacy of CV removal, two different types of adsorption test were performed, i.e., batch-mode and multi-step mode. Operating in the multi-step mode, the adsorption performance was larger both for cryogels and aerogels, since this adsorption method allowed a more effective contact between CV and agarose adsorbent. In particular, using 300 mg of cryogels, a removal efficiency of 74% was achieved; using the same quantity of aerogels, 96% of removal efficiency was reached after eight steps of adsorption. Desorption of CV from aerogels was realized using ascorbic acid and, after regeneration, 93% of removal efficiency was preserved, even after three cycles in multi-step filtration mode.

Novel PCR-Based Multiplex Assays for Detecting Major Quality and Biotic Stress in Commercial and Weedy Rice.

Simple SummaryRice, the staple food for more than half of humanity, is grown predominantly in Asia, the world’s most populous continent with the fastest-growing economy. The present-day rice industry must not only meet increasing demand but also changing consumer demands, with a strong emphasis placed on producing high-quality rice. While the rapid development of advanced genotyping methods can be useful for modern rice breeding programs, some methods (such as capillary electrophoresis or sequencing) can be costly to apply in laboratories with limited resources. To address this issue, we developed six novel multiplex polymerase chain reaction (PCR) assays that employ a standard agarose-based gel electrophoresis system to simultaneously detect at least two major grain quality (amylose content and fragrance) and biotic stress (blast, sheath blight, and bacterial leaf blight) genes in rice. One of these assays, which can detect all three targeted biotic stresses, was found to be useful in screening Malaysian weedy rice that may contain novel sources of disease resistance. The universal protocol described in this study can be used in routine molecular laboratories to aid rice breeding initiatives in Malaysia and other resource-constrained countries.While previous research has demonstrated that multiplex polymerase chain reaction (PCR) can be a cost-effective approach to detect various genes in crops, the availability of multiplex assays to simultaneously screen both grain quality and biotic stress resistance traits in rice (Oryza sativa) is limited. In this work, we report six novel multiplex assays that use a universal protocol to detect major rice grain quality (amylose content and fragrance) and biotic stress (blast, sheath blight, and bacterial leaf blight) traits with amplified products consisting of up to four primer pairs that can be analyzed using a standard agarose-based gel electrophoresis system. Recent studies have suggested that weedy rice has novel sources of disease resistance. However, an intensive screening of weedy biotypes has not been reported in Malaysia. Accordingly, we employed one of the developed multiplex assays to screen reported genes or quantitative trait loci (QTLs) associated with blast, sheath blight, and bacterial leaf blight diseases in 100 weedy rice biotypes collected from five local fields, with phenotyping performed to validate the genotyping results. In conclusion, our universal multiplex protocol is effective for the large-scale genotyping of rice genetic resources, and it can be employed in routine molecular laboratories with limited resources.

Performance of PEDOTOH/PEO-based Supercapacitors in Agarose Gel Electrolyte.

Poly(3,4-ethylenedioxythiophene) (PEDOT) is a prime example of conducting polymer materials for supercapacitor electrodes that offer ease of processability and sophisticated chemical stability during operation and storage in aqueous environments. Yet, continuous improvement of its electrochemical capacitance and stability upon long cycles remains a major interest in the field, such as developing PEDOT-based composites. This work evaluates the electrochemical performances of hydroxymethyl PEDOT (PEDOTOH) coupled with hydrogel additives, namely poly(ethylene oxide) (PEO), poly(acrylic acid) (PAA), and polyethyleneimine (PEI), fabricated via a single-step electrochemical polymerization method in an aqueous solution. The PEDOTOH/PEO composite exhibits the highest capacitance (195.2 F g-1 ) compared to pristine PEDOTOH (153.9 F g-1 ), PEDOTOH/PAA (129.9 F g-1 ), and PEDOTOH/PEI (142.3 F g-1 ) at a scan rate of 10 mV s-1 . The PEDOTOH/PEO electrodes were then assembled into a symmetrical supercapacitor in an agarose gel. The type of supporting electrolytes and salt concentrations were further examined to identify the optimal agarose-based gel electrolyte. The supercapacitors comprising 2 M agarose-LiClO4 achieved a specific capacitance of 27.6 F g-1 at a current density of 2 A g-1 , a capacitance retention of ∼94% after 10,000 charge/discharge cycles at 10.6 A g-1 , delivering a maximum energy and power densities of 11.2 Wh kg-1 and 17.28 kW kg-1 , respectively. The performance of the proposed supercapacitor outperformed several reported PEDOT-based supercapacitors, including PEDOT/carbon fiber, PEDOT/CNT, and PEDOT/graphene composites. This study provides insights into the effect of incorporated hydrogel in the PEDOTOH network and the optimal conditions of agarose-based gel electrolytes for high-performance PEDOT-based supercapacitor devices.

Western blotting of native proteins from agarose gels.

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(N-morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper isfocusedon theWestern blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane areintroduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in thetransfer of native GFP, native human plexin domain containing protein 2 (PLXDC2)and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.

Gel Chromatography for Separation of Single-Walled Carbon Nanotubes.

Carbon nanotubes (CNTs), having either metallic or semiconducting properties depending on their chirality, are advanced materials that can be used for different devices and materials (e.g., fuel cells, transistors, solar cells, reinforced materials, and medical materials) due to their excellent electrical conductivity, mechanical strength, and thermal conductivity. Single-walled CNTs (SWNTs) have received special attention due to their outstanding electrical and optical properties; however, the inability to selectively synthesize specific types of CNTs has been a major obstacle for their commercialization. Therefore, researchers have studied different methods for the separation of SWNTs based on their electrical and optical properties. Gel chromatography methods enable the large-scale separation of metallic/semiconducting (m/s) SWNTs and single-chirality SWNTs with specific bandgaps. The core principle of gel chromatography-based SWNT separation is the interaction between the SWNTs and gels, which depends on the unique electrical properties of the former. Controlled pore glass, silica gel, agarose-based gel, and allyl dextran-based gel have been exploited as mediums for gel chromatography. In this paper, the interaction between SWNTs and gels and the different gel chromatography-based SWNT separation technologies are introduced. This paper can serve as a reference for researchers who plan to separate SWNTs with gel chromatography.

Impact of the agarose ferrogel fine structure on magnetic heating efficiency

Magnetic nanoparticles-mediated hyperthermia was widely studied in the last decades as applicable in cancer therapy. The majority of magnetic hyperthermia research was devoted to improvement of heating efficiency by application of various nanomaterials. The influence of biocompatibility, magnetic properties, sizes, composition, and concentration of magnetic nanoparticles (MNPs) on hyperthermia efficiency was extensively studied. Also, the limits preserving biological safety and the chemical stability of MNPs delivered to the tissue were established. However, much less research concerned the impact of the physical interactions between the closest MNPs on the hyperthermia efficiency. Our goal was to demonstrate the relationships between the internal structure of soft tissue containing MNPs, and the thermal effects of an alternating magnetic field. Because agarose-based gel exhibits a tissue-like internal structure, we performed hyperthermia experiments in two types of such gel containing bare and polyethylene glycol-coated Fe3O4 nanoparticles. We described the structural difference and we estimated the ferrogels specific absorption rate coefficients (SAR) from calorimetric experiments. Magnetic measurements showed 11% lower magnetic saturation of PEG-coated MNPs than of the bare MNPs. While the SAR of ferrogel with PEG-coated MNPs was 15% greater than bare MNPs. The structural characteristics calculated from TEM and SEM images were significantly different. Particularly, we observed the nanoparticle agglomeration in the gel with bare MNPs. The bare MNPs uniformly packed and located inside agarose double helices were observed for the first time. The distribution of MNPs and their spatial configuration in gel influenced strongly the strength of bonds blocking the movement of MNPs and determined the ferrogels heating efficiency. As the tissues to which we delivered MNPs were composed of agarose gel-like structures, our results may be useful in further research on hyperthermia in vivo.

Simultaneous separation and quantification of acidic and basic dye specimens via a dual gel electro-membrane extraction from real environmental samples

In this work, an environmentally friendly and easy-in-performance dual gel electro-membrane extraction approach (dual G-EME) was combined with high performance liquid chromatography. Method was arranged for parallel isolation, enrichment and determination of brilliant green (BG) and reactive red (RR), serving as model basic and acidic dyes, respectively. A DIY (do-it-yourself) agarose-based gel membrane was fabricated, and served as green and highly selective filtering item. The extractions were hazards-free (i.e., there was no need for organic solvents or toxic regents) and membrane assembly is highly degradable as being of fully natural character. Thereby, setup has met the requirements of being a green analytical chemistry technique. By using an electric potential of 50 V during 20 min extraction time, the positively charged BG and negatively charged RR migrated selectively from a 7.0 mL aqueous sample (pH 7.0) to the 500 μL cathodic (pH 4.0) and 500 μL anodic (pH 9.0) aqueous acceptor phases, respectively. Under the optimal conditions, limits of detection were 0.020 and 0.025 ng mL−1, and extraction recoveries were of 75–78% and 67–71% for BG and RR, respectively. The linearity was in the range of 0.06–1.0 μg mL−1 with correlation coefficient higher than 0.996, obtained for both model analytes. Finally, the methodology was successfully applied for parallel quantification of BG and RR in complex environmental samples.

Cell-Laden Agarose-Collagen Composite Hydrogels for Mechanotransduction Studies.

The increasing investigation of cellular mechanotransduction mechanisms requires biomaterials combining biofunctionality and suitable mechanical properties. Agarose is a standard biomaterial for cartilage and intervertebral disc mechanobiology studies, but lacks adhesion motifs and the necessary cell-matrix interaction for mechanotransduction. Here, collagen type I was blended at two concentrations (2 and 4.5 mg/mL) with agarose 2% wt/vol. The composite hydrogels were characterized in terms of structural homogeneity, rheological properties and size stability. Nucleus pulposus (NP) cell viability, proliferation, morphology, gene expression, GAG production, adhesion and mechanotransduction ability were further tested. Blended hydrogels presented a homogenous network of the two polymers. While the addition of 4.5 mg/mL collagen significantly decreased the storage modulus and increased the loss modulus of the gels, blended gels containing 2 mg/mL collagen displayed similar mechanical properties to agarose. Hydrogel size was conserved over 21 days for all agarose-based gels. Embedded cells were viable (>80%) and presented reduced proliferation and a round morphology typical of NP cells in vivo. Gene expression of collagen types I and II and aggrecan significantly increased in blended hydrogels from day 1 to 7, further resulting in a significantly superior GAG/DNA ratio compared to agarose gels at day 7. Agarose-collagen hydrogels not only promoted cell adhesion, contrary to agarose gels, but also showed a 5.36-fold higher focal adhesion kinase phosphorylation (pFAK/β-tubulin) when not compressed, and increased pFAK/FAK values 10 min after compression. Agarose-collagen thus outperforms agarose, mimics native tissues constituted of non-fibrillar matrix and collagens, and allows exploring complex loading in a highly reproducible system.

Gel electromembrane extraction: Study of various gel types and compositions toward diminishing the electroendosmosis flow

For the first time, the effect of electroendosmosis (EEO) flow phenomenon was investigated in detail, on the gel electromembrane extraction (G-EME). When electric field is used as an actuator of ion transport in G-EME, the EEO flow phenomenon can occur within the agarose gel membrane interface and is generally considered the dominant issue. In this regard, various agarose-based gel membrane types with low-, medium- and high-EEO were fabricated and tested. To further diminish the EEO effect, the different gel membrane additives such as dextrin, chitosan and xylan with different concentrations i.e., 5%, 10% and 15% were also examined. The positively charged Cr(III) and negatively charged Cr(VI) were chosen as model analytes. The Cr(III) and Cr(VI) were simultaneously extracted from an aqueous sample (pH 3.0) via the cathodic and anodic gel membranes (pH 3.0 for both membranes), into the cathodic and anodic aqueous acceptors (200 µL each, pH 2.0), respectively. After extraction at the optimal conditions (i.e., voltage: 32 V, extraction time: 22 min), the both compounds were quantified by a cheap and easy-to-perform reader platform termed as microfluidic paper-based analytical device (μPAD). The results showed that high extraction recoveries such as 87% for Cr(III) and 75% for Cr(VI), were acquired when the low-EEO agarose gel membrane with 5% (w/v) dextrin was used. On the other hand, using the high-EEO agarose gel membrane with no additive lead to the high EEO flow and the increase of cathodic acceptor volume (equal to 350 µL) while the recovery depressed to about 50% due to the dilution effect. Simultaneously, the volume of anodic acceptor became almost vacant (~50 µL). Ultimately, by means of such simple gel membrane adjusting (i.e., low-EEO agarose with 5% dextrin), the both target compounds were quantitatively extracted at a single step achieving limits of detection (LODs) of 0.5 and 0.7 ng mL−1 for Cr(III) and Cr(VI), respectively.

Application of diffusive gradients in thin-films for in-situ monitoring of nitrochlorobenzene compounds in aquatic environments

Nitrochlorobenzene compounds (NCBs) are of key interest in environmental monitoring due to their high toxicity. To better understand the presence and fate of NCBs in aquatic environments, an in-situ sampling technique of diffusive gradients in thin films (DGT) based on hydrophilic-lipophilic-balanced (HLB) resin, combined with gas chromatography, was developed to measure four typical NCBs, e.g. meta-chloronitrobenzene (MNCB), para-chloronitrobenzene (PNCB), ortho-chloronitrobenzene (ONCB), and 2,4-dinitrochlorobenzene (CDNB). The diffusion coefficients of MNCB, PNCB, ONCB, and CDNB in agarose-based gel were firstly determined in diffusion cell experiments and ranged from 7.19 × 10−6 to 7.49 × 10−6 cm/s. The capacities of HLB-DGT for MNCB, PNCB, ONCB, and CDNB were higher than 114.65, 117.52, 117.72, and 37.58 μg/cm2, respectively. The HLB-DGT performance on NCBs determination was demonstrated to be independent of natural fluctuations in pH (3–9), ionic strength (0.001–0.5 M), and dissolved organic matter concentrations (0–20 mg/L) and of deployment time (0–120 h). In the field application, the DGT-based method to measure NCBs not only proved to be accurate and effective, but also performed better than the grab sampling method under the variable conditions. This study demonstrates that the newly developed in-situ method based on DGT can provide an attractive alternative for the routine monitoring of NCBs in aquatic environments.

Time lapse investigation of antibiotic susceptibility using a microfluidic linear gradient 3D culture device.

This study reports a novel approach to quantitatively investigate the antibacterial effect of antibiotics on bacteria using a three-dimensional microfluidic culture device. In particular, our approach is suitable for studying the pharmacodynamics effects of antibiotics on bacterial cells temporally and with a continuous range of concentrations in a single experiment. The responses of bacterial cells to a linear concentration gradient of antibiotics were observed using time-lapse photography, by encapsulating bacterial cells in an agarose-based gel located in a commercially available microfluidics chamber. This approach generates dynamic information with high resolution, in a single operation, e.g., growth curves and antibiotic pharmacodynamics, in a well-controlled environment. No pre-labelling of the cells is needed and therefore any bacterial sample can be tested in this setup. It also provides static information comparable to that of standard techniques for measuring minimum inhibitory concentration (MIC). Five antibiotics with different mechanisms were analysed against wild-type Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. The entire process, including data analysis, took 2.5-4 h and from the same analysis, high-resolution growth curves were obtained. As a proof of principle, a pharmacodynamic model of streptomycin against Salmonella Typhimurium was built based on the maximal effect model, which agreed well with the experimental results. Our approach has the potential to be a simple and flexible solution to study responding behaviours of microbial cells under different selection pressures both temporally and in a range of concentrations.

Optimal Enhancement Configuration of Silica Nanoparticles for Ultrasound Imaging and Automatic Detection at Conventional Diagnostic Frequencies

To experimentally investigate the acoustical behavior of silica nanoparticles within conventional diagnostic ultrasound fields and to determine a suitable configuration, in terms of particle size and concentration, for their employment as targetable contrast agents. We also assessed the effectiveness of a novel method for automatic detection of targeted silica nanoparticles for future tissue typing applications. Silica nanospheres of variable size (160, 330, and 660 nm in diameter) and concentration (10¹⁰-10¹³ part/mL) were dispersed in different custom-designed agarose-based gel samples and imaged at 7.5 MHz with a conventional echograph linked to a research platform for radiofrequency signal acquisition. Off-line analysis included evaluation of backscattered ultrasound amplitude, image brightness, and nanoparticle automatic detection through radiofrequency signal processing. Amplitude of nanoparticle-backscattered signals linearly increased with particle number concentration, but image brightness did not show the same trend, because the logarithmic compression caused the reaching of a "plateau" where brightness remained almost constant for further increments in particle concentration. On the other hand, both backscatter amplitude and image brightness showed significant increments when particle diameter was increased. Taking into account particle size constraints for tumor targeting (pore size of tumor endothelium and trapping effects because of reticulo-endothelial system limit the dimension of effectively employable particles to less than 380 nm), a suitable compromise is represented by the employment of 330-nm silica nanospheres at a concentration of about 1 to 2 x 10¹¹ part/mL. These particles, in fact, showed the best combination of number concentration and diameter value to obtain an effective enhancement on conventional echographic images. Furthermore, also the sensitivity of the developed method for automatic nanoparticle detection had a maximum (72.8%) with 330-nm particles, whereas it was lower with both bigger and smaller particles (being equal to 64.1% and 17.5%, respectively). Silica nanoparticles at a diameter of about 330 nm are very promising contrast agents for ultrasound imaging and specific tumor targeting at conventional diagnostic frequencies, being in particular automatically detectable with high sensitivity already at low doses. Future studies will be carried out to assess the acoustic behavior of nanoparticles with different geometries/sizes and to improve sensitivity of the automatic detection algorithm.

Polyelectrolyte Diode: Nonlinear Current Response of a Junction between Aqueous Ionic Gels

We demonstrate that a fixed junction between two aqueous gels containing oppositely charged polyelectrolytes could rectify electric current. The agarose-based gels were "doped" with sodium poly(styrene sulfonic acid) and poly(diallyl dimethylammonium chloride). The unidirectional current response of the interface between the cationic and anionic gels originates directly from anisotropy in the mobile ionic charges in the gels. The current depends on the concentration of polyelectrolyte, the background ionic concentration, and the distance traveled by the ions. The I-V curves from the devices demonstrated a combination of transient and stationary rectification effects. The current densities achieved were comparable to or higher than those obtained with previously reported organic semiconductor diodes. The diodes had good long-term stability in both DC and AC conduction modes. The materials and the process of preparation of these devices are simple, inexpensive, and scalable. They could be used in flexible and biocompatible electronic circuits.

It's Easy Being Green

Porous solids are widely used in chromatographic separations based on size and shape. Some pores are as large as proteins (for instance, in dextran- or agarose-based gel filtration), whereas others are as small as water molecules (as in molecular sieves used to keep organic solvents dry). Paukstelis has assessed the permeation properties of a self-assembled three-dimensional lattice constructed from four assembly strands [each 24 nucleotides (nt) long] and an 11-nt spacer strand. The estimated diameter of the largest internal solvent channel in crystals of this DNA array was 9 nm, which corresponds to a globular protein of 300 kD, but the measured size cutoff for negatively charged proteins appeared to be only one-10th this size (ovalbumin, no; carbonic anhydrase, yes), perhaps as a result of electrostatic interactions. Confocal microscopy revealed that the interior of a crystal soaked in a mixture of green fluorescent protein and a much bigger red maltose-binding protein was green and not red. — GJC

In vivo quantitative three-dimensional localization of tumor labeled with exogenous specific fluorescence markers.

We introduce a diffused optical detection system based on the administration of a fluorophore-antibody conjugate to diseased tissue. The conjugate interacts with the antigens expressed by the diseased tissue, resulting in fluorescent labeling of the antigen. By combining an optical detection system with a reconstruction algorithm developed on the basis of the random-walk model, we were able to determine the position of the fluorophore (and, thus, of the diseased cells) in the tissue. We present three-dimensional reconstructions of the location of a fluorophore (FITC-fluorescein isothiocyanate) in the tongues of mice. Measurements were performed with the fluorophore embedded at various simulated depths. The simulations were performed with agarose-based gel slabs applied to the tongue as tissuelike phantoms. Reconstructed fluorophore locations agree well with the actual values.

Shed Membrane Fragment-Associated Markers for Endometrial and Ovarian Cancers

Objective. The objective of this study was to assess circulating tumor-derived membrane fragments (MFs) and specific proteins associated with them as diagnostic and prognostic markers for ovarian and endometrial cancers.Methods. The level of shed tumor-derived plasma MFs was analyzed chromatographically on high exclusion limit agarose-based gels, using sera from non-cancer-bearing female controls (n = 50), women with ovarian disease (benign, n = 43, and stages III and IV ovarian cancer, n = 62), and women with early (n = 10) and late (n = 12) stage endometrial cancers. The presence of specific proteins on MFs associated with development and progression of cancer was examined by Western immunoblot, while the association of proteolytic enzymes with MFs was analyzed by zymography.Results. Shed MFs were demonstrated in the circulation of women with both ovarian (4.12 ± 1.46 mg/ml) and endometrial cancers (2.53 ± 0.34 mg/ml in women with stage I and 4.51 ± 1.33 mg/ml with late stage); however, they were not demonstrated in control sera or in sera from women with benign disease. In endometrial cancer, the level of MFs correlated with the tumor's progression. Specific proteins, including MMP-2, MMP-9, and Fas ligand (FasL), were present on MFs from both endometrial and ovarian cancer sera. However, FasL (3.2-fold) and MMP-2 and -9 (5.9× and 7.5×, respectively) levels were significantly elevated on MFs from late stage cancer.Conclusion. This study demonstrates the unique elevation of circulating MFs in ovarian and endometrial cancer patients. The levels of specific MF-associated proteins differentiate between early and late stage endometrial cancers and benign versus malignant ovarian disease.

Purification and characteristics of pectin lyase from Rhizoctonia solani

Pectin lyase was the predominant pectolytic enzyme produced by an anastomosis group 2-2 strain of Rhizoctonia solani both in culture and in infected sugar beet crowns. Sugar beet root cell walls were used as the carbon source in broth still culture. Cell walls from crowns induced more pectin lyase than cell walls from hypocotyl or root. Also, the yield of pectin lyase was higher from infected crown than from infected hypocotyl or root tissue. Rotted crown tissue had a pH of over 7, which was favourable for pectin lyase activity. Rotted root tissue, with a pH below 6, was not as favourable for pectin lyase activity. Healthy tissue had a pH of 6·5 ± 0·3. Pectin lyase was purified by affinity batch chromatography on cross-linked sodium polypectate followed by gel permeation on an agarose-based gel. Pectin lyase interacted with the agarose-based gel and eluted at or near the bed volume which resulted in a final effective purification. The molecular weight of pectin lyase from culture was determined as 35 kDa. The pI was 10.1. The purified enzyme caused wilt when injected into Rhizoctonia-susceptible sugar beet plants, but not when injected into resistant plants.