HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 microm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane-isopropanol with ethylene glycol, water or acetonitrile) are applied.     

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 μm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane–isopropanol with ethylene glycol, water or acetonitrile) are applied.     

Nitroarenes in Suimon River sediment

Mutagenic activity was observed in sediments of the Suimon River bed with and without S9 mix. The direct-acting mutagens in the sediment were investigated. The sediment was extracted with methanol and fractionated on a Silica gel column. The benzene fraction from the Silica gel column exhibited mutagenic activity without S9 mix in strain TA98, while it failed to show mutagenic activity in nitroreductase-deficient strain TA98NR. This observation led to the suspicion that nitro compounds were the direct-acting mutagens of these samples. The benzene fraction was treated by heptafluorobutyric anhydride (HFBA) and investigated with gas chromatography equipped with an electron capture detector (GC-ECD). 2-Nitrofluorene, 4,4'-dinitrobiphenyl, 2,7-dinitrofluorene and 1-nitropyrene were detected and measured quantitatively. The mutagenic activity of a mixture of these compounds was compared with that of the original fraction and the direct-acting mutagenicity of Suimon River sediment can be explained by these nitroarenes, especially 1-nitropyrene.     

IMPROVEMENTS IN THE ION-EXCHANGE METHOD OF PREPARING SILICA SOLS

SUMMARY: An improved ion-exchange column for producing stable silica sols is described. Best results were obtained with a sulphonic-polystyrene resin. The storage life of sols so produced may be extended to as much as two months by refrigeration and acidification with hydrochloric acid. Silica sol has a buffering action in the region of pH 2. Silica gel media prepared from such sols are comparable in cost with agar media.     

Reusability of avidin-biotinylated immunoglobulin Y columns in immunoaffinity chromatography

Reusability of avidin-biotinylated IgY columns for immunoaffinity chromatography was examined by repeated use and regeneration. An enzyme-linked immunosorbent assay-elution assay using CovaLink NH microtiter plates was used to find the optimal conditions for regeneration of columns. Actigel avidin-biotinylated IgY column retained about 90% of its initial IgG binding capacity after 50 cycles, with 0.1 M glycine-HCl buffer, pH 2.8, as eluent, requiring no regeneration. However, IgG binding capacity of UltraLink avidin-biotinylated IgY column gradually decreased to 75 and 65% after 10 and 20 cycles, respectively, with the commercial eluent, Actisep. Results from the CovaLink NH system agreed with those from UltraLink avidin-biotinylated IgY columns. The UltraLink avidin-biotinylated IgY column was regenerated twice, by applying 8 M guanidine-HCl, pH 1.6, to dissociate biotinylated IgY antibodies from the column. About 40 and 25% of IgG binding capacities remained after the first and second regeneration. By applying new biotinylated IgY to the treated columns, about 95 and 90% of the initial IgG binding capacity before any treatment were recovered. These results demonstrated that avidin-biotinylated IgY columns are reusable with or without regeneration depending on the avidin-immobilized matrix.     

A simple procedure for regeneration of an organomercurial agarose column

Organomercurial agarose has been used in the purification of various thiol compounds including enzymes (1). Thiol compounds are first adsorbed on a column of organomercurial agarose, and then eluted with a second thiol compound, e.g., 2-mercaptoethanol (2-ME)¹ and cysteine. Although this column can be used repeatedly, a usual method for regeneration of the column is to remove the second thiol by HgCl₂. It would be desirable to regenerate the column without using HgCl₂, since it is biohazardous. In the study of the purification of a thiol-containing enzyme, we found that organomercurial agarose, which had previously been treated with 2-ME, could adsorb the enzyme and that the enzyme was eluted with 2-ME. This finding led us to examine whether the column can be used repeatedly without the regeneration using HgCl₂.     

Canine mesenchymal stem cells are effectively labeled with silica nanoparticles and unambiguously visualized in highly autofluorescent tissues

ABSTRACT: BACKGROUND: Developing a long-term labeling method is critical and much needed to understand the fate, migration, and contribution in tissue regeneration. Silica nanoparticles have been developed recently and have been demonstrated to be biocompatible and to have high labeling capacity. Thus, this study was designed to assess the suitability of silica nanoparticles for canine MSCs and fluorescence efficiency in a highly autofluorescent tissue. RESULTS: Development of a method for long-term labeling of cells is critical to elucidate transplanted cell fate and migration as well as the contribution to tissue regeneration. Silica nanoparticles have been recently developed and demonstrated to be biocompatible with a high labeling capacity. Thus, our study was designed to assess the suitability of silica nanoparticles for labeling canine mesenchymal stem cells (MSCs) and the fluorescence efficiency in highly autofluorescent tissue.We examined the effect of silica nanoparticle labeling on stem cell morphology, viability and differentiation as compared with those of unlabeled control cells. After 4 h of incubation with silica nanoparticles, they were internalized by canine MSCs without a change in the morphology of cells compared with that of control cells. The viability and proliferation of MSCs labeled with silica nanoparticles were evaluated by a WST-1 assay and trypan blue exclusion. No effects on cell viability were observed, and the proliferation of canine MSCs was not inhibited during culture with silica nanoparticles. Furthermore, adipogenic and osteogenic differentiation of silica nanoparticle-labeled canine MSCs was at a similar level compared with that of unlabeled cells, indicating that silica nanoparticle labeling did not alter the differentiation capacity of canine MSCs. Silica nanoparticle-labeled canine MSCs were injected into the kidneys of BALB/c mice after celiotomy, and then the mice were sacrificed after 2 or 3 weeks. The localization of injected MSCs was closely examined in highly autofluorescent renal tissues. Histologically, canine MSCs were uniformly and completely labeled with silica nanoparticles, and were unambiguously imaged in histological sections. CONCLUSIONS: The results of the current study showed that silica nanoparticles are useful as an effective labeling marker for MSCs, which can elucidate the distribution and fate of transplanted MSCs.     

A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.     

Regeneration of commercial nucleic acid extraction columns without the risk of carryover contamination

Nucleic acid extraction is a basic requirement in a molecular biology laboratory. In terms of purity and yield, commercial nucleic acid extraction columns are superior; however they are expensive. We report here an efficient strategy to regenerate diverse commercial columns for several rounds without altering the binding capacity of the columns or changing the properties of the nucleic acids purified. Plasmids purified with regenerated columns were functionally identical in super-coiled nature, restriction analysis, expression of the encoded reporter genes, or amplification of the viral RNA in real-time PCR. To ensure that the regenerated columns were free of the residual DNA, we used two different plasmids with different drug-resistance markers. By colony plating and PCR amplification of the encoded genes, we show that the regeneration process is absolute. Using radiolabeled DNA, we demonstrate that DNA exposed to the regeneration reagent is fragmented to molecular weight below 36 bp. Our data collectively prove regeneration of the commercial columns without the concern of carryover contamination. A procedure to permit safe and efficient regeneration of the commercial columns is not only of great advantage to extend the lifetime of these columns but also makes them commercially more affordable, especially in a resource-poor setting.     

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50°C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.     

Evaluation of restricted access media for the purification of cell culture-derived Orf viruses

Recently, multimodal chromatography using restricted access media (RAM) for the purification of nanoparticles, such as viruses has regained increasing attention. These chromatography resins combine size exclusion on the particle shell and adsorptive interaction within the core. Accordingly, smaller process-related impurities, for example, DNA and proteins, can be retained, while larger product viruses can pass unhindered. We evaluated a range of currently available RAM, differing in the shells’ pore cut-off and the core chemistry, for the purification of a cell culture-derived clarified model virus, namely the Orf virus (ORFV). We examined impurity depletion and product recovery as relevant criteria for the evaluation of column performance, as well as scale-up robustness and regeneration potential for evaluating a multiple use application. The results indicate that some columns, for example, the Capto Core, enable both a high DNA and protein removal, while others, for example, the Monomix Core 60 (MC60), are more suitable for DNA depletion. Furthermore, column regeneration is facilitated by using columns with larger shell pores (5000 vs. 700 kDa) and weaker binding interactions (anion exchange vs. multimodal). According to these findings, the choice of RAM resins should be selected according to the respective feed sample composition and the planned number of application cycles.     

Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in patterning at the oral and aboral ends of hydra

Axial patterning of the aboral end of the hydra body column was examined using expression data from two genes. One, shin guard, is a novel receptor protein-tyrosine kinase gene expressed in the ectoderm of the peduncle, the end of the body column adjacent to the basal disk. The other gene, manacle, is a paired-like homeobox gene expressed in differentiating basal disk ectoderm. During regeneration of the aboral end, expression of manacle precedes that of shin guard. This result is consistent with a requirement for induction of peduncle tissue by basal disk tissue. Our data contrast with data on regeneration of the oral end. During oral end regeneration, markers for tissue of the tentacles, which lie below the extreme oral end (the hypostome), are detected first. Later, markers for the hypostome itself appear at the regenerating tip, with tentacle markers displaced to the region below. Additional evidence that tissue can form basal disk without passing through a stage as peduncle tissue comes from LiCl-induced formation of patches of ectopic basal disk tissue. While manacle is ectopically expressed during formation of basal disk patches, shin guard is not. The genes examined also provide new information on development of the aboral end in buds. Although adult hydra are radially symmetrical, expression of both genes in the bud's aboral end is initially asymmetrical, appearing first on the side of the bud closest to the parent's basal disk. The asymmetry can be explained by differences in positional information in the body column tissue that evaginates to form a bud. As predicted by this hypothesis, grafts reversing the orientation of evaginating body column tissue also reverse the orientation of asymmetrical gene expression.     

Toxic Effect of Silica Nanoparticles on Endothelial Cells through DNA Damage Response via Chk1-Dependent G2/M Checkpoint

Silica nanoparticles have become promising carriers for drug delivery or gene therapy. Endothelial cells could be directly exposed to silica nanoparticles by intravenous administration. However, the underlying toxic effect mechanisms of silica nanoparticles on endothelial cells are still poorly understood. In order to clarify the cytotoxicity of endothelial cells induced by silica nanoparticles and its mechanisms, cellular morphology, cell viability and lactate dehydrogenase (LDH) release were observed in human umbilical vein endothelial cells (HUVECs) as assessing cytotoxicity, resulted in a dose- and time- dependent manner. Silica nanoparticles-induced reactive oxygen species (ROS) generation caused oxidative damage followed by the production of malondialdehyde (MDA) as well as the inhibition of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Both necrosis and apoptosis were increased significantly after 24 h exposure. The mitochondrial membrane potential (MMP) decreased obviously in a dose-dependent manner. The degree of DNA damage including the percentage of tail DNA, tail length and Olive tail moment (OTM) were markedly aggravated. Silica nanoparticles also induced G2/M arrest through the upregulation of Chk1 and the downregulation of Cdc25C, cyclin B1/Cdc2. In summary, our data indicated that the toxic effect mechanisms of silica nanoparticles on endothelial cells was through DNA damage response (DDR) via Chk1-dependent G2/M checkpoint signaling pathway, suggesting that exposure to silica nanoparticles could be a potential hazards for the development of cardiovascular diseases.     

Acyl-coenzyme A: cholesterol acyltransferase assay: silica gel column separation of reaction products

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) assays are usually performed by incubation of the enzyme with a labeled substrate followed by thin-layer chromatography separation and subsequent quantification of cholesteryl esters (CE) formed. Herein, a method is described for rapid separation of CE from other lipids, by elution from a silica gel column with a solvent mixture of petroleum ether/diethyl ether (98:2, v/v). Silica gel column chromatography is reliable and more rapid and safer than TLC. The best results were obtained when the reaction was stopped by Dole extraction followed by CE separation on a silica gel column. Assays for ACAT from rat intestinal microsomes showed that the specific activity values obtained using this method were reproducible and in good agreement with those obtained by conventional TLC method.     

Bacterial motion in porous media

The motion of chemotactically different Escherichia coli C600, cheB287, and AW405 cells was studied using a column packed with silica gel. The model chemotaxis of bacteria in porous media seems to be adequate to natural bacterial chemotaxis in soils. The porous structure of silica gel prevents interfering convective flows. Silica gel columns make it possible to separate bacterial cells differing in motility and chemotaxis. Relevant physical phenomena are discussed. The concept of fast and slow chemotaxis is considered.     

Improved chromatographic separations on anion-exchange resins. II. Separation of uronic acids in acetate medium and detection with a noncorrosive reagent

An ion-exchange chromatographic system is described which is capable of separating complex uronic acid mixtures in about 2.5 hr. The system has the following advantages: (i) The resin is commercially available; (ii) no column temperature or buffer changes are needed during a run; (iii) the detection reagent is highly sensitive and also noncorrosive; and (iv) organic contaminants such as amino acids and reducing monosaccharides do not interfere with the separations. Several hundred samples can be run without column regeneration or equilibration. Furthermore, the system can be easily built by modification of existing conventional amino acid or sugar analyzers.     

广西甜茶苷柱纯化工艺研究

以含70%的广西甜茶粗提取物为原料,通过柱纯化与重结晶对甜茶苷进行提纯,从而制备高纯度的甜茶苷单体。以聚酰胺和LSA-10大孔吸附树脂树脂为吸附剂乙醇为洗脱剂进行柱纯化。实验表明,比较聚酰胺与大孔吸附树脂的吸附与脱附能力,皆为后者强;比较不同吸附材料处理所得甜茶苷单体的纯度,则是聚酰胺所吸附的样纯度高,特别是25%乙醇的处理样。     

Tentacular and oral-disc regeneration in the sea anemone,Aiptasia diaphana. I. Sequential morphological events in distal-end restitution

The complete regeneration of a new oral-disc and tentacles has been observed and described for Aiptasia diaphana. These structures are regenerated quite rapidly: seven to ten days at 20°C. At three days post-amputation, the new primary, secondary, and tertiary tentacle buds begin to develop in direct association with the underlying primary, secondary, and tertiary septae (respectively) of the column, suggesting that the latter organize the form of the regenerating oral-disc. Two days after amputation, the zooxanthellae of the presumptive oral disc arrange themselves into a ring which quite precisely delimits the area from which the tentacle buds will form. In spite of its suggestive proximity, this accumulation of algae plays no role in the induction of tentacle buds as was shown by studying regeneration in anemones which essentially lacked large quantities of these symbiotic algae. Cuts perpendicular to the longitudinal axis of the column result in an equal rate of tentacular regeneration around the entire circumference of the presumptive oral disc. Oblique amputations foster an asynchronous regeneration: the tentacle buds of the distal-most area of the severed column are larger and regenerate much sooner than those of the proximal region. Similar results were obtained by studying anemones which were cut perpendicular to their longitudinal axes at different levels along the column. The data suggest that an oral-aboral gradient exists concerning the time required for the initiation of tentacle budding and the rate of tentacle regeneration.     

Bacterial Motion in Porous Media

The motion of chemotactically different Escherichia coli C600, cheB287, and AW405 cells was studied using a column packed with silica gel. The model chemotaxis of bacteria in porous media seems to be adequate for natural bacterial chemotaxis in soils. The porous structure of silica gel prevents interfering convective flows. Silica gel columns make it possible to separate bacterial cells differing in motility and chemotaxis. Relevant physical phenomena are discussed. The concept of fast and slow chemotaxis is considered.     

Genic DNA methylation changes during in vitro organogenesis: organ specificity and conservation between parental lines of epialleles

During differentiation, in vitro organogenesis calls for the adjustment of the gene expression program toward a new fate. The role of epigenetic mechanisms including DNA methylation is suggested but little is known about the loci affected by DNA methylation changes, particularly in agronomic plants for witch in vitro technologies are useful such as sugar beet. Here, three pairs of organogenic and non-organogenic in vitro cell lines originating from different sugar beet (Beta vulgaris altissima) cultivars were used to assess the dynamics of DNA methylation at the global or genic levels during shoot or root regeneration. The restriction landmark genome scanning for methylation approach was applied to provide a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence that is particularly adapted for studies on crop plants without a fully sequenced genome. The cloned sequences had putative roles in cell proliferation, differentiation or unknown functions and displayed organ-specific DNA polymorphism for methylation and changes in expression during in vitro organogenesis. Among them, a potential ubiquitin extension protein 6 (UBI6) was shown, in different cultivars, to exhibit repeatable variations of DNA methylation and gene expression during shoot regeneration. In addition, abnormal development and callogenesis were observed in a T-DNA insertion mutant (ubi6) for a homologous sequence in Arabidopsis. Our data showed that DNA methylation is changed in an organ-specific way for genes exhibiting variations of expression and playing potential role during organogenesis. These epialleles could be conserved between parental lines opening perspectives for molecular markers.