Porous polyacrylamide monoliths in hydrophilic interaction capillary electrochromatography of oligosaccharides

Capillary electrochromatography (CEC) of oligosaccharides in porous polyacrylamide monoliths has been explored. While it is possible to alter separation capacity for various compounds by copolymerization of suitable separation ligands in the polymerization backbone, "blank" acrylamide matrix is also capable of sufficient resolution of oligosaccharides in the hydrophilic interaction mode. The "blank" acrylamide network, formed with a more rigid crosslinker, provides maximum efficiency for separations (routinely up to 350,000 theoretical plates/m for fluorescently-labeled oligosaccharides). These columns yield a high spatial resolution of the branched glycan isomers and large column permeabilities. From the structural point of view, some voids are observable in the monoliths at the mesoporous range (mean pore radius ca. 35 nm, surface area of 74 m2/g), as measured by intrusion porosimetry in the dry state.     

Enantioseparation by ligand-exchange using particle-loaded monoliths: Capillary-LC versus capillary electrochromatography

Particle-loaded monoliths containing a polymethacrylamide backbone were prepared by suspending a silica-based chiral phase in the mixture of the monomers followed by in-situ polymerization in the capillary. As chiral selector l-4-hydroxyproline chemically bonded to 3 μm silica particles was used following the separation principle of ligand-exchange. Electrolytes containing Cu(II) ions were used. Amino acid enantiomers were separated by capillary-LC and CEC, whereby the latter showed the better resolution properties. For the chiral separation of α-hydroxy acids the EOF was reversed by copolymerizing diallyldimethylammonium chloride instead of vinylsulfonic acid as charge providing agent. Short columns of 6 cm were found to be sufficient in the case of CEC for baseline separations of amino acids with α values up to 5.     

Design,synthesis and SAR study of 2-aminopyrimidines with diverse Michael addition acceptors for chemically tuning the potency against EGFRL858R/T790M

The covalent binding nature of irreversible kinase inhibitors potentially increases the severity of “off-target” toxicity. Based on our continual strategy of chemically tuning the Michael addition acceptors, herein, we further explore the relationship among the electronic nature of Michael addition acceptors and EGFR^T790M mutation selectivity as well as “off-target” toxicity balance. By perturbing the electronic nature of acrylamide moiety, compound 8a with a chloro-group at the α-position of the Michael addition acceptor was identified. It was found that 8a retained the excellent EGFR ^L858R/T790M potency (IC₅₀ = 3.9 nM) and exhibited good anti-proliferative activities against the gefitinib-resistant NCI-H1975 cells (IC₅₀ = 0.75 μM). Moreover, 8a displayed a significant EGFR^WT selectivity and much weaker inhibitory activity against non-EGFR dependent SW620 cell and COS7. Preliminary study showed that 8a could arrest NCI-H1975 cells in G0/G1 phase. This work provides a promising chemical tuned strategy for balancing the mutant-EGFR potency and selectivity as well as “off-target” toxicity.     

Faisabilité et intérêt d’un nouvel algorithme de reconstruction des images TEP-18FDG (AW OSEM DR) pour la délimitation des volumes cibles en radiothérapie. À propos d’un cas de néoplasie ORL

ObjectiveWe compared two reconstruction methods for 18fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography (PET) images with “attenuation weighted ordered subset expectation maximization” using either the manufacturer-provided (AW-OSEM) or a “Detector response” (AW-OSEM DR) tomographic operator. We looked at the feasibility of using the latter reconstruction for radiotherapy target volumes definition in cancers of the superior aero-digestive tract (VADS). In this preliminary study, we first assessed the spatial resolution of images obtained with AW-OSEM and AW-OSEM DR on a Biograph™ 6, and secondly target volumes of radiotherapy “Gross Tumor Volume” (GTV), “Clinical Target Volume” (CTV) and “Planning Target Volume” (PTV) obtained with each of these reconstruction methods.Material and methodsThe spatial resolution was measured on a test object containing 4 radioactive point sources. Furthermore, radiotherapy target volumes have been defined with the software Eclipse™ on injected scanner (CT IV) and PET/CT (PET AW-OSEM and PET AW-OSEM DR) images.ResultsSpatial resolution was improved with AW-OSEM DR algorithm reconstruction compared to images obtained with AW-OSEM reconstruction (from 7.5 mm down to 5.4 mm for the highest reduction). GTV from AW-OSEM DR reconstruction with 42 and 50% of the “Standard uptake value maximum” (SUVmax) semi-automatic threshold (1.2 and 0.7 cm³ respectively) were lower than those obtained with AW-OSEM (3.6 and 2.2 cm³ respectively). They were also lower than GTV defined with CT IV (5.5 cm³). It was the same for CTV and PTV.ConclusionThis study showed that AW-OSEM DR reconstruction method allows less impaired spatial resolution than AW-OSEM. In the case of radiotherapy target volumes delineation, AW-OSEM DR may decrease the GTV, CTV and PTV and therefore the risk of side effects associated with organs at risk.     

One-step purification of YLLIP2 isoforms from Candida sp. 99–125 by polyethyleneimine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith

The extracellular lipase Yarrowia lipolytica (YLLIP2) crude extract was efficiently separated and purified from Candida sp. 99–125 by one-step ion-exchange chromatography on polyethyleneimine (PEI) functionalized monolithic columns. The preparative conditions for the functionalization of monoliths were optimized, including PEI molecular mass, PEI concentration, modification time and temperature. The monolithic skeleton was prepared in situ by polymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) with a volume ratio of 8:2. Heptane was used as the porogen. PEI 30 kDa with the concentration of 10% (v/v) was applied for the modification of the monolith at 55 °C for 12 h. Lipase (EC.3.1.1.3) from Candida sp. 99–125 was separated to four isoforms (isoform A, isoform B, isoform C and isoform D). As analyzed on non-denaturing PAGE and MALDI-TOF–MS, the four isoforms are homogenous and have the same molecular mass of approximate 38 kDa. The monoliths can afford direct crude lipase loading without increasing too much back pressure, which explores the great potential of the application of monoliths for one-single step fast separation and purification of complicated proteins.     

Preparation of novel β-cyclodextrin functionalized monolith and its application in chiral separation

A novel β-cyclodextrin (β-CD) functionalized organic polymer monolith was prepared by covalently bonding ethylenediamine-β-CD (EDA-β-CD) to poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) monolith via ring opening reaction of epoxy groups. SEM characterization was performed to confirm the homogeneity of the monolithic polymer. The resulting monolith was then characterized by DSC and XPS elemental analysis to study the thermal stability of the monolith, and to prove the successful immobilization of β-CD on the polymer substrate. The β-CD ligand density of 0.68 mmol g^?1 was obtained for the modified monolith, indicating the high reactivity and efficiency of the EDA-β-CD modifier. The ethylenediamine-β-CD functionalized monoliths were used for the chiral separation of ibuprofen racemic mixture and showed promising results.     

Structural analysis of gellans produced by Sphingomonas elodea strains by electrospray tandem mass spectrometry

A commercial gellan sample (Gelrite) and a gellan-like polymer (JB3) obtained by exposure of the producing strain to chemical mutagenesis were subjected to partial acid hydrolysis and the resultant oligosaccharides were identified by Electrospray Mass Spectrometry (ESI-MS) and Tandem Mass Spectrometry (ESI-MS/MS and MSⁿ). In both gellans, the main fragments were in accordance with the tetrasaccharide repeating unit, d-Glucose (Glc)-d-Glucuronic acid (GlcA)-d-Glucose (Glc)-l-Rhamose (Rha), described for the wild-type gellan, showing the higher acid lability of the Rha-(1 → 3)-Glc linkage when compared to the (1 → 4) of the other residues. Under the experimental conditions used in the study, as expected, no acyl substituents were observed in the commercial gellan but in JB3 oligosaccharides a glyceryl moiety was identified, substituted in the 3-linked Glc residue. Furthermore, the analysis of the MS/MS and MSⁿ spectra of both gellans allowed the identification of structural details, some of them not yet reported for these exopolysaccharides. The presence of oligosaccharides with single Glc and Rha residues substituent of the tetrasaccharide unit of gellan may represent novel side chains of the backbone unit that to our knowledge have never been reported previously for the gellan exopolysaccharide.     

Datation d’enfouissement par 26Al/10Be et son application préliminaire à des sites du Paléolithique Inférieur en Chine et en France

The radionuclides ²⁶Al and ¹⁰Be in situ produced in quartz near the ground surface by secondary cosmic rays can be used for dating the sediment burial. This paper introduces the principles, preconditions and limitations of the recently established dating method. In China its first trial application is to the site of Peking man. The weighted mean of six significant results of samples from layers 7–10 is 770 ± 80 ka. This date provides strong support to an earlier and longer human presence at the site than once estimated. This method has also been applied for the first time to two Lower Paleolithic sites in the Cher Valley, central France, Lunery “la Terre-des-Sablons” and Brinay “la Noira”. The preliminary results attribute ages of 750 ± 240 and 730 ± 210 ka to the two sites, largely consistent with the previous age estimates based on geological and geomorphological studies and on ESR dating. With its well-founded basis in physics, its independence from other dating methods and its timescale filling a “blank period” in radioisotopic dating, the ²⁶Al/¹⁰Be burial dating will be widely applied and contribute substantially to the establishment of a reliable timescale for the earliest human occurrence and evolution in China and in Europe.     

Screening of yeast strains for transfructosylating activity

Fructooligosaccharides (FOSs) are functional food ingredients with prebiotic properties, and a recent increase in the use of oligosaccharides in the food industry has led to the search for “new” microorganisms and enzymes for the production of oligosaccharides. This paper focuses on the screening of yeasts obtained from fruits and flowers (from Brazilian tropical forests), and capable of secreting extra-cellular enzymes with high fructosyl transferase activity (FTA). The screening and isolation procedures resulted in four potentially interesting yeast strains: Candida sp. (LEB-I3), Rhodotorula sp. (LEB-U5.), Cryptococcus sp. (LEB-V2) and Rhodotorula sp. LEB-V10. All were able to produce more then 100 g l⁻¹ of FOS from a 500 g l⁻¹ sucrose solution, but only the last one, (LEB-V10), showed no hydrolytic activity with respect to the FOS produced, giving a continuous increase in FOS content up to the end of the reaction, when it was about 50% of the total carbohydrates.     

Enhanced immunostimulatory and antitumor activity of different derivatives of κ-carrageenan oligosaccharides from <Emphasis Type="Italic">Kappaphycus striatum</Emphasis>

Chemical modification of carbohydrates can lead to differences in their biological activities. We previously showed that κ-carrageenan oligosaccharides from Kappaphycus striatum have antitumor and immunomodulation effects on S180-bearing mice. In this study, we tested the hypothesis that different chemical modifications of carrageenan oligosaccharides enhance their activities. The mice inoculated with S180 cell suspension were treated p.o. with carrageenan oligosaccharides and their sulfated, acetylated, and phosphorylated derivatives (50, 100, and 200 μg g⁻¹) for 14 days. Transplantable tumor inhibition rate and macrophage phagocytosis, quantitative hemolysis of sheep red blood cells, lymphocyte proliferation, the activity of natural killer cells, production of interleukin-2, and tumor necrosis factor-α were also analyzed. As expected, treatment with different κ-carrageenan oligosaccharides derivatives resulted in an increase in tumor inhibition rate and macrophage phagocytosis and cellular immunity, especially on spleen lymphocyte proliferation. The sulfated derivative at the dose 200 μg g⁻¹ per day showed the highest antitumor activity with the 54.12% tumor weight inhibition and elicited an increase in nature killer cells activity up to 76.1% on S180-bearing mice, which were both significantly higher than the unmodified oligosaccharides. It suggested that chemical modification (especially sulfation) of carrageenan oligosaccharides can enhance their antitumor effect and boost their antitumor immunity.     

Strategy combining separation of isotope-labeled unfolded proteins and matrix-assisted laser desorption/ionization mass spectrometry analysis enables quantification of a wide range of serum proteins

A novel strategy for the quantitative profiling of serum proteome is described. It includes an ammonium sulfate depletion of the serum, an affordable stable isotope labeling chemistry for samples with a large amount of protein, separation of the unfolded proteins, and relative quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Labeling of unfolded proteins was performed using normal (D₀) acrylamide and deuterated (D₃) acrylamide. The workflow for separating the unfolded proteins includes whole gel elution and ion exchange liquid chromatography, and it combines electrophoretic separation based on the protein molecular weight followed by chromatographic separation in the presence of 8 M urea based on protein charge. This was followed by trypsinolysis and MALDI MS analysis, leading to the quantification of a large number of serum proteins, including those with an abundance of 10^-5 less than albumin. This robust and inexpensive workflow is suitable for the quantitative profiling of protein changes in serum associated with preanalytical variables.     

Flow‐injection determination of cysteine in pharmaceuticals based on luminol–persulphate chemiluminescence detection

A flow injection (FI) method is reported for the determination of l‐ cysteine, based on its enhancement on chemiluminescence (CL) emission of luminol oxidized by sodium persulphate in alkaline solution. The calibration graph was linear over the range 1.0 × 10^–9–5.0 × 10^–7 mol/L (r² = 0.9992), with relative standard deviations (RSDs) in the range 1.1–2.3% (n = 4). The limit of detection (3σ blank) was 5.0 × 10^–10 mol/L with a sample throughput of 120/h. The method was applied to pharmaceuticals and the results obtained were in reasonable agreement with the amount labelled. The proposed method was also applied to cysteine in synthetic amino acid mixtures. Calibration graphs of N‐acetylcysteine and glutathione over the range 1.0–50 × 10^–8 and 0.5–7.5 × 10^–7 mol/L were also established (r² = 0.998 and 0.9986) with RSDs in the range 1.0–2.0% (n = 4), and the limits of detection (3σ blank) were 5.0 × 10^–9 and 1.0 × 10^–8 mol/L, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Flow injection chemiluminescence determination of acetochlor and cartap-HCl in freshwater samples using an acidic KMnO4–rhodamine-B reaction system

A flow injection (FI) methodology using the acidic potassium permanganate (KMnO₄)–rhodamine-B (Rh-B) reaction with chemiluminescence (CL) detection was established to determine acetochlor and cartap-HCl pesticides in freshwater samples. Experimental parameters were optimized, and Chelex-100 cationic exchanger mini column and solid-phase extraction (SPE) were used as phase separation techniques. Linear calibration curves were observed for the standard solutions of acetochlor and cartap-HCl over the ranges 0.005–2.0 mg L⁻¹ [y = 1155.8x + 57.551, R² = 0.9999 (n = 8)] and 0.005–1.0 mg L⁻¹ [y = 979.76x + 14.491, R² = 0.9998 (n = 8)] with LODs and LOQs of 7.5 × 10⁻⁴ and 8.0 × 10⁻⁴ mg L⁻¹ (3σ blank) and 2.5 × 10⁻³ and 2.7 × 10⁻³ mg L⁻¹ (10σ blank), respectively, with an injection throughput of 140 h⁻¹. These methods were used to estimate acetochlor and cartap-HCl with or without the SPE procedure, respectively, in spiked freshwater samples. Results obtained were not significantly different at a 95% confidence level to those of other reported methods. Recoveries for acetochlor and cartap-HCl were obtained over the ranges 93–112% (RSD = 1.9–3.6%) and 98–109% (RSD = 1.7–3.8%), respectively. The most probable CL reaction mechanism was explored.     

Radiation damage effects at four specimen temperatures from 4 to 100 K

Radiation damage is the primary factor that limits resolution in electron cryo-microscopy (cryo-EM) of frozen-hydrated biological samples. Negative effects of radiation damage are attenuated by cooling specimens to cryogenic temperatures using liquid nitrogen or liquid helium. We have examined the relationship between specimen temperature and radiation damage across a broad spectrum of resolution by analyzing images of frozen-hydrated catalase crystal at four specimen temperatures: 4, 25, 42, and 100 K. For each temperature, “exposure series” were collected consisting of consecutive images of the same area of sample, each with 10 e^?/Ų exposure per image. Radiation damage effects were evaluated by examining the correlation between cumulative exposure and normalized amplitudes or IQ values of Bragg peaks across a broad range of resolution (4.0–173.5 Å). Results indicate that for sub-nanometer resolution, liquid nitrogen specimen temperature (100 K) provides the most consistent high-quality data while yielding statistically equivalent protection from radiation damage compared to the three lower temperatures. At lower resolution, suitable for tomography, intermediate temperatures (25 or 42 K) may provide a modest improvement in cryo-protection without introducing deleterious effects evident at 4 K.     

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts

Metschnikowia reukaufii is a widespread yeast able to grow in the plants’ floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l⁻¹, which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload-sucrose reactions. These products were purified and structurally characterized by MS-ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α-(1→1), α-(1→3) and α-(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l⁻¹, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l⁻¹ of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.     

Electrochemical immunosensor based on Pd–Au nanoparticles supported on functionalized PDDA-MWCNT nanocomposites for aflatoxin B1 detection

This paper reports a label-free electrochemical immunosensor for the determination of aflatoxin B1 (AFB1), which is based on a gold electrode modified by a biocompatible film of carbon nanotubes/poly(diallyldimethylammoniumchloride)/Pd–Au nanoparticles (CNTs/PDDA/Pd–Au). The nanocomposite was characterized by transmission electron microscopy and the electrochemical behavior of modified electrodes was investigated by cyclic voltammetry. The CNTs/PDDA/Pd–Au nanocomposites film showed good electron transfer ability, which ensured high sensitivity to detect AFB1 in a range from 0.05 to 25 ng mL⁻¹ with a detection limit of 0.03 ng mL⁻¹ obtained at 3σ (where σ is the standard deviation of the blank solution, n = 10). The proposed immunosensor provides a simple tool for AFB1 detection. This strategy can be extended to any other antigen detection by using the corresponding antibodies.     

Biological treatment of n-hexane and methanol in trickle bed air biofilters under acidic conditions

This study focuses on evaluating the degradation of n-hexane/methanol mixture in trickle-bed-air-biofilters (TBABs). Two different concentration ratios of methanol:n-hexane were evaluated (3:1) for TBAB “A” and (5:1) for TBAB “B”. Both TBABs were run and fed with nutrients buffered at pH 4 for encouraging the growth of fungi. The TBABs were loaded with pelletized diatomaceous earth support media and were run at an empty bed residence time of 120 s. n-Hexane loading rates (LRs) ranged from 0.9 to 13.2 g/m³ h for both TBABs. The corresponding methanol LRs varied from 2.3 to 37.7 g/m³ h and from 4.6 to 64.5 g/m³ h for TBABs “A” and “B”, respectively. Experimental results have shown that the degradation of n-hexane in presence of methanol is enhanced for n-hexane LRs less than 10.6 g/m³ h as compared to previous study for sole-fed n-hexane, but for n-hexane LRs of 13.2 g/m³ h, the performance of TBABs in eliminating n-hexane depended on the methanol to n-hexane ratios. The impact was less severe for TBAB “A” (RE 85%) as compared to TBAB “B” (RE 72%). This is attributed to the high LRs of methanol in TBAB “B”. n-Hexane performance stability was another advantage attained.     

Characterization of Acrylamidase Isolated from a Newly Isolated Acrylamide-Utilizing Bacterium, <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> AUM-01

A mesophilic bacterium capable of utilizing acrylamide was isolated, AUM-01, from soil collected from leaf litter at Picnic Point on the UW-Madison campus. In minimal medium with acrylamide as the sole carbon and nitrogen source, a batch culture of AUM-01 completely converted 28.0 mM acrylamide to acrylic acid in 8 h and reached a cell density of 0.3 (A₆₀₀). Afterward all the acrylic acid was degraded by 20 h with the cell density increasing to 1.9 (A₆₀₀). The acrylamide-utilizing bacterium was identified as Ralstonia eutropha based on morphological observations, the BiOLOG GN2 MicroPlate^TM identification system for Gram-negative bacteria, and additional physiological tests. An acrylamidase that hydrolyzes acrylamide to acrylic acid was purified from the strain AUM-01. The molecular weight of the enzyme from AUM-01 was determined to be 38 kDa by SDS–PAGE. The enzyme had pH and temperature optima of 6.3 and 55°C, and the influence of different metals and amino acids on the ability of the purified protein to transform acrylamide to acrylic acid was evaluated. The enzyme from AUM-01 was totally inhibited by ZnSO₄ and AgNO₃.     

Characterization of oligosaccharides of highly purified glycoprotein gC of herpes simplex virus type 1 (HSV-1)

The envelope membrane glycoprotein gC of HSV-1 was purified from Triton X-100 extracts of virus-infected BHK-21 or HEp-2 cells by a single step immuno-affinity column using monoclonal anti-gC antibody. The analysis of the purified [³H]G1cN labeled glycoprotein gC (by gel filtration on Bio-Gel P4) before and after digestion with endo-β-N-acetylglucosaminidase (endo D) indicated that gC contains Asn-linked “complex type” oligosaccharides. No “high mannose” type oligosaccharides were detected. Fractionation of radio-labeled glycopeptides of gC on a column of concanavalin A-sepharose suggested that glycopeptides have “diantennary” and “triantennary” and/or “tetra antennary” structures. Tunicamycin inhibited the incorporation of [¹⁴C]GalN or [³H]GlcN into gC in HSV-1 infected BHK-21 or HEp-2 cells. Gel filtration analysis of [³H]GlcN labeled gC following β-elimination reaction failed to indicate O-glycosidically linked oligosaccharides.     

Milk composition of captive vervet monkey (Chlorocebus pygerythrus) and rhesus macaque (Macaca mulatta) with observations on gorilla (Gorilla gorilla gorilla) and white handed gibbon (Hylobates lar)

The nutrient content and fatty acid composition of vervet monkey milk has been determined and is compared with rhesus macaque, and two hominoid apes, the white handed gibbon and gorilla. With 15.7 ± 4.1 g protein, 33.1 ± 9.4 g fat, and 85.1 ± 7.5 g lactose per kg milk, vervet monkey milk does not differ from that of rhesus macaque, and is within the range of other primates. Small amounts (> 1 g kg^− 1) of oligosaccharides, glucose, galactose and fucose were noted. In comparison, gorilla milk has a low fat content of 13.8 g kg^− 1, but contains high levels of oligosaccharides at 7.0 g kg^− 1 milk. The hominoid partner, the white handed gibbon, contains no oligosaccharides and a milk fat content similar to other hominoid species. Differences between vervet monkey and rhesus macaque milks were observed in the electrophoretic pattern of the milk proteins, mainly amongst the κ- and γ-caseins, which also differ from that of the hominids. The fatty acid contents of these milks differ from studies where a natural diet of leafy material was available in that a low content of α-linolenic acid (18:3n−3) was noted. A phylogenetic effect is observed for the content of 8:0, 10:0 fatty acids between the Cercopithecidae and Hominoidea, and a further phylogenetic effect suggested between the Hylobatidae and Hominidae.