Direct analysis of lipids on thin layer plates by matrix-assisted secondary ion mass spectrometry

A simple and rapid method for the analysis of lipids on a thin layer chromatography (TLC) plate by matrix-assisted secondary ion mass spectrometry (SI-MS) is reported. Analysis was performed without elution of the sample from the TLC plate. Mass spectra obtained by this method are free from interference due to the TLC plate absorbent and reagents used for the detection of the spots. About 1 micrograms of lipids applied on a TLC plate can be analyzed by this method. On scanning the plate, mass chromatograms of each lipid were obtained based on its migration distance along the plate.     

Investigating movement in the laboratory: dispersal apparatus designs and the red flour beetle,Tribolium castaneum

The natural dispersal of Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) has been emulated in the laboratory for more than 50 years, using a simple dispersal apparatus. This has typically comprised of a starting container (initial resource or patch) connected by tubing, which contains thread for the animals to climb into a tube and hence to an end container. That is, beetles move to a new viable resource or patch from an inter‐patch zone or non‐viable habitat. We modified this basic apparatus design to test the effect of tubing length and tubing insertion angle on the dispersal rate and proportion of successful dispersers. We expected that the proportion of successful dispersers would be repeatable within each apparatus design, and that increasing tubing length and steepness of the insertion angle would reduce dispersal rate and success across apparatus designs. Dispersal increased linearly through time, similarly so for both males and females. The design with the most vertical tubing insertion angle had a lower proportion of successful dispersers. Tubing length also had a negative relationship with dispersal success (as judged by insects reaching the end container), but a significant reduction in dispersal success was only apparent between the shortest and longest tubing between containers. We suggest that locating and climbing the vertical section of string before they can enter the tubing between containers restricts dispersal and that at higher densities, insects exhibit greater inclination to climb. This type of apparatus has flexible design tolerances and further potential to study the dispersal of other small insect species that primarily use pedestrian locomotion.     

Comparison of different procedures for the lipid extraction from HL-60 cells: a MALDI-TOF mass spectrometric study

A human leukaemia cell line--HL-60--can be differentiated into neutrophils or macrophages and both differentiation processes are accompanied by changes of the lipid composition. Various methods were described for the extraction of lipids from cellular systems, but only two of them were applied to the HL-60 cell line so far. In this study we compared five selected extraction methods for the lipid extraction from HL-60 cells with regard to their qualitative analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS): chloroform/methanol at volume ratios 2:1 and 1:2, isopropanol/ chloroform, isopropanol/hexane and butanol. In addition, the cholesterol and phospholipid concentrations in organic extracts were measured by colorimetric assays. Results can be summarized as follows: For the analysis of polar phospholipids obtained from HL-60 cells by MALDI-TOF MS, a chlorofom/methanol (1:2) or isopropanol/chloroform mixture or butanol can be applied as extraction systems On the other hand, if one would like to analyze changes in triacylglycerols, then chloroform/methanol (2:1) would be the method of choice.     

Polarity-dependent voltage-gated porin channels from Escherichia coli in lipid bilayer membranes.

A porin preparation from Escherichia coli 0111:B4 consisting of Omp F and Omp C (with Omp F in excess) was purified by salt extraction procedures and investigated in bilayer lipid membranes formed according to the Montal-Mueller technique. The porin preparation was added to the KCl electrolyte compartment of the Montal-Mueller cell which was connected to the voltage source. As the porin incorporated into the membrane, asymmetric, voltage-gated ion channels were formed. Transmembrane voltages greater than +50 mV (measured with respect to the side of porin addition) caused channel closing, while negative voltages, on the other hand, had no effect on channel behaviour but did increase the rate of porin incorporation at higher voltages. With porin added to both compartments voltage gating no longer occurred. Single-channel conductances corresponded to effective pore diameters of 1.5 nm for opening events and 1.18 nm for channel closing events. The number of charges involved in gating was approximately 2.     

High throughput flow cytometry

BACKGROUND: Conventional flow cytometry does not allow the rapid analysis of multiple samples. This has limited its uses in drug discovery, for which the standard for throughput is 100,000 samples per day. METHODS: We describe a simple method in which commercial peristaltic tubing is connected from a commercial autosampler to a flow cytometer. The samples are delivered via a peristaltic pump from source wells in a multiwell plate. The samples are separated by air bubbles. RESULTS: Throughput rates approach the limit of the autosampler (up to 100 wells per minute). Using optimal tubing and flow rates, particles remain within appropriate light scatter and fluorescence gates. The carryover between wells is typically less than 5% without and 1% with a wash step. The volumes of sample delivered are in the microliter scale. The approach has been validated with instruments from three manufacturers. CONCLUSIONS: Flow cytometry has potential throughput of 100,000 samples or more per day starting with the method described. The method is currently best suited to end-point assays. However, combined with high-speed sorting and single- cell assays, the number of assays could approach 1 billion per day.     

Efficacy of extraction methods for lipid and fatty acid composition from fungal cultures

The efficacy of three extraction methods for determining the lipid and fatty acid composition of six fungal cultures was studied. The extraction methods were: chloroform/methanol (2:1), hexane/isopropanol (3:2) and Soxhlet extraction by using hexane. The total lipid and fatty acid composition varied in fungal cultures depending on the extraction conditions. Of the three methods, chloroform/methanol (2:1) was found to be the best for extraction of lipid and fatty acids from fungal cultures.     

Stationary phase thickness determines the quality of thin-layer chromatography/matrix-assisted laser desorption and ionization mass spectra of lipids

Normal phase thin-layer chromatography (NP TLC) is an established method of (phospho)lipid analysis. The determination of the fatty acyl composition is, however, a more challenging task by NP TLC. The direct coupling of TLC separation with mass spectrometric detection (e.g., matrix-assisted laser desorption/ionization mass spectrometry, MALDI MS), however, enables a detailed characterization of complex lipid mixtures. Here we show that the thickness of the silica gel layer has a considerable effect on the quality of the mass spectra recorded directly from the TLC plate. In particular, the intensity of the matrix background signals can be reduced if “thinner” TLC layers are used.     

Controlled environment soil‐core microcosm unit for investigating fate,migration, and transformation of chemicals in soils

The controlled environment soil‐core microcosm unit (CESMU) methods embody a collection of techniques that began with soil sampling in the field and continued throughout the laboratory investigation of chemical fate, migration, and transformation in site‐specific soils; it was a cost‐effective investigative methodology that could be used to screen chemical materials before initiating high‐cost environmental field studies. Intact soil cores were collected in the field using a hydraulically controlled probe, delivering intact soil‐cores with minimal disturbance directly into high‐density polyethylene pipe (10.3‐cm ID). The inert polyethylene pipe was an effective hydrophobic barrier that remained an integral part of the soil‐core column, obviating subsequent transfers of soil. In the laboratory, each soil column was fitted with a porous ceramic plate and a polyethylene endcap containing fittings for teflon tubing, so that a tension could be applied at the bottom of each soil column (30–35 kPa) to mimic field conditions, thus preventing the undue buildup of water within columns that otherwise would change the chemical, physical, and biological properties of the soil. The intact soil‐cores were housed in the CESMU chamber, a controlled temperature unit with sufficient capacity for maintaining constant temperature within entire soil‐cores. Synthetic rain was added twice a week by peristaltic pump at rates simulating rainfall. Leachates were collected under tension via teflon tubing into flasks in darkness and kept at soil column temperature inside CESMU until harvested for analyses. Soil columns were harvested at intervals for sectioning by depth, extraction, and soil analyses. CESMU methods are applicable to investigations of water movement, soil chemistry, solute transport/transformation, and effects on plants.     

Phantom development for daily checks in electron intraoperative radiotherapy with a mobile linac

PurposeIORT with mobile linear accelerators is a well-established modality where the dose rate and, therefore, the dose per pulse are very high. The constancy of the dosimetric parameters of the accelerator has to be checked daily. The aim of this work is to develop a phantom with embedded detectors to improve both accuracy and efficiency in the daily test of an IORT linac at the surgery room.MethodsThe developed phantom is manufactured with transparent polymethyl methacrylate (PMMA), allocating 6 parallel-plate chambers: a central one to evaluate the on-axis beam output, another on-axis one placed at a fixed depth under the previous one to evaluate the energy constancy and four off-axis chambers to evaluate the flatness and symmetry. To analyse the readings a specific application has been developed.ResultsFor all chambers and energies, the mean saturation and polarization corrections were smaller than 0.7%. The beam is monitored at different levels of the clinical beam. Output, energy constancy and flatness correlate very well with the correspondent values with the complete applicator. During the first six months of clinical use the beam dosimetric parameters showed excellent stability.ConclusionsA phantom has been developed with embedded parallel plate chambers attached to the upper applicator part of an IORT linac. The phantom allows a very efficient setup reducing the time to check the parameters. It provides complete dosimetric information (output, energy and flatness) with just one shot and using ionization chambers with minimum saturation effect, as this highly pulsed beam requires.     

Quantitative analysis of major dibenzocyclooctane lignans in Schisandrae fructus by online TLC-DART-MS

Introduction – Direct analysis in real time (DART) ion source is a powerful ionising technique for the quick and easy detection of various organic molecules without any sample preparation steps, but the lack of quantitation capacity limits its extensive use in the field of phytochemical analysis. Objective – To improvise a new system which utilize DART‐MS as a hyphenated detector for quantitation. Methodology – A total extract of Schisandra chinensis fruit was analyzed on a TLC plate and three major lignan compounds were quantitated by three different methods of UV densitometry, TLC‐DART‐MS and HPLC‐UV to compare the efficiency of each method. To introduce the TLC plate into the DART ion source at a constant velocity, a syringe pump was employed. The DART‐MS total ion current chromatogram was recorded for the entire TLC plate. The concentration of each lignan compound was calculated from the calibration curve established with standard compound. Results – Gomisin A, gomisin N and schisandrin were well separated on a silica‐coated TLC plate and the specific ion current chromatograms were successfully acquired from the TLC‐DART‐MS system. The TLC‐DART‐MS system for the quantitation of natural products showed better linearity and specificity than TLC densitometry, and consumed less time and solvent than conventional HPLC method. Conclusion – A hyphenated system for the quantitation of phytochemicals from crude herbal drugs was successfully established. This system was shown to have a powerful analytical capacity for the prompt and efficient quantitation of natural products from crude drugs. Copyright © 2010 John Wiley & Sons, Ltd.     

Lipid sovent systems are not equivalent for analysis of lipid classes in the microeukaryotic green alga,Chlorella

A comparison of three lipid solvent system indicated that they are not equivalent for the analysis of lipid classes in the green alga, Chlorella. Soxhlet extraction (methylene chloride/methanol, 3 h refulx) recovers more neutral lipid than the other methods but is equivalent to the room-temperature Bligh and Dyer (chloroform/methanol/water) extraction modified with phosphate buffer in glycolipid and polar lipid recovery. The Soxhlet method, however, gave a significantly lower recovery of many polyunsaturated fatty acids. The hexane/isopropanol method is selective for algal neutral lipids with poor recovery of membrane lipids (glyco- and polar lipids). Although this selectivity may have some useful applications, for biochemical studies of lipid synthesis in Chlorella, the modified Bligh and Dyer provides the most quantitative and reproducible recovery of all Chlorella lipid classes while minimizing artifacts due to the extraction procedure.     

Systems for Maintenance of Urinary Sterility After Prostatectomy

Closed drainage is recommended for all patients after prostatectomy where hemostasis has been adequate. Although closed drainage can maintain sterility of the bladder, thereby fostering healing and reducing infectious complications, such drainage is not insisted upon at most hospitals because of the inconveniences associated with it. However, when closed drainage was used in 25 consecutive cases of transurethral resection, infection was reduced to 25 per cent (in contrast to the 85 to 100 per cent encountered with open drainage).The ideal closed system should incorporate:1. Fixed tubing to prevent contamination where the catheter joins the tubing and where the tubing is attached to the container;2. An aseptic method of emptying;3. A device to prevent reflux of the potentially contaminated urine in the container into the bladder;4. Free urinary flow from bladder to container; and5. Portability for the patient and convenience for the staff.A system is proposed that incorporates these features. Particularly effective are a fixed drip chamber with vents at the site of attachment of the tubing to the bag and a protected spigot for emptying.     

The resonance of the arterial system

The arterial system is assumed to consist of two elastic chambers connected by a conducting channel. It is assumed that a current of fluid enters one chamber, whereas the other chamber is drained by a pipe with a certain peripheral resistance. The continuity of the fluid is described by a differential equation for each chamber. The inertia resistance of the conducting channel is taken into consideration. It is shown that the system may possess a resonance frequency. The latter, if it exists, as well as the damping coefficients are expressed in terms of the elastic moduli of the chambers, the conductivity of the channel, and the peripheral resistance. It is shown that with plausible values of the latter variables the resonance frequency as determined theoretically has the right order of magnitude as found experimentally.     

Single streptomyces lividans K(+) channels: functional asymmetries and sidedness of proton activation.

Basic electrophysiological properties of the KcsA K(+) channel were examined in planar lipid bilayer membranes. The channel displays open-state rectification and weakly voltage-dependent gating. Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added. Addition of Na(+) to the trans chamber causes block of open-channel current, while addition to the cis side has no effect. Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH. To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer. Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side. Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively. Block by charybdotoxin, known to bind to the channel's external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive. These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein.     

Number of water molecules coupled to the transport of sodium, potassium and hydrogen ions via gramicidin, nonactin or valinomycin.

The number of water molecules (n) coupled to the transport of cations across lipid membranes was determined in two different wats: directly from the electro-osmotic volume flux per ion, and by the use of Onsager's relation, from the open circuit streaming potential produced by an osmotic pressure difference. The results of the two approaches were in general agreement. Monoolein membranes were formed on the ends of polyethylene or Teflon tubing connected to a microliter syringe and the volume change necessary to keep the membrane at a fixed position was measured. It was necessary to make corrections for unstirred layer effects. The results for gramicidin were: n approximately 12 for 0.15 M KCl and NaCl, n approximately 6 for 3.0 M KCl and NaCl, and n approximately 0 for 0.01 M HCl. For nonactin, n approximately 4 for both 0.15 and 3.0 M KCl and NaCl. Valinomycin (for 0.15 M KCl) behaved like nonactin. It is shown that for a channel mechanism, in general, n is less than or equal to the number of water molecules in a channel that does not contain any cations. Thus, the n of 12 for the 0.15 M salts implies that the gramicidin channel can hold at least 12 water molecules. This places an important constraint on models of the channel structure. The n of 0 for HCl is consistent with a process in which protons jump along a continuous row of water molecules. The decrease of n with the 3.0 M salts may indicate that the channel becomes multiply occupied at high salt concentrations. The n of 4 for nonactin and valinomycin means that at least four water molecules are associated with the carrier . cation complex, probably in the interstices between the complex and the disordered lipid.     

Diacylglycerols interfere in normal phase HPLC analysis of lipoxygenase products of docosahexaenoic or arachidonic acids

A methodological problem with the normal phase high performance liquid chromatography (HPLC) of hydroxylated products of docosahexaenoic and arachidonic acids is described. Diacylglycerols present in lipid extracts of rat retina co-elute with monohydroxy derivatives of docosahexaenoic or arachidonic acid, when samples are applied to uPorosil columns and eluted with hexane/isopropanol/acetic acid. Analysis of fatty acid composition of diacylglycerols which were acetone-extracted from the incubation medium showed a profile similar to diacylglycerols extracted from the tissue by hexane/isopropanol, although acetone extraction resulted in extremely variable recovery of diacylglycerols. This co-elution of diacylglycerols with monohydroxy polyunsaturated fatty acids can lead to a significant error in estimation of lipoxygenation activity by conversion of radiolabeled precursors, because the incorporation of fatty acids into diacylglycerols is very active in many tissues. An alternative extraction method and reverse phase HPLC procedures that result in the complete separation of hydroxy fatty acids and diacylglycerols are described.     

紫杉醇初分离工艺中的除杂方法比较

本文以除杂率、脱色率和紫杉醇回收率为定量指标,比较了萃取、碱液洗涤、酸碱液沉淀和离子交换树脂四种方法的优劣。对除杂率的评价,电导法结合TIC法较重量法更为客观。上述四种方法的电导法除杂率分别为73．4％、91．6％、30．7％和86．8％;脱色率分别为56．7％、75．9％、16．5％和71．8％,紫杉醇回收率分别为100％、159％、73．1％和152％。四种方法中,碱液洗涤法除杂效果最佳,紫杉醇回收率最高,此外,还具有操作简便、溶剂用量低、成本低和适合规模生产的优点。     

Automation of protein crystallization trials: use of a robot to deliver reagents to a novel multi-chamber vapor diffusion plate

We report here the automation of search procedures to rapidly screen a large number of reagents and incubation conditions that lead to the formation of protein crystals. The system consists of a Biomek 1000 Automated Laboratory Workstation from Beckman Instruments under the control of a custom user-interface program developed by Cryschem. A plate composed of twenty-four vapor diffusion chambers, each with its own reservoir well and protein drop holder was designed by Cryschem to fit the Biomek table. The Cryschem software manages a large data base of incubation conditions and generates instructions for the workstation to dispense the protein, buffers, detergents, cofactors and other reagents used to promote the formation of protein crystals. The plate is manually positioned on the Biomek table and then under program control additions are automatically made to each chamber as follows: Precipitating solution is added to each reservoir well and the protein solution along with the precipitating solution and various other reagents are added to each drop holder. The plate is removed from the table and a mylar tape is applied to simultaneously seal all the chambers. The plates are placed at a controlled temperature and periodically examined for crystal formation.     

Morphology of spiracles in adult Hyalomma truncatum ticks (Acari; Ixodidae)

Scanning electron microscopical investigations of fractures and corrosion casts of spirales in adult ticks of Hyalomma truncatum revealed a three-part structure consisting of the spiracular plate forming the outer part followed by the subostial space, which leads into the atrial chamber from which the main tracheal trunks arise. The spiracular plate sonsists of a thin surface plate perforated by aeropyles, an underlying interpedicellar space formed by pedicels and an inner thick base plate. The surface plate is subdivided into a porous and a non-porous area. The macula is surrounded by the porous area and cleft by the ostium, which is bounded by a lip. The lip rests on a stalk which passes through the subostial space and forms the lateral wall of the atrial chamber. The interpedicellar space is chambered comprising four types of chambers. Large pyriform chambers (type 1) open to the atmosphere via a large aeropyle and are connected at their base with a duct traversing the base plate. They correspond numerically and in their position with the large aeropyles and the ducts of the base plate. Each chamber is surrounded by four to six medium-sized tubular chambers (type 2) which are closed at both ends. Small tubular chambers (type 3) open to the atmosphere via a small aeropyle, are closed at their base and correspond in number and position to the small aeropyles. Elongated chambers (type 4) are arranged in two to three rows around the subostial space and are closed at both ends. The front row communicates with the subostial space via large gaps. All chambers interconnect with each other by slit-like fenestrations. Below the macula and surrounding the stalk is the subostial space. Over the medial half, the subostial space opens into the atrial chamber. The lateral wall of the atrial chamber is thick, whereas the opposite wall is thin, folded and can be everted and inverted. Inverted, the medial wall closes up the opening to the subostial space and the main tracheal trunks. The base of the atrial chamber sonsists of the openings of the main tracheal trunks only. It is concluded that the aeropyles constitute the functional openings of a spiracle, the interpedicellar space and the subostial space act as diffusion barrier and the atrial chamber is exclusively responsible for the motory process of in- and expiration and is the only closing device of the spiracle.     

Direct analysis of glycolipids on thin-layer plates by matrix-assisted secondary ion mass spectrometry: application for glycolipid storage disorders

The lipids accumulated in organs of patients with Gaucher's, Tay-Sachs, and Fabry's disease were identified by means of the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry. The total lipid extract of each lipidosis tissue was chromatographed on a TLC plate and then analyzed directly by mass spectrometry without elution of the sample from the TLC plate. The amount of material needed to obtain an adequate spectrum is in the order of a few micrograms of lipids per band for both positive and negative ion detection. By scanning the plates, mass spectral and chromatographic information can be obtained simultaneously, which was shown to be useful for the qualitative identification of the components on the plates.