A microfluidic device to establish concentration gradients using reagent density differences

Microfabrication has become widely utilized to generate controlled microenvironments that establish chemical concentration gradients for a variety of engineering and life science applications. To establish microfluidic flow, the majority of existing devices rely upon additional facilities, equipment, and excessive reagent supplies, which together limit device portability as well as constrain device usage to individuals trained in technological disciplines. The current work presents our laboratory-developed bridged μLane system, which is a stand-alone device that runs via conventional pipette loading and can operate for several days without need of external machinery or additional reagent volumes. The bridged μLane is a two-layer polydimethylsiloxane microfluidic device that is able to establish controlled chemical concentration gradients over time by relying solely upon differences in reagent densities. Fluorescently labeled Dextran was used to validate the design and operation of the bridged μLane by evaluating experimentally measured transport properties within the microsystem in conjunction with numerical simulations and established mathematical transport models. Results demonstrate how the bridged μLane system was used to generate spatial concentration gradients that resulted in an experimentally measured Dextran diffusivity of (0.82 ± 0.01) × 10(-6) cm(2)/s.     

A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D

BackgroundThe accurate measurement of 25-hydoxy vitamin D (25OH-D) in serum has been a challenge for many years. We developed a liquid chromatography tandem mass spectrometry (LC Tandem MS) assay for the quantitative determination of 25OH-D₂ and 25OH-D₃ in serum. The new method was compared with two widely used commercially available immunoassays.MethodsSample preparation involved protein precipitation with acetonitrile containing deuterated forms of the target species as internal standards. An API 5000 mass spectrometer coupled with a photoionization source was used for quantitation. The performance of the new LC Tandem MS assay was compared with a radioimmunoassay (RIA, Diasorin) and a chemiluminescence immunoassay (ECLIA, Roche Diagnostics), analysing serum obtained from 152 individuals.ResultsUsing 100 μl of serum, the LC Tandem MS assay had a limit of quantitation of 1.3 nmol/L for both 25OH-D₂ and 25OH-D₃ with a linear response between 1.3 and 625 nmol/L and accuracy of between 95 and 124%. Intra- and inter-assay precision were ≤7% and ≤4%, respectively. Measurement of 25OH-D levels in 152 serum samples gave run averages of 71, 56 and 62 nmol/L for LC Tandem MS, ECLIA and RIA, respectively. Correlations between the various methods were: LC Tandem MS vs. RIA: r = 0.931; LC Tandem MS vs. ECLIA: r = 0.784; RIA vs. ECLIA: r = 0.787. The LC Tandem MS method had a positive proportional bias of 26% over the RIA, whereas the ECLIA showed variable differences.ConclusionThe new LC Tandem MS assay is accurate and precise at physiologically relevant 25OH-D concentrations, and compares favourably with the RIA. In contrast, the ECLIA shows variable bias with the other assays tested.     

Characterization of single chain antibody targets through yeast two hybrid

Background  

Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.     

Newly discovered penicillin acylase activity of aculeacin A acylase from Actinoplanes utahensis

Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM(-1) s(-1). Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5 degrees C.     

Habitat utilization by sympatric European mink<Emphasis Type="Italic">Mustela lutreola</Emphasis> and polecats<Emphasis Type="Italic">Mustela putorius</Emphasis> in south-western France

The European minkMustela lutreola Linnaeus, 1761 and the European polecatMustela putorius Linnaeus, 1758 are related species sympatric in southwestern France. The European mink is rapidly disappearing whereas the polecat maintains good populations. Seasonal habitat use of both species was compared in the Landes de Gascogne region to identify if some vulnerability factors of the European mink were associated with habitats occupied by this mustelid. Potential habitats were mapped using a satellite picture and 12 main types of habitats were defined. Animal locations were recorded by radiotracking 9 European mink and 14 polecats from March 1996 to August 1999. Resting animals were located by triangulation, and, when possible, resting places were described. Animals in activity were tracked by continuous monitoring. Data collected revealed a strong preference of European mink for flooded habitats, particularly open marshes, flooded woodlands and moorlands. They seldom left the corridor of the riparian forest and their resting places were mainly in flooded environments, above ground (under herbs or bushes) or in cavities between tree roots. European polecats were less tightly linked to wetlands. Most of their locations were in the pine forests outside the valleys and their resting places were mainly in burrows. The strong specialisation of European mink in aquatic habitats is probably one of the main reasons for its decline because wetlands suffer drastic damages throughout all of its range. Maintaining adequate water levels is crucial for satisfying habitat requirements of mink.     

Combination therapy reduces self-injurious behavior in a chimpanzee (Pan Troglodytes Troglodytes): a case report

Self-injurious behavior (SIB) remains a severe and intractable abnormal behavior for nonhuman primates in diverse settings and is a significant concern for veterinarians and behavioral scientists. To date, no single pharmacological, behavioral, social, or environmental intervention method has emerged as a reliable permanent cure for treating SIB in all, or even most, individuals. Implementation and evaluation of a combination therapeutic approach to treating SIB for nonhuman primates is rare. In May 2004, a 25-year-old male chimpanzee with severe SIB (M = 2.09 episodes/day, range = 1-4 episodes/day) underwent intensive behavioral intervention that utilized a combination of techniques. The combination therapy approach entailed the following: (a) pharmacological intervention with a gamma-aminobutyric acid (GABA) analogue to treat suspected HIV-related sensory neuropathic pain, (b) positive reinforcement training, and (c) environmental enrichment, as well as social and environmental modification. The severity of SIB warranted immediate implementation of intensive combination therapy rather than a systematic evaluation of the individual treatment options. The individually tailored, multifaceted combination therapy resulted in the virtual elimination of SIB in this chimpanzee over a 2-year period.     

The Cytosolic pH of Individual Saccharomyces cerevisiae Cells Is a Key Factor in Acetic Acid Tolerance

It was shown recently that individual cells of an isogenic Saccharomyces cerevisiae population show variability in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the population level. In the current study, we investigated whether cell-to-cell variability in acetic acid tolerance could be explained by the observed differences in the cytosolic pHs of individual cells immediately before exposure to the acid. Results obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic pH drop after exposure to the acid. Moreover, only cells with a low initial cytosolic pH, which experienced a less severe drop in cytosolic pH, were able to proliferate. A similar correlation between initial cytosolic pH and cytosolic pH drop was also observed in the more acid-tolerant strain MUCL 11987-9. Interestingly, a fraction of cells in the MUCL 11987-9 population showed initial cytosolic pH values below the minimal cytosolic pH detected in cells of the strain CEN.PK113-7D; consequently, these cells experienced less severe drops in cytosolic pH. Although this might explain in part the difference between the two strains with regard to the number of cells that resumed proliferation, it was observed that all cells from strain MUCL 11987-9 were able to proliferate, independently of their initial cytosolic pH. Therefore, other factors must also be involved in the greater ability of MUCL 11987-9 cells to endure strong drops in cytosolic pH.     

TcPho91 is a contractile vacuole phosphate sodium symporter that regulates phosphate and polyphosphate metabolism in Trypanosoma cruzi

We have identified a phosphate transporter (TcPho91) localized to the bladder of the contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease. TcPho91 has 12 transmembrane domains, an N‐terminal regulatory SPX (named after SYG1, Pho81 and XPR1) domain and an anion permease domain. Functional expression in Xenopus laevis oocytes followed by two‐electrode voltage clamp showed that TcPho91 is a low‐affinity transporter with a K_m for P_i in the millimolar range, and sodium‐dependency. Epimastigotes overexpressing TcPho91‐green fluorescent protein have significantly higher levels of pyrophosphate (PP_i) and short‐chain polyphosphate (polyP), suggesting accumulation of P_i in these cells. Moreover, when overexpressing parasites were maintained in a medium with low P_i, they grew at higher rates than control parasites. Only one allele of TcPho91 in the CL strain encodes for the complete open reading frame, while the other one is truncated encoding for only the N‐terminal domain. Taking advantage of this characteristic, knockdown experiments were performed resulting in cells with reduced growth rate as well as a reduction in PP_i and short‐chain polyP levels. Our results indicate that TcPho91 is a phosphate sodium symporter involved in P_i homeostasis in T. cruzi.