Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO₂ coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO₂ coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.     

Description of Trichotokara nothriae n. gen. et sp. (Apicomplexa, Lecudinidae) - An intestinal gregarine of Nothria conchylega (Polychaeta, Onuphidae)

The trophozoites of a novel gregarine apicomplexan, Trichotokara nothriae n. gen. et sp., were isolated and characterized from the intestines of the onuphid tubeworm Nothria conchylega (Polychaeta), collected at 20 m depth from the North-eastern Pacific Coast. The trophozoites were 50-155 μm long with a mid-cell indentation that formed two prominent bulges (anterior bulge, 14-48 μm wide; posterior bulge, 15-55 μm wide). Scanning electron microscopy (SEM) demonstrated that approximately 400 densely packed, longitudinal epicytic folds (5 folds/μm) inscribe the surface of the trophozoites, and a prominently elongated mucron (14-60 μm long and 6-12 μm wide) was covered with hair-like projections (mean length, 1.97 μm; mean width, 0.2 μm at the base). Although a septum occurred at the junction between the cell proper and the mucron in most trophozoites, light microscopy (LM) demonstrated that the cell proper extended into the core of the mucron as a thin prolongation. A spherical nucleus (8-20 μm) was situated in the middle of the trophozoites, and gamonts underwent caudal syzygy. The small subunit (SSU) rDNA sequence and molecular phylogenetic position of T. nothriae was also characterized. The sequence from this species was the most divergent of all SSU rDNA sequences currently known from gregarines and formed a weakly supported clade with Lecudina polymorpha, which also possesses densely packed epicyctic folds (3-5 folds/μm) and a prominently elongated mucron.     

Multicomponent mesofluidic system for the detection of veterinary drug residues based on competitive immunoassay

An automated multicomponent mesofluidic system (MCMS) based on biorecognitions carried out on meso-scale glass beads in polydimethylsiloxane (PDMS) channels was developed. The constructed MCMS consisted of five modules: a bead introduction module, a bioreaction module, a solution handling module, a liquid driving module, and a signal collection module. The integration of these modules enables the assay to be automated and reduces it to a one-step protocol. The MCMS has successfully been applied toward the detection of veterinary drug residues in animal-derived foods. The drug antigen-coated beads (?250 μm) were arrayed in the PDMS channels (?300 μm). The competitive immunoassay was then carried out on the surface of the glass beads. After washing, the Cy3-labeled secondary antibody was introduced to probe the antigen-antibody complex anchored to the beads. The fluorescence intensity of each bead was measured and used to determine the residual drug concentration. The MCMS is highly sensitive, with its detection limits ranging from 0.02 (salbutamol) to 3.5 μg/L (sulfamethazine), and has a short assay time of 45 min or less. The experimental results demonstrate that the MCMS proves to be an economic, efficient, and sensitive platform for multicomponent detection of compound residues for contamination in foods or the environment.     

Fabrication of a circular PDMS microchannel for constructing a three-dimensional endothelial cell layer

We describe a simple and efficient fabrication method for generating microfluidic channels with a circular cross-sectional geometry by exploiting the reflow phenomenon of a thick positive photoresist. Initial rectangular shaped positive photoresist micropatterns on a silicon wafer, which were fabricated by a conventional photolithography process, were converted into a half-circular shape by tuning the temperature to around 105 °C. Through optimization of the reflow conditions, we could obtain a perfect circular micropattern of the positive photoresist, and control the diameter in a range from 100 to 400 μm. The resultant convex half-circular photoresist was used as a template for fabricating a concave polydimethylsiloxane (PDMS) through a replica molding process, and a circular PDMS microchannel was produced by bonding two half-circular PDMS layers. A variety of channel dimensions and patterns can be easily prepared, including straight, S-curve, X-, Y-, and T-shapes to mimic an in vivo vascular network. To form an endothelial cell layer, we cultured primary human umbilical vein endothelial cells inside circular PDMS microchannels, and demonstrated successful cell adhesion, proliferation, and alignment along the channel.     

Functional morphology of mucosal goblet cells based on spatial separation of orifice openings to the surface – Application to the rabbit bulbar conjunctiva

The purpose of the study was to assess spatial separation of goblet cell orifices observed at the surface of the rabbit bulbar conjunctiva by scanning electron microscopy (SEM) specimens of the bulbar conjunctiva from 8 healthy pigmented rabbits were obtained using a special preparation technique by which the tissue was carefully stretched out during glutaraldehyde fixation. On high magnification prints of SEM images of the conjunctival surface, the locations of goblet cell openings (orifices) to the apical surface were marked and the centre-to-centre spacing between all such orifices measured. Across the regions of interest (ROI), with averaged dimensions of 322 μm × 230 μm (adjusted for tissue shrinkage), the averaged value for the distances between all orifices was 196 μm (range 141–241 μm), with the calculated density of orifices being 412 mm⁻². A sequential order-based analysis of the spatial separation between orifices indicated a predictable value of 6 μm, a separation that showed a nearly linear inter-dependence over distances of at least 200 μm. The openings of goblet cells to the surface of unstimulated bulbar conjunctiva have a organized spatial distribution that is consistent with there being an organized control of goblet cell secretion.     

Steinernema brazilense n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Mato Grosso, Brazil

A new entomopathogenic nematode, Steinernema brazilense n. sp., was isolated from a single soil sample collected from a natural forest in Mato Grosso do Sul state, Brazil. S. brazilense n. sp. is characterized morphologically by features of infective juveniles (IJ), males and females. For the IJ, body length averaging 1157 (1023-1284) μm, distance from anterior end to excretory pore 95 (87-102) μm, from anterior end to end of esophagus 148 (139-153) μm, tail length 85 (80-104) μm, D% and E% values 63 (58-70) and 106 (95-118.0), respectively. Lateral field pattern variable; the formula for the arrangement of ridges from head to tail is: 2, 4, 6, 8, 6, 2. For the male, the diagnostic characters include spicule averaging 83 (75-89) μm; D% about 65; the ratio SW% about 192. The length of spicule head is greater than width. Lateral field with one narrow ridge. First generation females are characterized by the presence of a ventral postanal swelling. S. brazilense n. sp. is morphologically close to Steinernema diaprepesi. It can be differentiated from S.diaprepesi by its longer IJ body length (1157 vs 1002 μm), longer distance from anterior end to excretory pore (110 vs 75 μm), a longer tail length (103 vs 83 μm); males of the new species with longer spicule (83 vs 79 μm). The new species can be distinguished further from other members of Steinernema glaseri group by characteristics of rDNA of ITS and D2D3 regions.     

SU-8-carbon composite as conductive photoresist for biochip applications

A composite photoresist has been developed for the direct photopatterning of electrodes useful as biochip substrates. The material is composed of SU-8 polymer added with graphite carbon filler which enables patterning of conductive thin films (22μm) on both glass substrate and transparency flexible film with a standard UV photolithography protocol. The resolution obtained using the conductive composite compared well with the bare resist, with lateral resolutions of 5 and 10μm for bare and conductive resists, respectively. The obtained electrodes, after an electrochemical pre-treatment, exhibited very good electrochemical behaviors, opening the path to various electrochemical detections and grafting possibilities. In order to demonstrate the potentialities of the developed material in the biosensors and biochips field, DNA probes were electrografted, using diazonium chemistry, directly at the composite photoresist surface. Target oligonucleotide interactions were detected using chemiluminescent labeling and a satisfactory detection limit of 0.25nM target sequence was demonstrated with a detection ranging over three orders of magnitude.     

Planar microfluidic chamber for generation of stable and steep chemoattractant gradients

The extracellular availability of growth factors, hormones, chemokines, and neurotransmitters under gradient conditions is required for directional cellular responses such as migration, axonal pathfinding, and tissue patterning. These responses are, in turn, important in disease and developmental processes. This article addresses critical barriers toward devising a chemotaxis assay that is broadly applicable for different kinds of cancer cells through the design of a microfluidic chamber that produces a steep gradient of chemoattractant. Photolithography was used to create microchannels for chemoattractant delivery, flow diversion barriers/conduits, and small outlets in the form of apertures. The 1-μm apertures were made at the active surface by uncapping a thin (1.5 μm) layer of AZ1518. This process also created a vertical conduit that diverted the flow such that it occurred perpendicularly to the active, experimental surface where the gradients were measured. The other side of the vertical conduit opened to underlying 20-μm deep channels that carried microfluidic flows of tracer dyes/growth factors. Modeled data using computational fluid dynamics produced gradients that were steep along the horizontal, active surface. This simulation mirrors empirically derived gradients obtained from the flow analyses of fluorescent compounds. The open chamber contains a large buffer volume, which prevents chemoattractant saturation and permits easy cell and compound manipulation. The technique obviates the use of membranes or laminar flow that may hinder imaging, rinsing steps, cell seeding, and treatment. The utility of the chamber in the study of cell protrusion, an early step during chemotaxis, was demonstrated by growing cancer cells in the chamber, inducing a chemoattractant gradient using compressed air at 0.7 bar, and performing time-lapse microscopy. Breast cancer cells responded to the rapidly developed and stable gradient of epidermal growth factor by directing centroid positions toward the gradient and by forming a leading edge at a speed of 0.45 μm/min.     

Long and short distance movements of β2-adrenoceptor in cell membrane assessed by photoconvertible fluorescent protein dendra2-β2-adrenoceptor fusion

Local movements of receptors in the plasma membrane have been extensively studied, as it is generally believed that the dynamics of membrane distribution of receptors regulate their functions. However, the properties of large-scale (> 5 μm) receptor movements in the membrane are relatively obscure. In the present study, we addressed the question as to whether the large-scale movement of receptor in the plasma membrane at the whole cell level can be explained quantitatively by its local diffusive properties. We used HEK 293 cells transfected with human β2-adrenoceptor fused to photoconvertible fluorescent protein dendra2 as a model system; and found that 1) functional integrity of the dendra2-tagged receptor remains apparently intact; 2) in a mesoscopic scale (~ 4 μm), ~ 90% of the receptors are mobile on average, and receptor influx to, and out-flux from a membrane area can be symmetrically explained by a diffusion-like process with an effective diffusion coefficient of ~ 0.1 μm²/s; 3) these mobility parameters are not affected by the activity state of the receptor (assessed by using constitutively active receptor mutants); 4) in the macroscopic scale (4-40 μm), although a slowly diffusing fraction of receptors (with D < 0.01 μm²/s) is identifiable in some cases, the movement of the predominant fraction is perfectly explained by the same effective diffusion process observed in the mesoscopic scale, suggesting that the large scale structure of the cell membrane as felt by the receptor is apparently homogeneous in terms of its mesoscopic properties. We also showed that intracellular compartments and plasma membrane are kinetically connected even at steady-state.     

The effect of porcine luteinizing hormone in the synchronization of ovulation and corpus luteum development in nonlactating cows

The objective of this study was to determine the effects of different doses of porcine luteinizing hormone (pLH) versus 100 μg gonadotropin-releasing hormone (GnRH) on ovulatory response (during diestrus and proestrus) and corpus luteum (CL) development in nonlactating cows. In Experiment 1, 75 cows received an intravaginal insert containing 1.9 g progesterone (P4) for 10 d to synchronize estrus (Day 0), with prostaglandin F_2α (PGF) at insert removal. On Day 5, all follicles ≥8 mm were ablated, and on Day 12, cows received 8, 12.5, or 25 mg pLH or 100 μg GnRH. Mean (±SEM) plasma P4 concentrations on Day 12 did not differ among treatments (5.6 ± 0.2 ng/mL). Mean plasma LH concentration was greatest (P < 0.01) in cows given 25 mg pLH (4.3 ± 0.4 ng/mL). The ovulatory response to 25 mg pLH (84%) or 100 μg GnRH (72%) was greater (P < 0.05) than that to 8 mg pLH (32%), but not different from that of 12.5 mg pLH (58%). In Experiment 2, 68 cows were given two injections of PGF 10 d apart to synchronize estrus (Day 0). On Day 7, cows received PGF, and, 36 h later, pLH or GnRH (as in Experiment 1). The interval from treatment to ovulation was most variable in cows given 8 mg pLH; only 65% of these cows ovulated during the initial 27 h versus 88% of cows given 25 mg pLH (P < 0.05). Cows given 25 mg pLH or 100 μg GnRH had larger CL area and greater plasma P4 concentrations (P < 0.05) than that of those given 8 mg pLH. In summary, diestrous cows given 25 mg pLH had the greatest plasma luteinizing hormone concentrations, but ovulatory response did not differ from that of those given 100 μg GnRH. Proestrous cows given 25 mg pLH or 100 μg GnRH had greater CL area and P4 concentrations than that of those given 8 mg pLH.     

Development of a high-throughput method for the determination of ethosuximide in human plasma by liquid chromatography mass spectrometry

A simple, rapid, sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the quantification of ethosuximide in human plasma is described. Analyte was chromatographed on a Hypersil Gold C₁₈ column (100 mm × 2.1 mm, i.d., 1.9 μm) with isocratic elution at a flow rate of 0.250 mL/min and pravastatin was used as the internal standard. The assay involves a simple solid-phase extraction procedure of 0.25 mL human plasma and the analysis was performed on a triple-quadrupole tandem mass spectrometer by MRM mode via electrospray ionization (ESI). The method was linear in the concentration range of 0.25–60.0 μg/mL. The lower limit of quantification (LLOQ) was 0.25 μg/mL. The within- and between-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 95.1% and 94.4% for ethosuximide and pravastatin, respectively. The analysis time for each sample was 1.8 min. The method was highly reproducible and gave peaks with excellent chromatography properties.     

The relationship between water availability and anatomical characters in Carex hirta

The effects of edaphic moisture in anatomical characters were evaluated in two different populations of Carex hirta L. with three watering treatment for 6 months to evaluate stability, and determined taxonomic value. Water availability increased (p < 0.001) leaf thickness from 239 to 289 μm, metaxylem vessel diameter from 17 to 23 μm, air cavity size from 10 to 24% and adaxial epidermal cell height from 18 to 34 μm, and abaxial from 11 to 16 μm, adaxial epidermal cell length from 54 to 105 μm, and abaxial from 35 to 86 μm, and adaxial epidermal cell width from 20 to 33 μm, and abaxial from 15 to 23 μm. Stomatal index and the number of cells in the girder of sclerenchyma did not vary with water availability, hence these traits have taxonomic value. Other characters (the length and amplitude of wall undulations in the epidermal cells, the number of bulliform cells) have a doubtful relation with water availability, because they are variable even in constant homogeneous conditions.     

Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes

During a recent soil sample survey in Eastern China, a new entomopathogenic nematode species, collected from the Chongming Islands in the southern-eastern area of Shanghai, was discovered. Morphological characteristics of different developmental stages of the nematode combined with molecular data showed that this nematode is a new genus of Rhabditidae, and described as Heterorhabditidoides chongmingensis gen. nov., sp. nov., for that it shares more morphological characteristics with heterorhabditids than with steinernematids. For males, the papillae formula of bursa is 1, 2, 3, 3, with constant papillae number in the terminal group, stoma tubular-shaped and about 1.5 head width; cheilorhabdions cuticularized, esophageal collar present and long, median bulb present. For infective juveniles, EP = 90 (80-105) μm, ES = 104 (92-120) μm, tail length = 111 (89-159) μm, and a = 19.1 (15-21). The percentages of the nucleotides A, T, C and G in the ITS1 regions of the new species are significantly different from those of heterorhabditids and other rhabditids. Molecular phylogenetic trees based on 18S rDNA and the internal transcribed spacer (ITS) sequences data revealed that the new entomopathogenic nematode species forms a monophyletic group, which is a sister group of the clade comprised of some genera of Rhabditidae.     

Development and validation of a liquid chromatography/mass spectrometry method for pharmacokinetic studies of OZ78, a fasciocidal drug candidate

Fascioliasis is a zoonotic disease of considerable public health and great veterinary significance and new drugs are needed. OZ78 is a promising fasciocidal drug candidate. In order to support the development of OZ78, including pharmacokinetic (PK) studies an accurate, precise, and selective liquid chromatography/mass spectrometry (LC/MS) method for OZ78 was developed for sheep plasma and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. Protein precipitation was used for sample clean up. Separation was performed through a Phenomenex C8(2) analytical column (50.0 mm × 2.0 mm, 5 μm) with a mobile phase of acetonitrile (buffer B) and 5 mM ammonium formate (buffer A) at a flow-rate of 0.3 mL/min and a gradient from 20% to 95% acetonitrile. The mass spectrometer was operated under selected ion monitoring, and orifice voltage set to −4.1 kV and ion spray temperature to 400 °C. Nitrogen was used as a nebulizer, curtain, and collision gas. OZ78 was monitored at 321.4 m/z (deprotonated parent compound, M-). The validated linear dynamic range was between 156.25 ng/mL and 5 μg/mL and the achieved correlation coefficient (r²) was greater than 0.99. The validation results demonstrated that the developed LC/MS method is precise, accurate, and selective for the determination of OZ78 in sheep plasma. The method was successfully applied to the evaluation of the PK profile of OZ78 in sheep.     

Recovery of fat snook, Centropomus parallelus (Teleostei: Perciformes) after subchronic exposure to copper

We studied the recovery of juvenile fat snook (Centropomus parallelus) after subchronic exposure to different concentrations of copper. Healthy juveniles (1.98 g) were exposed to 25 or 50 μg Cu/L for 30 days (12 replicates with 5 fish in each one), and recovery was observed at 0, 4, 10, and 30 days after exposure (3 replicates with 5 fish in each one). Copper genotoxicity in exposed individuals was observed using a micronucleus assay, and recovery was not observed even 30 days post-exposure. Copper accumulation was observed in fish exposed to 25 or 50 μg/L of copper in the gills (14.4 and 34.4 μg/g, respectively) and muscle (5.7 and 5.5 μg/g, respectively), and a return to normal copper levels (6.0 μg/g for gills and 2.5 μg/g for muscle) was observed 4 and 30 days post-exposure in the gills and muscle tissues, respectively. Glutathione S-transferase (GST) was 80% inhibited in individuals exposed to copper and returned to normal levels for fish exposed to basal concentrations within 10 days. Although copper accumulation in tissues dispersed 30 days post-exposure, no recovery from genotoxicity was observed during this time. Thirty days was not enough to recover juvenile fat snook following subchronic exposure to copper.     

Intracellular ciliated protozoal infection in silverlip pearl oysters, Pinctada maxima (Jameson, 1901)

The pathology associated with an intracellular ciliate infection in the digestive gland of pearl oysters Pinctada maxima (Jameson, 1901) is described. Histopathological and transmission electron microscopic examination were used to characterise the organism and its location within host cells. The parasite is tear-drop shaped measuring 5.53 μm (range of 2.73-7.47 μm, n = 9) in width and 11.15 μm (range of 9.02-16.2 μm) in length with a centrally located lobulated nucleus and a large nucleus:cytoplasmic ratio. The ciliate has nine evenly spaced rows of cilia running obliquely along the length of cell, converging on the pointed end. Infected digestive glands typically had a moderate to severe infiltration with mononuclear hemocyte. A strong correlation existed between the burden of ciliates and the host response; (p < 0.001, C = 0.315 Pearson Correlation). The use of a single tissue section upon microscopic examination was found to detect only 38-50% of the infections. However, examination of serial haematoxylin and eosin stained sections improved the reliability of detecting infection.     

Steinernema ichnusae sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from Sardinia Island (Italy)

A new Steinernema species was isolated from three different sandy soil samples along the Platamona Beach, in the north-west coast of Sardinia Island (Italy). This new species is characterized by the following morphological characters: infective third-stage juvenile with a body length of 866 ± 61 (767-969) μm, distance from head to excretory pore of 63 ± 2.7 (59-68) μm, tail length of 81 ± 3.2 (76-89) μm, ratio E (%) 77 ± 3.4 (68-83); male tail with a mucron only in the second generation, spicule length of 66 ± 1.4 (64-67) μm and gubernaculum length of 44 ± 1.4 (43-46) μm in the first generation male; female of first generation with a slight vulval protrusion and ratio D (%) of 53 ± 4.0 (47-63). The new species differs distinctly from the related species (S. feltiae, S. kraussei, S. litorale, S. oregonense and S. cholashanense) in some morphometric values such as percentage of hyaline portion, ratios of gubernaculum/spicule length, spicule head length/width. The DNA analyses of the internal transcribed spacers and D2D3 regions show that the studied nematode isolates are a new species. Cross hybridisation tests with S. feltiae, S. kraussei, S. litorale, S. weiseri and S. oregonense showed that these species were reproductively isolated.     

Trophic interactions within the microbial food web in the South China Sea revealed by size-fractionation method

To define nanoflagellate-bacteria interactions and potential trophic levels within the microbial food web in the oligotrophic South China Sea, we conducted fourteen size-fractionation experiments in which seawater was filtered through 1, 2, 5, 10, 20, 60, and 200 μm membranes or meshes and the growth of four groups of picoplankton, Prochlorococcus, Synechococcus, high DNA heterotrophic bacteria, and low DNA heterotrophic bacteria were monitored in each filtrate after 24 hours of incubation. Removing grazers by filtration would relieve the grazing pressure on lower trophic levels which finally influenced the net growth rates of picoplankton. The growth patterns of Prochlorococcus and Synechococcus were similar, with higher growth rates in the < 1 μm or < 2 μm treatments, a second peak in the < 10 μm treatments and often a third peak in the < 200 μm treatments. The net growth rates of low DNA heterotrophic bacteria were little influenced by size-fractionation. Due to a subgroup of high DNA heterotrophic bacteria with larger size and higher DNA content which appeared to resist the grazing by < 5 μm nanoflagellates, the net growth rates of high DNA heterotrophic bacteria were higher in the < 2 μm or < 5 μm treatments with a second peak in the < 60 μm treatments. A general pattern of five potential trophic levels (< 2 μm, 2-5 μm, 5-10 μm, 10-60 μm, 60-200 μm) was revealed combining all the experiments, confirming the existence of multiple trophic levels within the microbial food web in the oligotrophic South China Sea.     

Copper accumulation and oxidative stress in the sea anemone, Aiptasia pallida, after waterborne copper exposure

Copper is a common marine pollutant yet its effects on symbiotic cnidarians are largely understudied. To further understand the impact of elevated copper concentrations on marine symbiotic organisms, toxicity tests were conducted using the model sea anemone, Aiptasia pallida, with and without its zooxanthellae symbiont. Symbiotic and aposymbiotic A. pallida were exposed to sublethal copper concentrations (0, 5, 15, and 50 µg/L) for 7 d and copper accumulation, behavior, and the activity of the oxidative stress enzymes, superoxide dismutase (SOD), and catalase (CAT) were measured. Additionally, acute 96-h toxicity tests were conducted to determine LC₅₀ values of the organisms after copper exposure. Both symbiotic and aposymbiotic A. pallida rapidly accumulated copper in a time and dose dependent manner. However, higher copper concentrations accumulated in the aposymbiotic as compared to the symbiotic A. pallida. In response to the highest two copper exposures (15 and 50 µg/L) symbiotic A. pallida upregulated CAT activity to combat the damaging effects of hydrogen peroxide. Contrary to these results, SOD activity significantly decreased during the highest copper exposure, when compared to controls. CAT activity was not detected and SOD was substantially (> 10 fold) reduced in aposymbiotic A. pallida, suggesting that the zooxanthellae are associated with the oxidative stress response. Copper exposure as low as 5 µg/L caused tentacle retraction and increased mucus production in both symbiotic and aposymbiotic anemones. The LC₅₀ values for symbiotic and aposymbiotic A. pallida exposed to copper for 96 h were 148 µg/L (95% confidence interval = 126.4, 173.8) and 206 µg/L (95% confidence interval = 175.2, 242.2), respectively. Understanding the varying responses of symbiotic and aposymbiotic A. pallida to copper stress may advance our comprehension of the functional roles of zooxanthellae and host. Although the mechanism of copper toxicity has not been fully elucidated, it is clear that A. pallida accumulate copper and are sensitive, as effects were detected at environmentally relevant copper concentrations. Likewise, A. pallida may be useful in biomonitoring copper polluted environments.     

Simultaneous determination of isoniazid,rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry

A rapid and selective high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250 mm × 4.6 mm, 5.0 μm, USA) at 25 °C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0 ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11–5.42 μg/g in tissues and 0.18–9.04 μg/ml in plasma, for RFP were 0.12–1200 μg/g in tissues and 4.0–200 μg/ml in plasma, and for LVX were 0.13–26.2 μg/g in tissues and 0.09–4.53 μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13 μg/g and for those in mouse plasma were 18.1, 20.0 and 21.8 ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05 μg/g and for those in mouse plasma were 5.5, 6.0 and 6.6 ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.