Wafer-scale nanowell array patterning based electrochemical impedimetric immunosensor

We have reported that nanowell array (NWA) can enhance electrochemical detection of molecular binding events by controlling the binding sites of the captured molecules. Using NWA biosensor based amperometric analysis, we have detected biological macromolecules such as DNA, protein or aptamers at low concentrations. In this research, we developed an impedimetric immunosensor based on wafer-scale NWA for electrochemical detection of stress-induced-phosphoprotein-1 (STIP-1). In order to develop NWA sensor through the cost-effective combination of high-throughput nanopattern, the NWA electrode was fabricated on Si wafer by krypton-fluoride (KrF) stepper semiconductor process. Finally, 12,500,000 ea nanowell with a 500 nm diameter was fabricated on 4 mm × 2 mm substrate. Next, by using these electrodes, we measured impedance to quantify antigen binding to the immunoaffinity layer. The limit of detection (LOD) of the NWA was improved about 100-fold compared to milli-sized electrodes (4 mm × 2 mm) without an NWA. These results suggest that wafer-scale NWA immunosensor will be useful for biosensing applications because their interface response is appropriate for detecting molecular binding events.     

Genetic predisposition to obesity and risk of subclinical atherosclerosis

Obesity has been associated with increased common carotid artery (CCA) intima–media thickness (IMT), a measure of subclinical atherosclerosis. We assessed the association between genetic predisposition to obesity and CCA IMT. The study included 428 young Chinese adults with CCA IMT measured using a high-resolution B-mode tomographic ultrasound system. We created a genetic risk score (GRS) by summing the risk alleles of 6 obesity-associated genetic variants confirmed in our previous analyses. The GRS was significantly associated with greater CCA IMT (p < 0.001) after adjustment for age and gender. Per 2 alleles of the GRS was related to 0.023 mm increment in IMT. The association was attenuated by one half with additional adjustment for obesity status, but remained significant (p = 0.009). In addition, we found that blood pressure significantly modified the association between the GRS and CCA IMT (p for interaction = 0.001). The associations between the GRS and CCA IMT were stronger in participants with systolic blood pressure (SBP) ≥ 120 mm Hg and/or diastolic blood pressure (DBP) ≥ 80 mm Hg (per 2 allele increment of the GRS relating to 0.028 mm greater CCA IMT, p for trend < 0.001) than those with SBP < 120 mm Hg and DBP < 80 mm Hg (per 2 allele increment of the GRS relating to 0.001 smaller CCA IMT, p for trend = 0.930). Our data provides suggestive evidence supporting the potential causal relation between obesity and development of subclinical atherosclerosis. Elevated blood pressure might amplify the adverse effect of obesity on cardiovascular risk.     

Application of simultaneous saccharification and fermentation (SSF) from viscosity reducing of raw sweet potato for bioethanol production at laboratory, pilot and industrial scales

The aim of this work was to research a bioprocess for bioethanol production from raw sweet potato by Saccharomyces cerevisiae at laboratory, pilot and industrial scales. The fermentation mode, inoculum size and pressure from different gases were determined in laboratory. The maximum ethanol concentration, average ethanol productivity rate and yield of ethanol after fermentation in laboratory scale (128.51 g/L, 4.76 g/L/h and 91.4%) were satisfactory with small decrease at pilot scale (109.06 g/L, 4.89 g/L/h and 91.24%) and industrial scale (97.94 g/L, 4.19 g/L/h and 91.27%). When scaled up, the viscosity caused resistance to fermentation parameters, 1.56 AUG/g (sweet potato mash) of xylanase decreased the viscosity from approximately 30000 to 500 cp. Overall, sweet potato is a attractive feedstock for be bioethanol production from both the economic standpoints and environmentally friendly.     

Echinococcus multilocularis: Single hepatic lesion experimentally established without metastasis in rats

We herein describe the establishment of single hepatic lesions of Echinococcus multilocularis in rats. A 3 mm incision was made on the liver with a surgical knife, and one small round vesicle of E. multilocularis (between 1 × 1 mm and <2 × 2 mm in diameter) was transplanted into the incision and covered with absorbable hemostat gauze. The presence and growth of the transplanted vesicle was monitored for 12 weeks using magnetic resonance imaging (MRI). Hepatic lesions, the metacestode of this parasite were confirmed in 12 of 17 infected rats (70.6%) by MRI and macroscopic examinations. The average size of the metacestodes with brood capsules at 12 weeks after the experimental transplantation of a single vesicle was 6.1 ± 2.5 mm × 4.4 ± 1.5 mm. The smallest size of the metacestodes detected by MRI was approximately 3 × 3 mm. This new approach of establishing single hepatic metacestodes of E. multilocularis in experimental animals is expected to be useful for analyzing the immune-pathological mechanisms of hepatic AE.     

An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-Retinol (Vitamin A) and α-Tocopherol (Vitamin E) in human serum: Comparison of different particulate reversed-phase HPLC columns

A novel, simple and fast reversed-phase HPLC/UV method was developed, optimized for various chromatographic conditions, and validated according to international guidelines for simultaneous determination of all-trans-retinol and α-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5 μg/ml. A liquid-phase extraction was applied to the 250 μl of serum with n-hexane–dichloromethane mixture (70:30, v/v), in two steps, using ethanol–methanol mixture (95:5, v/v) for protein precipitation and BHT (butylated hydroxy toluene) as stabilizer for sample preparation. Both analytes were analyzed on Kromasil 100 C₁₈ column (150 mm × 4.6 mm, 5 μm), Brownlee analytical (Perkin Elmer) C₁₈ column (150 mm × 4.6 mm, 5 μm), and Supelco (Supelcosil) LC-18 column (150 mm × 3 mm, 3 μm), protected by a Perkin Elmer C₁₈ (30 mm × 4.6 mm, 10 μm; Norwalk, USA) pre-column guard cartridge, at 292 nm wavelength, using methanol–water (99:1, v/v), in isocratic mode as mobile phase applied at flow rate of 1.5 ml/min and 1 ml/min for both 5 μm and 3 μm columns, respectively. Complete separation of all the analytes was achieved in 3 and 6 min on 3 μm and 5 μm columns, respectively by injecting 20 μl of sample into the HPLC system by autosampler, keeping column oven temperature at 25 °C. Different particulate reversed-phase chromatographic columns were evaluated in order to select the best column in terms of sensitivity, selectivity, resolution and short run time of both the analytes and it was concluded that 3 μm columns are better to be used in clinical set up as well as in laboratories for the separation of these analytes in a shorter time as compared with 5 μm columns. The method was validated and applied for the analysis of all-trans-retinol and α-tocopherol in the serum of human volunteers.     

Olive (Olea europaea) leaf extract effective in patients with stage-1 hypertension: Comparison with Captopril

A double-blind, randomized, parallel and active-controlled clinical study was conducted to evaluate the anti-hypertensive effect as well as the tolerability of Olive leaf extract in comparison with Captopril in patients with stage-1 hypertension. Additionally, this study also investigated the hypolipidemic effects of Olive leaf extract in such patients. It consisted of a run-in period of 4 weeks continued subsequently by an 8-week treatment period. Olive (Olea europaea L.) leaf extract (EFLA^®943) was given orally at the dose of 500 mg twice daily in a flat-dose manner throughout the 8 weeks. Captopril was given at the dosage regimen of 12.5 mg twice daily at start. After 2 weeks, if necessary, the dose of Captopril would be titrated to 25 mg twice daily, based on subject's response to treatment. The primary efficacy endpoint was reduction in systolic blood pressure (SBP) from baseline to week-8 of treatment. The secondary efficacy endpoints were SBP as well as diastolic blood pressure (DBP) changes at every time-point evaluation and lipid profile improvement. Evaluation of BP was performed every week for 8 weeks of treatment; while of lipid profile at a 4-week interval. Mean SBP at baseline was 149.3 ± 5.58 mm Hg in Olive group and 148.4 ± 5.56 mm Hg in Captopril group; and mean DBPs were 93.9 ± 4.51 and 93.8 ± 4.88 mm Hg, respectively. After 8 weeks of treatment, both groups experienced a significant reduction of SBP as well as DBP from baseline; while such reductions were not significantly different between groups. Means of SBP reduction from baseline to the end of study were −11.5 ± 8.5 and −13.7 ± 7.6 mm Hg in Olive and Captopril groups, respectively; and those of DBP were −4.8 ± 5.5 and −6.4 ± 5.2 mm Hg, respectively. A significant reduction of triglyceride level was observed in Olive group, but not in Captopril group. In conclusion, Olive (Olea europaea) leaf extract, at the dosage regimen of 500 mg twice daily, was similarly effective in lowering systolic and diastolic blood pressures in subjects with stage-1 hypertension as Captopril, given at its effective dose of 12.5-25 mg twice daily.     

Improving the specific activity of β-mannanase from Aspergillus niger BK01 by structure-based rational design

β-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic β-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57 Å crystal structure of ManBK. The protein adopts a typical (β/α)₈ fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18 ± 2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher k_cat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications.     

Transpiration and canopy conductance in an inner alpine Scots pine (Pinus sylvestris L.) forest

Canopy transpiration (E_c) of a 150-year-old Pinus sylvestris L. stand in an inner Alpine dry valley, Tyrol, Austria was estimated throughout two growing seasons 2011 and 2012 by means of xylem sap flow measurements. Although there were prolonged periods of limited soil water availability, E_c did not show a clear trend with respect to soil water availability and averaged 0.4 ± 0.19 mm day⁻¹ under conditions of non-limiting soil water availability and 0.37 ± 0.17 mm day⁻¹ when soil water availability was limited. This is because canopy conductance declined significantly with increasing evaporative demand and thus significantly reduced tree water loss. The growing season total of E_c was 74 mm and 88 mm in 2011 and 2012, respectively, which is significantly below the values estimated for other P. sylvestris forest ecosystems in Central Europe, and thus reflecting a strong adaptation to soil drought during periods of high evaporative demands.     

Determination of the γ-secretase inhibitor MK-0752 in human plasma by online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS)

A sensitive and rapid HTLC–ESI-MS/MS method with an advanced online sample preparation was developed for determination of the γ-secretase inhibitor MK-0752 in human plasma using an internal standard. Plasma samples (100 μL) were diluted and injected directly onto an online extraction column (Cohesive Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed out with an aqueous solution, and retained analytes were eluted out and transferred directly to the analytical column (Phenomenex Gemini 3μ C18 110A, 50 mm × 2.0 mm at 50 °C) for separation using a gradient mobile phase. The eluted analytes were then detected on an API-3000 LC–MS/MS System with ESI and a negative multiple reaction monitoring mode. The monitored ion transitions were m/z 441 → 175 for MK-0752 and 496 → 175 for the internal standard. Online extraction recoveries were 81%. The method was validated and was linear in the range of 0.05–50 μg/mL. Within-day and between-day precisions were < 8.6%, and accuracies were 0.7 and 7.1%. This method was applied to the measurement of plasma MK-0752 levels in a Phase I study of pediatric patients with recurrent or refractory brain tumors.     

Determination of cefazedone in human plasma by high performance liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study on Chinese volunteers

A rapid, sensitive and simple high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for determination of cefazedone in human plasma using metronidazole as internal standard (IS). The chromatographic separation was achieved on an Ultimate XB-CN column (2.1 mm × 150 mm, 5 μm) with an isocratic mobile phase of acetonitrile and 20 mM ammonium acetate in 0.1% formic acid in water (15:85, v/v). Detection was performed using electrospray ionization in positive ion multiple reaction-monitoring mode (SRM), monitoring the transitions m/z 548.2 → 344.1 for cefazedone and m/z 172.2 → 128.1 for IS. Calibration curves were linear over a wide range of 0.20–401.12 μg/mL for cefazedone in plasma. The lower limit of quantification (LLOQ) was 0.20 μg/mL. The intra- and inter-day precisions were less than 7.2%. The average recovery of cefazedone was 90.8–91.0%. The validated method was successfully applied to the pharmacokinetic study of cefazedone in Chinese healthy volunteers following intravenous (IV) administration of 500, 1000 and 2000 mg cefazedone injection.     

A microfluidic microbial fuel cell fabricated by soft lithography

Here we report a new microfluidic microbial fuel cell (MFC) platform built by soft-lithography techniques. The MFC design includes a unique sub-5 μL polydimethylsiloxane soft chamber featuring carbon cloth electrodes and microfluidic delivery of electrolytes. Bioelectricity was generated using Shewanella oneidensis MR-1 cultivated on either complex organic substrates or lactate-based minimal medium. These micro-MFCs exhibited fast start-ups, reproducible current generation, and enhanced power densities up to 62.5 W m⁻³ that represents the best result for sub-100 μL MFCs. Systematic comparisons of custom-made MFC reactors having different chamber sizes indicate volumetric power density is inversely correlated with chamber size in our systems: i.e., the smaller the chamber, the higher the power density is achieved.     

Lambing rates and litter size following carazolol administration prior to insemination in Kivircik ewes

The effect of carazolol on the ease of penetrating the cervix during artificial insemination, lambing rate and litter size was studied using 1.5–4.0-year old Kivircik ewes in an incomplete 3 × 2 × 2 experimental design. All of the ewes in this study were synchronized for oestrus by insertion of a progesterone impregnated vaginal sponge for 12 days and administration of 400 IU PMSG at sponge withdrawal. Three methods of service were compared: natural service, artificial insemination (AI) with fresh semen, or AI with frozen semen. Two times of insemination (fixed time AI versus AI at observed oestrus) were compared on the fresh and frozen AI treatments. The absence (control) or use of carazolol (carazolol; 0.5 mg/ewe i.m. 30 min before mating) was the third factor in the design and penetration of the cervix by the insemination pipette was assessed as shallow (<10 mm), middle (10–20 mm) or deep (>20 mm). Natural service ewes were only mated at observed oestrus. Consequently, the factorial design was incomplete and there were a total of 10 treatments each represented by 30 ewes. Natural service resulted in a significantly (P < 0.05) higher lambing rate and litter size (86%; 2.0 ± 0.05 lambs/ewe) than AI using fresh (65%; 1.6 ± 0.1 lambs/ewe) or frozen (40%; 1.4 ± 0.14 lambs/ewe) semen. For AI animals the lambing rate and litter size were not significantly different when service was at a fixed time (50%; 1.5 ± 0.12 lambs/ewe) or at observed oestrus (56%; 1.5 ± 0.12 lambs/ewe). Carazolol did not permit complete cervical penetration in any ewe. Deep penetration of the cervix at AI was achieved in 33% of untreated (control) and 48% of carazolol treated ewes (P < 0.05). However, the proportion of ewes in which penetration of the cervix and semen deposition was greater than shallow was similar for control (82%) and carazolol (85%), and lambing rate and litter size were similar for both treatments. Over the three service methods, the lambing rate was 56% for control and 63% for carazolol (NS) and litter size was similar for both treatments. It was concluded that the carazolol treatment used prior to natural mating or AI in this experiment did not improve lambing rate or litter size in Kivircik ewes.     

Deposit- and suspension-feeding sea cucumbers (Echinodermata) ingest plastic fragments

Weathering of plastic bottles, bags, fishing line, and other products discarded in the ocean causes tiny fragments to break off. These plastic fragments may accumulate biofilms, sink, and become mixed with sediment, where benthic invertebrates may encounter and ingest them. Here we show that four species of deposit-feeding and suspension-feeding sea cucumbers (Echinodermata, Holothuroidea) not only ingest small (0.25 mm < maximum dimension < 15 mm) nylon and polyvinyl chloride (PVC) fragments along with sediment, but also ingest significantly more plastic fragments than predicted given the ratio of plastic to sand grains in the sediment. During four-hour feeding trials, holothurians ingested between 2- and 20-fold more plastic per individual than expected for PVC fragments, and between 2- and 138-fold more for nylon line. In addition, two species ingested 4 mm diameter PVC pellets. The ecological relevance of plastic ingestion was assessed in the laboratory by counting and characterizing small plastic particles discovered in sediment samples from the same field sites where our holothurians were collected. Substantial numbers of plastic fragments (105 to 214 fragments per liter of sediment) were found in samples from three different locations along the east coast of the U.S.A. In addition, plastic collected from the sediment from two of our field sites was analyzed for polychlorinated biphenyls (PCBs). Plastic from one site tested positive for Aroclor 1254 at a concentration of 0.0106 μg g^-1. While the negative effects of macroscopic marine plastic debris on a host of organisms are well documented, ingestion of small plastic debris by a wide range of benthic organisms, including both primary and secondary consumers, has received little attention. Given that plastics readily adsorb PCBs and other organic pollutants in marine environments, ingestion of plastic from sediment may provide a heretofore-undescribed pathway of exposure for benthic marine invertebrates.     

Ecotypes of wild rooibos (Aspalathus linearis (Burm. F) Dahlg., Fabaceae) are ecologically distinct

Aspalathus linearis (Burm. F) Dahlg., Fabaceae is cultivated by small- and large-scale commercial farmers of the Cederberg and Bokkeveld Plateau in the Cape Floristic Region of South Africa, for the production of an herbal beverage called ‘rooibos’ or ‘rooibos tea’. Small-scale farmers also harvest A. linearis from the wild and market the tea as an organic and fair-trade certified product. However, little is known about the apparent ecotypes of wild A. linearis. We hypothesized that 1) rooibos ecotypes are ecologically distinct (occurring in different plant communities defined by environmental variables); 2) rooibos ecotypes are functionally distinct due to variance in water use efficiency; and 3) rooibos ecotypes are associated with threatened vegetation types/species, making populations of wild rooibos ecotypes worthy of conservation. Ecotypes of wild rooibos were identified based on plant habit and local knowledge. Plant communities were classified using Twinspan, environmental factors were tested as predictors of ecotype distribution and water use efficiency of ecotypes growing across a rainfall gradient was determined from foliar natural abundance of ¹³C. Wild rooibos was not generally associated with endangered vegetation types but was associated with plant species having endangered status. Wild rooibos occurred in four plant communities and comprised five wild rooibos ecotypes: shrub, tree, upright, salignus and prostrate types. Although some ecotypes clearly co-occurred, evidence is provided for habitat preference between the ecotypes: Prostrate and upright ecotypes occurred at higher elevations (> 400-600 m). Shrub ecotypes occurred at lower rainfall sites (< 200 mm p.a.) and the salignus ecotype occurred at higher rainfall sites (> 500 mm p.a.). Foliar ¹³C indicated greater water use efficiencies by ecotypes in relatively drier areas. The extent to which this is a plastic or inherent response requires further investigation. Considering that wild rooibos ecotypes differ both ecologically and possibly also functionally and genetically, it is concluded that populations of wild rooibos ecotypes should be considered as distinct and worthy of conservation. This distinctness should be considered when farmers apply for both harvesting and ploughing rights on land with wild rooibos ecotypes.     

Effect of formation of biofilms and chemical scale on the cathode electrode on the performance of a continuous two-chamber microbial fuel cell

A two-chamber MFC system was operated continuously for more than 500 days to evaluate effects of biofilm and chemical scale formation on the cathode electrode on power generation. A stable power density of 0.57 W/m² was attained after 200 days operation. However, the power density decreased drastically to 0.2 W/m² after the cathodic biofilm and chemical scale were removed. As the cathodic biofilm and chemical scale partially accumulated on the cathode, the power density gradually recovered with time. Microbial community structure of the cathodic biofilm was analyzed based on 16S rRNA clone libraries. The clones closely related to Xanthomonadaceae bacterium and Xanthomonas sp. in the Gammaproteobacteria subdivision were most frequently retrieved from the cathodic biofilm. Results of the SEM-EDX analysis revealed that the cation species (Na⁺ and Ca²⁺) were main constituents of chemical scale, indicating that these cations diffused from the anode chamber through the Nafion membrane. However, an excess accumulation of the biofilm and chemical scale on the cathode exhibited adverse effects on the power generation due to a decrease in the active cathode surface area and an increase in diffusion resistance for oxygen. Thus, it is important to properly control the formation of chemical scale and biofilm on the cathode during long-term operation.     

Scalable air cathode microbial fuel cells using glass fiber separators, plastic mesh supporters, and graphite fiber brush anodes

The combined use of brush anodes and glass fiber (GF1) separators, and plastic mesh supporters were used here for the first time to create a scalable microbial fuel cell architecture. Separators prevented short circuiting of closely-spaced electrodes, and cathode supporters were used to avoid water gaps between the separator and cathode that can reduce power production. The maximum power density with a separator and supporter and a single cathode was 75 ± 1 W/m³. Removing the separator decreased power by 8%. Adding a second cathode increased power to 154 ± 1 W/m³. Current was increased by connecting two MFCs connected in parallel. These results show that brush anodes, combined with a glass fiber separator and a plastic mesh supporter, produce a useful MFC architecture that is inherently scalable due to good insulation between the electrodes and a compact architecture.     

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.     

Quantification of CLR1401, a novel alkylphosphocholine anticancer agent,in rat plasma by hydrophilic interaction liquid chromatography–tandem mass spectrometric detection

A rapid and specific LC–MS/MS based bioanalytical method was developed and validated for the determination of 18-(p-iodophenyl)octadecyl phosphocholine (CLR1401), a novel phosphocholine drug candidate, in rat plasma. The optimal chromatographic behavior of CLR1401 was achieved on a Kromasil silica column (50 mm × 3 mm, 5 μm) under hydrophilic interaction chromatography. The total LC analysis time per injection was 2.8 min with a flow rate of 1.5 mL/min under gradient elution. Liquid–liquid extraction in a 96-well format using ethyl acetate was developed and applied for method validation and sample analysis. The method validation was conducted over the curve range of 2.00–1000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 5.9% relative standard deviation (RSD) and −10.8 to −1.4% relative error (RE). The method was successfully applied to determine the toxicokinetics of CLR1401 in rats from three dose groups of 0.4, 4.0, and 10.0 mg/kg/day via intravenous administration.