Suitable Buffer Research Articles

\u03b3-Secretase modulators exhibit selectivity for modulation of APP cleavage but inverse \u03b3-secretase modulators do not

Backgroundγ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs.MethodsFor cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry.ResultThese studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM.ConclusionCollectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.

Stable all-solid-state battery enabled with Li6.25PS5.25Cl0.75 as fast ion-conducting electrolyte

The Li 6.25 PS 5.25 Cl 0.75 fast ion-conducting electrolyte together with LiNbO 3 coated LiCoO 2 as cathode and lithium foil as the anode can realize improved performance. All-solid-state batteries (ASSB) with lithium anode have attracted ever-increasing attention towards developing safer batteries with high energy densities. While great advancement has been achieved in developing solid electrolytes (SE) with superb ionic conductivity rivalling that of the current liquid technology, it has yet been very difficult in their successful application to ASSBs with sustaining rate and cyclic performances. Here in this work, we have realized a stable ASSB using the Li 6.25 PS 5.25 Cl 0.75 fast ion-conducting electrolyte together with LiNbO 3 coated LiCoO 2 as cathode and lithium foil as the anode. The effective diffusion coefficient of Li-ions in the battery is higher than 10 −12 cm 2 s −1 , and the significantly enhanced electrochemical matching at the cathode-electrolyte interface was essential to enable long-term stability against high oxidation potential, with the LCO@LNO/Li 6.25 PS 5.25 Cl 0.75 /Li battery to retain 74.12% capacity after 430 cycles at 100 μA cm −2 and 59.7% of capacity after 800 cycles at 50 μA cm −2 , at a high charging cut-off voltage of 4.2 V. This demonstrates that the Li 6.25 PS 5.25 Cl 0.75 can be an excellent electrolyte for the realization of stable ASSBs with high-voltage cathodes and metallic lithium as anode, once the electrochemical compatibility between cathode and electrolyte can be addressed with a suitable buffer coating.

Surface Molecular Imprinting with Bacteria: Visualizing Re-Binding and Selectivity

Introduction Surface molecular imprinting [1,2] is one of the preferred techniques when it comes to generating biomimetic recognition for sensing bioanalytes. Limiting oneself to the surface of the respective molecularly imprinted polymer (MIP) usually is no problem, because biospecies are usually large enough that few binding events lead to measurable sensor responses, e.g. for mass-sensitive bacteria detection [3]. However, the detection limits of quartz crystal microbalances (QCM) do usually not meet the needs for detecting contamination of real-life samples when it comes to pathogen detection. The (geometrical) limitation of surface MIP in that regard makes it necessary to think about suitable systems for pre-concentration prior to the sensor measurement. Herein, we report Raman Microscopy studies on bacteria surface MIP aiming at visualizing different areas of the polymer and distinguishing between different species bound to the surface. Furthermore, we assess the binding properties of MIP resulting from Pickering emulsion polymerization [4] as a possible approach to selectively pre-concentrate bacteria. Experimental For molecular imprinting, we chose a non-pathogenic strain of Escherichia coli (E. coli) as a model species. We prepared surface MIP of two different morphologies based on polystyrenes cross-linked with divinyl benzene utilizing azo-bisisobutyronitrile (AIBN) as a radical initiator. For MIP thin films, we prepared respective mixtures of monomer and crosslinker, pre-polymerized at 70°C, and then spin-coated onto the respective devices. In parallel, we prepared stamps comprising E. coli on their surfaces. Then, we pressed the stamp into the oligomer thin films on the respective surface and cured the system at 80°C over night. We took all spectra on a confocal Raman Microscopy system (WiTec alpha300RAS) at an excitation wavelength of l=532nm. Chemometric data evaluation relied on the software package Solo&Mia by Eigenvector. For Pickering emulsion, we prepared mixtures of styrene and divinylbenzene and used bacteria to emulsify them in either distilled water, or a suitable buffer system (e.g. 10mM PBS) and polymerized the bacteria stabilized droplets at 37°C. Then, we removed E. coli cells from the polymer surface via solvent extractions in acetic acid + SDS, water and methanol. Results and Discussion In a first step it was essential to clarify if one can use the Raman spectra of different bacteria species and polymer, to differentiate between those, respectively. Polymer and bacteria spectra obviously differ from each other enough to distinguish them by the naked eye. However, this is not the case for the Raman emission spectra of different bacteria species, because they all contain nucleic acids, proteins, and lipids. Therefore it is necessary to rely on chemometric strategies – modified partial least squares discriminant analysis (PLS-DA) in the concrete case – to achieve such goal. This is indeed possible: Fig. 1A shows the outcome for four different bacteria species, namely Escherichia coli, Lactococcus lactis, Bacillus cereus and Staphylococcus epidermidis that clearly demonstrates separation of the species according to their spectral properties. Knowing the specific spectral ranges also allows for generating false-color images showing which areas of a bacteria MIP surfaces reveal pure polymer and which parts contain bacteria of different species. It is even possible to go one step further: Comparing AFM and false color so-called RamanTV images (see Fig. 1B) for an example of the latter) demonstrates that the Raman microscope is even useful for distinguishing polymer surface and imprinted cavities. For such micrometer-sized analytes, the combination of Raman microscopy and AFM hence allows for generating maps showing all possible types of surfaces, i.e. polymer matrix as well as both unoccupied cavities and different bacteria species present at the polymer surface. This opens the perspective of directly visualizing occupancy and selectivity of the respective MIP.AFM turned out useful to visualize such imprints not only on flat surfaces, but also on curved ones: Figure 1C) and 1D) reveal an imprinted site on the surface of a miroparticle resulting from emulsion polymerization, Fig. 1E) an SEM image showing occupied and empty cavities. One can generate such cavities in different polymer matrices, for instance when replacing divinyl benzene as a cross-linker by trimethyolylpropane trimethacrylate (TRIM), or ethylene glycol dimethacrylate (EGDMA). In both cases, the respective imprinted surface reveal cavities that fit the size and shape of the template bacteria. Scanning electron microscopy also revealed that bacteria re-occupy those cavities if one exposes the particles to to bacteria suspensions. This leads to two conclusions: first, one can tune polymer properties without destroying the emulsion structure necessary for Pickering emulsion. This is of interest for bringing the artificial system closer to physiological conditions. Second, those particles are inherently suitable for selectively pre-concentrating those species from larger samples.

High‐Efficiency CsPbI2Br Perovskite Solar Cells with Dopant‐Free Poly(3‐hexylthiophene) Hole Transporting Layers

AbstractCsPbI2Br is emerging as a promising all‐inorganic material for perovskite solar cells (PSCs) due to its more stable lattice structure and moisture resistance compared to CsPbI3, although its device performance is still much behind this counterpart. Herein, a preannealing process is developed and systematically investigated to achieve high‐quality CsPbI2Br films by regulating the nucleation and crystallization of perovskite. The preannealing temperature and time are specifically optimized for a dopant‐free poly(3‐hexylthiophene) (P3HT)‐based device to target dopant‐induced drastic performance degradation for spiro‐OMeTAD‐based devices. The resulting P3HT‐based device exhibits comparable power conversion efficiency (PCE) to spiro‐OMeTAD‐based devices but much enhanced ambient stability with over 95% PCE after 1300 h. A diphenylamine derivative is introduced as a buffer layer to improve the energy‐level mismatch between CsPbI2Br and P3HT. A record‐high PCE of 15.50% for dopant‐free P3HT‐based CsPbI2Br PSCs is achieved by alleviating the open‐circuit voltage loss with the buffer layer. These results demonstrate that the preannealing processing together with a suitable buffer layer are applicable strategies for developing dopant‐free P3HT PSCs with high efficiency and stability.

Solubilization of Human Interferon β-1b Inclusion Body Proteins by Organic Solvents.

Purposes: Solubilization of inclusion bodies expressed in E. coli is a critical step during manufacturing of recombinant proteins expressed as inclusion bodies. So far, various methods have been used for solubilization and purification of inclusion body proteins to obtain active proteins with high purity and yield. The aim of this study was to examine the benefit of organic solvents such as alcohols in solubilization of recombinant interferon β-1b inclusion bodies. Methods: Effect of important parameters inclusion pH, concentration and type of denaturant and concentration of alcoholic solvents were optimized to formulate a suitable solubilization buffer and investigate their effect on solubilization of interferon β-1b inclusion bodies. Results: Our findings showed the acidic pH in the range of 2-3 is more suitable than alkaline pH >12 for solubilization and achieving higher content of interferon β-1beta and pure recombinant protein. We have also demonstrated that 1% SDS acts better than 2M urea to solubilize Inclusion body proteins of interferon β-1b at pH of 2-3. The interferon concentration was 2.35 mg per 100 mg IB when we used 40% (v/v) 1-propanol and 20% (v/v) 2-butanol into the buffer solution as well. Conclusion: The optimized method provides gentile condition for solubilization of inclusion body at high protein concentration and purity with a degree of retention of native secondary structure which makes this method valuable to be used in production and research area.

Rooibos (Aspalathus linearis) Genome Size Estimation Using Flow Cytometry and K-Mer Analyses.

Plant genomes provide information on biosynthetic pathways involved in the production of industrially relevant compounds. Genome size estimates are essential for the initiation of genome projects. The genome size of rooibos (Aspalathus linearis species complex) was estimated using DAPI flow cytometry and k-mer analyses. For flow cytometry, a suitable nuclei isolation buffer, plant tissue and a transport medium for rooibos ecotype samples collected from distant locations were identified. When using radicles from commercial rooibos seedlings, Woody Plant Buffer and Vicia faba as an internal standard, the flow cytometry-estimated genome size of rooibos was 1.24 ± 0.01 Gbp. The estimates for eight wild rooibos growth types did not deviate significantly from this value. K-mer analysis was performed using Illumina paired-end sequencing data from one commercial rooibos genotype. For biocomputational estimation of the genome size, four k-mer analysis methods were investigated: A standard formula and three popular programs (BBNorm, GenomeScope, and FindGSE). GenomeScope estimates were strongly affected by parameter settings, specifically CovMax. When using the complete k-mer frequency histogram (up to 9 × 105), the programs did not deviate significantly, estimating an average rooibos genome size of 1.03 ± 0.04 Gbp. Differences between the flow cytometry and biocomputational estimates are discussed.

A 0.85% saline as alternative detection buffer for SD-bioline HIV rapid test in resource limited setting

Accuracy in the diagnosis is a key step to identify HIV infected individuals for appropriate management. Insufficient supply of manufacturer´s buffers in relation to the number of strips per kit has negative impact on patient´s results hence improper patient´s management. In resource limited settings, some laboratory staff use different substitute buffers which has never been validated on their reliability. This study aimed at comparing the performance of 0.85% saline and SD-Bioline manufacturer´s buffer in detection of HIV antibodies. A total of 220 whole blood specimens: 110 HIV positive specimens from patients attending care and treatment center (CTC) and 110 HIV negative specimens from blood donors were re-tested for HIV status using SD-Bioline HIV rapid test using manufacturer´s buffer and 0.85% saline separately. Data and laboratory results were recorded in Microsoft excel sheet followed by analysis using STATA version 13. For all tested samples, the level of agreement between 0.85% saline and manufacturer´s buffer was 98.64% (kappa=0.9727). The value of kappa indicates very good agreement between 0.85% saline and manufacturer´s buffer. In incidents where manufacturer´s buffer is not sufficient, 0.85% saline can give reliable results. Further studies to evaluate the suitable buffer for other rapid tests for HIV and other diseases are recommended especially in resource limited settings.

Theoretical investigation on enhancement of output performance of CZTSSe based solar cell

CZTSSe offers cheap, environmental friendly and earth abundant material for solar devices, yet the record power conversion efficiency for such solar cells has stagnated at about 12 percent over the years. The main reason for low output voltage and overall performance is because of the extreme non radiative recombination in the heterojunction region. To find the overall optimization of solar cell and suitable buffer layer numerical simulation has been performed by SCAPS-1D software. Here, CZTSSe was used as a absorber layer, Al doped ZnO as a transparent conducting layer and ZnO as a window layer. CdS, Cd0.4Zn0.6S and ZnSe was used as a different buffer layer. The impact of thickness of absorber layer and buffer layer on Voc, Jsc, FF and efficiency has been investigated. Also, the effect of temperature on the above-mentioned parameter of the solar cell was studied.

Rapid methodology for basal system selection of therapeutic proteins during the early stage biopharmaceutical development

This study aimed to introduce a quick method for developing formulations during the early stages of biopharmaceutical drug development, starting from the isoelectric point (pI). Human insulin and etanercept, an Fc fusion protein, were used as model proteins to observe the effects of pH, buffer, and buffer concentration on their physicochemical properties. For biophysical analysis, dynamic light scattering (DLS), micro differential scanning calorimetry (μDSC), and circular dichroism (CD) were utilized to measure zeta average size with zeta potential, transition melting temperature, and secondary structure, respectively. Furthermore, an accelerated storage test was performed at 4 °C, 25 °C, and 40 °C using size exclusion chromatography (SEC) to evaluate storage stability. Zeta potential measurements showed that the practical pI of human insulin in solution was 5.8 to 6.0, and the pI of etanercept was 5.0 to 5.1. Acetate and phosphate were selected as buffering agents, and the pH range was pH 3.4 to 7.4 with different buffer concentrations, 10 mM and 50 mM. Protein stability was increased when the pH was away from the pI with a relatively high absolute zeta potential, resulting in increased conformational stability and storage stability. Response surface methodology (RSM) was also used to identify a suitable pH and buffer. The determined pH and buffer were able to suppress protein aggregation and to sustain the monomer fraction, thereby increasing protein stability. Therefore, the approach in selecting basal system could be very useful during the early stage biopharmaceutical development.

Synthesis and application of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base derivative

Discrimination and quantification of intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is significant for academic research and disease diagnosis. A new fluorescent probe (complex 1-Cu2+) for discriminate detection of GSH was prepared by copper ions coordinate with coumarin carbohydrazide Schiff base derivative 1. In suitable buffer solution (CH3CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2–7.4), the UV–vis spectroscopy experiments showed that compound 1 and copper ion exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence quenching of compound 1 was observed when it complexed with Cu2+ ions. An obviously fluorescence restoration appeared after addition of GSH to the solution of probe, which also exhibited a highly selectivity relative to cysteine (Cys) and homocysteine (Hcy) in the amino acid competitive experiments. The minimum detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly below the physiological concentration of GSH in live cells. Its biological application to detect the endogenous GSH was further proved by the HepG2 cell fluorescence image test.

Radiochemical and radiopharmacological characterization of a 64 Cu-labeled α-MSH analog conjugated with different chelators.

Radiolabeled α-melanocyte-stimulating hormone (α-MSH) derivatives have a high potential for diagnosis and treatment of melanoma, because of high specificity and binding affinity to the melanocortin-1 receptor (MC1R). Hence, the α-MSH-derived peptide NAP-NS1 with a β-Ala linker (ε-Ahx-β-Ala-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 ) was conjugated to different chelators: either to NOTA (p-SCN-Bn-1,4,7-triazacyclononane-1,4,7-triacetic acid), to a hexadentate bispidine carbonate derivative (dimethyl-9-(((4-nitrophenoxy)carbonyl)oxy)-2,4-di(pyridin-2-yl)-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate), or to DMPTACN (p-SCN-Ph-bis(2-pyridyl-methyl)-1,4,7-triaza-cyclononane), labeled with 64 Cu, and investigated in terms of radiochemical and radiopharmacological properties. For the three 64 Cu-labeled conjugates negligible transchelation, suitable buffer and serum stability, as well as appropriate water solubility, was determined. The three conjugates exhibited high binding affinity (low nanomolar range) in murine B16F10, human MeWo, and human TXM13 cells. The Bmax values of [64 Cu]Cu-bispidine-NAP-NS1 ([64 Cu]Cu-2) and [64 Cu]Cu-DMPTACN-NAP-NS1 ([64 Cu]Cu-3) were higher than those of [64 Cu]Cu-NOTA-NAP-NS1 ([64 Cu]Cu-1), implying that different charged chelate units might have an impact on binding capacity. Preliminary in vivo biodistribution studies suggested the main excretion pathway of [64 Cu]Cu-1 and [64 Cu]Cu-3 to be renal, while that of [64 Cu]Cu-2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60minutes. All three 64 Cu-labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.

Method for guiding crowd evacuation at exit: The buffer zone

To control overcrowded pedestrians at the exit, buffer zones are very common in densely populated areas. However, little focus has been paid on the mechanisms of buffer zone. In this paper, a tentative experiment was designed to preliminarily reveal the role of buffer zone in crowd evacuation. Then a social force based simulation model was established by Massmotion according to the properties of the experiment. The simulated results show that buffer zones may have a potential positive effect on evacuation through comparing with the experimental results. After that, via changing the buffer zone length and the desired velocity of agents in further simulations, the data, such as evacuation time, density distribution, individual waiting time, instantaneous velocity and average velocity were investigated in detail. It is found that after the buffer zone set, the evacuation efficiency is significantly improved at high desired velocity, and the evacuation time is shortened with the increase in buffer zone length. Setting buffer zone in front of the exit can effectively reduce the crowd density at the exit, alleviate the surgent transmission of density waves in crowd, so as to reduce the risk of crowd squeezing and trampling. Besides, when the desired velocity is relatively high, the relationship between the buffer zone length and the average velocity is obtained specifically, indicating that the longer the buffer zone, the more likely pedestrians can escape as quickly as possible. Based on this work, some suggestions were put forward on how to control the dense evacuation via setting suitable buffer zones.

Towards high-efficiency CZTS solar cell through buffer layer optimization

Cu2ZnSnS4 (CZTS)-based solar cells show a promising performance in the field of sunlight-based energy production system. To increase the performance of CZTS-based solar cell, buffer layer optimization is still an obstacle. In this work, numerical simulations were performed on structures based on CZTS absorber layer, ZnO window layer, and transparent conducting layer n-ITO with different buffer layers using SCAPS-1D software to find a suitable buffer layer. Cadmium sulfide (CdS), zinc sulfide (ZnS) and their alloy cadmium zinc sulfide (Cd0.4Zn0.6S) were used as potential buffer layers to investigate the effect of buffer thickness, absorber thickness and temperature on open-circuit voltage (Voc), short-circuit current (Jsc), fill factor (FF) and efficiency (η) of the solar cell. The optimum efficiencies using these three buffer layers are around 11.20%. Among these three buffers, Cd0.4Zn0.6S is more preferable as CdS suffers from toxicity problem and ZnS shows drastic change in performance parameters. The simulation results can give important guideline for the fabrication of high-efficiency CZTS solar cell.

Characterization of performance parameters of organic solar cells with a buffer ZnO layer**Invited talk at 9th International Workshop on Advanced Materials Science and Nanotechnology (IWAMSN2018), 7-11 November 2018, Ninh Binh City, Vietnam

By embedding a thin ZnO layer sandwiched between the hole transport and photoactive layers, organic solar cells (OSC) based on poly(3-hexylthiophene) (P3HT) were prepared by spincoating. UV–vis spectra of the composite films showed that ZnO exhibited a suitable buffer layer that could block holes movement throughout the heterojunction of ITO/ZnO. The enhancement in the fill factor (FF) of the buffer-OSC (BOSC) is attributed to the presence of nanoheterojunctions of ZnO/PCBM and ZnO/ITO. For the normal temperature, the increase of the open-circuit potential and short-circuit current resulted in an overall increase of the energy conversion efficiency. Comparing to OSCs without buffer layer (WOSC), the laminar structure of ITO/ZnO/P3HT/PCBM/Li/Al cells possess a much larger photovoltaic energy conversion efficiency, namely 2.12% (for BOSC) compared to 1.75% (for WOSC).

Evaluation of in-line particle measurement with an SFT-probe as monitoring tool for process automation using a new time-based buffer approach

The Process Analytical Technology (PAT) initiative published by the Food and Drug administration (FDA) describes PAT systems as important tools to optimize pharmaceutical processes in order to ensure the final product quality. Real-time particle size measurements provide a promising approach for process automation of fluid bed granulation processes. This work focuses on the introduction and evaluation of a new time-based buffer approach for in-line spatial filtering technique (SFT) in order to provide a minimum time delay of the real-time measurement. Comparing the new buffer system to the conventionally used number-based approach, a reduced influence of the particle rate and a constant response time was found. A performance qualification of the Parsum probe IPP 80 using this buffer approach according to the ICH guideline Q2 was performed to investigate whether common variations of process parameters influence the real-time particle size measurement. Focussing on the main requirements for process control, hereby a good precision and specificity as well as an adequate robustness was found. Summarized, this study identified the time-based buffer system as more suitable buffer system for process automation.

Preparation of Proliferated Bovine Primary Skeletal Muscle Cells for Bottom-Up Proteomics by LC-MSMS Analysis.

This chapter outlines a method for sample preparation for bottom-up proteomics by LC-MSMS analysis of in vitro proliferated bovine primary skeletal muscle cells. The protocol describes the isolation of bovine primary skeletal muscle cells, extraction of proteins, proteolytic digestion of proteins, and desalting of the final peptide samples. The final peptide samples can be analyzed using various LC-MSMS systems after reconstitution in a suitable elution buffer.

Plasmonic Biosensor in NIR with Chalcogenide Glass Material: On the Role of Probe Geometry, Wavelength, and 2D Material

Samarium doped chalcogenide core, perfluorinated polymer clad with Ag metal and plasmonic 2D materials based plasmonic fiber-optic sensor is simulated and analyzed in near-infrared (NIR) wavelength regime. Proposed sensor is directed at the detection of the malignancy stages of liver tissues. The performance analysis (in terms of figure-of-merit, i.e., FOM) was carried out taking into account the MoS2 and graphene monolayers as performance enhancing 2D materials. The analysis suggests that FOM values of MoS2-based sensor probe are better than graphene-based probe. Further, a comparative study shows that fiber-optic probe is able to provide much better performance than prism-based probes. The FOM gets better for longer wavelength. The specificity of the biosensor can be improved by employing a suitable buffer layer (1–15 nm) as a bio-recognition element.

Landscape context is more important than wetland buffers for farmland amphibians

In regions with high rates of wetland loss, remnant wetlands and constructed ponds can provide important breeding habitat for amphibians. However, such wetlands are often embedded in a matrix of agricultural fields, potentially putting species within these wetlands at risk. One recommendation for conservation of amphibians in agriculture-dominated landscapes is to maintain a buffer of permanent vegetation around the wetland. However, it is not clear how wide wetland buffers must be to effectively conserve amphibians in agriculture-dominated landscapes or what vegetation types are suitable buffer vegetation. Furthermore, it is not clear whether wetland buffers produce similar—or better—conservation outcomes for amphibians than actions conducted at larger spatial extents. We addressed these questions using data from anuran (frog and toad) breeding call surveys in 36 wetlands in rural eastern Ontario, Canada. First, we tested for the effects of 49 different wetland buffer measurements on anuran richness, relative anuran abundance, and the abundance/probability of occurrence of individual species. These 49 measurements represented all combinations of seven different ways to measure the wetland buffer size and seven types of buffer vegetation. Wetland buffer size was measured as the minimum width of buffer vegetation contiguous with the wetland and proportion of the area within 5, 16, 30, 50, 120, or 300 m of the wetland containing buffer vegetation that was contiguous with the wetland. Then, to compare the strength of effect of wetland buffers versus landscape context on anurans, we compared the wetland buffer measurement with the strongest positive effect on each anuran response (from the previous analysis) to the effects of three landscape-scale variables: area of woodland; area of wetlands, streams, rivers, and lakes; and road density. We did not detect positive effects of wetland buffers on anuran richness or relative anuran abundance. This is because positive effects of wetland buffers on individual species were rare, i.e. positive effects were only supported for two of the six species with enough data to model individually: American toads (Anaxyrus americanus) and green frogs (Lithobates clamitans). Furthermore, we found that the landscape context had much stronger effects on relative anuran abundance than the wetland buffer, with effect sizes ranging from 4 (road density) to 14 (woodland cover) times that of the wetland buffer. These findings suggest that guidelines for anuran conservation in agricultural landscapes should generally focus on protection of terrestrial habitat at the landscape scale rather than on maintenance of wetland buffers.

Arbuscular Mycorrhizal Fungi (AMF) as Buffer for Heavy Metals Phytoextraction by <i>Cucurbita maxima</i> Duch. Grown on Crude Oil Contaminated Soil

This study evaluated the influence of Arbuscular Mycorrhizal (Rhizophagus irregularis) fungi inoculation (M) on the growth ofCucurbita maximaand as a buffer against phytoextraction of selected heavy metals (HM) (Zn, Cu, Cr, Cd and Pb) from a soil contaminated with crude oil (C). The experiment was set up using four soil treatments, each with three replicates C+ M-, C+ M+, C-M+ and C-M- (control without oil and inoculum). The shoot length, petiole length, number of nodes, leaf area and percentage germination ofC. maximawere significantly (p=0.05) reduced in uninoculated crude oil treatment (C+ M-), unpolluted mycorrhizal inoculated treatments (C-M+) showed remarkable response in growth parameters above the control (C-M-), while the polluted and inoculated treatment (C+ M+) showed significant (p=0.05) increase in growth parameters when compared to the polluted uninoculated treatment (C+ M-). Heavy metals analysis revealed a significant (p=0.05) difference in the heavy metal accumulation ofC. maxima. The heavy metals analyzed in this study are present thus inC. maxima; Zn>Cu>Cr>Pb>Cd. Crude oil polluted uninoculated treatment (C+ M-) recorded the highest concentrations of heavy metals than crude oil polluted inoculated (R. irregularis) treatment (C+ M+). Mycorrhizal inoculated unpolluted treatment (C-M+) and unpolluted uninoculated treatment (C-M-) indicated the lowest heavy metal concentrations. Inoculation withR. irregularissignificantly (p=0.05) reduced heavy metals uptake byC. maximaas observed in this study. Also, the negative effect of crude oil on AMF root colonization ofC. maximabyR. irregulariswas observed in polluted and inoculated treatment. HM often accumulate in the top layer of soil, therefore, are available for uptake by plants via roots, which is a major entry point of HM that ultimately affects different physiological processes. AM fungi can impinge on the chemical properties of heavy metals in the soil, their absorption by the host plant, and their allocation to different plant parts, affecting plant growth and the bioremediation process, thus making the AM fungi a suitable buffer for mitigating heavy metal stress onC. maxima.

Development of ultra-thin chips for immobilization of Caenorhabditis elegans in microfluidic channels during irradiation and selection of buffer solution to prevent dehydration

BackgroundTargeted microbeam irradiation of Caenorhabditis elegans allows the effective knockdown of specific regions, thus helping to identify their roles in processes such as locomotion. We previously employed on-chip immobilization of individuals without anesthesia; however, this method was limited by the thickness of the chip, which prevented the detection of ions passing through the animal, and by dehydration of the animals after prolonged immobilization. New MethodWe developed ultra-thin, ion-penetrable, polydimethylsiloxane microfluidic chips, referred to as Worm Sheets, with and without wettability (hydrophilicity/hydrophobicity), and identified suitable buffer conditions for maintaining moisture in the microfluidic channels. ResultsUsing a collimating microbeam system, we demonstrated that carbon ions (with a range of ∼1 mm) could pass through the chip, thus allowing the ions to be detected and the applied radiation dose to therefore by measured accurately. We also examined the locomotion of C. elegans following on-chip immobilization in different buffers. Locomotion was decreased in certain buffers on unwettable chips as a result of dehydration due to evaporation, but not on wettable chips. However, locomotion was unaffected on either chip in the presence of a gelatin-based wash buffer. Comparison with Existing Method(s)We developed 300-μm-ultra-thin, wettable, ion-penetrable chips for immobilizing C. elegans and provided initial guidance regarding suitable buffer solutions to maintain moisture in microfluidic channels. ConclusionsThis improved, wettable chip, together with the identification of suitable buffer conditions, will become a powerful tool for prolonged immobilizing C. elegans, and is widely applicable not only to microbeam irradiation but also to neurobiological assays.