Reproducible Conditions Research Articles

Evaluation of microflow configurations for scale inhibition and serial X-ray diffraction analysis of crystallization processes.

The clean and reproducible conditions provided by microfluidic devices are ideal sample environments for in situ analyses of chemical and biochemical reactions and assembly processes. However, the small size of microchannels makes investigating the crystallization of poorly soluble materials on-chip challenging due to crystal nucleation and growth that result in channel fouling and blockage. Here, we demonstrate a reusable insert-based microfluidic platform for serial X-ray diffraction analysis and examine scale formation in response to continuous and segmented flow configurations across a range of temperatures. Under continuous flow, scale formation on the reactor walls begins almost immediately on mixing of the crystallizing species, which over time results in occlusion of the channel. Depletion of ions at the start of the channel results in reduced crystallization towards the end of the channel. Conversely, segmented flow can control crystallization, so it occurs entirely within the droplet. Consequently, the spatial location within the channel represents a temporal point in the crystallization process. Whilst each method can provide useful crystallographic information, time-resolved information is lost when reactor fouling occurs and changes the solution conditions with time. The flow within a single device can be manipulated to give a broad range of information addressing surface interaction or solution crystallization.

Mid-infrared radiation technique for direct pyroelectric and electrocaloric measurements.

A mid-infrared radiometric (MIR) method for precise in situ temperature measurements when studying pyroelectric and electrocaloric properties of bulk and film materials is presented. The method uses new MIR-temperature sensors based on narrowband high-speed and high-sensitive uncooled immersion lens A3B5 photodiodes with a precalibration procedure. They are completely insensitive to the background illumination with λ ≤ 1 µm and provide contactless temperature measurements directly in the area of laser heating action. An accuracy of 50 mK at the temperature around 20 °C, rapidly improving up to 1 mK at 200 °C, is achieved at the operation speed of 1 ms. The reliable and reproducible conditions of measurements of pyroelectric and electrocaloric properties of various samples are formulated, and the novel experimental setup is described in detail. The experimental verification of the method is performed by the measurements of pyroelectric properties of single crystals, bulk ceramics, and AlN film. The results of joint measurements of the pyroelectric and electrocaloric properties of the ferroelectric relaxor ceramics are also presented.

An experimental study of methods for the analysis of variance components in the inference of laboratory information

Measurement uncertainty (MU) can be estimated and calculated by different procedures, representing different aspects and intended use. It is appropriate to distinguish between uncertainty determined under repeatability and reproducibility conditions, and to distinguish causes of variation using analysis of variance components. The intra-laboratory MU is frequently determined by repeated measurements of control material(s) of one or several concentrations during a prolonged period of time. We demonstrate, based on experimental results, how such results can be used to identify the repeatability, ‘pure’ reproducibility and intra-laboratory variance as the sum of the two. Native patient material was used to establish repeatability using the Dahlberg formula for random differences between measurements and an expanded Dahlberg formula if a non-random difference, e.g. bias, was expected. Repeatability and reproducibility have different clinical relevance in intensive care compared to monitoring treatment of chronic diseases, comparison with reference intervals or screening.

Supercritical fluid chromatography–mass spectrometry in routine anti-doping analyses: Estimation of retention time variability under reproducible conditions

The aim of this study was to estimate the retention time variability under reproducible conditions of an SFC-MS analytical method for routine anti-doping analyses. For this purpose, a set of 51 doping agents, as neat standards and spiked in diluted urine, was used to assess their retention times variability over a period of four months, as well as the column inter-batch reproducibility. Three UHPSFC stationary phases have been employed, the Acquity UPC2 Torus 2-Picolylamine (2-PIC), UPC2 Viridis BEH and Acquity UPLC HSS C18 SB. Four columns, per column chemistry, have been purchased to represent three different production lots, with a total of twelve columns employed in this study. The two columns from the same lot were applied to the first part of the study (repeatability), whereas the representative of three different lots were employed in the second part (robustness). In terms of organic modifier, a mixture of 98% MeOH and 2% water containing 20 mM ammonium formate was selected in order to limit the formation of methyl-silyl ethers on the surface of the silica particles, thus potentially improving the repeatability of retention times. A comparison with an UHPLC reference analytical method was made with the same set of analytes.The average relative standard deviations (RSD%), represented in split violin plots, illustrate how two of the UHPSFC columns assessed in this study were able to generate an excellent repeatability of retention times, with results that are in a similar range of those generated by UHPLC. Moreover, the Torus 2-PIC has proven to be the best of the three stationary phases, with an impressive RSD% of 0.5% in diluted urine relative to the inter-month variability. Finally, the inter-batch reproducibility assessment has highlighted a good reproducibility of the same stationary phase belonging to different production lots for all three column chemistries assessed, with the Viridis BEH silica generating an RSD% of 0.7% in diluted urine. Higher values of RSD (%) were found for Torus 2-PIC and HSS C18 SB, respectively of 1.0% and 1.6%.

Lab-On-A-Chip for the Development of Pro-/Anti-Angiogenic Nanomedicines to Treat Brain Diseases.

There is a huge demand for pro-/anti-angiogenic nanomedicines to treat conditions such as ischemic strokes, brain tumors, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Nanomedicines are therapeutic particles in the size range of 10–1000 nm, where the drug is encapsulated into nano-capsules or adsorbed onto nano-scaffolds. They have good blood–brain barrier permeability, stability and shelf life, and able to rapidly target different sites in the brain. However, the relationship between the nanomedicines’ physical and chemical properties and its ability to travel across the brain remains incompletely understood. The main challenge is the lack of a reliable drug testing model for brain angiogenesis. Recently, microfluidic platforms (known as “lab-on-a-chip” or LOCs) have been developed to mimic the brain micro-vasculature related events, such as vasculogenesis, angiogenesis, inflammation, etc. The LOCs are able to closely replicate the dynamic conditions of the human brain and could be reliable platforms for drug screening applications. There are still many technical difficulties in establishing uniform and reproducible conditions, mainly due to the extreme complexity of the human brain. In this paper, we review the prospective of LOCs in the development of nanomedicines for brain angiogenesis–related conditions.

How to ‘train’ your CVD to grow large-area 2D materials

In recent years 2D materials like monolayer of transition metal dichalcogenides (TMDs) have caught enormous interest due to their potential applications in electronic, optoelectronic, spintronic, valleytronic and twistronic devices. To grow large-area monolayer of these materials, chemical vapor deposition (CVD) is one of the most widely used techniques. But the size and quality of the samples are critically dependent on the various growth conditions. As a result, the CVD parameters vary from lab to lab and often it is difficult to find a stable and reproducible growth condition. Here we have demonstrated a methodology for the quick and easy optimization of various growth parameters of 2D materials using CVD. We have taken MoS2 as a model 2D system and discussed the way to achieve an optimum recipe for the growth of large-area monolayer reproducibly. We have done thorough electrical and optoelectrical characterization to show the device performance of the samples grown in various conditions. The methodology will help to optimize the growth condition of any other 2D materials easily and quickly.

Estimation of PM10 Levels and Sources in Air Quality Networks by Digital Analysis of Smartphone Camera Images Taken from Samples Deposited on Filters.

This paper explores the performance of smartphone cameras as low-cost and easily accessible tools to provide information about the levels and origin of particulate matter (PM) in ambient air. We tested the concept by digital analysis of the images of daily PM10 (particles with diameters 10 µm and smaller) samples captured on glass fibre filters by high-volume aerosol samplers at urban and rural locations belonging to the air quality monitoring network of Extremadura (Spain) for one year. The images were taken by placing the filters inside a box designed to maintain controlled and reproducible light conditions. Digital image analysis was carried out by a mobile colour-sensing application using red, green, blue/hue, saturation, value/hue, saturation, luminance (RGB/HSV/HSL) parameters, that were processed through statistical procedures, directly or transformed to greyscale. The results of the study show that digital image analysis of the filters can roughly estimate the concentration of PM10 within an air quality network, based on a significant linear correlation between the concentration of PM10 measured by an official gravimetric method and the colour parameters of the filters’ images, with better results in the case of the saturation parameter (SHSV). The methodology based on digital analysis can discriminate urban and rural sampling locations affected by different local particle-emitting sources and is also able to identify the presence of remote sources such as Saharan dust outbreaks in both urban and rural locations. The proposed methodology can be considered as a useful complement to the aerosol sampling equipment of air quality network field units for a quick estimation of PM10 in the ambient air, through a simple, accessible and low-cost procedure, with further miniaturization potential.

Robotized Fiber Laser Cladding of Steel Substrate by Metal Matrix Composite Powder at Cryogenic Conditions

Abstract The results of investigations on laser cladding of mild steel plates (0.17 wt.% carbon and 1.4 wt.% manganese) by nickel powder with addition 60 wt.% of tungsten carbides at conditions of forced cooling by liquid nitrogen are presented. To provide reproducible conditions and high accuracy, tests of cladding were carried out on a robotized stand equipped with a fiber laser generator and powder feeding system. The symmetrical circular laser beam with beam spot diameter of 300 μm was applied as the heat source for cladding of composite single stringer beads at different heat inputs and different cooling conditions. Free cooling of the steel substrate was conducted in the ambient air, while the forced cooling was achieved by partial immersion of the steel plate in liquid nitrogen at approximately −190°C. The results showed that the conditions of cooling have a strong influence on the cladding process, mechanism of a bead formation, quality, microstructure, and geometry of the beads. Laser cladding at forced cooling leads to a favorable fine-grained microstructure and increased microhardness of the metal matrix and also, lower dilution. In the case of stringer beads produced at cryogenic conditions, the heat-affected zone in the substrate of the mild steel is hardened with evident traces of bainitic microstructure. However, the heat input of laser cladding must be significantly increased at forced cooling to provide the proper shape of the bead and also to avoid cracks and a lack of fusion the steel substrate.

Standardized mounting method of (zebrafish) embryos using a 3D-printed stamp for high-content, semi-automated confocal imaging

BackgroundDevelopmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be easily observed and imaged using fluorescent proteins have become very popular. One limiting factor however is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. One way to improve this is by developing mounting methods to increase the number of embryos that can be imaged simultaneously in near-to-identical orientation.ResultsHere we present an improved mounting method allowing semi-automated and high-content imaging of zebrafish embryos. It is based on a 3D-printed stamp which is used to create a 2D coordinate system of multiple μ-wells in an agarose cast. Each μ-well models a negative of the average zebrafish embryo morphology between 22 and 96 h-post-fertilization. Due to this standardized and reproducible arrangement, it is possible to define a custom well plate in the respective imaging software that allows for a semi-automated imaging process. Furthermore, the improvement in Z-orientation significantly reduces post-processing and improves comparability of volumetric data while reducing light exposure and thus photo-bleaching and photo-toxicity, and improving signal-to-noise ratio (SNR).ConclusionsWe present here a new method that allows to standardize and improve mounting and imaging of embryos. The 3D-printed stamp creates a 2D coordinate system of μ-wells in an agarose cast thus standardizing specimen mounting and allowing high-content imaging of up to 44 live or mounted zebrafish embryos simultaneously in a semi-automated, well-plate like manner on inverted confocal microscopes. In summary, image data quality and acquisition efficiency (amount of data per time) are significantly improved. The latter might also be crucial when using the services of a microscopy facility.

Laser induced cavitation: Plasma generation and breakdown shockwave

Laser induced cavitation is one of the effective techniques to generate controlled cavitation bubbles, both for basic study and for applications in different fields of engineering and medicine. Unfortunately, control of bubble formation and symmetry is hardly achieved due to a series of concurrent causes. In particular, the need to focus the laser beam at the bubble formation spot leads, in general, to a conical region proximal to the light source where conditions are met for plasma breakdown. A finite sized region then exists where the electric field may fluctuate depending on several disturbing agents, leading to possible plasma fragmentation and plasma intensity variation. Such irregularities may induce asymmetry in the successive bubble dynamics, a mostly undesired effect if reproducible conditions are sought for. In the present paper, the structure of the breakdown plasma and the ensuing bubble dynamics are analyzed by means of high speed imaging and intensity measurements of the shockwave system launched at breakdown. It is found that the parameters of the system can be tuned to optimize repeatability and sphericity. In particular, symmetric rebound dynamics is achieved almost deterministically when a pointlike plasma is generated at the breakdown threshold energy. Spherical symmetry is also favored by a large focusing angle combined with a relatively large pulse energy, a process which, however, retains a significant level of stochasticity. Outside these special conditions, the elongated and often fragmented conical plasma shape is found to be correlated with anisotropic and multiple breakdown shockwave emission.

Mycelyso \u2013 high-throughput analysis of Streptomyces mycelium live cell imaging data

BackgroundStreptomycetes are filamentous microorganisms of high biotechnological relevance, especially for the production of antibiotics. In submerged cultures, the productivity of these microorganisms is closely linked to their growth morphology. Microfluidic lab-on-a-chip cultivation systems, coupled with automated time-lapse imaging, generate spatio-temporal insights into the mycelium development of streptomycetes, therewith extending the biotechnological toolset by spatio-temporal screening under well-controlled and reproducible conditions. However, the analysis of the complex mycelial structure formation is limited by the extent of manual interventions required during processing of the acquired high-volume image data. These interventions typically lead to high evaluation times and, therewith, limit the analytic throughput and exploitation of microfluidic-based screenings.ResultsWe present the tool mycelyso (MYCElium anaLYsis SOftware), an image analysis system tailored to fully automated hyphae-level processing of image stacks generated by time-lapse microscopy. With mycelyso, the developing hyphal streptomycete network is automatically segmented and tracked over the cultivation period. Versatile key growth parameters such as mycelium network structure, its development over time, and tip growth rates are extracted. Results are presented in the web-based exploration tool mycelyso Inspector, allowing for user friendly quality control and downstream evaluation of the extracted information. In addition, 2D and 3D visualizations show temporal tracking for detailed inspection of morphological growth behaviors. For ease of getting started with mycelyso, bundled Windows packages as well as Docker images along with tutorial videos are available.Conclusionmycelyso is a well-documented, platform-independent open source toolkit for the automated end-to-end analysis of Streptomyces image stacks. The batch-analysis mode facilitates the rapid and reproducible processing of large microfluidic screenings, and easy extraction of morphological parameters. The objective evaluation of image stacks is possible by reproducible evaluation workflows, useful to unravel correlations between morphological, molecular and process parameters at the hyphae- and mycelium-levels with statistical power.

The Dietary Addition of Fish Oil or Sunflower Oil Plus Starch Differently Modulates the Lipid Classes in Plasma of Lactating Cows and Goats

Plasma lipid content and composition are studied in dairy cows and goats fed similar diets inducing milk fat depression (MFD) only in cows. Twelve cows and 15 goats are fed a diet without additional lipids (control), or supplemented with fish oil (FO), or supplemented with sunflower oil plus starch (SOS) in a 3 × 3 Latin square design. A high‐performance thin‐layer chromatography (HP‐TLC) methodology is used to separate and quantify the following plasma lipid classes: cholesterol esters (CE), triglycerides (TG), free fatty acids (FFA), cholesterol (Chol), and phospholipids (PL). The plasma lipid composition reveales that TG concentration is 193% higher in the goats compared to the cows. For cows and goats, FO increases CE (+31%) and Chol levels (+33.7%) compared to the control. The TG concentration is differentially regulated by FO between cows (−23.5%) and goats (no change). In the goats, SOS increases CE (+25.7%), Chol (+46.7%), and FFA levels (+71.7%) compared to the control. These results suggest the existence of different mechanisms of lipid digestion, absorption, and transport between cows and goats and contribute to better understand the differences in the regulation of milk fat secretion responses to lipid supplementation between cows and goats.Practical Applications: A robust HP‐TLC methodology for plasma lipid separation and analysis in automated and reproducible conditions is developed which constitutes an original part of the present study. It has been shown that intake of plant‐derived lipids associated with starch‐rich diets decreases the milk fat content in cows but not in goats. Therefore, the direct species comparison model used in the present study constitutes an interesting tool to compare the responses of plasma lipid concentrations and FA composition in cows and goats fed fish oil or sunflower oil and starch supplemented diets. The direct comparison between cows and goats in plasma lipid class composition is reported for the first time in the present study, with in addition new information about the contribution of intermediary metabolism to differences between the two species in the regulation of milk fat secretion in response to lipid supplementation.Direct comparisons of plasma lipid classes between dairy cows and goats fed similar diets is studied using high‐performance thin‐layer chromatography (HP‐TLC) methodology and demonstrates significantly higher concentration of triglycerides (TG) in plasma of goats compared to cows, whereas the total analyzed lipid content is higher in cows than in goats. In addition, for both species, diets supplemented with fish oil or sunflower oil plus starch modulates the composition in plasma lipid classes.

Effect of methylene blue photodynamic therapy on human neutrophil functional responses

Photodynamic therapy (PDT) has become an emerging novel therapeutic approach for treating localized microbial infections, particularly those sustained by multidrug-resistant strains. Given the irreplaceable role played by professional phagocytes in limiting infections, such as polymorphonuclear neutrophils, any newly designed antimicrobial therapeutic approach must not interfere with their function. The present investigation presents a detailed analysis of the effect of PDT on the viability and several functional responses of human polymorphonuclear neutrophils loaded with methylene blue (MB), one of the more commonly used photosensitizers in antimicrobial PDT. Taking advantage of the use of a specifically-designed optical LED array for illuminating MB-loaded human polymorphonuclear neutrophils, a number of cell functions have been assayed under miniaturized, strictly controlled and reproducible experimental conditions. The major findings of this study are the following: (1) MB-PDT increases human neutrophils adhesion and does not modify myeloperoxidase release; (2) MB-PDT markedly enhances reactive oxygen species generation that is independent of superoxide-forming phagocytic oxidase and very likely ascribable to LED-dependent excitation of accumulated methylene blue; (3) MB-PDT almost abolishes human neutrophils candidacidal activity by hindering the engulfing machinery. This in vitro study may represent a valuable reference point for future research on PDT applications for treating localized microbial infections.

Numerical data quality in simulation research: A reflection and epistemic implications

Marsden and Pingry (2018) discuss different aspects of numerical data that are used in empirical research, with an aim to improve data quality and facilitate research replication in the IS field. However, the implications for numerical data from simulation research also need to be discussed. This article tries to join the discussion by using Marsden and Pingry's (2018) What, When, Where, How, Who, Which, and Why aspects to discuss how we can create the necessary, but not sufficient, conditions for data accuracy, validity and reproducibility in simulation research.

Alignment strategy for an optomechanical image derotator using a laser Doppler vibrometer.

When monitoring the condition of technical components, it is of particular interest to obtain information of the current condition of the component during operation. The main objectives are safety and machine efficiency. A special extension in this area is the derotator, since the derotator can be used to extend measuring methods designed for static components to rotating systems. In this paper, we present a new, to the best of our knowledge, approach to align a derotator using a laser Doppler vibrometer (LDV). When aligning the derotator, the optical axis must be coaxial with the rotating object to be measured. Current conventional approaches only ensure that the optical axis and the rotational axis intersect at the center of the measuring object. In our approach, a LDV is used to determine the angular deviation between the two axes, which cannot be determined by common methods. Our approach is easy to implement because the LDV is often already used in combination with the derotator as a measuring device. Experiments are carried out to show that this approach is feasible. In addition, several investigations are being performed to analyze how different parameters affect the result, creating reproducible conditions and deriving an alignment approach. By combining the approach developed in this work for determining a rotational deviation with common image-based approaches for determining a translational deviation of the axes, the derotator can be calibrated entirely. In this way, the quality of the measurement with the derotator can be further increased, as calibration errors can be reduced.

Interlaboratory comparisons’ measurement uncertainty in the field of environmental noise

This paper deals with the problem of measurement uncertainty in acoustic measurements regarding the results of interlaboratory comparison (ILC). The measurement results of ILC in the field of environmental noise have been analysed taking into account the proposed methods for standard deviations’ calculation in repeatability and reproducibility conditions. In the field of environmental noise measurements of point sound source, the measurement results as well as the precision and accuracy are discussed bearing in mind the microphone positions. The results for expanded measurement uncertainty calculations are obtained following the ISO 1996-2:2007 and ISO 1996-2017 Standard. The obtained results showed that expanded measurement uncertainty from interlaboratory comparison results calculated from probability density functions is considerably less when compared with the procedure in Standards. A good agreement is gained for the difference between A-weighted level and spectral values from logarithmically averaged measurement results on the reflection surface and close to the reflection surface.

Two Simple Methods for Optimizing the Production of "Difficult-to-Express" GnRH-DFF40 Chimeric Protein.

Purpose: GnRH-DFF40 (gonadotropin releasing hormone - DNA fragmentation factor 40) is a humanized recombinant immunotoxin and serves as a prospective candidate for targeted therapy of gonadotropin releasing hormone receptor (GnRHR) overexpressing malignancies. However, its production in Escherichia coli in a soluble and functional form still remains a challenge. Here we introduce two successful and reproducible conditions for production and purification of “difficult-to-express” GnRH-DFF40 protein. Methods: A synthetic codon optimized GnRH-DFF40 fusion gene was cloned in pET28a plasmid. Two methods including high cell density IPTG induction (HCDI) and autoinduction method (AIM) with a focus on obtaining high cell density have been investigated to enhance the protein production in (E. coli). Moreover, to obtain higher protein production several factors in the AIM method including carbon sources, incubation time and temperature, plasmid stability and double colony selection, were optimized. Results: Remarkable amounts of soluble GnRH-DFF40 protein were achieved by both methods. Cell density and protein yields in AIM was about 1.5 fold higher than that what obtained using HCDI. Initial screening showed that 25ºC is better to achieve higher protein production in both methods. pH alterations in AIM were maintained in a more constant level at 25ºC and 37ºC temperatures without any detrimental effects on cell growth during protein production phase up to 21 hours after incubation. Plasmid stability during growth and expression induction phase was maintained at a high level of 98% and 96% for AIM and HCDI methods, respectively. After parameter optimization and double colony selection in AIM, a very high yield of recombinant protein was achieved (528.3 mg/L). Conclusion: With the optimization of these high cell density expression methods, reproducible manifold enhancement of soluble protein yields can be achieved for “difficult-to-express” GnRH-DFF40 compared to conventional expression methods.

Dimensional Quality and Distortion Analysis of Thin-Walled Alloy Parts of AlSi10Mg Manufactured by Selective Laser Melting

The quality and reliability in additive manufacturing is an emerging area. To ensure process quality and reliability, the influence of all process parameters and conditions needs to be understood. The product quality and reliability characteristics, i.e., dimensional accuracy, precision, repeatability, and reproducibility are mostly affected by inherent and systematic manufacturing process variations. This paper presents research on dimensional quality and distortion analysis of AlSi10Mg thin-walled parts developed by a selective laser melting technique. The input process parameters were fixed, and the impact of inherent process variation on dimensional accuracy and precision was studied. The process stability and variability were examined under repeatability and reproducibility conditions. The sample length (horizontal dimension) results revealed a 0.05 mm maximum dimensional error, 0.0197 mm repeatability, and 0.0169 mm reproducibility. Similarly, in sample height (vertical dimension) results, 0.258 mm maximum dimensional error, 0.0237 mm repeatability, and 0.0863 mm reproducibility were observed. The effect of varying design thickness on thickness accuracy was analyzed, and regression analysis performed. The maximum 0.038 mm error and 0.018 mm standard deviation was observed for the 1 mm thickness sample, which significantly decreased for sample thickness ≥2 mm. The % error decreased exponentially with increasing sample thickness. The distortion analysis was performed to explore the effect of sample thickness on part distortion. The 0.5 mm thickness sample shows a very high distortion comparatively, and it is reduced significantly for >0.5 mm thickness samples. The study is further extended to examine the effect of solution heat treatment and artificial aging on the accuracy, precision, and distortion; however, it did not improve the results. Conclusively, the sample dimensions, i.e., length and height, have shown fluctuations due to inherent process characteristics under repeatability and reproducibility conditions. The ANOVA results revealed that sample length means are not statistically significantly different, whereas sample height means are significantly different. The horizontal dimensions in the xy-plane have better accuracy and precision compared to the vertical dimension in the z-axis. The accuracy and precision increased, whereas part distortion decreased with increasing thickness.

Chemical Synthesis, Proper Folding, Nav Channel Selectivity Profile and Analgesic Properties of the Spider Peptide Phlotoxin 1.

Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.

On the challenges for reliable measurements of convection in large aspect ratio Rayleigh-Bénard cells in air and sulfur-hexafluoride

Heat transfer through natural turbulent thermal convection is an important mechanism in geophysics and many engineering problems. A canonical well-known experiment is the Rayleigh-Bénard (RB) cell, which consists of an enclosure with adiabatic side walls uniformly heated from below and cooled from above. The direct correlation between the local momentum and heat transfer is currently under investigation, especially for large aspect ratios. In this situation the dominant large-scale circulation roll, as typically found for low aspect ratios, is substituted by a whole pattern of long-living rolls. Nowadays, commonly advanced optical methods are applied to investigate velocity fields, which makes a transparent heating or cooling plate necessary. For the current study glass plates were coated with a thin metal oxide layer enabling heating via the Joule effect. However, since the temperature homogeneity is crucial for the setup, two heating plates had to be combined to enable a homogeneous temperature distribution at the bottom of the convection cell. Additional difficulties arise from the large aspect ratio and thus the large observation angle. This may cause optical aberrations and systematic errors due to the perspective bias. Furthermore, the observation duration has to be in the order of minutes to hours to resolve the dynamics of the slowly evolving large-scale flow. This results in additional challenges for the seeding generation. To prove the reliability of the experimental approach, a convection cell with an aspect ratio Γ=l/h=10 was placed in the SCALEX (Scaled Convective Airflow Laboratory Experiment) facility. This facility enables experiments with sulfur-hexafluoride (SF6) and air under pressures of up to 10 bar to achieve very high Rayleigh numbers. Here, results of measurements for a Rayleigh number of 5×105 in SF6 at ambient pressure and in air at a pressure of 5.7 bar will be discussed and show the applicability of this approach. Stereoscopic particle image velocimetry (PIV) in horizontal planes covering the whole cross section of the cell (300×300mm2) was used for the estimation of all three components of the velocity vector. The optical access for the laser light sheet was provided by transparent sidewalls. The aim of the current study was to prove the possibility of reliable PIV measurements with reproducible homogenous temperature boundary conditions in the SCALEX facility. Issues of the temperature distribution at the heating plate, tracer particles, illumination and data evaluation will therefore be addressed in greater detail.