Optimization Of Experimental Procedure Research Articles

Experimental and numerical investigation of microdialysis probes for ethanol metabolism studies.

Microdialysis is an important technique for in vivo sampling of tissue's biochemical composition. Understanding the factors that affect the performance of the microdialysis probes and developing methods for sample analysis are crucial for obtaining reliable results. In this work, we used experimental and numerical procedures to study the performance of microdialysis probes having different configurations, membrane materials and dimensions. For alcohol research, it is important to understand the dynamics of ethanol metabolism, particularly in the brain and in other organs, and to simultaneously measure the concentrations of ethanol and its metabolites - acetaldehyde and acetate. Our work provides a comprehensive characterization of three microdialysis probes, in terms of recovery rates and backpressure, allowing for interpretation and optimization of experimental procedures. In vivo experiments were performed to measure the time course concentration of ethanol, acetaldehyde, and acetate in the rat brain dialysate. Additionally, the combination of in vitro experimental results with numerical simulations enabled us to calculate diffusion coefficients of molecules in the microdialysis membranes and study the extent of the depletion effect caused by continuous microdialysis sampling, thus providing additional insights for probe selection and data interpretation.

Optimization framework of sediment phosphate oxygen isotope pretreatment method based on large-scale application: A case study of Fuyang River basin

Phosphate oxygen isotope (δ18OP) technique is an effective tool to identify the source and transformation process of phosphorus. The poor applicability of existing δ18OP pretreatment methods for sediments hindered the large-scale application of δ18OP technology. This paper presents an optimization framework for the pretreatment of sediment δ18OP samples based on large-scale applications, using the Fuyang River Basin as a case study. The typical channel landscape outflow lake, South Lake, was selected as the most favorable point for assessing the applicability and optimizing the mainstream δ18OP pretreatment method, which was achieved by clarifying the sediment environmental characteristics of South Lake. To evaluate the suitability of the Blake and McLaughlin methods in South Lake, a comparative study was carried out based on five dimensions: phosphorus recovery rate, removal efficiency of organic matter, removal efficiency of extraction liquid impurity ion, experimental time, and reagent consumption cost. The findings demonstrated that the Blake method outperformed the McLaughlin method across all five dimensions. Based on the environmental characteristics of the sediments of South Lake, the Blake method was optimized from two perspectives, namely the substitution of reagents and adjustment and optimization of experimental procedures. This resulted in an enhancement of phosphorus recovery and organic matter removal efficiency, while also reducing the experimental time required. The optimized method also yielded satisfactory results when applied to the entire watershed. This research paper can thus offer valuable technical support for the widespread application of sediment δ18OP technology.

NMDA receptor modulation in protracted ethanol withdrawal

Functional dysregulation of the glutamate system during withdrawal from chronic drug exposure is a primary driver of drug craving and relapse. Animal models of psychostimulant use have demonstrated dynamic alterations in NMDA receptor function and expression that contribute to drug seeking behavior. These changes begin as early as 5 days following cessation of drug use are persistently expressed into long term withdrawal (>60d). Evidence of NMDA receptor dependent changes during short term withdrawal (24h) suggest that similar mechanisms may drive drug craving and relapse behaviors following chronic exposure to ethanol. To this end, we investigated NMDA receptor protein and surface expression during protracted withdrawal from chronic intermittent ethanol (CIE) exposure using western blot. We focused on the basolateral amygdala (BLA), as glutamatergic signaling in this region is robustly modulated by short term (24h) withdrawal from CIE (10d, 12hr/day) and regulates anxiety‐like behavior expressed during withdrawal. Adolescent male rats were exposed to repeated cycles of CIE (12hr/day, 4d on/3d off, 3 cycles) and brain tissue was collected during protracted withdrawal. Optimization of experimental procedures is currently being conducted and analysis of total and membrane surface expression will be analyzed by surface biotinylation for NMDAR subunits (GluN1, GluN2B, GluN3). Cell surface biotinylation will be used to isolate cell surface membrane proteins to detect and quantify expression of NMDAR subunits. We expect to identify an increase in membrane surface expression for NMDAR subunits, in agreement with functional data obtained in the laboratory.

Design and optimization of a fused-silica microfluidic device for separation of trivalent lanthanides by isotachophoresis.

Elemental analysis of rare earth elements is essential in a variety of fields including environmental monitoring and nuclear safeguards; however, current techniques are often labor intensive, time consuming, and/or costly to perform. The difficulty arises in preparing samples, which requires separating the chemically and physically similar lanthanides. However, by transitioning these separations to the microscale, the speed, cost, and simplicity of sample preparation can be drastically improved. Here, all fourteen non-radioactive lanthanides (lanthanum through lutetium minus promethium) are separated by ITP for the first time in a serpentine fused-silica microchannel (70µm wide×70µm tall×33cm long) in <10min at voltages ≤8kV with limits of detection on the order of picomoles. This time includes the 2min electrokinetic injection time at 2kV to load sample into the microchannel. The final leading electrolyte consisted of 10mM ammonium acetate, 7mM α-hydroxyisobutyric acid, 1% polyvinylpyrrolidone, and the final terminating electrolyte consisted of 10mM acetic acid, 7mM α-hydroxyisobutyric acid, and 1% polyvinylpyrrolidone. Electrophoretic electrodes are embedded in the microchip reservoirs so that voltages can be quickly applied and switched during operation. The limits of detection are quantified using a commercial capacitively coupled contactless conductivity detector (C4 D) to calculate ITP zone lengths in combination with ITP theory. Optimization of experimental procedures and reproducibility based on statistical analysis of subsequent experimental results are addressed. Percent error values in band length and conductivity are ≤8.1 and 0.37%, respectively.

Application of metabarcoding technology in studies of fungal diversity

Fungal diversity is threatened by climate change, land-use change, and environmental pollution, and requires urgent conservation action. Construction of the fungal species database is an important prerequisite for the study and conservation of fungal diversity. Recently developed DNA barcoding and metabarcoding technologies can provide accurate, rapid, and highly efficient identification on a large scale, and to a large extent compensate for the defects of traditional identification methods. In this paper, we review the application of metabarcoding in fungal species diversity assessment, the study of mechanisms underlying fungal diversity, and the reconstruction of fungal palaeoecology. We emphasize that the application of metabarcoding technology in fungal diversity studies is still in the primary phase, and greater efforts are needed in the construction of reliable reference databases, the optimization of experimental procedures, and updates of bioinformatics tools. Hence, we suggest enhancing cooperation among fungal taxonomists, ecologists, and computer technicians. They should work together to address problems in fungal diversity studies via metabarcoding, which would provide more sound scientific evidence for fungal diversity conservation on a large scale.

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

High-performance mass spectrometry (MS)-based proteomics enabled the construction of a detailed proteome atlas for Populus, a woody perennial plant model organism. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: (1) network-wide, (2) pathway-specific, and (3) protein-level viewpoints. Together, enhanced protein retrieval through a detergent-based lysis approach and maximized peptide sampling via the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have resulted in the identification of 63,056 tryptic peptides. The technological advancements, specifically spectral-acquisition and sequencing speed, afforded the deepest look into the Populus proteome, with peptide abundances spanning 6 orders of magnitude and mapping to ∼25% of the predicted proteome space. In total, tryptic peptides mapped to 11,689 protein assignments across four organ-types: mature (fully expanded, leaf plastichronic index (LPI) 10-12) leaf, young (juvenile, LPI 4-6) leaf, root, and stem. To resolve protein ambiguity, identified proteins were grouped by sequence similarity (≥ 90%), thereby reducing the protein assignments into 7538 protein groups. In addition, this large-scale data set features the first systems-wide survey of protein expression across different Populus organs. As a demonstration of the precision and comprehensiveness of the semiquantitative analysis, we were able to contrast two stages of leaf development, mature versus young leaf. Statistical comparison through ANOVA analysis revealed 1432 protein groups that exhibited statistically significant (p ≤ 0.01) differences in protein abundance. Experimental validation of the metabolic circuitry expected in mature leaf (characterized by photosynthesis and carbon fixation) compared with young leaf (characterized by rapid growth and moderate photosynthetic activities) strongly testifies to the credibility of the approach. Instead of quantitatively comparing a few proteins, a systems view of all the changes associated with a given cellular perturbation could be made.

Development of a sampling regime for research with environmental relevance based on variability of transgene expression in plants

AbstractTransgenic plants often produce proteins that are unknown in conventional crops. Therefore, it requires a fundamental analysis before approval of a new genetically modified plant is given. Since the EFSA-guidelines for risk assessment on transgenic plants provide only recommendations but no detailed instructions, we wanted to develop a valid test scheme that defines sample size and sampling organ to enable a statically valid and efficient analysis of transgene expression and its variability. The scheme will be based on data obtained for the variability in expression i. of different recombinant proteins in one variety; ii. of different integration sites of one transgene in one variety; iii. of the same recombinant protein between different varieties and between isogenic clones in a variety. The optimization of experimental procedures shall enable the development of cost and time efficient and valid sampling systems that will be used as a part of the Decision Support System (DSS) that is developed by BioOK.

Optimization of experimental procedure and statistical data treatment for kinetics of ethylene hydrogenation on a copper-magnesia catalyst

This study is an example of practical application of kinetic data treatment for simultaneous model discrimination and parameter estimation. The study is applied to the hydrogenation of ethylene on a copper-magnesia catalyst and brings a deepened analysis about the experimental strategy by comparing several alternative strategies using a priori and sequential experimental designs. The best model corresponds to a Langmuir–Hinshelwood mechanism with non-competing adsorption of hydrogen and ethylene and where the rate-determining step is either the addition of molecularly adsorbed hydrogen or the addition of the second atom of hydrogen adsorbed dissociatively. Furthermore, the important question of knowing in practice how many designs and how many measurements per design are actually necessary to determine accurate kinetic and physico-chemical parameters, is addressed. A data correction procedure is also presented that takes catalyst deactivation into account.

Estimating maximal EMG amplitude for the trapezius muscle: On the optimization of experimental procedure and electrode placement for improved reliability and increased signal amplitude

The amplitude of electromyographic (EMG) surface recordings on the upper trapezius muscle and its reproducibility during maximal voluntary contractions were examined. At different electrode positions 1.5–3.0 cm lateral to the midpoint between the seventh cervical vertebra (C7) and the acromion the mean maximal EMG amplitude estimates were within 90% of the highest EMG amplitude that was recorded, when the estimate was based on the highest value of two shoulder elevations and two arm abductions. Near the midpoint between C7 and the acromion the mean maximal EMG amplitude was only 42% of the highest value. The repeatability coefficient based on two estimates of the maximal EMG amplitude obtained with 2 h interval indicated that 95% of the differences in the recorded amplitudes should be less than 22% of the mean maximal amplitude. Increasing the averaging period during signal processing from 0.6–2.0 s decreased the maximal amplitude estimate by 8%. It is concluded that electrode positioning is an important factor to consider and that more than one shoulder elevation and arm abduction should be performed when estimating maximal amplitudes.