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Ultrasonics Sonochemistry Dec 2023This article proposes a substantive scope and scenario of a laboratory class that introduces students to the field of sonochemistry. The class requires only basic...
This article proposes a substantive scope and scenario of a laboratory class that introduces students to the field of sonochemistry. The class requires only basic laboratory equipment - typical laboratory glassware like graduated pipettes and conical flasks, as well as simple inorganic chemicals. It is designed to acquaint students with fundamental aspects of sonochemistry. In the qualitative aspect, they will conduct and observe some sonochemical reactions like a synthesis of hydrogen peroxide and ultrasound-assisted degradation of toxic chromates(VI) which will demonstrate the indirect consequences of water sonolysis which is the most basic sonochemical reaction, as well as they will illustrate the applications of sonochemistry. In the quantitative aspect, students will learn about how to measure the power of ultrasound and the sonochemical efficiency of the reaction and will conduct experiments allowing for the calculation of these parameters. Finally, an introduction to and demonstration of the sonocatalytic effect is planned. An evaluation system, consisting of a report and test, is also proposed.
PubMed: 37976564
DOI: 10.1016/j.ultsonch.2023.106691 -
Bio-protocol Nov 2023Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation. This protocol describes the measurement of...
Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation. This protocol describes the measurement of propagation velocity using a combination of somatic whole cell and axonal loose patch recordings in brain slice preparations. The axons of neurons filled with fluorescent dye via somatic whole-cell pipette can be targeted under direct optical control using the fluorophore-filled pipette. The propagation delays between the soma and 5-7 axonal locations can be obtained by analyzing the ensemble averages of 500-600 sweeps of somatic APs aligned at times of maximal rate-of-rise (dV/dtmax) and axonal action currents from these locations. By plotting the propagation delays against the distance, the location of the AP initiation zone becomes evident as the site exhibiting the greatest delay relative to the soma. Performing linear fitting of the delays obtained from sites both proximal and distal from the trigger zone allows the determination of the velocities of AP backward and forward propagation, respectively. Key features • Ultra-thin axons in cortical slices are targeted under direct optical control using the SBFI-filled pipette. • Dual somatic whole cell and axonal loose patch recordings from 5-7 axonal locations. • Ensemble averaging of 500-600 sweeps of somatic APs and axonal action currents. • Plotting the propagation delays against the distance enables the determination of the trigger zone's position and velocities of AP backward and forward propagation.
PubMed: 37969753
DOI: 10.21769/BioProtoc.4876 -
American Journal of Physiology. Cell... Jan 2024Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by Na1.7. We present evidence that this current is regulated by cAMP. ASM...
Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by Na1.7. We present evidence that this current is regulated by cAMP. ASM cells were isolated by enzymatic dispersal and studied using the whole cell patch clamp technique at room temperature. A fast sodium current, , was observed on holding cells under voltage clamp at -100 mV and stepping to -20 mV. This current was reduced in a concentration-dependent manner by denopamine (10 and 30 µM), a β-adrenergic agonist. Forskolin (1 µM), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 µM), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 µM), an activator of exchange protein directly activated by cAMP (Epac), reduced the current by 48%. The inhibitory effect of 007-AM was still observed in the presence of dantrolene (10 µM), an inhibitor of ryanodine receptors, and when cytosolic [Ca] was buffered by inclusion of 1,2-bis(-aminophenoxy)ethane-,,,-tetraacetic acid, Sigma (BAPTA) (50 µM) in the pipette solution, suggesting that the inhibition of was not due to Ca-release from intracellular stores. When 007-AM was tested on the current-voltage relationship, it reduced the current at potentials from -30 to 0 mV, but had no effect on the steady-state activation curve. However, the steady-state inactivation , the voltage causing inactivation of 50% of the current, was shifted in the negative direction from -76.6 mV to -89.7 mV. These findings suggest that cAMP regulates in mouse ASM via Epac, but not PKA. β-adrenergic agonists are commonly used in inhalers to treat asthma and chronic obstructive pulmonary disease. These work by causing bronchodilation and reducing inflammation. The present study provides evidence that these drugs have an additional action, namely, to reduce sodium influx into airway smooth muscle cells via fast voltage-dependent channels. This may have the dual effect of promoting bronchodilation and reducing remodeling of the airways, which has a detrimental effect in these diseases.
Topics: Mice; Animals; Sodium; Cyclic AMP; Guanine Nucleotide Exchange Factors; Myocytes, Smooth Muscle; Adrenergic beta-Agonists
PubMed: 37955124
DOI: 10.1152/ajpcell.00417.2023 -
Cells Oct 2023The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell...
The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell technologies. This work presents an original technique for fabricating the silver-coated pipette and its use for the cell analysis by combination with surface-enhanced Raman spectroscopy (SERS) and scanning ion-conducting microscopy (SICM). Unlike the majority of other designs, the pipette opening in our case remains uncovered, which is important for SICM. SERS-active Ag nanoparticles on the pipette surface are formed by vacuum-thermal evaporation followed by annealing. An array of nanoparticles had a diameter on the order of 36 nm and spacing of 12 nm. A two-particle model based on Laplace equations is used to calculate a theoretical enhancement factor (EF). The surface morphology of the samples is investigated by scanning electron microscopy while SICM is used to reveal the surface topography, to evaluate Young's modulus of living cells and to control an injection of the SERS-active pipettes into them. A Raman microscope-spectrometer was used to collect characteristic SERS spectra of cells and cell components. Local Raman spectra were obtained from the cytoplasm and nucleus of the same HEK-293 cancer cell. The EF of the SERS-active pipette was 7 × 10. As a result, we demonstrate utilizing the silver-coated pipette for both the SICM study and the molecular composition analysis of cytoplasm and the nucleus of living cells by SERS. The probe localization in cells is successfully achieved.
Topics: Humans; Silver; Metal Nanoparticles; HEK293 Cells; Microscopy, Electron, Scanning; Single-Cell Analysis; Ions
PubMed: 37947599
DOI: 10.3390/cells12212521 -
Crystal Growth & Design Nov 2023Controllable continuous release of functional materials from capsules is one of the unmet functions of theragnosis particles; on this way, understanding cargo-fluid...
Controllable continuous release of functional materials from capsules is one of the unmet functions of theragnosis particles; on this way, understanding cargo-fluid interactions in vitro is an essential milestone. We develop a flexible platform to investigate single particle-fluid interactions utilizing a glass micropipette as a highly localized flow source around an optically trapped particle. In proof-of-concept experiments, this microparticle is sensitive to local microflow distribution, thus serving as a probe. The very same flows are capable of the particle rotating (i.e., vaterite drug cargo) at frequencies dependent on the mutual particle-pipette position. Platform flexibility comes from different interactions of a tweezer (optical forces) and a pipette (mechanical/hydrodynamical) with a microparticle, which makes this arrangement an ideal microtool. We studied the vaterite dissolution kinetics and demonstrated that it can be controlled on demand, providing a wide cargo release dynamic rate. Our results promote the use of inorganic mesoporous nanoparticles as a nanomedicine platform.
PubMed: 37937190
DOI: 10.1021/acs.cgd.3c00799 -
Nature Microbiology Dec 2023Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across...
Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across disciplines, but it is time-intensive and resource-consuming. Here we describe the geometric viability assay (GVA) that replicates CFU measurements over 6 orders of magnitude while reducing over 10-fold the time and consumables required. GVA computes a sample's viable cell count on the basis of the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with Gram-positive and Gram-negative planktonic bacteria (Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis), biofilms and fungi (Saccharomyces cerevisiae). Laborious CFU experiments such as checkerboard assays, treatment time-courses and drug screens against slow-growing cells are simplified by GVA. The ease and low cost of GVA evinces that it can replace existing viability assays and enable viability measurements at previously impractical scales.
Topics: Colony Count, Microbial; Escherichia coli; Biofilms; Gram-Negative Bacteria; Pseudomonas aeruginosa
PubMed: 37919425
DOI: 10.1038/s41564-023-01513-9 -
Journal of Mass Spectrometry and... Nov 2023Pipettes are essential tools for biomedical and analytical laboratories, analogous to workstations for computer scientists. Variation in pipetting is a known unknown, as... (Review)
Review
Pipettes are essential tools for biomedical and analytical laboratories, analogous to workstations for computer scientists. Variation in pipetting is a known unknown, as it is generally accepted that variations exist, but thus far, there have been limited studies on the extent of these variations in practice. In this mini-review, we highlight how manual pipetting is a key technique in the laboratory, and, although simple, inaccuracy and imprecision exist. If variations are not adequately addressed, errors can be compounded and consequently compromise data quality. Determination of the accuracy and precision of manual pipetting is straightforward, and here we review two common approaches that use gravimetry and spectrophotometry as readouts. We also provide detailed protocols for determination of accuracy and precision using manual single and multi-channel pipettes. These simple-to-use methods can be used by any laboratory for competency training and regular checks. Having a common protocol for evaluation of variation will also enable cross-laboratory comparison and potentially facilitate establishment of a reference value of acceptable ranges for operator error. Such a value could be of relevance to the scientific community for benchmarking and assuring good laboratory practice.
PubMed: 37841753
DOI: 10.1016/j.jmsacl.2023.09.001 -
Sensors (Basel, Switzerland) Sep 2023A patch clamp is the "gold standard" method for studying ion-channel biophysics and pharmacology. Due to the complexity of the operation and the heavy reliance on...
A patch clamp is the "gold standard" method for studying ion-channel biophysics and pharmacology. Due to the complexity of the operation and the heavy reliance on experimenter experience, more and more researchers are focusing on patch-clamp automation. The existing automated patch-clamp system focuses on the process of completing the experiment; the detection method in each step is relatively simple, and the robustness of the complex brain film environment is lacking, which will increase the detection error in the microscopic environment, affecting the success rate of the automated patch clamp. To address these problems, we propose a method that is suitable for the contact between pipette tips and neuronal cells in automated patch-clamp systems. It mainly includes two key steps: precise positioning of pipettes and contact judgment. First, to obtain the precise coordinates of the tip of the pipette, we use the Mixture of Gaussian (MOG) algorithm for motion detection to focus on the tip area under the microscope. We use the object detection model to eliminate the encirclement frame of the pipette tip to reduce the influence of different shaped tips, and then use the sweeping line algorithm to accurately locate the pipette tip. We also use the object detection model to obtain a three-dimensional bounding frame of neuronal cells. When the microscope focuses on the maximum plane of the cell, which is the height in the middle of the enclosing frame, we detect the focus of the tip of the pipette to determine whether the contact between the tip and the cell is successful, because the cell and the pipette will be at the same height at this time. We propose a multitasking network CU-net that can judge the focus of pipette tips in complex contexts. Finally, we design an automated contact sensing process in combination with resistance constraints and apply it to our automated patch-clamp system. The experimental results show that our method can increase the success rate of pipette contact with cells in patch-clamp experiments.
Topics: Robotic Surgical Procedures; Robotics; Brain; Automation; Neurons
PubMed: 37836974
DOI: 10.3390/s23198144 -
Scientific Reports Oct 2023In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk:...
In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (μ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The μ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the μ-IMER method demonstrated superior sequence coverage for oxidized molecules in βCN (33 ± 1.5%) and βLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for βCN; 49 ± 2% for βLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for βCN; 60 ± 4% for βLG) and μ-IMER (41 ± 3% for βCN; 80 ± 5% for βLG). Additionally, phosphorylations were identified. For βCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With μ-IMER and μ-IMER-ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For βLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and μ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and μ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of μ-IMER systems for enhanced sequence coverage and post-translational modification detection.
Topics: Enzymes, Immobilized; Caseins; Lactoglobulins; Trypsin; Peptides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 37783762
DOI: 10.1038/s41598-023-43521-z -
Neuroprotection Sep 2023Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may...
OBJECTIVE
Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may contribute to mechanical brain damage. Glass pipettes with a thin tip may significantly reduce injection-associated brain damage but require access to prohibitively expensive programmable pipette pullers. This study is to remove the economic barrier to the application of minimally invasive delivery of therapeutics to the brain, such as chemical compounds, viral vectors, and cells.
METHODS
We took advantage of the rapid development of free educational online resources and emerging low-cost 3D printers by designing an affordable pipette puller (APP) to remove the cost obstacle.
RESULTS
We showed that our APP could produce glass pipettes with a sharp tip opening down to 20 μm or less, which is sufficiently thin for the delivery of therapeutics into the brain. A pipeline from pipette pulling to brain injection using low-cost and open-source equipment was established to facilitate the application of the APP.
CONCLUSION
In the spirit of frugal science, our device may democratize glass pipette-puling and substantially promote the application of minimally invasive and precisely controlled delivery of therapeutics to the brain for finding more effective therapies of brain diseases.
PubMed: 37771648
DOI: 10.1002/nep3.20