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Longitudinal diffusion barriers imposed by myofilaments and mitochondria in murine cardiac myocytes.The Journal of General Physiology Oct 2023Using optical and electrical methods, we document that diffusion in the cytoplasm of BL6 murine cardiomyocytes becomes restricted >20-fold as molecular weight increases...
Using optical and electrical methods, we document that diffusion in the cytoplasm of BL6 murine cardiomyocytes becomes restricted >20-fold as molecular weight increases from 30 to 2,000, roughly as expected for pores with porin channel dimensions. Bodipy-FL ATP diffuses >40-fold slower than in free water at 25°C. From several fluorophores analyzed, bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. Unbound fluorophores diffuse at 0.5-8 × 10-7 cm2/s (5-80 μm2/s). Analysis of Na/K pump and veratridine-modified Na channel currents suggests that Na diffusion is nearly unrestricted at 35°C (time constant for equilibration with the pipette tip, ∼20 s). Using multiple strategies, we estimate that at 35°C, ATP diffuses four to eight times slower than in free water. To address whether restrictions are caused more by protein or membrane networks, we verified first that a protein gel, 10 g% gelatin, restricts diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is less restricted than in intact myocytes. Notably, myofilaments extracted similarly from skeletal (diaphragm) myocytes are less restrictive. Solute diffusion in myocytes with sarcolemma permeabilized by β-escin (80 µM) is similar to diffusion in intact myocytes. Restrictions are strain-dependent, being twofold greater in BL6 myocytes than in CD1/J6/129svJ myocytes. Furthermore, longitudinal diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, VDAC1, than in WT CD1/J6/129svJ myocytes. Thus, mitochondria networks restrict long-range diffusion while presumably optimizing nucleotide transfer between myofilaments and mitochondria. We project that diffusion restrictions imposed by both myofilaments and the outer mitochondrial membrane are important determinants of total free cytoplasmic AMP and ADP (∼10 μM). However, the capacity of diffusion to deliver ATP to myofilaments remains ∼100-fold greater than ATP consumption.
Topics: Mice; Animals; Myocytes, Cardiac; Myofibrils; Mitochondria; Diffusion; Voltage-Dependent Anion Channels; Adenosine Triphosphate; Water
PubMed: 37555782
DOI: 10.1085/jgp.202213329 -
Diagnostic Microbiology and Infectious... May 2024The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic led to global shortages in laboratory consumables, in particular for automated PCR. The...
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic led to global shortages in laboratory consumables, in particular for automated PCR. The Technical University of Denmark supported Danish hospitals from 2020 to 2022, conducting SARS-CoV-2 RT-qPCR on around 10,000 patient samples daily. We encountered shortages of disposable pipette tips used with automated liquid handlers that transferred oropharyngeal swab samples to 96-well microplates before RNA extraction. To enable tip reuse, we developed an automated protocol for washing tips with a 0.5 % sodium hypochlorite solution. This effectively eliminated carry-over of genomic material and the wash solution remained effective when stored in an open reservoir at ambient temperatures for 24 h. A three-day validation setup demonstrated the robustness of the tip wash protocol. Reducing the number of tips used for transferring samples to 96-well microplates from 96 to 8 enabled us to mitigate pipette tip shortages, lower costs, and minimize plastic waste generation.
Topics: Humans; SARS-CoV-2; COVID-19; COVID-19 Testing; Laboratories; RNA, Viral
PubMed: 38452555
DOI: 10.1016/j.diagmicrobio.2024.116241 -
Materials (Basel, Switzerland) Oct 2018A detailed study of the production of polysaccharide aerogel (bio-aerogel) particles from lab to pilot scale is surveyed in this article. An introduction to various... (Review)
Review
A detailed study of the production of polysaccharide aerogel (bio-aerogel) particles from lab to pilot scale is surveyed in this article. An introduction to various droplets techniques available in the market is given and compared with the lab scale production of droplets using pipettes and syringes. An overview of the mechanisms of gelation of polysaccharide solutions together with non-solvent induced phase separation option is then discussed in the view of making wet particles. The main steps of particle recovery and solvent exchange are briefly described in order to pass through the final drying process. Various drying processes are overviewed and the importance of supercritical drying is highlighted. In addition, we present the characterization techniques to analyse the morphology and properties of the aerogels. The case studies of bio-aerogel (agar, alginate, cellulose, chitin, κ-carrageenan, pectin and starch) particles are reviewed. Potential applications of polysaccharide aerogel particles are briefly given. Finally, the conclusions summarize the prospects of the potential scale-up methods for producing bio-aerogel particles.
PubMed: 30384442
DOI: 10.3390/ma11112144 -
One Health (Amsterdam, Netherlands) Jun 2023Efficient and accurate diagnosis of Hendra virus (HeV), a biosafety level 4 (BSL-4) pathogen and zoonotic disease, is of primary importance for surveillance and outbreak...
Efficient and accurate diagnosis of Hendra virus (HeV), a biosafety level 4 (BSL-4) pathogen and zoonotic disease, is of primary importance for surveillance and outbreak control in the Australian equine industry. Sporadic HeV spillover events pose a serious public health concern and are predicted to expand geographically, aligning with the moving distribution of the main reservoir hosts, the flying-foxes. Here we describe the development of a low-resource rapid Hendra test. The test used a fast and simple sample processing protocol followed by reverse transcription isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results were obtained in 30 min and required only a heating block, ice, and pipettes for liquid handling. The one-step sample processing protocol inactivated HeV in 2 min, providing a simple protocol that could enable safe testing outside of a laboratory. Analytical sensitivity testing demonstrated a detection limit of 1000 copies/μL of synthetic HeV RNA, and analytical specificity testing indicated assays did not detect other pathogens. Gamma-irradiated HeV-spiked in viral transport medium was detected with high sensitivity, down to 10,000 TCID/mL, the equivalent of 18 RNA copies per reaction. Collectively, our data suggests that our rapid Hendra test offers a potential first-line screening on-site alternative to gold-standard RT-PCR detection, which requires samples to be shipped to central containment laboratories, thermocyclers and labour-intensive viral RNA purification, with testing time of approximately four hours. Our rapid Hendra test provided performance and speed without compromising sensitivity and specificity, and could become a promising more accessible tool for testing under resource-limited conditions for the veterinary community and thoroughbred industry.
PubMed: 37363221
DOI: 10.1016/j.onehlt.2023.100504 -
ENeuro Apr 2023Midbrain dopamine (DA) neurons are among the best characterized pacemaker neurons, having intrinsic, rhythmic firing activity even in the absence of synaptic input....
Midbrain dopamine (DA) neurons are among the best characterized pacemaker neurons, having intrinsic, rhythmic firing activity even in the absence of synaptic input. However, the mechanisms of DA neuron pacemaking have not been systematically related to how these cells respond to synaptic input. The input-output properties of pacemaking neurons can be characterized by the phase-resetting curve (PRC), which describes the sensitivity of interspike interval (ISI) length to inputs arriving at different phases of the firing cycle. Here we determined PRCs of putative DA neurons in the substantia nigra pars compacta in brain slices from male and female mice using gramicidin-perforated current-clamp recordings with electrical noise stimuli applied through the patch pipette. On average, and compared with nearby putative GABA neurons, DA neurons showed a low, nearly constant level of sensitivity across most of the ISI, but individual cells had PRCs showing relatively greater sensitivity at early or late phases. Pharmacological experiments showed that DA neuron PRCs are shaped by small-conductance calcium-activated potassium and Kv4 channels, which limit input sensitivity across early and late phases of the ISI. Our results establish the PRC as a tractable experimental measurement of individual DA neuron input-output relationships and identify two of the major ionic conductances that limit perturbations to rhythmic firing. These findings have applications in modeling and for identifying biophysical changes in response to disease or environmental manipulations.
Topics: Mice; Male; Female; Animals; Dopaminergic Neurons; Mesencephalon; Pars Compacta; Action Potentials
PubMed: 36973012
DOI: 10.1523/ENEURO.0445-22.2023 -
Heliyon May 2022The Bouyoucos method lacks sample pre-treatment whereby samples are dispersed for only 2 min after being soaked in 5% sodium hexametaphosphate (calgon) for 15-20 h. This...
The Bouyoucos method lacks sample pre-treatment whereby samples are dispersed for only 2 min after being soaked in 5% sodium hexametaphosphate (calgon) for 15-20 h. This study aimed to improve the accuracy of the Bouyoucos (Hydrometer) method of particle size analysis by proposing the best sample pre-treatment and analysis practices. The Pipette method was used as standard reference due to its precision and reproducibility. Treatments modified from the hydrometer method were compared with the Pipette method through Pearson's correlation. The treatments involved variation in the concentration of the pre-treating and dispersing material, time of hydrometer readings, method of agitation and that of dispersing. The proposed improvement suits the Food and Agriculture Organization (FAO) and United States Department of Agriculture (USDA) standard 2-50 μm system. Measurements made using the hydrometer variations overestimated the sand fraction in all samples. There was a positive correlation in the clay proportion between the Pipette method and Shaking + Stirring treatment (r = 0.644, p-value = 0.044). Treatments involving shaking instead of stirring, increased concentrations of hydrogen peroxide and calgon estimated the clay proportion with sufficient accuracy. These observations are indicative of the need for soil pre-treatment with hydrogen peroxide to remove organic matter and also the use 10% calgon as a dispersing agent.
PubMed: 35600453
DOI: 10.1016/j.heliyon.2022.e09395 -
Wellcome Open Research 2020Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to...
Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F containing internal solution together with a Ca containing external solution - so that a CaF crystal forms when the two solutions meet which 'plugs the holes' to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.
PubMed: 34805549
DOI: 10.12688/wellcomeopenres.15968.2 -
Biological & Pharmaceutical Bulletin Apr 2021To confirm that an increase in blood pressure induced by ketamine is mediated through the central nervous system, we examined the effect of ketamine, applied directly to...
To confirm that an increase in blood pressure induced by ketamine is mediated through the central nervous system, we examined the effect of ketamine, applied directly to the amygdala, on blood pressure. Six male Sprague-Dawley rats were used in the study. Under head-restrained and unanesthetized condition, 0.2 µL (5 mg/mL) of ketamine was injected in and around the amygdala at a flow rate of 0.2 µL/min through a glass pipette, and the blood pressure was recorded while monitoring the state of the animals by electroencephalogram and electromyogram. After ketamine injection, the injection site was marked by Pontamine Sky Blue infusion. Blood pressure was increased by ketamine injection into the basolateral and central nuclei of the amygdala, endopiriform nucleus and piriform cortex. In a total of 11 responses, an increase in blood pressure started with a mean latency of 193.5 ± 43.0 s, reached its peak 180.2 ± 23.3 s after the response onset, then gradually returned to the baseline with mean duration of 706.7 ± 113.5 s. The mean fluctuation was 17.1 ± 2.5 mmHg. We revealed that blood pressure fluctuations induced by ketamine are associated with the amygdala. Elucidation of the mechanism of ketamine-induced blood pressure increase will lead to understanding of the mechanism of side effects of ketamine, and will contribute to its appropriate use.
Topics: Amygdala; Animals; Blood Pressure; Injections; Ketamine; Male; Rats; Rats, Sprague-Dawley
PubMed: 33583871
DOI: 10.1248/bpb.b20-00881 -
Journal of Neuroscience Methods Dec 2019Recent advancements with induced pluripotent stem cell-derived (iPSC) retinal pigment epithelium (RPE) have made disease modeling and cell therapy for macular...
BACKGROUND
Recent advancements with induced pluripotent stem cell-derived (iPSC) retinal pigment epithelium (RPE) have made disease modeling and cell therapy for macular degeneration feasible. However, current techniques for intracellular electrophysiology - used to validate epithelial function - are painstaking and require manual skill; limiting experimental throughput.
NEW METHOD
A five-stage algorithm, leveraging advances in automated patch clamping, systematically derived and optimized, improves yield and reduces skill when compared to conventional, manual techniques.
RESULTS
The automated algorithm improves yield per attempt from 17% (manually, n = 23) to 22% (automated, n = 120) (chi-squared, p = 0.004). Specifically for RPE, depressing the local cell membrane by 6 μm and electroporating (buzzing) just prior to this depth (5 μm) maximized yield.
COMPARISON WITH EXISTING METHOD
Conventionally, intracellular epithelial electrophysiology is performed by manually lowering a pipette with a micromanipulator, blindly, towards a monolayer of cells and spontaneously stopping when the magnitude of the instantaneous measured membrane potential decreased below a predetermined threshold. The new method automatically measures the pipette tip resistance during the descent, detects the cell surface, indents the cell membrane, and briefly buzzes to electroporate the membrane while descending, overall achieving a higher yield than conventional methods.
CONCLUSIONS
This paper presents an algorithm for high-yield, automated intracellular electrophysiology in epithelia; optimized for human RPE. Automation reduces required user skill and training while, simultaneously, improving yield. This algorithm could enable large-scale exploration of drug toxicity and physiological function verification for numerous kinds of epithelia.
Topics: Algorithms; Electrophysiology; Humans; Patch-Clamp Techniques; Retinal Pigment Epithelium
PubMed: 31562888
DOI: 10.1016/j.jneumeth.2019.108442 -
Journal of Undergraduate Neuroscience... 2022Pipetting is an important technique used in almost every molecular neuroscience method including but not limited to, PCR, reverse transcription, immunohistochemistry,...
Pipetting is an important technique used in almost every molecular neuroscience method including but not limited to, PCR, reverse transcription, immunohistochemistry, chromatin immunoprecipitation, and cell culture. The COVID-19 pandemic has robbed the undergraduate population of time to practice in person laboratory techniques. In response, we have devised a standardized, quick, and fun way to instruct students on the fundamentals of pipetting, serial dilutions, and basic statistical analysis. Here, we offer a standardized protocol for instructors to use to teach undergraduates valuable skills while providing friendly competition. We also offer an example of an undergraduate performing the steps of this protocol with example results and the results from three separate undergrads' first two attempts. This exercise provides laboratories with a method to reintroduce undergraduates to lab basics while standardizing the training thereby saving time lost to the pandemic.
PubMed: 38322045
DOI: 10.59390/NOYQ7921