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The Journal of Physiology Apr 2024We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (i) in rat hippocampal mossy fibre boutons. Cox's...
We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (i) in rat hippocampal mossy fibre boutons. Cox's method for correcting space-clamp errors was extended to the case of an isopotential compartment with attached neurites. The method was applied to voltage-ramp experiments, in which i is assumed to gate instantaneously. The raw estimates of i led to predicted clamp currents that were at variance with observation, hence an algorithm was devised to improve these estimates. Optionally, the method also allows an estimate of the membrane specific capacitance, although values of the axial resistivity and seal resistance must be provided. Assuming that membrane specific capacitance and axial resistivity were constant, we conclude that seal resistance continued to fall after adding TTX to the bath. This might have been attributable to a further deterioration of the seal after baseline rather than an unlikely effect of TTX. There was an increase in the membrane specific resistance in TTX. The reason for this is unknown, but it meant that i could not be determined by simple subtraction. Attempts to account for i with a Hodgkin-Huxley model of the transient sodium conductance met with mixed results. One thing to emerge was the importance of voltage shifts. Also, a large variability in previously reported values of transient sodium conductance in mossy fibre boutons made comparisons with our results difficult. Various other possible sources of error are discussed. Simulations suggest a role for i in modulating the axonal attenuation of EPSPs. KEY POINTS: We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (i) in rat hippocampal mossy fibre boutons, using a KCl-based internal (pipette) solution and correcting for the liquid junction potential (2 mV). Space-clamp errors and deterioration of the patch-clamp seal during the experiment were corrected for by compartmental modelling. Attempts to account for i in terms of the transient sodium conductance met with mixed results. One possibility is that the transient sodium conductance is higher in mossy fibre boutons than in the axon shaft. The analysis illustrates the need to account for various voltage shifts (Donnan potentials, liquid junction potentials and, possibly, other voltage shifts). Simulations suggest a role for i in modulating the axonal attenuation of excitatory postsynaptic potentials, hence analog signalling by dentate granule cells.
Topics: Rats; Animals; Mossy Fibers, Hippocampal; Sodium; Presynaptic Terminals
PubMed: 38594842
DOI: 10.1113/JP284657 -
Advanced Materials (Deerfield Beach,... Apr 2024Accurate dosing for various liquids, especially for highly viscous liquids, is fundamental in wide-ranging from molecular crosslinking to material processing. Despite...
Accurate dosing for various liquids, especially for highly viscous liquids, is fundamental in wide-ranging from molecular crosslinking to material processing. Despite droppers or pipettes being widely used as pipetting devices, they are powerless for quantificationally splitting and dosing highly viscous liquids (>100 mPa s) like polymer liquids due to the intertwined macromolecular chains and strong cohesion energy. Here, a highly transparent photopyroelectric slippery (PS) platform is provided to achieve noncontact self-splitting for liquids with viscosity as high as 15 000 mPa s, just with the assistance of sunlight and a cooling source to provide a local temperature difference (ΔT). Moreover, to guarantee the accuracy for pipetting liquids (>80%), the ultrathin MXene film (within a thickness of 20 nm) is self-assembled as the photo-thermal layers, overcoming the trade-off between transparency and photothermal property. Compared with traditional pipetting strategies (≈1.3% accuracy for pipetting polymer liquids), this accurate microfluidic chip shows great potential in adhesive systems (bonding strength, twice than using the droppers or pipettes).
PubMed: 38594015
DOI: 10.1002/adma.202402779 -
Microsystems & Nanoengineering 2024Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic...
Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.
PubMed: 38590818
DOI: 10.1038/s41378-024-00664-x -
BioRxiv : the Preprint Server For... Mar 2024The small amount of human tissue available for testing is a paramount challenge in cancer drug development, cancer disease models, and personalized oncology....
The small amount of human tissue available for testing is a paramount challenge in cancer drug development, cancer disease models, and personalized oncology. Technologies that combine the microscale manipulation of tissues with fluid handling offer the exciting possibility of miniaturizing and automating drug evaluation workflows. This approach minimizes animal testing and enables inexpensive, more efficient testing of samples with high clinical biomimicry using scarce materials. We have developed an inexpensive platform based on an off-the-shelf robot that can manipulate microdissected tissues (µDTs) into user-programmed positions without using intricate microfluidic designs nor any other accessories such as a microscope or a pneumatic controller. The robot integrates complex functions such as vision and fluid actuation by incorporating simple items including a USB camera and a rotary pump. Through the robot's camera, the platform software optically recognizes randomly-seeded µDTs on the surface of a petri dish and positions a mechanical arm above the µDTs. Then, a custom rotary pump actuated by one of the robot's motors generates enough microfluidic lift to hydrodynamically pick and place µDTs with a pipette at a safe distance from the substrate without requiring a proximity sensor. The platform's simple, integrated construction is cost-effective and compact, allowing placement inside a tissue culture hood for sterile workflows. The platform enables users to select µDTs based on their size, place them in user-programmed arrays, such as multi-well plates, and control various robot motion parameters. As a case application, we use the robotic system to conduct semi-automated drug testing of mouse and human µDTs in 384-well plates. Our user-friendly platform promises to democratize microscale tissue research to clinical and biological laboratories worldwide.
PubMed: 38586030
DOI: 10.1101/2024.03.21.586169 -
Food Chemistry Aug 2024Ensuring the safety of animal-derived foods requires the reliable and swift identification of enrofloxacin residues to monitor the presence of antibiotics. In this...
Ensuring the safety of animal-derived foods requires the reliable and swift identification of enrofloxacin residues to monitor the presence of antibiotics. In this regard, we synthesized, tuned, and investigated the optical properties of a bimetallic metal-organic framework (Ce/Zr-UiO 66). The investigation was facilitated by employing a polydopamine-coated pipette tip with high adsorption efficiency, serving as an immunoreactive carrier. Subsequently, an immunofunctionalized variant of Ce/Zr-UiO 66, referred to as Ce/Zr-UiO 66@ Bovine serum albumin-enrofloxacin, was developed as an optical probe for the rapid and sensitive identification of enrofloxacin across a variety of samples. The method can accurately detect enrofloxacin at concentrations as low as 0.12 ng/mL, with a determination time of under 15 min; furthermore, it demonstrates exceptional efficacy when applied to food, environmental, and clinical samples. The implementation of this methodology offers a valuable means for cost-effective, rapid, and on-site enrofloxacin determination.
Topics: Enrofloxacin; Metal-Organic Frameworks; Animals; Milk; Food Contamination; Anti-Bacterial Agents; Cattle; Immunoassay; Biosensing Techniques; Limit of Detection
PubMed: 38581779
DOI: 10.1016/j.foodchem.2024.139050 -
Journal of Analytical Toxicology May 202411-Nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH) is the most frequently detected illicit drug metabolite in the military drug testing program. An increasing...
11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH) is the most frequently detected illicit drug metabolite in the military drug testing program. An increasing number of specimens containing unresolved Δ8-THCCOOH prompted the addition of this analyte to the Department of Defense drug testing panel. A method was developed and validated for the quantitative confirmation of the carboxylated metabolites of Δ8- and Δ9-THC in urine samples utilizing automated pipette tip dispersive solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Analytes were separated isocratically over an 8.5-min runtime and detected on an MS-MS equipped with an electrospray ionization source operated in negative mode. A single point calibrator (15 ng/mL) forced through zero demonstrated linearity from 3 to 1,000 ng/mL. Intra- and inter-day precision were ≤9.1%, and bias was within ±14.1% for Δ8-THCCOOH and Δ9-THCCOOH. No interferences were found after challenging the method with different over-the-counter drugs, prescription pharmaceuticals, drugs of abuse and several cannabinoids and cannabinoid metabolites, including Δ10-THCCOOH. Urine specimens presumptively positive by immunoassay (n = 2,939; 50 ng/mL Δ9-THCCOOH cutoff) were confirmed with this analytical method. Δ8-THCCOOH and Δ9-THCCOOH were present together above the 15 ng/mL cutoff in 33% of specimens. However, nearly one-third of the specimens analyzed were positive for Δ8-THCCOOH only. This manuscript describes the first validated automated extraction and confirmation method for Δ8- and Δ9-THCCOOH in urine that provides adequate analyte separation in urine specimens with extreme isomer abundance ratios.
Topics: Dronabinol; Humans; Tandem Mass Spectrometry; Substance Abuse Detection; Chromatography, Liquid; Solid Phase Extraction; Reproducibility of Results; Illicit Drugs; Limit of Detection; Isomerism; Liquid Chromatography-Mass Spectrometry
PubMed: 38581658
DOI: 10.1093/jat/bkae031 -
BMC Women's Health Apr 2024Women presenting with abnormal uterine bleeding needs careful and thorough assessment including ultrasound examination of endometrium and histopathological assessment of...
Rate and risk factors of inadequate endometrial tissues after endometrial sampling among Bhutanese women at the national referral hospital of Bhutan: a cross-sectional study.
INTRODUCTION
Women presenting with abnormal uterine bleeding needs careful and thorough assessment including ultrasound examination of endometrium and histopathological assessment of the endometrial tissues. The objective of this cross-sectional study was to determine the rate and the factors associated with inadequate endometrial tissues after endometrial sampling using MedGyn® pipette among Bhutanese women at the colposcopy clinic, Jigme Dorji Wangchuck National Referral Hospital (JDWNRH), Bhutan.
METHODS
This cross-sectional study was conducted at the colposcopy clinic, JDWNRH, Thimphu between October, 2021 and March, 2022. Women included in this study underwent endometrial sampling using MedGyn® pipette without anesthesia as an office procedure. Data were collected using an interviewer-administered questionnaire and results extracted into a structured pro forma. The histopathology reports were extracted from the Department of Pathology and Laboratory Medicine, JDWNRH using the unique Bhutanese citizenship identity card number of the study participants.
RESULTS
Inadequate endometrial tissues were noted in 27% (33 out of 122 cases). Among 89 patients with an adequate endometrial tissue, histologic results were normal in 30 (33.7%), benign pathology in 22 (24.7%), atrophy in 10 (8.2%), and hyperplasia in 27 (30.3%). In a univariate analysis, menopausal state (OR 1.6, 95% CI 0.708-3.765), overweight and obese (OR 1.6 95% CI 0.640-3.945), unemployed (OR 1.7, 95% CI 0.674-1.140), nulliparous (OR 1.7, 95% CI 0.183-15.816), primipara (OR 5.1, 95% CI 0.635-40.905) and use of hormonal contraception (OR 2.1, 95% CI 0.449-10.049) were associated with increased risk of inadequate endometrial tissues. On multivariate regression analysis, nulliparity (OR 1.1, 95% CI 0.101-12.061), overweight and obesity (OR 1.4, 95% CI 0.490-3.917), use of hormonal contraceptives (OR 2.2, 95% CI 0.347-13.889), and junior surgeons (OR 1.1, 95%CI 0.463-2.443) were found to be associated with inadequate endometrial tissues. However, the above associations were not statistically significant (p > 0.05).
CONCLUSION
The rate of inadequate endometrial tissue following endometrial sampling using MedGyn® pipette was 27.0%. Factors associated with an increased risk of inadequate endometrial tissue after endometrial sampling were menopausal state, overweight and obese, unemployed, nulliparous, primipara and use of hormonal contraception.
Topics: Humans; Female; Bhutan; Cross-Sectional Studies; Overweight; Endometrium; Obesity; Risk Factors; Referral and Consultation; Uterine Hemorrhage; Endometrial Neoplasms
PubMed: 38566186
DOI: 10.1186/s12905-024-03047-6 -
Micron (Oxford, England : 1993) Jun 2024Human red blood cells show submicron membrane fluctuations (CMFs) that have been mainly studied with optical microscopies. Although the functional role of this...
Human red blood cells show submicron membrane fluctuations (CMFs) that have been mainly studied with optical microscopies. Although the functional role of this phenomenon is still uncertain, the amplitude of membrane fluctuations is considered as an indicator of mechanical resilience to the stress encountered in the capillary beds. We investigate here the membrane fluctuations in red blood cells using the scanning ion conductance microscopy (SICM), a scanning probe technique that avoids the probe-sample contact. The ion current noise was recorded at a fixed distance from the cell and converted in terms of membrane fluctuation amplitude using as a converting factor the slope of the current-distance curve. We found that CMF amplitude was irreversibly reduced by membrane cross-link. Both whole cell and local increase of membrane tension induced a reduction of CMF amplitude. As for the biochemical regulation of membrane dynamics, we observed that the activation of the noradrenergic transduction pathway, via β-receptors, increased the CMF amplitude. We conclude that the CMFs recorded by SICM and those optically recorded on red blood cells share the main features. In addition SICM provides high spatial and temporal resolution as well as the possibility to apply through the glass pipette acting as probe chemical or physical stimuli to the membrane area where the CMFs are recorded.
PubMed: 38554487
DOI: 10.1016/j.micron.2024.103635 -
Sensors (Basel, Switzerland) Mar 2024This paper presents high-sensitivity sensors based on an open complementary square split-ring resonator and a modified open complementary split-ring resonator operating...
This paper presents high-sensitivity sensors based on an open complementary square split-ring resonator and a modified open complementary split-ring resonator operating at 4.5 GHz and 3.4 GHz, respectively. The sensors are designed for the detection of multiple liquid materials, including distilled water, methanol, and ethanol. The liquid under test is filled in a glass container loaded using a pipette. Compared to the conventional OCSSRR, the modified OCSSRR with multiple rings exhibits a higher frequency shift of 1200 MHz, 1270 MHz, and 1520 MHz for ethanol, methanol, and distilled water, respectively. The modified sensor also demonstrates a high sensitivity of 308 MHz/RIU for ethanol concentration which is the highest among the existing microwave sensors. The sensors in this manuscript are suitable for multiple liquid-material-sensing applications.
PubMed: 38544102
DOI: 10.3390/s24061840 -
Biophysical Journal Mar 2024Compaction is the first morphogenetic movement of the eutherian mammals and involves a developmentally regulated adhesion process. Previous studies investigated cellular...
Compaction is the first morphogenetic movement of the eutherian mammals and involves a developmentally regulated adhesion process. Previous studies investigated cellular and mechanical aspects of compaction. During mouse and human compaction, cells spread onto each other as a result of a contractility-mediated increase in surface tension pulling at the edges of their cell-cell contacts. However, how compaction may affect the mechanical stability of cell-cell contacts remains unknown. Here, we used a dual pipette aspiration assay on cell doublets to quantitatively analyze the mechanical stability of compacting mouse embryos. We measured increased mechanical stability of contacts with rupture forces growing from 40 to 70 nN, which was highly correlated with cell-cell contact expansion. Analyzing the dynamic molecular reorganization of cell-cell contacts, we find minimal recruitment of the cell-cell adhesion molecule Cdh1 (also known as E-cadherin) to contacts but we observe its reorganization into a peripheral adhesive ring. However, this reorganization is not associated with increased effective bond density, contrary to previous reports in other adhesive systems. Using genetics, we reduce the levels of Cdh1 or replace it with a chimeric adhesion molecule composed of the extracellular domain of Cdh1 and the intracellular domain of Cdh2 (also known as N-cadherin). We find that reducing the levels of Cdh1 impairs the mechanical stability of cell-cell contacts due to reduced contact growth, which nevertheless show higher effective bond density than wild-type contacts of similar size. On the other hand, chimeric adhesion molecules cannot form large or strong contacts indicating that the intracellular domain of Cdh2 is unable to reorganize contacts and/or is mechanically weaker than the one of Cdh1 in mouse embryos. Together, we find that mouse embryo compaction mechanically strengthens cell-cell adhesion via the expansion of Cdh1 adhesive rings that maintain pre-compaction levels of effective bond density.
PubMed: 38528761
DOI: 10.1016/j.bpj.2024.03.028