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Biomedical Journal Aug 2023Intercellular coupling is essential for the suprachiasmatic nucleus (SCN) to serve as a coherent central clock. Synaptic release of neurotransmitters and neuropeptides...
BACKGROUND
Intercellular coupling is essential for the suprachiasmatic nucleus (SCN) to serve as a coherent central clock. Synaptic release of neurotransmitters and neuropeptides is critical for synchronizing SCN neurons. However, intercellular coupling via non-synaptic mechanisms has also been demonstrated. In particular, the abundant perikaryal appositions with morphological specializations in the narrow extracellular space (ECS) may hinder molecular diffusion to allow for ion accumulation or depletion.
METHODS
The SCN neurons were recorded in the whole-cell current-clamp mode, with pipette filled with high (26 mM)-Na or low (6 mM)-Na solution.
RESULTS
Cells recorded with high-Na pipette solution could fire spontaneous action potentials (AP) with peak AHP more negative than the calculated value of K equilibrium potential (E) and with peak AP more positive than calculated E. Cells recorded with low-Na pipette solution could also have peak AHP more negative than calculated E. In contrast, the resting membrane potential (RMP) was always less negative to calculated E. The distribution and the averaged amplitude of peak AHP, peak AP, or RMP was similar between cells recorded with high-Na and low-Na solution pipette. In a number of cells, the peak AHP could increase from more positive to become more negative than calculated E spontaneously or after treatments to hyperpolarize the RMP. TTX blocked the Na -dependent APs and tetraethylammonium (TEA), but not Ba or Cd, markedly reduced the peak AHP. Perforated-patch cells could also but rarely fire APs with peak AHP more negative than calculated E.
CONCLUSION
The result of peak AHP negative to calculated E indicates that local [K] sensed by the TEA-sensitive AHP K channels must be lower than bulk [K], most likely due to K clearance from K diffusion-restricted ECS by the Na/K-ATPase. The K diffusion-restricted ECS may allow for K-mediated ionic interactions among neurons to regulate SCN excitability.
Topics: Humans; Extracellular Space; Membrane Potentials; Action Potentials; Suprachiasmatic Nucleus; Neurons; Tetraethylammonium
PubMed: 35863667
DOI: 10.1016/j.bj.2022.07.005 -
Biosensors & Bioelectronics Oct 2024Automation of liquid handling is indispensable to improve throughput and reproducibility in biochemical assays. However, the incorporation of automated systems into...
Automation of liquid handling is indispensable to improve throughput and reproducibility in biochemical assays. However, the incorporation of automated systems into laboratory workflows is often hindered by the high cost and complexity associated with building robotic liquid handlers. Here, we report a 3D-printed liquid handler based on a fluidic manifold, thereby obviating the need for complex robotic mechanisms. The fluidic manifold, termed a dispensing and aspirating (DA) device, comprises parallelized multi-pipette structures connected by distribution and aspiration channels, enabling the precise supply and removal of reagents, respectively. Leveraging the versatility of 3D printing, the DA device can be custom-designed and printed to fit specific applications. As a proof-of-principle, we engineered a 3D-printed liquid handler dedicated for 3D digital rolling circle amplification (4DRCA), an advanced biochemical assay involving multiple sample preparation steps such as antibody incubation, cell fixation, nucleic acid amplification, probe hybridization, and extensive washing. We demonstrate the efficacy of the 3D-printed liquid handler to automate the preparation of clinical samples for the simultaneous, in situ analysis of oncogenic protein and transcript markers in B-cell acute lymphoblastic leukemia cells using 4DRCA. This approach provides an effective and accessible solution for liquid handling automation, offering high throughput and reproducibility in biochemical assays.
Topics: Printing, Three-Dimensional; Humans; Biosensing Techniques; Nucleic Acid Amplification Techniques; Equipment Design; Automation
PubMed: 38905856
DOI: 10.1016/j.bios.2024.116503 -
European Journal of Human Genetics :... Apr 2024News stories and patient-facing material about genetic tests are often illustrated by images, but the content of such images and the messages they propagate are rarely...
News stories and patient-facing material about genetic tests are often illustrated by images, but the content of such images and the messages they propagate are rarely scrutinised. Stock image banks were searched to identify a hundred images relating to genetic tests and analysed using a multimodal critical discourse approach, aiming to identify what the images featured, how they were composed, and what they communicated about genetic testing. We found that images tended to focus on technical aspects of sample processing (for example, pipetting) and drew on older technologies (for example slab gel electrophoresis) when representing data arising from genetic tests. Composition choices like focussing images around pipette tips, or emphasising colour or brightness of electrophoretic bands, represented genetic testing as precise, unambiguous and illuminating. Only 7% of images featured a person having a genetic test, and only one image alluded to communication of genetic results. Current popular visual representations of genetic testing rarely highlight the possibility of uncertain or non-diagnostic outcomes, and may contribute to high public expectations of informativeness and certainty from such tests.
Topics: Humans; Genetic Testing; Gels
PubMed: 38066171
DOI: 10.1038/s41431-023-01508-4 -
Biotechnology Journal Feb 2024Here, we developed a field-deployable molecular diagnostic kit for the detection of RNA viruses that operates in a pipette-free manner. The kit is composed of acrylic...
Here, we developed a field-deployable molecular diagnostic kit for the detection of RNA viruses that operates in a pipette-free manner. The kit is composed of acrylic sticks, PCR tubes, and palm-sized three-dimensional(3D)-printed heaters operated by batteries. The kit performs RNA extraction, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), and visual detection in one kit. An acrylic stick was engraved with one shallow and one deep cylindrical chamber at each end for the insertion of an FTA card and ethidium homodimer-1 (EthD-1), respectively, to perform RNA extraction/purification and bimodal visual detection of the target amplicons. First, an intercalation of EthD-1 into the target DNA initially produces fluorescence upon UV illumination. Next, the addition of a strong oxidant, in this case sodium (meta) periodate (NaIO ), produces intense aggregates in the presence of EthD-1-intercalated DNA, realized by electrostatic interaction. In the absence of the target amplicon, no fluorescence or aggregates are observed. Using this kit, two major infectious viruses-severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus (SARS-CoV-2)-were successfully detected in 1 h, and the limits of detection (LOD) were approximately 1 virus μL for SFTSV and 10 copies μL for SARS-CoV-2 RNA. The introduced kit is portable, end-user-friendly, and can be operated in a pipette-free manner, paving the way for simple and convenient virus detection in resource-limited settings.
Topics: Humans; RNA, Viral; Pathology, Molecular; Sensitivity and Specificity; COVID-19; Virus Diseases; Nucleic Acid Amplification Techniques; DNA; COVID-19 Testing
PubMed: 38403439
DOI: 10.1002/biot.202300521 -
Journal of Analytical Toxicology Nov 2023Oral fluid (OF) is a valuable specimen for driving under the influence of drugs (DUID) applications. This study demonstrates the implementation of the first...
Oral fluid (OF) is a valuable specimen for driving under the influence of drugs (DUID) applications. This study demonstrates the implementation of the first comprehensive OF drug testing program in the United States, including approved roadside screening OF devices for law enforcement and validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation methods. Three roadside OF screening devices were evaluated: the Dräger DrugTest® 5000, Abbott SoToxa®, and Randox Evidence MultiSTAT™. Two qualitative LC-MS-MS confirmation methods were validated per ASB Standard 036. The first method utilized an automated dispersive pipette extraction extraction using Integra and Hamilton STARlet platforms for drugs of abuse. The second method used a liquid-liquid extraction to detect cannabinoids. The prevalence of drugs in blood and OF was monitored over 5 years of casework. Calibration curves were analyzed with each batch to monitor OF concentrations for research purposes. Three roadside OF screening devices were deemed fit for purpose. Devices demonstrated appropriate sensitivity, specificity, positive and negative predictive values, and accuracy above 80% for targeted drugs except for benzodiazepines (DrugTest® 5000) and amphetamine (SoToxa®). The validated LC-MS-MS OF confirmation methods met the National Safety Council-recommended cutoffs for 18/21 (86%) of the targets. Over 5 years of casework, THC and cocaine were detected at a positivity rate of 90% and 97% in OF versus 75% and 44% in blood, respectively. OF:blood ratios exceeded unity for parent drugs. Median concentrations of THC in OF and blood were 31 and 3.5 ng/mL, respectively. OF is a viable alternative or supplemental specimen for DUID investigations. Collecting OF close to the driving event increases the opportunity to identify pharmacologically active substances, and when combined with blood analysis results, an elevated OF:blood ratio provides valuable information for DUID investigation purposes.
Topics: Automobile Driving; Substance Abuse Detection; Saliva; Cannabinoids; Amphetamine
PubMed: 37526020
DOI: 10.1093/jat/bkad051 -
The Science of the Total Environment Jul 2023A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in...
A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in wastewater are necessary for applications in wastewater-based epidemiology. In this study, an automated filtration method using a concentrating pipette (CP Select; Innovaprep) was applied to detect SARS-CoV-2 in wastewater samples with several modifications to increase its sensitivity and throughput. The performance of the CP Select method was compared to other concentration methods (polyethylene glycol precipitation and direct capture using silica column) to evaluate its applicability to SARS-CoV-2 detection in wastewater. SARS-CoV-2 RNA was successfully detected in six of eight wastewater samples using the CP Select method, whereas other methods could detect SARS-CoV-2 RNA in all wastewater samples. Enteric viruses, such as noroviruses of genogroups I (NoVs-GI) and II (NoVs-GII) and enteroviruses, were tested, resulting in 100 % NoVs-GII detection using all concentration methods. As for NoVs-GI and enteroviruses, all methods gave comparable number of detected samples in wastewater samples. This study showed that the optimized CP Select method was less sensitive in SARS-CoV-2 detection in wastewater than other methods, whereas all methods were applicable to detect or recover other viruses in wastewater.
Topics: Humans; SARS-CoV-2; Wastewater; COVID-19; RNA, Viral; Viruses; Norovirus; Enterovirus
PubMed: 37068668
DOI: 10.1016/j.scitotenv.2023.163487 -
Analytical and Bioanalytical Chemistry Oct 2023Insulin-like growth factor 1 analogues are prohibited in sport for their ability to enhance athletic performance in several sport disciplines. Their detection presents... (Comparative Study)
Comparative Study
Detection of synthetic analogues of insulin-like growth factor 1 in different biological fluids by liquid chromatography quadrupole time-of-flight mass spectrometry: comparison of different immunoaffinity protocols.
Insulin-like growth factor 1 analogues are prohibited in sport for their ability to enhance athletic performance in several sport disciplines. Their detection presents several analytical challenges, mainly due to the minimum required performance limits fixed by the World Anti-Doping Agency. Here, we are presenting analytical workflows to detect IGF-1 and its analogues in different biological matrices. Several off-line immunocapture techniques and protocols were comparatively evaluated. Separation and detection were performed by using standard flow reverse-phase liquid chromatography coupled to a time-of-flight mass spectrometer. The best recoveries were obtained using magnetic beads or pipette tips functionalized with protein A. The analytical workflows were fully validated for qualitative determinations: all the target analytes were clearly distinguishable from the interference of the matrices, with limits of detection and identification in the range of 0.05-0.30 ng/mL in urine and 0.5-2.0 ng/mL in serum/plasma. The extraction efficiency proved to be repeatable (CV% < 10) with recoveries higher than 50%. Intra- and inter-day precision were found to be smaller than 10 and 15%, respectively. The method was successfully applied to the analysis of authentic matrix samples containing the target peptides at the minimum required performance limits, proving that the method developed can be successfully applied to detect and identify IGF-1 analogues for doping control purposes in all the matrices selected. The analytical workflow developed here to detect the target peptides in different matrices can be readily implemented in anti-doping laboratories and has the potential to be adapted for the simultaneous analysis of different similarly sized peptide hormones of doping relevance.
Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Doping in Sports; Insulin-Like Growth Factor I; Substance Abuse Detection; Tandem Mass Spectrometry
PubMed: 37566232
DOI: 10.1007/s00216-023-04885-3 -
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 -
BMC Chemistry Apr 20246-mercaptopurine (6-MP) is a chemotherapy drug mainly used to treat leukemia. It is a persistent organic pollutant and can remain in the environment for a long period of...
BACKGROUND
6-mercaptopurine (6-MP) is a chemotherapy drug mainly used to treat leukemia. It is a persistent organic pollutant and can remain in the environment for a long period of time. The presence of 6-MP in the environment poses a number of hazards and needs to be assessed to monitor its potential risk to human health and the environment. However, due to its trace amount in complicated matrices, a clean-up and preconcentration step before its determination is compulsory.
RESULTS
As a highly efficient adsorbent for the extrication of 6-mercaptopurine (6-MP), a novel carbon nanotube doped with camphor: decanoic acid deep eutectic solvent was synthesized and applied as a packing material for the pipette-tip micro solid phase extraction sorbent of 6-MP from tap, wastewater and seawater samples before its spectrophotometric determination. Characteristics and structure of this adsorbent was fully investigated. Factors affecting extraction, including type and volume of the eluent, ionic strength and pH of the sample solution, amount of adsorbent, and number of extraction and elution cycles were optimized using one-factor-at-a-time and response surface methodologies. The method was found to be linear in the range of 1 to 1000 µg/L with a limit of detection and quantification of 0.2 and 0.7 µg/L, respectively. Reproducibility as relative standard deviation was better than 4.6%.
CONCLUSION
Application of deep eutectic solvent modified carbon nanotube indicated suitable microextraction results and good potential for rapid extraction of trace amounts of 6-MP from different aqueous samples. The amount of sample required for the analysis was less than 10 mL and only 1.5 mg of the adsorbent was used. The total analysis time, including extraction was less than 15 min and the adsorbent could be used for at least 10 times, without significantly losing its adsorption ability. Compared to using unmodified usual carbon nanotubes, deep eutectic solvent doped carbon nanotubes showed 19.8% higher extraction ability.
PubMed: 38654336
DOI: 10.1186/s13065-024-01199-y -
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