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Journal of Chromatography. A Jul 2016Identifying recent cannabis intake is confounded by prolonged cannabinoid excretion in chronic frequent cannabis users. We previously observed detection times ≤2.1h...
Quantification of cannabinoids and their free and glucuronide metabolites in whole blood by disposable pipette extraction and liquid chromatography-tandem mass spectrometry.
Identifying recent cannabis intake is confounded by prolonged cannabinoid excretion in chronic frequent cannabis users. We previously observed detection times ≤2.1h for cannabidiol (CBD) and cannabinol (CBN) and Δ(9)-tetrahydrocannabinol (THC)-glucuronide in whole blood after smoking, suggesting their applicability for identifying recent intake. However, whole blood collection may not occur for up to 4h during driving under the influence of drugs investigations, making a recent-use marker with a 6-8h detection window helpful for improving whole blood cannabinoid interpretation. Other minor cannabinoids cannabigerol (CBG), Δ9-tetrahydrocannabivarin (THCV), and its metabolite 11-nor-9-carboxy-THCV (THCVCOOH) might also be useful. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry method for quantification of THC, its phase I and glucuronide phase II metabolites, and 5 five minor cannabinoids. Cannabinoids were extracted from 200μL whole blood via disposable pipette extraction, separated on a C18 column, and detected via electrospray ionization in negative mode with scheduled multiple reaction mass spectrometric monitoring. Linear ranges were 0.5-100μg/L for THC and 11-nor-9-carboxy-THC (THCCOOH); 0.5-50μg/L for 11-hydroxy-THC (11-OH-THC), CBD, CBN, and THC-glucuronide; 1-50μg/L for CBG, THCV, and THCVCOOH; and 5-500μg/L for THCCOOH-glucuronide. Inter-day accuracy and precision at low, mid and high quality control (QC) concentrations were 95.1-113% and 2.4-8.5%, respectively (n=25). Extraction recoveries and matrix effects at low and high QC concentrations were 54.0-84.4% and -25.8-30.6%, respectively. By simultaneously monitoring multiple cannabinoids and metabolites, identification of recent cannabis administration or discrimination between licit medicinal and illicit recreational cannabis use can be improved.
Topics: Biomarkers; Cannabidiol; Cannabinoids; Chromatography, Liquid; Dronabinol; Glucuronides; Humans; Marijuana Smoking; Substance Abuse Detection; Tandem Mass Spectrometry
PubMed: 27236483
DOI: 10.1016/j.chroma.2016.05.024 -
Journal of Neurophysiology Oct 2016Patch clamp is the main technique for measuring electrical properties of individual cells. Since its discovery in 1976 by Neher and Sakmann, patch clamp has been...
Patch clamp is the main technique for measuring electrical properties of individual cells. Since its discovery in 1976 by Neher and Sakmann, patch clamp has been instrumental in broadening our understanding of the fundamental properties of ion channels and synapses in neurons. The conventional patch-clamp method requires manual, precise positioning of a glass micropipette against the cell membrane of a visually identified target neuron. Subsequently, a tight "gigaseal" connection between the pipette and the cell membrane is established, and suction is applied to establish the whole cell patch configuration to perform electrophysiological recordings. This procedure is repeated manually for each individual cell, making it labor intensive and time consuming. In this article we describe the development of a new automatic patch-clamp system for brain slices, which integrates all steps of the patch-clamp process: image acquisition through a microscope, computer vision-based identification of a patch pipette and fluorescently labeled neurons, micromanipulator control, and automated patching. We validated our system in brain slices from wild-type and transgenic mice expressing channelrhodopsin 2 under the Thy1 promoter (line 18) or injected with a herpes simplex virus-expressing archaerhodopsin, ArchT. Our computer vision-based algorithm makes the fluorescent cell detection and targeting user independent. Compared with manual patching, our system is superior in both success rate and average trial duration. It provides more reliable trial-to-trial control of the patching process and improves reproducibility of experiments.
Topics: Algorithms; Animals; Automation, Laboratory; Calibration; Computer Graphics; Female; Fluorescent Dyes; Image Processing, Computer-Assisted; Immunohistochemistry; Male; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Neurons; Patch-Clamp Techniques; Time Factors; Tissue Culture Techniques; User-Computer Interface; Visual Cortex
PubMed: 27385800
DOI: 10.1152/jn.00386.2016 -
Analytical Chemistry Feb 2022HIV develops single nucleotide polymorphisms (SNPs), some of which lead to drug resistance mutations (DRMs) that prevent therapeutic viral suppression. Genomic...
HIV develops single nucleotide polymorphisms (SNPs), some of which lead to drug resistance mutations (DRMs) that prevent therapeutic viral suppression. Genomic sequencing enables healthcare professionals to select effective combination antiretroviral therapy (ART) to achieve and maintain viral suppression. However, sequencing technologies, which are resource-intensive, are limited in their availability. This report describes the first step toward a highly specific ligation-based SNP discrimination method with endpoint PCR detection, which is more suitable for resource-limited clinics. The approach is based on magnetic bead processing to maximize reaction product transfer and minimize the carryover of incompatible buffer for three consecutive enzymatic reactions─reverse transcription (RT), oligonucleotide ligation assay (OLA), and PCR. The method improved PCR detection following RT → OLA by 8.06 cycles (∼250-fold) compared to direct pipette processing and detected between 10 and 10 RNA copies per reaction. In studies with synthesized nucleic acids based on the well-studied HIV mutation, K103N, the assay successfully differentiated between wild-type and mutant for RNA targets with high specificity. With further development, this design provides a pathway for SNP detection with more accessible PCR instrumentation and is a step toward a self-contained processing approach that incorporates the SNP specificity of the ligation reaction for more effective clinical management of DRMs in resource-constrained settings.
Topics: Anti-HIV Agents; Drug Resistance, Viral; HIV Infections; HIV-1; Humans; Magnetic Phenomena; Mutation
PubMed: 35077642
DOI: 10.1021/acs.analchem.1c05040 -
Frontiers in Cell and Developmental... 2021Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early...
Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early embryo development . However, microinjection is an empirical technology. The cellular survival after microinjection is mainly dependent on the operator, and an experienced operator should be trained for a long time, from several months to years. Optimizing the microinjection to be highly efficient and quickly learned should be helpful for new operators and some newly established laboratories. Here, we combined the tip pipette and piezo-assisted micromanipulator to microinject the oocyte and early embryos at different stages of mouse. The results showed that the survival rate after microinjection was more than 85% for cumulus-oocyte complex, germinal vesicle oocyte, two-cell, and four-cell embryos, and close to 100% for MII oocyte and zygotes. The high-rate survival of microinjection can save many experimental samples. Thus, it should be helpful in studying some rare animal models such as aging and conditional gene knockout mice. Furthermore, our protocol is much easier to learn for new operators, who can usually master the method proficiently after several training times. Therefore, we would like to publicly share this experience, which will help some novices master microinjection skillfully and save many laboratory animals.
PubMed: 34540848
DOI: 10.3389/fcell.2021.735971 -
Molecules (Basel, Switzerland) Sep 2022Despite an outstanding agent for control of Lepidoptera, the diamide insecticide cyclaniliprole (CYCP) is a suspected carcinogen. In the present study, an analytical...
Determination of Cyclaniliprole in Fruits and Vegetables Using Disposable Pipette Extraction Cleanup and Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.
Despite an outstanding agent for control of Lepidoptera, the diamide insecticide cyclaniliprole (CYCP) is a suspected carcinogen. In the present study, an analytical method was developed for the determination of CYCP in six fruits and vegetables (apple, grape, peach, bell pepper, lettuce, and tomato) using ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry. Sample preparation was carried out by the acetonitrile-salting-out extraction followed by simple and fast cleanup of disposable pipette extraction tip containing styrene divinyl benzene and/or graphitized carbon black. Satisfactory linearity (r > 0.99) was obtained in the calibration range of 0.001−1 µg mL−1. Matrix effects decreased from −9.9−−17.9% to −1.0−−7.6% after the cleanup. The recoveries of CYCP at three spike levels (0.01, 0.1, and 1 mg kg−1) from different matrices were between 75.7% and 111.5%, with the intra-day (n = 5) and inter-day (n = 15) relative standard deviations lower than 12.1%. The limit of quantification was 0.01 mg kg−1. The developed method provides a good reference for routine monitoring of CYCP in these fruits and vegetables.
Topics: Acetonitriles; Carcinogens; Chromatography, High Pressure Liquid; Chromatography, Liquid; Diamide; Fruit; Insecticides; Pesticide Residues; Soot; Styrenes; Tandem Mass Spectrometry; Vegetables
PubMed: 36235002
DOI: 10.3390/molecules27196464 -
Biophysical Journal Jul 2021Within the nucleus of the eukaryotic cell, DNA is partitioned into domains of highly condensed, transcriptionally silent heterochromatin and less condensed,...
Within the nucleus of the eukaryotic cell, DNA is partitioned into domains of highly condensed, transcriptionally silent heterochromatin and less condensed, transcriptionally active euchromatin. Heterochromatin protein 1α (HP1α) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. Although the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin-modifying enzymes have been studied previously, here we measure how the material properties of the nuclei are modified after the knockdown of HP1α. These studies were inspired by the observation that poorly invasive MCF7 breast cancer cells become more invasive after knockdown of HP1α expression and that, indeed, in many solid tumors the loss of HP1α correlates with the onset of tumor cell invasion. Atomic force microscopy (AFM), optical tweezers (OT), and techniques based on micropipette aspiration (MA) were each used to characterize the mechanical properties of nuclei extracted from HP1α knockdown or matched control MCF7 cells. Using AFM or OT to locally indent nuclei, those extracted from MCF7 HP1α knockdown cells were found to have apparent Young's moduli that were significantly lower than nuclei from MCF7 control cells, consistent with previous studies that assert heterochromatin plays a major role in governing the mechanical response in such experiments. In contrast, results from pipette-based techniques in the spirit of MA, in which the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1α knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organization observed by various microscopy techniques between the MCF7 control and HP1α knockdown nuclei correlate well with the results of our measured mechanical responses and our hypotheses regarding their origin.
Topics: Cell Nucleus; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Heterochromatin; Humans; MCF-7 Cells; Transcription Factors
PubMed: 34087208
DOI: 10.1016/j.bpj.2021.05.017 -
Journal of Medical Entomology Jan 2021The purpose of this study was to determine the ecology of the common arboviral mosquito vectors in Mombasa, Kilifi and Malindi urban areas of coastal Kenya. Mosquito...
The purpose of this study was to determine the ecology of the common arboviral mosquito vectors in Mombasa, Kilifi and Malindi urban areas of coastal Kenya. Mosquito larvae were collected using standard dippers and pipettes. Egg survivorship in dry soil was evaluated by collecting soil samples from dry potential larval developmental sites, re-hydrating them for hatching and rearing of the eventual larvae to adults. Adult mosquitoes were collected with CDC light traps and BG-Sentinel traps. All blood-fed females were tested for bloodmeal origin. Mosquitoes were screened for arboviruses using RT-qPCR. Overall, the predominant species were Culex quinquefasciatus (Say) 72.4% (n = 2,364) and Aedes aegypti (L.), 25.7%, (n = 838). A total of 415 larval developmental sites were identified indoors (n = 317) and outdoors (n = 98). The most productive larval developmental sites, both indoors and outdoors, were assorted small containers, water tanks, drainages, drums, and jerricans. Overall, 62% (n = 18) of the soil samples collected were positive for larvae which were used as a proxy to measure the presence of eggs. The mosquitoes fed on humans (29.8%) and chickens (3.7%). Of 259 mosquitoes tested for viral infection, 11.6% were positive for Flavivirus only. The most productive larval developmental sites for arboviral vectors indoors were small containers, water tanks, jerricans, and drums whereas small containers, water tanks, drainage channels, buckets, tires, and water troughs were the productive larval developmental sites outdoors.
Topics: Animal Distribution; Animals; Arboviruses; Cities; Culicidae; Kenya; Larva; Longevity; Mosquito Vectors; Ovum; Pupa
PubMed: 32623459
DOI: 10.1093/jme/tjaa136 -
Scientific Reports Mar 2021A common electrophysiology technique used in neuroscience is patch clamp: a method in which a glass pipette electrode facilitates single cell electrical recordings from...
A common electrophysiology technique used in neuroscience is patch clamp: a method in which a glass pipette electrode facilitates single cell electrical recordings from neurons. Typically, patch clamp is done manually in which an electrophysiologist views a brain slice under a microscope, visually selects a neuron to patch, and moves the pipette into close proximity to the cell to break through and seal its membrane. While recent advances in the field of patch clamping have enabled partial automation, the task of detecting a healthy neuronal soma in acute brain tissue slices is still a critical step that is commonly done manually, often presenting challenges for novices in electrophysiology. To overcome this obstacle and progress towards full automation of patch clamp, we combined the differential interference microscopy optical technique with an object detection-based convolutional neural network (CNN) to detect healthy neurons in acute slice. Utilizing the YOLOv3 convolutional neural network architecture, we achieved a 98% reduction in training times to 18 min, compared to previously published attempts. We also compared networks trained on unaltered and enhanced images, achieving up to 77% and 72% mean average precision, respectively. This novel, deep learning-based method accomplishes automated neuronal detection in brain slice at 18 frames per second with a small data set of 1138 annotated neurons, rapid training time, and high precision. Lastly, we verified the health of the identified neurons with a patch clamp experiment where the average access resistance was 29.25 M[Formula: see text] (n = 9). The addition of this technology during live-cell imaging for patch clamp experiments can not only improve manual patch clamping by reducing the neuroscience expertise required to select healthy cells, but also help achieve full automation of patch clamping by nominating cells without human assistance.
Topics: Animals; Brain; Deep Learning; Image Processing, Computer-Assisted; Microdissection; Microscopy; Neurons
PubMed: 33727679
DOI: 10.1038/s41598-021-85695-4 -
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 -
Ulusal Travma Ve Acil Cerrahi Dergisi =... Sep 2018In our study, the effects of peritoneal fluid on some Gram-negative and Candida albicans in experimental peritonitis rats were studied. The primary objective of the...
BACKGROUND
In our study, the effects of peritoneal fluid on some Gram-negative and Candida albicans in experimental peritonitis rats were studied. The primary objective of the present study was to understand the effect of peritoneal fluid on microorganisms causing intra-abdominal infections.
METHODS
Twenty male Sprague-Dawley rats weighing between 250 and 300 g were used in the study. The rats were randomly divided into two groups consisting of 10 animals. The operative procedures were performed under sterile conditions. In group I, sham laparotomy was done. In group II, the distal part of the cecum was ligated, and cecum perforation was performed. Peritoneal fluid samples at baseline and 2 and 4 h were extracted using a Pasteur pipette during laparotomy under anesthesia.
RESULTS
Peritoneal fluid was ineffective on Citrobacter freundii, Proteus mirabilis, and Enterobacter aerogenes. It inhibited the growth of Klebsiella pneumoniae for 8 h. However, growth was significantly increased in the passages obtained after 24 h. The growth of C. albicans decreased in the passages that were extracted after 4 and 8 h and increased in the passages obtained after 24 h (p<0.05). It was found that the number of Escherichia coli and Pseudomonas aeruginosa colonies that were grown in 2 h decreased, and no growth was detected in the passages obtained after 2 h (p<0.05).
CONCLUSION
Proliferating colony counts of E. coli and P. aeruginosa decreased after 2 h, and there was no proliferation in subsequent cultures. Peritoneal fluid exhibits a bactericidal effect under appropriate conditions. It also exhibits peritoneal bactericidal activity against E. coli, the major pathogen in intra-abdominal infections.
Topics: Animals; Anti-Bacterial Agents; Ascitic Fluid; Disease Models, Animal; Enterobacteriaceae; Male; Peritonitis; Rats; Rats, Sprague-Dawley
PubMed: 30394489
DOI: 10.5505/tjtes.2018.10452