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Journal of Animal Science and Technology Mar 2021Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore,...
Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.
PubMed: 33987604
DOI: 10.5187/jast.2021.e41 -
Journal of International Society of... 2022The relevance of the study was explained by the fact that free radicals, known to be a product of lipid peroxidation, damage the integrity of cell membranes and...
AIM AND OBJECTIVE
The relevance of the study was explained by the fact that free radicals, known to be a product of lipid peroxidation, damage the integrity of cell membranes and corresponding intracellular structures, disrupting their functioning. The purpose of this cross-sectional study was to investigate the effect of free-radical lipid peroxidation in the blood on the body of dentists without diseases of the bronchi and lungs.
MATERIALS AND METHODS
The chemiluminescent properties of the blood hemolysate of 65 dentists were measured. Blood was collected in a test tube with an anticoagulant, and the plasma was aspirated with a Pasteur pipette. The hemolysate was aspirated two times. Distilled water was added to the sediment of erythrocytes, the mixture was shaken, and centrifuged. Statistical processing of morphometric indicators was carried out using the software package "Statistica 6.0."
RESULTS
The direct dependence of the spontaneous chemiluminescence (SCL) growth parameters in the blood hemolysate of dentists on their length of service was determined.
CONCLUSION
The conclusions indicate a direct correlation between the growth parameters of the SCL index in the blood hemolysate of dentists and their length of service. The applied value of this study lies in the possibility of practical application of the results obtained to qualitatively investigate the effect of lipid peroxidation processes on the body of dentists.
PubMed: 36312575
DOI: 10.4103/jispcd.JISPCD_7_22 -
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 -
Nanotechnology May 2015Carbon nanoelectrodes with tip diameters ranging from tens to hundreds of nanometers are fabricated by pyrolitic deposition of carbon films along the entire inner...
Carbon nanoelectrodes with tip diameters ranging from tens to hundreds of nanometers are fabricated by pyrolitic deposition of carbon films along the entire inner surfaces of pulled-glass pipettes. The pulled end of each glass pipette is then etched to expose a desired length (typically, a few micrometers) of carbon pipe. The carbon film provides an electrically conductive path from the nanoscopic carbon tip to the distal, macroscopic end of the pipette, bridging between the nanoscale tip and the macroscale handle, without a need for assembly. We used our nanoelectrodes to penetrate into individual cells and cell nuclei and measured the variations in the electrode impedance upon cell and nucleus penetration as well as the electrode impedance as a function of cell penetration depth. Theoretical predictions based on a simple circuit model were in good agreement with experimental data.
Topics: Carbon; Cell Line, Tumor; Cell Nucleus; Cell Physiological Phenomena; Electric Capacitance; Electric Impedance; Electrophysiological Phenomena; Humans; Microelectrodes; Nanotechnology; Osteosarcoma
PubMed: 25876625
DOI: 10.1088/0957-4484/26/18/185101 -
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 -
Communications Biology Sep 2022Microfluidic-based cell culture allows for precise spatio-temporal regulation of microenvironment, live cell imaging and better recapitulation of physiological...
Microfluidic-based cell culture allows for precise spatio-temporal regulation of microenvironment, live cell imaging and better recapitulation of physiological conditions, while minimizing reagents' consumption. Despite their usefulness, most microfluidic systems are designed with one specific application in mind and usually require specialized equipment and expertise for their operation. All these requirements prevent microfluidic-based cell culture to be widely adopted. Here, we designed and implemented a versatile and easy-to-use perfusion cell culture microfluidic platform for multiple applications (VersaLive) requiring only standard pipettes. Here, we showcase the multiple uses of VersaLive (e.g., time-lapse live cell imaging, immunostaining, cell recovery, cell lysis, plasmid transfection) in mammalian cell lines and primary cells. VersaLive could replace standard cell culture formats in several applications, thus decreasing costs and increasing reproducibility across laboratories. The layout, documentation and protocols are open-source and available online at https://versalive.tigem.it/ .
Topics: Animals; Cell Culture Techniques; Mammals; Microfluidics; Names; Reproducibility of Results
PubMed: 36175545
DOI: 10.1038/s42003-022-03976-8 -
The Journal of General Physiology May 2023Mechanosensitive PIEZO1 ion channels open in response to membrane stretch. Yet, the underlying microscopic mechanism of this activation remains unknown. To probe this...
Mechanosensitive PIEZO1 ion channels open in response to membrane stretch. Yet, the underlying microscopic mechanism of this activation remains unknown. To probe this mechanism, we used cell-attached pressure-clamp recordings to measure single channel currents at different steady-state negative pipette pressures, spanning the full range of the channel's pressure sensitivity. Pressure-dependent activation occurs through a sharp reduction of the mean shut duration and through a moderate increase of the mean open duration. Across all tested pressures, the distribution of open and shut dwell times best follows sums of two and three exponential components, respectively. As the magnitude of the pressure stimulus increases, the time constants of most of these exponential components gradually change, in opposite directions for open and shut dwell times, and to a similar extent. In addition, while the relative amplitudes of fast and slow components remain unchanged for open intervals, they fully reverse for shut intervals, further reducing the mean shut duration. Using two-dimensional dwell time analysis, Markov-chain modeling, and simulations, we identified a minimal five-states model which recapitulates essential characteristics of single channel data, including microscopic reversibility, correlations between adjacent open and shut intervals, and asymmetric modulation of dwell times by pressure. This study identifies a microscopic mechanism for the activation of PIEZO1 channels by pressure-induced membrane stretch and deepens our fundamental understanding of mechanotransduction by a vertebrate member of the PIEZO channel family.
Topics: Mechanotransduction, Cellular; Kinetics; Ion Channels
PubMed: 36715688
DOI: 10.1085/jgp.202213260 -
Journal of Mid-life Health 2018Endometrial evaluation is generally indicated in cases presenting with abnormal uterine bleeding (AUB), especially in women more than 35 years of age. AUB encompasses a...
UNLABELLED
Endometrial evaluation is generally indicated in cases presenting with abnormal uterine bleeding (AUB), especially in women more than 35 years of age. AUB encompasses a variety of presentation, for example, heavy menstrual bleeding, frequent bleeding, irregular vaginal bleeding, postcoital and postmenopausal bleeding to name a few. Many methods are used for the evaluation of such cases, with most common being sonography and endometrial biopsy with very few cases requiring more invasive approach like hysteroscopy. Endometrial aspiration is a simple and safe office procedure used for this purpose.
MATERIALS AND METHODS
We retrospectively analyzed cases of AUB where endometrial aspiration with Pipette (Medgyn) was done in outpatient department between January 2015 and April 2016. Case records (both paper and electronic) were used to retrieve data.
RESULTS
One hundred and fifteen cases were included in the study after applying inclusion and exclusion criteria. Most cases were between 46 and 50 years of age followed by 41-45 years. No cases were below 25 or more than 65 years of age. Heavy menstrual bleeding was the most common presentation of AUB. Adequate samples were obtained in 86% of cases while 13.9% of cases' sample was inadequate for opinion, many of which were later underwent hysteroscopy and/or dilatation and curettage (D and C) in operation theater; atrophic endometrium was the most common cause for inadequate sample. Uterine malignancy was diagnosed in three cases.
DISCUSSION
Endometrial aspiration has been compared with traditional D and C as well as postoperative histopathology in various studies with good results. Many such studies are done in India as well as in western countries confirming good correlation with histopathology and adequate tissue sample for the pathologist to give a confident diagnosis. No complication or side effect was noted with the use of this device.
CONCLUSION
Endometrial aspiration is a simple, safe, and effective method to sample endometrium in cases of AUB avoiding risk of anesthesia and is less time-consuming. Many similar devices are also available in the market and need to be popularized in all parts of the country.
PubMed: 29628726
DOI: 10.4103/jmh.JMH_109_17 -
Journal of Assisted Reproduction and... Jul 2022Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual...
PURPOSE
Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection.
METHODS
The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-μm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes).
RESULTS
Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection.
CONCLUSION
This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.
Topics: Animals; Blastocyst; Male; Mice; Microinjections; Oocytes; Polymerization; Semen; Sperm Injections, Intracytoplasmic
PubMed: 35552947
DOI: 10.1007/s10815-022-02485-1 -
The Journal of Physiological Sciences :... Jul 2018Hypotonic solutions can cause painful sensations in nasal and ocular mucosa through molecular mechanisms that are not entirely understood. We clarified the ability of...
Hypotonic solutions can cause painful sensations in nasal and ocular mucosa through molecular mechanisms that are not entirely understood. We clarified the ability of human TRPA1 (hTRPA1) to respond to physical stimulus, and evaluated the response of hTRPA1 to cell swelling under hypotonic conditions. Using a Ca-imaging method, we found that modulation of AITC-induced hTRPA1 activity occurred under hypotonic conditions. Moreover, cell swelling in hypotonic conditions evoked single-channel activation of hTRPA1 in a cell-attached mode when the patch pipette was attached after cell swelling under hypotonic conditions, but not before swelling. Single-channel currents activated by cell swelling were also inhibited by a known hTRPA1 blocker. Since pre-application of thapsigargin or pretreatment with the calcium chelator BAPTA did not affect the single-channel activation induced by cell swelling, changes in intracellular calcium concentrations are likely not related to hTRPA1 activation induced by physical stimuli.
Topics: Calcium; Cell Enlargement; HEK293 Cells; Humans; Hypotonic Solutions; Patch-Clamp Techniques; TRPA1 Cation Channel
PubMed: 28623463
DOI: 10.1007/s12576-017-0545-9