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Zygote (Cambridge, England) Feb 2024Despite the high level of standardization of the intracytoplasmic sperm injection (ICSI) technique, there are some aspects that deserve special attention and should...
Despite the high level of standardization of the intracytoplasmic sperm injection (ICSI) technique, there are some aspects that deserve special attention and should still be improved. The major drawback of the technique is its invasiveness, as during cytoplasmic aspiration different structures of the oocyte may be lost or damaged. This is partly because the microtools used in ICSI were not specially designed for assisted reproduction but for other medical-biological disciplines. In view of the above caveats, the aim of the study was to compare the results of ICSI with the traditional oocyte-holding pipette and the oocyte-holding pipette without aspiration (PiWA). In total, 155 patients and 1037 oocytes were included in the study. In each ICSI cycle, half of the oocytes were microinjected using a traditional holding pipette and the other half using a PiWA. In result, the PiWA technique produced a significant increase in the fertilization rate: 88.12% (95%CI: 84.62-90.92%); holding pipette: 73.33% (95%CI: 68.72-77.49%). Also, it produced a significant decrease in the embryo degeneration rate compared with the traditional holding pipette [PiWA: 2.07% (95%CI: 1.11-3.8%); holding pipette: 4.51% (95%CI: 3.06-6.59%)]. Pregnancy rate depended on the holding technique used, both in single embryo transfers ( = 59; χ = 4.608; -value = 0.032) and double embryo transfers ( = 156; χ = 4.344; -value = 0.037); with PiWA presenting a significantly higher pregnancy rate than the traditional holding technique. Based on current evidence and the present results, improvements should focus on decreasing the invasiveness of the microinjection itself by minimizing or avoiding aspiration and cytoplasmic disorganization, as is successfully achieved with PiWA.
Topics: Pregnancy; Female; Humans; Male; Sperm Injections, Intracytoplasmic; Semen; Infertility, Male; Pregnancy Rate; Oocytes
PubMed: 38173402
DOI: 10.1017/S0967199423000618 -
Analytical and Bioanalytical Chemistry Jul 2023In this work, we present an in situ droplet-based derivatization method for fast tissue lipid profiling at multiple isomer levels. On-tissue derivatization for isomer...
In this work, we present an in situ droplet-based derivatization method for fast tissue lipid profiling at multiple isomer levels. On-tissue derivatization for isomer characterization was achieved in a droplet delivered by the TriVersa NanoMate LESA pipette. The derivatized lipids were then extracted and analyzed by the automated chip-based liquid extraction surface analysis (LESA) mass spectrometry (MS) followed by tandem MS to produce diagnostic fragment ions to reveal the lipid isomer structures. Three reactions, i.e., mCPBA epoxidation, photocycloaddition catalyzed by the photocatalyst Ir[dF(CF)ppy](dtbbpy)PF, and Mn(II) lipid adduction, were applied using the droplet-based derivatization to provide lipid characterization at carbon-carbon double-bond positional isomer and sn-positional isomer levels. Relative quantitation of both types of lipid isomers was also achieved based on diagnostic ion intensities. This method provides the flexibility of performing multiple derivatizations at different spots in the same functional region of an organ for orthogonal lipid isomer analysis using a single tissue slide. Lipid isomers were profiled in the cortex, cerebellum, thalamus, hippocampus, and midbrain of the mouse brain and 24 double-bond positional isomers and 16 sn-positional isomers showed various distributions in those regions. This droplet-based derivatization of tissue lipids allows fast profiling of multi-level isomer identification and quantitation and has great potential in tissue lipid studies requiring rapid sample-to-result turnovers.
Topics: Mice; Animals; Tandem Mass Spectrometry; Isomerism; Lipids
PubMed: 37017722
DOI: 10.1007/s00216-023-04653-3 -
Methods in Molecular Biology (Clifton,... 2024Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus...
Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus playing a crucial role in unraveling the mechanistic processes governing brain function. Techniques for intracellular recordings of membrane potentials pioneered in the 1940s have witnessed significant advancements since their inception. Among these, whole-cell patch-clamp recording has emerged as a leading method for measuring neuronal membrane potentials due to its high stability and broad applicability ranging from cultured cells to brain slices and even behaving animals. This chapter provides a detailed protocol to acquire stable whole-cell recordings from neurons in the cerebral cortex of awake, head-restrained mice. Significant enhancements to our protocol include implanting a metal head-post using adhesive resin cement and preparing a recording pipette with a long shank for targeting deeper brain regions. This protocol, once implemented, enables whole-cell recordings up to 2.5 mM beneath the cortical surface.
Topics: Animals; Mice; Patch-Clamp Techniques; Neurons; Brain; Cerebral Cortex; Membrane Potentials
PubMed: 38630234
DOI: 10.1007/978-1-0716-3810-1_20 -
Analytica Chimica Acta Feb 2024In "shotgun" approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered...
BACKGROUND
In "shotgun" approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered as an indispensable tool. Despite the fact that CZE offers a high tolerance towards salts, most publications reported on the use of desalting. There seems to be no clear consensus on the utilization of desalting in the CZE-MS community, most probably due to the absence of works addressing the comparison of desalted and non-desalted digests. Our aim was to fill this research gap using protein samples of varying complexity in different sample matrices.
RESULTS
First, standard protein digests were analyzed to build the knowledge on the effect of sample clean-up by solid-phase extraction (SPE) pipette tips and the possible stacking phenomena induced by different sample matrices. Desalting led to a somewhat altered peptide profile, the procedure affected mostly the hydrophilic peptides (although not to a devastating extent). Nevertheless, desalting samples allowed remarkable stacking efficiency owing to their low-conductivity sample background, enabling a so-called field-amplified sample stacking phenomenon. Non-desalted samples also produced a stacking event, the mechanism of which is based on transient-isotachophoresis due to the presence of high-mobility ions in the digestion buffer itself. Adding either extra ammonium ions or acetonitrile into the non-desalted digests enhanced the stacking efficiency. A complex sample (yeast cell lysate) was also analyzed with the optimal conditions, which yielded similar tendencies.
SIGNIFICANCE
Based on these results, we propose that sample clean-up in the bottom-up sample preparation process prior to CZE-MS analysis can be omitted. The preclusion of desalting can even enhance detection sensitivity, separation efficiency or sequence coverage.
Topics: Peptide Mapping; Tandem Mass Spectrometry; Proteomics; Electrophoresis, Capillary; Peptides; Ions
PubMed: 38220294
DOI: 10.1016/j.aca.2023.342162 -
Journal of Synchrotron Radiation Jul 2023A sample environment and manipulation tool is presented for single-particle X-ray experiments in an aqueous environment. The system is based on a single water droplet,...
A sample environment and manipulation tool is presented for single-particle X-ray experiments in an aqueous environment. The system is based on a single water droplet, positioned on a substrate that is structured by a hydrophobic and hydrophilic pattern to stabilize the droplet position. The substrate can support several droplets at a time. Evaporation is prevented by covering the droplet by a thin film of mineral oil. In this windowless fluid which minimizes background signal, single particles can be probed and manipulated by micropipettes, which can easily be inserted and steered in the droplet. Holographic X-ray imaging is shown to be well suited to observe and monitor the pipettes, as well as the droplet surface and the particles. Aspiration and force generation are also enabled based on an application of controlled pressure differences. Experimental challenges are addressed and first results are presented, obtained at two different undulator endstations with nano-focused beams. Finally, the sample environment is discussed in view of future coherent imaging and diffraction experiments with synchrotron radiation and single X-ray free-electron laser pulses.
Topics: X-Rays; Radiography; Lasers; Synchrotrons; Holography; Water; X-Ray Diffraction
PubMed: 37233735
DOI: 10.1107/S1600577523003685 -
Analytica Chimica Acta Sep 2023A molecularly imprinted polymer (MIP) monolithic column was prepared in situ in a pipette tip using phenol and bisphenol A as dual templates, 4-vinyl pyridine and...
A molecularly imprinted polymer monolithic column with dual template and bifunctional monomers for selective extraction and simultaneous determination of eight phenolics from polycarbonate cups.
A molecularly imprinted polymer (MIP) monolithic column was prepared in situ in a pipette tip using phenol and bisphenol A as dual templates, 4-vinyl pyridine and β-cyclodextrin as bifunctional monomers. It was used for the selective and simultaneous solid phase extraction of eight phenolics, including phenol, m-cresol, p-tert-butylphenol, bisphenol A, bisphenol B, bisphenol E, bisphenol Z, and bisphenol AP. The MIP monolithic column was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis and nitrogen adsorption experiment. The results of selective adsorption experiments showed that the MIP monolithic column can selective recognize the phenolics and have excellent adsorption property. The imprinting factor for bisphenol A can be as high as 4.31, and the maximum adsorption capacity for bisphenol Z can reach 201.66 mg g. Under the optimal extraction conditions, a selective and simultaneous extraction and determination method for eight phenolics was established based on the MIP monolithic column and high-performance liquid chromatography with ultraviolet detection. The linear ranges (LRs) of the eight phenolics were 0.5-200 μg L, the limits of quantification (LOQs) and detection (LODs) were 0.5-2.0 μg L and 0.15-0.67 μg L. The method was applied to detect the migration quantity of the eight phenolics from polycarbonate cups and had satisfactory recovery. The method has the advantages of simple synthesis, short extraction time, as well as good repeatability and reproducibility, which provides a sensitive and reliable strategy for extracting and detecting phenolics from food contact material.
PubMed: 37423657
DOI: 10.1016/j.aca.2023.341493 -
Biochemical and Biophysical Research... Feb 2024With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited...
With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to -400 mV pulse between the patch pipette and ITO evoked focal Ca transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients ("Ca plumes") faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to -50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.
Topics: Longevity; Lipid Bilayers; Cell Membrane; Electroporation; Cell Division
PubMed: 38157631
DOI: 10.1016/j.bbrc.2023.149408 -
Environmental Science and Pollution... Oct 2023In this work, metal-organic frameworks (MOFs) including Fe-MIL-101 and Ti-MIL-125 were prepared and fixed on the melamine foam (MF) by polyvinylidene fluoride (PVDF) to...
In this work, metal-organic frameworks (MOFs) including Fe-MIL-101 and Ti-MIL-125 were prepared and fixed on the melamine foam (MF) by polyvinylidene fluoride (PVDF) to prepare MF/PVDF/MOFs, which was used as adsorbents in pipette-tip solid-phase extraction (PT-SPE) for rapid extraction of organophosphorus pesticides (OPPs). Then, a gas chromatograph-flame thermionic detector (GC-FTD) was used for simultaneous analysis of Dimethoate (DMT), Iprobenfos (IBF), Parathion-methyl (PAM), and Chlorpyrifos (CPF). The morphology, crystal structure, and functional groups of MF/PVDF/MOFs were characterized, indicating that Ti-MIL-125 and Fe-MIL-101 were successfully synthesized and distributed on MF. The Fe-MIL-101 and Ti-MIL-125 showed good extraction ability for OPPs, which was mainly due to the π-π interaction and the multiple porous structures. Under the optimal conditions, the limit of detection (LODs) of four OPPs was 0.03-0.14 μg L and the RSDs were less than 9.9%. The developed PT-SPE method showed a short extraction time (<3 min). The recoveries in fruits and vegetables (Celery, cabbages, and oranges) ranged from 75.3%-118.8% (RSDs<9.6%). The prepared MF/PVDF/MOFs demonstrated the efficient extraction performance of OPPs, contributing to the rapid pretreatment of OPPs from food and the environment.
Topics: Pesticides; Metal-Organic Frameworks; Organophosphorus Compounds; Vegetables; Fruit; Solid Phase Extraction; Limit of Detection
PubMed: 37755595
DOI: 10.1007/s11356-023-30055-0 -
Food Chemistry Jul 2023This study presents an in-pipette-tip kapok fiber-supported liquid extraction/in-situ derivatization (in-pipette-tip KF-SLE-ISD) method for simultaneous enrichment and...
In-pipette-tip kapok fiber-supported liquid extraction/in-situ derivatization coupled with high-performance liquid chromatography for conveniently determining three furfurals.
This study presents an in-pipette-tip kapok fiber-supported liquid extraction/in-situ derivatization (in-pipette-tip KF-SLE-ISD) method for simultaneous enrichment and derivatization of furfurals. Briefly, 3 mg of natural kapok fiber, which was loaded in an assembled pipette-tip, was used to support 12.5 μL of extractant (ethyl acetate/toluene, 75:25, v/v) containing 10 mM 2,4-dinitrophenylhydrazine. The in-pipette-tip KF-SLE-ISD procedure was conveniently conducted by aspirating/releasing 1 mL of sample solution 10 cycles, allowing simultaneous extraction and derivatization of furfurals. Then, 100 μL of acetonitrile was aspirated/released 5 cycles for elution, 10 μL of which was directly analyzed by high-performance liquid chromatography. The limits of quantitation were in ranges of 0.10-0.45 μg/mL. The method showed satisfied linearity (R > 0.99), precision (RSD < 8.53%) and relative recovery (90.34-114.71%), which was successfully applied to determine furfurals in various samples (e.g., honeys, juices and glucose injections). The proposed method has the merits of effectiveness, simplicity, low cost, wide availability and ease of automation.
Topics: Chromatography, High Pressure Liquid; Food; Furaldehyde; Solid Phase Extraction; Food Analysis
PubMed: 36854240
DOI: 10.1016/j.foodchem.2023.135788 -
Scientific Reports Jan 2024The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic...
The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.
Topics: Ultrasonics; Detergents; Neurons; Neurophysiology; Patch-Clamp Techniques
PubMed: 38238544
DOI: 10.1038/s41598-024-51837-7