Intracellular Injection Research Articles

Comparison of Clinical Outcomes in the Slow-Developing Blastocysts With or Without Preimplantation Genetic Testing-Aneuploidy on Day 6 in the Frozen–Thawed Cycle

ObjectivesThis study investigated the potential of the slow-developing blastocysts using preimplantation genetic testing-aneuploidy (PGT-A) in patients undergoing frozen–thawed embryo transfer, stratified by age. MethodsA retrospective analysis was performed including a total of 743 cycles, the first frozen embryo transfer (FET) cycle with single embryo transfer, who underwent treatment between January 2020 and July 2023 in a single fertility centre, Gangnam CHA Fertility Center. A total of 743 cycles, in which we performed intracellular sperm injection and freeze-all strategy, from 743 patients were included. The patient group was divided into 4 groups as follows: group 1 (G1), 208 FET on day 5; group 2 (G2), 177 FET with PGT-A on day 5; group 3 (G3), 220 FET on day 6; group 4 (G4), 138 FET with PGT-A on day 6. We also divided into 2 groups—under 35 years of age and over 35 years of age—and performed the analysis separately for each group. ResultsIn the under 35 years of age group, there were no significant differences in clinical pregnancy and miscarriage rates in G1 and G2 (67.2% vs. 63.8%, not statistically significantly different). Also, G4 had a higher clinical pregnancy rate than G3, but it was not significant (51.8% vs. 54.7%, not statistically significantly different). In the 35 years or older group, G2 had higher pregnancy rates than G1 and lower miscarriage rates (clinical pregnancy rate: 43.3% vs. 67.7%, P = 0.001, miscarriage rate: 22.5% vs. 3.4%, P = 0.001). In addition, G4 had a higher pregnancy rate than G3 and a lower miscarriage rate (clinical pregnancy rate: 31.8% vs. 46.9%, P = 0.003, miscarriage rate: 22.9% vs. 2.2%, P = 0.023). ConclusionsIn the under-35-year-old group, PGT-A on day 5 and day 6 showed a high pregnancy rate and a low miscarriage rate. Therefore, using PGT-A seems advantageous for patients of an advanced maternal age.

Spike Reliability is Cell-Type Specific and Shapes Excitation and Inhibition in the Cortex.

Neurons encode information in the highly variable spiking activity of neuronal populations, so that different repetitions of the same stimulus can generate action potentials that vary significantly in terms of the count and timing. How does spiking variability originate, and does it have a functional purpose? Leveraging the Allen Institute cell types dataset, we relate the spiking reliability of cortical neurons in-vitro during the intracellular injection of current resembling synaptic inputs to their morphologic, electrophysiologic, and transcriptomic classes. Our findings demonstrate that parvalbumin+ (PV) interneurons, a subclass of inhibitory neurons, show high reliability compared to other neuronal subclasses, particularly excitatory neurons. Through computational modeling, we predict that the high reliability of PV interneurons allows for strong and precise inhibition in downstream neurons, while the lower reliability of excitatory neurons allows for integrating multiple synaptic inputs leading to a spiking rate code. These findings illuminate how spiking variability in different neuronal classes affect information propagation in the brain, leading to precise inhibition and spiking rate codes.

Key morphological features of human pyramidal neurons.

The basic building block of the cerebral cortex, the pyramidal cell, has been shown to be characterized by a markedly different dendritic structure among layers, cortical areas, and species. Functionally, differences in the structure of their dendrites and axons are critical in determining how neurons integrate information. However, within the human cortex, these neurons have not been quantified in detail. In the present work, we performed intracellular injections of Lucifer Yellow and 3D reconstructed over 200 pyramidal neurons, including apical and basal dendritic and local axonal arbors and dendritic spines, from human occipital primary visual area and associative temporal cortex. We found that human pyramidal neurons from temporal cortex were larger, displayed more complex apical and basal structural organization, and had more spines compared to those in primary sensory cortex. Moreover, these human neocortical neurons displayed specific shared and distinct characteristics in comparison to previously published human hippocampal pyramidal neurons. Additionally, we identified distinct morphological features in human neurons that set them apart from mouse neurons. Lastly, we observed certain consistent organizational patterns shared across species. This study emphasizes the existing diversity within pyramidal cell structures across different cortical areas and species, suggesting substantial species-specific variations in their computational properties.

Spectroscopy of Testicular Tissues as a Tool for Azoospermia Visualization During Micro-TESE and IVF: a Feasibility Study

Non-obstructive azoospermia observed in 10% of infertile men and 0,6% of all men is the most severe form of male infertility. The histological structure of testicular tissue in patients with non-obstructive azoospermia is heterogeneous and is represented by various disorders of spermatogenesis. The only way to achieve pregnancy in the families of patients with non-obstructive azoospermia is the method of in vitro fertilization with intracellular sperm injection. The most effective method of detecting spermatozoa suitable for in vitro fertilization in testicles is the micro-TESE method. It is based on a subjective visual assessment of tissues and allows detecting spermatozoa in non-obstructive azoospermia only in 38-60% of cases. To increase the effectiveness of micro-TESE, the method used must be objective, high-specific, safe for reproductive cells, and also implemented during the operation in real time. The existing approaches to solving the problem of identifying seminal tubules with preserved spermatogenesis, such as multiphoton microscope, Raman spectroscopy, optical coherence tomography and etc., are either associated with the use of laser radiation with unproven safety or cannot be implemented intraoperatively and operationally. The paper considers the possibility of creating a specialized microsurgical system for intraoperative evaluation of testicular tissues histological structure using the spatial distribution of its spectral characteristics. We proposed to implement such a system based on multispectral imaging of the studied tissues with real-time data processing and displaying the results using augmented reality methods. The reflectance spectrum of testicular tissue with varying degrees of preservation of spermatogenesis in the visible and near infrared spectral ranges was studied. Spectral regions were identified that potentially provide the best differentiation of healthy tissues and tissues with impaired spermatogenesis. The described approach can be implemented intraoperatively and safely and may serve to automate and objectify the diagnosis of testicular tissues in micro-TESE.

An Integrated Dual‐Functional Nanotool Capable of Studying Single‐Cell Epigenetics and Programmable Gene Regulation

AbstractSingle‐cell epigenetics is envisioned to decipher manifold epigenetic phenomena and to contribute to our accurate knowledge about basic epigenetic mechanisms. Engineered nanopipette technology has gained momentum in single‐cell studies; however, solutions to epigenetic questions remain unachieved. This study addresses the challenge by exploring N6‐methyladenine (m6A)‐bearing deoxyribozyme (DNAzyme) confined within a nanopipette for profiling a representative m6A‐modifying enzyme, fat mass and obesity‐associated protein (FTO). Electroosmotic intracellular extraction of FTO could remove the m6A and cause DNAzyme cleavage, leading to the altered ionic current signal. Because the cleavage can release a DNA sequence, we simultaneously program it as an antisense strand against FTO‐mRNA, intracellular injection of which has been shown to induce early stage apoptosis. This nanotool thus features the dual functions of studying single‐cell epigenetics and programmable gene regulation.

An Integrated Dual-Functional Nanotool Capable of Studying Single-Cell Epigenetics and Programmable Gene Regulation.

Single-cell epigenetics is envisioned to decipher manifold epigenetic phenomena and to contribute to our accurate knowledge about basic epigenetic mechanisms. Engineered nanopipette technology has gained momentum in single-cell study, whereas solutions to epigenetic study remain unachieved. This study addresses the challenge by exploring N6-methyladenine (m6A)-bearing deoxyribozyme (DNAzyme) confined within nanopipette for profiling representative m6A modifying enzyme of fat mass and obesity-associated protein (FTO). Electroosmotic intracellular extraction of FTO could remove the m6A and cause DNAzyme cleavage, leading to the altered ionic current signal. Because the cleavage can release a DNA sequence, we simultaneously program it as an antisense strand against FTO-mRNA, intracellular injection of which has been shown to induce early stage apoptosis. This nanotool thus features the dual functions of studying single-cell epigenetics and programmable gene regulation.

Fasciclin 2 plays multiple roles in promoting cell migration within the developing nervous system of Manduca sexta

The coordination of neuronal and glial migration is essential to the formation of most nervous systems, requiring a complex interplay of cell-intrinsic responses and intercellular guidance cues. During the development of the enteric nervous system (ENS) in Manduca sexta (tobacco hornworm), the IgCAM Fasciclin 2 (Fas2) serves several distinct functions to regulate these processes. As the ENS forms, a population of 300 neurons (EP cells) undergoes sequential phases of migration along well-defined muscle pathways on the visceral mesoderm to form a branching Enteric Plexus, closely followed by a trailing wave of proliferating glial cells that enwrap the neurons. Initially, both the neurons and glial cells express a GPI-linked form of Fas2 (GPI-Fas2), which helps maintain cell-cell contact among the pre-migratory neurons and later promotes glial ensheathment. The neurons then switch isoforms, predominantly expressing a combination of transmembrane isoforms lacking an intracellular PEST domain (TM-Fas2 PEST−), while their muscle band pathways on the midgut transiently express transmembrane isoforms containing this domain (TM-Fas2 PEST+). Using intracellular injection protocols to manipulate Fas2 expression in cultured embryos, we found that TM-Fas2 promotes the directed migration and outgrowth of individual neurons in the developing ENS. Concurrently, TM-Fas2 expression by the underlying muscle bands is also required as a substrate cue to support normal migration, while glial expression of GPI-Fas2 helps support their ensheathment of the migratory neurons. These results demonstrate how a specific IgCAM can play multiple roles that help coordinate neuronal and glial migration in the developing nervous system.

Ex vivo, in situ perfusion protocol for human brain fixation compatible with microscopy, MRI techniques, and anatomical studies.

We present a method for human brain fixation based on simultaneous perfusion of 4% paraformaldehyde through carotids after a flush with saline. The left carotid cannula is used to perfuse the body with 10% formalin, to allow further use of the body for anatomical research or teaching. The aim of our method is to develop a vascular fixation protocol for the human brain, by adapting protocols that are commonly used in experimental animal studies. We show that a variety of histological procedures can be carried out (cyto- and myeloarchitectonics, histochemistry, immunohistochemistry, intracellular cell injection, and electron microscopy). In addition, ex vivo, ex situ high-resolution MRI (9.4T) can be obtained in the same specimens. This procedure resulted in similar morphological features to those obtained by intravascular perfusion in experimental animals, provided that the postmortem interval was under 10 h for several of the techniques used and under 4 h in the case of intracellular injections and electron microscopy. The use of intravascular fixation of the brain inside the skull provides a fixed whole human brain, perfectly fitted to the skull, with negligible deformation compared to conventional techniques. Given this characteristic of ex vivo, in situ fixation, this procedure can probably be considered the most suitable one available for ex vivo MRI scans of the brain. We describe the compatibility of the method proposed for intravascular fixation of the human brain and fixation of the donor's body for anatomical purposes. Thus, body donor programs can provide human brain tissue, while the remainder of the body can also be fixed for anatomical studies. Therefore, this method of human brain fixation through the carotid system optimizes the procurement of human brain tissue, allowing a greater understanding of human neurological diseases, while benefiting anatomy departments by making the remainder of the body available for teaching purposes.

Serotonin is a gap junction-permeable neuronal tracer in the mouse retina.

Gap junctions are dynamically modulated bridges allowing the transcellular passage of ions and small molecules with a molecular mass of up to 1 kDa, a mechanism utilized for molecular communication purposes by living cells. This same mechanism is also exploited by scientists to reveal the existence of gap junction contacts by the cell-to-cell movement of tracers. However, multiple labeling experiments require the availability of multiple gap junction-permeable tracers. To this end, we utilized the well-known transient OFF alpha retinal ganglion cell (RGC)-coupled array as a model system to study and compare the transjunctional movement of neurobiotin (NB), a commonly used tracer, and serotonin, a recently identified tracer. Although the transjunctional movement of serotonin has been established in cell cultures, here we show, for the first time, that serotonin is also a potent tracer in in vitro tissue. In addition, serotonin is lighter than the classical gap junction-permeable NB, and thus, we expected that tracer movement would be comparable to or better than that of serotonin. We found that intracellular serotonin injections result in the labeling of the coupled transient OFF alpha RGC array very similar to those of the classical NB-labeled arrays. Both serotonin and NB-injected transient OFF alpha RGCs displayed the well-known pattern with coupled RGCs and a cohort of coupled wide-field amacrine cells (ACs). By using morphological characteristics, we confirm that the serotonin and the NB-coupled AC arrays are identical, and thereby confirm that serotonin is a potent gap junction-permeable tracer and can be readily used as an alternative to NB in in vitro tissue. Moreover, serotonin can be utilized in parallel with other dyes or tracers, enabling the use of multiple labels in the same material.

Modulation of striatal glutamatergic, dopaminergic and cholinergic neurotransmission pathways concomitant with motor disturbance in rats with kaolin-induced hydrocephalus

BackgroundHydrocephalus is characterized by abnormal accumulation of cerebrospinal fluid in the cerebral ventricles and causes motor impairments. The mechanisms underlying the motor changes remain elusive. Enlargement of ventricles compresses the striatum of the basal ganglia, a group of nuclei involved in the subcortical motor circuit. Here, we used a kaolin-injection juvenile rat model to explore the effects of acute and chronic hydrocephalus, 1 and 5 weeks post-treatment, respectively on the three major neurotransmission pathways (glutamatergic, dopaminergic and cholinergic) in the striatum.MethodsRats were evaluated for motor impairments. Expressions of presynaptic and postsynaptic protein markers related to the glutamatergic, dopaminergic, and cholinergic connections in the striatum were evaluated. Combined intracellular dye injection and substance P immunohistochemistry were used to distinguish between direct and indirect pathway striatal medium spiny neurons (d and i-MSNs) for the analysis of their dendritic spine density changes.ResultsHydrocephalic rats showed compromised open-field gait behavior. However, male but not female rats displayed stereotypic movements and compromised rotarod performance. Morphologically, the increase in lateral ventricle sizes was greater in the chronic than acute hydrocephalus conditions. Biochemically, hydrocephalic rats had significantly decreased striatal levels of synaptophysin, vesicular glutamate transporter 1, and glutamatergic postsynaptic density protein 95, suggesting a reduction of corticostriatal excitation. The expression of GluR2/3 was also reduced suggesting glutamate receptor compositional changes. The densities of dendritic spines, morphological correlates of excitatory synaptic foci, on both d and i-MSNs were also reduced. Hydrocephalus altered type 1 (DR1) and 2 (DR2) dopamine receptor expressions without affecting tyrosine hydroxylase level. DR1 was decreased in acute and chronic hydrocephalus, while DR2 only started to decrease later during chronic hydrocephalus. Since dopamine excites d-MSNs through DR1 and inhibits i-MSNs via DR2, our findings suggest that hydrocephalus downregulated the direct basal ganglia neural pathway persistently and disinhibited the indirect pathway late during chronic hydrocephalus. Hydrocephalus also persistently reduced the striatal choline acetyltransferase level, suggesting a reduction of cholinergic modulation.ConclusionsHydrocephalus altered striatal glutamatergic, dopaminergic, and cholinergic neurotransmission pathways and tipped the balance between the direct and indirect basal ganglia circuits, which could have contributed to the motor impairments in hydrocephalus.

Contribution of parasol-magnocellular pathway ganglion cells to foveal retina in macaque monkey

Parasol-magnocellular pathway ganglion cells form an important output stream of the primate retina and make a major contribution to visual motion detection. They are known to comprise ON and OFF type response polarities but the relative numbers of ON and OFF parasol cells, and the overall contribution of parasol cells to high-acuity foveal vision are not well understood. Here we use antibodies against carbonic anhydrase 8 (CA8) and intracellular injections of the liphilic dye DiI to show that CA8 selectively labels OFF parasol cells in macaque retina. By combined labeling with CA8 antibodies and a previously-described marker for parasol cells (GABAA receptor antibodies), we show that ON and OFF parasol cells each comprise ∼ 6% of all ganglion cells in central retina (each peak density ∼ 3000 cells/mm2 at 5 deg.), and each population comprises ∼ 10% of all ganglion cells in peripheral temporal retina. Thus, the spatial density of parasol cells in central retina is greater than reported by previous anatomical studies, and the central-peripheral gradient in parasol cell density is shallower than previously reported. The data nevertheless predict decline in spatial acuity with visual field eccentricity for both midget-parvocellular pathway and parasol-magnocellular pathway mediated visual functions. The spatial resolving power of the OFF parasol array (peak ∼ 7 cpd) falls short of macaque behavioral grating acuity by at least a factor of three throughout the retina.

Intracellular Injection of Brain Extracts from Alzheimer's Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics.

Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimer's disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aβs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aβ oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract's ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aβ oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.

Endoscopic cyanoacrylate injection therapy for refractory high-risk peptic ulcer bleeding by conventional endoscopic therapy

Objective To evaluate the efficacy and safety of endoscopic cyanoacrylate injection therapy for refractory high-risk peptic ulcer bleeding by conventional endoscopic therapy. Methods 154 patients with refractory high-risk peptic ulcer bleeding by conventional endoscopic therapy at the Affiliated Hospital of Zunyi Medical Univesity and the People’s Hospital of Liupanshui City from January 2018 to December 2021were enrolled in this study. Endoscopic intracellular cyanoacrylate injection was first carried out. When failure, perintravascullar injection was carried out. The data were retrospectively collected. Results Among the 154 patients, 102 patients (66.23%) obtained successful intravascular injection and perivascular injection was performed in 52 patients (33.77%). Immediate hemostatic rate for active bleeding achieved 93.18%. Overall rebleeding rate within 30 days was 12.99% and successful hemostasis rate achieved 87.01%. Immediate hemostatic rate and successful hemostasis rate in intravascular injection patients were markedly superior over perivascular injection. Rebleeding rate in intravascular injection patients was markedly lower than that in perivascular injection patients. 14 patients complicated abdominal pain and no other complication occurred. Conclusion Endoscopic cyanoacrylate injection therapy, especial intravascular injection, was effective and safe, with high successful hemostasis rate for refractory high-risk peptic ulcer bleeding by conventional endoscopic therapy.

Cell Rover—a miniaturized magnetostrictive antenna for wireless operation inside living cells

An intracellular antenna can open up new horizons for fundamental and applied biology. Here, we introduce the Cell Rover, a magnetostrictive antenna which can operate wirelessly inside a living cell and is compatible with 3D biological systems. It is sub-mm in size, acoustically actuated by an AC magnetic field and resonantly operated at low MHz frequencies, which is ideal for living systems. We developed an injection scheme involving non-uniform magnetic fields for intracellular injection of the Cell Rovers and demonstrated their operation in fully opaque, stage VI Xenopus oocytes, for which real-time imaging with conventional technologies is challenging. We also show that they provide a pathway for multiplexing applications to individually address multiple cells or to tune to more than one antenna within the same cell for versatile functionalities. This technology forms the foundation stone that can enable the integration of future capabilities such as smart sensing, modulation as well as energy harvesting to power in-cell nanoelectronic computing and can potentially bring the prowess of information technology inside a living cell. This could lead to unprecedented opportunities for fundamental understanding of biology as well as diagnostics and therapeutics.

In vivo low-intensity magnetic pulses durably alter neocortical neuron excitability and spontaneous activity.

Magnetic brain stimulation is a promising treatment for neurological and psychiatric disorders. However, a better understanding of its effects at the individual neuron level is essential to improve its clinical application. We combined focal low-intensity repetitive transcranial magnetic stimulation (LI-rTMS) to the rat somatosensory cortex with intracellular recordings of subjacent pyramidal neurons in vivo. Continuous 10Hz LI-rTMS reliably evoked firing at ∼4-5Hz during the stimulation period and induced durable attenuation of synaptic activity and spontaneous firing in cortical neurons, through membrane hyperpolarization and a reduced intrinsic excitability. However, inducing firing in individual neurons by repeated intracellular current injection did not reproduce the effects of LI-rTMS on neuronal properties. These data provide a novel understanding of mechanisms underlying magnetic brain stimulation showing that, in addition to inducing biochemical plasticity, even weak magnetic fields can activate neurons and enduringly modulate their excitability. KEY POINTS: Repetitive transcranial magnetic stimulation (rTMS) is a promising technique to alleviate neurological and psychiatric disorders caused by alterations in cortical activity. Our knowledge of the cellular mechanisms underlying rTMS-based therapies remains limited. We combined in vivo focal application of low-intensity rTMS (LI-rTMS) to the rat somatosensory cortex with intracellular recordings of subjacent pyramidal neurons to characterize the effects of weak magnetic fields at single cell level. Ten minutes of LI-rTMS delivered at 10Hz reliably evoked action potentials in cortical neurons during the stimulation period, and induced durable attenuation of their intrinsic excitability, synaptic activity and spontaneous firing. These results help us better understand the mechanisms of weak magnetic stimulation and should allow optimizing the effectiveness of stimulation protocols for clinical use.

Mitochondria Transfer from Adipose Stem Cells Improves the Developmental Potential of Cryopreserved Oocytes.

Although it is not a well-established technology, oocyte cryopreservation is becoming prevalent in assisted reproductive technologies in response to the growing demands of patients’ sociological and pathological conditions. Oocyte cryopreservation can adversely affect the developmental potential of oocytes by causing an increase in intracellular oxidative stresses and damage to the mitochondrial structure. In this study, we studied whether autologous adipose stem cell (ASC) mitochondria supplementation with vitrified and warmed oocytes could restore post-fertilization development that decreased due to mitochondrial damage following cryopreservation. ASC mitochondria showed similar morphology to oocytes’ mitochondria and had a higher ATP production capacity. The vitrified-warmed oocytes from juvenile mice were supplemented with ASC mitochondria at the same time as intracellular sperm injection (ICSI), after which we compared their developmental capacity and the mitochondria quality of 2-cell embryos. We found that, compared to their counterpart, mitochondria supplementation significantly improved development from 2-cell embryos to blastocysts (56.8% vs. 38.2%) and ATP production in 2-cell embryos (905.6 & 561.1 pmol), while reactive oxygen species levels were comparable. With these results, we propose that ASC mitochondria supplementation could restore the quality of cryopreserved oocytes and enhance the embryo developmental capacity, signifying another possible approach for mitochondrial transplantation therapy.

P-218 Mitochondria transfer from adipose stem cell ameliorates the development potential of cryopreserved oocytes

Abstract Study question Could autologous adipose stem cell (ASC) mitochondria supplementation restore the post-fertilization development potentialin vitrified-thawed oocytes? Summary answer Autologous ASC mitochondria supplementation could rejuvenate the quality of vitrified-thawed oocytes and enhance the embryo’s developmental capacity. What is known already Pre- and post-implantation developmental potential of embryos derived from vitrified oocytes are remarkedly lower than those of fresh oocytes. Despite its’ low efficacy, oocyte cryopreservation is becoming prevalent in assisted reproductive technologies to cater the growing demands due to patients' sociological and pathological conditions. Unfavorable effects to mitochondrial membrane potential, mitochondrial structure, ATP production, reactive oxygen species (ROS), meiotic spindle and microfilaments, and Ca2+ ion regulation are reported as detrimental damages in oocytes following the cryopreservation, that adversely affect the development potential. Furthermore, autologous stem cell mitochondria supplementation can rescue the aging-related oocyte mitochondrial damages. Study design, size, duration The mature oocytes, autologous ASC, and mitochondria were collected from young mice and analyzed at Osaka City University, Japan. In total, 600 young mouse mature oocytes were occupied in this prospective study. Participants/materials, setting, methods Young C57BL/6JJmsmouse (8weeks) mature oocytes that have been pre-vitrified were thawed before the start of the ICSI procedure.ASC specificity and ASC mitochondria function and ultrastructure were pre-analyzed. ASC mitochondria were isolated on the same day and supplemented with intracellular sperm injection (ICSI) and as the control, the mitochondrial buffer was injected. The survival rate, fertilization rate, blastulation, mitochondria function, ROS level in 2 cell embryos, and live birth rates were compared between the 2 groups. Main results and the role of chance The ASC mitochondriashowed higher membrane potential compared to the somatic cells and were spherical in shape with low cristae numbers. The survival rate and the fertilization ratewere comparable in both mitochondria supplemented and control groups. However, theASC mitochondria supplementation seemed to havesignificantly improvedthe blastocyst development capacity from 2cell embryos compared to the control group (P < 0.05;56.8% & 38.2%, respectively).And interestingly, a significantly higher ATP level was found in the mitochondria supplemented group’s 2 cell embryos thanin the control group (P < 0.05;905.6pmol & 561.1pmol respectively). And though it was not statistically significant, a higher potential ofgetting live birth was found in the mitochondria supplemented group thanthe control group after 2 cell embryo transfer. Limitations, reasons for caution We acknowledge that the absence of compared data with fresh oocytes’ ICSI, the detailed cellular mechanism behind theimprovement of embryo development, and transgenerational safety in offspring developed fromthe mitochondria supplementation werethe limitations of this study. Wider implications of the findings With these results, we propose that ASC mitochondria supplementation could rejuvenate the quality of cryopreserved oocytes and enhance the embryo developmental capacity, signifying another possible approach of mitochondrial transplantation therapy. Trial registration number not applicable

Extending applications of AFM to fluidic AFM in single living cell studies.

In this article, a review of a series of applications of atomic force microscopy (AFM) and fluidic Atomic Force Microscopy (fluidic AFM, hereafter fluidFM) in single-cell studies is presented. AFM applications involving single-cell and extracellular vesicle (EV) studies, colloidal force spectroscopy, and single-cell adhesion measurements are discussed. FluidFM is an offshoot of AFM that combines a microfluidic cantilever with AFM and has enabled the research community to conduct biological, pathological, and pharmacological studies on cells at the single-cell level in a liquid environment. In this review, capacities of fluidFM are discussed to illustrate (1) the speed with which sequential measurements of adhesion using coated colloid beads can be done, (2) the ability to assess lateral binding forces of endothelial or epithelial cells in a confluent cell monolayer in an appropriate physiological environment, and (3) the ease of measurement of vertical binding forces of intercellular adhesion between heterogeneous cells. Furthermore, key applications of fluidFM are reviewed regarding to EV absorption, manipulation of a single living cell by intracellular injection, sampling of cellular fluid from a single living cell, patch clamping, and mass measurements of a single living cell.

Central projections of cercal giant interneurons in the adult field cricket, Gryllus bimaculatus.

The structures of neurons, such as dendrites and axonal projections, are closely related to their response properties and their specific functions in neural circuits. Identified neurons, having genetically determined morphological features and pre- and postsynaptic partners, play significant roles in specific behaviors. Giant interneurons (GIs) are identified in the terminal abdominal ganglion of the cricket as mechanosensory projection neurons and are sensitive to airflow stimulation of the cerci. GIs are classified into ventral GIs (vGIs) or dorsal GIs (dGIs) depending on the location of their axons running within the connective nerve cord. Based on their response properties to airflow, vGIs are presumed to be involved in triggering the wind-elicited escape response, whereas dGIs are thought to be airflow direction-encoding neurons. The previous findings regarding airflow sensitivity point to possible differences in the morphology of the central projections that may correspond to their neural functions. However, the detailed morphologies of the GIs in the cephalic and thoracic ganglia of adult crickets remain unclear. In this study, we stained six GIs, namely, GI 8-1 (medial giant interneuron, MGI), 9-1 (lateral giant interneuron, LGI), 9-2, 9-3, 10-2, and 10-3, using intracellular iontophoretic or pressure injection of dyes. Staining revealed remarkable differences in the axonal branching patterns between vGIs and dGIs. The dGIs were further divided into subgroups based on the profiles of their axon collaterals and projection sites in the brain. The anatomical differences between the GIs' central projections seemed to be related to their information encodement and behavioral functions.

Structural Analysis of Human and Mouse Dendritic Spines Reveals a Morphological Continuum and Differences across Ages and Species.

Dendritic spines have diverse morphologies, with a wide range of head and neck sizes, and these morphologic differences likely generate different functional properties. To explore how this morphologic diversity differs across species and ages we analyzed 3D confocal reconstructions of ∼8000 human spines and ∼1700 mouse spines, labeled by intracellular injections in fixed tissue. Using unsupervised algorithms, we computationally separated spine heads and necks and systematically measured morphologic features of spines in apical and basal dendrites from cortical pyramidal cells. Human spines had unimodal distributions of parameters, without any evidence of morphologic subtypes. Their spine necks were longer and thinner in apical than in basal spines, and spine head volumes of an 85-year-old individual were larger than those of a 40-year-old individual. Human spines had longer and thicker necks and larger head volumes than mouse spines. Our results indicate that human spines form part of a continuum, are larger and longer than those of mice, and become larger with increasing adult age. These morphologic differences in spines across species could generate functional differences in biochemical and electrical spine compartmentalization, or in synaptic properties, across species and ages.