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Journal of Visualized Experiments : JoVE Mar 2024Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent,...
Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent, temporally restricted, and transiently reversible only in a short, well-defined critical period. The directionality of brain circuit synaptic connectivity remodeling is determined by the specific odorant acting on the respondent receptor class of olfactory sensory neurons. In general, each neuron class expresses only a single odorant receptor and innervates a single olfactory synaptic glomerulus. In the Drosophila genetic model, the full array of olfactory glomeruli has been precisely mapped by odorant responsiveness and behavioral output. Ethyl butyrate (EB) odorant activates Or42a receptor neurons innervating the VM7 glomerulus. During the early-life critical period, EB experience drives dose-dependent synapse elimination in the Or42a olfactory sensory neurons. Timed periods of dosed EB odorant exposure allow investigation of experience-dependent circuit connectivity pruning in juvenile brain. Confocal microscopy imaging of antennal lobe synaptic glomeruli is done with Or42a receptor-driven transgenic markers that provide quantification of synapse number and innervation volume. The sophisticated Drosophila genetic toolkit enables the systematic dissection of the cellular and molecular mechanisms mediating brain circuit remodeling.
Topics: Animals; Olfactory Receptor Neurons; Brain; Olfactory Bulb; Receptors, Odorant; Drosophila; Butyrates
PubMed: 38497653
DOI: 10.3791/66629 -
Biomedical Optics Express Mar 2024In curative-intent cancer surgery, intraoperative fluorescence imaging of both diseased and healthy tissue can help to ensure the successful removal of all gross and...
In curative-intent cancer surgery, intraoperative fluorescence imaging of both diseased and healthy tissue can help to ensure the successful removal of all gross and microscopic diseases with minimal damage to neighboring critical structures, such as nerves. Current fluorescence-guided surgery (FGS) systems, however, rely on bulky and rigid optics that incur performance-limiting trade-offs between sensitivity and maneuverability. Moreover, many FGS systems are incapable of multiplexed imaging. As a result, clinical FGS is currently limited to millimeter-scale detection of a single fluorescent target. Here, we present a scalable, lens-less fluorescence imaging chip, VISION, capable of sensitive and multiplexed detection within a compact form factor. Central to VISION is a novel optical frontend design combining a low-numerical-aperture fiber optic plate (LNA-FOP) and a multi-bandpass interference filter, which is affixed to a custom CMOS image sensor. The LNA-FOP acts as a planar collimator to improve resolution and compensate for the angle-sensitivity of the interference filter, enabling high-resolution and multiplexed fluorescence imaging without lenses. We show VISION is capable of detecting tumor foci of less than 100 cells at near video framerates and, as proof of principle, can simultaneously visualize both tumors and nerves in prostate tissue.
PubMed: 38495694
DOI: 10.1364/BOE.509235 -
Journal of Visualized Experiments : JoVE Feb 2024Plant-derived cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of...
Plant-derived cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as its ability to effectively support osteogenic differentiation. The present study further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and energy-dispersive spectroscopy (EDS) confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young's modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.
Topics: Rats; Animals; Tissue Engineering; Osteogenesis; Malus; Tissue Scaffolds; Cells, Cultured; Bone and Bones; Cell Differentiation; Cellulose; Cell Proliferation; Mammals
PubMed: 38465930
DOI: 10.3791/65226 -
Heliyon Mar 2024Microfluidic blood flow models have been instrumental to study the functions of blood platelets in hemostasis and arterial thrombosis. However, they are not suited to...
Microfluidic blood flow models have been instrumental to study the functions of blood platelets in hemostasis and arterial thrombosis. However, they are not suited to investigate the interactions of platelets with the foreign surfaces of medical devices such as stents, mainly because of the dimensions and geometry of the microfluidic channels. Indeed, the channels of microfluidic chips are usually rectangular and rarely exceed 50 to 100 μm in height, impairing the insertion of clinically used stents. To fill this gap, we have developed an original macrofluidic flow system, which precisely reproduces the size and geometry of human vessels and therefore represents a biomimetic perfectly suited to insert a clinical stent and study its interplay with blood cells. The system is a circular closed loop incorporating a macrofluidic flow chamber made of silicone elastomer, which can mimic the exact dimensions of any human vessel, including the coronary, carotid or femoral artery. These flow chambers allow the perfect insertion of stents as they are implanted in patients. Perfusion of whole blood anticoagulated with hirudin through the device at relevant flow rates allows one to observe the specific accumulation of fluorescently labeled platelets on the stent surface using video-microscopy. Scanning electron microscopy revealed the formation of very large thrombi composed of tightly packed activated platelets on the stents.
PubMed: 38463800
DOI: 10.1016/j.heliyon.2024.e26550 -
Journal of Visualized Experiments : JoVE Feb 2024Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. Consequently, loss...
Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).
Topics: Animals; Axonal Transport; Caenorhabditis elegans; Axons; Microscopy, Fluorescence; Lissamine Green Dyes
PubMed: 38436410
DOI: 10.3791/66236 -
Acta Neurochirurgica Mar 2024The surgical 3D exoscopes have recently been introduced as an alternative to the surgical microscopes in microneurosurgery. Since the exoscope availability is still...
BACKGROUND
The surgical 3D exoscopes have recently been introduced as an alternative to the surgical microscopes in microneurosurgery. Since the exoscope availability is still limited, it is relevant to know whether even a short-term exoscope training develops the skills needed for performing exoscope-assisted surgeries.
METHODS
Ten participants (six consultants, four residents) performed two laboratory bypass test tasks with a 3D exoscope (Aesculap Aeos®). Six training sessions (6 h) were performed in between (interval of 2-5 weeks) on artificial models. The participants were divided into two groups: test group (n = 6) trained with the exoscope and control group (n = 4) with a surgical microscope. The test task was an artificial end-to-side microsurgical anastomosis model, using 12 interrupted 9-0 sutures and recorded on video. We compared the individual as well as group performance among the test subjects based on suturing time, anastomosis quality, and manual dexterity.
RESULTS
Altogether, 20 bypass tasks were performed (baseline n = 10, follow-up n = 10). The median duration decreased by 28 min and 44% in the exoscope training group. The decrease was steeper (29 min, 45%) among the participants with less than 6 years of microneurosurgery experience compared to the more experienced participants (13 min, 24%). After training, the participants with at least 1-year experience of using the exoscope did not improve their task duration. The training with the exoscope led to a greater time reduction than the training with the microscope (44% vs 17%).
CONCLUSIONS
Even short-term training with the exoscope led to marked improvements in exoscope-assisted bypass suturing among novice microneurosurgeons. For the more experienced participants, a plateau in the initial learning curve was reached quickly. A much longer-term effort might be needed to witness further improvement in this user group.
Topics: Humans; Prospective Studies; Microsurgery; Neurosurgical Procedures; Microscopy
PubMed: 38427127
DOI: 10.1007/s00701-024-05975-6 -
Microbiome Feb 2024Global warming is causing large-scale disruption of cnidarian-Symbiodiniaceae symbioses fundamental to major marine ecosystems, such as coral reefs. However, the...
BACKGROUND
Global warming is causing large-scale disruption of cnidarian-Symbiodiniaceae symbioses fundamental to major marine ecosystems, such as coral reefs. However, the mechanisms by which heat stress perturbs these symbiotic partnerships remain poorly understood. In this context, the upside-down jellyfish Cassiopea has emerged as a powerful experimental model system.
RESULTS
We combined a controlled heat stress experiment with isotope labeling and correlative SEM-NanoSIMS imaging to show that host starvation is a central component in the chain of events that ultimately leads to the collapse of the Cassiopea holobiont. Heat stress caused an increase in catabolic activity and a depletion of carbon reserves in the unfed host, concurrent with a reduction in the supply of photosynthates from its algal symbionts. This state of host starvation was accompanied by pronounced in hospite degradation of algal symbionts, which may be a distinct feature of the heat stress response of Cassiopea. Interestingly, this loss of symbionts by degradation was concealed by body shrinkage of the starving animals, resulting in what could be referred to as "invisible" bleaching.
CONCLUSIONS
Overall, our study highlights the importance of the nutritional status in the heat stress response of the Cassiopea holobiont. Compared with other symbiotic cnidarians, the large mesoglea of Cassiopea, with its structural sugar and protein content, may constitute an energy reservoir capable of delaying starvation. It seems plausible that this anatomical feature at least partly contributes to the relatively high stress tolerance of these animals in rapidly warming oceans. Video Abstract.
Topics: Animals; Ecosystem; Symbiosis; Cnidaria; Heat-Shock Response; Coral Reefs; Dinoflagellida; Anthozoa
PubMed: 38424629
DOI: 10.1186/s40168-023-01738-0 -
Biophysical Journal Apr 2024The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII...
The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII autophosphorylation. The autophosphorylation in turn increases the CaMKII affinity for CAM. Here, we studied the ATP dependence of CAM association with the actin-binding CaMKIIβ isoform using single-molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface-immobilized Venus-CaMKIIβ were resolved with 0.5 s resolution from video records, batch-processed with a custom algorithm. CAM occupancy was determined simultaneously with spot-photobleaching measurement of CaMKII holoenzyme stoichiometry. We show the ATP-dependent increase of the CAM association requires dimer formation for both the α and β isoforms. The study of mutant β holoenzymes revealed that the ATP-dependent increase in CAM affinity results in two distinct states. The phosphorylation-defective (T287.306-307A) holoenzyme resides only in the low-affinity state. CAM association is further reduced in the T287A holoenzyme relative to T287.306-307A. In the absence of ATP, the affinity of CAM for the T287.306-307A mutant and the wild-type monomer are comparable. The affinity of the ATP-binding impaired (K43R) mutant is even weaker. In ATP, the K43R holoenzyme resides in the low-affinity state. The phosphomimetic mutant (T287D) resides only in a 1000-fold higher-affinity state, with mean CAM occupancy of more than half of the 14-mer holoenzyme stoichiometry in picomolar CAM. ATP promotes T287D holoenzyme disassembly but does not elevate CAM occupancy. Single Poisson distributions characterized the ATP-dependent CAM occupancy of mutant holoenzymes. In contrast, the CAM occupancy of the wild-type population had a two-state distribution with both low- and high-affinity states represented. The low-affinity state was the dominant state, a result different from published in vitro assays. Differences in assay conditions can alter the balance between activating and inhibitory autophosphorylation. Bound ATP could be sufficient for CaMKII structural function, while antagonistic autophosphorylations may tune CaMKII kinase-regulated action-potential frequency decoding in vivo.
Topics: Calmodulin; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium; Single Molecule Imaging; Adenosine Triphosphate; Holoenzymes; Phosphorylation
PubMed: 38414237
DOI: 10.1016/j.bpj.2024.02.021 -
Journal of Visualized Experiments : JoVE Feb 2024Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse...
Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab's resources and demands.
Topics: Humans; Neutrophils; Extracellular Traps; Phagocytosis; Cytodiagnosis; Immunity, Innate
PubMed: 38407215
DOI: 10.3791/65329 -
Intensive Care Medicine Experimental Feb 2024Several studies have demonstrated associations between greater rate/volume of intravenous (IV) fluid administration and poorer clinical outcomes. One postulated... (Review)
Review
INTRODUCTION
Several studies have demonstrated associations between greater rate/volume of intravenous (IV) fluid administration and poorer clinical outcomes. One postulated mechanism for harm from exogenous fluids is shedding of the endothelial glycocalyx (EG).
METHODS
A systematic review using relevant search terms was performed using Medline, EMBASE and Cochrane databases from inception to October 2023. Included studies involved humans where the exposure was rate or volume of IV fluid administration and the outcome was EG shedding. The protocol was prospectively registered on PROSPERO: CRD42021275133.
RESULTS
The search yielded 450 articles, with 20 articles encompassing 1960 participants included in the review. Eight studies were randomized controlled clinical trials. Half of studies examined patients with sepsis and critical illness; the remainder examined perioperative patients or healthy subjects. Almost all reported blood measurements of soluble EG components; one study used in vivo video-microscopy to estimate EG thickness. Four of 10 sepsis studies, and 9 of 11 non-sepsis studies, found a positive relationship between IV fluid rate/volume and measures of EG shedding.
CONCLUSIONS
A trend toward an association between IV fluid rate/volume and EG shedding was found in studies of stable patients, but was not consistently observed among studies of septic and critically ill patients.
PubMed: 38403742
DOI: 10.1186/s40635-024-00602-1