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ELife Dec 2023Microglia surveillance manifests itself as dynamic changes in cell morphology and functional remodeling. Whether and how microglia surveillance is coupled to brain state...
Microglia surveillance manifests itself as dynamic changes in cell morphology and functional remodeling. Whether and how microglia surveillance is coupled to brain state switches during natural sleep-wake cycles remains unclear. To address this question, we used miniature two-photon microscopy (mTPM) to acquire time-lapse high-resolution microglia images of the somatosensory cortex, along with EEG/EMG recordings and behavioral video, in freely-behaving mice. We uncovered fast and robust brain state-dependent changes in microglia surveillance, occurring in parallel with sleep dynamics and early-onset phagocytic microglial contraction during sleep deprivation stress. We also detected local norepinephrine fluctuation occurring in a sleep state-dependent manner. We showed that the locus coeruleus-norepinephrine system, which is crucial to sleep homeostasis, is required for both sleep state-dependent and stress-induced microglial responses and β-adrenergic receptor signaling plays a significant role in this process. These results provide direct evidence that microglial surveillance is exquisitely tuned to signals and stressors that regulate sleep dynamics and homeostasis so as to adjust its varied roles to complement those of neurons in the brain. In vivo imaging with mTPM in freely behaving animals, as demonstrated here, opens a new avenue for future investigation of microglia dynamics and sleep biology in freely behaving animals.
Topics: Mice; Animals; Microglia; Sleep; Sleep Deprivation; Brain; Norepinephrine
PubMed: 38132088
DOI: 10.7554/eLife.86749 -
MBio Feb 2024The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R...
The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R endolysin, and the Rz/Rz1 spanin complex targeting the inner membrane, cell wall or peptidoglycan, and the outer membrane, respectively. Video microscopy has shown that in most infections, lysis occurs as a sudden, explosive event at a cell pole, such that the initial product is a less refractile ghost that retains rod-shaped morphology. Here, we investigate the molecular basis of polar lysis using time-lapse fluorescence microscopy. The results indicate that the holin determines the morphology of lysis by suddenly forming two-dimensional rafts at the poles about 100 s prior to lysis. Given the physiological and biochemical similarities between the lambda holin and other class I holins, dynamic redistribution and sudden concentration may be common features of holins, probably reflecting the fitness advantage of all-or-nothing lysis regulation.IMPORTANCEIn this study, we use fluorescent video microscopy to track -green fluorescent protein (GFP)-labeled holin in the minutes prior to phage lysis. Our work contextualizes prior genetic and biochemical data, showing when hole formation starts and where holin oligomers form in relation to the site of lytic rupture. Furthermore, prior work showed that the morphology of lambda-infected cells is characterized by an explosive event starting at the cell pole; however, the basis for this was not clear. This study shows that holin most often oligomerizes at cell poles and that the site of the oligomerization is spatially correlated with the site of lytic blowout. Therefore, the holin is the key contributor to polar lysis morphology for phage lambda.
Topics: Viral Proteins; Bacteriophage lambda; Cell Death; Cell Wall; Bacteriolysis
PubMed: 38126784
DOI: 10.1128/mbio.01290-23 -
Journal of Visualized Experiments : JoVE Dec 2023Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy...
Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 µm. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 µm level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.
Topics: Hyperspectral Imaging; Microscopy; Radionuclide Imaging; Bioengineering; Biomedical Engineering
PubMed: 38108388
DOI: 10.3791/65388 -
BioRxiv : the Preprint Server For... Dec 2023Biallelic mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect approximately 10,000 patients in North America and...
Biallelic mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect approximately 10,000 patients in North America and Europe alone. Pathogenic loss-of-function changes in the encoded enzyme sorbitol dehydrogenase result in abnormally high sorbitol levels in cells and serum. How sorbitol accumulation leads to peripheral neuropathy remains to be elucidated. A reproducible animal model for SORD neuropathy is essential to illuminate the pathogenesis of SORD deficiency and for preclinical studies of potential therapies. Therefore, we have generated a knockout (KO), , Sprague Dawley rat, to model the human disease and to investigate the pathophysiology underlying SORD deficiency. We have characterized the phenotype in these rats with a battery of behavioral tests as well as biochemical, physiological, and comprehensive histological examinations. rats had remarkably increased levels of sorbitol in serum, cerebral spinal fluid (CSF), and peripheral nerve. Moreover, serum from rats contained significantly increased levels of neurofilament light chain, NfL, an established biomarker for axonal degeneration. Motor performance significantly declined in animals starting at ∼7 months of age. Gait analysis evaluated with video motion tracking confirmed abnormal gait patterns in the hindlimbs. Motor nerve conduction velocities of the tibial nerves were slowed. Light and electron microscopy of the peripheral nervous system revealed degenerating myelinated axons, de- and remyelinated axons, and a likely pathognomonic finding - enlarged "ballooned" myelin sheaths. These findings mainly affected myelinated motor axons; myelinated sensory axons were largely spared. In summary, rats develop a motor-predominant neuropathy that closely resembles the human phenotype. Our studies revealed novel significant aspects of SORD deficiency, and this model will lead to an improved understanding of the pathophysiology and the therapeutic options for SORD neuropathy.
PubMed: 38106042
DOI: 10.1101/2023.12.05.570001 -
Cell Communication and Signaling : CCS Dec 2023Ephrin (EPH) receptors have been implicated in tumorigenesis and metastasis, but the functional understanding of mutations observed in human cancers is limited. We...
BACKGROUND
Ephrin (EPH) receptors have been implicated in tumorigenesis and metastasis, but the functional understanding of mutations observed in human cancers is limited. We previously demonstrated reduced cell compartmentalisation for somatic EPHB1 mutations found in metastatic colorectal cancer cases. We therefore integrated pan-cancer and pan-EPH mutational data to prioritise recurrent EPHB1 mutations for functional studies to understand their contribution to cancer development and metastasis.
METHODS
Here, 79,151 somatic mutations in 9,898 samples of 33 different tumour types were analysed with a bioinformatic pipeline to find 3D-mutated cluster pairs and hotspot mutations in EPH receptors. From these, 15 recurring EPHB1 mutations were stably expressed in colorectal cancer followed by confocal microscopy based in vitro compartmentalisation assays and phospho-proteome analysis.
RESULTS
The 3D-protein structure-based bioinformatics analysis resulted in 63% EPHB1 mutants with compartmentalisation phenotypes vs 43% for hotspot mutations. Whereas the ligand-binding domain mutations C61Y, R90C, and R170W, the fibronectin domain mutation R351L, and the kinase domain mutation D762N displayed reduced to strongly compromised cell compartmentalisation, the kinase domain mutations R743W and G821R enhanced this phenotype. While mutants with reduced compartmentalisation also had reduced ligand induced receptor phosphorylation, the enhanced compartmentalisation was not linked to receptor phosphorylation level. Phosphoproteome mapping pinpointed the PI3K pathway and PIK3C2B phosphorylation in cells harbouring mutants with reduced compartmentalisation.
CONCLUSIONS
This is the first integrative study of pan-cancer EPH receptor mutations followed by in vitro validation, a robust way to identify cancer-causing mutations, uncovering EPHB1 mutation phenotypes and demonstrating the utility of protein structure-based mutation analysis in characterization of novel cancer genes. Video Abstract.
Topics: Humans; Colorectal Neoplasms; Ligands; Mutation; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Receptor Protein-Tyrosine Kinases
PubMed: 38102712
DOI: 10.1186/s12964-023-01378-9 -
Cell Communication and Signaling : CCS Dec 2023Type 2 diabetes mellitus (T2DM) induced diabetes-associated cognitive dysfunction (DACD) that seriously affects the self-management of T2DM patients, is currently one of...
BACKGROUND
Type 2 diabetes mellitus (T2DM) induced diabetes-associated cognitive dysfunction (DACD) that seriously affects the self-management of T2DM patients, is currently one of the most severe T2DM-associated complications, but the mechanistic basis remains unclear. Mitochondria are highly dynamic organelles, whose function refers to a broad spectrum of features such as mitochondrial dynamics, mitophagy and so on. Mitochondrial abnormalities have emerged as key determinants for cognitive function, the relationship between DACD and mitochondria is not well understood.
METHODS
Here, we explored the underlying mechanism of mitochondrial dysfunction of T2DM mice and HT22 cells treated with high glucose/palmitic acid (HG/Pal) focusing on the mitochondrial fission-mitophagy axis with drug injection, western blotting, Immunofluorescence, and electron microscopy. We further explored the potential role of caveolin-1 (cav-1) in T2DM induced mitochondrial dysfunction and synaptic alteration through viral transduction.
RESULTS
As previously reported, T2DM condition significantly prompted hippocampal mitochondrial fission, whereas mitophagy was blocked rather than increasing, which was accompanied by dysfunctional mitochondria and impaired neuronal function. By contrast, Mdivi-1 (mitochondrial division inhibitor) and urolithin A (mitophagy activator) ameliorated mitochondrial and neuronal function and thereafter lead to cognitive improvement by inhibiting excessive mitochondrial fission and giving rise to mitophagy, respectively. We have previously shown that cav-1 can significantly improve DACD by inhibiting ferroptosis. Here, we further demonstrated that cav-1 could not only inhibit mitochondrial fission via the interaction with GSK3β to modulate Drp1 pathway, but also rescue mitophagy through interacting with AMPK to activate PINK1/Parkin and ULK1-dependent signlings.
CONCLUSIONS
Overall, our data for the first time point to a mitochondrial fission-mitophagy axis as a driver of neuronal dysfunction in a phenotype that was exaggerated by T2DM, and the protective role of cav-1 in DACD. Graphic Summary Illustration. In T2DM, excessive mitochondrial fission and impaired mitophagy conspire to an altered mitochondrial morphology and mitochondrial dysfunction, with a consequent neuronal damage, overall suggesting an unbalanced mitochondrial fission-mitophagy axis. Upon cav-1 overexpression, GSK3β and AMPK are phosphorylated respectively to activate Drp1 and mitophagy-related pathways (PINK1 and ULKI), ultimately inhibits mitochondrial fission and enhances mitophagy. In the meantime, the mitochondrial morphology and neuronal function are rescued, indicating the protective role of cav-1 on mitochondrial fission-mitophagy axis. Video Abstract.
Topics: Humans; Mice; Animals; Mitophagy; Mitochondrial Dynamics; Diabetes Mellitus, Type 2; Caveolin 1; AMP-Activated Protein Kinases; Glycogen Synthase Kinase 3 beta; Mitochondrial Diseases; Neurons; Cognitive Dysfunction; Ubiquitin-Protein Ligases
PubMed: 38102662
DOI: 10.1186/s12964-023-01328-5 -
Fungal Biology Dec 2023Unlike the mechanism of ballistospore discharge, which was not solved until the 1980s, the operation of asci as pressurized squirt guns is relatively straightforward and... (Review)
Review
Unlike the mechanism of ballistospore discharge, which was not solved until the 1980s, the operation of asci as pressurized squirt guns is relatively straightforward and was understood in the nineteenth century. Since then, mycologists have sought to understand how structural adaptations to asci have allowed the ascomycetes to expel spores of different shapes and sizes over distances ranging from a few millimeters to tens of centimeters. These modifications include the use of valves at the tips of asci that maintain ascus pressure and expel spores at the highest speeds, and gelatinous appendages that connect spores after release and create larger projectiles with greater momentum than single spores. Clever experiments in the twentieth century coupled with meticulous microscopic studies led investigators to understand how asci with complicated apical structures worked and mathematical models produced estimates of launch speeds. With the recent application of high-speed video microscopy, these inferences about ascus function have been tested by imaging the motion of spores on a microsecond timescale. These experiments have established that ascospore discharge is the fastest fungal movement and is among the fastest movements in biology. Beginning with the history of the study of asci, this review article explains how asci are pressurized, how spores are released, and how far spores travel after their release. We also consider the efficiency of ascospore discharge relative to the mechanism of ballistospore discharge and examine the way that the squirt gun mechanism has limited the morphological diversity of ascomycete fruit bodies.
Topics: Firearms; Ascomycota; Spores, Fungal
PubMed: 38097323
DOI: 10.1016/j.funbio.2023.11.001 -
Communications Biology Dec 2023Interrogation of subcellular biological dynamics occurring in a living cell often requires noninvasive imaging of the fragile cell with high spatiotemporal resolution...
Interrogation of subcellular biological dynamics occurring in a living cell often requires noninvasive imaging of the fragile cell with high spatiotemporal resolution across all three dimensions. It thereby poses big challenges to modern fluorescence microscopy implementations because the limited photon budget in a live-cell imaging task makes the achievable performance of conventional microscopy approaches compromise between their spatial resolution, volumetric imaging speed, and phototoxicity. Here, we incorporate a two-stage view-channel-depth (VCD) deep-learning reconstruction strategy with a Fourier light-field microscope based on diffractive optical element to realize fast 3D super-resolution reconstructions of intracellular dynamics from single diffraction-limited 2D light-filed measurements. This VCD-enabled Fourier light-filed imaging approach (F-VCD), achieves video-rate (50 volumes per second) 3D imaging of intracellular dynamics at a high spatiotemporal resolution of ~180 nm × 180 nm × 400 nm and strong noise-resistant capability, with which light field images with a signal-to-noise ratio (SNR) down to -1.62 dB could be well reconstructed. With this approach, we successfully demonstrate the 4D imaging of intracellular organelle dynamics, e.g., mitochondria fission and fusion, with ~5000 times of observation.
Topics: Imaging, Three-Dimensional; Microscopy, Fluorescence; Mitochondria
PubMed: 38086994
DOI: 10.1038/s42003-023-05636-x -
Nature Communications Dec 2023Autonomous migration is essential for the function of immune cells such as neutrophils and plays an important role in numerous diseases. The ability to routinely measure...
Autonomous migration is essential for the function of immune cells such as neutrophils and plays an important role in numerous diseases. The ability to routinely measure or target it would offer a wealth of clinical applications. Video microscopy of live cells is ideal for migration analysis, but cannot be performed at sufficiently high-throughput (HT). Here we introduce ComplexEye, an array microscope with 16 independent aberration-corrected glass lenses spaced at the pitch of a 96-well plate to produce high-resolution movies of migrating cells. With the system, we enable HT migration analysis of immune cells in 96- and 384-well plates with very energy-efficient performance. We demonstrate that the system can measure multiple clinical samples simultaneously. Furthermore, we screen 1000 compounds and identify 17 modifiers of migration in human neutrophils in just 4 days, a task that requires 60-times longer with a conventional video microscope. ComplexEye thus opens the field of phenotypic HT migration screens and enables routine migration analysis for the clinical setting.
Topics: Humans; Microscopy; Microscopy, Video; Lenses; Lens, Crystalline; Cell Movement
PubMed: 38081825
DOI: 10.1038/s41467-023-43765-3 -
International Journal of Surgery... Mar 2024Emerging three-dimensional digital visualization technology (DVT) provides more advantages than traditional microscopy in microsurgery; however, its impact on...
Comparing the impact of three-dimensional digital visualization technology versus traditional microscopy on microsurgeons in microsurgery: a prospective self-controlled study.
BACKGROUND
Emerging three-dimensional digital visualization technology (DVT) provides more advantages than traditional microscopy in microsurgery; however, its impact on microsurgeons' visual and nervous systems and delicate microsurgery is still unclear, which hinders the wider implementation of DVT in digital visualization for microsurgery.
METHODS AND MATERIAL
Forty-two microsurgeons from the Zhongshan Ophthalmic Center were enrolled in this prospective self-controlled study. Each microsurgeon consecutively performed 30 min conjunctival sutures using a three-dimensional digital display and a microscope, respectively. Visual function, autonomic nerve activity, and subjective symptoms were evaluated before and immediately after the operation. Visual functions, including accommodative lag, accommodative amplitude, near point of convergence and contrast sensitivity function (CSF), were measured by an expert optometrist. Heart rate variability was recorded by a wearable device for monitoring autonomic nervous activity. Subjective symptoms were evaluated by questionnaires. Microsurgical performance was assessed by the video-based Objective Structured Assessment of Technical Skill (OSATS) tool.
RESULTS
Accommodative lag decreased from 0.63 (0.18) diopters (D) to 0.55 (0.16) D ( P =0.014), area under the log contrast sensitivity function increased from 1.49 (0.15) to 1.52 (0.14) ( P =0.037), and heart rate variability decreased from 36.00 (13.54) milliseconds (ms) to 32.26 (12.35) ms ( P =0.004) after using the DVT, but the changes showed no differences compared to traditional microscopy ( P >0.05). No statistical significance was observed for global OSATS scores between the two rounds of operations [mean difference, 0.05 (95% CI: -1.17 to 1.08) points; P =0.95]. Subjective symptoms were quite mild after using both techniques.
CONCLUSIONS
The impact of DVT-based procedures on microsurgeons includes enhanced accommodation and sympathetic activity, but the changes and surgical performance are not significantly different from those of microscopy-based microsurgery. Our findings indicate that short-term use of DVT is reliable for microsurgery and the long-term effect of using DVT deserve more consideration.
Topics: Humans; Microscopy; Microsurgery; Prospective Studies; Wearable Electronic Devices; Technology
PubMed: 38079600
DOI: 10.1097/JS9.0000000000000950