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Biophysical Reports Jun 2024We present a method for tracking densely clustered, high-velocity, indistinguishable objects being spawned at a high rate and moving in a directed force field using only...
We present a method for tracking densely clustered, high-velocity, indistinguishable objects being spawned at a high rate and moving in a directed force field using only object centroids as inputs and no other image information. The algorithm places minimal restrictions on the velocities or accelerations of the objects being tracked and uses a methodology based on a scoring function and a backtracking refinement process. This combination leads to successful tracking of hundreds of particles in challenging environments even when the displacement of the individual objects at successive times approaches the separation between neighboring objects in any one frame. We note that these cases can be particularly difficult to handle by existing methods. The performance of the algorithm is methodically examined by comparison to simulated trajectories, which vary the temporal and spatial densities, velocities, and accelerations of the objects in motion, as well as the signal/noise ratio. Also, we demonstrate its capability by analyzing data from experiments with superparamagnetic microspheres moving in an inhomogeneous magnetic field in aqueous buffer at room temperature. Our method should be widely applicable since trajectory determination problems are ubiquitous in video microscopy applications in biology, materials science, physics, and engineering.
PubMed: 38505834
DOI: 10.1016/j.bpr.2024.100148 -
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 -
Cell Communication and Signaling : CCS Sep 2023While the increased screening, changes in lifestyle, and recent advances in treatment regimen have decreased colorectal cancer (CRC) mortality, metastatic disease and...
BACKGROUND
While the increased screening, changes in lifestyle, and recent advances in treatment regimen have decreased colorectal cancer (CRC) mortality, metastatic disease and recurrence remains a major clinical challenge. In the era of precision medicine, the identification of actionable novel therapeutic targets could ultimately offer an alternative treatment strategy for CRC.
METHODS
RNA-Seq was conducted using the illumina platform, while bioinformatics analyses were conducted using CLC genomics workbench and iDEP.951. Colony forming unit, flow cytometry, and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. The growth potential of CRC cells under 3-dimensional (3D) conditions was assessed using Matrigel. STRING database (v11.5) and Ingenuity Pathway Analysis (IPA) tool were used for network and pathway analyses. CRISPR-Cas9 perturbational effects database was used to identify potential therapeutic targets for CRC, through integration with gene-drug interaction database. Structural modeling and molecular docking were used to assess the interaction between candidate drugs and their targets.
RESULTS
In the current study, we investigated the therapeutic potential of targeting TPX2, TTK, DDX39A, and LRP8, commonly upregulated genes in CRC identified through differential expression analysis in CRC and adjacent non-cancerous tissue. Targeted depletion of TPX2 and TTK impaired CRC proliferation, cell cycle progression, and organoid formation under 3D culture conditions, while suppression of DDX39A and LRP8 had modest effects on CRC colony formation. Differential expression analysis and bioinformatics on TPX2 and TTK-deficient cells identified cell cycle regulation as the hallmark associated with loss of TPX2 and TTK. Elevated expression of TPX2 and TTK correlated with an oncogenic state in tumor tissue from patients with colon adenocarcinoma, thus corroborating an oncogenic role for the TPX2/TTK network in the pathogenesis of CRC. Gene set enrichment and pathway analysis of TPX2/TTK CRC identified numerous additional gene targets as integral components of the TPX2/TTK network. Integration of TPX2/TTK enriched network with CRISPR-Cas9 functional screen data identified numerous novel dependencies for CRC. Additionally, gene-drug interaction analysis identified several druggable gene targets enriched in the TPX2/TTK network, including AURKA, TOP2A, CDK1, BIRC5, and many others.
CONCLUSIONS
Our data has implicated an essential role for TPX2 and TTK in CRC pathogenesis and identified numerous potential therapeutic targets and their drug interactions, suggesting their potential clinical use as a novel therapeutic strategy for patients with CRC. Video Abstract.
Topics: Humans; Colonic Neoplasms; Colorectal Neoplasms; Adenocarcinoma; Molecular Docking Simulation; Cell Proliferation; Gene Expression Regulation, Neoplastic; Microtubule-Associated Proteins; Cell Cycle Proteins; Protein-Tyrosine Kinases; Protein Serine-Threonine Kinases
PubMed: 37770979
DOI: 10.1186/s12964-023-01290-2 -
BioRxiv : the Preprint Server For... Aug 2023Structured illumination microscopy (SIM) can double the spatial resolution of a fluorescence microscope and video rate live cell imaging in a two-dimensional format has...
Structured illumination microscopy (SIM) can double the spatial resolution of a fluorescence microscope and video rate live cell imaging in a two-dimensional format has been demonstrated. However, rapid implementations of 2D SIM typically only cover a narrow slice of the sample immediately at the coverslip, with most of the cellular volume out of reach. Here we implement oblique plane structured illumination microscopy (OPSIM) in a projection format to rapidly image an entire cell in a 2D SIM framework. As no mechanical scanning of the sample or objective is involved, this technique has the potential for rapid projection imaging with doubled resolution. We characterize the spatial resolution with fluorescent nanospheres, compare projection and 3D imaging using OPSIM and image mitochondria and ER dynamics across an entire cell at up to 2.7 Hz. To our knowledge, this represents the fastest whole cell SIM imaging to date.
PubMed: 37609312
DOI: 10.1101/2023.08.08.552447 -
Cell Communication and Signaling : CCS Aug 2023Cerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary...
BACKGROUND
Cerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI.
METHODS
SH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett's T3 test (for unequal homogeneity) were used for statistical tests.
RESULTS
ERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1 or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed.
CONCLUSION
Inhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model. Video Abstract.
Topics: Humans; Oxygen; Extracellular Signal-Regulated MAP Kinases; Apoptosis; Glucose; Neuroblastoma; Dynamins; Autophagy
PubMed: 37580749
DOI: 10.1186/s12964-023-01211-3 -
Trends in Cell Biology Nov 2023The growth of artificial intelligence (AI) has led to an increase in the adoption of computer vision and deep learning (DL) techniques for the evaluation of microscopy... (Review)
Review
The growth of artificial intelligence (AI) has led to an increase in the adoption of computer vision and deep learning (DL) techniques for the evaluation of microscopy images and movies. This adoption has not only addressed hurdles in quantitative analysis of dynamic cell biological processes but has also started to support advances in drug development, precision medicine, and genome-phenome mapping. We survey existing AI-based techniques and tools, as well as open-source datasets, with a specific focus on the computational tasks of segmentation, classification, and tracking of cellular and subcellular structures and dynamics. We summarise long-standing challenges in microscopy video analysis from a computational perspective and review emerging research frontiers and innovative applications for DL-guided automation in cell dynamics research.
PubMed: 38030542
DOI: 10.1016/j.tcb.2023.10.010 -
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 -
Frontiers in Pediatrics 2023Primary ciliary dyskinesia (PCD) is a rare genetic disorder that can result in significant morbidity and mortality if left untreated. Clinical manifestations of PCD... (Review)
Review
INTRODUCTION
Primary ciliary dyskinesia (PCD) is a rare genetic disorder that can result in significant morbidity and mortality if left untreated. Clinical manifestations of PCD include recurrent respiratory infections, laterality defects, and infertility, all of which arise from impaired or absent ciliary motility. Diagnostic approaches for PCD may include high-speed video microscopy, measurement of nasal nitric oxide levels, and genetic testing; however, no single definitive diagnostic test exists. The present study aims to highlight the lack of standardized diagnostic and treatment guidelines for PCD in Latin America (Central and South America, and the Caribbean). To this effect, we compared North American and European recommendations for the diagnosis and management of PCD and found that certain diagnostic tools and treatment options mentioned in these guidelines are not readily accessible in many Latin American countries.
METHODS & RESULTS
This review gathers disease information in North America, Europe, and Latin America organizing guideline results into tables for clarity and potential interventions. Management information for Latin America is inferred from case reports, as most findings are from North American recommendations and studies on PubMed, Google Scholar, and Scopus. Treatment and management information is based on North American and European standards.Among 5,774 publications reviewed, only 15 articles met the inclusion criteria (focused on PCD management, peer-reviewed, and located in America). No clinical guideline for PCD in Latin America was found, but recommendations on respiratory management from Colombia and Chile were discovered. The lack of guidelines in Latin America may originate from limited resources and research on the disease in those countries.
DISCUSSION
PCD lacks documentation, research, and recommendations regarding its prevalence in Latin America, likely due to unfavorable economic conditions. This disadvantage results in limited access to diagnostic tests available in North America and Europe. The PICADAR score, discussed in this review, can be used in low-income nations as a screening tool for the disorder.
PubMed: 37744431
DOI: 10.3389/fped.2023.1091173 -
Cell Communication and Signaling : CCS Aug 2023Extracellular vesicles (EVs) are membrane-coated nanoparticles secreted by almost all cell types in living organisms. EVs, as paracrine mediators, are involved in...
BACKGROUND
Extracellular vesicles (EVs) are membrane-coated nanoparticles secreted by almost all cell types in living organisms. EVs, as paracrine mediators, are involved in intercellular communication, immune response, and several reproductive events, including the maintenance of pregnancy. Using a domestic animal model (Sus scrofa) with an epitheliochorial, superficial type of placentation, we focused on EV biogenesis pathway at the embryo-maternal interface, when the embryonic signaling occurs for maternal recognition and the maintenance of pregnancy.
RESULTS
Transmission electron microscopy was used during early pregnancy to visualize EVs and apocrine and/or merocrine pathways of secretion. Immunofluorescent staining localized proteins responsible for EV biogenesis and cell polarization at the embryo-maternal interface. The expression profiles of genes involved in biogenesis and the secretion of EVs pointed to the possible modulation of endometrial expression by embryonic signals. Further in vitro studies showed that factors of embryonic origin can regulate the expression of the ESCRT-II complex and EV trafficking within endometrial luminal epithelial cells. Moreover, miRNA-mediated rapid negative regulation of gene expression was abolished by delivered embryonic signals.
CONCLUSIONS
Our findings demonstrated that embryonic signals are potent modulators of ESCRT-dependent EV-mediated secretory activity of the endometrium during the critical stages of early pregnancy. Video Abstract.
Topics: Animals; Female; Pregnancy; Biological Transport; Cell Communication; Endosomes; Extracellular Vesicles; Endosomal Sorting Complexes Required for Transport
PubMed: 37596609
DOI: 10.1186/s12964-023-01221-1 -
Computer Methods and Programs in... Nov 2023Primary ciliary dyskinesia (PCD) is a rare genetic disorder causing a defective ciliary structure, which predominantly leads to an impaired mucociliary clearance and...
BACKGROUND AND OBJECTIVE
Primary ciliary dyskinesia (PCD) is a rare genetic disorder causing a defective ciliary structure, which predominantly leads to an impaired mucociliary clearance and associated airway disease. As there is currently no single diagnostic gold standard test, PCD is diagnosed by a combination of several methods comprising genetic testing and the examination of the ciliary structure and function. Among the approved diagnostic methods, only high-speed video microscopy (HSVM) allows to directly observe the ciliary motion and therefore, to directly assess ciliary function. In the present work, we present our recently developed freely available open-source software - termed "Cilialyzer", which has been specifically designed to support and facilitate the analysis of the mucociliary activity in respiratory epithelial cells captured by high-speed video microscopy.
METHODS
In its current state, the Cilialyzer software enables clinical PCD analysts to load, preprocess and replay recorded image sequences as well as videos with a feature-rich replaying module facilitating the commonly performed qualitative visual assessment of ciliary function (including the assessment of the ciliary beat pattern). The image processing methods made accessible through an intuitive user interface allow clinical specialists to comfortably compute the ciliary beating frequency (CBF), the activity map and the "frequency correlation length" - an observable getting newly introduced. Furthermore, the Cilialyzer contains a simple-to-use particle tracking interface to determine the mucociliary transport speed.
RESULTS
Cilialyzer is fully written in the Python programming language and freely available under the terms of the MIT license. The proper functioning of the computational analysis methods constituting the Cilialyzer software is demonstrated by using simulated and representative sample data from clinical practice. Additionally, the software was used to analyze high-speed videos showing samples obtained from healthy controls and genetically confirmed PCD cases (DNAI1 and DNAH11 mutations) to show its clinical applicability.
CONCLUSIONS
Cilialyzer serves as a useful clinical tool for PCD analysts and provides new quantitative information awaiting to be clinically evaluated using cohorts of PCD. As Cilialyzer is freely available under the terms of a permissive open-source license, it serves as a ground frame for further development of computational methods aiming at the quantification and automation of the analysis of mucociliary activity captured by HSVM.
Topics: Humans; Respiratory Rate; Software; Programming Languages; Automation; Genetic Testing; Rare Diseases
PubMed: 37598471
DOI: 10.1016/j.cmpb.2023.107744