Video Microscopy Research Articles

Primary ciliary dyskinesia in children: clinical, laboratory-instrumental and genetic characteristics

Background. Primary ciliary dyskinesia (PCD) is an orphan disease, and diagnosis is difficult because there is no gold standard for diagnosis. Aim. Clinical, laboratory-instrumental, genetic characteristics of PCD in children. Materials and methods. From 2009 to 2024, 31 patients with a genetically confirmed diagnosis of PCD were observed as part of a multicenter, open-ended, descriptive pilot longitudinal study. Examination methods: clinical and anamnestic method; X-ray examination and computed tomography of the chest organs and paranasal sinuses, tracheobronchoscopy; sputum/aspirate cultures of the tracheobroncheal tree with determination of sensitivity to antibiotics; transmission electron microscopy, high-speed video microscopy of the ciliated epithelium, cytological examination of bronchoalveolar lavage; monitoring computer pulse oximetry, echocardiography, audiometry, spirometry with bronchodilator test. Results. Respiratory symptoms in the neonatal period have 80% of children with PCD, lateralization defects – 35%, congenital heart defects – 13%, bronchiectasis – 68%, purulent endobronchitis – 62%, year-round rhinitis – 84%, hearing loss, otitis – 65%. The average age of onset of symptoms was 1 [1; 1] weeks, and the verification of diagnosis was 6 [2,5; 8] years. The main pathogens of chronic respiratory infection with PCD are Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus. The most common cause of PCD was biallelic variants of the DNAH5 gene. Conclusion. The diagnosis of PCD should be based on the application of the maximum number of diagnostic tests.

Sidestream dark field video microscopy demonstrates shelf-stable blood products preserve the endothelial glycocalyx in a canine hemorrhagic shock model.

To utilize sidestream dark field video microscopic technology to evaluate the endothelium in a canine hemorrhagic shock and resuscitation model. 6 purpose-bred adult dogs were anesthetized, instrumented, and subjected to hemorrhagic shock from September 2021 through June 2022. Each dog was resuscitated with 5 resuscitation strategies in an experimental crossover design study: (1) lactated Ringer's solution (LRS) and hydroxyethyl starch (HES) solution; (2) canine chilled whole blood (CWB); (3) canine fresh frozen plasma (FFP) and packed RBCs (pRBC); (4) canine freeze-dried plasma (FDP) and hemoglobin-based oxygen carrier (HBOC); or (5) HBOC/FDP and canine lyophilized platelets. Sidestream dark field video microscopic evaluation was performed at 5 time points: commencement, after hemorrhage, after shock, after resuscitation (T135), and conclusion (T180). There was a significant difference between the perfused boundary region (PBR) measurements when comparing the LRS/HES resuscitation arm to the CWB and FFP/pRBC resuscitation arms at T180. A significant difference in PBR was appreciated in the LRS/HES arm at T135 and T180 compared to its baseline. No other significant differences in PBR were appreciated when resuscitation arms were compared longitudinally or to each other. Shelf-stable blood products preserved the endothelial glycocalyx similarly to CWB and pRBC/FFP as evaluated by sidestream dark field video microscopy. Lactated Ringer and HES solutions did not adequately preserve the endothelial glycocalyx compared to CWB and pRBC/FFP. Shelf-stable blood products are a viable option to preserve the endothelial glycocalyx when used during hemorrhagic resuscitation in dogs.

Flocculation of fiber suspensions studied by Rheo-OCT

When dealing with papermaking fiber suspensions, particle flocculation takes place even before the paper web is formed. The particle flocculation depends on several aspects, including particle mass concentration (consistency), particle collisions, electrochemical interactions promoted by chemical additives, etc. Due to its impor-tance, fiber suspension flocculation has been studied for a long time in papermaking, and several methods have been developed for this purpose. The traditional techniques include, for example, focused beam reflectance micros-copy (FBRM) and high-speed video imaging (HSVI). Recently, a new optical method, optical coherence tomography (OCT), has emerged for flocculation analysis. The advantages of OCT are the possibility to study opaque suspensions, its micron-level resolution, and its high data acquisition speed. The OCT measurements can be combined with rheological (Rheo) measurements, allowing simul-taneous measurement of both the time evolution of the floc size and the suspension viscosity. In this work, we used this approach, Rheo-OCT, to study the flocculation of suspensions of various papermaking furnishes. We analyzed the time evolution of the floc size and the fiber suspension viscosity when the studied paper-making suspensions were treated with highly refined furnish (HRF) — a furnish that contained a significant amount of micofibrillated cellulose (MFC)-type fibrils — and/or chemical additives. Such studies can lead to a better under-standing of the impact of flocculation on the produced paper web in terms of qualities like formation, drainage potential, and strength behavior.

Impact of porin deficiency on the synergistic potential of colistin in combination with β-lactam/β-lactamase inhibitors against ESBL- and carbapenemase-producing Klebsiella pneumoniae.

Combinations of colistin and β-lactam/β-lactamase inhibitors (BLBLIs) have shown in vitro synergy against β-lactamase-producing strains. However, data are limited and conflicting, potentially attributed to variations among the examined strains. This study investigated whether loss of porins OmpK35 and OmpK36 impacts the synergistic potential of colistin in combination with ceftazidime-avibactam or meropenem-avibactam against β-lactamase-producing Klebsiella pneumoniae. Genetically modified strains were constructed by introducing blaCTX-M-15, blaKPC-2, and blaOXA-48 chromosomally into K. pneumoniae ATCC 35657, in which the major porin-encoding genes (ompK35, ompK36) were either intact or knocked out. The in vitro activity of colistin in combination with ceftazidime-avibactam or meropenem-avibactam was evaluated by time-lapse microscopy screening and in static time-kill experiments. The deletion of porins in the β-lactamase-producing strains resulted in 2- to 128-fold increases in MICs for the β-lactams and BLBLIs. The activity of avibactam was concentration-dependent, and 4- to 16-fold higher concentrations were required to achieve similar inhibition of the β-lactamases in strains with porin loss. In the screening, synergy was observed for colistin and ceftazidime-avibactam against the CTX-M-15-producing strains and colistin and meropenem-avibactam against the KPC-2- and OXA-48-producing strains. The combination effects were less pronounced in the time-kill experiments, where synergy was rarely detected. No apparent associations were found between the loss of OmpK35 and OmpK36 and combination effects with colistin and BLBLIs, indicating that additional factors determine the synergistic potential of such combinations.

Visualizing the fine structure and dynamics of living cells with time-lapse polychromatic digital holographic microscopy

Visualizing the fine structure and dynamics of living cells with time-lapse polychromatic digital holographic microscopy

Visualization of membrane localization and the functional state of CB2R pools using matched agonist and inverse agonist probe pairs.

The diversity of physiological roles of the endocannabinoid system has turned it into an attractive yet elusive therapeutic target. However, chemical probes with various functionalities could pave the way for a better understanding of the endocannabinoid system at the cellular level. Notably, inverse agonists of CB2R - a key receptor of the endocannabinoid system - lagged behind despite the evidence regarding the therapeutic potential of its antagonism. Herein, we report a matched fluorescent probe pair based on a common chemotype to address and visualize both the active and inactive states of CB2R, selectively. Alongside extensive cross-validation by flow cytometry, time-lapse confocal microscopy, and super-resolution microscopy, we successfully visualize the intracellular localization of CB2R pools in live cells. The synthetic simplicity, together with the high CB2R-selectivity and specificity of our probes, turns them into valuable tools in chemical biology and drug development that can benefit the clinical translatability of CB2R-based drugs.

Inertial effect of cell state velocity on the quiescence-proliferation fate decision

Energy landscapes can provide intuitive depictions of population heterogeneity and dynamics. However, it is unclear whether individual cell behavior, hypothesized to be determined by initial position and noise, is faithfully recapitulated. Using the p21-/Cdk2-dependent quiescence-proliferation decision in breast cancer dormancy as a testbed, we examined single-cell dynamics on the landscape when perturbed by hypoxia, a dormancy-inducing stress. Combining trajectory-based energy landscape generation with single-cell time-lapse microscopy, we found that a combination of initial position and velocity on a p21/Cdk2 landscape, but not position alone, was required to explain the observed cell fate heterogeneity under hypoxia. This is likely due to additional cell state information such as epigenetic features and/or other species encoded in velocity but missing in instantaneous position determined by p21 and Cdk2 levels alone. Here, velocity dependence manifested as inertia: cells with higher cell cycle velocities prior to hypoxia continued progressing along the cell cycle under hypoxia, resisting the change in landscape towards cell cycle exit. Such inertial effects may markedly influence cell fate trajectories in tumors and other dynamically changing microenvironments where cell state transitions are governed by coordination across several biochemical species.

Self-inspired learning for denoising live-cell super-resolution microscopy.

Every collected photon is precious in live-cell super-resolution (SR) microscopy. Here, we describe a data-efficient, deep learning-based denoising solution to improve diverse SR imaging modalities. The method, SN2N, is a Self-inspired Noise2Noise module with self-supervised data generation and self-constrained learning process. SN2N is fully competitive with supervised learning methods and circumvents the need for large training set and clean ground truth, requiring only a single noisy frame for training. We show that SN2N improves photon efficiency by one-to-two orders of magnitude and is compatible with multiple imaging modalities for volumetric, multicolor, time-lapse SR microscopy. We further integrated SN2N into different SR reconstruction algorithms to effectively mitigate image artifacts. We anticipate SN2N will enable improved live-SR imaging and inspire further advances.

CCL2 may contribute to the damage of fallopian tube epithelium in women with tubal endometriosis

Research QuestionHow does tubal endometriosis (TEM) impact the morphology and ultrastructure of the fallopian tube epithelium? What are the underlying pathophysiological mechanisms of TEM? DesignEpithelial samples of the fallopian tubes from patients with (TEM group) and without (non-TEM group) TEM were collected. The morphological characteristics, ultrastructure, ciliated cell percentage, and ciliary beat frequency (CBF) were assessed via hematoxylin-eosin staining, electron microscopy, and high-speed video microscopy. mRNA microarray analysis was conducted to identify differentially expressed genes (DEGs) in the fallopian tube epithelium, which were further validated using qPCR, immunohistochemistry, and Western blotting. The effects of recombinant CCL2 protein on primary human fallopian tube epithelial cells (FTEC) were examined in vitro. ResultsPatients with TEM exhibited significant abnormalities in the morphology and ultrastructure of the fallopian tube epithelium with reduced percentage of ciliated cells and CBF, compared to the non-TEM patients. Among the 765 DEGs identified in the fallopian tube epithelium, 512 genes were upregulated and 253 genes were downregulated in the TEM group. qPCR, immunohistochemistry, and Western blotting validation confirmed a significant upregulation of CCL2 expression in the TEM group. In vitro experiments revealed that recombinant CCL2 protein significantly reduced ciliated cell differentiation, length of cilia, and CBF in FTEC, suggesting a role for CCL2 in the development of the TEM-related pathological phenotype. ConclusionsThese findings indicate that CCL2 may contribute to the pathological phenotype associated with TEM and could be a crucial signaling molecule in the damage of fallopian tube epithelium in patients with TEM.

Association between the lactate-albumin ratio and microcirculation changes in Pediatric Septic patients

A lactate/albumin ratio (LAR) greater than 0.5 measured early in the course of pediatric critical illness is associated with greater mortality. Whether the elevated LAR can be explained by microcirculation disorders in children with sepsis is not known. In this longitudinal retrospective study (January 2021-January 2024), serum albumin and lactate were measured on admission to the pediatric intensive care unit (PICU), with sublingual video microscopy performed simultaneously to measure microcirculation. A total of 178 children were included, 37% of whom had septic shock measured with the Phoenix Sepsis Score. Patients with remote sepsis had greater odds of an elevated LAR (aOR 6.87: 95% CI 1.98–23.73; p < 0.01). Children with an elevated LAR had more microvascular blood flow abnormalities (aOR 1.31 95% CI 1.08–1.58; p < 0.01), lower 4-6-micron capillary density (aOR 1.03 95% CI 1.01–1.05; p < 0.01) and greater odds of dying (aOR 3.55 95% CI 1.21–10.38; p = 0.02) compared to those with a low LAR. We found no association between LAR and endothelial glycocalyx degradation. A normal LAR is associated with less risk of microcirculatory injury (aOR 0.77 95% CI 0.65–0.93; p < 0.01). In children with sepsis, an elevated LAR is associated with microcirculation abnormalities (microvascular density and flow). The lactate/albumin ratio is a potentially useful biomarker for microcirculatory injury in sepsis.

Assessing the Effects of Surgical Irrigation Solutions on Human Neutrophil Interactions with Nascent Staphylococcus aureus Biofilms.

Staphylococcus aureus (S. aureus) is the leading cause of surgical site infections (SSIs) and is capable of biofilm growth on implanted foreign devices. The use of surgical irrigation solutions has become a common strategy to combat bacterial contamination events that occur during surgery. Despite their antimicrobial activity, SSI rates remain consistent, suggesting that low-level contamination persists. In these cases, circulating neutrophils must traffic from the blood to contamination sites to aid in bacterial clearance. The influence of irrigation solutions on neutrophils' ability to engage with bacteria has not been explored. The effects of three commonly used irrigation solutions: Xperience (sodium lauryl sulfate), Irrisept (chlorhexidine gluconate), and Betadine® (povidone-iodine) on nascent S. aureus biofilms alone and in the presence of human neutrophils were assessed at manufactured and diluted concentrations. All three solutions, at a 10% dilution, inhibited bacterial growth as demonstrated by culture assays and confocal video microscopy of bacterial aggregate formation. The effects of 10% dilutions of each of these solutions on neutrophil membrane integrity (by flow cytometry and propidium iodide staining) and motility (by confocal video microscopy of neutrophil track length) were investigated with differing outcomes for each irrigation solution. At this concentration only Irrisept preserved neutrophil membrane integrity and motility. Together, this study examines an overlooked aspect of surgical irrigation solutions by investigating their impact on innate immunity and highlights the feasibility of formulations wherein solution effectiveness is complemented by neutrophil function to reduce risks of infection.

Empagliflozin attenuates hypoxia-induced heart failure of zebrafish embryos via influencing MMP13 expression

BackgroundToday, sodium glucose co-transporter 2 (SGLT2) inhibitors are more than diabetes drugs. They are also indicated in chronic heart failure (HF) treatment in both diabetic and non-diabetic patients, independently of the ejection fraction. Multiple mechanisms have been suggested behind the cardioprotective effects of SGLT2 inhibitors. However, the underlying mechanisms still remain largely unexplored. Here, we used a zebrafish embryo model to search for new potential players whereby SGLT2 inhibitors attenuate HF. MethodsHF in zebrafish embryos was caused exposing them to chemically induced hypoxia. As a SGLT2 inhibitor, we used empagliflozin. Its effect on hypoxia-induced HF of the embryos was evaluated using video microscopy and calculation of fractional shortening (FS) of embryos´ hearts. RT-qPCR of brain natriuretic peptide (bnp) expression was also used to examine empagliflozin´s effect on HF. Transcriptome analysis of total RNA of the embryos was performed to search for new potential mechanisms contributing to the beneficial effect of empagliflozin on HF. ResultsEmpagliflozin significantly attenuated hypoxia-induced HF of zebrafish embryos as shown with improved FS of the hearts and decreased bnp expression. Transcriptome analysis revealed that the improvement of HF in response to empagliflozin was accompanied with decreased matrix metalloproteinase 13a (mmp13a) expression. Treatment of hypoxia-induced embryos with MMP13 inhibitor ameliorated the impaired heart function accordingly to the effect of empagliflozin. MMP13 inhibitor was not toxic to the embryos. ConclusionsOur study shows that empagliflozin´s favorable effect on attenuating HF is mediated via MMP13. MMP13 provides a novel option when developing new therapeutics for HF treatment.

The Innovative Role of Nuclear Receptor Interaction Protein in Orchestrating Invadosome Formation for Myoblast Fusion.

Nuclear receptor interaction protein (NRIP) is versatile and engages with various proteins to execute its diverse biological function. NRIP deficiency was reported to cause small myofibre size in adult muscle regeneration, indicating a crucial role of NRIP in myoblast fusion. The colocalization and interaction of NRIP with actin were investigated by immunofluorescence and immunoprecipitation assay, respectively. The participation of NRIP in myoblast fusion was demonstrated by cell fusion assay and time-lapse microscopy. The NRIP mutants were generated for mechanism study in NRIP-null C2C12 (termed KO19) cells and muscle-specific NRIP knockout (NRIP cKO) mice. A GEO profile database was used to analyse NRIP expression in Duchenne muscular dystrophy (DMD) patients. In this study, we found that NRIP directly and reciprocally interacted with actin both invitro and in cells. Immunofluorescence microscopy showed that the endogenous NRIP colocalized with components of invadosome, such as actin, Tks5, and cortactin, at the tips of cells during C2C12 differentiation. The KO19 cells were generated and exhibited a significant deficit in myoblast fusion compared with wild-type C2C12 cells (3.16% vs. 33.67%, p < 0.005). Overexpressed NRIP in KO19 cells could rescue myotube formation compared with control (3.37% vs. 1.00%, p < 0.01). We further confirmed that NRIP directly participated in cell fusion by using a cell-cell fusion assay. We investigated the mechanism of invadosome formation for myoblast fusion, which depends on NRIP-actin interaction, by analysing NRIP mutants in NRIP-null cells. Loss of actin-binding of NRIP reduced invadosome (enrichment ratio, 1.00 vs. 2.54, p < 0.01) and myotube formation (21.82% vs. 35.71%, p < 0.05) in KO19 cells and forced NRIP expression in KO19 cells and muscle-specific NRIP knockout (NRIP cKO) mice increased myofibre size compared with controls (over 1500 μm2, 61.01% vs. 20.57%, p < 0.001). We also found that the NRIP mRNA level was decreased in DMD patients compared with healthy controls (18 072 vs. 28 289, p < 0.001, N = 10 for both groups). NRIP is a novel actin-binding protein for invadosome formation to induce myoblast fusion.

Primary ciliary dyskinesia: a case report of double DNAH11 mutant alleles

Primary ciliary dyskinesia (PCD), a rare disorder, is genetically varied. Mutations in proteins involved in the structure, function, or assembly of cilia are known to determine situs inversus, male infertility, and chronic destructive airway disease. PCD is inherited by an autosomal recessive pattern of inheritance in most cases. Nonetheless, patterns of autosomal dominant and X-linked inheritance have been mentioned. A history of recurrent upper and lower respiratory tract infections raised clinical suspicion of primary ciliary dyskinesia in a 10-year-old patient. Genetic tests were performed using next-generation sequencing technology (Illumina NextGen) with the multiplex ligation-dependent probe amplification technique for primary ciliopathies and syndromes subject to differential diagnosis. Genetic testing identified two pathogenic variants, not previously associated with a case report in the literature, c.7727A&gt;G (p.Asp2576Gly) and c.8578G&gt;A (p.Gly2860Ser), within the DNAH11 gene, which is associated with autosomal recessive PCD. The result also reported mutations in other genes involved in autosomal recessive PCD (DNAH8, DNAH9 and ZMYND10), which were classified as variants with uncertain clinical significance. Transmission electron microscopy of respiratory cilia and nasal nitric oxide measurement cannot be used to diagnose PCD in patients with DNAH11 mutations because the structure of cilia is normal, and the levels of NO are not constantly low. High-speed video microscopy analysis can be helpful because DNAH11 mutations cause a distinct phenotype of PCD. Nevertheless, the mutation analysis of various PCD-causing genes remains the easiest to conduct and with good results. Genetic research on PCD has identified a number of significant ciliary genes in recent years, offering fresh perspectives on the molecular processes underlying cilia assembly and function. This facilitates the development of new methods for the diagnosis, prevention, and treatment of PCD. However, because it is a highly complex and heterogeneous disease, the field of gene diagnosis and therapy in PCD is still in its infancy.

Da_Tracker: Automated workflow for high throughput single cell and single phagosome tracking in infected cells.

Time-lapse microscopy has emerged as a crucial tool in cell biology, facilitating a deeper understanding of dynamic cellular processes. While existing tracking tools have proven effective in detecting and monitoring objects over time, the quantification of signals within these tracked objects often faces implementation constraints. In the context of infectious diseases, the quantification of signals at localized compartments within the cell and around intracellular pathogens can provide even deeper insight into the interactions between the pathogen and host cell organelles. Existing quantitative analysis at a single-phagosome level remains limited and dependent on manual tracking methods. We developed a near-fully automated workflow that performs with limited bias, high-throughput cell segmentation and quantitative tracking of both single cell and single bacterium/phagosome within multi-channel, z-stack, time-lapse confocal microscopy videos. We took advantage of the PyImageJ library to bring Fiji functionality into a Python environment and combined deep-learning-based segmentation from Cellpose with tracking algorithms from Trackmate. The 'da_tracker' workflow provides a versatile toolkit of functions for measuring relevant signal parameters at the single-cell level (such as velocity or bacterial burden) and at the single-phagosome level (i.e. assessment of phagosome maturation over time). Its capabilities in both single-cell and single-phagosome quantification, its flexibility and open-source nature should assist studies that aim to decipher for example the pathogenicity of bacteria and the mechanism of virulence factors that could pave the way for the development of innovative therapeutic approaches.

229 Awardee Talk: The quest for an ideal in-field embryo evaluation tool in cattle: Opportunities and challenges

Abstract Accurately evaluating bovine embryos to select the most viable ones for transfer and successful pregnancy has been a challenge since embryo transfer began in cattle over six decades ago. Despite more than 3,100 peer-reviewed studies on bovine embryo evaluation published in PubMed from 1964 to 2023, there is still no consensus or gold standard method for assessing their developmental potential. The evaluation “problem” in assisted reproduction extends beyond embryos to gametes as well. Numerous microscopic techniques (e.g., differential interference contrast, electron, fluorescent, time-lapse, and artificial intelligence-based microscopy) and non-microscopic methodologies (e.g., genomics, transcriptomics, epigenomics, proteomics, metabolomics, and nuclear magnetic resonance) have been tested to find an embryo evaluation technique superior to morphological evaluation. While many of these research tools can accurately determine embryo quality and viability, most are invasive, expensive, labor-intensive, technically complex, and time-consuming, making them impractical for in-field embryo evaluation. Employing complex methods like RNA sequencing, SNP chips, mass spectrometry, and multiphoton microscopy at thousands of embryo production and collection facilities is unrealistic, no matter how accurate they may be. Therefore, future research should focus on innovating field-friendly, simple benchtop tests using existing findings, particularly from omics-based research methodologies. Time-lapse monitoring and artificial intelligence-based automated image analysis also potentially provide accurate embryo evaluation. However, further research is necessary to develop economically feasible options for in-field applications. Our recent work has examined possible applications of nuclear magnetic resonance (NMR) imaging, label-free microscopy, particularly holographic microscopy techniques like gradient light interference microscopy (GLIM), spatial light interference microscopy (SLIM), and multiphoton holographic microscopy in IVF labs. The ideal embryo evaluation tool should be accurate, objective, non-invasive, affordable, and simple enough for widespread adoption by embryo production and collection facilities worldwide. Under current circumstances, it is doubtful that transcriptomics, proteomics, metabolomics, GLIM, SLIM, NMR, multiphoton holographic microscopy, and similar techniques can be directly used for in-field embryo evaluation. However, biomarkers identified by these methodologies could be used for in-field embryo evaluation by devising simple, affordable benchtop techniques. Further investigation on time-lapse microscopy (TLM) and AI-based automated image processing may make these methods more affordable and potentially adoptable by the masses. There is great potential for these methodologies to become widely used, provided they can be made more economically viable. In conclusion, the next generation of microscopy capable of providing objective markers for gamete and embryo quality is on the horizon.

CYTOSKELETON DYNAMICS AND SPATIAL ORGANIZATION DURING EPITHELIAL-TO-MESENCHYMAL TRANSITION

Epithelial-to-mesenchymal transition (EMT) is a crucial process that occurs during normal development, such as embryogenesis and organ formation. If it is inappropriately activated, it can lead to pathological processes like metastasis. Although EMT is well studied at the morphological and transcriptome level, cytoskeleton changes during this process are less understood. In our work we aimed to describe the morphological changes that occurred in MCF-7, A-549 and HaCaT cells after EMT, analyze microtubule dynamics, spatial distribution and its contribution to cell motility, identify changes in actin filament organization and study focal adhesion turnover in the cells after EMT. We hypothesized that the dynamics of microtubules in cells undergoing EMT might change. Cells undergoing EMT were expected to have more dynamic microtubules. Also, cells undergoing EMT are expected to adhere more efficiently to diverse substrates, and therefore spread and move more easily. Focal contacts in cells undergoing EMT were expected to be more pronounced and dynamic than in cells that not undergoing EMT. The research methods used in the study included inducing EMT in modified MCF-7 cells through an inserted inducible Tet-on system and stimulation of EMT in A-549 and HaCaT cells using TGF-β. The cells were observed using bright field microscopy, and immunofluorescence analysis was conducted to visualize microtubules and actin filaments. Transfection with EB-3–RFP protein was done to describe and measure microtubule dynamics, while transduction with Talin-RFP and transient transfection with Ptag-RFP-vinculin was done to visualize focal adhesions. Time-lapse fluorescent microscopy was used to record films, and the Fiji Image J program was used to analyze the data. All statistical analysis was performed using GraphPad Prism (Dotmatics, USA), and a nonparametric Mann-Whitney U test or parametric t-test with Welch correction. The actin filament measurements were completed using Matlab scripts. The major research findings: In all three-cell models after EMT, cell morphology changed. Cells increased in size. MCF-7 and HaCaT became spread out, while A-549 became elongated. Some of the cells lost cell-cell contacts. All three cell models after EMT had alterations in microtubule organization and dynamics. MCF-7 and HaCaT cells showed more frequently individual MTs at cell edges, while A-549 had less covered nucleus by MTs after EMT. Microtubule dynamics (growth velocity) increased, and the length of microtubule growth tracks became longer. The average angle of MT growth trajectories to cell radius decreased. Actin fibers rearranged into more pronounced stress fibers after EMT. Alterations in the focal adhesion behaviors were different in three models used. These results indicate that cytoskeletal changes during EMT mainly include increased microtubule dynamics.

Cell-Penetrating Peptides Translocate across the Plasma Membrane by Inducing Vesicle Budding and Collapse.

Cell-penetrating peptides (CPPs) enter the cell by two different mechanisms-endocytosis followed by endosomal escape and direct translocation at the plasma membrane. The mechanism of direct translocation remains unresolved. In this work, the direct translocation of nonaarginine (R9) and two cyclic CPPs (CPP12 and CPP17) into Jurkat cells was monitored by time-lapse confocal microscopy. Our results provide direct evidence that all three CPPs translocate across the plasma membrane by a recently discovered vesicle budding-and-collapse (VBC) mechanism. Membrane translocation is preceded by the formation of nucleation zones. Up to four different types of nucleation zones and three variations of the VBC mechanism were observed. The VBC mechanism reconciles the enigmatic and conflicting observations in the literature.

In vivo video microscopy of the rupturing process of thin blood vessels to clarify the mechanism of bruising caused by blunt impact: an animal study

BackgroundThe thresholds of mechanical inputs for bruising caused by blunt impact are important in the fields of machine safety and forensics. However, reliable data on these thresholds remain inadequate owing to a lack of in vivo experiments, which are crucial for investigating the occurrence of bruising. Since experiments involving live human participants are limited owing to ethical concerns, finite-element method (FEM) simulations of the bruising mechanism should be used to compensate for the lack of experimental data by estimating the thresholds under various conditions, which requires clarifying the mechanism of formation of actual bruises. Therefore, this study aimed to visualize the mechanism underlying the formation of bruises caused by blunt impact to enable FEM simulations to estimate the thresholds of mechanical inputs for bruising.MethodsIn vivo microscopy of a transparent glass catfish subjected to blunt contact with an indenter was performed. The fish were anesthetized by immersing them in buffered MS-222 (75–100 mg/L) and then fixed on a subject tray. The indenter, made of transparent acrylic and having a rectangular contact area with dimensions of 1.0 mm × 1.5 mm, was loaded onto the lateral side of the caudal region of the fish. Blood vessels and surrounding tissues were examined through the transparent indenter using a microscope equipped with a video camera. The contact force was measured using a force-sensing table.ResultsOne of the processes of rupturing thin blood vessels, which are an essential component of the bruising mechanism, was observed and recorded as a movie. The soft tissue surrounding the thin blood vessel extended in a plane perpendicular to the compressive contact force. Subsequently, the thin blood vessel was pulled into a straight configuration. Next, it was stretched in the axial direction and finally ruptured.ConclusionThe results obtained indicate that the extension of the surrounding tissue in the direction perpendicular to the contact force as well as the extension of the thin blood vessels are important factors in the bruising mechanism, which must be reproduced by FEM simulation to estimate the thresholds.

Gradient-induced instability in tumour spheroids unveils the impact of microenvironmental nutrient changes

Tumours often display invasive behaviours that induce fingering, branching and fragmentation processes. The phenomenon, known as diffusional instability, is driven by differential cell proliferation, migration, and death due to the presence of metabolite and catabolite concentration gradients. An understanding of the intricate dynamics of this spatially heterogeneous process plays a key role in the investigation of tumour growth and invasion. In this study, we developed an in vitro tumour invasion assay to investigate cell invasiveness in tumour spheroids under a chemotactic stimulus. Our method, employing tumour spheroids seeded in a 3D collagen gel within a microfluidic chemotaxis chamber, focuses on the role of diffusive gradients. Using Time-Lapse Microscopy, the dynamic evolution of tumour spheroids was monitored in real-time, providing a comprehensive view of the morphological changes and cell migration patterns under different chemotactic conditions. Specifically, we explored the impact of fetal bovine serum (FBS) gradients on the behaviour of CT26 mouse colon carcinoma cells and compared the effects of varying FBS concentrations to two isotropic control conditions. Furthermore, a finite element in silico model was developed to quantify the diffusive flow of nutrients in the chemotaxis chamber and obtain a detailed understanding of tumour dynamics. Our findings reveal that the presence of a chemotactic gradient significantly influences tumour invasiveness, with higher concentrations of nutrients associated with increased cancer growth and cell migration.