Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing.

Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca2+) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (- 2.94 to 0 mmH2O) and a pneumatic high-speed pressure clamp (- 25 to 0 mmHg). To visualize Ca2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca2+ levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.

Ultra-stable Biomembrane Force Probe for Accurately Determining Slow Dissociation Kinetics of PD-1 Blockade Antibodies on Single Living Cells.

Immune checkpoint blockade with monoclonal antibodies (mAbs) that target programmed cell death protein-1 (PD-1) has remarkably revolutionized cancer therapy. Their binding kinetics measured by surface plasmon resonance does not always correlate well with their immunotherapeutic efficacies, mainly due to the lack of two-dimensional cell plasma membrane and the capability of force sensing and manipulation. In this regard, based on a more suitable and ultra-sensitive biomechanical nanotool, biomembrane force probe (BFP), we developed a Double-edge Smart Feedback control system as an ultra-stable platform to characterize ultra-long bond lifetimes of receptor-ligand binding on living cells. We further benchmarked the dissociation kinetics for three clinically approved PD-1 blockade mAbs (Nivolumab, Pembrolizumab, and Camrelizumab), intriguingly correlating well with the objective response rates in the hepatocellular carcinoma second-line treatment. This ultra-stable BFP potentially provides a compelling kinetic platform to direct the screening, optimization, and clinical selection of therapeutic antibodies in the future.

Partial loss of actin nucleator actin-related protein 2/3 activity triggers blebbing in primary T lymphocytes.

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.

Evaluating Recalibrating AI Models for Breast Cancer Diagnosis in a New Context: Insights from Transfer Learning, Image Enhancement and High-Quality Training Data Integration.

This paper investigates the adaptability of four state-of-the-art artificial intelligence (AI) models to the Australian mammographic context through transfer learning, explores the impact of image enhancement on model performance and analyses the relationship between AI outputs and histopathological features for clinical relevance and accuracy assessment. A total of 1712 screening mammograms (n = 856 cancer cases and n = 856 matched normal cases) were used in this study. The 856 cases with cancer lesions were annotated by two expert radiologists and the level of concordance between their annotations was used to establish two sets: a 'high-concordances subset' with 99% agreement of cancer location and an 'entire dataset' with all cases included. The area under the receiver operating characteristic curve (AUC) was used to evaluate the performance of Globally aware Multiple Instance Classifier (GMIC), Global-Local Activation Maps (GLAM), I&H and End2End AI models, both in the pretrained and transfer learning modes, with and without applying the Contrast Limited Adaptive Histogram Equalization (CLAHE) algorithm. The four AI models with and without transfer learning in the high-concordance subset outperformed those in the entire dataset. Applying the CLAHE algorithm to mammograms improved the performance of the AI models. In the high-concordance subset with the transfer learning and CLAHE algorithm applied, the AUC of the GMIC model was highest (0.912), followed by the GLAM model (0.909), I&H (0.893) and End2End (0.875). There were significant differences (p < 0.05) in the performances of the four AI models between the high-concordance subset and the entire dataset. The AI models demonstrated significant differences in malignancy probability concerning different tumour size categories in mammograms. The performance of AI models was affected by several factors such as concordance classification, image enhancement and transfer learning. Mammograms with a strong concordance with radiologists' annotations, applying image enhancement and transfer learning could enhance the accuracy of AI models.

The iron chelator, deferasirox, as a novel strategy for cancer treatment: oral activity against human lung tumor xenografts and molecular mechanism of action.

Deferasirox is an orally effective iron (Fe) chelator currently used for the treatment of iron-overload disease and has been implemented as an alternative to the gold standard chelator, desferrioxamine (DFO). Earlier studies demonstrated that DFO exhibits anticancer activity due to its ability to deplete cancer cells of iron. In this investigation, we examined the in vitro and in vivo activity of deferasirox against cells from human solid tumors. To date, there have been no studies to investigate the effect of deferasirox on these types of tumors in vivo. Deferasirox demonstrated similar activity at inhibiting proliferation of DMS-53 lung carcinoma and SK-N-MC neuroepithelioma cell lines compared with DFO. Furthermore, deferasirox was generally similar or slightly more effective than DFO at mobilizing cellular (59)Fe and inhibiting iron uptake from human transferrin depending on the cell type. However, deferasirox potently inhibited DMS-53 xenograft growth in nude mice when given by oral gavage, with no marked alterations in normal tissue histology. To understand the antitumor activity of deferasirox, we investigated its effect on the expression of molecules that play key roles in metastasis, cell cycle control, and apoptosis. We demonstrated that deferasirox increased expression of the metastasis suppressor protein N-myc downstream-regulated gene 1 and upregulated the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) while decreasing cyclin D1 levels. Moreover, this agent increased the expression of apoptosis markers, including cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1. Collectively, we demonstrate that deferasirox is an orally effective antitumor agent against solid tumors.

4D-flow MRI derived wall shear stress for the risk stratification of bicuspid aortic valve aortopathy: A systematic review.

Purpose: Current intervention guidelines for bicuspid aortic valve (BAV) associated ascending aorta (AAo) dilatation are suboptimal predictors of clinical outcome. There is growing interest in identifying better biomarkers such as wall shear stress (WSS) to help risk stratify BAV aortopathy. The aim of the systematic review is to synthesize existing evidence of the relationship between WSS and aortopathy in the BAV population. Methods: A comprehensive literature search of available major databases was performed in May 2022 to include studies that used four-dimensional flow cardiac magnetic resonance (4D-flow) MRI to quantify WSS in the AAo in adult BAV populations. Summary results and statistical analysis were provided for key numerical results. A narrative summary was provided to assess similarities between studies. Results: A total of 26 studies that satisfied selection criteria and quality assessment were included in the review. The presence of BAV resulted in significantly elevated WSS magnitude and circumferential WSS, but not axial WSS. The presence of aortic stenosis had additional impact on WSS and flow alterations. BAV phenotypes were associated with different WSS distributions and flow profiles. Altered protein expression in the AAo wall associated with WSS supported the contribution of altered hemodynamics to aortopathy in addition to genetic factors. Conclusion: WSS has the potential to be a valid biomarker for BAV aortopathy. Future work would benefit from larger study cohorts with longitudinal evaluations to further characterize WSS association with aortopathy, mortality, and morbidities. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022337077, identifier CRD42022337077.

Hybrid HCNN-KNN Model Enhances Age Estimation Accuracy in Orthopantomography.

Age estimation in dental radiographs Orthopantomography (OPG) is a medical imaging technique that physicians and pathologists utilize for disease identification and legal matters. For example, for estimating post-mortem interval, detecting child abuse, drug trafficking, and identifying an unknown body. Recent development in automated image processing models improved the age estimation's limited precision to an approximate range of +/- 1 year. While this estimation is often accepted as accurate measurement, age estimation should be as precise as possible in most serious matters, such as homicide. Current age estimation techniques are highly dependent on manual and time-consuming image processing. Age estimation is often a time-sensitive matter in which the image processing time is vital. Recent development in Machine learning-based data processing methods has decreased the imaging time processing; however, the accuracy of these techniques remains to be further improved. We proposed an ensemble method of image classifiers to enhance the accuracy of age estimation using OPGs from 1 year to a couple of months (1-3-6). This hybrid model is based on convolutional neural networks (CNN) and K nearest neighbors (KNN). The hybrid (HCNN-KNN) model was used to investigate 1,922 panoramic dental radiographs of patients aged 15 to 23. These OPGs were obtained from the various teaching institutes and private dental clinics in Malaysia. To minimize the chance of overfitting in our model, we used the principal component analysis (PCA) algorithm and eliminated the features with high correlation. To further enhance the performance of our hybrid model, we performed systematic image pre-processing. We applied a series of classifications to train our model. We have successfully demonstrated that combining these innovative approaches has improved the classification and segmentation and thus the age-estimation outcome of the model. Our findings suggest that our innovative model, for the first time, to the best of our knowledge, successfully estimated the age in classified studies of 1 year old, 6 months, 3 months and 1-month-old cases with accuracies of 99.98, 99.96, 99.87, and 98.78 respectively.

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy.

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

Imaging and quantifying drug delivery in skin - Part 2: Fluorescence andvibrational spectroscopic imaging methods.

Understanding the delivery and diffusion of topically-applied drugs on human skin is of paramount importance in both pharmaceutical and cosmetics research. This information is critical in early stages of drug development and allows the identification of the most promising ingredients delivered at optimal concentrations to their target skin compartments. Different skin imaging methods, invasive and non-invasive, are available to characterize and quantify the spatiotemporal distribution of a drug within ex vivo and in vivo human skin. The first part of this review detailed invasive imaging methods (autoradiography, MALDI and SIMS). This second part reviews non-invasive imaging methods that can be applied in vivo: i) fluorescence (conventional, confocal, and multiphoton) and second harmonic generation microscopies and ii) vibrational spectroscopic imaging methods (infrared, confocal Raman, and coherent Raman scattering microscopies). Finally, a flow chart for the selection of imaging methods is presented to guide human skin ex vivo and in vivo drug delivery studies.

Imaging and quantifying drug delivery in skin - Part 1: Autoradiography and mass spectrometry imaging.

In this two-part review we present an up-to-date description of different imaging methods available to map the localization of drugs on skin as a complement of established ex-vivo absorption studies. This first part deals with invasive methods which are grouped in two classes according to their underlying principles: i) methods using radioactivity such as autoradiography and ii) mass spectrometry methods such as MALDI and SIMS. For each method, a description of the principle is given along with example applications of imaging and quantifying drug delivery in human skin. Thanks to these techniques a better assessment of the fate of drugs is obtained: its localization on a particular skin structure, its potential accumulation, etc. A critical comparison in terms of capabilities, sensitivity and practical applicability is included that will help the reader to select the most appropriate technique depending on the particular problem to be solved.

Research Techniques Made Simple: Two-Photon Intravital Imaging of the Skin.

Over the last few years, intravital two-photon microscopy has matured into a powerful technology helping basic and clinical researchers obtain quantifiable details of complex biological mechanisms in live and intact tissues. Two-photon microscopy provides high spatial and temporal resolution in vivo with little phototoxicity that is unattainable by other optical tools like confocal microscopy. Using ultrashort laser pulses, two-photon microscopy allows the visualization of molecules, cells, and extracellular structures up to depths of 1 mm within tissues. Consequently, real-time imaging of the individual skin layers under both physiological and pathological conditions has revolutionized our understanding of cutaneous homeostasis, immunity, and tumor biology. This review provides an overview to two-photon microscopy of the skin by covering the basic concepts and current applications in diverse preclinical and clinical settings.

Effect of surface chemistry on tropomyosin binding to actin filaments on surfaces.

Reconstitution of actin filaments on surfaces for observation of filament-associated protein dynamics by fluorescence microscopy is currently an exciting field in biophysics. Here we examine the effects of attaching actin filaments to surfaces on the binding and dissociation kinetics of a fluorescence-labeled tropomyosin, a rod-shaped protein that forms continuous strands wrapping around the actin filament. Two attachment modalities of the actin to the surface are explored: where the actin filament is attached to the surface at multiple points along its length; and where the actin filament is attached at one end and aligned parallel to the surface by buffer flow. To facilitate analysis of actin-binding protein dynamics, we have developed a software tool for the viewing, tracing and analysis of filaments and co-localized species in noisy fluorescence timelapse images. Our analysis shows that the interaction of tropomyosin with actin filaments is similar for both attachment modalities. © 2016 Wiley Periodicals, Inc.

Novel second-generation di-2-pyridylketone thiosemicarbazones show synergism with standard chemotherapeutics and demonstrate potent activity against lung cancer xenografts after oral and intravenous administration in vivo.

We developed a series of second-generation di-2-pyridyl ketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands to improve the efficacy and safety profile of these potential antitumor agents. Two novel DpT analogues, Dp4e4mT and DpC, exhibited pronounced and selective activity against human lung cancer xenografts in vivo via the intravenous and oral routes. Importantly, these analogues did not induce the cardiotoxicity observed at high nonoptimal doses of the first-generation DpT analogue, Dp44mT. The Cu(II) complexes of these ligands exhibited potent antiproliferative activity having redox potentials in a range accessible to biological reductants. The activity of the copper complexes of Dp4e4mT and DpC against lung cancer cells was synergistic in combination with gemcitabine or cisplatin. It was demonstrated by EPR spectroscopy that dimeric copper compounds of the type [CuLCl](2), identified crystallographically, dissociate in solution to give monomeric 1:1 Cu:ligand complexes. These monomers represent the biologically active form of the complex.

NKG2D and its ligands.

NKG2D is an activating immunoreceptor, first recognized on NK cells but subsequently found on gammadelta T cells, CD8(+) alphabeta T cells and macrophages. In NK cells, inhibitory signals are generally dominate over activating signals. However, activating signals mediated through engagement of NKG2D by its ligands on target cells can bypass signals transmitted through inhibitory NK receptors, allowing NKG2D to function as a "master-switch" in determining the activation status of NK cells. NKG2D is important for T cell and NK cell-mediated immunity to viruses and tumours, and has roles in autoimmune disease, allogeneic transplantation, and xenotransplantation. Depending upon the situation, development of strategies to either block or to enhance the interactions between NKG2D and its ligands may have important implications for human health and disease.