Discrete protein metric (DPM): A new image similarity metric to calculate accuracy of deep learning-generated cell focal adhesion predictions.

Understanding cell behaviors can provide new knowledge on the development of different pathologies. Focal adhesion (FA) sites are important sub-cellular structures that are involved in these processes. To better facilitate the study of FA sites, deep learning (DL) can be used to predict FA site morphology based on limited microscopic datasets (e.g., cell membrane images). However, calculating the accuracy score of these predictions can be challenging due to the discrete/point pattern like nature of FA sites. In the present work, a new image similarity metric, discrete protein metric (DPM), was developed to calculate FA prediction accuracy. This metric measures differences in distribution (d), shape/size (s), and angle (a) of FA sites between predicted and ground truth microscopy images. Performance of the DPM was evaluated by comparing it to three other commonly used image similarity metrics: Pearson correlation coefficient (PCC), feature similarity index (FSIM), and Intersection over Union (IoU). A sensitivity analysis was performed by comparing changes in each metric value due to quantifiable changes in FA site location, number, aspect ratio, area, or orientation. Furthermore, accuracy score of DL-generated predictions was calculated using all four metrics to compare their ability to capture variation across samples. Results showed better sensitivity and range of variation for DPM compared to the other metrics tested. Most importantly, DPM had the ability to determine which FA predictions were quantitatively more accurate and consistent with qualitative assessments. The proposed DPM hence provides a method to validate DL-generated FA predictions and has the potential to be used for investigation of other sub-cellular protein aggregates relevant to cell biology.

Human dermal microvascular endothelial cell morphological response to fluid shear stress.

As the cells that line the vasculature, endothelial cells are continually exposed to fluid shear stress by blood flow. Recent studies suggest that the morphological response of endothelial cells to fluid shear stress depends on the endothelial cell type. Thus, the present study characterizes the morphological response of human dermal microvascular endothelial cells (HMEC-1) and nuclei to steady, laminar, and unidirectional fluid shear stress. Cultured HMEC-1 monolayers were exposed to shear stress of 0.3 dyn/cm2, 16 dyn/cm2, or 32 dyn/cm2 for 72 h with hourly live-cell imaging capturing both the nuclear and cellular morphology. Despite changes in elongation and alignment occurring with increasing fluid shear stress, there was a lack of elongation and alignment over time under each fluid shear stress condition. Conversely, changes in cellular and nuclear area exhibited dependence on both time and fluid shear stress magnitude. The trends in cellular morphology differed at shear stress levels above and below 16 dyn/cm2, whereas the nuclear orientation was independent of fluid shear stress magnitude. These findings show the complex morphological response of HMEC-1 to fluid shear stress.

In situ fixation and subsequent collection of cultured endothelial cells in a shear flow.

In situ fixation of adherent cells is a necessary process for downstream assays. Current methods to dissociate adherent endothelial cells require the use of a cell scraper that may introduce variability in nuclear morphology. Also, a cell scraper is not an option for experiments using sealed flow chambers. HMEC-1 cells were sheared at 5 dyn/cm2 for 24 h and then fixed in situ, quenched, and dissociated at the same shear rate. Analysis revealed no statistically significant change in nuclear shape between the steps of fixation and dissociation. This method outlines an alternative for the dissociation of adherent sheared endothelial cells after being fixed in situ in a micro-scale channel without causing a change in the nuclear morphology. •This method can be used with any commercially available, or custom-made, flow chamber and flow system.•Allows for downstream experimentation with adherent cells fixed in situ, such as Hi-C analysis, without impacting nuclear morphology or chromatin organization.•Cells are cultured, fixed, and dissociated at the same shear rate. Using the same shear rate for each step yields results that are not influenced by variable forces.

Migration through a small pore disrupts inactive chromatin organization in neutrophil-like cells.

BACKGROUND: Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. The nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through confined spaces, where the nuclear shape must change in order to fit through a constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration. Here we characterized the effects of constricted migration in neutrophil-like cells. RESULTS: Total RNA sequencing identified that migration of neutrophil-like cells through 5- or 14-µm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeletal remodeling. Hi-C was used to capture the genome organization in control and migrated cells. Limited switching was observed between the active (A) and inactive (B) compartments after migration. However, global depletion of short-range contacts was observed following migration with constriction compared to migration without constriction. Regions with disrupted contacts, TADs, and compartments were enriched for inactive chromatin. CONCLUSION: Short-range genome organization is preferentially altered in inactive chromatin, possibly protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in relation to human health and disease.

Serum levels of endothelial glycocalyx constituents in women at 20†weeks' gestation who later develop gestational diabetes mellitus compared to matched controls: a pilot study.

OBJECTIVES: The aim of this pilot study was to determine the serum concentration of heparan sulfate, hyaluronan, chondroitin sulfate and syndecan-1 and if these serum concentrations can be used to identify women at 20 weeks' gestation who later develop gestational diabetes mellitus (GDM). DESIGN: Nested case-control study from Auckland, New Zealand participants in the prospective cohort Screening for Pregnancy Endpoints study. SETTING: Auckland, New Zealand. PARTICIPANTS: 20 pregnant women (70% European, 15% Indian, 10% Asian, 5% Pacific Islander) at 20 weeks' gestation without any hypertensive complications who developed GDM by existing New Zealand criteria defined as a fasting glucose ≥5.5 mmol/L and/or 2 hours ≥9.0 mmol/L after a 75 g Oral Glucose Tolerance Test. Women not meeting these criteria were excluded from this study. The patients with GDM were matched with 20 women who had uncomplicated pregnancies and negative screening for GDM and matched for ethnicity, maternal age and BMI. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary measures were the serum concentrations of syndecan-1, heparan sulfate, hyaluronan and chondroitin sulfate determined by quantitative ELISA. There were no secondary outcome measures. RESULTS: Binary logistic regression was performed to determine if serum concentrations of endothelial glycocalyx layer constituents in women at 20 weeks' gestation would be useful in predicting the subsequent diagnosis of GDM. The model was not statistically significant χ2=12.5, df=8, p=0.13, which indicates that the model was unable to distinguish between pregnant women at 20 weeks' gestation who later developed GDM and those who did not. CONCLUSIONS: Serum concentrations of syndecan-1, heparan sulfate, hyaluronan and chondroitin sulfate in pregnant women at 20 weeks' gestation were not associated with later development of GDM. To further explore whether there is any relationship between endothelial glycocalyx constituents and GDM, the next step is to evaluate serum concentrations at the time diagnosis of GDM.

A potential role for genome structure in the translation of mechanical force during immune cell development.

Immune cells react to a wide range of environments, both chemical and physical. While the former has been extensively studied, there is growing evidence that physical and in particular mechanical forces also affect immune cell behavior and development. In order to elicit a response that affects immune cell behavior or development, environmental signals must often reach the nucleus. Chemical and mechanical signals can initiate signal transduction pathways, but mechanical forces may also have a more direct route to the nucleus, altering nuclear shape via mechanotransduction. The three-dimensional organization of DNA allows for the possibility that altering nuclear shape directly remodels chromatin, redistributing critical regulatory elements and proteins, and resulting in wide-scale gene expression changes. As such, integrating mechanotransduction and genome architecture into the immunology toolkit will improve our understanding of immune development and disease.

Culture and detection of primary cilia in endothelial cell models.

BACKGROUND: The primary cilium is a sensor of blood-induced forces in endothelial cells (ECs). Studies that have examined EC primary cilia have reported a wide range of cilia incidence (percentage of ciliated cells). We hypothesise that this variation is due to the diversity in culture conditions in which the cells are grown. We studied two EC types: human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Both cell types were grown in media containing foetal bovine serum (FBS) at high (20 % FBS and 10 % FBS for HUVECs and HMEC-1s, respectively) or low (2 % FBS) concentrations. Cells were then either fixed at confluence, serum-starved or grown post-confluence for 5 days in corresponding expansion media (cobblestone treatment). For each culture condition, we quantified cilia incidence and length. RESULTS: HUVEC ciliogenesis is dependent on serum concentration during the growth phase; low serum (2 % FBS) HUVECs were not ciliated, whereas high serum (20 % FBS) confluent HUVECs have a cilia incidence of 2.1 ± 2.2 % (median ± interquartile range). We report, for the first time, the presence of cilia in the HMEC-1 cell type. HMEC-1s have between 2.2 and 3.5 times greater cilia incidence than HUVECs (p < 0.001). HMEC-1s also have shorter cilia compared to HUVECs (3.0 ± 1.0 µm versus 5.1 ± 2.4 µm, at confluence, p = 0.003). CONCLUSIONS: We demonstrate that FBS plays a role in determining the prevalence of cilia in HUVECs. In doing so, we highlight the importance of considering a commonly varied parameter (% FBS), in the experimental design. We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs. For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.

Computational models of the primary cilium and endothelial mechanotransmission.

In endothelial cells (ECs), the mechanotransduction of fluid shear stress is partially dependent on the transmission of force from the fluid into the cell (mechanotransmission). The role of the primary cilium in EC mechanotransmission is not yet known. To motivate a framework towards quantifying cilia contribution to EC mechanotransmission, we have reviewed mechanical models of both (1) the primary cilium (three-dimensional and lower-dimensional) and (2) whole ECs (finite element, non-finite element, and tensegrity). Both the primary cilia and whole EC models typically incorporate fluid-induced wall shear stress and spatial geometry based on experimentally acquired images of cells. This paper presents future modelling directions as well as the major goals towards integrating primary cilium models into a multi-component EC mechanical model. Finally, we outline how an integrated cilium-EC model can be used to better understand mechanotransduction in the endothelium.

Microscope-based near-infrared stereo-imaging system for quantifying the motion of the murine epicardial coronary arteries in vivo.

Atherosclerosis is a leading cause of mortality in industrialized countries. In addition to "traditional" systemic risk factors for atherosclerosis, the geometry and motion of coronary arteries may contribute to individual susceptibility to the development and progression of disease in these vessels. To be able to test this, we have developed a high-speed (∼40 frames per second) microscope-based stereo-imaging system to quantify the motion of epicardial coronary arteries of mice. Using near-infrared nontargeted quantum dots as an imaging contrast agent, we synchronously acquired paired images of a surgically exposed murine heart, from which the three-dimensional geometry of the coronary arteries was reconstructed. The reconstructed geometry was tracked frame by frame through the cardiac cycle to quantify the in vivo motion of the vessel, from which displacements, curvature, and torsion parameters were derived. Illustrative results for a C57BL/6J mouse are presented.

Characterizing 3-D geometry of mouse aortic arch using light stereo-microscopic imaging.

The vascular geometry may play an important role in the development of atherosclerosis by modulating the local hemodynamics and mechanical stresses of the vessel wall. The mouse is now the most popular animal model to study cardiovascular disease. Here, we present a method to characterize the 3-D geometry of mouse aortic arches by casting and light stereo-microscopic imaging. After calibration of the stereo-microscopic imaging system, the 3-D axis of an aortic cast is reconstructed using two stereo images. Based on the analysis of this 3-D curve, a geometry-based definition of the arch segment is given, and some geometric features, including curvature, torsion, and symmetry, have been derived. Casts from a C57BL6 and a SVEV mouse have been processed. This method will be used to detect the geometric difference of the aortic arch among different mouse strains.

Microviscometry reveals reduced blood viscosity and altered shear rate and shear stress profiles in microvessels after hemodilution.

We show that many salient hemodynamic flow properties, which have been difficult or impossible to assess in microvessels in vivo, can be estimated by using microviscometry and fluorescent microparticle image velocimetry in microvessels >20 microm in diameter. Radial distributions in blood viscosity, shear stress, and shear rate are obtained and used to predict axial pressure gradient, apparent viscosity, and endothelial-cell surface-layer thickness in vivo. Based solely on microparticle image velocimetry data, which are readily obtainable during the course of most intravital microscopy protocols from systemically injected particle tracers, we show that the microviscometric method consistently predicted a reduction in local and apparent blood viscosity after isovolemic hemodilution. Among its clinical applications, hemodilution is a procedure that is used to treat various pathologies that require reduction in peripheral vascular-flow resistance. Our results are directly relevant in this context because they suggest that the fractional decrease in systemic hematocrit is approximately 25-35% greater than the accompanying fractional decrease in microvascular-flow resistance in vivo. In terms of its fundamental usefulness, the microviscometric method provides a comprehensive quantitative analysis of microvascular hemodynamics that has applications in broad areas of medicine and physiology and is particularly relevant to quantitative studies of angiogenesis, tumor growth, leukocyte adhesion, vascular-flow resistance, tissue perfusion, and endothelial-cell mechanotransduction.

Near-wall micro-PIV reveals a hydrodynamically relevant endothelial surface layer in venules in vivo.

High-resolution near-wall fluorescent microparticle image velocimetry (micro-PIV) was used in mouse cremaster muscle venules in vivo to measure velocity profiles in the red cell-depleted plasma layer near the endothelial lining. micro-PIV data of the instantaneous translational speeds and radial positions of fluorescently labeled microspheres (0.47 microm) in an optical section through the midsagittal plane of each vessel were used to determine fluid particle translational speeds. Regression of a linear velocity distribution based on near-wall fluid-particle speeds consistently revealed a negative intercept when extrapolated to the vessel wall. Based on a detailed three-dimensional analysis of the local fluid dynamics, we estimate a mean effective thickness of approximately 0.33 micro m for an impermeable endothelial surface layer or approximately 0.44 micro m assuming the lowest hydraulic resistivity of the layer that is consistent with the observed particle motions. The extent of plasma flow retardation through the layer required to be consistent with our micro-PIV data results in near complete attenuation of fluid shear stress on the endothelial-cell surface. These findings confirm the presence of a hydrodynamically effective endothelial surface layer, and emphasize the need to revise previous concepts of leukocyte adhesion, stress transmission to vascular endothelium, permeability, and mechanotransduction mechanisms.