Bovine Pulmonary Artery Endothelial Cells Research Articles

Oxidant-Dependent Sensitizing, Protective, and Mitigative Effects in X-Ray-Irradiated Pulmonary Endothelial Cells.

The primary response of proliferating bovine pulmonary artery endothelial cells (BPAECs) after X-ray irradiation [≤10 gray (Gy)] is shown to be transient cell-cycle arrest. Accompanying oxidant-linked functional changes within the mitochondria are readily measured, but increased autophagy is not. Radiation-induced apoptosis is negligible in this line-important because cells undergoing apoptosis release oxygen-derived species that can overwhelm/mask the radiation-associated species and their effects that we wish to investigate. Cells irradiated and cultured at 3% oxygen exhibited delayed cell-cycle arrest (6-8 hours after 10 Gy irradiation) compared with those maintained at 20% oxygen (2-4 hours after 10 Gy irradiation). At 3% oxygen, either only during or only after irradiation, results intermediate between 20% and 3% oxygen throughout were obtained. No variability in cell-cycle distribution was observed for unirradiated cells cultured under different prevailing oxygen levels. Mitochondrially localized manganese superoxide dismutase delayed the X-ray-induced cell-cycle changes when over-expressed in BPAEC, indicating superoxide to be one of the key oxygen-derived cytotoxic species involved in the radiobiological response. Also, the peroxynitrite biomarker 3-nitrotyrosine was elevated, whereas hydrogen peroxide levels were not. Lastly, the utility of the BPAEC for screening potential countermeasures to ionizing radiation is demonstrated with some quinoline derivatives. Three of the five compounds appeared mitigative, and all were protective. It is suggested that the oxidation-reduction chemistry of these compounds probably offers a reasonable explanation for their observed ameliorative properties. Furthermore, the results suggest a promising new direction in the search for lead compounds as countermeasures to the effects of ionizing radiation. SIGNIFICANCE STATEMENT: The primary radiological response of proliferating bovine pulmonary artery endothelial cells is cell-cycle arrest, starting soon after X-ray irradiation (1-10 Gy) at 20% O2 but delayed by 4 hours at systemic (3%) O2. Oxygen/superoxide is found to be radio-sensitizing in at least two distinct time windows, during and after the irradiation, with both responses antagonized by various hydroxyquinoline derivatives. Similar responses in many other cell lines are likely to be masked by elevated oxidants associated with apoptosis.

Fabrication of a multifaceted mapping mirror using two-photon polymerization for a snapshot image mapping spectrometer.

A design and fabrication technique for making high-precision and large-format multifaceted mapping mirrors is presented. The method is based on two-photon polymerization, which allows more flexibility in the mapping mirror design. The mirror fabricated in this paper consists of 36 2D tilted square pixels, instead of the continuous facet design used in diamond cutting. The paper presents a detailed discussion of the fabrication parameters and optimization process, with particular emphasis on the optimization of stitching defects by compensating for the overall tilt angle and reducing the printing field of view. The fabricated mirrors were coated with a thin layer of aluminum (93nm) using sputter coating to enhance the reflection rate over the target wave range. The mapping mirror was characterized using a white light interferometer and a scanning electron microscope, which demonstrates its optical quality surface (with a surface roughness of 12nm) and high-precision tilt angles (with an average of 2.03% deviation). Finally, the incorporation of one of the 3D printed mapping mirrors into an image mapping spectrometer prototype allowed for the acquisition of high-quality images of the USAF resolution target and bovine pulmonary artery endothelial cells stained with three fluorescent dyes, demonstrating the potential of this technology for practical applications.

Resolution Improvement of Structured Illumination Microscopy using Non‐Diffraction‐Limited Point‐Scanned Fluorescence Fringe

AbstractTo further improve the resolving ability of structured illumination microscopy (SIM), a space‐time‐modulation–based laser‐scanning structured illumination microscopy (LSSIM) technique is presented in which a non‐diffraction‐limited non‐sinusoidal cumulative fluorescence fringe is generated in samples by point scanning the modulated excitation beam. The higher‐frequency information of samples can be mixed using the cumulative fringes with unlimited frequency into the passband of a confocal system. The non‐sinusoidal intensity‐modulated function used in LSSIM is specifically designed as a combination of cosine functions with different orders. The resolving ability of LSSIM experimentally by imaging fluorescent beads and bovine pulmonary artery endothelial cells with stained F‐actin is demonstrated. The results demonstrate three‐fold resolution enhancement over the confocal system, surpassing the resolving ability of conventional SIM. Moreover, the optical system is a simple modification of laser‐scanning confocal microscopy, making LSSIM more compatible with other techniques and suitable for biomedical research without the specific requirements for fluorescent dyes.

Optical projection tomography of fluorescent microscopic specimens using lateral translation of tube lens.

Optical projection tomography (OPT) is a three-dimensional (3D) fluorescence imaging technique, in which projection images are acquired for varying orientations of a sample using a large depth of field. OPT is typically applied to a millimeter-sized specimen, because the rotation of a microscopic specimen is challenging and not compatible with live cell imaging. In this Letter, we demonstrate fluorescence optical tomography of a microscopic specimen by laterally translating the tube lens of a wide-field optical microscope, which allows for high-resolution OPT without rotating the sample. The cost is the reduction of the field of view to about halfway along the direction of the tube lens translation. Using bovine pulmonary artery endothelial cells and 0.1 µm beads, we compare the 3D imaging performance of the proposed method with that of the conventional objective-focus scan method.

Growth hormone-releasing hormone antagonists counteract hydrogen peroxide – induced paracellular hyperpermeability in endothelial cells

Growth Hormone-Releasing Hormone (GHRH) is a hypothalamic peptide which regulates the release of Growth Hormone from the anterior pituitary gland, and has been involved in inflammatory processes. On the other hand, GHRH antagonists (GHRHAnt) were developed to counteract those effects. Herein we demonstrate for the first time that GHRHAnt can suppress hydrogen peroxide (H2O2) - induced paracellular hyperpermeability in bovine pulmonary artery endothelial cells. Increased production of reactive oxygen species (ROS) and barrier dysfunction have been associated with the development of potentially lethal disorders, including sepsis and acute respiratory distress syndrome (ARDS). Our study supports the protective actions of GHRHAnt in the impaired endothelium, and suggests that those compounds represent an exciting therapeutic possibility towards lung inflammatory disease.

Growth hormone-releasing hormone antagonists protect against hydrochloric acid-induced endothelial injury in vitro

Growth hormone‐releasing hormone (GHRH) regulates the synthesis of growth hormone from the anterior pituitary gland, and it is involved in inflammatory responses. On the other hand, GHRH antagonists (GHRHAnt) exhibit the opposite effects, resulting in endothelial barrier enhancement. Exposure to hydrochloric acid (HCL) is associated with acute and chronic lung injury. In this study, we investigate the effects of GHRHAnt in HCL-induced endothelial barrier dysfunction, utilizing commercially available bovine pulmonary artery endothelial cells (BPAEC). Cell viability was measured by utilizing 3-(4,5-dimethylthiazol2-yl)− 2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, fluorescein isothiocyanate (FITC)-dextran was used to assess barrier function. Our observations suggest that GHRHAnt exert protective effects against HCL-induced endothelial breakdown, since those peptides counteract HCL-triggered paracellular hyperpermeability. Based on those findings, we propose that GHRHAnt represent a new therapeutic approach towards HCL-induced endothelial injury.

Small training dataset convolutional neural networks for application-specific super-resolution microscopy.

Machine learning (ML) models based on deep convolutional neural networks have been used to significantly increase microscopy resolution, speed [signal-to-noise ratio (SNR)], and data interpretation. The bottleneck in developing effective ML systems is often the need to acquire large datasets to train the neural network. We demonstrate how adding a "dense encoder-decoder" (DenseED) block can be used to effectively train a neural network that produces super-resolution (SR) images from conventional microscopy diffraction-limited (DL) images trained using a small dataset [15 fields of view (FOVs)]. The ML helps to retrieve SR information from a DL image when trained with a massive training dataset. The aim of this work is to demonstrate a neural network that estimates SR images from DL images using modifications that enable training with a small dataset. We employ "DenseED" blocks in existing SR ML network architectures. DenseED blocks use a dense layer that concatenates features from the previous convolutional layer to the next convolutional layer. DenseED blocks in fully convolutional networks (FCNs) estimate the SR images when trained with a small training dataset (15 FOVs) of human cells from the Widefield2SIM dataset and in fluorescent-labeled fixed bovine pulmonary artery endothelial cells samples. Conventional ML models without DenseED blocks trained on small datasets fail to accurately estimate SR images while models including the DenseED blocks can. The average peak SNR (PSNR) and resolution improvements achieved by networks containing DenseED blocks are and , respectively. We evaluated various configurations of target image generation methods (e.g., experimentally captured a target and computationally generated target) that are used to train FCNs with and without DenseED blocks and showed that including DenseED blocks in simple FCNs outperforms compared to simple FCNs without DenseED blocks. DenseED blocks in neural networks show accurate extraction of SR images even if the ML model is trained with a small training dataset of 15 FOVs. This approach shows that microscopy applications can use DenseED blocks to train on smaller datasets that are application-specific imaging platforms and there is promise for applying this to other imaging modalities, such as MRI/x-ray, etc.

Classic Phytochemical Antioxidant and Lipoxygenase Inhibitor, Nordihydroguaiaretic Acid, Activates Phospholipase D through Oxidant Signaling and Tyrosine Phosphorylation Leading to Cytotoxicity in Lung Vascular Endothelial Cells.

Nordihydroguaiaretic acid (NDGA), a dicatechol and phytochemical polyphenolic antioxidant and an established inhibitor of human arachidonic acid (AA) 5-lipoxygenase (LOX) and 15-LOX, is widely used to ascertain the role of LOXs in vascular endothelial cell (EC) function. As the modulatory effect of NDGA on phospholipase D (PLD), an important lipid signaling enzyme in ECs, thus far has not been reported, here we have investigated the modulation of PLD activity and its regulation by NDGA in the bovine pulmonary artery ECs (BPAECs). NDGA induced the activation of PLD (phosphatidic acid formation) in cells in a dose- and time-dependent fashion that was significantly attenuated by iron chelator and antioxidants. NDGA induced the formation of reactive oxygen species (ROS) in cells in a dose- and time-dependent manner as evidenced from fluorescence microscopy and fluorimetry of ROS and electron paramagnetic resonance spectroscopy of oxygen radicals. Also, NDGA caused a dose-dependent loss of intracellular glutathione (GSH) in BPAECs. Protein tyrosine kinase (PTyK)-specific inhibitors significantly attenuated NDGA-induced PLD activation in BPAECs. NDGA also induced a dose- and time-dependent phosphorylation of tyrosine in proteins in cells. NDGA caused in situ translocation and relocalization of both PLD1 and PLD2 isoforms, in a time-dependent fashion. Cyclooxygenase (COX) inhibitors were ineffective in attenuating NDGA-induced PLD activation in BPAECs, thus ruling out the activation of COXs by NDGA. NDGA inhibited the AA-LOX activity and leukotriene C4 (LTC4) formation in cells. On the other hand, the 5-LOX-specific inhibitors, 5, 8, 11, 14-eicosatetraynoic acid and kaempferol, were ineffective in activating PLD in BPAECs. Antioxidants and PTyK-specific inhibitors effectively attenuated NDGA cytotoxicity in BPAECs. The PLD-specific inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI), significantly attenuated and protected against the NDGA-induced PLD activation and cytotoxicity in BPAECs. For the first time, these results demonstrated that NDGA, the classic phytochemical polyphenolic antioxidant and LOX inhibitor, activated PLD causing cytotoxicity in ECs through upstream oxidant signaling and protein tyrosine phosphorylation.

Protective effects of GHRH antagonists against hydrogen peroxide-induced lung endothelial barrier disruption.

PurposeGrowth hormone-releasing hormone (GHRH) is a hypothalamic hormone, which regulates growth hormone release from the anterior pituitary gland. GHRH antagonists (GHRHAnt) are anticancer agents, which also exert robust anti-inflammatory activities in malignancies. GHRHAnt exhibit anti-oxidative and anti-inflammatory effects in vascular endothelial cells, indicating their potential use against disorders related to barrier dysfunction (e.g. sepsis). Herein, we aim to investigate the effects of GHRHAnt against lung endothelial hyperpermeability.MethodsThe in vitro effects of GHRHAnt in H2O2-induced endothelial barrier dysfunction were investigated in bovine pulmonary artery endothelial cells (BPAEC). Electric cell-substrate impedance sensing (ECIS) was utilized to measure transendothelial resistance, an indicator of barrier function.ResultsOur results demonstrate that GHRHAnt protect against H2O2-induced endothelial barrier disruption via P53 and cofilin modulation. Both proteins are crucial modulators of vascular integrity. Moreover, GHRHAnt prevent H2O2 – induced decrease in transendothelial resistance.ConclusionsGHRHAnt represent a promising therapeutic intervention towards diseases related to lung endothelial hyperpermeability, such as acute respiratory distress syndrome - related or not to COVID-19 - and sepsis. Targeted medicine for those potentially lethal disorders does not exist.

Activating transcription factor 6 protects against endothelial barrier dysfunction

BackgroundEndothelial hyperpermeability is associated with sepsis and acute respiratory distress syndrome (ARDS). The identification of molecular pathways involved in barrier dysfunction; may reveal promising therapeutic targets to combat ARDS. Unfolded protein response (UPR) is a highly conserved molecular pathway, which ameliorates endoplasmic reticulum stress. The present work focuses on the effects of ATF6, which is a UPR sensor, in lipopolysaccharides (LPS)-induced endothelial hyperpermeability. MethodsThe in vitro effects of AA147 and Ceapin-A7 in LPS-induced endothelial barrier dysfunction were investigated in bovine pulmonary artery endothelial cells (BPAEC). Small interfering (si) RNA was utilized to “silence” ATF6, and electric cell-substrate impedance sensing (ECIS) measured transendothelial resistance. Fluorescein isothiocyanate (FITC)-dextran assay was utilized to assess paracellular permeability. Protein expression levels were evaluated with Western blotting, and cell viability with MTT assay. ResultsWe demonstrated that AA147 prevents LPS-induced barrier disruption by counteracting Cofilin and myosin light chain 2 (MLC2) activation, as well as VE-Cadherin phosphorylation. Moreover, this ATF6 inducer opposed LPS-triggered decrease in transendothelial resistance (TEER), as well as LPS-induced paracellular hyperpermeability. On the other hand, ATF6 suppression due to Ceapin-A7 or small interfering RNA exerted the opposite effects, and potentiated LPS-induced endothelial barrier disruption. Moderate concentrations of both ATF6 modulators did not affect cell viability. ConclusionsATF6 activation protects against endothelial barrier function, suggesting that this UPR sensor may serve as a therapeutic target for sepsis and ARDS.

Evaluation and optimization of PolyJet 3D-printed materials for cell culture studies.

Use of 3D printing for microfluidics is a rapidly growing area, with applications involving cell culture in these devices also becoming of interest. 3D printing can be used to create custom-designed devices that have complex features and integrate different material types in one device; however, there are fewer studies studying the ability to culture cells on the various substrates that are available. This work describes the effect of PolyJet 3D-printing technology on cell culture of two cell lines, bovine pulmonary artery endothelial cells (BPAECs) and Madin-Darby Canine Kidney (MDCK) cells, on two different types of printed materials (VeroClear or MED610). It was found that untreated devices, when used for studies of 1day or more, led to unsuccessful culture. A variety of device treatment methodologies were investigated, with the most success coming from the use of sodium hydroxide/sodium metasilicate solution. Devices treated with this cleaning step resulted in culture of BPAECs and MDCK cells that were more similar to what is obtained in traditional culture flasks (in terms of cell morphology, viability, and cell density). LC-MS/MS analysis (via Orbitrap MS) was used to determine potential leachates from untreated devices. Finally, the use of a fiber scaffold in the devices was utilized to further evaluate the treatment methodology and to also demonstrate the ability to perform 3D culture in such devices. This study will be of use for researchers wanting to utilize these or other cell types in PolyJet-based 3D-printed devices.

Deep-learning-assisted Fourier transform imaging spectroscopy for hyperspectral fluorescence imaging

Hyperspectral fluorescence imaging is widely used when multiple fluorescent probes with close emission peaks are required. In particular, Fourier transform imaging spectroscopy (FTIS) provides unrivaled spectral resolution; however, the imaging throughput is very low due to the amount of interferogram sampling required. In this work, we apply deep learning to FTIS and show that the interferogram sampling can be drastically reduced by an order of magnitude without noticeable degradation in the image quality. For the demonstration, we use bovine pulmonary artery endothelial cells stained with three fluorescent dyes and 10 types of fluorescent beads with close emission peaks. Further, we show that the deep learning approach is more robust to the translation stage error and environmental vibrations. Thereby, the He-Ne correction, which is typically required for FTIS, can be bypassed, thus reducing the cost, size, and complexity of the FTIS system. Finally, we construct neural network models using Hyperband, an automatic hyperparameter selection algorithm, and compare the performance with our manually-optimized model.

Correction of out-of-focus microscopic images by deep learning

MotivationMicroscopic images are widely used in basic biomedical research, disease diagnosis and medical discovery. Obtaining high-quality in-focus microscopy images has been a cornerstone of the microscopy. However, images obtained by microscopes are often out-of-focus, resulting in poor performance in research and diagnosis. ResultsTo solve the out-of-focus issue in microscopy, we developed a Cycle Generative Adversarial Network (CycleGAN) based model and a multi-component weighted loss function. We train and test our network in two self-collected datasets, namely Leishmania parasite dataset captured by a bright-field microscope, and bovine pulmonary artery endothelial cells (BPAEC) captured by a confocal fluorescence microscope. In comparison to other GAN-based deblurring methods, the proposed model reached state-of-the-art performance in correction. Another publicly available dataset, human cells dataset from the Broad Bioimage Benchmark Collection is used for evaluating the generalization abilities of the model. Our model showed excellent generalization capability, which could transfer to different types of microscopic image datasets. Availability and ImplementationCode and dataset are publicly available at: https://github.com/jiangdat/COMI.

Suppression of reactive oxygen species in endothelial cells by an antagonist of growth hormone-releasing hormone.

Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone, which regulates the secretion of growth hormone (GH) from the anterior pituitary gland. The effects of GHRH extend beyond the GH-insulin-like growth factor I axis, and that neuropeptide has been involved in the potentiation of several malignancies and other inflammatory disorders. The development of GHRH antagonists (GHRHAnt) delivers an exciting possibility to counteract the pathogenesis of the GHRH-related effects in human pathophysiology, especially when considered that GHRHAnt support endothelial barrier integrity. Those GHRHAnt-mediated effects are exerted at least in part due to the suppression of major inflammatory pathways, and the modulation of major cytoskeletal components. In the present study, we measured the production of reactive oxygen species (ROS) in bovine pulmonary artery endothelial cells, human cerebral microvascular endothelial cells, and human lung microvascular endothelial cells exposed to GHRH or a commercially available GHRHAnt. Our findings reveal the antioxidative effects of GHRHAnt in all three cell lines, which express GHRH receptors. The redox status of NIH/3T3 cells, which do not produce GHRH receptors, was not significantly affected by GHRH or GHRHAnt. Hence, the application of GHRHAnt in pathologies related to increased ROS production should be further investigated.

Severe COVID-19 Pneumonia is Associated with Increased Plasma Immunoglobulin G Agonist Autoantibodies Targeting the 5-Hydroxytryptamine 2A Receptor.

To test whether plasma autoantibodies targeting the 5-hydroxytryptamine 2A receptor increase in COVID-19 infection; and to characterize the pharmacologic specificity, and signaling pathway activation occurring downstream of receptor binding in mouse neuroblastoma N2A cells and cell toxicity of the autoantibodies. Plasma obtained from nineteen, older COVID-19 patients having mild or severe infection was subjected to protein-A affinity chromatography to obtain immunoglobulin G fraction. One-fortieth dilution of the protein-A eluate was tested for binding to a linear synthetic peptide QN.18 corresponding to the second extracellular loop of the human 5-hydroxytryptamine 2A receptor. Mouse neuroblastoma N2A cells were incubated with COVID-19 IgG autoantibodies in the presence or absence of selective inhibitors of G-protein coupled receptors, signaling pathway antagonists, or a novel decoy receptor peptide. 5-hydroxytryptamine 2A receptor autoantibody binding occurred in 17 of 19 (89%) patients with acute COVID-19 infection and increased level was significantly correlated with increased severity of COVID-19 infection. The agonist autoantibodies mediated acute neurite retraction in mouse neuroblastoma cells by a mechanism involving Gq11/PLC/IP3R/Ca2+ activation and RhoA/Rho kinase pathway signaling occurring downstream of receptor binding which had pharmacologic specificity consistent with binding to the 5-HT2A receptor. A novel synthetic peptide 5-HT2AR fragment, SN..8, dose-dependently blocked autoantibody-induced neurotoxicity. The COVID-19 autoantibodies displayed acute toxicity in bovine pulmonary artery endothelial cells (stress fiber formation, contraction) and modulated proliferation in a manner consistent with known 'biased agonism' on the 5-HT2A receptor. These data suggest that 5-HT2AR targeting autoantibodies are highly prevalent may contribute to pathophysiology in acute, severe COVID-19 infection.

Mutifocal image scanning microscopy based on double-helix point spread function engineering

Confocal laser scanning microscopy (CLSM) is a powerful imaging tool providing high resolution and optical sectioning. In its standard optical configuration, a pair of confocal pinholes is used to reject out-of-focus light. The diffraction limited resolution can be broken by reducing the confocal pinhole size. But this comes at the cost of extremely low signal-to-noise ratio (SNR). The limited SNR problem can be solved by image scanning microscopy (ISM), in which the single-point detector of a regular point-scanning confocal microscopy is substituted with an array detector such as CCD or CMOS, thus the two-fold super-resolution imaging can be achieved by pixel reassignment and deconvolution. However, the practical application of ISM is challenging due to its limited image acquisition speed. Here, we present a hybrid microscopy technique, named multifocal refocusing after scanning using helical phase engineering microscopy (MRESCH), which combines the double-helix point spread function (DH-PSF) engineering with multifocal structured illumination to dramatically improve the image acquisition speed. In the illumination path, sparse multifocal illumination patterns are generated by a digital micromirror device for parallel imaging information acquisition. In the detection path, a phase mask is introduced to modulate the conventional PSF to the DH-PSF, which provides volumetric information, and meanwhile, we also present a digital refocusing strategy for processing the collected raw data to recover the wild-filed image from different sample layers. To demonstrate imaging capabilities of MRESCH, we acquire the images of mitochondria in live HeLa cells and make a detailed comparison with those from the wide-field microscopy. In contrast to the conventional wide-field approach, the MRESCH can expand the imaging depth in a range from –1 μm to 1 μm. Next, we sample the F-actin of bovine pulmonary artery endothelial cells to characterize the lateral resolution of the MRESCH. The results show that the MRESCH has a better resolution capability than the conventional wide-field illumination microscopy. Finally, the proposed image scanning microscopy can record three-dimensional specimen information from a single multi-spot two-dimensional scan, which ensures faster data acquisition and larger field of view than ISM.

Metformin in acute respiratory distress syndrome: An opinion

Senior individuals are more susceptible to the irreversible outcomes of endothelial barrier dysfunction, the hallmark of Acute Respiratory Distress Syndrome (ARDS). The Severe Acute Respiratory Syndrome Coronovirus 2 (SARS-CoV-2) - inflicted ARDS delivers the devastating outcomes of the COVID-19 worldwide. Endothelial hyperpermeability has been associated with both the progression and establishment of the COVID-19 - related respiratory failure. In the present study we investigated the in vitro effects of Metformin in the permeability of bovine pulmonary artery endothelial cells. Our preliminary results suggest that moderate doses (0.1, 0.5, 1.0 mM) of this anti-diabetic agent enhance the vascular barrier integrity, since it produces an increase in the transendothelial resistance of endothelial monolayers. Thus, we speculate that Metformin may deliver a new therapeutic possibility in ARDS, alone or in combination with other barrier enhancers.

Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method

Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.

Modelling early events in Mycobacterium bovis infection using a co-culture model of the bovine alveolus

Bovine tuberculosis (bTB), a zoonosis mainly caused by Mycobacterium bovis has severe socio-economic consequences and impact on animal health. Host–pathogen interactions during M. bovis infection are poorly understood, especially early events which are difficult to follow in vivo. This study describes the utilisation of an in vitro co-culture model, comprising immortalised bovine alveolar type II (BATII) epithelial cells and bovine pulmonary arterial endothelial cells (BPAECs). When cultured at air–liquid interface, it was possible to follow the migration of live M. bovis Bacille Calmette-Guérin (BCG) and to observe interactions with each cell type, alongside cytokine release. Infection with BCG was shown to exert a detrimental effect primarily upon epithelial cells, with corresponding increases in IL8, TNFα, IL22 and IL17a cytokine release, quantified by ELISA. BCG infection increased expression of CD54, MHC Class I and II molecules in endothelial but not epithelial cells, which exhibited constitutive expression. The effect of peripheral blood mononuclear cell conditioned medium from vaccinated cattle upon apical-basolateral migration of BCG was examined by quantifying recovered BCG from the apical, membrane and basolateral fractions over time. The numbers of recovered BCG in each fraction were unaffected by the presence of PBMC conditioned medium, with no observable differences between vaccinated and naïve animals.

The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission.

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV’s alternating-host transmission cycle on the virus’s genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV’s genome. To understand the role of alternating hosts in BTV’s genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV.