Viability Measurements Research Articles

Lymphoma and Leukemia Cell Vulnerabilities and Resistance Identified by Compound Library Screens.

Response to anticancer agents is often restricted to subsets of patients. Recognition of factors underlying this heterogeneity and identification of biomarkers associated with response to drugs would greatly improve the efficacy of drug treatment. Platforms that can comprehensively map cellular response to compounds in high-throughput provide a unique tool to identify associated biomarkers and provide hypotheses for mechanisms underlying variable response. Such screens can be performed on cell lines and short-term cultures of primary cells to take advantage of the respective models' strength, which include, e.g., the ability to silence genes or introduce somatic mutations to cell lines. Cohorts of patient samples represent the natural diversity of cancers, including rarer mutations and combinatorial patterns of mutations that are often absent from existing cell lines. We here summarize a simple and scalable method for the measurement of viability after drug exposure based on ATP measurements as a surrogate for viability, which we use to measure and understand drug response in cell lines and primary cells.

Non-destructive viability assessment of cancer cell spheroids using dynamic optical coherence tomography with trypan blue validation

3D cell cultures are widely used in biomedical research for the recapitulation of in vivo microenvironments. Viability assessment and monitoring of these intricate conformations remain an open problem as standard cell viability protocols based on colorimetry or microscopy are not directly applicable to intact 3D samples. Optical coherence tomography (OCT) has been explored extensively for subsurface structural and quasi-functional analysis of 3D cell cultures and tissue. Recent studies of dynamic OCT as a source of cellular contrast have found qualitative associations with necrosis in cell spheroids, suggesting potential as a viability marker. We present empirical and validated evidence for dynamic OCT as a quantitative indicator of cell viability in 3D cultures. We analysed over 240 MCF-7 cancer cell spheroids with dynamic OCT and corresponding viability measurements using the trypan blue exclusion assay. Significant effects of common reagents dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS) on OCT readouts were noted. We proposed a regression-based OCT brightness normalisation technique that removed reagent-induced OCT intensity biases and helped improve correspondence to the viability assay. These results offer a quantitative biological foundation for further advances of dynamic OCT as a novel non-invasive modality for 3D culture monitoring.

Mechanism of Guishao Yigong decoction in treating colorectal cancer based on network pharmacology and experimental validation.

To explore the effective components of Guishao Yigong decoction (GYD) in the treatment of colorectal cancer and reveal its potential mechanism of action. Through network pharmacology, the main target and signaling pathway of GYD therapy for colorectal cancer (CRC) were found. Subsequently, the effect of GYD was verified by in vitro cell viability measurements, colony formation, and scratch healing tests. The effects of GYD on metabolic pathways in vivo were found through plasma metabolomics. Finally, flow cytometry and qPCR experiments were used to verify the cycle-blocking effect of GYD on CRC cells. Based on the network pharmacological analysis and molecular docking technology, it was found that GYD could restrain the growth of CRC cells by affecting lipid metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathways. A series of cell experiments showed that GYD could inhibit the proliferation, migration and clonogenic ability of CRC cells. Furthermore, the plasma metabolomics results showed that GYD could affect the production of unsaturated fatty acids in mice. Flow cytometry and qPCR experiments further proved that GYD blocked the CRC cells in the G1 phase and modulated the expression of cell cycle-related targets, such as AKT, TP53, CDKN1A, and CDK2. All the results indicated that GYD could regulate the related metabolism of unsaturated fatty acids. Thus, the cell cycle was blocked and the expressions of the key proteins such as AKT and TP53 were regulated, which achieved the purpose of intervention in colorectal cancer.

High glucose promotes lipopolysaccharide-induced macrophage pyroptosis through GSDME O-GlcNAcylation.

The high glucose (HG) environment in diabetic periodontitis aggravates the damage of periodontal tissue. Pyroptosis has been shown to be positively correlated with the severity of periodontitis, including macrophage pyroptosis. O-GlcNAcylation is a posttranslational modification that is involved in the pathogenesis of periodontitis. However, whether HG regulates macrophage pyroptosis through O-GlcNAcylation remains uncertain. This study aimed to investigate the effect of HG on the O-GlcNAcylation level of a pyroptosis regulator GSDME in macrophages to further probe the mechanisms of diabetic periodontitis. Blood samples were collected from patients with diabetic periodontitis. THP-1 monocytes were induced to differentiate into macrophages by phorbol 12-myristate 13-acetate and then treated with HG to simulate periodontitis invitro. GSDME expression of blood samples and macrophages was measured by quantitative real-time PCR. Pyroptosis was assessed by propidium iodide staining, measurement of cell viability, cytotoxicity, protein levels of inflammation factors, and pyroptosis-related proteins. O-GlcNAcylation of GSDME was analyzed using co-immunoprecipitation (co-IP), IP, and western blot. The results showed that GSDME expression was elevated in patients with periodontitis and HG-treated macrophages. HG inhibited cell viability but increased LDH content, levels of IL-1β, IL-18, TNF-α, NLRP3, GSDMD, and Caspase-1, indicating that HG promoted pyroptosis of macrophages, which was reversed by GSDME knockdown. HG treatment increased O-GlcNAcylation in macrophages. Mechanically, GSDME interacted with OGT, and OGT knockdown suppressed O-GlcNAcylation of GSDME at Ser (S)339 site. Knockdown of OGT inhibited pyroptosis in HG-treated macrophages, while GSDME overexpression partially reversed this inhibition. HG treatment enhanced OGT-mediated GSDME O-GlcNAcylation, thereby augmenting pyroptosis in LPS-induced macrophages. These results may provide a novel sight for the treatment of periodontitis.

Abstract A023: Patient-Derived Organoid Cultures for Personalized Therapies and Targeted Drug Screening Applications

Abstract Patient-derived organoids (PDOs) have emerged as a powerful tool for modeling human cancers due to their ability to closely mirror in vivo architecture and preserve the genetic landscape of tumors. They offer a crucial platform for studying disease progression and treatment responses. In this study, we present a comprehensive characterization of a unique collection of PDO cultures derived from pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy often diagnosed in advanced stages with limited treatment options and poor prognosis. Our investigation explores morphological and genetic profiling of 10 PDAC PDOs, specifically identifying distinct drug-response patterns. The morphological characteristics of these PDO cultures were investigated by microscopy, while the mutational status was acquired by panel sequencing. Subsequently, PDOs were treated with a panel of 10 inhibitors, either individually or in combination. The response to these treatments was assessed through live-cell imaging (Incucyte®) to capture and quantify changes in organoid morphology and endpoint measurements of cellular viability using a CellTiter-Glo® assay. Our detailed in vitro drug sensitivity testing revealed a remarkable diversity in the responses of the PDAC PDOs to different chemotherapeutic agents, including those currently under clinical trials. Of notable significance is the depth of understanding we have achieved, allowing us to establish potential correlations between PDO morphology, genetic mutations, and drug reactions. This finding provides a key insight into the complex interplay of these factors and their impact on treatment outcomes, offering valuable insights into understanding individual tumor subtypes' sensitivity or resistance to treatments. The PDOs exhibited a spectrum of cancer-associated mutations, including KRAS, TP53, SMAD4, CDKN2A, and ARID1A, reflecting the heterogeneity observed in PDAC patients. Morphologically, the organoids displayed various growth patterns, ranging from cystic and regular structures to filled or unfilled, granular, dark, and light membrane structures. Drug treatments underscored the diverse and unique drug-response profiles within the PDOs, with some demonstrating sensitivity to multiple treatments. In contrast, others exhibited resistance, highlighting the intricate nature of drug responses within this model. Overall, our findings emphasize the ability of PDAC organoids to faithfully capture the complexity and heterogeneity of the disease, making them promising platforms for guiding personalized oncology approaches. The diverse drug response profiles observed in PDOs advocate leveraging this model to develop precision medicine strategies tailored to patients' characteristics, ultimately paving the way for more effective and personalized treatment regimens in the fight against PDAC. Citation Format: Constanza Tapia Contreras, Günter Schneider, Denise Müller, Kimia Mirzakhani, Karly Conrads, Silke Kaulfuß, Tim Beißbarth, Gabriela Salinas, Nils Beyer, Sophia Reinländer, Ulrix Sax, Elisabeth Heßmann, Volker Ellenrieder, Philipp Ströbel, Michael Ghadimi. Patient-Derived Organoid Cultures for Personalized Therapies and Targeted Drug Screening Applications [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A023.

LINC01614 activated by SP1 promoted malignant behavior of triple-negative breast cancer cells via the WNT/b-Catenin signaling pathway.

Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer (BC), characterized by a dismal prognosis. Dysregulated long non-coding RNA LINC01614 might be a potential biomarker for BC as previously reported. Nevertheless, its functions and mechanism in TNBC cells are unclear. The study aimed to study the effects of LINC01614 on TNBC cell migration, invasion, and epithelial-mesenchymal transition (EMT) process as well as the related mechanism. Reverse transcription quantitative polymerase chain reaction was performed to detect the expression of LINC01614 and SP1 in TNBC cells and tissues. The cellular localization of LINC01614 was determined by subcellular fraction assays. Cell counting kit-8 and Transwell invasion assays were conducted for measurement of TNBC cell viability and invasive ability. Cell migration was performed using wound healing assays and Transwell migration assays. Chromatin immunoprecipitation assays and luciferase reporter assays were used to explore the interaction between SP1 and LINC01614. Western blotting was used to assess protein levels of factors involved in EMT process and Wnt/ß-catenin signaling in TNBC cells. LINC01614 expression was elevated in TNBC tissues and cells. LINC01614 knockdown inhibited cell viability as well as migratory and invasive abilities of TNBC cells. LINC01614 knockdown also obstructed EMT process, as shown by E-cadherin upregulation and vimentin downregulation in TNBC cells. SP1 directly bound to the promoter of LINC01614 and activated LINC01614 expression. SP1 overexpression reversed the suppressive effect of LINC01614 knockdown on TNBC cell migration, invasion, and EMT process. Protein levels of Wnt and ß-catenin were diminished by LINC01614 knockdown, and the trend was partially rescued by SP1 overexpression. SP1-induced LINC01614 promoted malignant behavior of TNBC cells by activating the Wnt/ß-catenin signaling pathway.

Superhydrophobicity Effects on Spheroid Formation and Polarization of Macrophages.

The interaction of biomaterials with the immune system is ruled by the action of macrophages. The surface features of these biomaterials, like wettability, which is an expression of chemical composition, texture, and geometry, can affect macrophages response. Such surface parameters can be then efficiently exploited to improve biocompatibility by lowering undesired immunological reactions and at the same time creating the substrate for positive interactions. In this work, the preparation and physicochemical characterization of highly water-repellent surfaces to develop and characterize 3D spheroids derived from monocyte-macrophages (RAW 264.7 cell line) has been carried out. As a measure of cell viability over time, the obtained aggregates have been transferred under standard 2D cell culture conditions. Significant changes on the morphology-associated polarization of the derived cellular entities have been evaluated at the nanoscale through 3D profilometry. The results suggested that the spheroid formation using highly repellent substrates induced the activation of M2-type cells. This simple and cost-effective approach can be used for preparing M2-based macrophages for regenerative purposes.

Differential Mitochondrial Bioenergetics in Neurons and Astrocytes Following Ischemia-Reperfusion Injury and Hypothermia.

The close interaction between neurons and astrocytes has been extensively studied. However, the specific behavior of these cells after ischemia-reperfusion injury and hypothermia remains poorly characterized. A growing body of evidence suggests that mitochondria function and putative transference between neurons and astrocytes may play a fundamental role in adaptive and homeostatic responses after systemic insults such as cardiac arrest, which highlights the importance of a better understanding of how neurons and astrocytes behave individually in these settings. Brain injury is one of the most important challenges in post-cardiac arrest syndrome, and therapeutic hypothermia remains the single, gold standard treatment for neuroprotection after cardiac arrest. In our study, we modeled ischemia-reperfusion injury by using in vitro enhanced oxygen-glucose deprivation and reperfusion (eOGD-R) and subsequent hypothermia (HPT) (31.5 °C) to cell lines of neurons (HT-22) and astrocytes (C8-D1A) with/without hypothermia. Using cell lysis (LDH; lactate dehydrogenase) as a measure of membrane integrity and cell viability, we found that neurons were more susceptible to eOGD-R when compared with astrocytes. However, they benefited significantly from HPT, while the HPT effect after eOGD-R on astrocytes was negligible. Similarly, eOGD-R caused a more significant reduction in adenosine triphosphate (ATP) in neurons than astrocytes, and the ATP-enhancing effects from HPT were more prominent in neurons than astrocytes. In both neurons and astrocytes, measurement of reactive oxygen species (ROS) revealed higher ROS output following eOGD-R, with a non-significant trend of differential reduction observed in neurons. HPT after eOGD-R effectively downregulated ROS in both cells; however, the effect was significantly more effective in neurons. Lipid peroxidation was higher after eOGD-R in neurons, while in astrocytes, the increase was not statistically significant. Interestingly, HPT had similar effects on the reduction in lipoperoxidation after eOGD-R with both types of cells. While glutathione (GSH) levels were downregulated after eOGD-R in both cells, HPT enhanced GSH in astrocytes, but worsened GSH in neurons. In conclusion, neuron and astrocyte cultures respond differently to eOGD-R and eOGD-R + HTP treatments. Neurons showed higher sensitivity to ischemia-reperfusion insults than astrocytes; however, they benefited more from HPT therapy. These data suggest that given the differential effects from HPT in neurons and astrocytes, future therapeutic developments could potentially enhance HPT outcomes by means of neuronal and astrocytic targeted therapies.

Taxus chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu overcomes the resistance to osimertinib in EGFR-mutant non-small-cell lung cancer via suppression of ERK1/2-related cholesterol biosynthesis

Ethnopharmacological relevanceAcquired resistance to osimertinib limits its clinical efficacy in non-small cell lung cancer (NSCLC) with EGFR mutations. The widespread recognition of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu (Chinese yew) as a natural anti-cancer medication is well-established. However, the specific contribution of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu in addressing resistance to osimertinib is still uncertain. Aim of the studyBased on the biological behaviors and lipid metabolism, we investigated whether aqueous extract of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu (AETC) could enhance the antitumor effect of osimertinib in NSCLC with an investigation on the precise mechanisms. Materials and methodsThe effect of AETC on enhancing osimertinib sensitivity was assessed via cell viability measurements, levels of reactive oxygen species (ROS), apoptosis, and lipid levels. Western blotting was used to verify the mechanisms of AETC responsible for overcoming the resistance to osimertinib via ERK1/2 overexpression and knockdown models. In vivo validation was conducted using subcutaneous xenografts from osimertinib-resistant cells in nude mice. ResultsOsimertinib-resistant cells exhibited altered cholesterol biosynthesis, which was induced by ERK1/2 activation. The combination of AETC and osimertinib can synergistically decrease the levels of ROS in cells, enhance apoptosis, and inhibit the growth of osimertinib-resistant cells. Mechanistic experiments demonstrated that AETC can downregulate the key regulators of cholesterol biosynthesis by regulating ERK1/2, inhibiting the endogenous synthesis rate of cholesterol, and suppressing the level of lipids in osimertinib-resistant cells and xenograft tumors when combined with osimertinib, ultimately reversing resistance to osimertinib. ConclusionsThe resistance to osimertinib is significantly influenced by cholesterol biosynthesis, highlighting its pivotal role in this context. AETC can enhance osimertinib sensitivity via ERK/SREBP-2/HMGCR-mediated cholesterol biosynthesis. These results provide a promising therapeutic target and potential treatment option for resistance to osimertinib.

A molecular method to assess viability of Phytophthora in infected wood following heat treatment

International trade in wood products is an important component of the global economy. However, wood and wood products may have pests associated with them that could be introduced into importing countries, posing phytosanitary risks, and leading to the implementation of regulatory restrictions that affect wood trade. The application of heat to kill wood-associated pests has been a successful phytosanitary method to reduce their spread. To evaluate the efficacy of wood heat treatment to kill fungal and fungus-like pathogens, the method of choice has been to grow organisms in cultures for subsequent identification. However, some plant pathogens can be difficult or impossible to grow in axenic cultures and a molecular method can still be useful for assessing pathogen viability after heat-treatment. RNA is a single stranded molecule that is responsible for the transcription of genes. Since it becomes rapidly unstable after cell death, it provides a measure of viability. We therefore designed and tested RNA-based molecular diagnostic assays targeting essential genes and assessed their presence after heat treatment in wood colonized by four Phytophthora species of phytosanitary concern (P. xmultiformis, P. cinnamomi, P. lateralis and P. ramorum) through reverse transcription and real-time polymerase chain reaction (RT-qPCR). Our assays differentiate between genomic and mRNA as the TaqMan probes span exon-intron junctions. We validated these RT-qPCR assays to assess heat treatment efficacy of Phytophthora-inoculated wood. These assays can be very useful tools to assess effectiveness of current and emerging phytosanitary wood treatments.

High-resolution magic angle spinning nuclear magnetic resonance of donor pancreatic tissue may predict islet viability prior to isolation.

For patients with type 1 diabetes mellitus complicated by severe hypoglycemia, clinical islet transplantation is an efficacious alternative to whole pancreas transplantation. While islet transplantation has improved over the last few years, there remain questions regarding its cost-effectiveness and donor allosensitization, which is exacerbated when islets from more than one donor are required. Understanding the features of a pancreas that would provide viable islets prior to isolation may lead to development of an accurate assay that could identify suitable pancreases and provide significant cost savings to a clinical islet transplantation program. In this pilot study, solid-state high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy was used to assess samples of convenience of human pancreatic tissue taken prior to islet isolation both before and after incubation using the two-layer perfluorocarbon (PFC)/University of Wisconsin (UW) solution cold-storage method. We observed that, prior to incubation, human pancreatic tissue exhibited evidence of hypoxia with decreased peak integrals associated with glucose and increased peak integrals corresponding to lactate and free fatty acids. After incubation, we observed a reversal of the hypoxia-induced damage, as integrals corresponding to glucose increased, and those corresponding to lactate and free fatty acid resonances decreased. Interestingly, a significant correlation between the ratio of the glucose integral (at 3.0-4.5 ppm) to the sum of the fatty acid (at 0.9 ppm) and lactate + fatty acid (at 1.3 ppm) integrals and glucose responsiveness, a measure of islet viability, of the isolated islets, was observed after incubation in PFC/UW solution for pancreases that responded to PFC/UW solution incubation (p = 0.02). Notably, pancreases with little or no change in the integral ratio after PFC/UW solution incubation had poor recovery. These results suggest that tissue recovery is a key feature for determining islet cell viability, and further that HRMAS NMR may be a practical method to quickly assess human donor pancreatic tissue prior to islet isolation for clinical transplantation.

Effects of Copper on Legionella pneumophila Revealed via Viability Assays and Proteomics.

Cu is an antimicrobial that is commonly applied to premise (i.e., building) plumbing systems for Legionella control, but the precise mechanisms of inactivation are not well defined. Here, we applied a suite of viability assays and mass spectrometry-based proteomics to assess the mechanistic effects of Cu on L. pneumophila. Although a five- to six-log reduction in culturability was observed with 5 mg/L Cu2+ exposure, cell membrane integrity only indicated a <50% reduction. Whole-cell proteomic analysis revealed that AhpD, a protein related to oxidative stress, was elevated in Cu-exposed Legionella relative to culturable cells. Other proteins related to cell membrane synthesis and motility were also higher for the Cu-exposed cells relative to controls without Cu. While the proteins related to primary metabolism decreased for the Cu-exposed cells, no significant differences in the abundance of proteins related to virulence or infectivity were found, which was consistent with the ability of VBNC cells to cause infections. Whereas the cell-membrane integrity assay provided an upper-bound measurement of viability, an amoebae co-culture assay provided a lower-bound limit. The findings have important implications for assessing Legionella risk following its exposure to copper in engineered water systems.

Inducing Effect of Corylus avellana on Cytotoxic Activity in Lung and Breast Cancer Cells via Apoptosis

Turkish hazelnut (Corylus avellana L. cv Tombul) is a widely used nut in the chocolate industry and is also rich in polyphenol content, which promises anticancer effects. The anti-cancer and apoptotic effects of hazelnut leaves extracts examined on lung and breast cancer cells. Sulforhodamine B (SRB) and Adenosine 5’- triphosphate (ATP) assays were carried out for cell viability measurement. The mode of cell death was shown morphologically by the double fluorescence staining. Apoptosis was determined by performing caspase-mediated cytokeratin 18 (M30 ELISA) and western blot analysis. PARP, caspase 3, caspase 8, DR4, and GAPHD (Glyceraldehyde-3-phosphate Dehydrogenase) protein bands were visualized as markers of apoptosis. A wound healing test was employed to measure cell migration. Methanol extract of hazelnut leaf exhibited inhibition of cell growth activities in a dose-dependent manner. IC50 values were determined as 32.17 µg/ml in MCF-7, 32.16 µg/ml in MDA-MB-231, 20.40 µg/ml in A549 and 12.04 µg/ml in H1299 cells for ethanol extract while it was determined as 21.08 µg/ml in MCF-7, 40.16 µg/ml in MDA-MB-231, 22.04 µg/ml in A549 and 5.91 µg/ml in H1299 cells in methanol extract. In comparison, methanol leaf extracts were more effective in H1299 cells (IC50 value was 5.91 µg/ml).In comparison, ethanol leaf extracts were more effective in H1299 cells (IC50 value was 9.722 µg/ml). Western blot analysis demonstrated that hazelnut leaf extract treatment of cancer cells led to cell death via apoptosis and inhibited cell migration in lung and breast cancer cell lines. The cytotoxic effects of hazelnut extract on breast and lung cancer cells might be valuable and promising in elucidating cell death mechanisms for the development of new methods in cancer treatment.

A Second Wind for Inorganic APIs: Leishmanicidal and Antileukemic Activity of Hydrated Bismuth Oxide Nanoparticles.

American cutaneous leishmaniasis is a disease caused by protozoa of the genus Leishmania. Currently, meglumine antimoniate is the first-choice treatment for the disease. The limited efficacy and high toxicity of the drug results in the necessity to search for new active principles. Nanotechnology is gaining importance in the field, since it can provide better efficacy and lower toxicity of the drugs. The present study aimed to synthesize, characterize, and evaluate the in vitro leishmanicidal and antileukemic activity of bismuth nanoparticles (BiNPs). Promastigotes and amastigotes of L. (V.) guyanensis and L. (L.) amazonensis were exposed to BiNPs. The efficacy of the nanoparticles was determined by measurement of the parasite viability and the percentage of infected cells, while the cytotoxicity was characterized by the colorimetry. BiNPs did not induce cytotoxicity in murine peritoneal macrophages and showed better efficacy in inhibiting promastigotes (IC50 < 0.46 nM) and amastigotes of L. (L.) amazonensis. This is the first report on the leishmanicidal activity of Bi-based materials against L. (V.) guayanensis. BiNPs demonstrated significant cytotoxic activity against K562 and HL60 cells at all evaluated concentrations. While the nanoparticles also showed some cytotoxicity towards non-cancerous Vero cells, the effect was much lower compared to that on cancer cells. Treatment with BiNPs also had a significant effect on inhibiting and reducing colony formation in HL60 cells. These results indicate that bismuth nanoparticles have the potential for an inhibitory effect on the clonal expansion of cancer cells.

A DIO2 missense mutation and its impact on fetal response to PRRSV infection

BackgroundPorcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection. Our objectives were to identify mutation(s) within the porcine DIO2 gene and to determine if they were associated with fetal outcomes after PRRSV-2 challenge. Sequencing of the DIO2, genotyping identified variants, and association of DIO2 genotypes with fetal phenotypes including DIO2 mRNA levels, viability, survival, viral loads, cortisol and thyroid hormone levels, and growth measurements were conducted.ResultsA missense variant (p.Asn91Ser) was identified in the parental populations from two independent PRRSV-2 challenge trials. This variant was further genotyped to determine association with fetal PRRS outcomes. DIO2 mRNA levels in fetal heart and kidney differed by the genotypes of Asn91Ser substitution with significantly greater DIO2 mRNA expression in heterozygotes compared with wild-type homozygotes (P < 0.001 for heart, P = 0.002 for kidney). While Asn91Ser did not significantly alter fetal viability and growth measurements, interaction effects of the variant with fetal sex or trial were identified for fetal viability or crown rump length, respectively. However, this mutation was not related to dysregulation of the hypothalamic-pituitary-adrenal and thyroid axis, indicated by no differences in circulating cortisol, T4, and T3 levels in fetuses of the opposing genotypes following PRRSV-2 infection.ConclusionsThe present study suggests that a complex relationship among DIO2 genotype, DIO2 expression, fetal sex, and fetal viability may exist during the course of fetal PRRSV infection. Our study also proposes the increase in cortisol levels, indicative of fetal stress response, may lead to fetal complications, such as fetal compromise, fetal death, or premature farrowing, during PRRSV infection.

Development and characterisation of a novel complex triple cell culture model of the human alveolar epithelial barrier

Owing to increased pressure from ethical groups and the public to avoid unnecessary animal testing, the need for new, responsive and biologically relevant in vitro models has surged. Models of the human alveolar epithelium are of particular interest since thorough investigations into air pollution and the effects of inhaled nanoparticles and e-cigarettes are needed. The lung is a crucial organ of interest due to potential exposures to endogenous material during occupational and ambient settings. Here, an in vitro model of the alveolar barrier has been created in preparation for use in the quasi-air liquid interface (qALI) and (aerosol) air–liquid interface (ALI) exposures. The model consists of an alveolar type 1-like cell line (TT1), an alveolar type 2-like cell line (NCI-H441) and a model of (alveolar) macrophages (dTHP-1). The model formulates a complex, multi-cellular system, cultured at the air–liquid interface, that mimics the apical layer of the alveolar epithelial region in the human lung. Characterisation data has shown that both TT1 and NCI-H441 epithelial cells are able to be cultured together in addition to dTHP-1 cells through imaging (morphology), pro-inflammatory response and viability measurements. This dataset also demonstrates evidence of a reasonable barrier created by the cell culture in comparison to negative controls. Furthermore, it shows that while maintaining a low baseline of (pro)-inflammatory mediator expression during normal conditions, the model is highly responsive to inflammatory stimuli. This model is proposed to be suitable for use in toxicology testing of inhaled exogenous agents.

Potential of electrospun fibrous membranes involving tri-n-octylphosphine oxide for selective clearance of uremic toxin p-cresol over urea and creatinine

In this study, various electrospun and electrosprayed fibrous membranes from polyethersulfone (PES) and polyvinylpyrrolidone (PVP) were fabricated, to which a common solvating extractant tri-n-octylphosphine oxide (TOPO) was incorporated. Such TOPO-involved PES membranes were attempted to selectively clear p-cresol, one of trace amounts of small uremic toxins, from simulated serum via hydrogen bonding. Morphological and physicochemical properties of the as-synthesized fibrous membranes were first studied. Batch tests displayed that the equilibrium of p-cresol adsorption on all fibrous membranes were attained within 2 h. Analysis of adsorption isotherms of p-cresol by the Langmuir equation revealed that the adsorption was improved in the presence of TOPO; moreover, the distribution state of TOPO powders along the fibers played an important role in membrane performance. Among the six as-prepared TOPO-incorporated membranes, the highest adsorption capacity of p-cresol in simulated serum at 37oC and pH 7.4 reached to be 21.7 mmol per gram of TOPO. The operational stability of as-prepared fibrous membranes was evaluated by checking their adsorption ability, membrane weight, water contact angle, structural changes by SEM imaging, and PVP dissolution using UV–Vis spectroscopy, both before and after exposure to PBS solution. Crossflow permeation-adsorption of a multicomponent mixture (0.46 mmol/L of p-cresol, 1.33 mmol/L of creatinine, and 38.3 mmol/L of urea) for 4 h indicated that the prepared membranes yielded a high selectivity toward p-cresol against creatinine and urea of 21.2–43.0 and 10.8–18.5, respectively. Hemolysis tests and viability measurements of both human umbilical vein endothelial cells and THP 1 monocytic leukemia cells demonstrated that the proposed configurations of the TOPO-incorporated fibrous membranes were hemo- and bio-compatible. Our results on the composition and systematic design of these fibrous membranes suggested that they can serve as an advanced hemoperfusion unit, maintaining high p-cresol removal and bio-compatibility. These well-designed membrane-based devices could be connected in series with the conventional hemodialysis devices to further improve the overall removal of small uremic toxins.

#1955 Ferroptotic proteins ACSL4 and ALOX15 as therapeutic targets in AKI

Abstract Background and Aims Ferroptosis is a regulated form of necrosis which is dependent on cellular iron and is characterized by the accumulation of lipid peroxides and failure of cellular antioxidant defences. We have previously described in vivo that ferroptosis is the primary cause of folic acid-induced acute kidney injury (FA-AKI) and that necroinflammation secondary to ferroptosis may further worsen kidney injury, since ferroptosis inhibition improved kidney function and decreased tubular cell death and oxidative stress. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is involved in the incorporation of polyunsaturated fatty acid (PUFA) into membranes, while 15-Lipoxygenase (Alox15) catalyzes the regio- and enantioselective peroxidation of membrane-esterified PUFAs, forming the ultimate peroxidized species that induce ferroptosis. In the present work, we aim to explore the potential of both proteins as therapeutic targets in AKI, as well as the molecular characterization of kidney ferroptosis in cultured tubular cells. Method Animal model: Female 12- to 14-week-old C57BL/6J wild type mice received a single intraperitoneal (i.p) injection of folic acid or vehicle and were sacrificed 48 hours later. Troglitazone was intravenously administered for in vivo ACSL4 inhibition, and Compound 1 was i.p administered for in vivo ALOX15 inhibition. In vitro characterization of ferroptosis: HK2 human tubular kidney cells were incubated with arachidonic acid (AA) prior to sublethal RSL3 administration for sensitization to ferroptosis. We used Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lpx-1) as inhibitors of ferroptosis. The thiazolidinedione family members Rosiglitazone, Pioglitazone and Troglitazone were used as pharmacological inhibitors of ACSL4. We also performed transcriptional silencing of ACSL4 using a specific siRNA. Compound 1 and PD1646 were administered for in vitro Alox15 inhibition. Cellular lipid peroxidation was analyzed by flow cytometry with Bodipy 581/591 staining. To characterize the lipid signature of ferroptotic cells, supernatants were collected and 15-HETEs levels were measured by ELISA. Oxido-lipidomic analysis of ferroptotic cells was performed by LC-MS. Real Time PCR and Western Blot of whole kidneys and cultured cells were performed for the detection and quantification of ACSL4 and ALOX15. Cell death was assessed by measurement of cell viability (MTT assay) and cytotoxicity (LDH assay). Tissue cell death was assessed by TUNEL. Results Kidney transcriptomics identified Acsl4 as the most upregulated member of Acsl family during FA-AKI. This was validated at mRNA and protein levels. Supplementation with AA, the preferred ACSL4 substrate, sensitized HK2 tubular cells to ferroptosis under sublethal RSL3 conditions, and this was prevented with specific ferroptosis inhibitors Fer-1 and Lpx-1. Pharmacological inhibition of ACSL4 with Troglitazone and a specific siRNA protected from cell death and lipid peroxidation induced by co-stimulation of AA and RSL3 in HK2 cells. Likewise, ALOX15 proteins levels were also increased in FA-AKI at 48 hours. PD1646 and Compound 1, both used to inhibit ALOX15, protected from ferroptotic cell death and lipid peroxidation in HK2 cells. Lipidomic analysis on HK2 tubular cells stimulated with AA+RSL3 uncovered an increased content of key peroxidized lipid species involved in ferroptosis execution, which was alleviated by targeting ACSL4 or ALOX15. Additionally, renal function of FA-AKI in mice was improved by ACSL4 pharmacological inhibition with Troglitazone and with ALOX15 inhibition with Compound 1. Conclusion Our preliminary results suggest therapeutic potential of ACSL4 and ALOX15 as ferroptotic targes during AKI. High PUFA content sensitizes human tubular HK2 cells to cell death and lipid peroxidation in the presence of sublethal ferroptotic triggers, and this was prevented by targeting ACSL4 or ALOX15 both in vitro and in vivo in FA-AKI. Closely related lipid peroxide species were decreased by ACSL4 or ALOX15 inhibitors.

#1457 Human renal proximal tubule-on-a-chip model to study prevention of ischemia and reperfusion injury

Abstract Background and Aims Renal ischemia/reperfusion injury (rIRI) is characterized by restriction of blood supply followed by restoration of blood flow and re-oxygenation and is a leading cause of acute kidney injury (AKI). While animal models are useful for studying systemic manifestations of AKI, their translational relevance is limited. Human in vitro models provide valuable mechanistic insights into rIRI, but often fail to incorporate crucial aspects such as reperfusion injury. To address these limitations, this study aims to develop an advanced in vitro model to study rIRI comprising a perfused three-dimensional (3D) human renal proximal tubule-on-a-chip. Method Our proximal tubule-on-a-chip model comprises a renal proximal tubule alongside a endothelial vessel, separated by extracellular matrix. A microfluidic platform, the OrganoPlate 3-Lane 40, was used to establish the model. The resulting coculture was characterized in terms of its three-dimensional structure, protein expression, and response to nephrotoxins. Subsequently, we simulated rIRI by manipulating oxygen levels, nutrient availability, and perfusion flow settings. The potential protective effect of adenosine was evaluated in the coculture model during ischemia and reperfusion. The extent of injury was assessed through morphological evaluation, caspase 3/7 activation, and cell viability measurements. Results The combination of low oxygen, reduced glucose, and interrupted flow exerted a potent disruptive effect on the proximal tubules, which was strongly amplified upon reperfusion. Endothelial vessels were less sensitive to the ischemia–reperfusion parameters. Adenosine treatment showed a protective effect on the disruption of the epithelium and on the caspase-3/7 activation. Conclusion We successfully developed a human in vitro model to study rIRI utilizing a coculture of a proximal tubule and a blood vessel on-a-chip. This model allowed us to characterize the renoprotective effect of adenosine. The robustness of the model and assays, coupled with the platform's high throughput capabilities, position it as a promising tool for advancing pathophysiological research and facilitating the development of innovative therapeutic modalities aimed at treating rIRI and improving AKI outcomes.

The Proteoglycans Biglycan and Decorin Protect Cardiac Cells against Irradiation-Induced Cell Death by Inhibiting Apoptosis

Radiation-induced heart disease (RIHD), a common side effect of chest irradiation, is a primary cause of mortality among patients surviving thoracic cancer. Thus, the development of novel, clinically applicable cardioprotective agents which can alleviate the harmful effects of irradiation on the heart is of great importance in the field of experimental oncocardiology. Biglycan and decorin are structurally related small leucine-rich proteoglycans which have been reported to exert cardioprotective properties in certain cardiovascular pathologies. Therefore, in the present study we aimed to examine if biglycan or decorin can reduce radiation-induced damage of cardiomyocytes. A single dose of 10 Gray irradiation was applied to induce radiation-induced cell damage in H9c2 cardiomyoblasts, followed by treatment with either biglycan or decorin at various concentrations. Measurement of cell viability revealed that both proteoglycans improved the survival of cardiac cells post-irradiation. The cardiocytoprotective effect of both biglycan and decorin involved the alleviation of radiation-induced proapoptotic mechanisms by retaining the progression of apoptotic membrane blebbing and lowering the number of apoptotic cell nuclei and DNA double-strand breaks. Our findings provide evidence that these natural proteoglycans may exert protection against radiation-induced damage of cardiac cells.