Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Single-Cell Study of Two Rat Models of Pulmonary Arterial Hypertension Reveals Connections to Human Pathobiology and Drug Repositioning.

Rationale: The cellular and molecular landscape and translational value of commonly used models of pulmonary arterial hypertension (PAH) are poorly understood. Single-cell transcriptomics can enhance molecular understanding of preclinical models and facilitate their rational use and interpretation.Objectives: To determine and prioritize dysregulated genes, pathways, and cell types in lungs of PAH rat models to assess relevance to human PAH and identify drug repositioning candidates.Methods: Single-cell RNA sequencing was performed on the lungs of monocrotaline (MCT), Sugen-hypoxia (SuHx), and control rats to identify altered genes and cell types, followed by validation using flow-sorted cells, RNA in situ hybridization, and immunofluorescence. Relevance to human PAH was assessed by histology of lungs from patients and via integration with human PAH genetic loci and known disease genes. Candidate drugs were predicted using Connectivity Map.Measurements and Main Results: Distinct changes in genes and pathways in numerous cell types were identified in SuHx and MCT lungs. Widespread upregulation of NF-κB signaling and downregulation of IFN signaling was observed across cell types. SuHx nonclassical monocytes and MCT conventional dendritic cells showed particularly strong NF-κB pathway activation. Genes altered in SuHx nonclassical monocytes were significantly enriched for PAH-associated genes and genetic variants, and candidate drugs predicted to reverse the changes were identified. An open-access online platform was developed to share single-cell data and drug candidates (http://mergeomics.research.idre.ucla.edu/PVDSingleCell/).Conclusions: Our study revealed the distinct and shared dysregulation of genes and pathways in two commonly used PAH models for the first time at single-cell resolution and demonstrated their relevance to human PAH and utility for drug repositioning.

Secreted Extracellular Cyclophilin A Is a Novel Mediator of Ventilator-induced Lung Injury.

Rationale: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator-induced lung injury. eCypA (extracellular CypA [cyclophilin A]) is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to coronavirus disease (COVID-19). Objectives: To explore the involvement of eCypA in the pathophysiology of ventilator-induced lung injury. Methods: Mice were ventilated with a low or high Vt for up to 3 hours, with or without blockade of eCypA signaling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretching to explore the cellular source of eCypA, and CypA concentrations were measured in BAL fluid from patients with acute respiratory distress syndrome to evaluate the clinical relevance. Measurements and Main Results: High-Vt ventilation in mice provoked a rapid increase in soluble CypA concentration in the alveolar space but not in plasma. In vivo ventilation and in vitro stretching experiments indicated the alveolar epithelium as the likely major source. In vivo blockade of eCypA signaling substantially attenuated physiological dysfunction, macrophage activation, and MMPs (matrix metalloproteinases). Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated concentrations of eCypA within BAL fluid. Conclusions: CypA is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. eCypA represents an exciting novel target for pharmacological intervention.

Nitrogen, phosphorus and high CO2 modulate photosynthesis, biomass and lipid production in the green alga Chlorella vulgaris.

Climate change could impact nutrient bioavailability in aquatic environment. To understand the interaction of nutrient bioavailability and elevated CO2, Chlorella vulgaris cells were grown in ambient air or 5% CO2 in different concentrations of nitrogen and phosphorus in a photobioreactor. The chlorophyll content, photosynthesis and respiration rates increased in 5% CO2 to support higher biomass production. The nutrient limitation in the growth media resulted in reduced photosynthetic rates of the algal cells and their PSI, PSII, and whole chain electron transport rates and biomass production. Conversely, their lipid content increased partly due to upregulation of expression of several lipid biosynthesis genes. The order of downregulation of photosynthesis and upregulation in lipid production due to nutrient limitation was in the order of N > P. The N-50 and 5% CO2 culture had only 10% reduction in biomass and 32% increase in lipids having 85% saturated fat required for efficient biofuel production. This growth condition is ideal for generation of biodiesel required to reduce the consumption of fossil fuel and combat global warming.

Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1.

Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.

TWIST1 Drives Smooth Muscle Cell Proliferation in Pulmonary Hypertension via Loss of GATA-6 and BMPR2.

Rationale: The bHLH (basic helix-loop-helix) transcription factor TWIST1 (Twist-related protein 1) controls cell proliferation and differentiation in tissue development and disease processes. Recently, endothelial TWIST1 has been linked to pulmonary hypertension (PH) and endothelial-to-mesenchymal transition, yet the role of TWIST1 in smooth muscle cells (SMCs) remains so far unclear.Objectives: To define the role of TWIST1 in SMCs in the pathogenesis of PH.Methods: SMC-specific TWIST1-deficient mice, SMC-specific TWIST1 silencing in rats, mass spectrometry, immunoprecipitation, and chromatin immunoprecipitation were used to delineate the role of SMC TWIST1 in PH.Measurements and Main Results: In pulmonary vessels from patients with PH and rodent PH models, TWIST1 expression was markedly increased and predominantly localized to SMCs. SMC-specific TWIST1 deficiency or silencing attenuated the development of PH and distal vessel muscularization in chronically hypoxic mice and in monocrotaline-treated rats. In vitro, TWIST1 inhibition or silencing prevented pulmonary artery SMC proliferation and migration. Mechanistically, the observed effects were mediated, at least in part, by TWIST1-dependent degradation of GATA-6 (GATA-binding protein 6). BMPR2 (bone morphogenetic protein receptor-2) was identified as a novel downstream target of GATA-6, which directly binds to its promoter. Inhibition of TWIST1 promoted the recruitment of GATA-6 to the BMPR2 promoter and restored BMPR2 functional expression.Conclusions: Our findings identify a key role for SMC TWIST1 in the pathogenesis of lung vascular remodeling and in PH that is partially mediated via reduced GATA-6-dependent BMPR2 expression. Inhibition of SMC TWIST1 may constitute a new therapeutic strategy for the treatment of PH.

Collecting e-cigarette aerosols for in vitro applications: A survey of the biomedical literature and opportunities to increase the value of submerged cell culture-based assessments.

Electronic nicotine delivery systems (ENDS) are being developed as potentially reduced-risk alternatives to the continued use of combustible tobacco products. Because of the widespread uptake of ENDS-in particular, e-cigarettes-the biological effects, including the toxic potential, of their aerosols are under investigation. Preclinically, collection of such aerosols is a prerequisite for testing in submerged cell culture-based in vitro assays; however, despite the growth in this research area, there is no apparent standardized collection method for this application. To this end, through an Institute for in vitro Sciences, Inc. workshop initiative, we surveyed the biomedical literature catalogued in PubMed® to map the types of methods hitherto used and reported publicly. From the 47 relevant publications retrieved, we identified seven distinct collection methods. Bubble-through (with aqueous solvents) and Cambridge filter pad (CFP) (with polar solvents) collection were the most frequently cited methods (57% and 18%, respectively), while the five others (CFP + bubble-through; condensation; cotton filters; settle-upon; settle-upon + dry) were cited less often (2-10%). Critically, the collected aerosol fractions were generally found to be only minimally characterized chemically, if at all. Furthermore, there was large heterogeneity among other experimental parameters (e.g., vaping regimen). Consequently, we recommend that more comprehensive research be conducted to identify the method(s) that produce the fraction(s) most representative of the native aerosol. We also endorse standardization of the aerosol generation process. These should be regarded as opportunities for increasing the value of in vitro assessments in relation to predicting effects on human health.

Cellular effects of nicotine salt-containing e-liquids.

"Pod-based" e-cigarettes such as JUUL are currently the most prevalent electronic nicotine delivery systems (ENDS) in the United States. JUUL-type ENDS utilize nicotine salts protonated with benzoic acid rather than freebase nicotine. However, limited information is available on the cellular effects of these products. Cytoplasmic Ca2+ is a universal second messenger that controls many cellular functions including cell growth and cell death. Of note, dysregulation of cell Ca2+ homeostasis has been linked with several disease processes including autoimmune disease and several types of cancer. We exposed HEK293T cells and THP-1 macrophage-like cells to different JUUL e-liquids. We evaluated their effects on cellular viability and Ca2+ signaling by measuring fluorescence from calcein-AM/propidium iodide and Fluo-4, respectively. E-liquid autofluorescence was used to look for e-liquid permeation into cells. To identify the mechanisms behind the Ca2+ responses, different inhibitors of Ca2+ channels and phospholipase C signaling were used. JUUL e-liquids caused significant cytotoxic effects, with "Mint" flavor being the most cytotoxic. The Mint flavored e-liquid also caused a significant elevation in cytoplasmic Ca2+ . Using autofluorescence, the permeation of JUUL e-liquids into live cells was confirmed, indicating that intracellular organelles are directly exposed to e-liquids. Further studies identified the endoplasmic reticulum as being the source of e-liquid-induced changes in cytoplasmic Ca2+ . Nicotine salt-based e-liquids cause cytotoxicity and elevate cytoplasmic Ca2+ , indicating that they can exert biological effects beyond what would be expected with nicotine alone. These effects are flavor-dependent, and we propose that flavored e-liquids be reassessed for potential lung toxicity.

Demonstration of the first-pass metabolism in the skin of the hair dye, 4-amino-2-hydroxytoluene, using the Chip2 skin-liver microphysiological model.

We used TissUse's HUMIMIC Chip2 microfluidic model, incorporating reconstructed skin models and liver spheroids, to investigate the impact of consumer-relevant application scenarios on the metabolic fate of the hair dye, 4-amino-2-hydroxytoluene (AHT). After a single topical or systemic application of AHT to Chip2 models, medium was analysed for parent and metabolites over 5 days. The metabolic profile of a high dose (resulting in a circuit concentration of 100 µM based on 100% bioavailability) of AHT was the same after systemic and topical application to 96-well EpiDerm™ models. Additional experiments indicated that metabolic capacity of EpiDerm™ models were saturated at this dose. At 2.5 µM, concentrations of AHT and several of its metabolites differed between application routes. Topical application resulted in a higher Cmax and a 327% higher area under the curve (AUC) of N-acetyl-AHT, indicating a first-pass effect in the EpiDerm™ models. In accordance with in vivo observations, there was a concomitant decrease in the Cmax and AUC of AHT-O-sulphate after topical, compared with systemic application. A similar alteration in metabolite ratios was observed using a 24-well full-thickness skin model, EpiDermFT™, indicating that a first-pass effect was also possible to detect in a more complex model. In addition, washing the EpiDermFT™ after 30 min, thus reflecting consumer use, decreased the systemic exposure to AHT and its metabolites. In conclusion, the skin-liver Chip2 model can be used to (a) recapitulate the first-pass effect of the skin and alterations in the metabolite profile of AHT observed in vivo and (b) provide consumer-relevant data regarding leave-on/rinse-off products.

Comparing the effects of an exposure to a polycyclic aromatic hydrocarbon mixture versus individual polycyclic aromatic hydrocarbons during monocyte to macrophage differentiation: Mixture exposure results in altered immune metrics.

Polycyclic aromatic hydrocarbons (PAHs) are generated by the incomplete combustion of carbon. Exposures correlate with systemic immune dysfunction and overall immune suppression. Real-world exposures to PAHs are almost always encountered as mixtures; however, research overwhelmingly centers on isolated exposures to a single PAH, benzo[a]pyrene (B[a]P). Here, a human monocyte line (U937) was exposed to B[a]P, benz[a]anthracene (B[a]A), or a mixture of six PAHs (6-MIX) to assess the differential toxicity on monocytes. Further, monocytes were exposed to PAHs with and without CYP1A1 inhibitors during macrophage differentiation to delineate PAH exposure and PAH metabolism-driven alterations to the immune response. U937 monocytes exposed to B[a]P, B[a]A, or 6-MIX had higher levels of cellular health and growth not observed following equimolar exposures to other individual PAHs. PAH exposures during differentiation did not alter monocyte-derived macrophage (MDM) numbers; however, B[a]A and 6-MIX exposures significantly altered M1/M2 polarization in a CYP1A1-dependent manner. U937-MDM adherence was differentially suppressed by all three PAH treatments with 6-MIX exposed U937-MDM having significantly more adhesion than U937-MDM exposed to either individual PAH. Finally, 6-MIX exposures during differentiation reduced U937-MDM endocytic function significantly less than B[a]A exposed cells. Exposure to a unique PAH mixture during U937-MDM differentiation resulted in mixture-specific alterations of pro-inflammatory markers compared to individual PAH exposures. While subtle, these differences highlight the probability that using a model PAH, B[a]P, may not accurately reflect the effects of PAH mixture exposures. Therefore, future studies should include various PAH mixtures that encompass probable real-world PAH exposures for the endpoints under investigation.

Cytotoxic and immunological responses of fish leukocytes to nodularin exposure in vitro.

Nodularin (NOD) is a cyclic peptide released by bloom-forming toxic cyanobacteria Nodularia spumigena commonly occurring in brackish waters throughout the world. Although its hepatotoxic effects are well known, other negative effects of NOD have not yet been completely elucidated. The present study aims were to evaluate and compare the cytotoxic and immunotoxic effects of the toxin on primary leukocytes (from head kidney [HK]) and stable fish leukocytes (carp leucocyte cell line [CLC] cells). The cells were incubated with the cyanotoxin at concentrations of 0.001, 0.01, 0.05, or 0.1 µg/ml. After 24 h of exposure, the concentrations ≥0.05 µg/ml of toxin resulted in cytotoxicity in the primary cells, while in CLC cells, the toxic effect was obtained only with the highest concentration. Similarly, depending on the concentration, exposure to NOD causes a significant inhibition of chemotaxis of the phagocytic abilities of primary leukocytes and a significant reduction in the proliferation of lymphocytes isolated from the HKs. Moreover, CLC cells and HK leukocytes incubated with this toxin at all the mentioned concentrations showed an increased production of reactive oxygen and nitrogen species. NOD also evidently influenced the expression of genes of cytokine TNF-α and IL-10 and, to a minor extent, IL-1ß and TGF-ß. Notably, the observed changes in the mRNA levels of cytokines in NOD-exposed cells were evident, but not clearly dose-dependent. Interestingly, NOD did not affect the production and release of IL-1ß of the CLC cells. This study provides evidence that NOD may exert cytotoxicity and immune-toxicity effects depending on cell type and toxin concentration.

Effect of amphotericin B-deoxycholate (Fungizone) on the mitochondria of Wistar rats' renal proximal tubules cells.

Amphotericin B-deoxycholate (Fungizone [FZ]) is a widely used potent antimycotic drug in spite of its nephrotoxic effect via different mechanisms. The effect of FZ on renal cell bioenergetics is not clear. The current study evaluated the effect of FZ on the bioenergetics of albino rats' isolated renal proximal tubule cells (PTCs). The cytotoxic effect of FZ on the isolated renal cells was assessed by MTT and lactate dehydrogenase (LDH) assays. The effect of FZ on the PTCs uptake (OAT1 and OCT2) and efflux (P-gp and MRP2) transporters was evaluated. Then, the effect of FZ on mitochondria was assessed by studying complexes I-IV activities, lactate assay, oxygen consumption rates (OCR), and western blotting for all mitochondrial complexes. Moreover, the effect of FZ on mitochondrial membrane fluidity (MMF) and fatty acids composition was evaluated. Additionally, the protective effect of coenzyme q10 was studied. Outcomes showed that FZ was cytotoxic to the PTCs in a concentration and time-dependent patterns. FZ significantly inhibited the studied uptake and efflux tubular transporters with inhibition of the mitochondrial complexes activities and parallel increase in lactate production and decrease in OCRs. Finally, FZ significantly reduced the expression of all mitochondrial complexes in addition to significant increase in MMF and MMFA concentration. Coenzyme Q10 was found to significantly decrease FZ-induced cytotoxicity and transporters impairment in the PTC. FZ significantly inhibits bioenergetics of PTC, which may stimulate the cascade of cell death and clinical nephrotoxicity.

Effects of methylparaben on in vitro maturation of porcine oocytes.

Parabens (PBs) are compounds widely used in industry for food and personal care products as antimicrobials and preservatives. Their indiscriminate use has resulted in their detection in different ecosystems so that humans and other organisms are highly exposed. Methylparaben (MePB), compared with other PBs, is mostly detected in food, personal care and baby care products. PBs could be linked to the generation of hormonal disorders and fertility impairment since their recent classification as endocrine disruptors. The knowledge of the effects that MePB can exert is of great importance as, in terms of reproduction, information is limited. Therefore, the objective of the present study was to evaluate the effect of MePB on porcine oocyte viability and in vitro maturation (IVM), as well as to determine the lethal concentration at 50% (LC50 ) and the maturation inhibition concentration at 50% (MIC50 ). Oocytes were exposed to different MePB concentrations 0 (control), 50, 100, 500, 750 and 1000 µm during 44 h of IVM. Cytoplasmic alterations and reduced cumulus cell expansion were observed in oocytes exposed to MePB; however, viability was not affected. In addition, oocyte maturation decreased in a concentration-dependent manner after exposure to MePB. The estimated LC50 was 2028.38 µm, whereas MIC50 was 780.31 µm. To our knowledge, this is the first study that demonstrates that MePB altered porcine oocyte morphology, and caused a reduction in cumulus cell expansion, both of which resulted in decreased oocyte maturation. Therefore, MePB exposure may be one of the factors involved in fertility impairment in mammals, including that of humans.

Activation of forkhead box O3a by mono(2-ethylhexyl)phthalate and its role in protection against mono(2-ethylhexyl)phthalate-induced oxidative stress and apoptosis in human cardiomyocytes.

Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of di(2-ethylhexyl)phthalate (DEHP), is known to exert cardiotoxicity. The aim of the present study was to investigate the role of forkhead box O3a (FOXO3a) in MEHP-induced human AC16 cardiomyocyte injuries. MEHP reduced cell viability and mitochondrial membrane potential (ΔΨm), whereas it increased lactate dehydrogenase (LDH) leakage, production of reactive oxygen species (ROS), and apoptosis in cardiomyocytes. The expression of FOXO3a and its target genes, mitochondrial superoxide dismutase (Mn-SOD) and apoptosis repressor with caspase recruitment domain (ARC), increased after MEHP exposure, but the expression of p-FOXO3a protein was decreased. Overexpression of FOXO3a decreased the production of ROS and the apoptosis rate induced by MEHP, and the expression of Mn-SOD and ARC was further increased after MEHP exposure. In contrast, knockdown of FOXO3a resulted in increased ROS production and apoptosis and suppressed the expression of Mn-SOD and ARC in the presence of MEHP. However, overexpression or knockdown of FOXO3a did not affect MEHP-induced loss of ΔΨm. In conclusion, the loss of ΔΨm and apoptosis are involved in MEHP-induced cardiomyocyte toxicity. Activation of FOXO3a defends against MEHP-induced oxidative stress and apoptosis by upregulating the expression of Mn-SOD and ARC in AC16 cardiomyocytes.

Structural and biochemical modifications of model and native membranes of human immune cells in response to the action of zinc oxide nanoparticles.

The development of nanotechnology has led to the increased production of zinc oxide nanoparticles (ZnO-NPs) and their application in a wide variety of everyday products. It creates the need for a full assessment of their safety for humans. The aim of the study was to assess the toxic effects of ZnO-NPs on model human cells of the immune system: U-937, HL-60, HUT-78, and COLO-720L. Particular attention was paid to the direct interaction of the nanoparticles with membrane lipids and the role of zinc ions in the mechanism of their toxicity. Cell viability, lipid peroxidation, cell membrane integrity, and the amount of zinc ions released from nanoparticles were tested. Disruption in cell metabolism was noted for ZnO-NPs concentrations from 6.25 mg/L. Contact with ZnO-NPs caused lipid peroxidation of all cells and correlated with membrane disruption of HL-60, HUT-78, and COLO-720L cells. Model monolayers (Langmuir technique) were used to assess the interaction of the nanoparticles with the studied lipids. Physicochemical parameters, such as the area per molecule at maximal layer compression, the pressure at which the monolayer collapses, and the static compression modulus, were calculated. The models of the HL-60 and U-937 cell membranes under ZnO-NPs treatment reacted in a different way. It has also been shown that Zn2+ are not the main causative factor of ZnO-NPs toxicity. Investigating the early stages of mechanism of nanoparticles toxicity will allow for a more complete risk assessment and development of methods for a safer synthesis of engineering nanomaterials.

Genetic Toxicity Studies of the Ketogenic Ester Bis Hexanoyl (R)-1,3-Butanediol.

A series of studies was conducted to assess the genetic toxicity of a novel ketone ester, bis hexanoyl (R)-1,3-butanediol (herein referred to as BH-BD), according to Organization for Economic Co-operation and Development testing guidelines under the standards of Good Laboratory Practices. In bacterial reverse mutation tests, there was no evidence of mutagenic activity in any of the Salmonella typhimurium strains tested or in Escherichia coli strain WP2uvrA, at dose levels up to 5,000 µg/plate in the presence or absence of Aroclor 1254-induced rat liver (S9 mix) for metabolic activation. In the in vitro micronucleus test using human TK6 cells, BH-BD did not show a statistically significant increase in the number of cells containing micronuclei when compared with concurrent control cultures at all time points and at any of the concentrations analyzed (up to 100 µg/mL, final concentration in culture medium), with and without S9 mix activation. In the in vivo micronucleus test using Sprague Dawley rats, BH-BD did not show a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control group. Therefore, BH-BD was concluded to be negative in all 3 tests. These results support the safety assessment of BH-BD for potential use in food.

Evaluation of the Effect of Hyaluronic Acid-Based Biomaterial Enriched With Tenascin-C on the Behavior of the Neural Stem Cells.

One of the most important natural extracellular constituents is hyaluronic acid (HA) with the potential to develop a highly organized microenvironment. In the present study, we enriched HA hydrogel with tenascin-C (TN-C) and examined the viability and survival of mouse neural stem cells (NSCs) using different biological assays. Following NSCs isolation and expansion, their phenotype was identified using flow cytometry analysis. Cell survival was measured using MTT assay and DAPI staining after exposure to various concentrations of 50, 100, 200, and 400 nM TN-C. Using acridine orange/ethidium bromide staining, we measured the number of live and necrotic cells after exposure to the combination of HA and TN-C. MTT assay revealed the highest NSCs viability rate in the group exposed to 100 nM TN-C compared to other groups, and a combination of 1% HA + 100 nM TN-C increased the viability of NSCs compared to the HA group after 24 hours. Electron scanning microscopy revealed the higher attachment of these cells to the HA + 100 nM TN-C substrate relative to the HA substrate. Epifluorescence imaging and DAPI staining of loaded cells on HA + 100 nM TN-C substrate significantly increased the number of NSCs per field over 72 hours compared to the HA group (P < 0.05). Live and dead assay revealed that the number of live NSCs significantly increased in the HA + 100 TN-C group compared to HA and control groups. The enrichment of HA substrate with TN-C promoted viability and survival of NSCs and could be applied in neural tissue engineering approaches and regenerative medicine.

Nonclinical Development of SRK-181: An Anti-Latent TGFß1 Monoclonal Antibody for the Treatment of Locally Advanced or Metastatic Solid Tumors.

Checkpoint inhibitors offer a promising immunotherapy strategy for cancer treatment; however, due to primary or acquired resistance, many patients do not achieve lasting clinical responses. Recently, the transforming growth factor-ß (TGFß) signaling pathway has been identified as a potential target to overcome primary resistance, although the nonselective inhibition of multiple TGFß isoforms has led to dose-limiting cardiotoxicities. SRK-181 is a high-affinity, fully human antibody that selectively binds to latent TGFß1 and inhibits its activation. To support SRK-181 clinical development, we present here a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies showed that SRK-181 has no effect on human platelet function and does not induce cytokine release in human peripheral blood. Four-week toxicology studies with SRK-181 showed that weekly intravenous administration achieved sustained serum exposure and was well tolerated in rats and monkeys, with no treatment-related adverse findings. The no-observed-adverse-effect levels levels were 200 mg/kg in rats and 300 mg/kg in monkeys, the highest doses tested, and provide a nonclinical safety factor of up to 813-fold (based on Cmax) above the phase 1 starting dose of 80 mg every 3 weeks. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that SRK-181 is a selective inhibitor of latent TGFß1 that does not produce the nonclinical toxicities associated with nonselective TGFß inhibition. These data support the initiation and safe conduct of a phase 1 trial with SRK-181 in patients with advanced cancer.

Effects of Reverse Transcriptase Inhibitors on Proliferation, Apoptosis, and Migration in Breast Carcinoma Cells.

High telomerase activity in human breast cancer is associated with aggressive tumors resulting in decreased survival. Recent studies have shown that telomerase inhibitors may display anticancer properties in some human cancer cell lines. In the present study, we examined the effects of 4 reverse transcriptase inhibitors (RTIs), used for the treatment of HIV; Abacavir (AC), Lamivudine (LV), Stavudine (SV), and Tenofovir (TF) on proliferation, apoptosis, and migration in the normal human mammary epithelial cell line, hTERT-HME1, and the human breast cancer cell line, MCF-7. Cells were treated with AC, LV, SV, or TF alone or in combination with paclitaxel (PAC), a known drug used to treat breast cancer. Conduct of the thiazolyl blue tetrazolium bromide assay demonstrated that AC, SV, and TF had stronger cytotoxic effects on MCF-7 cells than in hTERT-HME1 cells. The combined treatment of RTIs and PAC caused high rates of cell death in MCF-7 and low rates of cell death in HTERT-HME1 by apoptosis. The percentages of apoptotic cells in the treatment of AC and SV in combination with PAC for 48 and 72 hours were higher than PAC. Significantly increased apoptosis and decreased migration levels were found in MCF-7 cells treated with AC and co-treatment of AC+PAC or SV+PAC than HME1 cells. These treatments can also prevent migration capacity more than PAC. Therefore, a combination strategy based on telomerase inhibitors such as AC or SV and anticancer drugs may be more effective in the treatment of certain breast cancers.

Individual and combined hepatocytotoxicity of DDT and cadmium in vitro.

The organochlorine insecticide dichlorodiphenyltrichloroethane (DDT) and heavy metal cadmium (Cd) are widespread environmental pollutants. They are persistent in the environment and can accumulate in organisms. Although the individual toxicity of DDT and Cd has been well documented, their combined toxicity is still not clear. Since liver is their common target, in this study, the individual and combined toxicity of DDT and Cd in human liver carcinoma HepG2 and human normal liver THLE-3 cell lines were investigated. The results showed that DDT and Cd inhibited the viability of HepG2 and THLE-3 cells dose-dependently and altered lysosomal morphology and function. Intracellular reactive oxygen species and lipid peroxidation levels were induced by DDT and Cd treatment. The combined cytotoxicity of DDT and Cd was greater than their individual cytotoxicity, and the interaction between Cd and DDT was additive on the inhibition of cell viability and lysosomal function of HepG2 cells. The interaction was antagonistic on the inhibition of cell viability of THLE-3 cells. These results may facilitate the evaluation of the cumulative risk of pesticides and heavy metal residues in the environment.