High-throughput screening of respiratory hazards: Exploring lung surfactant inhibition with 20 benchmark chemicals.

As environmental air quality worsens and respiratory health injuries and diseases increase, it is essential to enhance our ability to develop better methods to identify potential hazards. One promising approach in emerging toxicology involves the utilization of lung surfactant as a model that addresses the limitations of conventional in vitro toxicology methods by incorporating the biophysical aspect of inhalation. This study employed a constrained drop surfactometer to assess 20 chemicals for potential surfactant inhibition. Of these, eight were identified as inhibiting lung surfactant function: 1-aminoethanol, bovine serum albumin, maleic anhydride, propylene glycol, sodium glycocholate, sodium taurocholate, sodium taurodeoxycholate, and Triton X-100. These results are consistent with previously reported chemical-induced acute lung dysfunction in vivo. The study provides information on each chemical's minimum and maximum surface tension conditions and corresponding relative area and contact angle values. Isotherms and box plots are reported for selected chemicals across doses, and vector plots are used to summarize and compare the results concisely. This lung surfactant bioassay is a promising non-animal model for hazard identification, with broader implications for developing predictive modeling and decision-making tools.

Selenium-Priming mediated growth and yield improvement of turnip under saline conditions.

Salt toxicity is one of the foremost environmental stresses that declines nutrient uptake, photosynthetic activity and growth of plants resulting in a decrease in crop yield and quality. Seed priming has become an emergent strategy to alleviate abiotic stress and improve plant growth. During the current study, turnip seed priming with sodium selenite (Na2SeO3) was investigated for its ability to mitigate salt stress. Turnip (Brassica rapa L. var. Purple Top White Globe) seeds primed with 75, 100, and 125 µML-1 of Se were subjected to 200 mM salt stress under field conditions. Findings of the current field research demonstrated that salt toxicity declined seed germination, chlorophyll content, and gas exchange characteristics of B. rapa seedling. Whereas, Se-primed seeds showed higher germination rate and plant growth which may be attributed to the decreased level of hydrogen peroxide (H2O2) and malondialdehyde (MDA) decreased synthesis of proline (36%) and besides increased total chlorophyll (46%) in applied turnip plants. Higher expression levels of genes encoding antioxidative activities (CAT, POD, SO,D and APX) mitigated oxidative stress induced by the salt toxicity. Additionally, Se treatment decreased Na+ content and enhanced K+ content resulting in elevated K+/Na+ ratio in the treated plants. The in-silico assessment revealed the interactive superiority of Se with antioxidant enzymes including CAT, POD, SOD, and APX as compared to sodium chloride (NaCl). Computational study of enzymes-Se and enzymes-NaCl molecules also revealed the stress ameliorative potential of Se through the presence of more Ramachandran-favored regions (94%) and higher docking affinities of Se (-6.3). The in-silico studies through molecular docking of Na2SeO3, NaCl, and ROS synthesizing enzymes (receptors) including cytochrome P450 (CYP), lipoxygenase (LOX), and xanthine oxidase (XO), also confirmed the salt stress ameliorative potential of Se in B. rapa. The increased Ca, P, Mg, and Zn nutrients uptake nutrients uptake in 100 µML-1 Se primed seedlings helped to adjust the stomatal conductivity (35%) intercellular CO2 concentration (32%), and photosynthetic activity (41%) resulting in enhancement of the yield attributes. More number of seeds per plant (6%), increased turnip weight (115 gm) root length (17.24 cm), root diameter (12 cm) as well as turnip yield increased by (9%tons ha-1) were recorded for 100 µML-1 Se treatment under salinity stress. Findings of the current research judiciously advocate the potential of Se seed priming for salt stress alleviation and growth improvement in B. rapa.

According to our best of knowledge, it is the first time that seed primed with Selenium have been evaluated regarding NaCl stress mitigation in turnip. Salinity toxicity negatively affected physiochemical activities and growth of B.rapa.Seed priming with Selenium (Na₂SeO₃) mitigated salinity stress.Selenium (Se) enhanced nutrition, photosynthetic and antioxidant activity of applied plants.Selenium treated plants exhibited improved growth and reduced salinity content.

Toxicity assessment of CeO2 and CuO nanoparticles at the air-liquid interface using bioinspired condensational particle growth.

CeO2 and CuO nanoparticles (NPs) are used as additives in petrodiesel to enhance engine performance leading to reduced diesel combustion emissions. Despite their benefits, the additive application poses human health concerns by releasing inhalable NPs into the ambient air. In this study, a bioinspired lung cell exposure system, Dosimetric Aerosol in Vitro Inhalation Device (DAVID), was employed for evaluating the toxicity of aerosolized CeO2 and CuO NPs with a short duration of exposure (≤10 min vs. hours in other systems) and without exerting toxicity from non-NP factors. Human epithelial A549 lung cells were cultured and maintained within DAVID at the air-liquid interface (ALI), onto which aerosolized NPs were deposited, and experiments in submerged cells were used for comparison. Exposure of the cells to the CeO2 NPs did not result in detectable IL-8 release, nor did it produce a significant reduction in cell viability based on lactate dehydrogenase (LDH) assay, with a marginal decrease (10%) at the dose of 388 µg/cm2 (273 cm2/cm2). In contrast, exposure to CuO NPs resulted in a concentration dependent reduction in LDH release based on LDH leakage, with 38% reduction in viability at the highest dose of 52 µg/cm2 (28.3 cm2/cm2). Cells exposed to CuO NPs resulted in a dose dependent cellular membrane toxicity and expressed IL-8 secretion at a global dose five times lower than cells exposed under submerged conditions. However, when comparing the ALI results at the local cellular dose of CuO NPs to the submerged results, the IL-8 secretion was similar. In this study, we demonstrated DAVID as a new exposure tool that helps evaluate aerosol toxicity in simulated lung environment. Our results also highlight the necessity in choosing the right assay endpoints for the given exposure scenario, e.g., LDH for ALI and Deep Blue for submerged conditions for cell viability.

Potassium silicate and zinc oxide nanoparticles modulate antioxidant system, membranous H+-ATPase and nitric oxide content in faba bean (Vicia faba) seedlings exposed to arsenic toxicity.

Current research focused on the potential role of zinc oxide nanoparticles (ZnONPs) and potassium (K+ ) in mitigation of arsenic (As) toxicity in Vicia faba L. seedlings. Faba bean seedlings were grown for 30days in potted soil. As stress curtailed root and shoot length, chlorophyll (Chl) content and net photosynthetic rate in V. faba seedlings. However, ZnONPs and K+ curtailed As stress in faba bean seedling through enhanced activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) enzyme. Furthermore, ZnONPs and K+ significantly enhanced cysteine (Cys) content and serine acetyletransferase (SAT) activity in faba bean seedling exposed to As-toxificated soil. Application of ZnONPs and K+ curtailed superoxide ionic content and hydrogen peroxide (H2 O2 ) accumulation in V. faba seedlings exposed to As-polluted soil. Nitric oxide (NO) content also increased in faba bean seedlings treated with ZnONPs and K+ in normal and As-polluted soil. As stress alleviation was credited to reduce As uptake in faba bean seedlings treated with synergistic application of ZnONPs and K+ . It is proposed that K+ interaction with nanoparticles can be exploited at molecular level to understand the mechanisms involved in abiotic stress tolerance.

In-silico Studies Calculated a New Chitin Oligomer Binding Site Inside Vicilin: A Potent Antifungal and Insecticidal Agent.

Vicilins are major seed storage proteins and show differential binding affinities toward sugar moieties of fungal cell wall and insect gut epithelium. Hence, purpose of study is the thorough in-silico characterization of interactions between vicilin and chitin oligomer followed by fungal and insecticidal bioassays. This work covers the molecular simulation studies explaining the interactions between Pisum sativum vicilin (PsV) and chitin oligomer followed by protein bioassay against different pathogens. LC-MS/MS of purified PsV (∼50 kDa) generated residual data along high pea vicilin homology (UniProtKB ID; P13918). Predicted model (PsV) indicated the characteristic homotrimer joined through head-to-tail association and each monomer is containing a bicupin domain. PsV site map analysis showed a new site (Site 4) into which molecular docking confirmed the strong binding of chitin oligomer (GlcNAc)4. Molecular dynamics simulation data (50 ns) indicated that chitin-binding site was comprised of 8 residues (DKEDRNEN). However, aspartate and glutamate significantly contributed in the stability of ligand binding. Computational findings were further verified via significant growth inhibition of Aspergillus flavus, A. niger, and Fusarium oxysporum against PsV. Additionally, the substantial adult population of Brevicoryne brassicae was reduced and different life stages of Tribolium castaneum also showed significant mortality.

Comprehensive Structural and Functional Characterization of a Seed γ-thionin as a Potent Bioactive Molecule Against Fungal Pathogens and Insect Pests.

BACKGROUND: Fungi and insect pests ruin stored crop grain, which results in millions of dollars of damage, presenting an ongoing challenge for farmers in addition to diminishing the safety of stored food. A wide-range defensive system against pathogens is needed to reduce or even eliminate the dependence of the crop yield upon the use of pesticides. Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) that are a component of the host defense system. They are known to interact with cell membranes to exhibit antifungal and insecticidal activity. They exhibit a broad range of activities against fungi and insects and are effective at low concentrations. Thionins act on membranes, greatly reducing the development of pathogen resistance. OBJECTIVE: The aim of this study is to investigate a bioactive molecule that acts against fungal pathogens and stored grain insect pests. METHODS: γ-thionin protein was extracted from Brassica oleracea L. var. capitata f. alba (white cabbage) seed powder in phosphate buffer (100 mM, pH 7.0) and was identified by MALDI-TOF/TOF. The crude extract was subjected to 70% ammonium sulfate saturation followed by gel filtration chromatography. The disc diffusion assay along with a microtiter bioassay was used to determine the antifungal activity of the protein against phytopathogenic fungi. The insecticidal efficacy was evaluated by feeding insect pests with food contaminated with the purified protein. Additionally, an in silico molecular structure prediction study of the protein was performed using Auto Dock Vina for molecular docking of the protein with either fungal membrane moieties or α-amylase from Tenebrio molitor L. MD simulations of protein-ligand complexes were conducted using Schrodinger's Desmond module. RESULTS: γ-Thionin (BoT) was purified from white cabbage seeds and showed 100% homology with thionin (Brassica oleracea L. var. viridis) and 80% homology with defensin-like protein 1 (Raphanus sativus L.), respectively. BoT significantly inhibited the mycelial growth of Aspergillus niger van Tieghem and Aspergillus flavus Link at a concentration of 2 µM. Similarly, 0.12 µM BoT treatment resulted in significant mortality of Tribolium castaneum Herbst and Sitophilus oryzae L. Molecular docking and MD simulation of BoT confirmed the strong binding affinity with fungal membrane moieties (phosphatidylinositol 4,5-bisphosphate and phosphatidic acid), which causes disruption of the cell membrane and leakage of the cellular contents, leading to cell death. BoT blocked the active site of α-amylase, and as a result of the inactivation of this gut enzyme, the digestive systems of insects were disturbed, resulting in their deaths. CONCLUSION: This study revealed that γ-thionin is a good antifungal and insecticidal agent that could be used as an alternate to fungicides and insecticides.

The impact of newly synthesized sulfonamides on soil microbial population and respiration in rhizospheric soil of wheat (Triticum aestivum L.).

Antibiotics released into agricultural fields through the manure of grazing animals could exert harmful impacts on soil microbes and plants. Antibiotics exert high impacts on environment than other pharmaceuticals due to their higher biological activity. However, little is known about their impacts on plants, despite indications that antibiotics exert negative effects on soil microorganisms, which ultimately harm the plants. It has been demonstrated that beneficial microorganisms promote plant growth and development under various stresses. This study evaluated the toxicity of four newly derived sulfonamides (SAs), i.e., 2-(phenylsulfonyl) hydrazine carbothioamide (TSBS-1), N, 2-bis phenyl hydrazine carbothioamide (TSBS-2), aminocarbonyl benzene sulfonamide (UBS-1), and N, N'-carbonyl dibenzene sulfonamide (UBS-2) on bacterial growth and soil microbial respiration. Each SA was tested at four different concentrations (i.e., 2.25, 2.5, 3, 4 mg/ml) against five rhizospheric bacterial strains, including AC (Actinobacteria sp.), RS-3a (Bacillus sp.), RS-7a (Bacillus subtilis), RS-4a (Enterobacter sp.), and RS-5a (Enterobacter sp.). Antimicrobial activity was checked by disc diffusion method, which showed that inhibition zone increased with increasing concentration of SAs. The UBS-1 resulted in the highest inhibition zone (11.47 ± 0.90 mm) against RS-4a with the highest concentration (4 mg/ml). Except TSBS-1, all sulfonamide derivatives reduced CO2 respiration rates in soil. Soil respiration values significantly increased till 6th day; however, exposure of sulfonamide derivatives suppressed microbial respiration after 6th day. On the 20th day, poor respiration activity was noted at 0.23, 0.2, and 0.4 (CO2 mg/g dry soil) for TSBS-1, UBS-1, and UBS-2, respectively. Our results demonstrate that sulfonamides, even in small concentrations, significantly affect soil microbial population and respiration. Soil microbial respiration changes mediated by sulfonamides were dependent on length of exposure and concentration. It is recommended that antibiotics should be carefully watched and their impact on plant growth should be tested in the future studies.

3D printed transwell-integrated nose-on-chip model to evaluate effects of air flow-induced mechanical stresses on mucous secretion.

While there are many chip models that simulate the air-tissue interface of the respiratory system, only a few represent the upper respiratory system. These chips are restricted to unidirectional flow patterns that are not comparable to the highly dynamic and variable flow patterns found in the native nasal cavity. Here we describe the development of a tunable nose-on-chip device that mimics the air-mucosa interface and is coupled to an air delivery system that simulates natural breathing patterns through the generation of bi-directional air flow. Additionally, we employ computational modeling to demonstrate how the device design can be tuned to replicate desired mechanical characteristics within specific regions of the human nasal cavity. We also demonstrate how to culture human nasal epithelial cell line RPMI 2650 within the lab-on-chip (LOC) device. Lastly, Alcian Blue histological staining was performed to label mucin proteins, which play important roles in mucous secretion. Our results revealed that dynamic flow conditions can increase mucous secretion for RPMI 2650 cells, when compared to no flow, or stationary, conditions.

Selenium seed priming enhanced the growth of salt-stressed Brassica rapa L. through improving plant nutrition and the antioxidant system.

Various abiotic stresses may affect the germination, growth, and yield of direct-seeded vegetable crops. Seed priming with effective antioxidant mediators may alleviate these environmental stresses by maintaining uniformity in seed germination and improving the subsequent health of developing seedlings. Salt-induced stress has become a limiting factor for the successful cultivation of Brassica rapa L., especially in Southeast Asian countries. The present study was performed to elucidate the efficacy of seed priming using selenium (Se) in mitigating salt-induced oxidative stress in turnip crops by reducing the uptake of Na+. In this study, we administered three different levels of Se (Se-1, 75 µmol L-1; Se-2, 100 µmol L-1; and Se-3, 125 µmol L-1) alone or in combination with NaCl (200 mM). Conspicuously, salinity and Se-2 modulated the expression levels of the antioxidant genes, including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX). The upregulated expression of stress-responsive genes alleviated salt stress by scavenging the higher reactive oxygen species (ROS) level. The stress ameliorative potential of Se (Se-2 = 100 µmol L-1) enhanced the final seed germination percentage, photosynthetic content, and seedling biomass production up to 48%, 56%, and 51%, respectively, under stress. The advantageous effects of Se were attributed to the alleviation of salinity stress through the reduction of the levels of malondialdehyde (MDA), proline, and H2O2. Generally, treatment with Se-2 (100 µmo L-1) was more effective in enhancing the growth attributes of B. rapa compared to Se-1 (75 µmo L-1) and Se-3 (125 µmo L-1) under salt-stressed and non-stressed conditions. The findings of the current study advocate the application of the Se seed priming technique as an economical and eco-friendly approach for salt stress mitigation in crops grown under saline conditions.

A Dual Approach of an Oil-Membrane Composite and Boron-Doped Diamond Electrode to Mitigate Biofluid Interferences.

Electrochemical biosensors promise a simple method to measure analytes for both point-of-care diagnostics and continuous, wearable biomarker monitors. In a liquid environment, detecting the analyte of interest must compete with other solutes that impact the background current, such as redox-active molecules, conductivity changes in the biofluid, water electrolysis, and electrode fouling. Multiple methods exist to overcome a few of these challenges, but not a comprehensive solution. Presented here is a combined boron-doped diamond electrode and oil-membrane protection approach that broadly mitigates the impact of biofluid interferents without a biorecognition element. The oil-membrane blocks the majority of interferents in biofluids that are hydrophilic while permitting passage of important hydrophobic analytes such as hormones and drugs. The boron-doped diamond then suppresses water electrolysis current and maintains peak electrochemical performance due to the foulant-mitigation benefits of the oil-membrane protection. Results show up to a 365-fold reduction in detection limits using the boron-doped diamond electrode material alone compared with traditional gold in the buffer. Combining the boron-doped diamond material with the oil-membrane protection scheme maintained these detection limits while exposed to human serum for 18 h.

Oil-Membrane Protection of Electrochemical Sensors for Fouling- and pH-Insensitive Detection of Lipophilic Analytes.

To take full advantage of the reagent- and label-free sensing capabilities of electrochemical sensors, a frequent and remaining challenge is interference and degradation of the sensors due to uncontrolled pH or salinity in the sample solution or foulants from the sample solution. Here, we present an oil-membrane sensor protection technique that allows for the permeation of hydrophobic (lipophilic) analytes into a sealed sensor compartment containing ideal salinity and pH conditions while simultaneously blocking common hydrophilic interferents (proteins, acids, bases, etc.) In this paper, we validate the oil-membrane sensor protection technique by demonstrating continuous cortisol detection via electrochemical aptamer-based (EAB) sensors. The encapsulated EAB cortisol sensor exhibits a 5 min concentration-on rise time and maintains a measurement signal of at least 7 h even in the extreme condition of an acidic solution of pH 3.

Examining cellular responses to reconstituted antibody protein liquids.

Protein ionic liquids (PIL) are a new class of biologic stabilizers designed to protect the functionality and extend the shelf-life of biotechnological and therapeutic agents making them more readily available, and resistant to austere environments. Protein biorecognition elements such as monoclonal antibodies are commonly utilized therapeutics that require the robust stabilization offered by PILs, but biocompatibility remains an important issue. This study has focused on characterizing the biocompatibility of an antibody based PIL by exposing multiple cells types to a cationized immunoglobulin suspended in an anionic liquid (IgG-IL). The IgG-IL caused no significant alterations in cellular health for all three cell types with treatments < 12.5 µg/mL. Concentrations ≥ 12.5 µg/mL resulted in significant necrotic cell death in A549 and HaCaT cells, and caspase associated cell death in HepG2 cells. In addition, all cells displayed evidence of oxidative stress and IL-8 induction in response to IgG-IL exposures. Therapeutic Ig can be utilized with a wide dose range that extends into concentrations we have found to exhibit cytotoxicity raising a toxicity concern and a need for more extensive understanding of the biocompatibility of IgG-ILs.

Engineered aluminum nanoparticle induces mitochondrial deformation and is predicated on cell phenotype.

The main role of mitochondria is to generate the energy necessary for the cell to survive and adapt to different environmental stresses. Energy demand varies depending on the phenotype of the cell. To efficiently meet metabolic demands, mitochondria require a specific proton homeostasis and defined membrane structures to facilitate adenosine triphosphate production. This homeostatic environment is constantly challenged as mitochondria are a major target for damage after exposure to environmental contaminants. Here we report changes in mitochondrial structure profiles in different cell types using electron microscopy in response to particle stress exposure in three different representative lung cell types. Endpoint analyses include nanoparticle intracellular uptake; quantitation of mitochondrial size, shape, and ultrastructure; and confirmation of autophagosome formation. Results show that low-dose aluminum nanoparticles exposure (1 ppm; 1 µg/mL; 1.6 × 1 0-7 µg/cell)) to primary and asthma cells incurred significant mitochondrial deformation and increases in mitophagy, while cancer cells exhibited only slight changes in mitochondrial morphology and an increase in lipid body formation. These results show low-dose aluminum nanoparticle exposure induces subtle changes in the mitochondria of specific lung cells that can be quantified with microscopy techniques. Furthermore, within the lung, cell type by the nature of origin (i.e. primary vs. cancer vs. asthma) dictates mitochondrial morphology, metabolic health, and the metabolic stress response of the cell.

Biofabrication of endothelial cell, dermal fibroblast, and multilayered keratinocyte layers for skin tissue engineering.

The skin serves a substantial number of physiological purposes and is exposed to numerous biological and chemical agents owing to its large surface area and accessibility. Yet, current skin models are limited in emulating the multifaceted functions of skin tissues due to a lack of effort on the optimization of biomaterials and techniques at different skin layers for building skin frameworks. Here, we use biomaterial-based approaches and bioengineered techniques to develop a 3D skin model with layers of endothelial cell networks, dermal fibroblasts, and multilayered keratinocytes. Analysis of mechanical properties of gelatin methacryloyl (GelMA)-based bioinks mixed with different portions of alginate revealed bioprinted endothelium could be better modeled to optimize endothelial cell viability with a mixture of 7.5% GelMA and 2% alginate. Matrix stiffness plays a crucial role in modulating produced levels of Pro-Collagen I alpha-1 and matrix metalloproteinase-1 in human dermal fibroblasts and affecting their viability, proliferation, and spreading. Moreover, seeding human keratinocytes with gelatin-coating multiple times proved to be helpful in reducing culture time to create multiple layers of keratinocytes while maintaining their viability. The ability to fabricate selected biomaterials for each layer of skin tissues has implications in the biofabrication of skin systems for regenerative medicine and disease modeling.

Model prediction of a Kunitz-type trypsin inhibitor protein from seeds of Acacia nilotica L. with strong antimicrobial and insecticidal activity.

A Kunitz-type trypsin inhibitor protein has been purified and characterized from seeds of Acacia nilotica L. LC-MS/MS analysis of Acacia nilotica trypsin inhibitor (AnTI) provided the N-terminal fragment of 11 amino acids which yielded 100% identity with already reported Kunitz-type trypsin inhibitor protein of Acacia confusa (AcTI) in UniProtKB database search. SDS-PAGE showed a single band of ~21 kDa under nonreduced condition and appearance of a daughter band (17 kDa) in the presence of ß-mercaptoethanol indicating the presence of interchain disulfide linkage typical for Kunitz-type trypsin inhibitors. AnTI was purified from seed extract by using a combination of anion exchange and gel filtration chromatography. Since AnTI showed maximum homology with AcTI, a molecular structure of AcTI was predicted which showed highly ß-sheeted molecular conformation similar to crystallographic structure of Enterolobium contortisiliquum trypsin inhibitor (EcTI). AnTI (20 µg) produces significant population inhibition against different human pathogenic bacteria along strong antifungal activity (50 µg). Entomotoxin potential of AnTI was evaluated against two stored grain insect pests Tribolium castaneum (Herbst) (Tenebrionidae: Coleoptera) and Sitophilus oryzae (Linnaeus) (Curculionidae: Coleoptera). Statistically significant mortality of T. castaneum adults was observed at 1.5 mg after 15 days in comparison to control. Additionally, number of total eggs, larvae, pupae, adults, and their male/female ratio were also severely reduced in comparison to control. Similarly, two generation progeny of S. oryzae was studied after mixing AnTI with rice kernels. Mean percent mortality of adult population was significantly higher after 9 days of exposure in comparison to control group. AnTI significantly reduced the F1 generation while little mortality was observed for F2 generation. Exploration of such potent molecules is the prerequisite of our time regarding the anticipation of postantibiotic era and the development of insect resistance against chemical pesticides.

Human Nontumorigenic Microglia Synthesize Strongly Fluorescent Au/Fe Nanoclusters, Retaining Bioavailability.

The ability for cells to self-synthesize metal-core nanoclusters (mcNCs) offers increased imaging and identification opportunities. To date, much work has been done illustrating the ability for human tumorigenic cell lines to synthesize mcNCs; however, this has not been illustrated for nontumorigenic cell lines. Here, we present the ability for human nontumorigenic microglial cells, which are the major immune cells in the central nervous system, to self-synthesize gold (Au) and iron (Fe) core nanoclusters, following exposures to metallic salts. We also show the ability for cells to internalize presynthesized Au and Fe mcNCs. Cellular fluorescence increased in most exposures and in a dose dependent manner in the case of Au salt. Scanning transmission electron microscopic imaging confirmed the presence of the metal within cells, while transmission electron microscopy images confirmed nanocluster structures and self-synthesis. Interestingly, self-synthesized nanoclusters were of similar size and internal structure as presynthesized mcNCs. Toxicity assessment of both salts and presynthesized NCs illustrated a lack of toxicity from Au salt and presynthesized NCs. However, Fe salt was generally more toxic and stressful to cells at similar concentrations.

Interparticle Molecular Exchange of Surface Chemical Components of Native High-Density Lipoproteins to Complementary Nanoparticle Scaffolds.

High-density lipoproteins (HDL) are constitutionally dynamic nanoparticles that circulate in the blood. The biological functions of HDLs are impacted by interchangeable surface chemical components, like cholesterol and HDL-associated proteins. Current methods to quantify the chemical constituents of HDL are largely restricted to clinical or academic laboratories and require expensive instrumentation, and there is no commonality to the techniques required to detect and quantify different analytes (e.g., cholesterol versus HDL-associated protein). To potentially facilitate and streamline the analysis of HDL composition, we hypothesized that mixing native HDLs with similarly sized gold nanoparticles whose surfaces are endowed with phospholipids, called complementary nanoparticle scaffolds (CNS), would enable interparticle exchange of surface components. Then, easy isolation of the newly formed particles could be accomplished using benchtop centrifugation for subsequent measurement of HDL components exchanged to the surface of the CNS. As proof-of-concept, data demonstrate that CNS incubated with only a few microliters of human serum rapidly (1 h) sequester cholesterol and HDL-associated proteins with direct correlation to native HDLs. As such, data show that the CNS assay is a single platform for rapid isolation and subsequent detection of the surface components of native HDLs.

A Glial-Silicon Nanowire Electrode Junction Enabling Differentiation and Noninvasive Recording of Slow Oscillations from Primary Astrocytes.

The correct human brain function is dependent on the activity of non-neuronal cells called astrocytes. The bioelectrical properties of astrocytes in vitro do not closely resemble those displayed in vivo and the former are incapable of generating action potential; thus, reliable approaches in vitro for noninvasive electrophysiological recording of astrocytes remain challenging for biomedical engineering. Here it is found that primary astrocytes grown on a device formed by a forest of randomly oriented gold coated-silicon nanowires, resembling the complex structural and functional phenotype expressed by astrocytes in vivo. The device enables noninvasive extracellular recording of the slow-frequency oscillations generated by differentiated astrocytes, while flat electrodes failed on recording signals from undifferentiated cells. Pathophysiological concentrations of extracellular potassium, occurring during epilepsy and spreading depression, modulate the power of slow oscillations generated by astrocytes. A reliable approach to study the role of astrocytes function in brain physiology and pathologies is presented.

Determining the Biological Mechanisms of Action for Environmental Exposures: Applying CRISPR/Cas9 to Toxicological Assessments.

Toxicology is a constantly evolving field, especially in the area of developing alternatives to animal testing. Toxicological research must evolve and utilize adaptive technologies in an effort to improve public, environmental, and occupational health. The most commonly cited mechanisms of toxic action after exposure to a chemical or particle test substance is oxidative stress. However, because oxidative stress involves a plethora of genes and proteins, the exact mechanism(s) are not commonly defined. Exact mechanisms of toxicity can be revealed using an emerging laboratory technique referred to as CRISPR (clustered regularly interspaced short palindromic repeats). This article reviews the most common CRISPR techniques utilized today and how each may be applied in Toxicological Sciences. Specifically, the CRISPR/CRISPR-associated protein complex is used for single gene knock-outs, whereas CRISPR interference/activation is used for silencing or activating (respectively) ribonucleic acid. Finally, CRISPR libraries are used for knocking-out entire gene pathways. This review highlights the application of CRISPR in toxicology to elucidate the exact mechanism through which toxicants perturb normal cellular functions.

In Vitro Aerosol Exposure to Nanomaterials: From Laboratory to Environmental Field Toxicity Testing.

Exposure to nanomaterials (NMs) is inevitable, requiring robust toxicological assessment to understand potential environmental and human health effects. NMs are favored in many applications because of their small size; however, this allows them to easily aerosolize and, subsequently, expose humans via inhalation. Toxicological assessment of NMs by conventional methods in submerged cell culture is not a relevant way to assess inhalation toxicity of NMs because of particle interference with bioassays and changes in particokinetics when dispersed in medium. Therefore, an in vitro aerosol exposure chamber (AEC) was custom designed and used for direct deposition of NMs from aerosols in the environment to the air-liquid interface of lung cells. Human epithelial lung cell line, A549, was used to assess the toxicity of copper, nickel, and zinc oxide nanopowders aerosolized by acoustic agitation in laboratory study. Post optimization, the AEC was used in the field to expose the A549 cells to NM aerosols generated from firing a hand gun and rifle. Toxicity was assessed using nondestructive assays for cell viability and inflammatory response, comparing the biologic effect to the delivered mass dose measured by inductively coupled plasma-mass spectrometry. The nanopowder exposure to submerged and ALI cells resulted in dose-dependent toxicity. In the field, weapon exhaust from the M4 reduced cell viability greater than the M9, while the M9 stimulated inflammatory cytokine release of IL-8. This study highlights the use of a portable chamber with the capability to assess toxicity of NM aerosols exposed to air-liquid interface in vitro lung cell culture.