CRISPR/Cas9-mediated editing of ACTB in induced pluripotent stem cells: A model for investigating human ACTB loss-of-function and genetic adaptive responses.

Heterozygous beta-actin (ACTB) indel and nonsense mutations are linked to developmental disorders. We generated two CRISPR/Cas9 human induced pluripotent stem cell (iPSC) lines, WTSIi018-B-19 and WTSIi018-B-20, carrying heterozygous and homozygous indel mutations in ACTB exon 4. Both iPSCs exhibited normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. While iPSCs with a heterozygous ACTB mutation maintain genome integrity, homozygous mutants showed a loss of heterozygosity in chromosome three. These mutants provide a powerful model to study the onset, progression, and complex interplay of genetic compensation and phenotypic variation of ACTB-related diseases.

Prime-Editing of human ACTB in induced pluripotent stem cells to model human ACTB Loss-of-Function diseases and compensatory mechanisms.

Beta-actin (ACTB) heterozygous loss-of-function mutations are associated with pleiotropic developmental disorders entailing intellectual disability and frequent organ malformations in affected individuals. We generated two CRISPR/Cas9 prime-edited human induced pluripotent stem cell (iPSC) lines, IUFi004-A-1 and IUFi004-A-2, carrying a heterozygous missense mutation in exon 4 of ACTB. Mutant iPSCs exhibited normal cell morphology and genomic integrity, maintained expression of pluripotency markers, and differentiated into the three primary germ layers. The mutants offer a valuable platform for examining the molecular and functional consequences of ACTB haploinsufficiency, developing effective treatments, and exploring mechanisms underlying phenotypic variability and genetic compensation observed in monogenic diseases.

Biological impact of sequential exposures to allergens and ultrafine particle-rich combustion aerosol on human bronchial epithelial BEAS-2B cells at the air liquid interface.

The prevalence of allergic diseases is constantly increasing since few decades. Anthropogenic ultrafine particles (UFPs) and allergenic aerosols is highly involved in this increase; however, the underlying cellular mechanisms are not yet understood. Studies observing these effects focused mainly on singular in vivo or in vitro exposures of single particle sources, while there is only limited evidence on their subsequent or combined effects. Our study aimed at evaluating the effect of subsequent exposures to allergy-related anthropogenic and biogenic aerosols on cellular mechanism exposed at air-liquid interface (ALI) conditions. Bronchial epithelial BEAS-2B cells were exposed to UFP-rich combustion aerosols for 2 h with or without allergen pre-exposure to birch pollen extract (BPE) or house dust mite extract (HDME). The physicochemical properties of the generated particles were characterized by state-of-the-art analytical instrumentation. We evaluated the cellular response in terms of cytotoxicity, oxidative stress, genotoxicity, and in-depth gene expression profiling. We observed that single exposures with UFP, BPE, and HDME cause genotoxicity. Exposure to UFP induced pro-inflammatory canonical pathways, shifting to a more xenobiotic-related response with longer preincubation time. With additional allergen exposure, the modulation of pro-inflammatory and xenobiotic signaling was more pronounced and appeared faster. Moreover, aryl hydrocarbon receptor (AhR) signaling activation showed to be an important feature of UFP toxicity, which was especially pronounced upon pre-exposure. In summary, we were able to demonstrate the importance of subsequent exposure studies to understand realistic exposure situations and to identify possible adjuvant allergic effects and the underlying molecular mechanisms.

Construction of an In Vitro Air-Liquid Interface Exposure System to Assess the Toxicological Impact of Gas and Particle Phase of Semi-Volatile Organic Compounds.

Anthropogenic activities and industrialization render continuous human exposure to semi-volatile organic compounds (SVOCs) inevitable. Occupational monitoring and safety implementations consider the inhalation exposure of SVOCs as critically relevant. Due to the inherent properties of SVOCs as gas/particle mixtures, risk assessment strategies should consider particle size-segregated SVOC association and the relevance of released gas phase fractions. We constructed an in vitro air-liquid interface (ALI) exposure system to study the distinct toxic effects of the gas and particle phases of the model SVOC dibutyl phthalate (DBP) in A549 human lung epithelial cells. Cytotoxicity was evaluated and genotoxic effects were measured by the alkaline and enzyme versions of the comet assay. Deposited doses were assessed by model calculations and chemical analysis using liquid chromatography tandem mass spectrometry. The novel ALI exposure system was successfully implemented and revealed the distinct genotoxic effects of the gas and particle phases of DBP. The empirical measurements of cellular deposition and the model calculations of the DBP particle phase were concordant.The model SVOC DBP showed that inferred oxidative DNA damage may be attributed to particle-related effects. While pure gas phase exposure may follow a distinct mechanism of genotoxicity, the contribution of the gas phase to total aerosol was comparably low.

Exposure to naphthalene and ß-pinene-derived secondary organic aerosol induced divergent changes in transcript levels of BEAS-2B cells.

The health effects of exposure to secondary organic aerosols (SOAs) are still limited. Here, we investigated and compared the toxicities of soot particles (SP) coated with ß-pinene SOA (SOAßPin-SP) and SP coated with naphthalene SOA (SOANap-SP) in a human bronchial epithelial cell line (BEAS-2B) residing at the air-liquid interface. SOAßPin-SP mostly contained oxygenated aliphatic compounds from ß-pinene photooxidation, whereas SOANap-SP contained a significant fraction of oxygenated aromatic products under similar conditions. Following exposure, genome-wide transcriptome responses showed an Nrf2 oxidative stress response, particularly for SOANap-SP. Other signaling pathways, such as redox signaling, inflammatory signaling, and the involvement of matrix metalloproteinase, were identified to have a stronger impact following exposure to SOANap-SP. SOANap-SP also induced a stronger genotoxicity response than that of SOAßPin-SP. This study elucidated the mechanisms that govern SOA toxicity and showed that, compared to SOAs derived from a typical biogenic precursor, SOAs from a typical anthropogenic precursor have higher toxicological potency, which was accompanied with the activation of varied cellular mechanisms, such as aryl hydrocarbon receptor. This can be attributed to the difference in chemical composition; specifically, the aromatic compounds in the naphthalene-derived SOA had higher cytotoxic potential than that of the ß-pinene-derived SOA.

A comparative study of persistent DNA oxidation and chromosomal instability induced in vitro by oxidizers and reference airborne particles.

Adverse health effects driven by airborne particulate matter (PM) are mainly associated with reactive oxygen species formation, pro-inflammatory effects, and genome instability. Therefore, a better understanding of the underlying mechanisms is needed to evaluate health risks caused by exposure to PM. The aim of this study was to compare the genotoxic effects of two oxidizing agents (menadione and 3-chloro-1,2-propanediol) with three different reference PM (fine dust ERM-CZ100, urban dust SRM1649, and diesel PM SRM2975) on monocytic THP-1 and alveolar epithelial A549 cells. We assessed DNA oxidation by measuring the oxidized derivative 8-hydroxy-2'-deoxyguanosine (8-OHdG) following short and long exposure times to evaluate the persistency of oxidative DNA damage. Cytokinesis-block micronucleus cytome assay was performed to assess chromosomal instability, cytostasis, and cytotoxicity. Particles were characterized by inductively coupled plasma mass spectrometry in terms of selected elemental content, the release of ions in cell medium and the cellular uptake of metals. PM deposition and cellular dose were investigated by a spectrophotometric method on adherent A549 cells. The level of lipid peroxidation was evaluated via malondialdehyde concentration measurement. Despite differences in the tested concentrations, deposition efficiency, and lipid peroxidation levels, all reference PM samples caused oxidative DNA damage to a similar extent as the two oxidizers in terms of magnitude but with different oxidative DNA damage persistence. Diesel SRM2975 were more effective in inducing chromosomal instability with respect to fine and urban dust highlighting the role of polycyclic aromatic hydrocarbons derivatives on chromosomal instability. The persistence of 8-OHdG lesions strongly correlated with different types of chromosomal damage and revealed distinguishing sensitivity of cell types as well as specific features of particles versus oxidizing agent effects. In conclusion, this study revealed that an interplay between DNA oxidation persistence and chromosomal damage is driving particulate matter-induced genome instability.

In vitro genotoxicity of dibutyl phthalate on A549 lung cells at air-liquid interface in exposure concentrations relevant at workplaces.

The ubiquitous use of phthalates in various materials and the knowledge about their potential adverse effects is of great concern for human health. Several studies have uncovered their role in carcinogenic events and suggest various phthalate-associated adverse health effects that include pulmonary diseases. However, only limited information on pulmonary toxicity is available considering inhalation of phthalates as the route of exposure. While in vitro studies are often based on submerged exposures, this study aimed to expose A549 alveolar epithelial cells at the air-liquid interface (ALI) to unravel the genotoxic and oxidative stress-inducing potential of dibutyl phthalate (DBP) with concentrations relevant at occupational settings. Within this scope, a computer modeling approach calculating alveolar deposition of DBP particles in the human lung was used to define in vitro ALI exposure conditions comparable to potential occupational DBP exposures. The deposited mass of DBP ranged from 0.03 to 20 ng/cm2 , which was comparable to results of a human lung particle deposition model using an 8 h workplace threshold limit value of 580 µg/m3 proposed by the Scientific Committee on Occupational Exposure Limits for the European Union. Comet and Micronucleus assay revealed that DBP induced genotoxicity at DNA and chromosome level in sub-cytotoxic conditions. Since genomic instability was accompanied by increased generation of the lipid peroxidation marker malondialdehyde, oxidative stress might play an important role in phthalate-induced genotoxicity. The results highlight the importance of adapting in vitro studies to exposure scenarios relevant at occupational settings and reconsidering occupational exposure limits for DBP.

The cytokine network in acute myeloid leukemia (AML): A focus on pro- and anti-inflammatory mediators.

Cytokines exert profound effects on the progression of hematopoietic malignancies such as acute myeloid leukemia (AML). Critical roles of cytokines in the context of inflammation have gained special interest. While pro-inflammatory mediators such as IL-1ß, TNF-α and IL-6 tend to increase AML aggressiveness, anti-inflammatory mediators such as TGF-ß and IL-10 appear to impede AML progression. Dysregulation of the complex interactions between pro- and anti-inflammatory cytokines in AML may create a pro-tumorigenic microenvironment with effects on leukemic cell proliferation, survival and drug-resistance. This article summarizes current knowledge about the functions of pro- and anti-inflammatory cytokines in AML, their modes of action, and therapeutic interventions with potential to improve clinical outcomes for AML patients.

Increasing VLBW deliveries at subspecialty perinatal centers via perinatal outreach.

OBJECTIVES: To test the hypothesis that the promotion of national guidelines recommending the transfer of high-risk mothers to subspecialty perinatal centers reduces mortality and morbidity through the reduction of preterm infants delivered at nontertiary maternity hospitals. METHODS: After implementation of hospital-based educational and communication programs emphasizing the importance of maternal transfer to subspecialty perinatal centers, we conducted a population-based cohort study of all live births delivered at maternity hospitals in greater Cincinnati from 2003 through 2007 (n = 1825). Birth weights measured between 500 and 1499 g and gestational ages were less than 32 weeks. Risk-adjusted outcomes were measured by multivariate logistic regression in 2 stages. We compared these findings with those from a similar study conducted at our institution that included infants with birth weights less than 1500 g born between September 1, 1995, and December 31, 1997 (n = 848). The primary outcome was the percentage decrease in infants born with very low birth weights at nontertiary centers compared with our previous study. RESULTS: The number of infants born with birth weights less than 1500 g and at less than 32 weeks' gestation delivered at hospitals without tertiary perinatal and neonatal care decreased from 25% to 11.8% between the 2 study periods. The odds of death or major morbidity for infants born with very low birth weights at nontertiary perinatal centers is 3 times that of infants born at subspecialty perinatal centers after controlling for demographic variations (odds ratio: 3.05 [95% confidence interval: 2.1-4.4]). CONCLUSIONS: Local promotion of national guidelines by neonatologists coincided with a significant reduction in the percentage of infants born with birth weights less than 1500 g and at less than 32 weeks' gestation who were not delivered at subspecialty perinatal centers, and, at 88.2%, this nearly achieves the Healthy People 2010 objective to deliver 90% of infants born with very low birth weights in subspecialty perinatal centers.

Is there a link between children's motor abilities and unintentional injuries?

PROBLEM: The common view is that clumsy children experience unintentional injury more frequently. Empirical evidence supporting this position is mixed. METHOD: One hundred 6- and 8-year-olds completed a battery of nine tasks designed to assess motor ability. Mothers completed a lifetime injury history measure about their children and families completed a 2-week injury diary assessing frequency and severity of daily injuries. RESULTS: Internal reliability for the motor ability battery was good. Correlations between motor ability measures and injury risk were nonsignificant and near zero. DISCUSSION: Motor ability does not appear to be directly related to injury risk. Possible explanations include: (a) coordinated and clumsy children engage in hazardous activities with differing frequency; or (b) other individual difference factors may interact with motor ability to explain children's injury risk. IMPACT ON INDUSTRY: Children's motor abilities do not appear to be directly linked to rate of unintentional injury, but instead may influence risk for injury in conjunction with other factors. Results could have implications to the engineering of children's toys and playground equipment and to the design of appropriate supervision strategies for children engaging in potentially dangerous activities.

Using an injury diary to describe the ecology of children's daily injuries.

PROBLEM: Two problems were addressed in this study. First, are daily injury diaries an effective means to gather information about children's unintentional injuries? Second, what are the circumstances of children's injuries as recorded through the diary method? METHOD: Two studies were conducted with a total of 172 children to describe the ecology of children's daily unintentional injuries. Families completed a daily diary for 14 days, describing the circumstances surrounding the injuries children experienced each day. RESULTS: Descriptive data is provided on the locations, causes, and types of injuries children incurred, as well as who was present when they were injured. Daily injury rate was modestly related to the number of major injuries children had incurred in their lifetimes. DISCUSSION: The diary methodology was an effective means to study the ecology of children's daily injuries. Children's injuries occur in a wide range of circumstances that can be quantified through diary techniques. IMPACT ON INDUSTRY: Data obtained from daily injury diaries may be useful in a variety of areas, including study of the etiology of childhood injury, design of interventions to prevent injury, and engineering of toys and playground equipment for children.