Integrating gene expression and splicing dynamics across dose-response oxidative modulators.

Toxicological risk assessment increasingly utilizes transcriptomics to derive point of departure (POD) and modes of action (MOA) for chemicals. One essential biological process that allows a single gene to generate several different RNA isoforms is called alternative splicing. To comprehensively assess the role of splicing dysregulation in toxicological evaluation and elucidate its potential as a complementary endpoint, we performed RNA-seq on A549 cells treated with five oxidative stress modulators across a wide dose range. Differential gene expression (DGE) showed limited pathway enrichment except at high concentrations. However, alternative splicing analysis revealed variable intron retention events affecting diverse pathways for all chemicals in the absence of significant expression changes. For instance, diazinon elicited negligible gene expression changes but progressive increase in the number of intron retention events, suggesting splicing alterations precede expression responses. Benchmark dose modeling of intron retention data highlighted relevant pathways overlooked by expression analysis. Systematic integration of splicing datasets should be a useful addition to the toxicogenomic toolkit. Combining both modalities paint a more complete picture of transcriptomic dose-responses. Overall, evaluating intron retention dynamics afforded by toxicogenomics may provide biomarkers that can enhance chemical risk assessment and regulatory decision making. This work highlights splicing-aware toxicogenomics as a possible additional tool for examining cellular responses.

Increasing confidence in new approach methodologies for inhalation risk assessment with multiple end point assays using 5-day repeated exposure to 1,3-dichloropropene.

New Approach Methodologies (NAMs) are being widely used to reduce, refine, and replace, animal use in studying toxicology. For respiratory toxicology, this includes both in silico and in vitro alternatives to replace traditional in vivo inhalation studies. 1,3-Dichloropropene (1,3-DCP) is a volatile organic compound that is widely used in agriculture as a pre-planting fumigant. Short-term exposure of humans to 1,3-DCP can result in mucous membrane irritation, chest pain, headache, and dizziness. In our previous work, we exposed differentiated cells representing different parts of the respiratory epithelium to 1,3-DCP vapor, measured cytotoxicity, and did In Vitro to In Vivo Extrapolation (IVIVE). We have extended our previous study with 1,3-DCP vapors by conducting transcriptomics on acutely exposed nasal cultures and have implemented a separate 5-day repeated exposure with multiple endpoints to gain further molecular insight into our model. MucilAir™ Nasal cell culture models, representing the nasal epithelium, were exposed to six sub-cytotoxic concentrations of 1,3-DCP vapor at the air-liquid interface, and the nasal cultures were analyzed by different methodologies, including histology, transcriptomics, and glutathione (GSH) -depletion assays. We observed the dose-dependent effect of 1,3-DCP in terms of differential gene expression, change in cellular morphology from pseudostratified columnar epithelium to squamous epithelium, and depletion of GSH in MucilAir™ nasal cultures. The MucilAir™ nasal cultures were also exposed to 3 concentrations of 1,3-DCP using repeated exposure 4 h per day for 5 days and the histological analyses indicated changes in cellular morphology and a decrease in ciliated bodies and an increase in apoptotic bodies, with increasing concentrations of 1,3-DCP. Altogether, our results suggest that sub-cytotoxic exposures to 1,3-DCP lead to several molecular and cellular perturbations, providing significant insight into the mode-of-action (MoA) of 1,3-DCP using an innovative NAM model.

The Effects of Race/Ethnicity, Age, and Area Deprivation Index (ADI) on COVID-19 Disease Early Dynamics: Washington, D.C. Case Study.

The COVID-19 pandemic and its associated mitigation strategies have significant psychosocial, behavioral, socioeconomic, and health impacts, particularly in vulnerable US populations. Different factors have been identified as influencers of the transmission rate; however, the effects of area deprivation index (as a measure of social determinants of health, SDoH) as a factor on COVID-19 disease early dynamics have not been established. We determined the effects of area deprivation index (ADI) and demographic factors on COVID-19 outcomes in Washington, D.C. This retrospective study used publicly available data on COVID-19 cases and mortality of Washington, D.C., during March 31st-July 4th, 2020. The main predictors included area deprivation index (ADI), age, and race/ethnicity. The ADI of each census block groups in D.C. (n=433) were obtained from Neighborhood Atlas map. Using a machine learning-based algorithm, the outcome variables were partitioned into time intervals: time duration (Pi, days), rate of change coefficient (Ei), and time segment load (Pi×Ei) for transmission rate and mortality. Correlation analysis and multiple linear regression models were used to determine associations between predictors and outcome variables. COVID-19 early transmission rate (E1) was highly correlated with ADI (SDoH; r= 0.88, p=0.0044) of the Washington, D.C. community. We also found positive association between ADI, age (0-17 years, r=0.91, p=0.0019), and race (African American/Black, r=0.86; p=0.0068) and COVID-19 outcomes. There was high variability in early transmission across the geographic regions (i.e., wards) of Washington, D.C., and this variability was driven by race/ethnic composition and ADI. Understanding the association of COVID-19 disease early transmission and mortality dynamics and key socio-demographic risk factors such as age, race, and ADI, as a measure of social determinants, will contribute to health equity/equality and distribution of economic resources/assistance and is essential for future predictive modeling of the COVID-19 pandemic to limit morbidity and mortality.

Data-Driven Forecasting of Agitation for Persons with Dementia: A Deep Learning-Based Approach.

The World Health Organization estimates that approximately 10 million people are newly diagnosed with dementia each year and a global prevalence of nearly 50 million persons with dementia (PwD). The vast majority of PwD living at home receive the majority of their care from informal familial caregivers. The quality of life (QOL) of familial caregivers may be significantly impacted by their caregiving responsibilities and resultant caregiver burden. A major contributor to caregiver burden is the random occurrence of agitation in PwD and familial caregivers' lack of preparedness to manage these episodes. Caregiver burden may be reduced if it is possible to forecast impending agitation episodes. In this study, we leverage data-driven deep learning models to predict agitation episodes in PwD. We used Long Short-Term Memory (LSTM), a deep learning class of algorithms, to forecast agitations up to 30 min before actual agitation events. In particular, we managed the missing data by estimating the missing values and compensated for the class imbalance challenge by down-sampling the majority class. The simulations were based on real-world data from Alzheimer's disease (AD) caregivers and PwD dyads home environments, including ambient noise level, illumination, room temperature, atmospheric pressure (Pa), and relative humidity. Our results show the efficacy of data-driven deep learning models in predicting agitation episodes in community-dwelling AD dyads with accuracy of 98.6% and recall (sensitivity) of 84.8%.

Radioactive waste: A review.

The reviewed papers presented here provide a general overview of worldwide radioactive waste-related studies conducted in 2019. The current review includes studies related to safety assessments, decommission and decontamination of nuclear facilities, fusion facilities, and transportation. Further, the review highlights radioactive wastewater decontamination, management solutions for the final disposal of low- and high-level radioactive wastes (LLRW and HLRW), interim storage and final disposal options for spent fuel (SF), and tritiated wastes, with a focus on environmental impacts due to the mobility of radionuclides in the ecosystem, water and soil along with other research progress made in the management of radioactive waste. PRACTITIONER POINTS: The release of radionuclides and their subsequent fate and transport in the environment poses public health concern and has stimulated recent research on the waste management techniques. Seeking a safe and environmental-friendly solution is the current trend for existing and projected inventories of radioactive waste. Significant progress in the field of geological disposal of radioactive waste has been made in the last two decades.

Textiles wastewater treatment technology: A review.

The following is a review of published literature on textile wastewater in 2019. Presented are the sections described for the review: concise introduction on the textiles wastewater, followed by a review of present textile treatment technologies organized by physicochemical, biological, and combined processes. Lastly, a discussion of the future topics is presented. PRACTITIONER POINTS: The discharge of textile dye wastewater represents a serious environmental problem and public health concern. Effluents from textile manufacturing, dyeing, and finishing processes contain high-concentration recalcitrant chemicals that are resistant to biodegradation. The textile wastewater needs environmental-friendly and cost-effective combined treatment process.

Prominent metalware from pelvic surgery causing dyspareunia.

We present a case of female dyspareunia secondary to metalware placement during extensive pelvic surgery following a motor vehicle accident. The patient initially had an uneventful recovery from her operations. However, she noticed pain with vaginal intercourse, due to a screw tip which was palpable on vaginal examination. X-ray imaging confirmed long screws in the medial part of an anterior column plate, which were impacting on the anterior vaginal wall. Subsequent percutaneous removal of two screws resulted in resolution of her symptoms of painful vaginal intercourse. While the pain from mechanical irritation of the vagina was resolved, the patient continues to have difficulty with intercourse, which is related to hip pain as a result of her initial injury and complex pelvic surgery.

Osteoconductive protamine-based polyelectrolyte multilayer functionalized surfaces.

The integration of orthopedic implants with host bone presents a major challenge in joint arthroplasty, spinal fusion and tumor reconstruction. The cellular microenvironment can be programmed via implant surface functionalization allowing direct modulation of osteoblast adhesion, proliferation, and differentiation at the implant--bone interface. The development of layer-by-layer assembled polyelectrolyte multilayer (PEM) architectures has greatly expanded our ability to fabricate intricate nanometer to micron scale thin film coatings that conform to complex implant geometries. The in vivo therapeutic efficacy of thin PEM implant coatings for numerous biomedical applications has previously been reported. We have fabricated protamine-based PEM thin films that support the long-term proliferation and differentiation of pre-osteoblast cells on non-cross-linked film-coated surfaces. These hydrophilic PEM functionalized surfaces with nanometer-scale roughness facilitated increased deposition of calcified matrix by osteoblasts in vitro, and thus offer the potential to enhance implant integration with host bone. The coatings can make an immediate impact in the osteogenic culture of stem cells and assessment of the osteogenic potential of new therapeutic factors.

Tunable dual growth factor delivery from polyelectrolyte multilayer films.

A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein-2) and angiogenic rhVEGF165 (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(ß-amino ester)/polyanion/growth factor/polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF165 eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF165 upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF165 PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.

Tissue integration of growth factor-eluting layer-by-layer polyelectrolyte multilayer coated implants.

Drug eluting coatings that can direct the host tissue response to implanted medical devices have the potential to ameliorate both the medical and financial burden of complications from implantation. However, because many drugs useful in this arena are biologic in nature, a paucity of delivery strategies for biologics, including growth factors, currently limits the control that can be exerted on the implantation environment. Layer-by-Layer (LbL) polyelectrolyte multilayer films are highly attractive as ultrathin biologic reservoirs, due to the capability to conformally coat difficult geometries, the use of aqueous processing likely to preserve fragile protein function, and the tunability of incorporation and release profiles. Herein, we describe the first LbL films capable of microgram-scale release of the biologic Bone Morphogenetic Protein 2 (BMP-2), which is capable of directing the host tissue response to create bone from native progenitor cells. Ten micrograms of BMP-2 are released over a period of two weeks in vitro; less than 1% is released in the first 3 h (compared with commercial collagen matrices which can release up to 60% of BMP-2, too quickly to induce differentiation). BMP-2 released from LbL films retains its ability to induce bone differentiation in MC3T3 E1S4 pre-osteoblasts, as measured by induction of alkaline phosphatase and stains for calcium (via Alizarin Red) and calcium matrix (via Von Kossa). In vivo, BMP-2 film coated scaffolds were compared with film coated scaffolds lacking BMP-2. BMP-2 coatings implanted intramuscularly were able to initiate host progenitor cells to differentiate into bone, which matured and expanded from four to 9 weeks as measured by MicroCT and histology. Such LbL films represent new steps towards controlling and tuning host response to implanted medical devices, which may ultimately increase the success of implanted devices, provide alternative new approaches toward bone wound healing, and lay the foundation for development of a multi-therapeutic release coating.

The effectiveness of the controlled release of gentamicin from polyelectrolyte multilayers in the treatment of Staphylococcus aureus infection in a rabbit bone model.

While the infection rate of orthopedic implants is low, the required treatment, which can involve six weeks of antibiotic therapy and two additional surgical operations, is life threatening and expensive, and thus motivates the development of a one-stage re-implantation procedure. Polyelectrolyte multilayers incorporating gentamicin were fabricated using the layer-by-layer deposition process for use as a device coating to address an existing bone infection in a direct implant exchange operation. The films eluted about 70% of their payload in vitro during the first three days and subsequently continued to release drug for more than four additional weeks, reaching a total average release of over 550 microg/cm(2). The coatings were demonstrated to be bactericidal against Staphylococcus aureus, and degradation products were generally nontoxic towards MC3T3-E1 murine preosteoblasts. Film-coated titanium implants were compared to uncoated implants in an in vivo S. aureus bone infection model. After a direct exchange procedure, the antimicrobial-coated devices yielded bone homogenates with a significantly lower degree of infection than uncoated devices at both day four (p < 0.004) and day seven (p < 0.03). This study has demonstrated that a self-assembled ultrathin film coating is capable of effectively treating an experimental bone infection in vivo and lays the foundation for development of a multi-therapeutic film for optimized, synergistic treatment of pain, infection, and osteomyelitis.

Systemic hypoxia alters gene expression levels of structural proteins and growth factors in knee joint cartilage.

We investigated the effects of short- (8- and 24-h) and long-term (3 weeks) exposure to systemic normobaric hypoxia (13%) on the gene expression level of structural proteins and growth factors in knee joint cartilage of rabbits. Collagen type Ia2, II, and Va1, TGF-beta1, and b-FGF were upregulated after short-term hypoxia in both menisci, but not in articular cartilage. In contrast, long-term hypoxia downregulated gene expression level of collagens, aggrecan, and growth factors in articular cartilage and meniscal fibrocartilage. Interestingly, gene expression levels of non-collagenous proteins biglycan, decorin, and versican were not affected by short-term or by long-term hypoxia in knee joint cartilage. The present study suggests that changes in oxygen level differentially affect gene expression levels of growth factors, collagens, and non-collagenous proteins in normal knee joint cartilage in rabbits.

Differential expression of VEGF isoforms and receptors in knee joint menisci under systemic hypoxia.

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. We examined the expression levels of VEGF isoforms and their receptors in the medial and lateral meniscus of rabbits under normal physiologic conditions as well their expression levels after 8 and 24 h of systemic normobaric hypoxia (13%). VEGF121 is the most abundant VEGF isoform in the medial and lateral meniscus, followed by VEGF165, VEGF189, and VEGF183. While the soluble VEGF121 and VEGF165 are only upregulated at 8 h of hypoxia, the membrane-bound VEGF183 and VEGF189 are further increased at 24 h. VEGFR-2 is expressed at a much higher level than VEGFR-1 under normal conditions, and both receptors are upregulated under hypoxia. Differential expression levels under normoxia as well as a differential response to hypoxia may indicate different functions of VEGF isoforms in the meniscus.