Biological system considerations for application of toxicogenomics in next-generation risk assessment and predictive toxicology.

There is increasing interest in using modern 'omics technologies, such as whole transcriptome sequencing, to inform decisions about human health safety and chemical toxicity hazard. High throughput methodologies using in vitro assays offer a path forward in reducing or eliminating animal testing. However, many aspects of these technologies need assessment before they will gain the trust of regulators and the public as viable alternative test methods for human health and safety. We used a high throughput whole transcriptome sequence assay (TempO-Seq) to assess the use of three widely used cancer cell lines (HepG2, MCF7, and Ishikawa cells) as in vitro systems for determination of cellular modes of action for two well studied compounds with canonical liver responses: ketoconazole and phenobarbital. We evaluated transcriptomic data to infer points of departure for use in risk analyses of compounds. Both compounds displayed shortcomings in evidence for canonical liver-related responses in any cell line, despite a strong dose response in all three. This raises questions about the competence of simple, mono-cultured cancer cell lines as appropriate surrogates for some adverse effects or toxic endpoints. Points of departure derived from benchmark doses were highly consistent across all three cell lines however, indicating the use of transcriptomic BMD analyses for such purposes would be a reliable and consistent approach.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets.

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

In vivo assessment of bisphenol A induced histopathological alterations and inflammatory gene expression in lungs of male Wistar rats.

Bisphenol A (BPA), an imperative environmental contaminant used in polycarbonate plastics. Due to limited information concerning the effect of BPA on lungs, this study design to assess whether BPA cause alterations in histopathology and trace metal content in lungs of rats. They were divided into five groups with five rats per group. Group I was named as control group. Group L6 and L12 were received BPA (10 mg/kg body weight/day) for 6 weeks and 12 weeks respectively. Group H6 and L12 were given BPA (25 mg/kg body weight/day) for 6 weeks and 12 weeks respectively. Considerable alteration in Cu, Zn and Fe was detected in experimental groups. BPA also caused significant increase in the expression of tumor necrosis factor α that mediate the pulmonary inflammatory response. Comparative study of resolved proteins i.e. 72 KDa (matrix metalloproteinase 2 fragment) and 109 KDa (nucleolin) on SDS-PAGE showed their altered expression in experimental groups. Histopathology of experimental groups revealed altered architecture of lungs. Special staining of BPA treated groups showed significant number of mast cells in alveoli and bronchioles. Prolonged administration of BPA causes deleterious aggravating lung damage even at extremely low dose, so the use of BPA should be prohibited in plastic synthesizing industries.

Assessment of eugenol inhibitory effect on biofilm formation and biofilm gene expression in methicillin resistant Staphylococcus aureus clinical isolates in Egypt.

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm infection is a major threat in Healthcare facilities. The search for biofilm inhibitors is essential to overcome the antibiotic resistance. Eugenol is a phyto-compound that possesses many biological properties. In this study, the aim was to estimate the effect of eugenol on biofilms of MRSA through quantifying the level of gene expression of three genes (IcaA, IcaD and SarA) involved in biofilm development.. Fifty MRSA biofilm producers collected from the microbiology lab at Theodor Bilharz Research Institute were incubated with different concentrations of eugenol for 24 h. The minimum inhibitory concentration of eugenol (MIC) that eradicates the biofilms growth was detected. mRNA was extracted from all isolates before and after the application of eugenol at 0.5 x MIC, and then subjected to quantitative real-time PCR (qPCR). Results showed that fourteen isolates out of 50 (28%) exhibited intermediate biofilm formation ability, and 36 out of 50 (72%) were strong biofilm producers. The MIC values of eugenol for MRSA ranged from 3.125% to 0.01%. The mean values of MIC in both strong and intermediate biofilm forming MRSA isolates were statistically comparable (p = 0.202). qPCR results revealed that the levels of expression of the studied genes IcaA, IcaD, and SarA were decreased after eugenol treatment when compared with their corresponding values before treatment (p = 0.001). Eugenol inhibited the formation of biofilm of MRSA isolates, indicating it could be used to control infections associated with MRSA biofilms.

Withaferin A exerts an anti-obesity effect by increasing energy expenditure through thermogenic gene expression in high-fat diet-fed obese mice.

BACKGROUND: The enhancement of energy expenditure has attracted attention as a therapeutic target for the management of body weight. Withaferin A (WFA), a major constituent of Withania somnifera extract, has been reported to possess anti-obesity properties, however the underlying mechanism remains unknown. PURPOSE: To investigate whether WFA exerts anti-obesity effects via increased energy expenditure, and if so, to characterize the underlying pathway. METHODS: C57BL/6 J mice were fed a high-fat diet (HFD) for 10 weeks, and WFA was orally administered for 7 days. The oxygen consumption rate of mice was measured at 9 weeks using an OxyletPro™ system. Hematoxylin and eosin (H&E), immunohistochemistry, immunoblotting, and real-time PCR methods were used. RESULTS: Treatment with WFA ameliorated HFD-induced obesity by increasing energy expenditure by improving of mitochondrial activity in brown adipose tissue (BAT) and promotion of subcutaneous white adipose tissue (scWAT) browning via increasing uncoupling protein 1 levels. WFA administration also significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the BAT of obese mice. Additionally, WFA activated mitogen-activated protein kinase (MAPK) signaling, including p38/extracellular signal-regulated kinase MAPK, in both BAT and scWAT. CONCLUSION: WFA enhances energy expenditure and ameliorates obesity via the induction of AMPK and activating p38/extracellular signal-regulated kinase MAPK, which triggers mitochondrial biogenesis and browning-related gene expression.

Validation of an air/liquid interface device for TiO2 nanoparticle toxicity assessment on NR8383 cells: preliminary results.

nvestigations on adverse biological effects of nanoparticles (NP) are performed usually either in vivo on rodents or in vitro under submerged conditions where NP are in suspension into cell culture media. However, sedimentation of NP in vitro is a continuous process and to assess the exact deposited cellular dose remains difficult, as the cellular internal dose is a function of time. Moreover, the cellular responses to NP under submerged culture conditions or by exposing rodents by nose-only to NP aerosols might differ from those observed at physiological settings at the air-liquid interface (ALI). Rat alveolar NR8383 macrophages were exposed to aerosols at the air-liquid interface. We studied TiO2 NM105, a mixture of anatase and rutile. NR8383 cells were exposed to a single dose of 3.0 cm2/cm2 of TiO2 aerosol. Following RNA extraction, transcriptome allowing full coverage of the rat genome was performed, and differentially expressed genes were retrieved. Their products were analyzed for functions and interaction with String DB. Only 126 genes were differentially expressed and 98 were recognized by String DB and give us the gene expression signature of exposed rat alveolar NR8383 macrophages. Among them, 13 display relationships at a high confidence level and the ten most differentially expressed compared to unexposed cells were: Chac1, Ccl4, Zfp668, Fam129b, Nab2, Txnip, Id1, Cdc42ep3, Dusp6 and Myc, ranked from the most overexpressed to the most under-expressed. Some of them were previously described as over or under-expressed in NP exposed cell systems. We validated in our laboratory an easy-to-use device and a physiological relevant paradigm for studying the effects of cell exposure to TiO2. Ccl4 gene expression seems to be a positive marker of exposure evidenced as well as in vivo or in both in vitro conditions.

Goat's Milk Intake Prevents Obesity, Hepatic Steatosis and Insulin Resistance in Mice Fed A High-Fat Diet by Reducing Inflammatory Markers and Increasing Energy Expenditure and Mitochondrial Content in Skeletal Muscle.

Goat's milk is a rich source of bioactive compounds (peptides, conjugated linoleic acid, short chain fatty acids, monounsaturated and polyunsaturated fatty acids, polyphenols such as phytoestrogens and minerals among others) that exert important health benefits. However, goat's milk composition depends on the type of food provided to the animal and thus, the abundance of bioactive compounds in milk depends on the dietary sources of the goat feed. The metabolic impact of goat milk rich in bioactive compounds during metabolic challenges such as a high-fat (HF) diet has not been explored. Thus, we evaluated the effect of milk from goats fed a conventional diet, a conventional diet supplemented with 30% Acacia farnesiana (AF) pods or grazing on metabolic alterations in mice fed a HF diet. Interestingly, the incorporation of goat's milk in the diet decreased body weight and body fat mass, improved glucose tolerance, prevented adipose tissue hypertrophy and hepatic steatosis in mice fed a HF diet. These effects were associated with an increase in energy expenditure, augmented oxidative fibers in skeletal muscle, and reduced inflammatory markers. Consequently, goat's milk can be considered a non-pharmacologic strategy to improve the metabolic alterations induced by a HF diet. Using the body surface area normalization method gave a conversion equivalent daily human intake dose of 1.4 to 2.8 glasses (250 mL per glass/day) of fresh goat milk for an adult of 60 kg, which can be used as reference for future clinical studies.

An assessment of serum-dependent impacts on intracellular accumulation and genomic response of per- and polyfluoroalkyl substances in a placental trophoblast model.

Per- and polyfluoroalkyl substances (PFAS), a class of environmental contaminants, have been detected in human placenta and cord blood. The mechanisms driving PFAS-induced effects on the placenta and adverse pregnancy outcomes are not well understood. This study investigated the impact of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and a replacement PFAS known as hexafluoropropylene oxide dimer acid (HFPO-DA, tradename GenX) on placental trophoblasts in vitro. Several key factors were addressed. First, PFAS levels in cell culture reagents at baseline were quantified. Second, the role of supplemental media serum in intracellular accumulation of PFAS in a human trophoblast (JEG3) cell line was established. Finally, the impact of PFAS on the expression of 96 genes involved in proper placental function in JEG3 cells was evaluated. The results revealed that serum-free media (SFM) contained no detectable PFAS. In contrast, fetal bovine serum-supplemented media (SSM) contained PFNA, PFUdA, PFTrDA, and 6:2 FTS, but these PFAS were not detected internally in cells. Intracellular accumulation following 24 hr treatments was significantly higher when cultured in SFM compared to SSM for PFOS and PFOA, but not HFPO-DA. Treatment with PFAS was associated with gene expression changes (n = 32) in pathways vital to placental function, including viability, syncytialization, inflammation, transport, and invasion/mesenchymal transition. Among the most robust PFAS-associated changes were those observed in the known apoptosis-related genes, BAD and BAX. These results suggest a complex relationship between PFAS, in vitro culture conditions, and altered expression of key genes necessary for proper placentation.

Video-based assessment of drug-induced effects on contractile motion properties using human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Drug-induced inotropic change is a risk factor in drug development; thus, de-risking is desired in the early stages of drug development. Unlike proarrhythmic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), few in vitro models were validated to predict cardiac contractility. Motion field imaging (MFI), a high-resolution block matching-based optical flow technique, was expected to possess high quantitative predictivity in the detection of contraction speed. We aimed to establish an in vitro model to assess drug-induced contractile changes using hiPSC-CMs and MFI. METHODS: MFI was designed to noninvasively characterize cardiomyocyte contractile behavior by analyzing light microscope video images, and maximum contraction speed (MCS) was used as the index of contractility. Using MFI, 9 inactive compounds, 10 negative inotropes, and 10 positive inotropes were tested. Two negative chronotropes, ZD7288 and ivabradine, were also tested. To determine the sensitivity and specificity of the assay, the minimum effective concentration of the MCS was compared with the human effective total therapeutic concentration for 28 compounds in clinical use. RESULTS: For 8 negative and 8 positive inotropes, the effects observed in in vivo and clinical studies were detected in MFI assay. MFI assay showed negative chronotropic changes without inotropic changes. MFI assay presented sufficient specificity (83%) and sensitivity (88%), and RNA-sequence analysis provided an insight into the relationship between occurrence of the false compounds and target gene expression. DISCUSSION: We demonstrated the utility of MFI assay using hiPSC-CMs to assess drug-induced contractile function. These results will facilitate the de-risking of compounds during early drug development.

Incorporation of in vitro techniques for botanicals dietary supplement safety assessment - Towards evaluation of developmental and reproductive toxicity (DART).

As complex mixtures, botanicals present unique challenges when assessing safe use, particularly when endpoint gaps exist that cannot be fully resolved by existing toxicological literature. Here we explore in vitro gene expression as well receptor binding and enzyme activity as alternative assays to inform on developmental and reproductive toxicity (DART) relevant modes of action, since DART data gaps are common for botanicals. Specifically, botanicals suspected to have DART effects, in addition to those with a significant history of use, were tested in these assays. Gene expression changes in a number of different cell types were analysed using the connectivity mapping approach (CMap) to identify modes of action through a functional read across approach. Taken together with ligand affinity data obtained using a set of molecular targets customised towards known DART relevant modes of action, it was possible to inform DART risk using functional analogues, potency comparisons and a margin of internal exposure approach.

The potential role of nicotinamide on Leishmania tropica: An assessment of inhibitory effect, cytokines gene expression and arginase profiling.

Leishmaniasis represents a major health concern worldwide which has no effective treatment modality. Nicotinamide (NAm) has been used for a wide range of applications from anticancer to antimicrobial usage. This study aimed to assess the effect of NAm combination on Leishmania tropica Inhibition, as well as on cytokines gene expression and arginase (ARG) activity in L. tropica-infected macrophages in an in vitro model. The leishmanicidal effects of NAm and Glucantime (meglumine antimoniate, MA) alone and in combination (NAm/MA) were evaluated using a colorimetric assay and macrophage model. Additionally, immunomodulatory effects and enzymatic activity were assessed by analyzing Th1 and Th2 cytokines gene expression and ARG level, respectively, in infected macrophages treated with NAm and MA, alone and in combination. Findings indicated that the NAm/MA combination demonstrated greater inhibitory effects on L. tropica promastigotes and amastigotes compared with each drug individually. Docking results proved the affinity of NAm to IFN-γ, which can affirm the increased levels of IFN-γ, IL-12p40 and TNF-α as well as reductions in IL-10 secretion with a dose-response effect, especially in the combination group. The NAm/MA combination also showed a significant reduction in the level of ARG activity at all concentrations used compared to each drug individually. These findings indicate higher effectiveness of NAm plus MA in reducing parasite growth, promoting immune response and inhibiting ARG level. This combination should be considered as a potential therapeutic regimen for treatment of volunteer patients with anthroponotic cutaneous leishmaniasis (ACL) in future control programs.

New approach for reproductive toxicity assessment: chromatoid bodies as a target for methylmercury and polychlorinated biphenyls in prepubertal male rats.

This study investigated the reproductive toxicity of methylmercury (MeHg) and Aroclor (Sigma-Aldrich), alone or in combination, following exposure of prepubertal male rats considering the chromatoid body (CB) as a potential target. The CB is an important molecular regulator of mammalian spermatogenesis, primarily during spermatid cytodifferentiation. Male Wistar rats were exposed to MeHg and/or Aroclor , according the following experimental design: control group, which was administered in corn oil (vehicle) only; MeHg-treated group, which was administered 0.5mg kg-1 day-1 MeHg; Aroclor-treated group, which was administered 1mg kg-1 day-1 Aroclor; Mix-LD, group which was administered a low-dose mixture of MeHg (0.05mg kg-1 day-1) and Aroclor (0.1mg kg-1 day-1); and Mix-HD group, which was administered a high-dose mixture of MeHg (0.5mg kg-1 day-1) and Aroclor (1.0mg kg-1 day-1). MeHg was diluted in distilled water and Aroclor was made up in corn oil (volume 1mL kg-1). Rats were administered the different treatments from PND23 to PND53 by gavage, . The morphophysiology of CBs was analysed, together with aspects of steroid hormones status and regulation, just after the last treatment on PND53. In addition, the long-term effects on sperm parameters were assessed in adult animals. MeHg exposure increased mouse VASA homologue (MVH) protein levels in seminiferous tubules, possibly affecting the epigenetic status of germ cells. Aroclor produced morphological changes to CB assembly, which may explain the observed morphological defects to the sperm flagellum and the consequent decrease in sperm motility. There were no clear additive or synergistic effects between MeHg and Aroclor when administered in combination. In conclusion, this study demonstrates that MeHg and Aroclor have independent deleterious effects on the developing testis, causing molecular and morphological changes in CBs. To the best of our knowledge, this is the first study to show that CBs are targets for toxic agents.

Shikonin Attenuates Hepatic Steatosis by Enhancing Beta Oxidation and Energy Expenditure via AMPK Activation.

Shikonin, a natural plant pigment, is known to have anti-obesity activity and to improve insulin sensitivity. This study aimed to examine the effect of shikonin on hepatic steatosis, focusing on the AMP-activated protein kinase (AMPK) and energy expenditure in Hepa 1-6 cells and in high-fat fed mice. Shikonin increased AMPK phosphorylation in a dose- and time-dependent manner, and inhibition of AMPK with compound C inhibited this activation. In an oleic acid-induced steatosis model in hepatocytes, shikonin suppressed oleic acid-induced lipid accumulation, increased AMPK phosphorylation, suppressed the expression of lipogenic genes, and stimulated fatty acid oxidation-related genes. Shikonin administration for four weeks decreased body weight gain and the accumulation of lipid droplets in the liver of high-fat fed mice. Furthermore, shikonin promoted energy expenditure by activating fatty acid oxidation. In addition, shikonin increased the expression of PPARγ coactivator-1α (PGC-1α), carnitine palmitoyltransferase-1 (CPT1) and other mitochondrial function-related genes. These results suggest that shikonin attenuated a high fat diet-induced nonalcoholic fatty liver disease by stimulating fatty acid oxidation and energy expenditure via AMPK activation.

Biosynthesis of NiFe2 O4 @Ag Nanocomposite and Assessment of Its Effect on Expression of norA Gene in Staphylococcus aureus.

Activity of norA efflux pump has been known as a resistance mechanism to antibiotics like ciprofloxacin in Staphylococcus aureus. This study was carried out to assess the effect of biosynthesized NiFe2 O4 @Ag nanocomposite on expression of norA gene in Staphylococcus aureus. In this experimental study, 30 clinical samples were collected from patients hospitalized at different hospitals in Guilan Province, Iran. Then, clinical isolates of S. aureus were identified by standard microbiological tests. Antimicrobial susceptibility tests of clinical and standard strains of S. aureus were done by disk diffusion method according to CLSI guideline. Fourier transform infrared spectroscopy (FT-IR) was used to analyze the various functional groups present in the biosynthesized NiFe2 O4 @Ag nanocomposite. This analysis confirmed the formation of alga proteins coated on magnetite nanocomposite. X-ray diffraction (XRD) verified the crystalline structure of NiFe2 O4 @Ag and the deposition of silver on the surface of NiFe2 O4 . Energy dispersive X-ray mapping (EDX-map) analysis confirmed the existence of Ag, Ni, Fe and O in the final product. Scanning electron microscopy (SEM) confirmed that the nanocomposites were spherical in shape and Transmission electron microscopy (TEM) results revealed that the NiFe2 O4 @Ag had the particle size about 100 nm. Antibacterial activity of NiFe2 O4 @Ag alone and combined with ciprofloxacin was evaluated using the disk assay method, and minimum inhibitory concentration (MIC) by broth dilution method. Afterwards, the expression of norA efflux pump gene with and without of NiFe2 O4 @Ag nanocomposite and ciprofloxacin was evaluated by Real-Time PCR. Real-Time PCR results demonstrated that the expression of norA gene in the strains exposed to both NiFe2 O4 @Ag nanocomposite (1/4 MIC) and ciprofloxacin (1/8 MIC) significantly reduced in comparison to untreated strains. This study reveals that, when NiFe2 O4 @Ag nanocomposite is combined with ciprofloxacin, the inhibitory effect of ciprofloxacin increases against growth of S. aureus. Therefore, NiFe2 O4 @Ag nanocomposite can be considered as an effective factor to decrease the growth of S. aureus along with ciprofloxacin.

Impact of ketamine on the behavior and immune system of adult medaka (Oryzias latipes) at environmentally relevant concentrations and eco-risk assessment in surface water.

This work for the first time investigated the bioconcentration factor (BCF), toxicity, and eco-risk of KET using adult medaka fish (Oryzias latipes) as model organism after exposure at environmental concentrations (0.05-0.5 µg L-1) and higher levels (5-100 µg L-1) for 90 days. The BCF of KET was approximately 1.07- to 10.94- folds. The behavioral functions, including swimming properties, feeding rate, and food preference, were significantly impacted by KET (≥0.05 µg L-1). After 90-days exposure, KET induced histological abnormalities in liver and kidney tissue at 0.1 and 0.2 µg L-1, respectively. Additionally, the condition factor, hepatic-somatic index (HSI), and nephric-somatic index (NSI) of medaka were markedly impacted by KET treatment at 0.5, 0.5, and 0.1 µg L-1, respectively. Morphological inflammation (i.e., haemorrhage and erosion) in the fish body was observed exposed to KET, and the EC10 value was 0.407 µg L-1. Alterations in the expressions of genes (i.e., cacna1c, oxtr, erk1, and c-fos) and proteins (i.e., OXT and PKA), involved in in calcium ion channels induced by KET, could partly elucidate the underlying mechanism of the toxicity. The inflammatory risk to fish posed by KET in some rivers in southern China was at high level, suggesting the long-term concentration monitoring was required.

Assessment of Gene Expression Biomarkers in the Chilean Pencil Catfish, Trichomycterus areolatus, from the Choapa River Basin, Coquimbo Chile.

The objective of this study was to describe changes in the gene expression in the Chilean catfish, Trichomycterus areolatus, based on their geographic location within the Choapa River. Genes of choice included those that are biomarkers of exposure to metals, oxidative stress, and endocrine disruption. Male and female T. areolatus were sampled from four sites in January 2015 differently impacted by human activities. In males, but not females, hepatic gene expression of heat shock protein (HSP70) and cytochrome P450 1A (CYP1A) were significantly elevated at the site adjacent to the small city of Salamanca, relative to the other sites. In females, hepatic HSP70, the aryl hydrocarbon receptor (AHR), and the estrogen responsive genes, vitellogenin (VTG) and estrogen receptor alpha (ERα), were significantly lower at the site located furthest downstream. A similar downstream pattern of lower expression levels also was found in ovarian tissue for the genes, HSP70 and ERα. Gill gene expression showed a unique pattern in females as levels of metallothionein were elevated at the site furthest downstream. While analytical chemistry of water samples provided limited evidence of agrichemical contamination, the gene expression data are consistent with an exposure to agrichemicals and metals. T. areolatus may be a valuable sentinel organism and its use as a bioindicator species in some rivers within Chile can provide considerable insight, particularly in situations analytical chemistry is limited by environmental constraints.

Gold nanoparticles for the in situ polymerization of near-infrared responsive hydrogels based on fibrin.

Non-invasiveness and relative safety of photothermal therapy, which enables local hyperthermia of target tissues using a near infrared (NIR) laser, has attracted increasing interest. Due to their biocompatibility, amenability of synthesis and functionalization, gold nanoparticles have been investigated as therapeutic photothermal agents. In this work, hollow gold nanoparticles (HGNP) were coated with poly-l-lysine through the use of COOH-Poly(ethylene glycol)-SH as a covalent linker. The functionalized HGNP, which peak their surface plasmon resonance at 800 nm, can bind thrombin. Thrombin-conjugated HGNP conduct in situ fibrin polymerization, facilitating the process of generating photothermal matrices. Interestingly, the metallic core of thrombin-loaded HGNP fragmentates at physiological temperature. During polymerization process, matrices prepared with thrombin-loaded HGNP were loaded with genetically-modified stem cells that harbour a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation of resulting cell constructs in the presence of ligand successfully triggered transgene expression in vitro and in vivo. STATEMENT OF SIGNIFICANCE: Current technological development allows synthesis of gold nanoparticles (GNP) in a wide range of shapes and sizes, consistently and at scale. GNP, stable and easily functionalized, show low cytotoxicity and high biocompatibility. Allied to that, GNP present optoelectronic properties that have been exploited in a range of biomedical applications. Following a layer-by-layer functionalization approach, we prepared hollow GNP coated with a positively charged copolymer that enabled thrombin conjugation. The resulting nanomaterial efficiently catalyzed the formation of fibrin hydrogels which convert energy of the near infrared (NIR) into heat. The resulting NIR-responsive hydrogels can function as scaffolding for cells capable of controlled gene expression triggered by optical hyperthermia, thus allowing the deployment of therapeutic gene products in desired spatiotemporal frameworks.

Development of an ENPP1 Fluorescence Probe for Inhibitor Screening, Cellular Imaging, and Prognostic Assessment of Malignant Breast Cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein that is involved in bone metabolism and insulin resistance, hydrolyzes 2',3'-cGAMP (a STING ligand that promotes innate immunity), and is associated with cancer stemness in breast cancers and glioblastoma. Therefore, ENPP1 is considered a candidate therapeutic target and/or biomarker for early diagnosis of malignant tumors. In this study, we designed and synthesized a sensitive ENPP1 fluorescence probe, Tokyo Green (TG) mAMP. We used it to screen a chemical library for non-phosphate ENPP1 inhibitors. Structural optimization of a selected hit afforded a potent and specific ENPP1 inhibitor. We further found that ENPP1 mRNA expression in tissue samples from patients with triple-negative breast cancer was significantly inversely related to recurrence-free survival (RFS) and overall survival (OS), and TG-mAMP assay revealed a significant difference in ENPP1 activity between ENPP1 high-expressing and ENPP1 low-expressing samples. Our results suggest that TG-mAMP assay might be a rapid and inexpensive tool for predicting the prognosis of patients with malignant breast cancers.

Regulation of Energy Expenditure by Brainstem GABA Neurons.

Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.

Hexabromocyclododecane (HBCD): A case study applying tiered testing for human health risk assessment.

Current global efforts are aiming to increase use of mechanistic information in regulatory testing. In tiered testing paradigms, in vitro, in silico, and in vivo studies are employed progressively to identify and classify health hazards, which are then compared against human equivalent doses. We used data from three companion papers on the brominated flame retardant hexabromocyclododecane (HBCD) to conduct a case study on tiered testing. We included ToxCast™ and in vitro-in vivo extrapolation (Tier 1), rat liver transcriptomic (Tier 2), and conventional rat (Tier 3) data. Bioactivity-exposure ratios (BERs) were derived by comparing human administered dose equivalents of the measured effects to Canadian exposure levels. Biological perturbations were highly aligned between Tiers 1/2, and consistent with apical effects in Tier 3. Tier 1 had the smallest BERs, and Tiers 2/3 were similar. The study demonstrates the promise of using physiologically-based pharmacokinetic modeling and mechanistic analyses in a tiered framework to identify pathways through which chemicals exert toxicological effects; however, they also point to some shortcomings associated with in vitro and in silico approaches. Additional case studies of chemicals from multiple classes are required to define optimal tiered screening procedures to reduce future in vivo requirements in health hazard assessments.