Paradoxical Role for Wild-Type p53 in Driving Therapy Resistance in Melanoma.

Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.

Brain simulation as a cloud service: The Virtual Brain on EBRAINS.

The Virtual Brain (TVB) is now available as open-source services on the cloud research platform EBRAINS (ebrains.eu). It offers software for constructing, simulating and analysing brain network models including the TVB simulator; magnetic resonance imaging (MRI) processing pipelines to extract structural and functional brain networks; combined simulation of large-scale brain networks with small-scale spiking networks; automatic conversion of user-specified model equations into fast simulation code; simulation-ready brain models of patients and healthy volunteers; Bayesian parameter optimization in epilepsy patient models; data and software for mouse brain simulation; and extensive educational material. TVB cloud services facilitate reproducible online collaboration and discovery of data assets, models, and software embedded in scalable and secure workflows, a precondition for research on large cohort data sets, better generalizability, and clinical translation.

microRNAs signatures as potential biomarkers of structural cardiotoxicity in human-induced pluripotent stem-cell derived cardiomyocytes.

Identification of early biomarkers of heart injury and drug-induced cardiotoxicity is important to eliminate harmful drug candidates early in preclinical development and to prevent severe drug effects. The main objective of this study was to investigate the expression of microRNAs (miRNAs) in human-induced pluripotent stem cell cardiomyocytes (hiPSC-CM) in response to a broad range of cardiotoxic drugs. Next generation sequencing was applied to hiPSC-CM treated for 72 h with 40 drugs falling into the categories of functional (i.e., ion channel blockers), structural (changes in cardiomyocytes structure), and general (causing both functional and structural) cardiotoxicants as well as non-cardiotoxic drugs. The largest changes in miRNAs expression were observed after treatments with structural or general cardiotoxicants. The number of deregulated miRNAs was the highest for idarubicin, mitoxantrone, and bortezomib treatments. RT-qPCR validation confirmed upregulation of several miRNAs across multiple treatments at therapeutically relevant concentrations: hsa-miR-187-3p, hsa-miR-146b-5p, hsa-miR-182-5p (anthracyclines); hsa-miR-365a-5p, hsa-miR-185-3p, hsa-miR-184, hsa-miR-182-5p (kinase inhibitors); hsa-miR-182-5p, hsa-miR-126-3p and hsa-miR-96-5p (common some anthracyclines, kinase inhibitors and bortezomib). Further investigations showed that an upregulation of hsa-miR-187-3p and hsa-miR-182-5p could serve as a potential biomarker of structural cardiotoxicity and/or an additional endpoint to characterize cardiac injury in vitro.

Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening.

A metabolic biomarker-based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery-phase detection of potential human developmental toxicants. In this study, metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure-based biomarker assay using these metabolites, along with a cytotoxicity endpoint, was then developed using a 9-point dose-response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity, an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy, but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity, 100% specificity). The assay had a high concordance (≥75%) with existing in vivo models, demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests.

Intoxication by intraperitoneal injection or oral gavage equally potentiates postburn organ damage and inflammation.

The increasing prevalence of binge drinking and its association with trauma necessitate accurate animal models to examine the impact of intoxication on the response and outcome to injuries such as burn. While much research has focused on the effect of alcohol dose and duration on the subsequent inflammatory parameters following burn, little evidence exists on the effect of the route of alcohol administration. We examined the degree to which intoxication before burn injury causes systemic inflammation when ethanol is given by intraperitoneal (i.p.) injection or oral gavage. We found that intoxication potentiates postburn damage in the ileum, liver, and lungs of mice to an equivalent extent when either ethanol administration route is used. We also found a similar hematologic response and levels of circulating interleukin-6 (IL-6) when either ethanol paradigm achieved intoxication before burn. Furthermore, both i.p. and gavage resulted in similar blood alcohol concentrations at all time points tested. Overall, our data show an equal inflammatory response to burn injury when intoxication is achieved by either i.p. injection or oral gavage, suggesting that findings from studies using either ethanol paradigm are directly comparable.

First insights into human mobility in Neolithic Belgium using strontium isotopic analysis and proteomics: A case study of Grotte de La Faucille (Sclayn, province of Namur).

OBJECTIVES: So far, no 87 Sr/86 Sr mobility studies have been done for Neolithic remains from Belgium and information on the Sr isotopic variability in the region is scarce. This study aims to explore mobility in a Final Neolithic population from the funerary cave 'Grotte de La Faucille', contribute to the understanding of the isotopic composition of bioavailable Sr in Belgium, assess evidence for male mobility using proteomic analysis, and explore possible places of origin for nonlocal individuals. MATERIALS AND METHODS: The 87 Sr/86 Sr isotope ratio of dental enamel from six adults and six juveniles was determined. Liquid chromatography mass spectrometry-based protein analysis was employed to identify individuals of male biological sex. 87 Sr/86 Sr of micromammal teeth, snail shells, and modern plants from three geological areas in Belgium were measured to establish isotopic signatures for bioavailable strontium. Nonlocality was assessed by comparing human 87 Sr/86 Sr isotope ratios to the 87 Sr/86 Sr range for bioavailable Sr. RESULTS: Four individuals yielded 87 Sr/86 Sr isotope ratios consistent with a nonlocal origin. No statistical differences were found between adults and juveniles. Three males were detected in the sample set, of which two show nonlocal 87 Sr/86 Sr values. DISCUSSION: This study provides evidence for mobility in Final Neolithic Belgium. The four nonlocal 87 Sr/86 Sr signatures correspond with the 87 Sr/86 Sr of bio-available Sr in Dutch South Limburg, the Black Forest in Southwest Germany, and regions of France, such as parts of the Paris Basin and the Vosges. The results support the ruling hypothesis of connections with Northern France, brought to light by archeological research.

Aging and innate immunity in the mouse: impact of intrinsic and extrinsic factors.

Aging affects every innate immune cell, including changes in cell numbers and function. Defects in the function of some cells are intrinsic, whereas for other cells, defects are extrinsic and possibly the consequence of the complex interactions with other cell types or the environmental milieu that is altered with aging. Abnormal function contributes to worsened outcomes after injury or infection and leads to diseases observed in the elderly. Knowing the mechanisms responsible for the aberrant function of innate immune cells might lead to the development of therapeutic strategies designed to improve innate immunity in aged individuals. Herein, advances in the field of innate immunity and aging with a focus on neutrophils, macrophages and dendritic cells in laboratory animals are discussed.

Metabolic biomarkers of prenatal alcohol exposure in human embryonic stem cell-derived neural lineages.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. The mechanisms underlying FASD are incompletely understood, and biomarkers to identify those at risk are lacking. Here, we perform metabolomic analysis of embryoid bodies and neural lineages derived from human embryonic stem (hES) cells to identify the neural secretome produced in response to ethanol (EtOH) exposure. METHODS: WA01 and WA09 hES cells were differentiated into embryoid bodies, neural progenitors, or neurons. Cells along this progression were cultured for 4 days with 0, 0.1, or 0.3% EtOH. Supernatants were subjected to C18 chromatography followed by ESI-QTOF-MS. Features were annotated using public databases, and the identities of 4 putative biomarkers were confirmed with purified standards and comparative MS/MS. RESULTS: EtOH treatment induced statistically significant changes to metabolite abundance in human embryoid bodies (180 features), neural progenitors (76 features), and neurons (42 features). There were no shared significant features between different cell types. Fifteen features showed a dose-response to EtOH. Four chemical identities were confirmed: L-thyroxine, 5'-methylthioadenosine, and the tryptophan metabolites, L-kynurenine and indoleacetaldehyde. One feature with a putative annotation of succinyladenosine was significantly increased in both EtOH treatments. Additional features were selective to EtOH treatment but were not annotated in public databases. CONCLUSIONS: EtOH exposure induces statistically significant changes to the metabolome profile of human embryoid bodies, neural progenitors, and neurons. Several of these metabolites are normally present in human serum, suggesting their usefulness as potential serum FASD biomarkers. These findings suggest the biochemical pathways that are affected by EtOH in the developing nervous system and delineate mechanisms of alcohol injury during human development.

Genetic gatekeepers: regulating direct-to-consumer genomic services in an era of participatory medicine.

Should consumers be able to obtain information about their own bodies, even if it has no proven medical value? Direct-to-consumer ("DTC") genomic companies offer consumers two services: generation of the consumer's personal genetic sequence, and interpretation of that sequence in light of current research. Concerned that consumers will misunderstand genomic information and make ill-advised health decisions, regulators, legislators and scholars have advocated restricted access to DTC genomic services. The Food and Drug Administration, which has historically refrained from regulating most genetic tests, has announced its intent to treat DTC genomic services as medical devices because they make "medical claims." This Article argues that FDA regulation of genomic services as medical devices would be counterproductive. Clinical laboratories conducting genetic tests are already overseen by a federal regime administered by the Centers for Medicare and Medicaid Services. While consumers and clinicians would benefit from clearer communication of test results and their health implications, FDA's gatekeeping framework is ill-suited to weigh the safety and efficacy of genomic information that is not medically actionable in traditional ways. Playing gatekeeper would burden FDA's resources, conflict with the patient-empowering policies promoted by personalized medicine initiatives, impair individuals' access to information in which they have powerful autonomy interests, weaken novel participatory research infrastructures, and set a poor precedent for the future regulation of medical information. Rather than applying its risk-based regulatory framework to genetic information, FDA should ameliorate regulatory uncertainty by working with the Federal Trade Commission and Centers for Medicare and Medicaid Services to ensure that DTC genomic services deliver analytically valid data, market and implement their services in a truthful manner, and fully disclose the limitations of their services. Federal agencies with relevant expertise should collaborate on standards and best practices for interpreting genetic information in light of scientific uncertainty, and an adverse event reporting system should be established to collect empirical data verifying or disproving the speculative harms resulting from individual access to genetic information. Most of all, FDA should take advantage of this opportunity to adapt its regulatory process to an increasingly informational health ecosystem.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Predict the Cardiotoxicity Potential of Next Generation Nicotine Products.

Combustible cigarette smoking is an established risk factor for cardiovascular disease. By contrast, the cardiotoxicity potential of non-combustible next generation nicotine products (NGPs), which includes heated tobacco products (HTPs) and electronic vaping products (EVPs), and how this compares relative to combustible cigarettes is currently an area of scientific exploration. As such, there is a need for a rapid screening assay to assess this endpoint. The Cardio quickPredict is a metabolomics biomarker-based assay that uses human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to screen for potential structural and functional cardiac toxicants based on the changes of four metabolites, lactic acid, arachidonic acid, thymidine, and 2'-deoxycytidine. The study aims were to investigate the cardiotoxicity potential of NGPs compared to cigarettes, in addition to nicotine. To accomplish this, hiPSC-CM were exposed to smoke or aerosol bubbled PBS samples: reference cigarette (1R6F); three variants of HTP; and three EVP variants. The 1R6F bPBS was the most active, having cardiotoxic potential at 0.3-0.6% bPBS (0.4-0.9 µg/mL nicotine), followed by HTP, which displayed cardiotoxic potential at a 10 times higher concentration, 3.3% bPBS (4.1 µg/mL nicotine). Both 1R6F and HTP bPBS (at 10-fold higher concentration than 1R6F) affected all four predictive metabolites, whereas none of the EVP bPBS samples were active in the assay up to the maximal concentration tested (10% bPBS). Nicotine tested on its own was predicted to have cardiotoxic potential at concentrations greater than 80 µg/mL, which is higher than expected physiological levels associated with combustible cigarette smoking. The application of this rapid screening assay to NGP research and the associated findings adds to the weight-of-evidence indicating that NGPs have a tobacco harm reduction potential when compared to combustible cigarettes. Additionally, this technique was shown to be sensitive and robust for the assessment of different NGPs and may be considered as part of a larger overall scientific framework for NGP assessments.

Quantitative in vitro to in vivo extrapolation for developmental toxicity potency of valproic acid analogues.

BACKGROUND: The developmental toxicity potential (dTP) concentration from the devTOX quickPredict (devTOXqP ) assay, a metabolomics-based human induced pluripotent stem cell assay, predicts a chemical's developmental toxicity potency. Here, in vitro to in vivo extrapolation (IVIVE) approaches were applied to address whether the devTOXqP assay could quantitatively predict in vivo developmental toxicity lowest effect levels (LELs) for the prototypical teratogen valproic acid (VPA) and a group of structural analogues. METHODS: VPA and a series of structural analogues were tested with the devTOXqP assay to determine dTP concentration and we estimated the equivalent administered doses (EADs) that would lead to plasma concentrations equivalent to the in vitro dTP concentrations. The EADs were compared to the LELs in rat developmental toxicity studies, human clinical doses, and EADs reported using other in vitro assays. To evaluate the impact of different pharmacokinetic (PK) models on IVIVE outcomes, we compared EADs predicted using various open-source and commercially available PK and physiologically based PK (PBPK) models. To evaluate the effect of in vitro kinetics, an equilibrium distribution model was applied to translate dTP concentrations to free medium concentrations before subsequent IVIVE analyses. RESULTS: The EAD estimates for the VPA analogues based on different PK/PBPK models were quantitatively similar to in vivo data from both rats and humans, where available, and the derived rank order of the chemicals was consistent with observed in vivo developmental toxicity. Different models were identified that provided accurate predictions for rat prenatal LELs and conservative estimates of human safe exposure. The impact of in vitro kinetics on EAD estimates is chemical-dependent. EADs from this study were within range of predicted doses from other in vitro and model organism data. CONCLUSIONS: This study highlights the importance of pharmacokinetic considerations when using in vitro assays and demonstrates the utility of the devTOXqP human stem cell-based platform to quantitatively assess a chemical's developmental toxicity potency.

Pre- and post-surgery brain tumor multimodal magnetic resonance imaging data optimized for large scale computational modelling.

We present a dataset of magnetic resonance imaging (MRI) data (T1, diffusion, BOLD) acquired in 25 brain tumor patients before the tumor resection surgery, and six months after the surgery, together with the tumor masks, and in 11 controls (recruited among the patients' caregivers). The dataset also contains behavioral and emotional scores obtained with standardized questionnaires. To simulate personalized computational models of the brain, we also provide structural connectivity matrices, necessary to perform whole-brain modelling with tools such as The Virtual Brain. In addition, we provide blood-oxygen-level-dependent imaging time series averaged across regions of interest for comparison with simulation results. An average resting state hemodynamic response function for each region of interest, as well as shape maps for each voxel, are also contributed.

A novel role for NKT cells in cutaneous wound repair.

Here, we report the novel observation that natural killer T (NKT) cells contribute to the cutaneous wound repair process. Using an excisional wound model in wild-type versus NKT cell-deficient mice, this report shows that when NKT cells are absent, initial wound closure is markedly accelerated. We report here for the first time that NKT cells are a significant constituent of early wound inflammation and that they regulate the local production of a key subset of neutrophil and monocyte/macrophage chemokines, as well as TGF-ß1 content and collagen deposition. Together, our findings support the concept that NKT cells regulate the early inflammatory and fibroproliferative phases of nonpathologic healing wounds, positioning the NKT cell as an attractive potential therapeutic target for modulation of impaired wound healing.

Prevention of NKT cell activation accelerates cutaneous wound closure and alters local inflammatory signals.

We previously reported that in the absence of NKT cells, wound closure was accelerated in a murine excisional punch wound model. Here, we explored whether purposefully inhibiting NKT cell activation had similar effects on wound closure and the dermal inflammatory response to injury. We found that prevention of NKT cell activation accelerated wound closure in a dose-responsive manner. If anti-CD1d was administered before wounding, NKT cell infiltration into cutaneous wounds was diminished without quantitative changes in cellular infiltrates. Furthermore, prevention of NKT cell activation transiently enhanced the local production of a subset of chemokines, including MIP-2, MCP-1, MIP-1α, and MIP-1ß, and altered the relative expression of CD69 and CXCR2 on the surface of both circulating and wound NKT cells. Taken together, these findings suggest that wounding activates NKT cells via CD1d presentation of glycolipid antigen and help further define a role for NKT cells in the regulation of wound inflammation and closure. Many soluble factors have been targeted as potential wound healing therapies, but their clinical success has been limited. Given our findings, the NKT cell may be an attractive target for wound healing therapies.

The adaptive significance of crustacean hyperglycaemic hormone (CHH) in daily and seasonal migratory activities of the Christmas Island red crab Gecarcoidea natalis.

The Christmas Island red crab Gecarcoidea natalis undergoes extreme changes in metabolic status, ranging from inactivity during the dry season, to a spectacular annual breeding migration at the start of the wet season. The dramatic change in metabolic physiology that this polarisation entails should be reflected in changes in endocrine physiology, particularly that of the crustacean hyperglycaemic hormone (CHH), of which we know relatively little. CHH levels were measured using a novel ultrasensitive time-resolved fluoroimmunoassay (TR-FIA), together with metabolites (glucose, lactate), in the field at several scales of temporal resolution, during migratory activities (wet season) and during the inactive fossorial phase (dry season). Release patterns of CHH were measured during extreme (forced) exercise, showing for the first time an unexpectedly rapid pulsatile release of this hormone. A seasonally dependent glucose-sensitive negative-feedback loop was identified that might be important in energy mobilisation during migration. Haemolymph lactate levels were strongly correlated with CHH levels in both field and experimental animals. During migration, CHH levels were lower than during the dry season and, during migration, daytime CHH levels (when most locomotor activity occurred) increased. However, the intense dawn activity in both dry and wet seasons was not always associated with repeatable hyperglycaemia or CHH release. The results obtained are discussed in relation to the life history and behaviour of G. natalis.

Interleukin-6 contributes to age-related alteration of cytokine production by macrophages.

Here, we studied in vitro cytokine production by splenic macrophages obtained from young and aged BALB/c wild type (WT) and IL-6 knockout (IL-6 KO) mice. Relative to macrophages obtained from young WT mice given lipopolysaccharide (LPS), those from aged WT mice had decreased production of proinflammatory cytokines. In contrast, when compared to macrophages from young IL-6 KO mice, LPS stimulation yielded higher levels of these cytokines by cells from aged IL-6 KO mice. Aging or IL-6 deficiency did not affected the percentage of F4/80(+) macrophages, or the surface expression of Toll-like receptor 4 (TLR4) and components of the IL-6 receptor. Overall, our results indicate that IL-6 plays a role in regulating the age-related defects in macrophages through alteration of proinflammatory cytokines, adding to the complexity of IL-6-mediated impairment of immune cell function with increasing age.

A Targeted Metabolomics-Based Assay Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Identifies Structural and Functional Cardiotoxicity Potential.

Implementing screening assays that identify functional and structural cardiotoxicity earlier in the drug development pipeline has the potential to improve safety and decrease the cost and time required to bring new drugs to market. In this study, a metabolic biomarker-based assay was developed that predicts the cardiotoxicity potential of a drug based on changes in the metabolism and viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Assay development and testing was conducted in 2 phases: (1) biomarker identification and (2) targeted assay development. In the first phase, metabolomic data from hiPSC-CM spent media following exposure to 66 drugs were used to identify biomarkers that identified both functional and structural cardiotoxicants. Four metabolites that represent different metabolic pathways (arachidonic acid, lactic acid, 2'-deoxycytidine, and thymidine) were identified as indicators of cardiotoxicity. In phase 2, a targeted, exposure-based biomarker assay was developed that measured these metabolites and hiPSC-CM viability across an 8-point concentration curve. Metabolite-specific predictive thresholds for identifying the cardiotoxicity potential of a drug were established and optimized for balanced accuracy or sensitivity. When predictive thresholds were optimized for balanced accuracy, the assay predicted the cardiotoxicity potential of 81 drugs with 86% balanced accuracy, 83% sensitivity, and 90% specificity. Alternatively, optimizing the thresholds for sensitivity yields a balanced accuracy of 85%, 90% sensitivity, and 79% specificity. This new hiPSC-CM-based assay provides a paradigm that can identify structural and functional cardiotoxic drugs that could be used in conjunction with other endpoints to provide a more comprehensive evaluation of a drug's cardiotoxicity potential.

Survival of a serotype 4b strain and a serotype 1/2a strain of Listeria monocytogenes, isolated from a stone fruit outbreak investigation, on whole stone fruit at 4 °C.

In the summer of 2014, a multistate outbreak of listeriosis associated with contaminated stone fruit (peach and nectarine) was reported. A serotype 4b variant Listeria monocytogenes (Lm) strain of singleton Sequence Type (ST) 382 was isolated from clinical samples and stone fruit associated with the outbreak. A serotype 1/2b Lm strain of ST5, Clonal Complex 5 was isolated only from outbreak-associated stone fruit, not from clinical samples. Here we investigated the fate of the serotype 4b and 1/2b strains, at two inoculation levels (high level at 3.7 logCFU/fruit and low level at 2.7 logCFU/fruit), on the surfaces of white peach, yellow peach and yellow nectarine stored at 4 °C for 26 days. After rinsing the fruits, we determined the Lm levels in the rinsates and on the peels. We enumerated Lm using a direct plating method and compared two chromogenic agars. The Lm populations rapidly declined in the first 3 days and then declined more slowly until Day 19/21. The maximum decline was 1.6 logCFU/fruit on yellow peach inoculated with serotype 4b at high level. For fruits inoculated with high-level Lm, the lowest level of Lm (1.7 logCFU/fruit) was observed on for white peach inoculated with serotype 1/2b, and the highest level of Lm (2.6 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with the serotype 1/2b strain. For fruits inoculated with low-level Lm, the lowest level of Lm (1.3 logCFU/fruit) was observed on yellow nectarine inoculated with either the serotype 4b or 1/2b strain, and the highest level of Lm (1.7 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with ST382. The D-values ranged from 15 days to 28 days. Lm remained viable until the end of storage (Day 26), but the levels were not significantly different from those on Day 19/21. The types of stone fruit and Lm strain did not significantly affect the survival of Lm. These results demonstrate that contaminated stone fruit can carry a potential risk for causing listeriosis in susceptible populations. Comparison of direct plating results using two chromogenic agars showed that RAPID' L. mono and Agar Listeria Ottavani & Agosti performed equivalently for enumerating Lm on stone fruit. The fruit rinsing recovered 80% to 84% of Lm from fruit surfaces.