Transient Exposure Research Articles

Gravitational slope-deformation of a resurgent caldera: New insights from the mechanical behaviour of Mt. Nuovo tuffs (Ischia Island, Italy)

Ischia Island (Italy) is an impressive example of the rare phenomenon of caldera resurgence. The emplacement and replenishment of magmas at shallow depth resulted in a vertical uplift of about 900m, concentrated in the western portion of Mt. Epomeo (789m a.s.l.). As a consequence of this uplift, the island has experienced several slope instabilities at different scales since the Holocene, from shallow mass movements to large rock and debris avalanches. These mass wasting events, which mobilised large volumes of greenish alkali-trachytic tuff (the Mt. Epomeo Green Tuff, MEGT), were strictly related to volcano-tectonic activity and the interaction between the volcanic slopes and the hydrothermal system beneath the island. Deep-Seated Gravitational Slope Deformation (DSGSD) at Mt. Nuovo, located adjacent to densely populated coastal villages, is an ongoing process that covers an area of 1.6km2. The Mt. Nuovo DSGSD involves a rock mass volume of 190Mm3 and is accommodated by a main shear zone and a series of sub-vertical fault zones associated with high-angle joint sets. To improve our understanding of this gravity-induced process, we performed a physical (porosity and permeability) and mechanical (uniaxial and triaxial deformation experiments) characterisation of two ignimbrite deposits - both from the MEGT - that form a significant component of the NW sector of Mt. Epomeo. The main conclusions drawn from our experiments are twofold. First, the presence of water dramatically reduces the strength of the tuffs, suggesting that the movement of fluids within the hydrothermal system could greatly impact slope stability. Second, the transition from brittle (dilatant) to ductile (compactant) behaviour in the tuffs of the MEGT occurs at a very low effective pressure, analogous to a depth of a couple of hundred metres, and that this transition is likely moved closer to the surface in the presence of water. We hypothesise that compactant (porosity decreasing) behaviour at the base of the layer could therefore facilitate slope instability. Although our results show that transient exposure to 300°C does not influence the short-term strength of the tuff, we speculate that the high in-situ temperature could increase the efficiency of brittle and compactant creep and therefore increase the rate of slope deformation. Taken together, our experimental data highlight a potentially important role for the hydrothermal system (that reaches a minimum depth of ~1km) in dictating the DSGSD at Mt. Nuovo. An understanding of this deformation process is not only important for the proximal coastal villages, at risk of engulfment by a large debris avalanche, but also for the towns and cities along the coast of the Gulf of Naples that are at risk to a secondary consequence of such an avalanche - a tsunami wave.

Feruloylserotonin inhibits hydrogen peroxide-induced melanogenesis and apoptosis in B16F10 and SK-Mel-2 melanoma cells

Feruloylserotonin (FS) is a major bioactive component of safflower seeds, with documented strong antibacterial, anti-inflammatory, and free radical scavenging activities. Reactive oxygen species (ROS) can strongly induce melanogenesis and cell apoptosis. The present study aimed to investigate the ability of FS in preventing hydrogen peroxide (H2O2)-induced melanogenesis and cell apoptosis. Melanogenesis and apoptotic cell death were induced by transient exposure to H2O2 in B16F10 and SK-Mel-2 melanoma cells. FS significantly inhibited melanogenesis and cell death in both cell lines. FS inhibited H2O2-induced melanin production by down-regulating CREB/MITF/TYR signaling via inhibited intracellular cAMP accumulation. Additionally, FS induced extracellular regulated kinase activation, which led to the degradation of MITF and consequently decreased TYR expression and melanin production in H2O2-stimulated cells. Furthermore, FS inhibited H2O2-induced apoptotic cell death by maintaining mitochondrial membrane potential. Therefore, FS might have potential use for cosmetic whitening and as a therapeutic agent for hyperpigmentation disorder.

Transcriptional changes in chick wing bud polarization induced by retinoic acid.

Background: Retinoic acid is implicated in the induction of the gene encoding Sonic hedgehog (Shh) that specifies anteroposterior positional values and promotes growth of the developing limb bud. However, because retinoic acid is involved in limb initiation, it has been difficult to determine if it could have additional roles in anteroposterior patterning. To investigate this, we implanted retinoic acid–soaked beads to the anterior margin of the chick wing bud and performed microarray analyses prior to onset of Shh expression. Results: Retinoic acid up‐regulates expression of Hoxd11‐13 that encode transcription factors implicated in inducing Shh transcription and that are involved in digit development. In our assay, retinoic acid induces Shh transcription and, consequently, a new pattern of digits at a much later stage than anticipated. Retinoic acid represses many anteriorly expressed genes, including Bmp4, Lhx9, Msx2, and Alx4. We provide evidence that retinoic acid influences transcription via induction of dHAND and inhibition of Gli3 to establish a new anteroposterior pre‐pattern. We show that transient exposure to retinoic acid can suppress distal development and expedite cells to transcriptionally respond to Shh. Conclusions: Our findings reveal how retinoic acid and Shh signaling could cooperate in anteroposterior patterning of the limb. Developmental Dynamics 246:682–690, 2017. © 2017 Wiley Periodicals, Inc.

Abstract 15: Glucose-Mediated Remodeling of Cardiac DNA Methylation

To identify the role of glucose in the development of diabetic cardiomyopathy, we had directly assessed glucose delivery to the intact heart on alterations of DNA methylation and gene expression using both an inducible heart-specific transgene (glucose transporter 4; mG4H) and streptozotocin-induced diabetes (STZ) mouse models. We aimed to determine whether long-lasting diabetic complications arise from prior transient exposure to hyperglycemia via a process termed “glycemic memory.” We had identified DNA methylation changes associated with significant gene expression regulation. Comparing our results from STZ, mG4H, and the modifications which persist following transgene silencing, we now provide evidence for cardiac DNA methylation as a persistent epigenetic mark contributing to glycemic memory. To begin to determine which changes contribute to human heart failure, we measured both RNA transcript levels and whole-genome DNA methylation in heart failure biopsy samples (n = 12) from male patients collected at left ventricular assist device placement using RNA-sequencing and Methylation450 assay, respectively. We hypothesized that epigenetic changes such as DNA methylation distinguish between heart failure etiologies. Our findings demonstrated that type 2 diabetic heart failure patients (n = 6) had an overall signature of hypomethylation, whereas patients listed as ischemic (n = 5) had a distinct hypermethylation signature for regulated transcripts. The focus of this initial analysis was on promoter-associated CpG islands with inverse changes in gene transcript levels, from which diabetes (14 genes; e.g. IGFBP4) and ischemic (12 genes; e.g. PFKFB3) specific targets emerged with significant regulation of both measures. By combining our mouse and human molecular analyses, we provide evidence that diabetes mellitus governs direct regulation of cellular function by DNA methylation and the corresponding gene expression in diabetic mouse and human hearts. Importantly, many of the changes seen in either mouse type 1 diabetes or human type 2 diabetes were similar supporting a consistent mechanism of regulation. These studies are some of the first steps at defining mechanisms of epigenetic regulation in diabetic cardiomyopathy.

Metabolic Disruption Early in Life is Associated With Latent Carcinogenic Activity of Dichloroacetic Acid in Mice.

Early-life environmental factors can influence later-life susceptibility to cancer. Recent evidence suggests that metabolic pathways may mediate this type of latency effect. Previously, we reported that short-term exposure to dichloroacetic acid (DCA) increased liver cancer in mice 84 weeks after exposure was stopped. Here, we evaluated time course dynamics for key events related to this effect. This study followed a stop-exposure design in which 28-day-old male B6C3F1 mice were given the following treatments in drinking water for up to 93 weeks: deionized water (dH2O, control); 3.5 g/l DCA continuously; or 3.5 g/l DCA for 4-52 weeks followed by dH2O. Effects were evaluated at eight interim time points. A short-term biomarker study was used to evaluate DCA effects at 6, 15, and 30days. Liver tumor incidence was higher in all DCA treatment groups, including carcinomas in 82% of mice previously treated with DCA for only 4 weeks. Direct effects of DCA in the short-term study included decreased liver cell proliferation and marked mRNA changes related to mitochondrial dysfunction and altered cell metabolism. However, all observed short-term effects of DCA were ultimately reversible, and prior DCA treatment did not affect liver cell proliferation, apoptosis, necrosis, or DNA sequence variants with age. Key intermediate events resulting from transient DCA exposure do not fit classical cytotoxic, mitogenic, or genotoxic modes of action for carcinogenesis, suggesting a distinct mechanism associated with early-life metabolic disruption.

Activation of WEE1 confers resistance to PI3K inhibition in glioblastoma.

Oncogenic activation of phosphatidylinositol-3 kinase (PI3K) signaling plays a pivotal role in the development of glioblastoma (GBM). However, pharmacological inhibition of PI3K has so far not been therapeutically successful due to adaptive resistance through a rapid rewiring of cancer cell signaling. Here we identified that WEE1 is activated after transient exposure to PI3K inhibition and confers resistance to PI3K inhibition in GBM. Patient-derived glioma-initiating cells and established GBM cells were treated with PI3K inhibitor or WEE1 inhibitor alone or in combination, and cell proliferation was evaluated by CellTiter-Blue assay. Cell apoptosis was analyzed by TUNEL, annexin V staining, and blotting of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Both subcutaneous xenograft and orthotropic xenograft studies were conducted to evaluate the effects of the combination on tumorigenesis; the tumor growth was monitored by bioluminescence imaging, and tumor tissue was analyzed by immunohistochemistry to validate signaling changes. PI3K inhibition activates WEE1 kinase, which in turn phosphorylates cell division control protein 2 homolog (Cdc2) at Tyr15 and inhibits Cdc2 activity, leading to G2/M arrest in a p53-independent manner. WEE1 inhibition abrogated the G2/M arrest and propelled cells to prematurely enter into mitosis and consequent cell death through mitotic catastrophe and apoptosis. Additionally, combination treatment significantly suppressed tumor growth in a subcutaneous model but not in an intracranial model due to limited blood-brain barrier penetration. Our findings highlight WEE1 as an adaptive resistant gene activated after PI3K inhibition, and inhibition of WEE1 potentiated the effectiveness of PI3K targeted inhibition, suggesting that a combinational inhibition of WEE1 and PI3K might allow successful targeted therapy in GBM.

Ultraviolet radiation accelerates NRas-mutant melanomagenesis: A cooperative effect blocked by sunscreen.

To mitigate melanoma risk, sunscreen use is widely advocated; yet, the ability of sunscreens to prevent melanoma remains controversial. Here, we test the tenet that sunscreens limit melanoma risk by blocking ultraviolet radiation (UV)-induced DNA damage using murine models that recapitulate the genetics and spontaneous evolution of human melanoma. We find that a single, non-erythematous dose of UV dramatically accelerates melanoma onset and increases tumor multiplicity in mice carrying an endogenous, melanocyte-specific NRas61R allele. By contrast, transient UV exposure does not alter tumor onset in mice lacking p16INK4a or harboring an NRas12D allele. To block the rapid onset of melanoma cooperatively caused by UV and NRas61R , we employed a variety of aerosol sunscreens. While all sunscreens delayed melanoma formation and blocked UV-induced DNA damage, differences in aerosol output (i.e., amount applied/cm2 ) caused variability in the cancer preventative efficacy of products with identical sunburn protection factor (SPF) ratings.

Abstract 4358: The immunomodulatory anticancer agent RRx-001 induces a vaccine-like interferon response through epigenetic induction of viral mimicry

Abstract RRx-001, sourced from the aerospace industry and currently in phase II clinical trials, is a novel anti-cancer agent that mediates immunomodulatory effects, either directly through polarization of tumor associated macrophages or indirectly through vascular normalization and increased T-lymphocyte infiltration. With multiple additional mechanisms of action including upregulation of oxidative stress, depletion of GSH and NADPH, anti-angiogenesis and epigenetic modulation, RRx-001 is being studied as a single chemotherapeutic agent to resensitize tumors to prior therapy and to prime tumors to respond to radiation, chemotherapy and immunotherapy in combination therapy studies. In this study, we identified another mechanism, viral mimicry, which refers to the ‘unsilencing’ of epigenetically repressed viral genes present in the tumor that provokes an immune response and may contribute to the anti-cancer activity of RRx-001. Specifically, RRx-001 inhibited the growth of human colon cancer cells (HCT 116) and decreased levels of the DNA methyltransferases Dnmt1 and Dnmt3a in a time and dose-dependent manner. Treatment of HCT 116 cells with 0.5 μM RRx-001 for 24 hours significantly increased transcripts of interferon (IFN)-responsive genes and this induction was sustained for up to 4 weeks after transient exposure to RRx-001. ELISA assays showed that RRx-001 increased secretion of type I and III IFNs by HCT 116 cells, and these IFNs were confirmed to be bioactive. Transcription of endogenous retroviruses was induced by RRx-001 through demethylation of the promoter of endogenous retrovirus genes as determined by methylation-specific polymerase chain reaction and combined bisulfite restriction analysis. Immunofluorescence staining with J2 antibody confirmed induction of double-stranded RNA. In conclusion, transient exposure of HCT 116 cells to low-dose RRx-001 induced transcription of silenced retroviral genes present in the cancer cell DNA with subsequent synthesis of IFN in response to this “pseudo-pathogenic” stimulus, mimicking an antiviral defense. RRx-001-mediated IFN induction may have the potential to improve the efficacy of immunotherapies as well as radiotherapy, standard chemotherapies and molecularly targeted agents when used in combination. Citation Format: Hongjuan Zhao, Shoucheng Ning, Rosalie Nolley, Jan Scicinski, Bryan Oronsky, Susan J. Knox, Donna M. Peehl. The immunomodulatory anticancer agent RRx-001 induces a vaccine-like interferon response through epigenetic induction of viral mimicry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4358. doi:10.1158/1538-7445.AM2017-4358

Abstract 122: Preclinical update on targeting KRAS wild-type colorectal cancer with an EGFR-targeted monoclonal tri-body mixture, MM-151

Abstract Within KRAS wild-type colorectal cancer (KRAS-wt CRC), standard of care treatment includes EGFR-directed monoclonal antibodies (mAb) in combination with an irinotecan-based chemotherapy regimen. While such combinations have clinically established synergy, the underlying mechanisms of synergy remain elusive, and resistance invariably develops against either the EGFR-targeted therapy or irinotecan, leaving patients with limited treatment options. MM-151 consists of three anti-EGFR IgG1 mAbs that bind simultaneously to non-overlapping epitopes on the EGFR extracellular domain (ECD). Preclinical and clinical studies have demonstrated that oligoclonal targeting of EGFR with MM-151 results in blockade of the EGFR pathway via potent inhibition of ligand-driven signal amplification and receptor downregulation. Critically, these activities are uniquely maintained in the presence of high-affinity EGFR ligands as well as mutations in the ECD of EGFR, both of which drive resistance to cetuximab and panitumumab. Here, we tested the preclinical hypothesis that comprehensive inhibition of the EGFR pathway by MM-151 leads to enhanced antitumor activity when combined with chemotherapy in models of KRAS-wt CRC. In vitro studies to investigate the activity of MM-151 in combination with SN-38, the active metabolite of irinotecan, showed that MM-151 potentiates SN-38-induced cell death in KRAS-wt CRC models. Treatment with MM-151 potently suppresses EGFR pathway activation caused by SN-38, and the combination enhances stress and pro-apoptotic signaling, leading to increased apoptosis. Notably, these synergistic activities are maintained in the presence of two key mechanisms of resistance to cetuximab: high-affinity ligands and EGFR ECD mutations. Dose scheduling studies demonstrated that sustained treatment with MM-151 following transient SN-38 exposure - a hallmark of small molecule therapy - partially overcomes the sub-optimal activity of chemotherapy alone. Finally, data from in vivo xenograft and PDX studies strongly supports the addition of MM-151 to irinotecan-based chemotherapy regimens for KRAS-wt CRC. In summary, we present data supporting the hypothesis that comprehensive antagonism of the EGFR pathway via oligoclonal targeting of EGFR with MM-151 leads to antitumor activity when combined with chemotherapy in preclinical KRAS-wt CRC models. The ability of MM-151 and SN-38 to overcome both de novo and acquired mechanisms of resistance within CRC models supports further clinical evaluation of this combination in metastatic CRC patients. Citation Format: Shawn P. Carey, Hongfang Wang, Erika Handly, Brittany Ahlstedt, Daniel Gaddy, Jeffrey D. Kearns, Greg Finn, Birgit Schoeberl, Rachel Nering. Preclinical update on targeting KRAS wild-type colorectal cancer with an EGFR-targeted monoclonal tri-body mixture, MM-151 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 122. doi:10.1158/1538-7445.AM2017-122

Cytocompatible cell encapsulation via hydrogel photopolymerization in microfluidic emulsion droplets

Encapsulating cells within biocompatible materials is a widely pursued and promising element of tissue engineering and cell-based therapies. Recently, extensive interest in microfluidic-enabled cell encapsulation has emerged as a strategy to structure hydrogels and establish custom cellular microenvironments. In particular, it has been shown that the microfluidic-enabled photoencapsulation of cells within PEG diacrylate (PEGDA)-based microparticles can be performed cytocompatibly within gas-permeable, nitrogen-jacketed polydimethylsiloxane microfluidic devices, which mitigate the oxygen inhibition of radical chain growth photopolymerization. Compared to bulk polymerization, in which cells are suspended in a static hydrogel-forming solution during gelation, encapsulating cells via microfluidic processing exposes cells to a host of potentially deleterious stresses such as fluidic shear rate, transient oxygen depletion, elevated pressures, and UV exposure. In this work, we systematically examine the effects of these factors on the viability of cells that have been microfluidically photoencapsulated in PEGDA. It was found that the fluidic shear rate during microdroplet formation did not have a direct effect on cell viability, but the flow rate ratio of oil to aqueous solution would impart harmful effects to cells when a critical threshold was exceeded. The effects of UV exposure time and intensity on cells, however, are more complex, as they contribute unequally to the cumulative rate of peroxy radical generation, which is strongly correlated with cell viability. A reaction-diffusion model has been developed to calculate the cumulative peroxy radical concentration over a range of UV light intensity and radiation times, which was used to gain further quantitative understanding of experimental results. Conclusions drawn from this work provide a comprehensive guide to mitigate the physical and biochemical damage imparted to cells during microfluidic photoencapsulation and expands the potential for this technique.

Abstract 3180: Chemotherapeutical agent triggers transient resistance of cancer cells by promoting ABCB1 intercellular transfer

Abstract Multidrug resistance remains a major impediment to successful chemotherapy. In clinical chemotherapy, some patients often develop chemoresistance quite rapidly, the underlying mechanism remains elusive. Herein, we report that a transient exposure to chemotherapeutic agents can acutely promote the intercellular transfer of ABCB1 through stimulating Rab8B-mediated secretion of microvesicles (MVs) containing ABCB1 in drug-resistant donor cells, and promoting parallelly the endocytic recycling of ABCB1 in recipient tumor cells via the downregulation of Rab5. Sensitive recipient cells take up these ABCB1-incorporated MVs by a clathrin- and dynamin 2- dependent endocytic pathway, thereby acquiring a transient resistance to chemotherapeutic agents which are ABCB1 substrates. More fascinatingly, ABCB1 molecules can locally or distantly transfer to recipient tumor cells and respond to chemotherapeutical drug in the xenograft tumor models. In some cases of patients with NSCLC, a variable increase of ABCB1 surface expression was also detected in the peripheral blood monocyte subpopulations after they received the first chemotherapy. Our findings proposed a novel molecular mechanism of how sensitive cancer cells rapidly acquire the emergency resistance to chemotherapeutic agents by stimulating intercellular transfer of ABCB1. The precise modulation of this process may provide a valid therapeutic strategy to alleviate the MDR phenotype for successful treatment. Citation Format: Xiaokun Wang, Dongjuan Qiao, Likun Chen, Liyan Huang, Meng Xu, Zhen Chen, Fang Wang, Zhesheng Chen, Musheng Zeng, Li-Wu Fu. Chemotherapeutical agent triggers transient resistance of cancer cells by promoting ABCB1 intercellular transfer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3180. doi:10.1158/1538-7445.AM2017-3180

Bilayer silk fibroin grafts support functional oesophageal repair in a rodent model of caustic injury.

Surgical repair of caustic oesophageal injuries with autologous gastrointestinal segments is often associated with dysmotility, dysphagia and donor site morbidity, and therefore alternative graft options are needed. Bilayer silk fibroin (BLSF) scaffolds were assessed for their ability to support functional restoration of damaged oesophageal tissues in a rat model of onlay oesophagoplasty. Transient exposure of isolated oesophageal segments with 40% NaOH led to corrosive oesophagitis and a 91% reduction in the luminal cross-sectional area of damaged sites. Oesophageal repair with BLSF matrices was performed in injured rats (n = 27) as well as a nondiseased cohort (n = 12) for up to 2 months after implantation. Both implant groups exhibited >80% survival rates, displayed similar degrees of weight gain, and were capable of solid food consumption following a 3-day liquid diet. End-point μ-computed tomography of repaired sites demonstrated a 4.5-fold increase in luminal cross-sectional area over baseline injury levels. Reconstructed oesophageal conduits from damaged and nondiseased animals produced comparable contractile responses to KCl and electric field stimulation while isoproterenol generated similar tissue relaxation responses. Histological and immunohistochemical evaluations of neotissues from both implant groups showed formation of a stratified, squamous epithelium with robust cytokeratin expression as well as skeletal and smooth muscle layers positive for contractile protein expression. In addition, synaptophysin positive neuronal junctions and vessels lined with CD31 positive endothelial cells were also observed at graft sites in each setting. These results provide preclinical validation for the use of BLSF scaffolds in reconstructive strategies for oesophageal repair following caustic injury.

Sustained Intracellular Acidosis Triggers the Na⁺/H⁺ Exchager-1 Activation in Glutamate Excitotoxicity.

The Na+/H+ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-β (PKC-β) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to 100 μM glutamate or 20 mM NH4Cl. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-β, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to NH4Cl. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-β, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-β/ERK1/2/p90RSK pathway in neuronal cells.

Luteolin attenuates interleukin-6-mediated astrogliosis in human iPSC-derived neural aggregates: A candidate preventive substance for maternal immune activation-induced abnormalities.

Maternal infection during pregnancy increases the risk of neurodevelopmental conditions such as autism spectrum disorders and schizophrenia in offspring. Several previous animal studies have indicated that maternal immune activation (MIA), rather than a specific pathogen, alters fetal brain development. Among them, prenatal exposure to interleukin-6 (IL-6) has been associated with behavioral and neuropathological abnormalities, though such findings remain to be elucidated in humans. We developed a human cell-based model of MIA by exposing human induced pluripotent stem cells (hiPSCs)-derived neural aggregates to IL-6 and investigated whether luteolin-a naturally occurring flavonoid found in edible plants-could prevent MIA-induced abnormalities. We generated neural aggregates from hiPSCs using the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) method, following which aggregates were cultured in suspension. We then exposed the aggregates to IL-6 (100ng/ml) for 24h at day 51. Transient IL-6 exposure significantly increased the area ratio of astrocytes (GFAP-positive area ratio) and decreased the area ratio of early-born neurons (TBR1-positive or CTIP2-positive area ratio) relative to controls. In addition, western blot analysis revealed that levels of phosphorylated STAT3 were significantly elevated in IL-6-exposed neural aggregates. Luteolin treatment inhibited STAT3 phosphorylation and counteracted IL-6-mediated increases of GFAP-positive cells and reductions of TBR1-positive and CTIP2-positive cells. Our observations suggest that the flavonoid luteolin may attenuate or prevent MIA-induced neural abnormalities. As we observed increased apoptosis at high concentrations of luteolin, further studies are required to determine the optimal intake dosage and duration for pregnant women.

Conformational Flexibility Differentiates Naturally Occurring Bet v 1 Isoforms.

The protein Bet v 1 represents the main cause for allergic reactions to birch pollen in Europe and North America. Structurally homologous isoforms of Bet v 1 can have different properties regarding allergic sensitization and Th2 polarization, most likely due to differential susceptibility to proteolytic cleavage. Using NMR relaxation experiments and molecular dynamics simulations, we demonstrate that the initial proteolytic cleavage sites in two naturally occurring Bet v 1 isoforms, Bet v 1.0101 (Bet v 1a) and Bet v 1.0102 (Bet v 1d), are conformationally flexible. Inaccessible cleavage sites in helices and strands are highly flexible on the microsecond-millisecond time scale, whereas those located in loops display faster nanosecond-microsecond flexibility. The data consistently show that Bet v 1.0102 is more flexible and conformationally heterogeneous than Bet v 1.0101. Moreover, NMR hydrogen-deuterium exchange measurements reveal that the backbone amides in Bet v 1.0102 are significantly more solvent exposed, in agreement with this isoform’s higher susceptibility to proteolytic cleavage. The differential conformational flexibility of Bet v 1 isoforms, along with the transient exposure of inaccessible sites to the protein surface, may be linked to proteolytic susceptibility, representing a potential structure-based rationale for the observed differences in Th2 polarization and allergic sensitization.

Catch and Release of Cytokines Mediated by Tumor Phosphatidylserine Converts Transient Exposure into Long-Lived Inflammation

Immune cells constantly survey the host for pathogens or tumors and secrete cytokines to alert surrounding cells of these threats. Invivo, activated immune cells secrete cytokines for several hours, yet an acute immune reaction occurs over days. Given these divergent timescales, we addressed how cytokine-responsive cells translate brief cytokine exposure into phenotypic changes that persist over long timescales. We studied melanoma cell responses to transient exposure to the cytokine interferon γ (IFNγ) by combining a systems-scale analysis of gene expression dynamics with computational modeling and experiments. We discovered that IFNγ is captured by phosphatidylserine (PS) on the surface of viable cells both invitro and invivo then slowly released to drive long-term transcription of cytokine-response genes. This mechanism introduces an additional function for PS in dynamically regulating inflammation across diverse cancer and primary cell types and has potential to usher in new immunotherapies targeting PS and inflammatory pathways.

A Class of Environmental and Endogenous Toxins Induces BRCA2 Haploinsufficiency and Genome Instability

SummaryMutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.

General Anesthesia Causes Epigenetic Histone Modulation of c-Fos and Brain-derived Neurotrophic: Factor, Target Genes Important for Neuronal Development in the Immature Rat Hippocampus

(Anesthesiology. 2016;124(6):1311–1327) Research studies have shown that general anesthesia (GA) exposure among young animals causes cognitive impairments in not only first generation offspring but also nonexposed second generation offspring born to dams. Studies on rodents have revealed that GA exposure in utero has proven this to be true, thus confirming that transient GA exposure during a critical period of neuronal remodeling does result in changes that become embedded in the genetic information thus causing cognitive impairments. This study hypothesized that during critical stages of synaptogenesis, transient exposure to GA results in epigenetic changes in chromatin with adverse effects on transcription of target genes which are important for proper synapse formation and cognitive development.

Heavy metal-induced reactive oxygen species and cell death in barley root tip

Transient exposure of roots to Cd, Pb, Hg or Cu for 30min reduced root growth in a concentration-dependent manner. While the lower concentrations of metals evoked a marked root growth inhibition accompanied by a visible radial root expansion, the higher concentrations caused root growth arrest without the radial expansion of root cells. At lower metal concentrations, evoking mild stress, the amount of reactive oxygen species (ROS) increased linearly after the treatments. They probably function in both stress signalization and metabolic adaptation processes without damaging the root cells. In turn, at high metal concentrations, causing moderate or severe stress, their production has a biphasic character. The amount of ROS, mainly superoxide, increased rapidly in a metal dose-dependent manner, causing a marked cell death at the site of their generation in the root tips. After this transient burst, ROS generation increased again, together with highly ROS, during the destructive processes associated with cell death in the root tips. Our results show that despite the different origin of ROS induced by these heavy metals, the rate of their generation, depending on the metal concentration, determines the development of typical mild stress-activated morphogenic changes or severe stress-caused cell death within a few minutes after the exposure of roots to toxic level of metals.

Transient enalapril attenuates the reduction in glomerular filtration rate in prenatally programmed rats.

A maternal low‐protein diet has been shown to program hypertension and a reduction in glomerular filtration rate in adult offspring. This study examined the effect of continuous administration of enalapril in the drinking water and transient administration of enalapril administered from 21 to 42 days of age on blood pressure and glomerular filtration rate (GFR) in male rats whose mothers were fed a 20% protein diet (control) or a 6% protein diet (programmed) during the last half of pregnancy. After birth all rats were fed a 20% protein diet. Programmed rats (maternal 6% protein diet) were hypertensive at 15 months of age compared to control rats and both continuous and transient administration of enalapril had no effect on blood pressure on control offspring, but normalized the blood pressure of programmed offspring. GFR was 3.2 ± 0.1 mL/min in the control group and 1.7 ± 0.1 mL/min in the programmed rats at 17 months of age (P < 0.001). The GFR was 3.0 ± 0.1 mL/min in the control and 2.7 ± 0.1 mL/min in the programmed group that received continuous enalapril in their drinking water showing that enalapril can prevent the decrease in GFR in programmed rats. Transient administration of enalapril had no effect on GFR in the control group (3.2 ± 0.1 mL/min) and prevented the decrease in GFR in the programmed group (2.9 ± 0.1 mL/min). In conclusion, transient exposure to enalapril for 3 weeks after weaning can prevent the hypertension and decrease in GFR in prenatal programmed rats.