Transient Exposure Research Articles

Effects on prostate cancer cells of targeting RNA polymerase III.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

4-D Computational Modeling of Cardiac Outflow Tract Hemodynamics over Looping Developmental Stages in Chicken Embryos.

Cardiogenesis is interdependent with blood flow within the embryonic system. Recently, a number of studies have begun to elucidate the effects of hemodynamic forces acting upon and within cells as the cardiovascular system begins to develop. Changes in flow are picked up by mechanosensors in endocardial cells exposed to wall shear stress (the tangential force exerted by blood flow) and by myocardial and mesenchymal cells exposed to cyclic strain (deformation). Mechanosensors stimulate a variety of mechanotransduction pathways which elicit functional cellular responses in order to coordinate the structural development of the heart and cardiovascular system. The looping stages of heart development are critical to normal cardiac morphogenesis and have previously been shown to be extremely sensitive to experimental perturbations in flow, with transient exposure to altered flow dynamics causing severe late stage cardiac defects in animal models. This paper seeks to expand on past research and to begin establishing a detailed baseline for normal hemodynamic conditions in the chick outflow tract during these critical looping stages. Specifically, we will use 4-D (3-D over time) optical coherence tomography to create in vivo geometries for computational fluid dynamics simulations of the cardiac cycle, enabling us to study in great detail 4-D velocity patterns and heterogeneous wall shear stress distributions on the outflow tract endocardium. This information will be useful in determining the normal variation of hemodynamic patterns as well as in mapping hemodynamics to developmental processes such as morphological changes and signaling events during and after the looping stages examined here.

The changes of hydrological environment recorded in the section of Tianluoshan Site in Zhejiang Province

The Tianluoshan Site of Yuyao City in Zhejiang Province is a typical Neolithic site with a complete preservation of the Hemudu culture (ca. 7–5 ka). This culture is well-known for its important role in studies on early rice domestication. Studying samples from the Tianluoshan Site provides a good opportunity to understand regional paleoenvironmental changes and their impact on Neolithic settlement. In this study, 263 cm samples were obtained from the T005W profile in the Tianluoshan Site Museum. The profile samples were subjected to lithologic stratigraphic description, accelerated mass spectrometry (AMS)14C dating, high-resolution X-ray fluorescence (XRF) spectrometry, elemental scanning (2 mm/sample), diatom (20 cm/sample) and particle size analysis (2 cm/sample). The paleoenvironmental changes reflected by this profile could be divided into three stages based on multi-proxy analysis. (1) In the first stage (263–211 cm, about 7.0–6.5 cal ka BP), or the first and second cultural periods of Hemudu, the cultural layer was stacked and mixed with high contents of sand, gravel, and pottery pieces, thereby showing obvious disturbance from human activities. The diatom assemblage was dominated by aerophilous species, such as Hantzschia amphioxys , which live in shallow swamp. These findings indicated that this period was dominated mainly by a freshwater swamp environment. The geomorphic condition provided Neolithic people with a suitable environment to colonize and develop rice cultivation. (2) In the second stage (211–28 cm, around 4.2–4.0 cal ka BP), the layer had a thickness of approximately 180 cm. However, five AMS 14C dates from this stage were between ca. 4.2 and 4.0 cal ka BP, and the lithology of these overlying strata were inconsistent with the cultural layer underneath. As such, a hiatus existed between the two stages. The lithological feature of this stage mainly consisted of a parallel layer with a couplet of interbedded clay silt and white-gray sand layer, which represented deposition under hydrostatic conditions after flooding. This part also contained multiple yellow oxide layers, possibly indicating transient exposure. The intensity of Ca between 211 and 166 cm was low, but this value increased rapidly above 166 cm and stabilized at a higher level than that of the first period. The dominant diatom assemblage changed from a freshwater group ( H. amphioxys ) at the bottom of this part to a marine and brackish group (e.g., Cyclotella striata var. striata and Actionptychus undulates ). In this period, the rapid deposition of flooding events likely occurred as initiated by heavy precipitation (211–166 cm), which was indicated by low Ca and freshwater aerophilous diatom assemblage. Afterward, multiple storm surges resulted in sedimentation (166–50 cm) with marine-brackish characteristics. (3) In the third stage (28–0 cm, no date), the layer was characterized by a mixture of brown clay, silt, sand, and gravel with low contents of water, iron, and manganese oxide spots. Ca was weak, and clay content was high. This period was mainly dominated by freshwater epiphytic diatom species, such as Pinnularia vividis and Navicula cuspidate , and affected by modern farming. This study revealed the background of the hydrological environment during the first and second cultural periods of the Tianluoshan Site and the flooding events around 4.2–4.0 cal ka BP, which were essential for the understanding of the environmental background of Neolithic people and the extreme regional events in the Tianluoshan Site. The stratigraphic deposits of different sections in the site varied because of the influence of microtopography. Therefore, studies on the paleoenvironment of the Tianluoshan Site should involve a systematic analysis of multiple sections and cores around this site.

Effect of transient exposure to carbaryl wettable powder on the gut microbial community of honey bees

Bees are important pollinators in agriculture. The bee population has recently begun to decline possibly due to pesticides. The bee gut microbiota strongly influences the health of bees. The gut microbiota of bees is composed of distinct members belonging to selective taxa. Chemicals like pesticides can alter the gut microbiota. The present study investigated the effect of carbaryl pesticides on gut microbiota of honey bees, which had come in contact with rapeseed plants (Brassica napus) sprayed with carbaryl wettable powder during the honey bee brood test under semi-field condition. Molecular techniques (conventional and quantitative polymerase chain reaction (PCR), clone library method, and DNA sequencing) were employed to analyze changes in the microbial communities between the pesticide-exposed and unexposed bees. Phylogenetic analysis of 16S rRNA genes of the clones from both groups, showed differences in their respective compositions of core and non-core bacteria. Both groups contained carbohydrate-degrading bacteria such as Gilliamella apicola and Lactobacillus. However, the unexposed bees harbored Alphaproteobacteria, which were absent in the exposed bees. Microorganisms found in honey bee guts such as Snodgrassella alvi and L. kullabergensis, however, were observed only in the exposed bees, but not in the unexposed bees. The difference between the two groups was distinctly recognized when copy numbers of 16S rRNA genes were compared by quantitative PCR. Results showed that the average gene copy number for the unexposed bees was higher than that for the exposed bees. This may indicate the toxic effect of pesticides on bees and gut microbiota.

Hypoxic tachycardia is not a result of increased respiratory activity in healthy subjects.

What is the central question of this research? Does increased ventilation contribute to the increase in heart rate during transient exposure to hypoxia in humans? What is the main finding and its importance? Voluntary suppression of the ventilatory response to transient hypoxia does not affect the magnitude of the heart rate response to the stimulus. This indicates that hypoxic tachycardia is not secondary to hyperpnoea in humans. Better understanding of the physiology underlying the cardiovascular response to hypoxia might help in identification of new markers of elevated chemoreceptor activity, which has been proposed as a target in treatment of sympathetically mediated diseases. Animal data suggest that hypoxic tachycardia is secondary to hyperpnoea, and for years this observation has been extrapolated to humans, despite a lack of experimental evidence. We addressed this issue in 17 volunteers aged 29±7(SD) years. A transient hypoxia test, comprising several nitrogen-breathing episodes, was performed twice in each subject. In the first test, the subject breathed spontaneously (spontaneous breathing). In the second test, the subject was repeatedly asked to adjust his or her depth and rate of breathing according to visual (real-time inspiratory flow) and auditory (metronome sound) cues, respectively (controlled breathing), to maintain respiration at the resting level during nitrogen-breathing episodes. Hypoxic responsiveness, including minute ventilation [Hyp-VI; in liters per minute per percentage of blood oxygen saturation ( )], tidal volume [Hyp-VT; in litres per ], heart rate [Hyp-HR; in beats per minute per ], systolic [Hyp-SBP; in millimetres of mercury per ] and mean blood pressure [Hyp-MAP; in millimetres of mercury per ] and systemic vascular resistance [Hyp-SVR; in dynes seconds (centimetres)-5 per ] was calculated as the slope of the regression line relating the variable to , including pre- and post-hypoxic values. The Hyp-VI and Hyp-VT were reduced by 69±25 and 75±10%, respectively, in controlled versus spontaneous breathing (Hyp-VI, -0.30±0.15 versus -0.11±0.09; Hyp-VT, -0.030±0.024 versus -0.007±0.004; both P<0.001). However, the cardiovascular responses did not differ between spontaneous and controlled breathing (Hyp-HR, -0.62±0.24 versus -0.71±0.33; Hyp-MAP, -0.43±0.19 versus -0.47±0.21; Hyp-SVR, 9.15±5.22 versus 9.53±5.57; all P≥0.22), indicating that hypoxic tachycardia is not secondary to hyperpnoea. Hyp-HR was correlated with Hyp-SVR (r=-074 and -0.80 for spontaneous and controlled breathing, respectively; both P<0.05) and resting barosensitivity assessed with the sequence technique (r=-0.60 for spontaneous breathing; P<0.05). This might suggest that the baroreflex mechanism is involved.

ADAM10 sheddase activation is controlled by cell membrane asymmetry.

Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer’s disease. The mechanisms underlying ADAM10 sheddase activation are incompletely understood. Here, we show that transient exposure of the negatively charged phospholipid phosphatidylserine (PS) is necessarily required. The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage. Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6 (ANO6) led to increased PS externalization and substrate release. Transfection with a constitutively active form of ANO6 resulted in maximum sheddase activity in the absence of any stimulus. Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in ANO6. A putative PS-binding motif was identified in the conserved stalk region. Replacement of this motif resulted in strong reduction of sheddase activity. In conjunction with the recently described 3D structure of the ADAM10 extracellular domain, a model is advanced to explain how surface-exposed PS triggers ADAM10 sheddase function.

Ketamine increases the expression of GluR1 and GluR2 α-amino-3-hydroxy-5-methy-4-isoxazole propionate receptor subunits in human dopaminergic neurons differentiated from induced pluripotent stem cells.

The mechanisms underlying the prolonged antidepressant effects after a single exposure to ketamine are only partially understood. Converging findings indicate a critical role of structural neuroplasticity, recently also proposed for dopaminergic (DA) neurons known to be involved in a depression core symptom, anhedonia. We recently showed that ketamine induces dendritic outgrowth in human DA neurons differentiated in-vitro from induced pluripotent stem cells of healthy donors, a phenomenon blocked by the α-amino-3-hydroxy-5-methy-4-isoxazole propionate receptor antagonist NBQX. As changes in the expression of AMPA receptor subunits GluR1 and GluR2 were observed in neuroplasticity of rodent DA neurons, we aimed to explore this phenomenon in human DA neurons. Using specific antibodies against GluR1 and GluR2 α-amino-3-hydroxy-5-methy-4-isoxazole propionate receptor subunits, we showed that GluR1 levels were significantly higher in soma than in dendrites, whereas for GluR2, levels were significantly higher in dendrites than in soma. One hour exposure to 1 µM ketamine increased the signal of both subunits in dendrites, but only of GluR2 in soma, at 24, 48, and 72 h. Nonlinear polynomial fitting of dendritic expression indicated that the two curves were significantly different, with stronger and more sustained effects on GluR2 expression. Overall, these data support a role for GluR1 and GluR2 dendritic upregulation in driving structural plasticity in human DA neurons depending on ketamine transient exposure, indicating translationally relevant downstream mechanism possibly involved in antidepressant effects.

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor-binding head domains.

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The “breathing” of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.

Varying the exposure period and duration of neuroactive pharmaceuticals and their metabolites modulates effects on the visual motor response in zebrafish (Danio rerio) larvae

Pharmaceuticals and personal care products are emerging contaminants that are increasingly detected in surface waters around the world. Despite the rise in environmental detections, measured concentrations are still typically low, raising the importance of environmental risk assessments that focus on ecologically relevant sublethal endpoints, such as altered behavior. Neuroactive pharmaceuticals, like mental health medications, pain killers, etc., may be particularly potent in this regard as they are specifically designed to cause behavioral changes without causing physiologic impairment in mammalian systems. We screened 15 different popular neuroactive pharmaceuticals, ranging from antidepressants (including 3 major antidepressant metabolites), anxiety medications, and pain killers, under three different exposure scenarios (repeated, late acute and early transient exposure) to look for behavioral effects in larval zebrafish using the visual motor response (VMR). Drugs were screened at 0, 1, 10, and 100 μg/L in the repeated exposure scenario, and at 0 and 100 μg/L in the late acute and early transient exposure scenarios. Eight of the 15 compounds tested, specifically the antidepressants amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, sertraline, nor-sertraline, venlafaxine, and the antipsychotic drug haloperidol decreased swimming activity by 25% to 40% under repeated exposure conditions. Five of the compounds (amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, and sertraline) also significantly decreased activity by 17% to 31% in the late acute exposure paradigm. Three compounds (fluoxetine, paroxetine and venlafaxine) significantly altered swimming activity with early transient exposure, however creating a hyperactive response and increasing activity from 24% to 28%, while haloperidol significantly decreased activity by 31%. This paper is, to our knowledge, the first to screen so many neuroactive pharmaceuticals, including major metabolites, in parallel under multiple exposure conditions. We show that antidepressants most consistently alter VMR swimming activity. Additionally, we show that major antidepressant metabolites can potentially alter behavior as much as their parent compounds. Furthermore, we show that the magnitude and direction of behavioral effect is dependent on the exposure duration and period, indicating that a more diverse experimental approach might be needed to more accurately assess the risk these compounds pose to the environment.

Mechanisms underlying the regulation of intracellular and luminal pH in vaginal epithelium.

The vagina provides a characteristic low-Na+ and low-pH fluid microenvironment that is considered generally protective. Previous studies have shown that various types of epithelial cells harbor the capacity of intracellular pH (pHi) regulation. However, it remains elusive whether vaginal epithelium could actively regulate pHi by transporting acid-base ions. In this study, we verified that after transient exposure to NH4 Cl, the pHi values could rapidly recover from acidification via Na+ -H+ exchanger (NHE), Na+ -HCO3 - cotransporter (NBC), and carbonic anhydrase in human vaginal epithelial cell line VK2/E6E7. Positive expression of the main acid-base transporters including NHE1-2, NBCe1-2, and NBCn1 mRNA was also detected in VK2/E6E7 cells. Moreover, the in vivo study further showed that interfering with the function of V-type H+ -ATPase, NHE or NBC expressed in vagina impaired vaginal luminal pH homeostasis in rats. Taken together, our study reveals the property of pH regulation in vaginal epithelial cells, which might provide novel insights into the potential role of vaginal epithelium in the formation of the vaginal acidic microenvironment.

Activation of EGFR-DNA-PKcs pathway by IGFBP2 protects esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage

BackgroundThe incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett’s esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.MethodsReal-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.ResultsOur results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett’s cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.ConclusionsIGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

Physiology and metabolism of grafted bell pepper in response to low root-zone temperature.

Low temperature is a prominent limiting factor for tropical originated crops production in temperate regions, particularly during cool-season production. The diverse response of two rootstocks (Canon-sensitive and S103-tolerant to low root-zone temperature) was studied when exposed to aeroponically different temperature regimes at the root zone: constant low temperature of 14°C low root-zone temperature (LRZT), transient exposure to LRZT of 27-14-27°C and control temperature of 27°C. Gas exchange, shoot dry mass, and root morphology were measured. Shifts in central and secondary metabolite levels in the leaves and roots were examined by gas chromatography-mass spectrometry (GC-MS). Low root-zone temperature inhibited photosynthesis and transpiration of both grafted bell pepper plants; however, self-grafted Canon physiology was impeded to a greater extent compared with Canon grafted onto rootstock S103. Rootstock S103 demonstrated higher sink potential contributing to milder reduction of photosynthesis and transpiration during stress compared with self-grafted Canon. This reduction of gas exchange led to a significant reduction of root maximum length and root dry mass in self-grafted Canon in response to the stress at 14°C compared with Canon grafted onto rootstock S103. In response to stress, GC-MS metabolite profiling showed enhance metabolism in both cultivars' leaves, as well as in the roots irrespective of the developmental stage of the plant. This evidence combined indicates enhance gas exchange and carbon assimilation when bell pepper is grafted on S103 under low root-zone temperature.

Barrett's esophagus and esophageal cancer: Links to microbes and the microbiome.

Esophageal cancer represents a major problem globally, with an estimated 398,000 esophageal squamous cell cancers (ESCC) and 52,000 esophageal adenocarcinomas (EAC) diagnosed worldwide in 2012 [1]. In the United States, EAC is more common than ESCC, and EAC in the US increased dramatically between 1997 and 2006 before stabilizing [2]. Furthermore, esophageal cancer remains particularly deadly, with a five-year survival rate of less than 20% [3]. EAC is thought to arise from Barrett’s esophagus (BE), a metaplastic differentiation from normal squamous epithelium to a columnar phenotype. Accepted risk factors for BE and progression to EAC include age, male sex, obesity, gastroesophageal reflux disease (GERD), and smoking [4, 5]. Unfortunately, less is known about the association between esophageal dysbiosis, inflammation, and the pathogenesis of BE and EAC. Most of the existing data on esophageal microbiota in BE and esophageal cancer are derived from small cross-sectional studies with limited information on causality. As such, this represents an important area for future research because changes in the esophageal microbiota may relate to modifiable factors such as antibiotic use and proton pump inhibitors (PPIs). The esophageal microbiota was initially thought to be primarily related to transient esophageal exposure to the oral microbiota [6], but more recently, culture-independent studies have shown the esophageal microbiota to represent its own niche [7]. In addition to swallowed oral bacteria, the esophagus is also exposed to refluxed gastric microbes, and while the esophageal microbiota resembles both oral and gastric microbiota, it is equal to neither [7, 8]. This review provides an overview of differences that have been described between the normal esophageal microbiota, BE, and EAC. We also discuss novel approaches to esophageal microbial sampling and consider clinical exposures that may alter the esophageal microbiota.

Developmental Exposure to Psychostimulant Primes Activity-dependent Gene Induction in Frontal Cortex.

Perinatal neurodevelopment involves extensive formation of neural connections and onset of activity-dependent gene expression for synaptic function and plasticity. Exposure to psychostimulants at this stage imposes significant risks for developing cognitive and affective disorders later in life. However, how developmental exposure to psychostimulants may induce long-lasting molecular changes relevant to neural circuit function remains incompletely understood. In this study, we investigated the impacts of psychostimulant amphetamine on the activity-dependent induction of synaptic adaptor molecule Arc in the frontal cortex of neonatal mice. We found that transient exposure to amphetamine not only amplifies activity-dependent Arc mRNA expression immediately, but also potentiates subsequent induction of Arc mRNA in the absence of amphetamine. This priming effect is associated with a rapid and persistent increase in histone mono-methylation (H3K4me1), a marker for transcriptionally permissive chromatin, at the Arc locus, but not any long-lasting change in the phosphorylation of upstream transcription factor CREB. Furthermore, the increase in H3K4me1 at the Arc locus requires dopamine receptor signaling, and the priming of Arc expression correlates with the dopaminergic innervation pattern in the frontal cortex. Together, our results demonstrate that developmental exposure to psychostimulant amphetamine induces long-lasting chromatin changes and primes activity-dependent Arc gene induction. These findings reveal the molecular targets of psychostimulant during perinatal development that may contribute to long-term psychiatric risks.

Exploiting MCL1 Dependency with Combination MEK + MCL1 Inhibitors Leads to Induction of Apoptosis and Tumor Regression in KRAS-Mutant Non–Small Cell Lung Cancer

BH3 mimetic drugs, which inhibit prosurvival BCL2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL1 inhibitors, we demonstrate that concurrent MEK + MCL1 inhibition induces apoptosis and tumor regression in KRAS-mutant non-small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL1 or BCL-xL was determined by the differential binding of proapoptotic BCL2 proteins to MCL1 or BCL-xL, respectively. The efficacy of dual MEK + MCL1 blockade was augmented by prior transient exposure to BCL-xL inhibitors, which promotes the binding of proapoptotic BCL2 proteins to MCL1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL2 family proteins for KRAS-mutant NSCLC. SIGNIFICANCE: Defining the molecular basis for MCL1 versus BCL-xL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-xL and MCL1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS-mutant NSCLCs treated with MEK inhibitors.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.

Specifying the Anterior Primitive Streak by Modulating YAP1 Levels in Human Pluripotent Stem Cells

SummarySpecifying the primitive streak (PS) guides stem cell differentiation in vitro; however, much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1−/− human embryonic stem cells (hESCs) in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1−/− hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and induced pluripotent stem cell differentiation into distinct mesendodermal lineages.

Mammalian Susceptibility to a Neonicotinoid Insecticide after Fetal and Early Postnatal Exposure

Neonicotinoids have become the most widely used class of insecticides world-wide. Although numerous studies have documented neonicotinoid toxicity in bees and other insects, the effects of exposure during early development in mammals remain largely unexplored. We assessed the effects of the neonicotinoid imidacloprid (IMI) in adult male and female mice after in utero and early postnatal exposure. Pregnant mice were infused with IMI (0.5 mg/kg/day) from gestational day 4 to the end of nursing at postnatal day 21. The young adult offspring were studied in a series of biochemical and behavioral tests. To assess reproducibility, the behavioral analyses were conducted in three separate studies using multiple exposed litters. Exposure to IMI reduced fecundity, and in adult offspring, decreased body weight in male but not female pups. Offspring from IMI-treated mothers displayed lower triglycerides, elevated motor activity, enhanced social dominance, reduced depressive-like behavior, and a diminution in social aggression compared to vehicle treated controls. Low levels of IMI were detected in the brains and livers of the treated mothers, while trace levels were detected in some offspring. Our results demonstrate that transient exposure to a neonicotinoid over the early developmental period induces long-lasting changes in behavior and brain function in mice.

Heterogeneity in efflux pump expression predisposes antibiotic-resistant cells to mutation.

Antibiotic resistance is often the result of mutations that block drug activity; however, bacteria also evade antibiotics by transiently expressing genes such as multidrug efflux pumps. A crucial question is whether transient resistance can promote permanent genetic changes. Previous studies have established that antibiotic treatment can select tolerant cells that then mutate to achieve permanent resistance. Whether these mutations result from antibiotic stress or preexist within the population is unclear. To address this question, we focused on the multidrug pump AcrAB-TolC. Using time-lapse microscopy, we found that cells with higher acrAB expression have lower expression of the DNA mismatch repair gene mutS, lower growth rates, and higher mutation frequencies. Thus, transient antibiotic resistance from elevated acrAB expression can promote spontaneous mutations within single cells.

Efflux pumps and mutation

Antibiotic Resistance Antibiotic resistance is an alarming and growing challenge. Bacteria show great heterogeneity in growth and mutation rates. Such variability allows some cells to persist during transient antibiotic exposure. During this persistent phase, mutations accumulate, which can result in selection for full-blown antibiotic resistance. El Meouche and Dunlop found that increased expression of efflux pumps on some cells affords them some relief from antibiotic toxicity. But up-regulating efflux pumps is costly for the bacteria, reducing growth rate and expression of MutS, a protein involved in DNA mismatch repair. These changes thus lift the lid on increased levels of bacterial mutation. Science , this issue p. [686][1] [1]: /lookup/doi/10.1126/science.aar7981

Transient exposure of methylparaben to zebrafish (Danio rerio) embryos altered cortisol level, acetylcholinesterase activity and induced anxiety-like behaviour

Parabens are widely used as antimicrobial preservatives. Recent studies have reported the endocrine disrupting effects of these chemicals, especially methylparaben. Previously, we have reported the alteration in Vtg gene expression upon exposure to environmentally relevant doses of methylparaben in zebrafish (Danio rerio) embryos. However, studies reporting neurobehavioural outcomes on exposure to methylparaben are limited. Therefore, this study was aimed at investigating the methylparaben-induced effects on developmental and neurobehavioural endpoints. Zebrafish embryos were exposed to sub-lethal concentrations of methylparaben: 0.1 ppb, 1 ppb, 10 ppb and 100 ppb. Alterations in developmental landmarks such as heart rate and hatching percentage were observed in embryos exposed to 10 ppb and 100 ppb of methylparaben. Results obtained from the novel tank diving test established that anxiety-like behaviour is induced in larvae exposed to 0.1 ppb and 1 ppb of methylparaben. A significant inhibition in the acetylcholinesterase (AChE) activity was also recorded in methylparaben-exposed groups. An increase in cortisol levels was observed in the exposed groups, which further supports the observations made in the novel tank diving test, establishing methylparaben as an anxiogenic agent even at sub-lethal concentrations. The underlying molecular mechanism needs further elucidation to investigate whether the behavioural effects are proximally or distally induced by early developmental exposure to methylparaben.