Signal Adaptation Research Articles

Synaptic Microtubule-Associated Protein EB3 and SRC Phosphorylation Mediate Structural and Behavioral Adaptations During Withdrawal From Cocaine Self-Administration.

Addictive behaviors, including relapse, are thought to depend in part on long-lasting drug-induced adaptations in dendritic spine signaling and morphology in the nucleus accumbens (NAc). While the influence of activity-dependent actin remodeling in these phenomena has been studied extensively, the role of microtubules and associated proteins remains poorly understood. We report that pharmacological inhibition of microtubule polymerization in the NAc inhibited locomotor sensitization to cocaine and contextual reward learning. We then investigated the roles of microtubule end-binding protein 3 (EB3) and SRC kinase in the neuronal and behavioral responses to volitionally administered cocaine. In synaptoneurosomal fractions from the NAc of self-administering male rats, the phosphorylation of SRC at an activating site was induced after 1 d of withdrawal, while EB3 levels were increased only after 30 d of withdrawal. Blocking SRC phosphorylation during early withdrawal by virally overexpressing SRCIN1, a negative regulator of SRC activity known to interact with EB3, abolished the incubation of cocaine craving in both male and female rats. Conversely, mimicking the EB3 increase observed after prolonged withdrawal increased the motivation to consume cocaine in male rats. In mice, the overexpression of either EB3 or SRCIN1 increased dendritic spine density and altered the spine morphology of NAc medium spiny neurons. Finally, a cocaine challenge after prolonged withdrawal recapitulated most of the synaptic protein expression profiles observed at early withdrawal. These findings suggest that microtubule-associated signaling proteins such as EB3 cooperate with actin remodeling pathways, notably SRC kinase activity, to establish and maintain long-lasting cellular and behavioral alterations following cocaine self-administration.SIGNIFICANCE STATEMENT Drug-induced morphological restructuring of dendritic spines of nucleus accumbens neurons is thought to be one of the cellular substrates of long-lasting drug-associated memories. The molecular basis of these persistent changes has remained incompletely understood. Here we implicate for the first time microtubule function in this process, together with key players such as microtubule-bound protein EB3 and synaptic SRC phosphorylation. We propose that microtubule and actin remodeling cooperate during withdrawal to maintain the plastic structural changes initially established by cocaine self-administration. This work opens new translational avenues for further characterization of microtubule-associated regulatory molecules as putative drug targets to tackle relapse to drug taking.

Allostatic plasticity of cAMP system drives opioid induced adaptations in striatal dopamine signaling

Opioids generate euphoria by activating the reward system in the brain, however chronic exposure generates plastic changes in signal processing to program aversive reactions. A core element of the reward circuit involves the integration of dopamine in subpopulations of medium spiny striatal neurons, D1‐MSN and D2‐MSN, whereby these two parallel channels encode the opponent processes through cAMP‐dependent signaling. However, the precise mechanisms that bias circuit activity to encode a range of hedonic states during various stages of opioid exposure are not well understood. Uncovering the fundamental properties and molecular machinery for parsing GPCR signal integration in striatal neurons is a critical step in advancing our understanding of neurobiology necessary to comprehend the adaptations induced by opioids. By recording cAMP dynamics with nanomolar resolution from genetically defined striatal neurons in an intact neuronal circuit, we revealed elemental properties that promote D1‐MSN signaling during acute opioid exposure while favoring D2‐MSN signaling following chronic drug administration. Additionally, we identified two key molecular components that shape plasticity of cAMP signaling. We found that opioids enable phosphodiesterases to regulate D1‐MSN signaling capacity while reducing the role of presynaptic dopamine transporters to influence D2‐MSN response strength. Our results demonstrate the importance of balancing signal integration between concurrently activated subpopulations of striatal neurons while exploring pre‐ and postsynaptic mechanisms that impact accompanying computations performed by the circuit. We propose a quantitative model whereby differential exposure to opioids provides cell‐specific adjustments in cAMP signal strength that imbalances circuit activity to promote transitions between states of reward and aversion.Support or Funding InformationThis work was supported by NIH grants: DA041207 (to B.S.M.), DA036596 (to K.A.M.), and DA026405 (to K.A.M.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heat flow due to time-delayed feedback

Many stochastic systems in biology, physics and technology involve discrete time delays in the underlying equations of motion, stemming, e. g., from finite signal transmission times, or a time lag between signal detection and adaption of an apparatus. From a mathematical perspective, delayed systems represent a special class of non-Markovian processes with delta-peaked memory kernels. It is well established that delays can induce intriguing behaviour, such as spontaneous oscillations, or resonance phenomena resulting from the interplay between delay and noise. However, the thermodynamics of delayed stochastic systems is still widely unexplored. This is especially true for continuous systems governed by nonlinear forces, which are omnipresent in realistic situations. We here present an analytical approach for the net steady-state heat rate in classical overdamped systems subject to time-delayed feedback. We show that the feedback inevitably leads to a finite heat flow even for vanishingly small delay times, and detect the nontrivial interplay of noise and delay as the underlying reason. To illustrate this point, and to provide an understanding of the heat flow at small delay times below the velocity-relaxation timescale, we compare with the case of underdamped motion where the phenomenon of “entropy pumping” has already been established. Application to an exemplary (overdamped) bistable system reveals that the feedback induces heating as well as cooling regimes and leads to a maximum of the medium entropy production at coherence resonance conditions. These observations are, in principle, measurable in experiments involving colloidal suspensions.

KRAS G12C NSCLC Models Are Sensitive to Direct Targeting of KRAS in Combination with PI3K Inhibition.

KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy.Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C-activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models. Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K-AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT-mTOR pathway inhibitors. The G12Ci+PI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models. Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

Molecular basis of wax-based color change and UV reflection in dragonflies.

Many animals change their body color for visual signaling and environmental adaptation. Some dragonflies show wax-based color change and ultraviolet (UV) reflection, but the biochemical properties underlying the phenomena are totally unknown. Here we investigated the UV-reflective abdominal wax of dragonflies, thereby identifying very long-chain methyl ketones and aldehydes as unique and major wax components. Little wax was detected on young adults, but dense wax secretion was found mainly on the dorsal abdomen of mature males of Orthetrum albistylum and O. melania, and pruinose wax secretion was identified on the ventral abdomen of mature females of O. albistylum and Sympetrum darwinianum. Comparative transcriptomics demonstrated drastic upregulation of the ELOVL17 gene, a member of the fatty acid elongase gene family, whose expression reflected the distribution of very long-chain methyl ketones. Synthetic 2-pentacosanone, the major component of dragonfly's wax, spontaneously formed light-scattering scale-like fine structures with strong UV reflection, suggesting its potential utility for biomimetics.

Quantum and Non-Quantum Formulation of Eye’s Adaptation to Light’s Intensity Increments

Context and background: A quantum formulation of vision in vertebrates was proposed in the early 1940s. The number of quanta useful for enabling vision was found. The time interval required for their absorption, however, was never specified. In the early 1950s, experimental data on the effects of light’s intensity increment on vision indicated that the quantum formulation is true only at low light’s intensities. In this case, a vaguely described signaling adaptation mechanism was invoked to explain the separation between vision at low and high intensities, accompanied by the switch from rod to cones as photoreceptors. Motivation: In this article, we want to prove the validity of the non-totally-quantum formulation and unveil the nature of the signaling adaptation mechanism. Hypothesis: To accomplish our proof, we hypothesize that the amount of energy transferred and conserved in light’s interaction with the eyes is given by the product of light’s intensity (or power) times its period. Method: We construct and use the plots of the trends of light’s intensity increments and the corresponding changes in the axon’s membrane capacitance versus adapting intensity. Results: We find that 1) the average solar light’s intensity is the critical value that separates low from high light’s intensity regimes in vision, and 2) changes in the capacitance of the axon’s membrane enable the signaling adaptation of vision when light’s intensity changes. Conclusions: We prove the validity of the non-totally-quantum formulation and unveil the nature of the signaling adaptation mechanism. Our proof is supported by the model based on light’s intensity times period as being the energy conserved in light-matter interaction This model suggests that 1) all the waves in the electromagnetic spectrum, at the correct intensity for each frequency, could be used to produce the effects of optogenetics in diagnostics and therapy, and 2) it takes seconds to minutes to see details in the dark when light is switched off.

Gene Editing of BTK in Acute Myeloid Leukaemia Using CRISPR-Cas9

Acute myeloid leukemia (AML) blood cancer is found primarily in adults and the elderly, being the third biggest blood cancer killer. In the last decades there has been some improved rates in younger patients, but little improvement in older patients. AML survival rates are the worst of all blood cancers, therefore finding new therapeutic targets to treat AML is crucial.Bruton's tyrosine kinase (BTK) is involved in the signalling of multiple receptors including growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen receptors and integrins. BTK in turn activates many of the major downstream signalling pathways that control cell migration, adhesion, survival and proliferation. BTK is best known for its role in B-cell receptor signalling, but BTK is also expressed and constitutively active in AML cell lines and AML patient blasts. Targeting BTK in B-cell malignancies with the BTK inhibitor ibrutinib has shown clinical effectiveness and tolerability in recent patient trials.Ibrutinib is orally active and covalently binds at the Cys-481 in the ATP-binding domain of BTK, to irreversibly block its enzymatic activity. A proportion of CLL and MCL patients treated with ibrutinib relapse during the therapy, with secondary resistance developing against ibrutinib. This resistance is mainly due to a BTK cysteine to serine mutation at position 481 (C481S) which disrupts ibrutinib's covalent binding to BTK.As little is known about BTK's role and therapeutic potential in AML, we aimed to study the effects and functional consequences of pharmacological inhibition of BTK using ibrutinib. We furthermore generated the BTK-C481S mutation in AML cell lines using the CRISPR-Cas9 gene-editing system. Shown here for the first time is a gene-edited mutation introduced into a human leukemia cell clone that has been purified to homogeneity. We explored the functional impact of BTK-C481S mutation on ibrutinib sensitivity, and mutation-induced adaptation of the phosphoproteome and kinome in AML cells.HEK-293T cells were used for validation purposes to find the most efficient knock-in strategy. We tested the most commonly used CRISPR endonuclease spCas9 as well as two other Cas9 orthologs called SaCas9 and AsCas12a. The designed spacer sequences within the sgRNA that target the gene of interest were delivered either via plasmid or in vitro transcripts. A repair template for homology directed repair (HDR) was designed containing the necessary base changes to change the amino acid at position 481 in BTK from cysteine to serine. The HDR template was delivered via transfection as single-stranded oligodeoxynucleotide (ssODN) or incorporated into plasmid. All strategies tested were successful but due to the poor transfectability of AML cell lines, we used lentiviral delivery to introduce BTK-C481S mutation into AML cell lines.The BTK-C481S mutation was introduced in THP-1 and OCI-AML3 cells. Mutant-positive cells were found via clonal selection and Sanger sequencing. Wild-type (WT) as well as mutant cells were treated with various ibrutinib concentrations ranging from 1 nM to 1 µM and analysed for BTK expression/phosphorylation via Western blot, with effects on cell proliferation, viability and cell cycle measured via flow cytometry. We also performed phosphoproteomics and kinome assays comparing untreated as well as treated (30 nM ibrutinib, 2h) THP-1 WT and THP-1 BTK-C481S cells.Western blot results showed that the effective concentration required for BTK inhibition is 30-fold higher in BTK-C481S mutant compared to WT cells (300 nM vs 10 nM). Wash-out experiments revealed that ibrutinib binding can be reversed in BTK-C481S mutant cells but not WT cells. Little effect of ibrutinib on proliferation and viability were seen comparing BTK-C481S mutant and WT AML cells.These signalling adaptations in BTK-C481S cells are consistent with changes observed in ibrutinib-resistant leukemia patient samples. DisclosuresSlupsky:Verastem: Research Funding.

An increase in song pitch of eastern great tits (Parus minor) in response to urban noise at Seoul, Korea

Anthropogenic noise may mask effective vocal signal transmission by birds, particularly in notes occurring in lower frequencies. Previous studies have shown a correlation of increased urban noise levels, higher minimum frequency, and lowered bandwidth in Great Tit song (Parus major). We therefore studied the effect of urban noise pollution on the song characteristics of the closely related Eastern Great Tit (EGTs, Parus minor) at Hongneung experimental forests (HEFs) in the metropolitan urban of Seoul, Korea. We analysed the song characteristics of breeding territories of EGTS in total 13 individuals recorded including four individuals at high noise group. EGTs singing in noisier locations exhibited higher minimum & maximum frequency, and lower note & strophe length, and higher bandwidth, replicating the different findings of Great Tits. This data also suggests that several individuals at high noise location exhibited a non-typical song type indicative of structural adaptation to higher noise levels. Although the mechanisms of these correlations are as yet undetermined, it is likely that behavioural plasticity may play a role of vocal signal adaption in an increasingly urbanizing Korea.

PtpA, a secreted tyrosine phosphatase from Staphylococcus aureus, contributes to virulence and interacts with coronin-1A during infection

Secretion of bacterial signaling proteins and adaptation to the host, especially during infection, are processes that are often linked in pathogenic bacteria. The human pathogen Staphylococcus aureus is equipped with a large arsenal of immune-modulating factors, allowing it to either subvert the host immune response or to create permissive niches for its survival. Recently, we showed that one of the low-molecular-weight protein tyrosine phosphatases produced by S. aureus, PtpA, is secreted during growth. Here, we report that deletion of ptpA in S. aureus affects intramacrophage survival and infectivity. We also observed that PtpA is secreted during macrophage infection. Immunoprecipitation assays identified several host proteins as putative intracellular binding partners for PtpA, including coronin-1A, a cytoskeleton-associated protein that is implicated in a variety of cellular processes. Of note, we demonstrated that coronin-1A is phosphorylated on tyrosine residues upon S. aureus infection and that its phosphorylation profile is linked to PtpA expression. Our results confirm that PtpA has a critical role during infection as a bacterial effector protein that counteracts host defenses.

Visual mismatch response and fMRI signal adaptation correlate in the occipital-temporal cortex

Few rigorous longitudinal assessments have examined health-related quality of life (HRQoL) changes after smoking cessation, especially among recently-hospitalized smokers. We compared the change in HRQoL between those who did or did not quit smoking 6 months after hospital discharge. Participants were 1357 smokers recruited for a cessation trial between 2012 and 2014 while hospitalized at two hospitals in Massachusetts and one in Pennsylvania. Cessation was defined as biochemically confirmed 7-day point prevalence abstinence at 6 months or as self-reported continuous abstinence at 1, 3, or 6 months post discharge. HRQoL measures included a single-item global health measure (SF1); the Patient Health Questionnaire for Depression and Anxiety (PHQ-4) screening tool for psychological distress; and the EQ-5D-5L health utilities measure. Multivariable models controlled for age, sex, race, education, insurance, study site, study arm, discharge diagnoses, and baseline HRQoL. Improvements in HRQoL were evident in the first month after discharge among those achieving abstinence compared to continuing smokers. At 6 months post-discharge, those with biochemically confirmed cessation were 30% more likely to report at least good health by the SF1 (aRR 95% CI 1.14–1.45), 19% less likely to screen positive for psychological distress (aRR, 95% CI 0.68–0.93), and had EQ-5D-5L health utility scores 0.05 points (95% CI 0.02–0.08) higher than continuing smokers. Results were similar when assessed as a function of self-reported cessation. Hospital-initiated smoking cessation is associated with rapid statistically and clinically significant improvements in a range of HRQoL measures, providing an additional tool clinicians and health systems could use to encourage smoking cessation.

Elimination of the error signal in the superior colliculus impairs saccade motor learning

When movements become dysmetric, the resultant motor error induces a plastic change in the cerebellum to correct the movement, i.e., motor adaptation. Current evidence suggests that the error signal to the cerebellum is delivered by complex spikes originating in the inferior olive (IO). To prove a causal link between the IO error signal and motor adaptation, several studies blocked the IO, which, unfortunately, affected not only the adaptation but also the movement itself. We avoided this confound by inactivating the source of an error signal to the IO. Several studies implicate the superior colliculus (SC) as the source of the error signal to the IO for saccade adaptation. When we inactivated the SC, the metrics of the saccade to be adapted were unchanged, but saccade adaptation was impaired. Thus, an intact rostral SC is necessary for saccade adaptation. Our data provide experimental evidence for the cerebellar learning theory that requires an error signal to drive motor adaptation.

In Cocaine Dependence, Neural Prediction Errors During Loss Avoidance Are Increased With Cocaine Deprivation and Predict Drug Use

BackgroundIn substance-dependent individuals, drug deprivation and drug use trigger divergent behavioral responses to environmental cues. These divergent responses are consonant with data showing that short- and long-term adaptations in dopamine signaling are similarly sensitive to state of drug use. The literature suggests a drug state–dependent role of learning in maintaining substance use; evidence linking dopamine to both reinforcement learning and addiction provides a framework to test this possibility. MethodsIn a randomized crossover design, 22 participants with current cocaine use disorder completed a probabilistic loss-learning task during functional magnetic resonance imaging while on and off cocaine (44 sessions). Another 54 participants without Axis I psychopathology served as a secondary reference group. Within-drug state and paired-subjects’ learning effects were assessed with computational model–derived individual learning parameters. Model-based neuroimaging analyses evaluated effects of drug use state on neural learning signals. Relationships among model-derived behavioral learning rates (α+, α−), neural prediction error signals (δ+, δ−), cocaine use, and desire to use were assessed. ResultsDuring cocaine deprivation, cocaine-dependent individuals exhibited heightened positive learning rates (α+), heightened neural positive prediction error (δ+) responses, and heightened association of α+ with neural δ+ responses. The deprivation-enhanced neural learning signals were specific to successful loss avoidance, comparable to participants without psychiatric conditions, and mediated a relationship between chronicity of drug use and desire to use cocaine. ConclusionsNeurocomputational learning signals are sensitive to drug use status and suggest that heightened reinforcement by successful avoidance of negative outcomes may contribute to drug seeking during deprivation. More generally, attention to drug use state is important for delineating substrates of addiction.

Abstract 2439: The importance of cellular oxygenation measurements in the analysis of hypoxia-induced signaling and related metabolic adaptation

Abstract There is a growing appreciation that typical cell culture conditions reflect a hyperoxic condition for the majority of cell types. In an effort to more accurately reflect the in vivo condition, a growing number of researchers are conducting in vitro measurements at what are deemed to be more physiologically relevant oxygen concentrations (1-8% O2). The importance of oxygen concentration in in vitro model design is underscored by observations that O2 levels influence a wide range of cellular phenomena, including metabolic poise, differentiation and senescence while the development of localised hypoxic environments are implicated in a variety of diseases including stroke, and cancer. Impaired O2 supply can reduce O2 concentrations to below normal physiological levels, initiating a graded series of complex interconnected adaptive responses mediated by regulators including the hypoxia-inducible factor (HIF). These processes are particularly relevant within solid tumours, where oncogene-driven proliferation causes nutrient and oxygen deprivation, aberrant angiogenesis, and the activation of O2-sensitive survival pathways. Despite the importance of cellular oxygenation, research using in vitro models makes the assumption that oxygenation can be defined by simply altering the oxygen condition applied to the in vitro model. To test this key assumption, cellular oxygenation measurements are performed using a nanoparticulate intracellular oxygen probe (MitoXpress®-Intra), while tumour hypoxia/oygenation is modelled on a fluorescence plate reader equipped with an atmospheric control unit (CLARIOstar®). Data is presented illustrating that the depth of hypoxia experienced by the cell model is impacted significantly by respiratory activity, and that this additional oxygen deprivation is a dynamic process, effected by respiratory substrate availability and related metabolic poise. Data is also presented demonstrating that, in in vitro models, additional respiration-induced hypoxia directly impacts glycolytic flux and other relevant metabolic pathways. Together these data invalidate the assumption that cellular oxygenation can be inferred from ambient oxygen measurements. If ignored, the significant and dynamic deviations between ambient O2 and oxygenation at the cellular level leads to erroneous collusions regarding the relationship between oxygen concentration, HIF stabilisation and related metabolic adaptations. This in turn can impair the physiological relevance of experimental observations. Citation Format: Michelle Potter, James Hynes, Anke Nijhuis, Conn Carey, Christos Zois, Adrian Harris, Hector Keun, Karl Morten. The importance of cellular oxygenation measurements in the analysis of hypoxia-induced signaling and related metabolic adaptation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2439.

Abstract 3025: Ligand-independent EphA2 signaling drives an amoeboid phenotype that promotes melanoma brain metastasis development

Abstract The majority of melanoma patients treated with BRAF inhibitors (BRAFi) ultimately develop resistance and fail on therapy. It has previously been shown that BRAFi resistance is highly correlated with the adoption of a strongly invasive phenotype through ligand-independent EphA2 signaling. In this study, we used comprehensive mass spectrometry-based proteomic approaches to delineate the global signaling changes associated with the aggressive phenotype driven by ligand-independent EphA2 signaling. Preliminary data have identified new signaling adaptations associated with a mesenchymal-to-amoeboid transition (MAT) phenotype, which was confirmed in 3D collagen cultures. Functional experiments demonstrated that the amoeboid phenotype promoted cell migration and invasion, which was mediated through the interaction of EphA2 and CDC42. These findings were confirmed by expressing a constitutively activated form of CDC42. BRAFi resistant cell lines also exhibited the MAT phenotype and were more invasive compared to their treatment-naïve counterparts, in line with their dependence upon ligand-independent EphA2 signaling following BRAFi selection pressure. To further demonstrate the metastatic potential of these amoeboid cells driven by ligand-independent EpHA2 signaling in vivo, we performed intracardiac injections in mice with cell lines expressing either EpHA2 S897A (inactive) or S897E (constitutively active phosphomimetic). Interestingly, we found a preferential homing of EphA2 S897E cells to the brain, but no difference in metastasis to other organs including the lung and liver. An analysis of brain metastasis specimens from patients failing BRAF and BRAF/MEK inhibitor therapy showed strong staining for the amoeboid phenotype marker EphA2. To investigate whether the amoeboid phenotype may confer a survival advantage in circulation, we carried out shear stress assays. These experiments demonstrated that the amoeboid phenotype driven by ligand-independent EphA2 signaling promoted the survival of melanoma cells under shear stress. Cell attachment assays, trans-endothelial invasion assays and vascular permeability assays show that these cells are better suited to attach to and permeate an endothelial monolayer. We further show that inhibiting PI3K reversed the amoeboid phenotype and limited the EphA2-driven invasive capacity. In summary, we show for the first time that BRAFi resistance is associated with the adoption of an MAT phenotype that increases the metastatic seeding of melanoma cells to the brain, which can be reversed through inhibition of PI3K signaling. Citation Format: Chao Zhang, Inna Smalley, Ritin Sharma, Michael Emmons, Jane Messina, John Koomen, Keiran Smalley. Ligand-independent EphA2 signaling drives an amoeboid phenotype that promotes melanoma brain metastasis development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3025.

Identification of tumor necrosis factor receptor-associated factor 6 in the pearl mussel Hyriopsis cumingii and its involvement in innate immunity and pearl sac formation

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.

Metabolomics in Plant Priming Research: The Way Forward?

A new era of plant biochemistry at the systems level is emerging, providing detailed descriptions of biochemical phenomena at the cellular and organismal level. This new era is marked by the advent of metabolomics—the qualitative and quantitative investigation of the entire metabolome (in a dynamic equilibrium) of a biological system. This field has developed as an indispensable methodological approach to study cellular biochemistry at a global level. For protection and survival in a constantly-changing environment, plants rely on a complex and multi-layered innate immune system. This involves surveillance of ‘self’ and ‘non-self,’ molecule-based systemic signalling and metabolic adaptations involving primary and secondary metabolites as well as epigenetic modulation mechanisms. Establishment of a pre-conditioned or primed state can sensitise or enhance aspects of innate immunity for faster and stronger responses. Comprehensive elucidation of the molecular and biochemical processes associated with the phenotypic defence state is vital for a better understanding of the molecular mechanisms that define the metabolism of plant–pathogen interactions. Such insights are essential for translational research and applications. Thus, this review highlights the prospects of metabolomics and addresses current challenges that hinder the realisation of the full potential of the field. Such limitations include partial coverage of the metabolome and maximising the value of metabolomics data (extraction of information and interpretation). Furthermore, the review points out key features that characterise both the plant innate immune system and enhancement of the latter, thus underlining insights from metabolomic studies in plant priming. Future perspectives in this inspiring area are included, with the aim of stimulating further studies leading to a better understanding of plant immunity at the metabolome level.

Epigenetic alterations of the POMC promoter in tobacco dependence

Impaired regulation of the hypothalamic-pituitary-adrenal (HPA) axis is substantially involved in several psychiatric disorders. Smoking interferes with HPA axis by activating proopiomelanocortin (POMC) neurons and thus stimulating the expression of POMC. The POMC transcript is processed into several peptide hormones, such as adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH), that play a role in stress response and weight control. In alcohol dependence, POMC promoter methylation is associated with craving. Here, we describe evidence of altered POMC promoter methylation in smoking. To determine how tobacco dependence and its withdrawal affect POMC promoter-specific DNA methylation, we assessed blood samples of 36 tobacco dependent individuals at day 1, 7 and 14 of withdrawal compared to 41 healthy controls using direct bisulfite sequencing. We found that POMC promoter methylation is significantly higher in smokers than in non-smokers. Moreover, this methylation difference does not readapt within 14 days of abstinence. We offer two explanatory models: Smokers could have a higher methylation state before the onset of smoking and this premorbid status might be acquired by environmental factors in early life. Alternatively, smoking may activate POMC neurons and its protein expression. Therefore, increasing methylation status of its promoter might be an adjustment to keep homeostasis. In either way, altered POMC methylation in smokers seems to indicate an adaptation of stress signaling, thereby potentially serving as a marker for stress-related functions that support the addiction.

Receptor-interacting protein kinase 3 mediates macrophage/monocyte activation in autoimmune hepatitis and regulates interleukin-6 production.

The mechanisms of macrophages/monocytes in autoimmune hepatitis (AIH) remain unclear. We investigated the role of receptor-interacting protein kinase 3 (RIP3), a key inflammatory signal adapter, in macrophage/monocyte activation in AIH. Liver tissues and monocytes from patients were collected to evaluate the relationship between macrophage activation and RIP3 by double-immunofluorescence and Western blotting. RAW264.7 macrophages were used to study the regulation of RIP3 signaling on inflammatory cytokines. Compared to the hepatic cyst, the majority of accumulated macrophages expressed RIP3 in AIH liver tissues. Moreover, RIP3 expression of monocytes was correlated with the levels of serum hepatic enzyme in AIH. Furthermore, RIP3 signaling was activated by lipopolysaccharide in RAW264.7 macrophages, which was accompanied with upregulated interleukin (IL)-1β, IL-6, and IL-10 and downregulated IL-4 and transforming growth factor-β. Notably, necrostatin-1, the specific inhibitor of the RIP3 signaling pathway, and 6-thioguanine (6-TG), the active metabolite of azathioprine, predominantly reduced IL-6 production compared to other cytokines. Moreover, the gene level of IL-6 was dramatically increased in AIH liver tissues. RIP3 signaling is involved in macrophage/monocyte activation in AIH and mediates IL-6 production, and is a novel molecular mechanism of 6-TG, indicating that it might be a promising therapeutic target for AIH treatment.

Cell-type specific MyD88 signaling is required for intestinal tumor initiation and progression to malignancy

ABSTRACTThe signal adapter MyD88, an essential component of Toll-like receptor (TLR) signaling, is important for gut-microbiome interactions. However, its contribution to cancer and its cell-type specific functions are controversially discussed. Therefore, we generated new tissue-specific mouse models and analyzed the clinical importance in human colorectal cancer. A gene-trap was inserted into the murine Myd88 gene (Myd88LSL), yielding MyD88-deficient background with Cre-mediated re-expression in myeloid (MYEL) or intestinal epithelial cells (IECs). These lines were bred with the Apc1638N model that develops invasive adenocarcinoma and analyzed at 12 months. Further, two patient collectives of colorectal cancer (n = 61, and n = 633) were analyzed for expression of Myd88 and TLRs. MyD88 expression was significantly increased in carcinomas, and increased intratumoral levels of MyD88 and TLR pathway components were associated with significantly shorter disease-free (P = .011), and overall survival (P < .0001). In accordance, fully MyD88-deficient mice showed highly significantly decreased tumor incidence, tumor numbers, increased survival, and, importantly, fully lacked malignant lesions. Thus, MyD88 is essential for tumorigenesis and especially progression to malignancy. Tissue-specific re-expression of MyD88 highly significantly increased tumor initiation by differing mechanisms. In intestinal epithelia, MyD88 enhanced epithelial turnover, whereas in myeloid cells, it led to increased production of tumor- and stemness-enhancing cytokines, significantly associated with altered expression of adaptive immune genes. However, neither re-expression of MyD88 in IECs or myeloid cells was sufficient for malignant progression to carcinoma. Thus, MyD88 crucially contributes to colorectal cancer initiation and progression with non-redundant and cell-type specific functions, constituting an attractive therapeutic target.

Cellular differentiation of non-transformed intestinal epithelial cells is regulated by Lactobacillus rhamnosus and L. casei strains.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.