Microbiota-mediated fine-tuning of the threshold of intestinal inflammasome activation in host-microbial mutualism

The inflammasome is a complex of proteins that controls the activity of caspase-1, pro-IL-1b and pro-IL-18. It acts in inflammatory processes and in pyropoptosis. The lower intestine is densely populated by a community of commensal bacteria that, under healthy conditions, are beneficial to the host. Some evidence suggests that the gut microbiota influences regulation of the inflammasome. Components of inflammasomes have been shown to have a protective function against development of experimental colitis, dependent on IL-18 production. However the precise mechanisms and the role of the inflammasome in maintaining a healthy host-microbial mutualism remains unknown. To address this question, we have performed axenic (GF) and gnotobiotic in vivo experiments to investigate how the inflammasome components mainly at the level of intestinal epithelial cells (IECs) are regulated under different hygiene conditions. We have established that gene expression of the inflammasome components NLRC4, NLRP3, NLRP6, NLRP12, caspase-1, ASC and IL-18 do not differ between germ-free and colonised conditions under steady-state. In contrast, induction in IL-18 was observed following infection with the pathobiont Segmented Filamentous Bacteria or the pathogen C. rodentium. Additional preliminar findings suggest that a more diverse intestinal flora, like specific pathogen-free (SPF) flora, is more efficient in inducing basal activation of the inflammasome and especially production of IL-18 by IECs, shortly after colonisation. We are also in the process of testing if basal activation of the inflammasome upon intestinal colonization with commensal bacteria helps to protect the host from potential pathobiont bacteria, like C. rodentium, SFB, Prevotella and TM7.

Normal platelet activation profile in patients with peripheral arterial disease on aspirin.

BACKGROUND Peripheral arterial disease (PAD) is a progressive vascular disease associated with a high risk of cardiovascular morbidity and death. Antithrombotic prevention is usually applied by prescribing the antiplatelet agent aspirin. However, in patients with PAD aspirin fails to provide protection against myocardial infarction and death, only reducing the risk of ischemic stroke. Platelets may play a role in disease development, but this has not been tested by proper mechanistic studies. In the present study, we performed a systematic evaluation of platelet reactivity in whole blood from patients with PAD using two high-throughput assays, i.e. multi-agonist testing of platelet activation by flow cytometry and multi-parameter testing of thrombus formation on spotted microarrays. METHODS Blood was obtained from 40 patients (38 on aspirin) with PAD in majority class IIa/IIb and from 40 age-matched control subjects. Whole-blood flow cytometry and multiparameter thrombus formation under high-shear flow conditions were determined using recently developed and validated assays. RESULTS Flow cytometry of whole blood samples from aspirin-treated patients demonstrated unchanged high platelet responsiveness towards ADP, slightly elevated responsiveness after glycoprotein VI stimulation, and decreased responsiveness after PAR1 thrombin receptor stimulation, compared to the control subjects. Most parameters of thrombus formation under flow were similarly high for the patient and control groups. However, in vitro aspirin treatment caused a marked reduction in thrombus formation, especially on collagen surfaces. When compared per subject, markers of ADP- and collagen-induced integrin activation (flow cytometry) strongly correlated with parameters of collagen-dependent thrombus formation under flow, indicative of a common, subject-dependent regulation of both processes. CONCLUSION Despite of the use of aspirin, most platelet activation properties were in the normal range in whole-blood from class II PAD patients. These data underline the need for more effective antithrombotic pharmacoprotection in PAD.

Cdk7 is required for full activation of Drosophila heat shock genes and RNA polymerase II phosphorylation in vivo

TFIIH has been implicated in several fundamental cellular processes, including DNA repair, cell cycle progression, and transcription. In transcription, the helicase activity of TFIIH functions to melt promoter DNA; however, the in vivo function of the Cdk7 kinase subunit of TFIIH, which has been hypothesized to be involved in RNA polymerase II (Pol II) phosphorylation, is not clearly understood. Using temperature-sensitive and null alleles of cdk7, we have examined the role of Cdk7 in the activation of Drosophila heat shock genes. Several in vivo approaches, including polytene chromosome immunofluorescence, nuclear run-on assays, and, in particular, a protein-DNA cross-linking assay customized for adults, revealed that Cdk7 kinase activity is required for full activation of heat shock genes, promoter-proximal Pol II pausing, and Pol II-dependent chromatin decondensation. The requirement for Cdk7 occurs very early in the transcription cycle. Furthermore, we provide evidence that TFIIH associates with the elongation complex much longer than previously suspected.

Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins.

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment.

Inhibition of photosynthesis by high temperature in oak (Quercus pubescens L.) leaves grown under natural conditions closely correlates with a reversible heat-dependent reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase

Inhibition of the net photosynthetic CO2 assimilation rate (Pn) by high temperature was examined in oak (Quercus pubescens L.) leaves grown under natural conditions. Combined measurements of gas exchange and chlorophyll (Chl) a fluorescence were employed to differentiate between inhibition originating from heat effects on components of the thylakoid membranes and that resulting from effects on photosynthetic carbon metabolism. Regardless of whether temperature was increased rapidly or gradually, Pn decreased with increasing leaf temperature and was more than 90% reduced at 45 °C as compared to 25 °C. Inhibition of Pn by heat stress did not result from reduced stomatal conductance (gs), as heat-induced reduction of gs was accompanied by an increase of the intercellular CO2 concentration (Ci). Chl a fluorescence measurements revealed that between 25 and 45 °C heat-dependent alterations of thylakoid-associated processes contributed only marginally, if at all, to the inhibition of Pn by heat stress, with photosystem II being remarkably well protected against thermal inactivation. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased from about 90% at 25 °C to less than 30% at 45 °C. Heat stress did not affect Rubisco per se, since full activity could be restored by incubation with CO2 and Mg2+. Western-blot analysis of leaf extracts disclosed the presence of two Rubisco activase polypeptides, but heat stress did not alter the profile of the activase bands. Inhibition of Pn at high leaf temperature could be markedly reduced by artificially increasing Ci. A high Ci also stimulated photosynthetic electron transport and resulted in reduced non-photochemical fluorescence quenching. Recovery experiments showed that heat-dependent inhibition of Pn was largely, if not fully, reversible. The present results demonstrate that in Q. pubescens leaves the thylakoid membranes in general and photosynthetic electron transport in particular were well protected against heat-induced perturbations and that inhibition of Pn by high temperature closely correlated with a reversible heat-dependent reduction of the Rubisco activation state.

What do brain network interactions tell about cognitive processes? How to seek causality?

Optimal adjustment of brain networks allows the biased processing of information in response to the demand of environments and is therefore prerequisite for adaptive behaviour. It is widely shown that a biased state of networks is associated with a particular cognitive process. However, those associations were identified by backward categorization of trials and cannot provide a causal association with cognitive processes. This problem still remains a big obstacle to advance the state of our field in particular human cognitive neuroscience. In my talk, I will present two approaches to address the causal relationships between brain network interactions and behaviour. Firstly, we combined connectivity analysis of fMRI data and a machine leaning method to predict inter-individual differences of behaviour and responsiveness to environmental demands. The connectivity-based classification approach outperforms local activation-based classification analysis, suggesting that interactions in brain networks carry information of instantaneous cognitive processes. Secondly, we have recently established a brand new method combining transcranial alternating current stimulation (tACS), transcranial magnetic stimulation (TMS), and EEG. We use the method to measure signal transmission between brain areas while introducing extrinsic oscillatory brain activity and to study causal association between oscillatory activity and behaviour. We show that phase-matched oscillatory activity creates the phase-dependent modulation of signal transmission between brain areas, while phase-shifted oscillatory activity blunts the phase-dependent modulation. The results suggest that phase coherence between brain areas plays a cardinal role in signal transmission in the brain networks. In sum, I argue that causal approaches will provide more concreate backbones to cognitive neuroscience.

Activation of common signaling pathways during remodeling of the heart and the bladder

The heart and the urinary bladder are hollow muscular organs, which can be afflicted by pressure overload injury due to pathological conditions such as hypertension and bladder outlet obstruction. This increased outflow resistance induces hypertrophy, marked by dramatic changes in the organs' phenotype and function. The end result in both the heart and the bladder can be acute organ failure due to advanced fibrosis and the subsequent loss of contractility. There is emerging evidence that microRNAs (miRNAs) play an important role in the pathogenesis of heart failure and bladder dysfunction. MiRNAs are endogenous non-coding single-stranded RNAs, which regulate gene expression and control adaptive and maladaptive organ remodeling processes. This Review summarizes the current knowledge of molecular alterations in the heart and the bladder and highlights common signaling pathways and regulatory events. The miRNA expression analysis and experimental target validation done in the heart provide a valuable source of information for investigators working on the bladder and other organs undergoing the process of fibrotic remodeling. Aberrantly expressed miRNA are amendable to pharmacological manipulation, offering an opportunity for development of new therapies for cardiac and bladder hypertrophy and failure.

Functional interplay of SP family members and nuclear factor Y is essential for transcriptional activation of the human Calreticulin gene

Calreticulin (CALR) is a highly conserved, multifunctional protein involved in a variety of cellular processes including the maintenance of intracellular calcium homeostasis, proper protein folding, differentiation and immunogenic cell death. More recently, a crucial role for CALR in the pathogenesis of certain hematologic malignancies was discovered: in clinical subgroups of acute myeloid leukemia, CALR overexpression mediates a block in differentiation, while somatic mutations have been found in the majority of patients with myeloproliferative neoplasms with nonmutated Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL). However, the mechanisms underlying CALR promoter activation have insufficiently been investigated so far. By dissecting the core promoter region, we could identify a functional TATA-box relevant for transcriptional activation. In addition, we characterized two evolutionary highly conserved cis-regulatory modules (CRMs) within the proximal promoter each composed of one binding site for the transcription factors SP1 and SP3 as well as for the nuclear transcription factor Y (NFY) and we verified binding of these factors to their cognate sites in vitro and in vivo.

Spatio-temporal co-ordination of RhoA, Rac1 and Cdc42 activation during prototypical edge protrusion and retraction dynamics

The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility.

Fear Reduction Processes in Imaginal and In Vivo Flooding: A Comment on James' Review

The research comparing imaginal and in vivo exposure in the treatment of clinically significant fear, recently reviewed by James (1986), is reexamined from the perspective of bioinformational theory and the concept of emotional processing. Fear is assumed to be stored in long term memory as a network of propositionally-coded information, which has to be processed if treatment is to be successful. Emotional processing is indicated by activation of fear responses and their habituation within and across treatment sessions. Consistent with the theory, our review indicates that successful treatment via imaginal and in vivo exposure is indeed related to activation and habituation of fear responses; interference with processing has a negative impact upon fear reduction, regardless of the specific treatment techniques employed. Furthermore, some apparently discrepant findings in the available research literature can be understood in terms of the theories cited. These ideas provide a useful perspective from which to plan future research efforts and to advance our understanding of the processes underlying reduction of pathological fear.

The role of MAPK during the activation of NK cells

Although many clinical trials investigated the use of IL-2, IL-12, and LAK in adoptive immunotherapy to treat cancer, only limited clinical success has been achieved. Better understanding of the intracellular processes that IL-2 and IL-12 utilize to generate LAK and other functions in NK cells is necessary to improve this mode of therapy. IL-2 and IL-12 stimulate extracellular signal-regulated protein kinase (ERK) and p38 MAPK in mitogen-activated T lymphocytes. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK Kinase (MKK)/ERK and/or p38 MAPK pathways are necessary for IL-2 or IL-12 to activate NK cells. We established that IL-2 activates MKK1/2/ERK pathway in freshly isolated human NK cells without any prior stimulation. Furthermore, we determined that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK activity, IFN-γ secretion, and CD25 and CD69 expression. Treatment of NK cells with a specific inhibitor of MKK1/2 PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four activation parameters. Although activation of ERK was not detected in NK cells immediately after IL-12 stimulation, IL-12-induced functional activation was inhibited by the MKK1/2 inhibitor, as well. In contrast to what was observed by others in T lymphocytes, activation of p38 MAPK by IL-2 was not detected in NK cells. Additionally, a specific inhibitor of p38 MAPK (SB203850) did not inhibit IL-2-activated NK functions. These data reveal selective signaling differences between NK cells and T lymphocytes. Collectively, the data support that the MKK/ERK pathway plays a critical positive regulatory role in NK cells during activation by IL-2 and IL-12. ^

Studies of MEKK2 activation mechanisms and its biological role in LPS/TLR4 signaling

MEKK2 is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that controls the MAPK and IKK-NF-κB pathways. The MAPK and IKK pathways are intracellular signaling networks that are crucial for the Toll-like receptor (TLR) mediated innate immunity, cellular stress and many other physiological responses. Members of the MAP3K family are central to the activation of these processes. However, the molecular mechanisms underlying stimuli-mediated MAP3K activation remain largely unknown. In this study, we identified a key phosphoserine residue, Ser-519 in MEKK2, and its equivalent site Ser-526 in MEKK3 within their activation loop whose phosphorylation are essential for their optimal activation. Mutation of this regulatory serine to an alanine severely impaired MEKK2 activation and MEKK2 signaling to its downstream targets. To demonstrate that physiological stimuli induce this serine phosphorylation, we generated an antibody that specifically recognizes the phosphorylated serine residue. We found that many, but not all, of the MAPK agonists, including the TLR ligands, growth factors, cytokines and cellular stresses, induced this regulatory serine phosphorylation in MEKK2, suggesting an involvement of MEKK2 in the activation of the MAPK cascade leading to different cellular responses. We further investigated the specific role of MEKK2 in LPS/TLR4 signaling by using MEKK2−/− mice. We found that MEKK2 was selectively required for LPS-induced ERK1/2 activation, but not JNK, p38 or NF-κB activation. We also found that MEKK2 was involved in TLR4 dependent induction of proinflammatory cytokines and LPS-induced septic shock. In conclusion, we identified a key regulatory serine residue in the activation loop of MEKK2 whose phosphorylation is a key sensor of receptor- and cellular stress-mediated signals. We also demonstrated that MEKK2 is crucial for TLR4-mediated innate immunity. ^

The Role of K63-linked Ubiquitination Cycles in Akt Kinase Activation

Akt (also known as protein kinase B) serves a central regulator in PI3K/Akt signaling pathways to regulate numerous physiological functions including cell proliferation, survival and metabolism. Akt activation requires the binding of Akt to phospholipid PIP3 on the plasma membrane and subsequent phosphorylation of Akt by its kinases. Growth factor-mediated membrane recruitment of Akt is a crucial step for Akt activation. However, the mechanism of Akt membrane translocation is unclear. Protein ubiquitination is a significant posttranslational modification that controls many biological functions such as protein trafficking and signaling activation. Therefore, we hypothesize that ubiquitination may be involved in Akt signaling activation. We have demonstrated that Akt could be conjugated with non-proteolytic K63-linked ubiquitination by TRAF6 ubiquitin E3 ligase. This modification on Akt was required for membrane recruitment, phosphorylation and activation of Akt in response to growth factor stimulation. The human cancer-associated Akt E17K mutant exhibited an increase in K63-linked ubiquitination, which contributes to the enrichment of membrane recruitment and phosphorylation of Akt. Thus, we conclude that K63-linked ubiquitination is a critical step for oncogenic Akt activation and also involved in human cancer development. Notably, the process of protein ubiquitination can be reversed by deubiquitinating enzymes (DUBs), which play a critical role to terminate signaling activation induced by ubiquitination. To further investigate how ubiquitination cycles regulate Akt activation, we have identified that CYLD as a DUB for Akt, and CYLD inhibited growth factor-induced ubiquitination and activation of Akt. Under serum-depletion condition, CYLD interacts with Akt and keep Akt under inactive state by directly removing K63-linked ubiquitination of Akt. CYLD disassociates with Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. We also demonstrated that CYLD deficiency promoted cancer cell proliferation, survival, glucose metabolism and human prostate cancer development. Therefore, we conclude that CYLD plays a critical role for negatively regulating Akt signaling activation through deubiquitination of Akt. In summary, this study delineated the important mechanism of cycles of ubiquitination and deubiquitination of Akt in regulating membrane translocation and activation of Akt, and TRAF6 and CYLD as central switches for these processes.

Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells

The activation of cyclin-dependent kinases (cdks) has been implicated in apoptosis induced by various stimuli. We find that the Fas-induced activation of cdc2 and cdk2 in Jurkat cells is not dependent on protein synthesis, which is shut down very early during apoptosis before caspase-3 activation. Instead, activation of these kinases seems to result from both a rapid cleavage of Wee1 (an inhibitory kinase of cdc2 and cdk2) and inactivation of anaphase-promoting complex (the specific system for cyclin degradation), in which CDC27 homolog is cleaved during apoptosis. Both Wee1 and CDC27 are shown to be substrates of the caspase-3-like protease. Although cdk activities are elevated during Fas-induced apoptosis in Jurkat cells, general activation of the mitotic processes does not occur. Our results do not support the idea that apoptosis is simply an aberrant mitosis but, instead, suggest that a subset of mitotic mechanisms plays an important role in apoptosis through elevated cdk activities.

Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation

Electrical stimulation of neonatal cardiac myocytes produces hypertrophy and cellular maturation with increased mitochondrial content and activity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation [c-fos, c-jun, JunB, nuclear respiratory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome c (Cyt c), cytochrome oxidase], and mitochondrial differentiation [carnitine palmitoyltransferase I (CPT-I) isoforms] were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25–3 hr) and followed sequentially by c-jun (0.5–3 hr), JunB (0.5–6 hr), NRF-1 (1–12 hr), Cyt c (12–72 hr), and muscle-specific CPT-I (48–72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA, thus supporting the developmental fidelity of this pattern of gene regulation. Consistent with a transcriptional mechanism, electrical stimulation increased c-fos, β-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the NRF-1 and CRE sites inhibited the induction by electrical stimulation (5-fold and 2-fold, respectively) whereas mutation of the Sp-1 site maintained or increased the fold induction. This finding is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c and suggests that induction of these transcription factors is a prerequisite for the transcriptional activation of Cyt c expression. These results support a regulatory role for NRF-1 and possibly AP-1 in the initiation of mitochondrial proliferation.