Glucocorticoids unmask silent non-coding genetic risk variants for common diseases.

Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were 'unmasked' only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.

RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

Directed migration is essential for cell motility in many processes, including development and cancer cell invasion. RSKs (p90 ribosomal S6 kinases) have emerged as central regulators of cell migration; however, the mechanisms mediating RSK-dependent motility remain incompletely understood. We have identified a unique signaling mechanism by which RSK2 promotes cell motility through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 or Cdc42. We further show that epidermal growth factor or FBS stimulation induces association of endogenous RSK2 with LARG and LARG with RhoA. In response to these stimuli, RSK2 phosphorylates LARG at Ser1288 and thereby activates RhoA. Phosphorylation of RSK2 at threonine 577 is essential for activation of LARG-RhoA. Moreover, RSK2-mediated motility signaling depends on RhoA and -B, but not RhoC. These results establish a unique RSK2-dependent LARG-RhoA signaling module as a central organizer of directed cell migration and invasion.

Six-way decomposition of causal effects: Unifying mediation and mechanistic interaction.

The sufficient component cause (SCC) model and counterfactual model are two common methods for causal inference, each with their own advantages: the SCC model allows the mechanistic interaction to be detailed, whereas the counterfactual model features a systemic framework for quantifying causal effects. Hence, integrating the SCC and counterfactual models may facilitate the conceptualization of causation. Based on the marginal SCC (mSCC) model, we propose a novel counterfactual mSCC framework that includes the steps of definition, identification, and estimation. We further propose a six-way effect decomposition for assessing mediation and the mechanistic interaction. The results demonstrate that when all variables are binary, the six-way decomposition is an extension of four-way decomposition and that without agonism, the six-way decomposition is reduced to four-way decomposition. To illustrate the utility of the proposed decomposition, we apply it to a Taiwanese cohort to examine the mechanism of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) with liver inflammation measured by alanine aminotransferase (ALT) as a mediator. Among the HCV-induced HCC cases, 62.27% are not explained by either mediation or interaction in relation to ALT; 9.32% are purely mediated by ALT; 16.53% are caused by the synergistic effect of HCV and ALT; and 9.31% are due to the mediated synergistic effect of HCV and ALT. In summary, we introduce an SCC model framework based on counterfactual theory and detail the required identification assumptions and estimation procedures; we also propose a six-way effect decomposition to unify mediation and mechanistic interaction analyses.

Rit-mediated stress resistance involves a p38-mitogen- and stress-activated protein kinase 1 (MSK1)-dependent cAMP response element-binding protein (CREB) activation cascade.

The cAMP response element (CRE)-binding protein (CREB) is a key regulatory factor of gene transcription, and plays an essential role in development of the central nervous system and for neuroprotection. Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both ERK and p38 mitogen-activated protein (MAP) kinases cascades. Recent studies have identified the Ras-related small G-protein, Rit, as a central regulator of a p38-MK2-HSP27 signaling cascade that functions as a critical survival mechanism for cells adapting to stress. Here, we examine the contribution of Rit-p38 signaling to the control of stress-dependent gene transcription. Using a pheochromocytoma cell model, we find that a novel Rit-p38-MSK1/2 pathway plays a critical role in stress-mediated CREB activation. RNAi-mediated Rit silencing, or inhibition of p38 or MSK1/2 kinases, was found to disrupt stress-mediated CREB-dependent transcription, resulting in increased cell death. Furthermore, ectopic expression of active Rit stimulates CREB-Ser133 phosphorylation, induces expression of the anti-apoptotic Bcl-2 and Bcl(XL) proteins, and promotes cell survival. These data indicate that the Rit-p38-MSK1/2 signaling pathway may have an important role in the stress-dependent regulation of CREB-dependent gene expression.

Vacuolar H+-ATPase and BZR1 form a feedback loop to regulate the homeostasis of BR signaling in Arabidopsis.

Brassinosteroid (BR) is a vital plant hormone that regulates plant growth and development. BRASSINAZOLE RESISTANT 1 (BZR1) is a key transcription factor in BR signaling, and its nucleocytoplasmic localization is crucial for BR signaling. However, the mechanisms that regulate BZR1 nucleocytoplasmic distribution and thus the homeostasis of BR signaling remain largely unclear. The vacuole is the largest organelle in mature plant cells and plays a key role in maintenance of cellular pH, storage of intracellular substances, and transport of ions. In this study, we uncovered a novel mechanism of BR signaling homeostasis regulated by the vacuolar H+-ATPase (V-ATPase) and BZR1 feedback loop. Our results revealed that the vha-a2 vha-a3 mutant (vha2, lacking V-ATPase activity) exhibits enhanced BR signaling with increased total amount of BZR1, nuclear-localized BZR1, and the ratio of BZR1/phosphorylated BZR1 in the nucleus. Further biochemical assays revealed that VHA-a2 and VHA-a3 of V-ATPase interact with the BZR1 protein through a domain that is conserved across multiple species. VHA-a2 and VHA-a3 negatively regulate BR signaling by interacting with BZR1 and promoting its retention in the tonoplast. Interestingly, a series of molecular analyses demonstrated that nuclear-localized BZR1 could bind directly to specific motifs in the promoters of VHA-a2 and VHA-a3 to promote their expression. Taken together, these results suggest that V-ATPase and BZR1 may form a feedback regulatory loop to maintain the homeostasis of BR signaling in Arabidopsis, providing new insights into vacuole-mediated regulation of hormone signaling.

The combined effects of microplastics and the heavy metal cadmium on the marine periphytic ciliate Euplotes vannus.

Microplastics could be grazed by marine organisms and possibly transferred to higher trophic levels along the microbial loop. Due to their size and capacity to concentrate heavy metals that trigger joint toxic effects, microplastics (MPs) have already become a severe threat to marine organisms. The detrimental effects of MPs on large marine organisms have been studied, but the combined toxicity of MPs and cadmium (Cd) on protozoan ciliates remains unclear. In the present study, we selected different diameters and concentrations of polystyrene microspheres (PS-MPs) and Cd2+ as model MPs and heavy metals to evaluate their single and combined effects on the periphytic marine ciliate Euplotes vannus in relation to carbon biomass and oxidative stress. The MPs were indeed ingested by Euplotes vannus and significantly reduced the abundance and carbon biomass of ciliate populations. Combined exposure to MPs and Cd2+ not only increased the bioaccumulation of Cd2+ in ciliates but also exacerbated the decrease in ciliate biomass by increasing oxidative stress and membrane damage. In comparison, the effects of nano-sized plastics (0.22 µm) were more harmful than those of micro-sized plastics (1.07 µm, 2.14 µm and 5.00 µm). A smaller size represents a higher potential for penetrating biological members and a stronger adsorption capacity for cadmium. These results provide new insight into the combined toxicity of microplastics and heavy metals on ciliated protozoa and lay a foundation for higher trophic levels and ecosystems.

Depolarization increases phosphatidylinositol (PI) 4,5-bisphosphate level and KCNQ currents through PI 4-kinase mechanisms.

A growing body of evidence shows that membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P(2), PIP(2)) play an important role in cell signaling. The presence of PIP(2) is fundamentally important for maintaining the functions of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility, and endo- and exocytosis. PIP(2) levels in the membrane are dynamically modulated, which is an important signaling mechanism for modulation of PIP(2)-dependent cellular processes. In this study, we describe a novel mechanism of membrane PIP(2) modulation. Membrane depolarization induces an elevation in membrane PIP(2), and subsequently increases functions of PIP(2)-sensitive KCNQ potassium channels expressed in Xenopus oocytes. Further evidence suggests that the depolarization-induced elevation of membrane PIP(2) occurs through increased activity of PI4 kinase. With increased recognition of the importance of PIP(2) in cell function, the effect of membrane depolarization in PIP(2) metabolism is destined to have important physiological implications.

A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP38) stimulation results in the activation of G(s)alpha protein-coupled receptors to regulate neuronal differentiation in a cyclic AMP (cAMP)-dependent manner. These pathways involve protein kinase A (PKA)-dependent processes, but a growing body of evidence indicates that cAMP also regulates cellular functions through PKA-independent signaling cascades. Here we show that the Rit small GTPase is regulated by PACAP38 in a cAMP-dependent but PKA-independent fashion. Rit activation results from stimulation of the cAMP-activated guanine nucleotide exchange factor Epac but does not appear to rely upon the activation of Rap GTPases, the accepted cellular Epac substrates. Although RNA interference studies demonstrated that Epac is required for PACAP38-mediated Rit activation, neither Epac1 nor Epac2 activates Rit directly, indicating that Epac signals to Rit through a novel mechanism in which Rap signaling is not essential. Loss-of-function analysis demonstrated that Rit makes an important contribution to PACAP38-mediated neuronal differentiation. Surprisingly, although Rit is required for sustained extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase signaling following nerve growth factor stimulation of pheochromocytoma 6 (PC6) cells, Rit silencing selectively suppressed PACAP38-elicited activation of p38, without obvious effects on ERK signaling in the same cells. Moreover, the ability of PACAP38 to stimulate CREB-dependent transcription and to promote neurite outgrowth was inhibited by Rit knockdown. Together, these studies identify an unsuspected connection between cAMP and Rit signaling pathways and imply that Rit can function downstream of G(s)alpha/cAMP/Epac in a novel signal transduction pathway necessary for PACAP38-mediated neuronal differentiation and CREB signaling.

Specificity of Gbetagamma signaling depends on Galpha subunit coupling with G-protein-sensitive K(+) channels.

Many neurotransmitters activate G-protein-gated inwardly rectifying K(+) (Kir3) channels by stimulating G-protein-coupled receptors. However, in native systems, only receptors coupled to pertussis-toxin (PTX)-sensitive G proteins (Gi/Go) have been shown to be able to activate Kir3 channels through the betagamma subunits of G proteins (Gbetagamma), whereas activation of receptors coupled to PTX-insensitive G proteins such as Gq or Gs do not activate Kir3 channels. The question remains as to how signaling specificity is achieved and what are its key determinants. In this study, we have used the Xenopus oocyte expression system to investigate specific activation of Kir3 channels by heterotrimeric G proteins. We have demonstrated the activation of Kir3.4 channels by agonist stimulation of non-PTX-sensitive G proteins under conditions of Galpha subunit overexpression. We present evidence to suggest a key role for the coupling efficiency of Galpha subunits in determining the specificity of Gbetagamma signaling to the channel.

The novel GTPase Rit differentially regulates axonal and dendritic growth.

The Rit GTPase is widely expressed in developing and adult nervous systems, and our previous data with pheochromocytoma cells implicate Rit signaling in NGF-induced neurite outgrowth. In this study, we investigated a role for Rit in neuronal morphogenesis. Expression of a dominant-negative (dn) Rit mutant in hippocampal neurons inhibited axonal growth but potentiated dendritic growth. Conversely, a constitutively active (ca) Rit mutant promoted axonal growth but inhibited dendritic growth. Dendritogenesis is regulated differently in sympathetic neurons versus hippocampal neurons in that sympathetic neurons require NGF and bone morphogenetic proteins (BMPs) to trigger dendritic growth. Despite these differences, dnRit potentiated and caRit blocked BMP7-induced dendritic growth in sympathetic neurons. Biochemical studies indicated that BMP7 treatments that caused dendritic growth also decreased Rit GTP loading. Additional studies demonstrate that caRit increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and pharmacological inhibition of MEK1 (mitogen-activated protein kinase/ERK 1) blocked the axon-promoting and dendrite-inhibiting effects of caRit. These observations suggest that Rit is a convergence point for multiple signaling pathways and it functions to promote axonal growth but inhibit dendritic growth via activation of ERK1/2. Modulation of the activational status of Rit may therefore represent a generalized mechanism across divergent neuronal cell types for regulating axonal versus dendritic growth modes.

Rit mutants confirm role of MEK/ERK signaling in neuronal differentiation and reveal novel Par6 interaction.

Rit is a novel member of the Ras superfamily of small GTP-binding proteins that regulates signaling pathways controlling cellular fate determination. Constitutively activated mutants of Rit induce terminal differentiation of pheochromocytoma (PC6) cells resulting in a sympathetic neuron-like phenotype characterized by the development of highly-branched neurites. Rit signaling has been found to activate several downstream pathways including MEK/ERK, p38 MAPK, Ral-specific guanine nucleotide exchange factors (GEFs), and Rit associates with the Par6 cell polarity machinery. In this study, a series of Rit effector loop mutants was generated to test the importance of these cellular targets to Rit-mediated neuronal differentiation. We find that Rit-mediated neuritogenesis is dependent upon MEK/ERK MAP kinase signaling but independent of RalGEF activation. In addition, in vivo binding studies identified a novel mechanism of Par6 interaction, suggesting that the cell polarity machinery may serve to spatially restrict Rit signaling.

Rit signaling contributes to interferon-gamma-induced dendritic retraction via p38 mitogen-activated protein kinase activation.

The proinflammatory cytokine interferon-gamma (IFNgamma) alters neuronal connectivity via selective regressive effects on dendrites but the signaling pathways that mediate this effect are poorly understood. We recently demonstrated that signaling by Rit, a member of the Ras family of GTPases, modulates dendritic growth in primary cultures of sympathetic and hippocampal neurons. In this study, we investigated a role for Rit signaling in IFNgamma-induced dendritic retraction. Expression of a dominant negative Rit mutant inhibited IFNgamma-induced dendritic retraction in cultured embryonic rat sympathetic and hippocampal neurons. In pheochromacytoma cells and hippocampal neurons, IFNgamma caused rapid Rit activation as indicated by increased GTP binding to Rit. Silencing of Rit by RNA interference suppressed IFNgamma-elicited activation of p38 MAPK in pheochromacytoma cells, and pharmacological inhibition of p38 MAPK significantly attenuated the dendrite-inhibiting effects of IFNgamma in cultured sympathetic and hippocampal neurons without altering signal transducer and activator of transcription 1 activation. These observations identify Rit as a downstream target of IFNgamma and suggest that a novel IFNgamma-Rit-p38 signaling pathway contributes to dendritic retraction and may, therefore, represent a potential therapeutic target in diseases with a significant neuroinflammatory component.

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades.

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.

[Cloning and analyzing of rice blast resistance gene Pi-ta+ allele from Jinghong erect type of common wild rice (Oryza rufipogon Griff) in Yunnan].

A 4,672 bp DNA sequence including the whole coding region and partial non-coding region of rice blast resistance gene Pi-ta+ has been cloned from Jinghong erect type of common wild rice (Oryza rufipogon Griff) in Yunnan by polymerase chain reaction method. The coding region shares 99.86% and 98.78% identity with the corresponding regions of the reported cultivated rice Yashiro-mochi and Yuanjiang type of common wild rice respectively. There are 4 nucleotides difference in the coding region and 6 in intron of the cloned Pi-ta+ gene,compared with Pi-ta from Yashiro-mochi. Pi-ta+ gene in Jinghong erect type of common wild rice has been proved to be a rare existing Pi-ta+ allele, because there was a alanine rather than a serine at the position 918 within the predicted amino acid sequence of PITA. Pi-ta+ allele can cause disease resistance response to rice blast pathogens in plant cells. Differences in DNA sequence, deduced amino acid sequence and antibacterial spectrum may make the Pi-ta+ allele new resistant characteristics. Finding and cloning of Pi-ta+ allele from Jinghong erect type of common wild rice in Yunnan provides a basement for further utilization of the wild rice resources.

Selective inhibition of Kir currents by antihistamines.

In the present study, the effects of antihistamines on inwardly rectifying potassium (Kir) channels expressed in Xenopus oocyte were investigated using two-electrode voltage clamp technique. Firstly, effects of antihistamines on two members of Kir2.0 sub-family, Kir2.1 and Kir2.3 were compared. For antihistamines that selectively block histamine H(1) receptor, the first-generation antihistamines mepyramine and diphenhydramine inhibited Kir2.3 current by 25.0+/-2.9% and 17.3+/-0.7% at concentrations of 100 microM, respectively. In contrast, the second- and third-generation antihistamines astemizole and desloratadine were completely devoid of any inhibitory effect on Kir2.3 current. Histamine H(2) receptor antagonist cimetidine, at 100 microM, failed to inhibit Kir2.3 current. On the other hand, Kir2.1 current was not sensitive to any of these drugs. The mepyramine-induced inhibition of Kir2.3 current was significantly reduced by a single point mutation in Kir2.3 (Kir2.3(I213L)), which enhances Kir2.3-PIP(2) interaction. Secondly, the effect of mepyramine was also tested on Kir3.4*, another member of Kir family. 100 microM mepyramine produced a 30.3+/-4.6% inhibition on Kir3.4* current. These results suggest that the first-generation histamine H(1) receptor antagonists selectively inhibit Kir currents. The inhibitory effect of antihistamines on Kir currents may be involved in their neuronal and cardiac toxic effects caused by drug overdosing.

[Identification of white muscardin silkworms by infrared spectroscopy].

The infrared spectra of the ethanol extracts of well-living silkworms and white muscardin silkworms of different seasons and breeds were analyzed by means of the sequential analysis in which two indexes, i. e. common peak ratio and variant peak ratio, were applied. The results showed that the ethanol extracts of white muscardin silkworm have a stable and distinct infrared spectrum. The spectral differences of the ethanol extracts between white muscardin silkworms and well-living silkworms were so obvious that the common peak ratio of them was no more than 63. 0%, and the variant peak ratio amounted to 41. 2%. The spectra of different breeds and seasons conformed with each other with a few small differences. The minimum common peak ratio of the spectra of different breeds was 76. 0%, and the maximal ratio was 92. 0%. The common peak ratio of the spectra of different seasons was 73. 1%. Infrared spectrometry was proved to be good for the identification of white muscardin silkworms and the differentiation of white muscardin silkworms of different breeds and seasons.

[Study on the development of fetus and infant congenitally infected by Toxoplasma gondii and intervention].

Toxoplasma gondii infection during pregnancy can result in abortion, premature delivery, fetal death, deformity, and impact the physical and intellectual development of the newborns. This is an investigation on the consequences of pregnancy in Toxoplasma gondii-infected women, the development of their babies, and the effect of pyrimethamine treatment during 1990-1996 in Baoding City.

Analysis of Rit signaling and biological activity.

Rit (Ras-like expressed in many tissues) is the founding member of a novel subgroup within the larger Ras superfamily of small GTP-binding proteins. Although Rit shares more than 50% amino acid identity with Ras, it contains a unique effector domain in common with the closely related Rin and Drosophila Ric proteins and lacks the C-terminal lipidation motifs critical for the membrane association and biological activity of many Ras proteins. Interestingly, whereas Rit has only modest transforming ability when assayed in NIH 3T3 cells, Rit exhibits neuronal differentiation activities comparable to those of oncogenic mutants of Ras when assayed in PC12 and other neuronal cell lines. This cell-type specificity is explained in part by the ability of Rit to selectively activate the neuronal Raf isoform, B-Raf. Importantly, Rit seems to play a critical role in neurotrophin-mediated MAP kinase signaling, because Rit gene silencing significantly alters NGF-dependent MAP kinase signaling and neuronal differentiation. In this chapter, we discuss the reagents and methods used to characterize Rit-mediated signaling to MAP kinase-signaling pathways to determine the extracellular stimuli that regulate Rit activation and to characterize Rit-induced neuronal differentiation.

[Appraisal of the repair gastroschisis with autogenous umbilical cord].

OBJECTIVE: To review the treatment of repair gastroschisis with autogenous umbilical cord in 13-year and evaluate its effect. METHODS: Twenty-two newborns who underwent the repair gastroschisis with autogenous umbilical cord between 1992 and 2005. The physical growth, intelligence measuring, area of operation in abdomen in the survived 18 cases were observed and followed-up. RESULTS: Eighteen patients recovered uneventfully, survival rate is 82%, their growth is well. They all developed incisional hernia near the operation, 9 cases recovered himself, 2 cases was operated to repair the abdominal hernia, 7 cases is under observed. CONCLUSIONS: The material is adopted easily in the operating, autogenous umbilical cord is elastic tissue and no toxicity could relax the abdominal press effectively after the operation, the survival rate is high.