Mitogen-activated protein kinase signaling controls basal and oncostatin M-mediated JUNB gene expression.

The mitogen-activated protein kinase (MAPK) pathway is aberrantly activated in many human cancers, including breast cancer. Activation of MAPK signaling is associated with the increased expression of a wide range of genes that promote cell survival, proliferation, and migration. This report investigated the influence of MAPK signaling on the regulation and expression of JUNB in human breast cancer cell lines. JUNB has been associated with tumor suppressor and oncogenic functions, with most reports describing JUNB as an oncogene in breast cancer. Our results indicated that JUNB expression is elevated in MCF10A(met), SKBR3, and MDA-MB-231 human breast cancer cell lines compared to nontransformed MCF10A mammary epithelial cells. Increased RAS/MAPK signaling in MCF10A(met) cells correlates with the increased association of RNA polymerase II (Pol II) phosphorylated on serine 5 (Pol IIser5p) with the JUNB proximal promoter. Pol IIser5p is the "transcription initiating" form of Pol II. Treatment with U0126, a MAPK pathway inhibitor, reduces Pol IIser5p association with the JUNB proximal promoter and reduces JUNB expression. Oncostatin M (OSM) enhances MAPK and STAT3 signaling and significantly induces JUNB expression. U0126 treatment reduces OSM-induced Pol IIser5p binding to the JUNB proximal promoter and JUNB expression, but does not reduce pSTAT3 levels or the association of pSTAT3 with the JUNB proximal promoter. These results demonstrate that the MAPK pathway plays a primary role in the control of JUNB gene expression by promoting the association of Pol IIser5p with the JUNB proximal promoter.

JUNB promotes the survival of Flavopiridol treated human breast cancer cells.

Chemotherapy resistance is a major obstacle to achieving durable progression-free-survival in breast cancer patients. Identifying resistance mechanisms is crucial to the development of effective breast cancer therapies. Immediate early genes (IEGs) function in the initial cellular reprogramming response to alterations in the extracellular environment and IEGs have been implicated in cancer cell development and progression. The purpose of this study was to investigate the influence of kinase inhibitors on IEG expression in breast cancer cells. The results demonstrated that Flavopiridol (FP), a CDK9 inhibitor, effectively reduced gene expression. FP treatment, however, consistently produced a delayed induction of JUNB gene expression in multiple breast cancer cell lines. Similar results were obtained with Sorafenib, a multi-kinase inhibitor and U0126, a MEK1 inhibitor. Functional studies revealed that JUNB plays a pro-survival role in kinase inhibitor treated breast cancer cells. These results demonstrate a unique induction of JUNB in response to kinase inhibitor therapies that may be among the earliest events in the progression to treatment resistance.

Key roles for MED1 LxxLL motifs in pubertal mammary gland development and luminal-cell differentiation.

Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand-dependent interactions of the MED1 subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is little information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Toward this end, we have generated MED1 LxxLL motif-mutant knockin mice. Interestingly, these mice are both viable and fertile and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. Consistent with this phenotype, as well as loss of the strong ligand-dependent estrogen receptor (ER)alpha-Mediator interaction, expression of a number of known ERalpha-regulated genes was down-regulated in MED1-mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen-stimulated mammary duct growth in MED1-mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor- and cell-specific in vivo role for MED1 LxxLL motifs, through Mediator-ERalpha interactions, in mammary gland development.

Prediction Method of Coal Texture Considering Longitudinal Resolution of Logging Curves and Its Application-Taking No. 15 Coal Seam in the Shouyang Block as an Example.

The quantitative identification of the coal texture is of great importance as a crucial parameter for coalbed methane (CBM) reservoir evaluation. This study combined drilling core data, electrical imaging logging data, and four conventional logging data, namely, compensation density (DEN), natural γ (GR), deep lateral resistivity (RD), and acoustic time difference (AC), to achieve accurate inversion of coal texture in the Shouyang Block. Meanwhile, wavelet analysis and Fisher discriminant analysis were introduced to the inversion process to further improve the accuracy. Through the utilization of software packages, such as Matlab and SPSS, the establishment of the coal texture logging interpretation chart of the No. 15 coal seam in the Shouyang block was successfully realized. The outcome of this comprehensive study reveals that the coal texture logging interpretation chart is an effective tool for the identification and classification of each coal texture and gangue. Moreover, the validity and reliability of this method were tested and confirmed using wells CS-8 and CS-9 in the region, achieving an accuracy of 97.1 and 93.2%, respectively. This innovative method has significant prospects for predicting and evaluating the coal texture in the Shouyang Block, which can be further applied to other regions.

Monocyte NLRP3-IL-1ß Hyperactivation Mediates Neuronal and Synaptic Dysfunction in Perioperative Neurocognitive Disorder.

Perioperative neurocognitive disorder may develop in vulnerable patients following major operation. While neuroinflammation is linked to the cognitive effects of surgery, how surgery and immune signaling modulate neuronal circuits, leading to learning and memory impairment remains unknown. Using in vivo two-photon microscopy, Ca2+ activity and postsynaptic dendritic spines of layer 5 pyramidal neurons in the primary motor cortex of a mouse model of thoracic surgery are imaged. It is found that surgery causes neuronal hypoactivity, impairments in learning-dependent dendritic spine formation, and deficits in multiple learning tasks. These neuronal and synaptic alterations in the cortex are mediated by peripheral monocytes through the NLRP3 inflammasome-dependent IL-1ß production. Depleting peripheral monocytes or inactivating NLRP3 inflammasomes before surgery reduces levels of IL-1ß and ameliorates neuronal and behavioral deficits in mice. Furthermore, adoptive transfer of IL-1ß-producing myeloid cells from mice undertaking thoracic surgery is sufficient to induce neuronal and behavioral deficits in naïve mice. Together, these findings suggest that surgery leads to excessive NLRP3 activation in monocytes and elevated IL-1ß signaling, which in turn causes neuronal hypoactivity and perioperative neurocognitive disorder.

Inhibition of unfolded protein response prevents post-anesthesia neuronal hyperactivity and synapse loss in aged mice.

Delirium is the most common postoperative complication in older patients after prolonged anesthesia and surgery and is associated with accelerated cognitive decline and dementia. The neuronal pathogenesis of postoperative delirium is largely unknown. The unfolded protein response (UPR) is an adaptive reaction of cells to perturbations in endoplasmic reticulum function. Dysregulation of UPR has been implicated in a variety of diseases including Alzheimer's disease and related dementias. However, whether UPR plays a role in anesthesia-induced cognitive impairment remains unexplored. By performing in vivo calcium imaging in the mouse frontal cortex, we showed that exposure of aged mice to the inhalational anesthetic sevoflurane for 2 hours resulted in a marked elevation of neuronal activity during recovery, which lasted for at least 24 hours after the end of exposure. Concomitantly, sevoflurane anesthesia caused a prolonged increase in phosphorylation of PERK and eIF2α, the markers of UPR activation. Genetic deletion or pharmacological inhibition of PERK prevented neuronal hyperactivity and memory impairment induced by sevoflurane. Moreover, we showed that PERK suppression also reversed various molecular and synaptic changes induced by sevoflurane anesthesia, including alterations of synaptic NMDA receptors, tau protein phosphorylation, and dendritic spine loss. Together, these findings suggest that sevoflurane anesthesia causes abnormal UPR in the aged brain, which contributes to neuronal hyperactivity, synapse loss and cognitive decline in aged mice.

Investigation on the Structure and Fractal Characteristics of Nanopores in High-Rank Coal: Implications for the Methane Adsorption Capacity.

The structure and fractal characteristics of nanopores of high-rank coal were investigated using an approach that integrates N2 adsorption and field emission scanning electron microscopy (FE-SEM). The results indicated that the high-rank coal of the Shanxi Formation has a complex pore-fracture network composed of organic matter pores, mineral-related pores, and microfractures. The pore type of high-rank coal tends to be complicated, and the main pore types are inkbottle pores and open pores, which are more conducive to methane enrichment. The Ro,max has a negative relationship with the total pore volume. In addition, the ash and inertinite contents show a positive correlation with the average pore size (APS), while the fixed carbon content exhibits a negative relationship with the APS. The pore structure of high-rank coal is controlled not only by the degree of metamorphism but also by coal composition, which leads to the variation in pore structure becoming more complicated. With the increase in coal metamorphism, high-rank coal with high amounts of fixed carbon content generally possesses a higher irregularity in pore structure. No obvious relationship was observed between D2 and the coal components, which indicates that the pore structure, ash content, moisture content and other factors controlled by the metamorphism of coal have different effects on D2 that lead to this inapparent relationship. A negative relationship exists between adsorption volume and D1, which indicates that the high irregularity of the pore structure is not conducive to methane absorption and that no obvious correlation exists between the adsorption volume and D2. In the high-rank coal, the high D1 value represents the complexity and heterogeneity of the pore structure and represents a low adsorption affinity for methane molecules; in addition, D2 has no effect on the methane adsorption capacity.

Identification of epithelial gaps in human small and large intestine by confocal endomicroscopy.

BACKGROUND & AIMS: Confocal endomicroscopy is an emerging technology that poses the endoscopist with challenges for identifying epithelial structures in the human intestine. We have shown previously that the murine intestinal epithelium is punctuated by gaps caused by cell shedding. The goals of this study were to determine if confocal endomicroscopy could resolve the presence of human epithelial gaps and whether a proinflammatory cytokine could increase cell shedding. METHODS: Intestinal mucosa was imaged after staining with acriflavine. Confocal endomicroscopy of 17 patients yielded 6277 images from the human terminal ileum and rectum. Results were validated by parallel studies of anesthetized mice (wild-type and Math1(DeltaIntestine)) using rigid confocal probe microscopy, 2-photon/confocal microscopy, and scanning electron microscopy. RESULTS: Human terminal ileal and rectal epithelium revealed unstained areas with the diameter of an individual epithelial cell, with 2 distinct morphologies. One had a "target" appearance, shown by mouse studies to be goblet cells. The other morphology had no nucleus and was observed by rigid confocal probe microscopy and scanning electron microscopy in the villi of Math1(DeltaIntestine) mice, which lack goblet cells. In the mouse, tumor necrosis factor alpha (0.33 microg/g intraperitoneally) increases cell shedding by 27-fold and caused loss of barrier function across 20% of resultant gaps. CONCLUSIONS: Confocal endomicroscopy can distinguish between epithelial discontinuities (gaps) and goblet cells in human intestine. Results suggest that the sealing of epithelial gaps must be considered as a component of the intestinal barrier and has potential implications for intestinal barrier dysfunction in human disease.

Anti-inflammatory effects of rosmarinic acid-4-O-ß-D-glucoside in reducing acute lung injury in mice infected with influenza virus.

Rosmarinic acid-4-O-ß-D-glucoside (RAG) is a dicaffeoyl phenolic compound isolated from Sarcandra glabra (Thunb.) Nakai. Preliminary studies show that RAG has significant anti-inflammatory properties and can alleviate ear swelling in mice and the paw swelling in rats. Here, the anti-influenza effects of RAG were investigated in mice infected with A/FM/1/47 H1N1 virus. The survival rate and body weight were observed, the lung edema, virus copies, inflammatory cytokines (including IL-4, IL-5, TNF-α and IFN-γ) and oxidative damage indexes (including SOD, MDA, NO, and CAT) were measured. Moreover, immune cell recruitment in alveoli was measured with white blood cells and differential counts. Therapeutic RAG concentrations substantially improve the symptoms, mitigate body weight loss and alleviate lung edema induced by virus, thus improve survival protection effects. Furthermore, RAG was shown to regulate influenza virus-induced inflammatory cytokine expression, specifically by downregulating the Th1 cell cytokines IFN-γ, TNF-α and upregulating the Th2 cell cytokines IL-4, IL-5. Cell migration and infiltration were also diminished after RAG administration.

MicroRNA-150 protects the mouse heart from ischaemic injury by regulating cell death.

AIMS: Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs). For example, miR-150 is down-regulated in patients with acute myocardial infarction, atrial fibrillation, dilated and ischaemic cardiomyopathy as well as in various mouse heart failure (HF) models. Circulating miR-150 has been recently proposed as a better biomarker of HF than traditional clinical markers such as brain natriuretic peptide. We recently showed using the ß-arrestin-biased ß-blocker, carvedilol that ß-arrestin1-biased ß1-adrenergic receptor cardioprotective signalling stimulates the processing of miR-150 in the heart. However, the potential role of miR-150 in ischaemic injury and HF is unknown. METHODS AND RESULTS: Here, we show that genetic deletion of miR-150 in mice causes abnormalities in cardiac structural and functional remodelling after MI. The cardioprotective roles of miR-150 during ischaemic injury were in part attributed to direct repression of the pro-apoptotic genes egr2 (zinc-binding transcription factor induced by ischaemia) and p2x7r (pro-inflammatory ATP receptor) in cardiomyocytes. CONCLUSION: These findings reveal a pivotal role for miR-150 as a regulator of cardiomyocyte survival during cardiac injury.

The roots of Ilex asprella extract lessens acute respiratory distress syndrome in mice induced by influenza virus.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, the root of Ilex asprella (Hook. & Arn.) Champ. ex Benth. (IA) has been widely used to treat influenza, lung abscess and other diseases in South China for many years. The present study is aimed at investigating the treatment effect of IA on acute respiratory distress syndrome (ARDS) induced by the H1N1 virus in mice. MATERIALS AND METHODS: After being inoculated with several viral doses of influenza A/FM/1/47 H1N1 virus, mice were given oral administration of IA extract (500 mg/kg or 12 5mg/kg per day) for five or 10 consecutive days, respectively. Mice survival rate and clinical condition were observed for 15 days after inoculation. Lung weight, pathological analysis and arterial blood gas analysis were assessed. Lung viral load was quantified by RT-PCR. Moreover, immunological analysis was measured by leukocyte counts and the levels of inflammatory cytokines, including IL-6, IL-10, TNF-α, IFN-γ, MCP-1 and IL-12p 70 in serum of mice. RESULTS: We found that the extract of Ilex asprella at dosages of 500 mg/kg could effectively diminish mortality rate, and ameliorate lung edema and inflammation. Administration of IA extract significantly depressed the expression of IL-6, TNF-α and MCP-1, and significantly increased the expression of IL-10 and IFN-γ in serum. Simultaneously, the extract was also found to reduce the lung viral load and improve pulmonary ventilation. CONCLUSION: The present study shows that the extract of IA has the potential to treat ARDS, due to its abilities of attenuation of systemic and pulmonary inflammatory responses and inhibition of viral replication.

Endothelin-1/endothelin A receptor-mediated biased signaling is a new player in modulating human ovarian cancer cell tumorigenesis.

The endothelin-1 (ET-1)/endothelin A receptor (ETAR, a G protein-coupled receptor) axis confers pleiotropic effects on both tumor cells and the tumor microenvironment, modulating chemo-resistance and other tumor-associated processes by activating Gαq- and ß-arrestin-mediated pathways. While the precise mechanisms by which these effects occur remain to be elucidated, interference with ETAR signaling has emerged as a promising antitumor strategy in many cancers including ovarian cancer (OC). However, current clinical approaches using ETAR antagonists in the absence of a detailed knowledge of downstream signaling have resulted in multiple adverse side effects and limited therapeutic efficacy. To maximize the safety and efficacy of ETAR-targeted OC therapy, we investigated the role of other G protein subunits such as Gαs in the ETAR-mediated ovarian oncogenic signaling. In HEY (human metastatic OC) cells where the ET-1/ETAR axis is well-characterized, Gαs signaling inhibits ETAR-mediated OC cell migration, wound healing, proliferation and colony formation on soft agar while inducing OC cell apoptosis. Mechanistically, ET-1/ETAR is coupled to Gαs/cAMP signaling in the same ovarian carcinoma-derived cell line. Gαs/cAMP/PKA activation inhibits ETAR-mediated ß-arrestin activation of angiogenic/metastatic Calcrl and Icam2 expression. Consistent with our findings, Gαs overexpression is associated with improved survival in OC patients in the analysis of the Cancer Genome Atlas data. In conclusion, our results indicate a novel function for Gαs signaling in ET-1/ETAR-mediated OC oncogenesis and may provide a rationale for a biased signaling mechanism, which selectively activates Gαs-coupled tumor suppressive pathways while blocking Gαq-/ß-arrestin-mediated oncogenic pathways, to improve the targeting of the ETAR axis in OC.

Chloroplast genome of one brown seaweed, Saccharina japonica (Laminariales, Phaeophyta): its structural features and phylogenetic analyses with other photosynthetic plastids.

The chloroplast genome sequence of one brown seaweed, Saccharina japonica, was fully determined. It is characterized by 130,584 base pairs (bp) with a large and a small single-copy region (LSC and SSC), separated by two copies of inverted repeats (IR1 and IR2). The inverted repeat is 5015 bp long, and the sizes of SSC and LSC are 43,174 bp and 77,378 bp, respectively. The chloroplast genome of S. japonica consists of 139 protein-coding genes, 29 tRNA genes, and 3 ribosomal RNA genes. One intron was found in one tRNA-Leu gene in the chloroplast genome of S. japonica. Four types of overlapping genes were identified, ycf24 overlapped with ycf16 by 4 nucleotides (nt), ftrB overlapped with ycf12 by 6 nt, rpl4 and rpl23 overlapped by 8 nt, finally, psbC overlapped with psbD by 53 nt. With two sets of concatenated plastid protein data, 40-protein dataset and 26-protein dataset, the chloroplast phylogenetic relationship among S. japonica and the other photosynthetic species was evaluated. We found that the chloroplast genomes of haptophyte, cryptophyte and heterokont were not resolved into one cluster by the 40-protein dataset with amino acid composition bias, although it was recovered with strong support by the 26-protein dataset.

Altered cerebellar function in mice lacking CaV2.3 Ca2+ channel.

Voltage-dependent Ca(2+) channels play important roles in cerebellar functions including motor coordination and learning. Since abundant expression of Ca(V)2.3 Ca(2+) channel gene in the cerebellum was detected, we searched for possible deficits in the cerebellar functions in the Ca(V)2.3 mutant mice. Behavioral analysis detected in delayed motor learning in rotarod tests in mice heterozygous and homozygous for the Ca(V)2.3 gene disruption (Ca(V)2.3+/- and Ca(V)2.3-/-, respectively). Electrophysiological analysis of mutant mice revealed perplexing results: deficit in long-term depression (LTD) at the parallel fiber Purkinje cell synapse in Ca(V)2.3+/- mice but apparently normal LTD in Ca(V)2.3-/- mice. On the other hand, the number of spikes evoked by current injection in Purkinje cells under the current-clamp mode decreased in Ca(V)2.3 mutant mice in a gene dosage-dependent manner, suggesting that Ca(V)2.3 channel contributed to spike generation in Purkinje cells. Thus, Ca(V)2.3 channel seems to play some roles in cerebellar functions.

The carboxy-terminal tail region of human Cav2.1 (P/Q-type) channel is not an essential determinant for its subcellular localization in cultured neurones.

A recent report on the mechanism of synaptic targeting of Ca(v)2.2 channel suggested that this process depends upon the presence of long C-terminal tail and that protein interactions mediated by SH3-binding and PDZ-binding motifs in the tail region are important. To examine the possibility that C-terminal tail of the Ca(v)2.1 channel and the polyglutamine stretch therein are also involved in the mechanism for channel localization, we constructed several expression plasmids for human Ca(v)2.1 channel tagged with enhanced green fluorescent protein (EGFP) and introduced them into mouse hippocampal neuronal culture. HC construct encodes short version of Ca(v)2.1, and HS and HL encode Ca(v)2.1 channel with a long C-terminal tail, which contains polyglutamine tract of 13 (normal range) and 28 (SCA6 disease range) repeat units, respectively. Surprisingly, transfection with HC, HS, and HL gave essentially the same results: EGFP signal was observed in cell soma, dendrites, and the axon as well. Furthermore, mutation of the PDZ-binding motif located at the C-terminus of the long version of Ca(v)2.1, by adding FLAG tag, did not affect the localization patterns of HS and HL as well. Therefore, the C-terminal region is not indispensable for the subcellular localization of Ca(v)2.1 channel, nor expansion of polyglutamine length affected the localization of the channel. Thus, it is possible that the localization mechanism of Ca(v)2.1 channel is different from that of Ca(v)2.2, though these channels share various structural and functional characteristics.