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Dynamic regulation of intestinal epithelial cell (IEC) differentiation is crucial for both homeostasis and the response to helminth infection. SIRT6 belongs to the NAD+-dependent deacetylases and has established diverse roles in aging, metabolism and disease. Here, we report that IEC Sirt6 deletion leads to impaired tuft cell development and type 2 immunity in response to helminth infection, thereby resulting in compromised worm expulsion. Conversely, after helminth infection, IEC SIRT6 transgenic mice exhibit enhanced epithelial remodeling process and more efficient worm clearance. Mechanistically, Sirt6 ablation causes elevated Socs3 expression, and subsequently attenuated tyrosine 641 phosphorylation of STAT6 in IECs. Notably, intestinal epithelial overexpression of constitutively activated STAT6 (STAT6vt) in mice is sufficient to induce the expansion of tuft and goblet cell linage. Furthermore, epithelial STAT6vt overexpression remarkedly reverses the defects in intestinal epithelial remodeling caused by Sirt6 ablation. Our results reveal a novel function of SIRT6 in regulating intestinal epithelial remodeling and mucosal type 2 immunity in response to helminth infection.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss., Author summary More than 50% of deafness cases are due to genetic defects with no treatment available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common types of autosomal dominant non-syndromic deafness. Here, we established a novel mouse model with the exact Pou4f3 mutation identified in human patients. The mutant mouse display similar auditory pathophysiology as human patients and exhibit multiple hair cell abnormalities. The onset and severity of hearing loss in the mouse model is highly modifiable to environmental factors, such as aging, noise exposure or genetic backgrounds. Using a new knockout mouse model, we found Pou4f3 haploinsufficiency as the underlying mechanism of human DFNA15. Importantly, we identified Aldh inhibitor as a potent small molecule for upregulation of Pou4f3 and treatment of hearing loss in the mutant mouse. The identification of Aldh inhibitor for treatment of DFNA15 deafness represents a major advance in the unmet medical need for this common form of progressive hearing loss.

Intestinal barrier function is required for the maintenance of mucosal homeostasis. Barrier dysfunction is thought to promote progression of both intestinal and systemic diseases. In many cases, this barrier loss reflects increased permeability of the paracellular tight junction as a consequence of myosin light chain kinase (MLCK) activation and myosin II regulatory light chain (MLC) phosphorylation. Although some details about MLCK activation remain to be defined, it is clear that this triggers perijunctional actomyosin ring (PAMR) contraction that leads to molecular reorganization of tight junction structure and composition, including occludin endocytosis. In disease states, this process can be triggered by pro-inflammatory cytokines including tumor necrosis factor-α (TNF), interleukin-1β (IL-1β), and several related molecules. Of these, TNF has been studied in the greatest detail and is known to activate long MLCK transcription, expression, enzymatic activity, and recruitment to the PAMR. Unfortunately, toxicities associated with inhibition of MLCK expression or enzymatic activity make these unsuitable as therapeutic targets. Recent work has, however, identified a small molecule that prevents MLCK1 recruitment to the PAMR without inhibiting enzymatic function. This small molecule, termed Divertin, restores barrier function after TNF-induced barrier loss and prevents disease progression in experimental chronic inflammatory bowel disease.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or "enteroids". Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2'-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

Key points Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+/calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. Abstract Ca2+/calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1SM+/+, MYPT1SM-/− and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

Leiomyosarcoma is a rare malignant smooth muscle tumor which can be very unpredictable. Myosin II is involved in many functions, including cell contraction, migration, and adhesion. The phosphorylation of myosin regulatory light chain (MLC) by myosin light chain kinase (MLCK) determines the activity of Myosin II. However, it is still unclear whether MLC phosphorylation is involved in cell proliferation in leiomyosarcoma. In this study, we aimed to explore the role of MLCK-dependent MLC phosphorylation in leiomyosarcoma development. We found that the expression of MLCK, phosphorylated MLC, and Ki67 in leiomyosarcoma was significantly higher than in leiomyoma and adjacent normal smooth muscle cells. MLCK expression was significantly correlated with phosphorylated MLC level. Kaplan-Meier survival analysis revealed that patients with high expression of MLCK or phosphorylated MLC had shorter overall survival times compared with the patients with low expression of MLCK or phosphorylated MLC. In vitro studies revealed a causative link between MLC phosphorylation and cellular proliferation as expression of phosphomimetic MLC (T19D, S20D) increased cellular proliferation as assessed by Ki67 staining. In contrast, MLCK specific inhibitor reduced cellular proliferation. We concluded that MLCK, phosphorylated MLC and Ki67 were overexpressed in leiomyosarcoma. MLCK dependent MLC phosphorylation might be responsible for the high proliferative state in leiomyosarcoma. MLCK and phosphorylated MLC are potential prognostic indicators of leiomyosarcoma.

Decreased autophagy is accompanied by the development of a myeloproliferative state or acute myeloid leukemia (AML). AML cells are often sensitive to autophagy‑inducing stimuli, prompting the idea that targeting autophagy can be useful in AML cytotoxic therapy. AML NB4 cells overexpressing microtubule-associated protein 1 light chain 3-green fluorescent protein were screened with 69 inhibitors to analyze autophagy activity. AML cells were treated with the polo-like kinase 1 (PLK1) inhibitors RO3280 and BI2536 before autophagy analysis. Cleaved LC3 (LC3-II) and the phosphorylation of mammalian target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase, and Unc-51-like kinase 1 during autophagy was detected with western blotting. Autophagosomes were detected using transmission electron microscopy. Several inhibitors had promising autophagy inducer effects: BI2536, MLN0905, SK1-I, SBE13 HCL and RO3280. Moreover, these inhibitors all targeted PLK1. Autophagy activity was increased in the NB4 cells treated with RO3280 and BI2536. Inhibition of PLK1 expression in NB4, K562 and HL-60 leukemia cells with RNA interference increased LC3-II and autophagy activity. The phosphorylation of mTOR was reduced significantly in NB4 cells treated with RO3280 and BI2536, and was also reduced significantly when PLK1 expression was downregulated in the NB4, K562 and HL-60 cells. We demonstrate that PLK1 inhibition induces AML cell autophagy and that it results in mTOR dephosphorylation. These results may provide new insights into the molecular mechanism of PLK1 in regulating autophagy.

Emotional information is critical for our social life, in which attentional bias is now a focus in the study on attention. However, the attentional bias processing mechanism of emotional faces still arouses huge controversy. Using similar experimental paradigms and stimuli, the published studies have yielded contradictory results. Some studies suggest that angry faces could automatically stimulate attention, that is, there is an anger superiority effect. On the contrary, lines of growing evidence support the existence of a happiness superiority effect, suggesting that the superiority effect is shown in happy faces rather than angry faces. In the present paper, the behavioral and neuroscience studies of anger and happiness superiority effects are combined. It is found that there are three major reasons for the debate over the two types of effects, which include the choice of stimulus materials, the difference of paradigm setting, and the different stages of emotional processing. By comparatively integrating the previous published results, we highlight that the future studies should further control the experimental materials and procedures, and investigate the processing mechanism of anger and happiness superiority effects by combining cognitive neurobiology means to resolve the disputes.

The regulation of intestinal epithelial permeability requires phosphorylation of myosin regulatory light chain (MLC). The phosphorylation status of MLC is regulated by myosin light chain phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (PP1cδ) toward MLC depends on its regulatory subunit (MYPT1). In this study, we revealed the presence of two MYPT1 isoforms, full length and variant 2 in human intestinal (Caco-2) epithelial cells and isolated intestinal epithelial cells (IECs) from mice. In confluent Caco-2 cells, MYPT1 was distributed at cell-cell contacts and colocalized with F-actin. These results suggest that MYPT1 isoforms are expressed in intestinal epithelial cells and MYPT1 may be involved in the regulation of intestinal epithelial barrier function.

Key points The extent of myosin regulatory light chain phosphorylation (RLC) necessary for smooth muscle contraction depends on the respective activities of Ca(2+) /calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP), which contains a regulatory subunit MYPT1 bound to the phosphatase catalytic subunit and myosin. MYPT1 showed significant constitutive T696 and T853 phosphorylation, which is predicted to inhibit MLCP activity in isolated ileal smooth muscle tissues, with additional phosphorylation upon pharmacological treatment with the muscarinic agonist carbachol. Electrical field stimulation (EFS), which releases ACh from nerves, increased force and RLC phosphorylation but not MYPT1 T696 or T853 phosphorylation. The conditional knockout of MYPT1 or the knockin mutation T853A in mice had no effect on the frequency-maximal force responses to EFS in isolated ileal tissues. Physiological RLC phosphorylation and force development in ileal smooth muscle depend on myosin light chain kinase and MLCP activities without changes in constitutive MYPT1 phosphorylation. Abstract Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+) /calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in ileal smooth muscle in adult mice with (1) smooth muscle-specific deletion of MYPT1; (2) non-phosphorylatable MYPT1 containing a T853A knockin mutation; and (3) measurements of force and protein phosphorylation responses to cholinergic neurostimulation initiated by electric field stimulation. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted and relaxed rapidly with moderate differences in sustained responses to KCl and carbachol treatments and washouts, respectively. Similarly, measurements of regulatory proteins responsible for RLC phosphorylation during contractions also revealed moderate changes. There were no differences in contractile or RLC phosphorylation responses to carbachol between tissues from normal mice vs. MYPT1 T853A knockin mice. Quantitatively, there was substantial MYPT1 T696 and T853 phosphorylation in wild-type tissues under resting conditions, predicting a high extent of MLCP phosphatase inhibition. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Electric field stimulation increased RLC phosphorylation and force development in tissues from wild-type mice without an increase in MYPT1 phosphorylation. Thus, physiological RLC phosphorylation and force development in ileal smooth muscle appear to be dependent on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.

Key points Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. Abstract Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.

Background Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Methods Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Results Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. Conclusions We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence. Electronic supplementary material The online version of this article (doi:10.1186/s12935-017-0405-y) contains supplementary material, which is available to authorized users.

Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.

Although many scholars have utilized high-throughput microarrays to delineate gene expression patterns after spinal cord injury (SCI), no study has evaluated gene changes in raphe magnus (RM) and somatomotor cortex (SMTC), two areas in brain primarily affected by SCI. In present study, we aimed to analyze the differentially expressed genes (DEGs) of RM and SMTC between SCI model and sham injured control at 4, 24 h, 7, 14, 28 days, and 3 months using microarray dataset GSE2270 downloaded from gene expression omnibus and unpaired significance analysis of microarray method. Protein-protein interaction (PPI) network was constructed for DEGs at crucial time points and significant biological functions were enriched using DAVID. The results indicated that more DEGs were identified at 14 days in RM and at 4 h/3 months in SMTC after SCI. In the PPI network for DEGs at 14 days in RM, interleukin 6, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBJ murine osteosarcoma viral oncogene homolog (FOS), tumor necrosis factor, and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) were the top 5 hub genes; In the PPI network for DEGs at 3 months in SMTC, the top 5 hub genes were ubiquitin B, Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), FOS, Janus kinase 2 and vascular endothelial growth factor A. Hedgehog and Wnt signaling pathways were the top 2 significant pathways in RM. These hub DEGs and pathways may be underlying therapeutic targets for SCI.

Sphingosine kinase 1 (SphK1) is over-expressed in many cancers and therefore serves as a biomarker for cancer prognosis. SKI-5C is a new SphK1 inhibitor, and until now its molecular function in Wilms’ tumor cells remained unknown. Here, using CCK-8 and nude mice experiments we assessed cell growth in Wilms’ tumor cell lines (SK-NEP-1 and G401) in vitro and in vivo. We demonstrated that SphK1 is highly expressed in SK-NEP-1 and G401 cells, and through annexin V/propidium iodide staining and flow cytometry analysis, we detected cell apoptosis. Treatment with SKI-5C inhibited proliferation and induced apoptosis of SK-NEP-1 and G401 cells in a dose-dependent manner. Moreover, SKI-5C treatment inhibited the growth of SK-NEP-1 xenograft tumors in nude mice, with few side effects. Our microarray analysis revealed that SKI-5C-treated SK-NEP-1 cells mostly downregulated PRKACA and significantly inhibited phosphorylation of ERK1/2 and NF-κB p65. These results imply that SKI-5C induces apoptosis of SK-NEP-1 cells through the PRKACA/MAPK/NF-κB pathway. While, further research is required to determine the underlying details, these results provide new clues for the molecular mechanism of cell death induced by SKI-5C and suggest that SKI-5C may act as new candidate drug for Wilms’ tumor.

Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca2+ channels (VDCCs) or TMEM16A Ca2+-activated Cl− channels significantly changes global cytosolic Ca2+ concentration ([Ca2+]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca2+]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca2+]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence., The molecular basis of the basal tone generated by internal anal sphincters (IAS) is largely unknown. Here, the authors show that the tone arises from a global rise in intracellular Ca2+ in smooth muscle cells via a Ryanodine receptor-TMEM16A-L-type Ca2+ channel-MLC kinase pathway, suggesting a potential therapy for IAS motility disorders.

There is a current view that myosin light chain kinase (MLCK) plays a critical role in endothelial permeability. To investigate the functions of MLCK in endothelial cells in vivo, we generated a mouse model in which MLCK was selectively deleted by crossing Mylk1 floxed mice with Tie2/cre transgenic mice. Knocking out Mylk1 from endothelial cells had no effect on the global phenotype of the mice, including body weight and blood pressure. Lipopolysaccharide (LPS)-mediated septic death was also not altered in the knockout (KO) mice. Consistently, LPS-induced inflammatory injury and the increase in microvascular permeability in the main organs, including the lung and the kidney, was not significantly attenuated in KO mice as compared with wild-type mice. However, the LPS-induced microvascular hyperpermeability of the esophagus and the eyeballs was attenuated in KO mice. We also found that the LPS-mediated increase in the number of caveolae in the endothelial cells of the esophagus was significantly reduced in KO mice. Our results do not support a role for endothelial cell MLCK in the pathogenesis of inflammatory diseases.

Event-related potentials were used to investigate the neural correlates of two-digit exact and approximate addition calculations. The results showed that the dissociation between exact and approximate calculations began at approximately 150 ms after stimulus. In the left hemisphere, N170 amplitude elicited by exact calculation was larger than that elicited by approximate calculation; however, in the right hemisphere, N170 amplitudes elicited by the two calculation strategies were not significantly different. Moreover, a larger P3 amplitude was elicited by exact calculation than by approximate calculation. In the 130-210 ms windows, dipole source analysis of the difference wave (exact calculation minus approximate calculation) indicated that the generator of N170 was localized in the left fusiform gyrus.

Current approaches to generate lentiviral vectors, which have been used extensively for gene therapy, are time consuming and require a large expenditure. Here, we directly clone the full length myosin light chain kinase cDNA into enhanced green fluorescence protein (EGFP)-fused pLenti6/V5 expression vector in just one step with the use of Red-mediated recombination system, allowing for rapid and effective cloning of lentiviral expression vectors. In addition, the simultaneous expression of EGFP reporter provides a convenient monitoring mean for host cell infection and for localization of the target proteins.

Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.

Objective To analyse the temporal and spatial characteristics of traumatic brain injury and the distribution of combined injuries in the Wenchuan earthquake, and describe the treatment opportunities and preferences for therapy. Methods The diagnosis and treatment of 92 patients with traumatic brain injury who survived the massive earthquake (magnitude 8) in Wenchuan, Sichuan Province, on 12 May 2008 were systematically analysed. Results The patients all came from the plains northwest of Chengdu city. Seventy-six patients were admitted during the early stage (within 12 h) after the earthquake. Ten patients underwent surgery and three patients died. Conclusion Patients with traumatic brain injury during the early period accounted for a large proportion of the patients wounded in the Wenchuan earthquake, and their conditions changed quickly. The patients all came from the plain area which has convenient transportation. After admission, providing first-aid early had a significant effect on increasing the success of treatment for these patients.

The functions of long smooth muscle myosin light chain kinase (L-MLCK), a molecule with multiple domains, are poorly understood. To examine the existence of further potentially functional domains in this molecule, we analyzed its amino acid sequence with a tango program and found a putative aggregation domain located at the 4Ig domain of the N-terminal extension. To verify its aggregation capability in vitro, expressible truncated L-MLCK variants driven by a cytomegalovirus promoter were transfected into cells. As anticipated, only the overexpression of the 4Ig fragment led to particle formation in Colon26 cells. These particles contained 4Ig polymers and actin. Analysis with detergents demonstrated that the particles shared features in common with aggregates. Thus, we conclude that the 4Ig domain has a potent aggregation ability. To further examine this aggregation domain in vivo, eight transgenic mouse lines expressing the 4Ig domain (4Ig lines) were generated. The results showed that the transgenic mice had typical aggregation in the thigh and diaphragm muscles. Histological examination showed that 7.70 ± 1.86% of extensor digitorum longus myofibrils displayed aggregates with a 36.44% reduction in myofibril diameter, whereas 65.13 ± 3.42% of diaphragm myofibrils displayed aggregates and the myofibril diameter was reduced by 43.08%. Electron microscopy examination suggested that the aggregates were deposited at the mitochondria, resulting in structural impairment. As a consequence, the oxygen consumption of mitochondria in the affected muscles was also reduced. Macrophenotypic analysis showed the presence of muscular degeneration characterized by a reduction in force development, faster fatigue, decreased myofibril diameters, and structural alterations. In summary, our study revealed the existence of a novel aggregation domain in L-MLCK and provided a direct link between L-MLCK and aggregation. The possible significance and mechanism underlying the aggregation-based pathological processes mediated by L-MLCK are also discussed.

Diarrhea is a host response to enteric pathogens, but its impact on pathogenesis remains poorly defined. By infecting mice with the attaching and effacing bacteria Citrobacter rodentium, we defined the mechanisms and contributions of diarrhea and intestinal barrier loss to host defense. Increased permeability occurred within 2 days of infection and coincided with IL-22-dependent upregulation of the epithelial tight junction protein claudin-2. Permeability increases were limited to small molecules, as expected for the paracellular water and Na+ channel formed by claudin-2. Relative to wild-type, claudin-2-deficient mice experienced severe disease, including increased mucosal colonization by C. rodentium, prolonged pathogen shedding, exaggerated cytokine responses, and greater tissue injury. Conversely, transgenic claudin-2 overexpression reduced disease severity. Chemically induced osmotic diarrhea reduced colitis severity and C. rodentium burden in claudin-2-deficient, but not transgenic, mice, demonstrating that claudin-2-mediated protection is the result of enhanced water efflux. Thus, IL-22-induced claudin-2 upregulation drives diarrhea and pathogen clearance.

Objective To explore the operative method and therapeutic efficacy of surgical resection of large invasive pituitary adenomas with individualized approach under neuronavigator guidance.Methods Seventeen patients(10 males and 7 females,aged from 22 to 78 years with a mean of 39.2±9.2 years) suffering from large invasive pituitary adenoma of higher than Hardy IV grade hospitalized from 2004 to 2009 were involved in the present study.All procedures were performed with the assistance of neuronavigator via individualized pterion approach,subfrontal extradural approach,trans-sphenoidal approach,or combined approach.The dispersedly invasive pituitary adenomas were resected under the guidance of neuronavigator by fully utilizing the natural anatomical cleavages.All the patients received follow-up CT scanning 3 days after operation,MRI scanning 1 to 3 months after operation,and clinical follow-up ranged from 6 to 72 months.The resection extent and outcome were assessed by imaging examination and clinical results.Results Total tumor removal was achieved in 15 cases,subtotal removal in 1 case,and extensive partial removal in 1 case.The visual impairment and headache were ameliorated in most cases,but in 1 patient they were worsened.Transient diabetes insipidus occurred in 8 cases,electrolyte disturbances were observed in 2 cases,leakage of cerebrospinal fluid appeared in 2 cases,hyposmia in 2 cases,visual impairment aggravated in 1 case,oculomotor nerve and abducens nerve paralysis on the operative side in 1 case,epidural hematoma in occipital and parietal regions in 1 case.No patient died during the follow-up period.Conclusions Individualized surgical approach designed according to the growth direction of tumor under neuronavigator guidance is helpful for the operators to identify the vessels and nerves in the operative field distinctly during the operation,thus the total removal rate is improved,safely of the operation to remove large invasive pituitary adenomas is secured,and disability rate due to surgery is decreased.

Objective To explore the microsurgical technique of medial sphenoid ridge meningioma resectional therapy.Methods The clinical data were retrospectively analyzed of 29 patients(13 males and 16 females;aged 18-68 years with average of 42 years;duration of disease was 5 months to 8 years,averaged 28 months) with medial sphenoidal ridge meningioma and admitted from Jan.2005 to Jan.2010.The anatomical relationship of the tumor to surrounding structures was assessed intraoperatively,the tumor was then completely resected through cutting off the tumor supplying vessels,shrinking the tumor volume and separating the tumors from adjacent vessels and nerves.All the patients were followed up for 4 months to 4 years.Results Of the 29 cases,20 got total tumor removal,7 got subtotal and 2 got partial tumor removal.Of the 20 patients with obviously preoperative visual impairment,12 were obviously relieved,6 showed no improvement and 2 got symptoms aggravation.Hemiplegia occurred in 2 cases and oculomoter nerve palsy in 6 cases.There was no death after surgery.A 6 months to 4 years follow-up showed that no recurrence was found in 27 patients with tumor resection level of Simpson I and II,2 patients with tumor resection level of Simpson III received postoperative radiotherapy or gamma knife surgery,and 1 recurred and received reoperation.Conclusions Fine intraoperative assessment of the anatomical relationship of the tumor to surrounding structures,separating and excising tumor according to the assessed result is the key of medial sphenoid ridge meningioma resection,and the tumor resection is favorable to visual rehabilitation and tumor control.

The inefficiency in detection of early-stage impairment of nervous system,if ever,and the lack of measures to quantify the impairment have raised challenges to an early and effective treatment forcritically ill patients,and the establishment of neurosurgical intensive care unit(NSICU) has become necessary.The agreement has been reached on establishment of a NSICU with specific equipments for monitoring the function of nervous system,and on development of special therapeutic techniques.Besides the routine equipments,those facilities capable of monitoring the regional oxygen saturation(rSO2),metabolism and microenvironment of brain tissue,intracranial pressure,jugular bulb oxygen saturation(SjvO2),blood flow and evoked potential are necessary,and personnel who are specifically trained in neurological sciences should be recruited.Only timely discovery of potential nervous lesion will make it possible to accurately monitor for an efficient intervention for the lesions,thereby complications and mortality could be reduced.

Objective To evaluate the therapeutic effect of small craniotomic posterior fossa decompression combined with occipital-cervical bone graft fusion and internal fixation on Chiari malformation complicated with basilar impression.Methods The clinical data of 16 cases(7 males and 9 females,aged 17 to 65 years,mean 36.4) of Chiari malformation complicated with basilar impression from 2006 to 2010 were retrospectively analyzed.The diagnoses for all the patients were confirmed by radiology.Small craniotomic posterior fossa decompression was performed in all patients,cerebellar tonsils were resected,and then one-stage occipital-cervical bone graft fusion using autogenous iliac bone and internal wiring fixation were performed.Neck support was used for 3 months after surgery.Results Symptoms were significantly improved in all cases after surgical operation.No patient died or infected.Cerebrospinal fluid leakage was found at draining site in one case.Transient pain of scapular and chest was found in one case and disappeared spontaneously.A 6-months follow-up showed that 6 patients were cured,9 improved and 1 unchanged according to Symon and Lavender standard.Postoperative MRI showed the reconstructed cisterna magna was clear in all patients,no cerebellar ptosis was found,and the occipital-cervical graft bone was fused.Conclusion In patients with Chiari malformation complicated with basilar impression,small craniotomic posterior fossa decompression combined with one-stage occipital-cervical bone graft fusion and internal wiring fixation has a clear and definite effect,it can increase the volume of posterior fossa and alleviate the ventral brain stem compression simultaneously,and reconstruct the stability of cranio-cervical junction.

The objective of this study is to observe the adult growth hormone level in postoperative pituitary tumor patients of multi-centers, and explore the change of hypophyseal hormones in postoperative pituitary tumor patients. Sixty patients with pituitary tumor admitted during March, 2011–March, 2012 were selected. Postoperative hypophyseal hormone deficiency and the change of preoperative, intraoperative, and postoperative growth hormone levels were recorded. Growth hormone hypofunction was the most common hormonal hypofunction, which took up to 85.0 %. Adrenocortical hormone hypofunction was next to it and accounted for 58.33 %. GH + ACTH + TSH + Gn deficiency was the most common in postoperative hormone deficiency, which took up to 40.00 %, and GH + ACTH + TSH + Gn + AVP and GH deficiencies were next to it and accounted for 23.33 and 16.67 %, respectively. The hormone levels in patients after total pituitary tumor resection were significantly lower than those after partial pituitary tumor resection, and the difference was statistically significant; growth hormone and serum prolactin levels after surgery in two groups were decreased, and the difference was statistically significant. The incidence rate of growth hormone deficiency in postoperative pituitary tumor patients is high, which is usually complicated with deficiency of various hypophyseal hormones. In clinical, we should pay attention to the levels of the hypopnyseal hormones, and take timely measures to avoid postoperative complications.

Stroke, either ischemic or hemorrhagic, is the leading cause of death and morbidity worldwide. Identifying the risk factors is a prerequisite step for stroke prevention and treatment. It is believed that a major portion of the currently unidentified risk factors is of genetic origin. Consistent with this idea, numerous potential risk alleles for stroke have been reported, however, the genetic evidence so far is not conclusive. The major goal of this review is to update the current knowledge about the genetic predisposition to the common multifactorial stroke, and to provide a bird’s-eye view of this fast moving field. We selectively review and meta-analyze the related English literatures in public domain (PubMed) from 2000 onward, including the original reports and meta-analyses, to evaluate the genetic risk factors of common multifactorial stroke. The results indicated that we reviewed and meta-analyzed original reports and existing meta-analyses that studied the genetic predisposition to the common multifactorial stroke. Some original reports and meta-analyses were specific for ischemic stroke and others were for hemorrhagic stroke only. We also evaluated the major evolving issues in this field and discussed the future directions. In conclusion, strong evidences suggest that genetic risk factors contribute to common multifactorial stroke, and many genetic risk genes have been implicated in the literatures. However, not a single risk allele has been conclusively approved.

Three-dimensional cell culture techniques can better reflect the in vivo characteristics of tumor cells compared with traditional monolayer cultures. Compared with their 2D counterparts, 3D-cultured tumor cells showed enhanced resistance to the cytotoxic T cell-mediated immune response. However, it remains unclear whether 3D-cultured tumor cells have an enhanced resistance to NK cell cytotoxicity. In this study, a total of 363 differentially expressed proteins were identified between the 2D- and 3D-cultured U251 cells by comparative proteomics, and an immune-associated protein-protein interaction (PPI) network based on these differential proteins was constructed by bioinformatics. Within the network, HLA-E, as a molecule for inhibiting NK cell activation, was significantly up-regulated in the 3D-cultured tumor cells. Then, we found that the 3D-cultured U251 cells exhibited potent resistance to NK cell cytotoxicity in vitro and were prone to tumor formation in vivo. The resistance of the 3D-cultured tumor cells to NK cell lysis was mediated by the HLA-E/NKG2A interaction because the administration of antibodies that block either HLA-E or NKG2A completely eliminated this resistance and significantly decreased tumor formation. Taken together, our findings indicate that HLA-E up-regulation in 3D-cultured cells may result in enhanced tumor resistance to NK cell-mediated immune response.

Although many scholars have utilized high-throughput microarrays to delineate gene expression patterns after spinal cord injury (SCI), no study has evaluated gene changes in raphe magnus (RM) and somatomotor cortex (SMTC), two areas in brain primarily affected by SCI. In present study, we aimed to analyze the differentially expressed genes (DEGs) of RM and SMTC between SCI model and sham injured control at 4, 24 h, 7, 14, 28 days, and 3 months using microarray dataset GSE2270 downloaded from gene expression omnibus and unpaired significance analysis of microarray method. Protein-protein interaction (PPI) network was constructed for DEGs at crucial time points and significant biological functions were enriched using DAVID. The results indicated that more DEGs were identified at 14 days in RM and at 4 h/3 months in SMTC after SCI. In the PPI network for DEGs at 14 days in RM, interleukin 6, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBJ murine osteosarcoma viral oncogene homolog (FOS), tumor necrosis factor, and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) were the top 5 hub genes; In the PPI network for DEGs at 3 months in SMTC, the top 5 hub genes were ubiquitin B, Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), FOS, Janus kinase 2 and vascular endothelial growth factor A. Hedgehog and Wnt signaling pathways were the top 2 significant pathways in RM. These hub DEGs and pathways may be underlying therapeutic targets for SCI.

Metastasis associated in colon cancer 1 (MACC1) has been regarded as a novel potential therapeutic target for multiple cancers. However, the impact of MACC1 in glioma remains unclear. The aim of this study was to analyze the correlation of MACC1 expression with the clinicopathological features of glioma. MACC1 mRNA and protein expression levels in human glioma tissues were detected by quantitative real-time polymerase chain reaction and immunohistochemistry assays, respectively. MACC1 mRNA and protein expression were both significantly higher in glioma tissues than in corresponding noncancerous brain tissues (both P

Background: Malignant gliomas are the leading cause of morbidity and mortality in brain and central nervous system tumors. Recently, casticin has drawn wide attention to its critical role in tumor progression. However, the effect of casticin on glioma remains undefined. Methods: Following treatment with casticin, cell viability, apoptosis, and cell cycle arrest were examined in U251 glioma cells. Additionally, the involved molecular mechanism was assessed by western blotting and flow cytometry. Results: Casticin triggered an obvious dose-dependent decrease in U251, U87 and U373 glioma cell viability, and the growth inhibitory effect of casticin was correlated with cell cycle arrest and cell apoptosis. Further mechanistic analysis indicated that casticin induced G2/M phase arrest by attenuating the polymerization of tubulin. Furthermore, striking apoptosis was also confirmed, accompanied by the up-regulation of caspase-3, p53 and proapoptotic protein Bax. These effects were absent when the caspase inhibitor z-VAD-fmk or p53 inhibitor PFTα were applied, suggesting that casticin could trigger cell apoptosis in a caspase-3 and p53-dependent manner. Conclusion: These findings provide a prominent insight into how casticin abrogates the pathogenesis of glioma, and support its potential clinical prospect for further development of anti-brain cancer therapy.

Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca(2+)/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca(2+) sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.