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Anastomotic leakage (AL) is a common postoperative complication of rectal cancer, and transanal drainage tube (TDT) efficacy is still contentious. This study aimed to evaluate the TDT effect on AL prevention.All relevant papers were searched by using a predefined search strategy (two randomized controlled trials (RCTs), one prospective study, and four retrospective studies). Meta-analysis was conducted to estimate AL and re-operation pooled rates.A total of 7 studies (1556 patients) were included: No significant statistic difference was found between two groups on AL rate (odds ratio (OR) 0.61, P = 0.11) and re-operation rate (OR 0.52, P = 0.10). For subgroup analysis, significant statistic difference was found between two groups on AL rate (OR 0.29, P = 0.002) and re-operation rate (OR 0.15, P = 0.04) in patients without neoadjuvant therapy. As for patients without diverting stoma, the AL rate (OR 0.35, P = 0.002) was significantly lower than that in patients without TDT.TDT may reduce AL morbidity and re-operation rate for patients without high risk of AL, but may be useless for those in high-risk situations.

The forkhead box transcription factor genesFoxc1andFoxc2are expressed in the condensing mesenchyme of the developing skeleton prior to the onset of chondrocyte differentiation. To determine the roles of these transcription factors in limb development we deleted bothFoxc1andFoxc2in lateral plate mesoderm using the Prx1-cre mouse line. Resulting compound homozygous mice died shortly after birth with exencephaly, and malformations to this sternum and limb skeleton. Notably distal limb structures were preferentially affected, with the autopods displaying reduced or absent mineralization. The radius and tibia bowed and the ulna and fibula were reduced to an unmineralized rudimentary structure. Molecular analysis revealed reduced expression of Ihh leading to reduced proliferation and delayed chondrocyte hypertrophy at E14.5. At later ages, Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δembryos exhibited restored Ihh expression and an expanded COLX-positive hypertrophic chondrocyte region, indicating a delayed exit and impaired remodeling of the hypertrophic chondrocytes. Osteoblast differentiation and mineralization were disrupted at the osteochondral junction and in the primary ossification center (POC). Levels of OSTEOPONTIN were elevated in the POC of compound homozygous mutants, while expression of Phex was reduced, indicating that impaired OPN processing by PHEX may underlie the mineralization defect we observe. Together our findings suggest that Foxc1 and Foxc2 act at different stages of endochondral ossification. Initially these genes act during the onset of chondrogenesis leading to the formation of hypertrophic chondrocytes. At later stages Foxc1 and Foxc2 are required for remodeling of HC and for Phex expression required for mineralization of the POC.

A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence. Its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca(2+) signals (i.e., synchronized Ca(2+) oscillations (SCaOs) and asynchronized Ca(2+) oscillations (ACaOs)) that last for hours. RyRs, TMEM16A, L-type Ca(2+) channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca(2+) signals, leading to IAS’s full relaxation. Our results show that the oscillating nature of Ca(2+) signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.

The paracellular gap formed by endothelial cell (EC) contraction is fundamental for endothelium permeability, but the mechanism underlying EC contraction has yet to be determined. Here, we identified the zipper-interacting protein kinase (ZIPK) as the kinase for EC contraction and myosin light chain (MLC) phosphorylation. Inhibition of ZIPK activity by pharmacological inhibitors and small interfering RNAs led to a significant decrease in the mono- and diphosphorylation of MLCs along with a contractile response to thrombin, suggesting an essential role of ZIPK in EC paracellular permeability. To assess the role of ZIPK in vivo, we established mouse lines with conditional deletion of Zipk gene. The endothelium-specific deletion of Zipk led to embryonic lethality, whereas the UBC-Cre(ERT2)–mediated deletion of Zipk by tamoxifen induction at adulthood caused no apparent phenotype. The induced deletion of Zipk significantly inhibited ischemia-reperfusion–induced blood-brain barrier dysfunction and neuronal injuries from middle cerebral artery occlusion and reperfusion, as evidenced by reduced infarct and edema volume, attenuated Evans blue dye leakage, and improved neuronal behavior. We thus concluded that ZIPK and its phosphorylation of MLC were required for EC contraction and ischemic neuronal injuries. ZIPK may be a prospective therapeutic target for stroke.—Zhang, Y., Zhang, C., Zhang, H., Zeng, W., Li, S., Chen, C., Song, X., Sun, J., Sun, Z., Cui, C., Cao, X., Zheng, L., Wang, P., Zhao, W., Zhang, Z., Xu, Y., Zhu, M., Chen, H. ZIPK mediates endothelial cell contraction through myosin light chain phosphorylation and is required for ischemic-reperfusion injury.

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by thePrg4gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-β and EGFR signaling to inducePrg4expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-β and EGFR signals to inducePrg4expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to twoPrg4promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction ofPrg4by TGF-β. Our results indicate that Creb5 is a critical regulator ofPrg4/lubricin expression in the articular cartilage.

While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium.

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-β and EGFR signaling to induce Prg4 expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-β and EGFR signals to induce Prg4 expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction of Prg4 by TGF-β. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage., Zhang et al. identify Creb5 as a transcription factor that is required for induction of Prg4 expression by TGFβ and EGFR ligands. They show that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, working together with a more distal regulatory element to drive induction of Prg4 by TGFβ. These findings provide new insight into the molecular regulation of Prg4 expression in superficial zone articular chondrocytes.

Bitter taste receptors (TAS2Rs or T2Rs) belong to the superfamily of seven-transmembrane G protein–coupled receptors, which are the targets of >50% of drugs currently on the market. Canonically, T2Rs are located in taste buds of the tongue, where they initiate bitter taste perception. However, accumulating evidence indicates that T2Rs are widely expressed throughout the body and mediate diverse nontasting roles through various specialized mechanisms. It has also become apparent that T2Rs and their polymorphisms are associated with human disorders. In this review, we summarize the physiological and pathophysiological roles that extraoral T2Rs play in processes as diverse as innate immunity and reproduction, and the major challenges in this emerging field.

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.

Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness.We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness.We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the CaInflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene DA G protein-coupled receptor-TMEM16A-voltage-dependent Ca

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.

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.

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.

Background Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness. Objective We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness. Methods We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca 2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model. Results Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D 4 , are capable of inducing Ca 2+ -activated Cl − currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor–TMEM16A–voltage-dependent Ca 2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity. Conclusions A G protein-coupled receptor–TMEM16A–voltage-dependent Ca 2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.

A recent study from Naqvi et al1 in Cell shows that in mice during preadolescence, cardiomyocytes undergo a burst of proliferation that is related to a thyroid hormone surge. This study challenges the conventional model of the timing of terminal differentiation and provides a direction for further mechanistic research on cardiomyocyte proliferation and differentiation. In mice and rats, cardiomyocytes transition from the mono- to the binucleated phenotype in the first week of life.2,3 It is commonly thought that this indicates the transition from the proliferative state to permanent cell cycle exit. However, the precise molecular-genetic mechanisms of terminal differentiation are unknown. Reversing this process and stimulating the proliferation of terminally differentiated cardiomyocytes is a century-old challenge and considered by many scientists as a Holy Grail. It has been thought that heart growth after birth is exclusively through enlargement (hypertrophy) but not proliferation of cardiomyocytes.4 However, several lines of evidence challenge the absoluteness of the notion that new cardiomyocytes are not generated after birth. First, Poss et al5 found that adult zebrafish can completely regenerate heart defects by increasing cardiomyocyte proliferation, indicating that differentiated adult cardiomyocyte phenotypes exist that can re-enter the cell cycle. Second, we have demonstrated that human hearts show cardiomyocyte proliferation until the second decade of life.6 Third, it was demonstrated that some adult mammalian cardiomyocytes can be stimulated to re-enter the cell cycle.7–11 Fourth, we have demonstrated that neuregulin-stimulated cell cycle re-entry happens predominantly in the mononucleated portion.10 The causative connection between endogenous cardiomyocyte proliferation and myocardial regeneration was established in zebrafish5 and neonatal mice,12 although part of the latter has been challenged recently.13 Several reports indicate that stimulating cardiomyocyte proliferation in adult mammals improves myocardial repair.10,11 Thus, controlling postnatal …

To explore the risk factors for pulmonary metastasis after curative resection of colorectal cancer in order to improve the effectiveness of follow-up and the rate of early diagnosis for the high-risk patients.The clinicopathological and follow-up data of 268 patients with colorectal cancer undergoing radical resection from January 2004 to December 2006 in the Beijing Cancer Hospital were analyzed retrospectively. Patients were divided into study group including 16(6.0%) patients who developed lung metastasis and control group without lung metastasis. The high-risk variables associated with lung metastasis were reviewed by univariate analysis and multivariate analysis.Lung metastasis developed in 16 patients, including 10 cases with unilateral lung metastasis and 6 with bilateral. The median duration from colorectal surgery to identification of lung metastasis was 13.9 months. The diagnosis rate of pulmonary metastasis by enhanced chest CT was 81.3%(13/16). Univariate analysis identified the following associated with significant factors associated with pulmonary metastasis: primary tumor location(P=0.003), adjuvant chemotherapy(P=0.034), TNM stage(P=0.005) and preoperative serum carcinoembryonic antigen(CEA) level (P=0.001). Multivariate analysis revealed that primary tumor location(rectum) and preoperative serum CEA level(≥5 μg/L) were independent risk factors for pulmonary metastasis(both P0.05).Primary tumor location and elevated preoperative CEA level are independent risk factors for pulmonary metastasis. Strict postoperative follow-up and routine chest enhanced CT examination is necessary for this particular patient population.

To study the safety and feasibility of transorally inserted anvil (OrVil(TM)) in laparoscopic-assisted radical resection for Siewert type II adenocarcinoma of the esophagogastric junction (AEG).Clinical data (operative time, rate of thoracotomy, residual cancer in the proximal margin, and postoperative recovery) of 72 patients suffered from Siewert type II AEG were analyzed retrospectively, including 46 cases of applying OrVil(TM) in digestive tract reconstruction for laparoscopic-assisted radical resection and 26 cases of applying pouch clamp embedding anvil, between May 2009 and August 2012 in Department of Minimally Invasive Gastrointestinal Surgery at the Peking University Cancer Hospital and Institute.The length between proximal margin and superior border of tumor was (2.5±1.5) cm in OrVil(TM) group, significantly longer than that in the traditional group [(1.6±1.1) cm, P0.01]. Moreover, the intraoperative frozen pathological positive incidence of cancer remnant was 2.2% (1/46), and rate of thoracotomy was 0, both of which were significantly lower as compared to the traditional group [23.1% (6/26) and 15.4% (4/26) respectively, both P0.01]. However, intraoperative blood loss and postoperative complications did not differ between the two groups (both P0.05).As for laparoscopic-assisted Siewert type II AEG radical resection, application of OrVil(TM) in digestive tract reconstruction is a safe surgical procedure, and can effectively reduce the rate of intra-operative thoracotomy, which is beneficial to postoperative recovery.

Bitter tastants can activate bitter taste receptors on constricted smooth muscle cells to inhibit L-type calcium channels and induce bronchodilation., Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca2+ signals, as revealed in cultured ASM cells, to activate large-conductance Ca2+-activated K+ channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca2+ events nor alter spontaneous local Ca2+ transients. Interestingly, they increase global intracellular [Ca2+]i, although to a much lower level than bronchoconstrictors. We show that these Ca2+ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca2+]i induced by bronchoconstrictors, and this lowering of the [Ca2+]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca2+ channels (VDCCs), resulting in reversal in [Ca2+]i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca2+ signaling pathways via Gβγ to increase [Ca2+]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca2+]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants., Author Summary Bitter taste receptors (TAS2Rs), a G-protein-coupled receptor family long thought to be solely expressed in taste buds on the tongue, have recently been detected in airways. Bitter substances can activate TAS2Rs in airway smooth muscle to cause greater bronchodilation than β2 adrenergic receptor agonists, the most commonly used bronchodilators. However, the mechanisms underlying this bronchodilation remain elusive. Here we show that, in resting primary airway smooth muscle cells, bitter tastants activate a TAS2R-dependent signaling pathway that results in an increase in intracellular calcium levels, albeit to a level much lower than that produced by bronchoconstrictors. In bronchoconstricted cells, however, bitter tastants reverse the bronchoconstrictor-induced increase in calcium levels, which leads to the relaxation of smooth muscle cells. We find that this reversal is due to inhibition of L-type calcium channels. Our results suggest that under normal conditions, bitter tastants can activate TAS2Rs to modestly increase calcium levels, but that when smooth muscle cells are constricted, they can block L-type calcium channels to induce bronchodilation. We postulate that this novel mechanism could operate in other extraoral cells expressing TAS2Rs.

Rationale: Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca2+-activated Cl− [Cl(Ca)] channels are inferred to be involved in AHR, yet their molecular nature and the cell type they act within to mediate this response remain unknown. Objectives: Transmembrane protein 16A (TMEM16A) and TMEM16B are Cl(Ca) channels, and activation of Cl(Ca) channels in airway smooth muscle (ASM) contributes to agonist-induced airway contraction. We hypothesized that Tmem16a and/or Tmem16b encode Cl(Ca) channels in ASM and mediate AHR. Methods: We assessed the expression of the TMEM16 family, and the effects of niflumic acid and benzbromarone on AHR and airway contraction, in an ovalbumin-sensitized mouse model of chronic asthma. We also cloned TMEM16A from ASM and examined the Cl− currents it produced in HEK293 cells. We further studied the impacts of TMEM16A deletion on Ca2+ agonist–induced cell shortening, and on Cl(Ca) currents activated by Ca2+ sparks (localized, short-lived Ca2+ transients due to the opening of ryanodine receptors) in mouse ASM cells. Measurements and Main Results: TMEM16A, but not TMEM16B, is expressed in ASM cells and its expression in these cells is up-regulated in ovalbumin-sensitized mice. Niflumic acid and benzbromarone prevent AHR and contraction evoked by methacholine in ovalbumin-sensitized mice. TMEM16A produces Cl(Ca) currents with kinetics similar to native Cl(Ca) currents. TMEM16A deletion renders Ca2+ sparks unable to activate Cl(Ca) currents, and weakens caffeine- and methacholine-induced cell shortening. Conclusions: Tmem16a encodes Cl(Ca) channels in ASM and contributes to Ca2+ agonist–induced contraction. In addition, up-regulation of TMEM16A and its augmented activation contribute to AHR in an ovalbumin-sensitized mouse model of chronic asthma. TMEM16A may represent a potential therapeutic target for asthma.

Background & Aims The regulatory subunit of myosin light chain phosphatase, MYPT1, has been proposed to control smooth muscle contractility by regulating phosphorylation of the Ca 2+ -dependent myosin regulatory light chain. We generated mice with a smooth muscle–specific deletion of MYPT1 to investigate its physiologic role in intestinal smooth muscle contraction. Methods We used the Cre -loxP system to establish Mypt1 -floxed mice, with the promoter region and exon 1 of Mypt1 flanked by 2 loxP sites. These mice were crossed with SMA-Cre transgenic mice to generate mice with smooth muscle–specific deletion of MYPT1 ( Mypt1 SMKO mice). The phenotype was assessed by histologic, biochemical, molecular, and physiologic analyses. Results Young adult Mypt1 SMKO mice had normal intestinal motility in vivo, with no histologic abnormalities. On stimulation with KCl or acetylcholine, intestinal smooth muscles isolated from Mypt1 SMKO mice produced robust and increased sustained force due to increased phosphorylation of the myosin regulatory light chain compared with muscle from control mice. Additional analyses of contractile properties showed reduced rates of force development and relaxation, and decreased shortening velocity, compared with muscle from control mice. Permeable smooth muscle fibers from Mypt1 SMKO mice had increased sensitivity and contraction in response to Ca 2+ . Conclusions MYPT1 is not essential for smooth muscle function in mice but regulates the Ca 2+ sensitivity of force development and contributes to intestinal phasic contractile phenotype. Altered contractile responses in isolated tissues could be compensated by adaptive physiologic responses in vivo, where gut motility is affected by lower intensities of smooth muscle stimulation for myosin phosphorylation and force development.

To study the status of splenic hilar lymph nodes(No.4sa, No.10 or No.11d lymph nodes) metastasis and to investigate the proper dissection technique in patients with advanced gastric cancer.A retrospective study was performed to investigate 590 patients who underwent D2 curative proximal or total gastrectomy for gastric carcinoma from January 2006 to December 2009. Clinicopathological factors such as sex, age, location of the primary tumor, tumor sizes, gross type, depth of invasion, microscopic classification, neoadjuvant chemotherapy and the metastasis of adjacent lymph node were analyzed with univariate and multivariate analysis. Influence of combined splenectomy or pancreatectomy on lymph node dissection was also investigated.The overall ratio of metastatic lymph node(positive lymph nodes/lymph nodes harvested) in the splenic hilum was 17.5%(99/565). The positive rates of No.4sa, No.10, No.11d lymph nodes were 17.8% (41/230), 13.9%(29/209), and 22.8%(29/127), respectively. A total of 7.1%(42/590) of the patients had lymph node metastasis in the splenic hilum. Multivariable logistic regression analysis showed that age, tumor size, depth of tumor invasion, positive metastasis of No.4sb lymph node were independent risk factors for lymph node metastasis in the splenic hilum region. When comparing patients undergoing combined splenectomy or pancreatectomy(n=23) and those who did not undergo combined organ resection (n=553), the ratios of metastatic lymph node in the splenic hilum were 14.8%(4/27) and 17.2%(91/527), respectively, and the difference was not statistically significant(P0.05). The postoperative complication rates were 26.1%(6/23) and 5.4%(30/553), respectively, and the difference was statistically significant(P0.05). The operative mortality rates were 4.3% and 0.9%, respectively, and the difference was not statistically significant(P0.05).Metastasis to lymph nodes in the splenic hilum region in patients with gastric cancer possesses a certain pattern, and it is associated with tumor location, size, depth of invasion, and metastasis in No.4sb. Combined resection of the spleen or pancreas does not result in increased number of harvested lymph nodes or positive lymph nodes, yet is associated with higher complication rate. Therefore, combined organ resection should be meticulous.

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) but Ca2+-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCKSMKO) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCKSMKO mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCKSMKO mice may be a useful model of vascular failure and hypotension.

Zipper-interacting protein kinase (ZIPK) or DAPK3, is a member of the death-associated-protein kinase family associated with apoptosis in nonmuscle cells where it phosphorylates myosin regulatory light chain (RLC) to promote membrane blebbing. ZIPK mRNA is abundant in muscles, prompting our investigation of its role in the heart. A substrate search on mouse heart homogenates led to the discovery that cardiac RLC is a substrate for ZIPK. Enzyme kinetic studies revealed that both smooth and cardiac RLCs were good substrates, with a Vmax value two-fold greater for cardiac as compared to smooth/nonmuscle RLC. Moreover, ZIPK phosphorylated cardiac RLC at Ser15, the site responsible for modulating Ca2+ sensitivity of myofibrillar contraction. Knockdown of ZIPK in isolated neonatal cardiac myocytes by siRNA decreased the extent of RLC Ser15 phosphorylation. Localization studies using adenovirus- mediated overexpression of GFP-ZIPK in neonatal and adult rat cardiac myocytes showed it is in the nucleus, but also the cytoplasm where it may affect RLC phosphorylation. In adult cardiac myocytes, ZIPK appears to associate with myofilaments. ZIPK gene ablation specifically in mouse heart (ZKflox/Nkx2.5Cre) did not affect basal RLC phosphorylation, nor did it induce apparent pathological responses (assessed by histological analysis and heart weight measurements). Based on what is known about the DAPK family, we hypothesize that ZIPK function in the heart may be stress or death signal-dependent. Effects of physiological and pathophysiological stresses on hearts from ZKflox/Nkx2.5Cre mice are currently being investigated. Supported by NIH NHLBI (J. T. S.) and AHA Postdoctoral Fellowship (A.N.C.).

Different interacting signaling modules involving Ca(2+)/calmodulin-dependent myosin light chain kinase, Ca(2+)-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K(+)-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.

To express recombinant human interferon lambda2 in E.coli and to study its antiviral activities.According to preferred codons used in E.coli, the highly-expressed human interferon lambda2 gene was designed, synthesized and cloned into expression vector pBV220 and transfected into E.coli DH5alpha. The expressed product was purified by using CM FF and size exclusion chromatography. Its antiviral activities were tested on different cells.The expressed product was calculated about 15% of the total E.coli protein. The purified protein reached about 90% purity. Its specific antiviral activity was about 1.5 x 10(6) IU/mg on WISH/VSV test system. It was shown that the antiviral activity of the product on primates-origin cells seemed to be much higher than that on other non-primates-origin cells, indicating that interferon lambda2 possessed more stringent species specificity as compared with interferon-alpha2b. New interferon lambda2 showed similar anti-HBV activity as interferon-alpha2b.Recombinant human interferon lambda2 could be expressed on E.coli. The purified product showed more stringent species specificity and similar anti-HBV activity as compared with interferon-alpha2b.

To clone KGF-2 gene, get hKGF-2 protein and detemine its activity. The cNDA of human KGF-2 was isolated from fetal lung by RT-PCR and cloned into pBV220 plasmid. The recombinant pBV220-hKGF-2 plasmid was transformed into E. coli (BL21), induced at 42 degrees C for the expression of hKGF-2. Recombinant human KGF-2 was purified from the ultrasonic-treated BL21 by heparin-Sepharose CL-6B treated column chromatography and cation exchange column chromatography. MTT method was used for the determination of its biological activity. SDS-PAGE showed that rhKGF-2 was expressed in E. coli BL21 as soluble protein of approximately 20kD. The rhKGF-2 protein can stimulate the proliferation of NIH3T3 cells significantly from 1 ng/mL to 10 ng/mL. HKGF-2 cDNA wasclned and highly expressed in E. coli BL21 and the purified rhKGF-2 showed the mitogenic activity on NIH3T3 cells.

To study the anti-SARS virus activities of different recombinant human interferons on the cell culture system.Anti-SARS virus activities of interferons were determined by using CPE inhibition test in human skeletal muscle sarcoma (Rda) cell culture.The average minimum amount of interferon alpha 2b, alpha 1b, beta 1b or omega 1b to inhibit 50% CPE in Rda cell culture was (160.5+/-129.5) IU/ml, (149.0+/-71.7) IU/ml, (69.5+/-61.5) IU/ml, (87.3+/-47.1) IU/ml, respectively or (0.6+/-0.5) ng/ml, (10.6+/-5.1) ng/ml, (3.5+/-3.1) ng/ml, (0.9+/-0.5) ng/ml, respectively.All the tested recombinant interferons showed anti-SARS virus activities on the Rda cell culture with different sensitivities.

Publisher Summary This chapter discusses the three phase flash calculation under high-pressure separator in hydrocracking system by using continuous thermodynamics method. Continuous thermodynamics method has great advantages in the aspects of accuracy, efficiency, and reliability. Continuous thermodynamics method is also a time saving method. The flash of 27-multicomponent mixtures in the high pressure hydrocracking separator are calculated in an experiment described in the chapter. For polar components, special treatment was used. It was found that the distribution of liquid phase petroleum fraction was similar to that of feed, because there were little amounts of petroleum fraction which were dissolved in water phase even though the system pressure is very high. Other examples were also calculated using this method and similar results were obtained. Continuous thermodynamic method is a good approach for the three-phase flash calculation of continuous or semicontinuous mixtures.

From 1958 to 1973, 682 patients with lung cancer were treated by radiation therapy in the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China. The clinical presentation, sex, age, histology and stage and palliative effects of radiation therapy were reported. The 1-, 3- and 5-year survival rates were 40.62, 8.94 and 3.81%, respectively. The prognostic factors such as staging, histology, combination with chemotherapy and response of tumours are discussed. The causes of failure are also analysed. It is suggested that improving the local control rate of squamous cell carcinoma would increase the survival rate.