MiR-489 regulates chemoresistance in breast cancer via epithelial mesenchymal transition pathway

To investigate the role of microRNAs in the development of chemoresistance and related epithelial–mesenchymal transition (EMT), we examined the effect of miR-489 in adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM). MiR-489 was significantly suppressed in MCF-7/ADM cells compared with chemosensitive parental control MCF-7/WT cells. Forced-expression of miR-489 reversed chemoresistance. Furthermore, Smad3 was identified as the target of miR-489 and is highly expressed in MCF-7/ADM cells. Forced expression of miR-489 both inhibited Smad3 expression and Smad3 related EMT properties. Finally, the interactions between Smad3, miR-489 and EMT were confirmed in chemoresistant tumor xenografts and clinical samples, indicating their potential implication for treatment of chemoresistance.     

Determining water level management strategies for lake protection at the ecosystem level

To prevent lake degradation, water level management has been a major focus of research in the past several decades. There are, however, some shortcomings in the traditional studies, and the protection of entire ecosystems is difficult to achieve in practice. In this paper, the framework of a new method for determining ecosystem-based water level regimes (WLRs) for lake protection is proposed. First, historical WLRs are divided into several sub-stages. Then, ecosystem statuses corresponding to different WLRs are quantified and compared. Finally, parameters of optimal and acceptable WLRs are used to determine water level management goals. The proposed method was applied to Baiyangdian Lake, the largest shallow lake in the North China Plain, to test its effectiveness. Results showed that to protect the ecosystem at the optimal status, 50% of the parameter values should fall within the range of the 25th and 75th percentiles of Stage I; and to protect the ecosystem from reverse succession, 50% of the water level parameter values should fall within the range of the 25th and 75th percentiles of Stage II. This method takes ecosystem status into account, and has high practicability in water resources management.     

Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion

Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) degrading enzymes and have complex and specific regulation networks. This includes activation interactions, where one MMP family member activates another. ECM degradation and MMP activation can be initiated by several different stimuli including changes in ECM mechanical properties or intracellular contractility. These mechanical stimuli are known enhancers of metastatic potential. MMP-14 facilitates local ECM degradation and is well known as a major mediator of cell migration, angiogenesis and invasion. Recently, function blocking antibodies have been developed to specifically block MMP-14, providing a useful tool for research as well as therapeutic applications. Here we utilize a selective MMP-14 function blocking antibody to delineate the role of MMP-14 as an activator of other MMPs in response to changes in cellular contractility and ECM stiffness. Inhibition using function blocking antibodies reveals that MMP-14 activates soluble MMPs like MMP-2 and -9 under various mechanical stimuli in the pancreatic cancer cell line, Panc-1. In addition, inhibition of MMP-14 abates Panc-1 cell extension into 3D gels to levels seen with non-specific pan-MMP inhibitors at higher concentrations. This strengthens the case for MMP function blocking antibodies as more potent and specific MMP inhibition therapeutics.     

Active Site Coupling in PDE:PKA Complexes Promotes Resetting of Mammalian cAMP Signaling

Cyclic 3′5′ adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (K_D ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.     

Numerical simulation of the effect of superparamagnetic nanoparticles on microwave rewarming of cryopreserved tissues

In this study, the microwave rewarming process of cryopreserved samples with embedded superparamagnetic (SPM) nanoparticles was numerically simulated. The Finite Element Method (FEM) was used to calculate the coupling of the electromagnetic field and the temperature field in a microwave rewarming system composed of a cylindrical resonant cavity, an antenna source, and a frozen sample phantom with temperature-dependent properties. The heat generated by the sample and the nanoparticles inside the electromagnetic field of the microwave cavity was calculated. The dielectric properties of the biological tissues were approximated using the Debye model, which is applicable at different temperatures. The numerical results showed that, during the rewarming process of the sample phantom without nanoparticles, the rewarming rate was 29.45 °C/min and the maximum temperature gradient in the sample was 3.58 °C/mm. If nanoparticles were embedded in the sample, and the cavity power was unchanged, the rewarming rate was 47.76 °C/min and the maximum temperature gradient in the sample was 1.64 °C/mm. In the presence of SPM nanoparticles, the rewarming rate and the maximum temperature gradient were able to reach 20.73 °C/min and 0.68 °C/mm at the end of the rewarming under the optimized cavity power setting, respectively. The ability to change these temperature behaviors may prevent devitrification and would greatly diminish thermal stress during the rewarming process. The results indicate that the rewarming rate and the uniformity of temperature distribution are increased by nanoparticles. This could be because nanoparticles generated heat in the sample homogeneously and the time-dependent parameters of the sample improved after nanoparticles were homogeneously embedded within it. We were thus able to estimate the positive effect of SPM nanoparticles on microwave rewarming of cryopreserved samples.     

The atypical Rho GTPase,RhoU, regulates cell-adhesion molecules during cardiac morphogenesis

The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.     

Bioproduction of chiral mandelate by enantioselective deacylation of α-acetoxyphenylacetic acid using whole cells of newly isolated Pseudomonas sp. ECU1011

Substrate-directed screening was carried out to find bacteria that could deacylate O-acetylated mandelic acid from environmental samples. From more than 200 soil isolates, we identified for the first time that Pseudomonas sp. ECU1011 biocatalytically deacylated (S)-α-acetoxyphenylacetic acid with high enantioselectivity (E > 200), yielding (S)-mandelic acid with 98.1% enantiomeric excess (ee) at a 45.5% conversion rate. The catalytic deacylation of (S)-α-acetoxyphenylacetic acid by the resting cell was optimized using a single-factor method to yield temperature and pH optima of 30°C and 6.5, respectively. These optima help to reduce the nonselective spontaneous hydrolysis of the racemic substrate. It was found that substrate concentrations up to 60 mM could be used. 2-Propanol was used as a moderate cosolvent to help the substrate disperse in the aqueous phase. Under optimized reaction conditions, the ee of the residual (R)-α-acetoxyphenylacetic acid could be improved further, to greater than 99%, at a 60% conversion rate. Furthermore, using this newly isolated strain of Pseudomonas sp. ECU1011, three kinds of optically pure analogs of (S)-mandelic acid and (R)-α-acetoxyphenylacetic acid were successfully prepared at high enantiomeric purity.     

Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization

Earlier studies have established the importance of five disulfide bridges (DBs) in Aspergillus niger phytase. In this study, the relative importance of each of the individual disulfide bridge is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene. Individually, these mutant phytases were expressed in a Pichia expression system and their product purified and characterized. The removal of disulfide bridge 2 yielded a mutant phytase with a complete loss of catalytic activity. The other disulfide mutants displayed a broad array of altered catalytic properties including a lower optimum temperature from 58°C to 53°C for bridge number 1, 37°C for bridge number 3 and 4, and 42°C for bridge number 5. The pH versus activity profile was also modified in the DB mutants. The pH profile of the wild-type phytase was modified by the DB mutations. In bridge number 1, 3, and 4, the second peak at pH 2.5 was abolished, and in bridge number 5, the peak at pH 5.0 was abolished completely leaving only the pH 2.5. While the K _m was not affected drastically, the turnover number was lowered significantly in bridge number 3, 4, and 5.     

Inhibition of human lung adenocarcinoma growth using survivint34a by low-dose systematic administration

Anti-apoptosis plays an important role in tumour formation and development. Survivin is a member of the inhibitor of apoptosis (IAP) family, which is a target for anti-cancer drug exploitation was replaced as development. We investigated the role of the homo dominant-negative mutant Survivin-T34A in suppressing human lung adenocarcinomas (A549). The anti-tumour activity of HSurvivinT34A plasmid was evaluated in the A549 cell line and nude mice bearing A549 subcutaneous tumours. Low-dose systemic administration was continuously used. The HSurvivinT34A plasmid (5 µg/one) complexed with a cationic liposome (DOTAP/Chol) significantly inhibited tumour growth in our model. We observed microvessel density degradation by CD31 immunohistochemistry and apoptotic cell increase by TUNEL assay, PI staining and flow cytometric analysis in the treated group. The present findings suggest that the HSurvivinT34A plasmid complexed with a cationic liposome may provide an effective approach to inhibit the growth of human lung adenocarcinomas in vitro and in vivo.