Identification of a PCSK9-LDLR disruptor peptide with in vivo function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employ an affinity-based screen of 1013in vitro-translated macrocyclic peptides to identify high-affinity PCSK9 ligands that utilize a unique, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction. Structure-based design led to molecules with enhanced function and pharmacokinetic properties (e.g., 13PCSK9i). In mice, 13PCSK9i reduces plasma cholesterol levels and increases hepatic LDLR density in a dose-dependent manner. 13PCSK9i functions by a unique, allosteric mechanism and is the smallest molecule identified to date with in vivo PCSK9-LDLR disruptor function.

Discovery of a Novel Piperidine-Based Inhibitor of Cholesteryl Ester Transfer Protein (CETP) That Retains Activity in Hypertriglyceridemic Plasma.

Herein we describe the discovery and characterization of a novel, piperidine-based inhibitor of cholesteryl ester transfer protein (CETP) with a core structure distinct from other reported CETP inhibitors. A versatile synthesis starting from 4-methoxypyridine enabled an efficient exploration of the SAR, giving a lead molecule with potent CETP inhibition in human plasma. The subsequent optimization focused on improvement of pharmacokinetics and mitigation of off-target liabilities, such as CYP inhibition, whose improvement correlated with increased lipophilic efficiency. The effort led to the identification of an achiral, carboxylic acid-bearing compound 16 (TAP311) with excellent pharmacokinetics in rats and robust efficacy in hamsters. Compared to anacetrapib, the compound showed substantially reduced lipophilicity, had only modest distribution into adipose tissue, and retained potency in hypertriglyceridemic plasma in vitro and in vivo. Furthermore, in contrast to torcetrapib, the compound did not increase aldosterone secretion in human adrenocortical carcinoma cells nor in chronically cannulated rats. On the basis of its preclinical efficacy and safety profile, the compound was advanced into clinical trials.

R-HPDC Process with Forced Convection Mixing Device for Automotive Part of A380 Aluminum Alloy.

The continuing quest for cost-effective and complex shaped aluminum castings with fewer defects for applications in the automotive industries has aroused the interest in rheological high pressure die casting (R-HPDC). A new machine, forced convection mixing (FCM) device, based on the mechanical stirring and convection mixing theory for the preparation of semisolid slurry in convenience and functionality was proposed to produce the automotive shock absorber part by R-HPDC process. The effect of barrel temperature and rotational speed of the device on the grain size and morphology of semi-solid slurry were extensively studied. In addition, flow behavior and temperature field of the melt in the FCM process was investigated combining computational fluid dynamics simulation. The results indicate that the microstructure and pore defects at different locations of R-HPDC casting have been greatly improved. The vigorous fluid convection in FCM process has changed the temperature field and composition distribution of conventional solidification. Appropriately increasing the rotational speed can lead to a uniform temperature filed sooner. The lower barrel temperature leads to a larger uniform degree of supercooling of the melt that benefits the promotion of nucleation rate. Both of them contribute to the decrease of the grain size and the roundness of grain morphology.

[Fingertip replantation with anastomosis of palm vein and retaining the nail].

OBJECTIVE: To study the replantation methods and clinical results of amputated fingertip. METHODS: From October 2007 to June 2011, 18 fingers of 13 cases were replanted with anastomosis of palm vein and retaining the nail, including 9 males and 4 females,with an average age of 26 years old ranging from 17 to 45 years old. The time from injury to therapy was from 30 min to 5 h, time of broken finger ischemia was from 1.5 to 7 h. All broken fingers were preservation under normal temperature. RESULTS: All fingers were survived, no vascular crisis happened. All cases were followed up from 3 to 24 months with an average of 14 months. The length and shape of replanted fingers were similar to that of the healthy side. The new nails were smooth, the function was perfect,the sense of pain and touched sensation had been recovered. Their two-piont discriminations ranged from 3 to 6 mm with an average of 5 mm. According to the assessment standard of Chinese Medical Association of Hand Surgery, the results were excellent in 14 cases, good in 3 cases, poor in 1 case. CONCLUSION: Fingertip replantation with anastomosis of palm vein and retaining the nail is regained satisfactory appearance and function of the digits with a high survival rate.

miR-495 and miR-551a inhibit the migration and invasion of human gastric cancer cells by directly interacting with PRL-3.

The phosphatase of regenerating liver-3 (PRL-3) gene is associated with metastasis in gastric cancer, and is believed to play a causative role by promoting tumor cell motility, invasion, and metastasis, but little is known of the mechanisms involved. We previously reported that PRL-3 expression is significantly higher in the tissues of primary gastric carcinomas with peritoneal metastasis. In the present study, we found that two microRNAs (miRNAs), miR-495 and miR-551a, predicted to target PRL-3, are downregulated in gastric carcinoma samples. The validation of this interaction between those two miRNAs and PRL-3 was confirmed by western blotting and quantitative real-time PCR (qPCR) in GC cell lines transfected with miR-495 and miR-551a mimics. Furthermore, the migration and invasion of GC cells were significantly inhibited by transfection with miR-495 or -551a mimics, and the mRNA and protein levels of PRL-3 were reduced in cells overexpressing miR-495 or -551a. Collectively, our findings suggest that miR-495 and miR-551a both act as tumor suppressors by targeting the PRL-3 oncogene and inhibiting gastric cancer cell migration and invasion. The findings of this study contribute to current understanding of the functions of miRNA mimics in GC gene therapy.

ATP-sensitive potassium channels are involved in adenosine-induced reduction of blood pressure variability in spontaneously hypertensive rats.

With a computerized analysis system, blood pressure was recorded continuously in conscious unrestrained spontaneously hypertensive rats. The effects of different adenosine receptor agonists and ATP-sensitive potassium channel opener and blocker on blood pressure variability in spontaneously hypertensive rats were studied. It was found that adenosine, 5'-N-cyclopropyl-carboxamidoadenosine (CPCA, a selective adenosine A2-receptor agonist) and pinacidil (a nonselective ATP-sensitive potassium channel opener) decreased blood pressure variability when one of them was used alone, whereas N -cyclopentyladenosine (CPA, a selective adenosine A1-receptor agonist) had no significant effects on blood pressure variability. When pretreated with glibenclamide (a nonselective ATP-sensitive potassium channel blocker), the inhibitory effects of adenosine and CPCA on blood pressure variability were significantly prevented. By itself, however, glibenclamide had no influence on blood pressure variability. These results suggest that the effect of adenosine on blood pressure variability in spontaneously hypertensive rats is due to activation of ATP-sensitive potassium channels mediated by adenosine A2-receptor.

A system for the enhancement of adenovirus mediated gene transfer to uro-epithelium.

PURPOSE: Recombinant adenovirus has been used widely as an in vivo gene transfer vector, although its transfection efficiency in bladder tissue is limited. Several studies have indicated that the bladder surface glycosaminoglycan (GAG) layer functions as a nonspecific anti-adherence factor and possibly as a first line anti-infection defense mechanism. We determined whether recombinant adenovirus mediated gene transfer could be enhanced in intact bladders by HCl pretreatment and by alterations in the GAG layer. MATERIALS AND METHODS: In vitro viral transfection efficiencies with and without the GAG analog pentosan polysulfate (Sigma Chemical Co., St. Louis, Missouri) were determined in bladder muscle and urothelial cells. Immunocytochemical studies and Western blot analysis were performed to determine whether urothelial cells possessed the Coxsackievirus and adenovirus receptor. Rat bladders were intravesically pretreated with HCl at various concentrations and for various periods. After 60 mM. HCl pretreatment for 10 minutes 2 x 109 pfu of recombinant adenovirus carrying the Escherichia coli LacZ gene were intravesically instilled into the bladders. RESULTS: Adenoviral infection of urothelial cells was significantly reduced in the presence of pentosan polysulfate in vitro. Coxsackievirus and adenovirus receptor expression was detected in urothelial cells in vivo and in vitro. Bladders pretreated with HCl resulted in an alteration of the bladder GAG layers. After intravesical gene instillation reporter gene analyses using X-5-bromo-4-chloro-3-inodolyl beta-D-galactopyranoside (Sigma Chemical Co.) showed approximately 80% urothelial cell transfection efficiency in bladders pretreated with HCl. However, less than 10% of the urothelial cells expressed the transfected gene in control HCl untreated bladders. CONCLUSIONS: Primary urothelial cells and bladder carcinoma cells can be efficiently transfected using an adenoviral vector with similar infectivity. In vitro viral infection shows that the efficiency of adenoviral transfection is significantly reduced in the presence of pentosan polysulfate, a GAG analog. Adenoviral mediated gene transfer to bladder urothelium is enhanced by HCl pretreatment.