6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, K_i = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA’s S1β subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.     

In silico biophysics and hemorheology of blood hyperviscosity syndrome

Hyperviscosity syndrome (HVS) is characterized by an increase of the blood viscosity by up to seven times the normal blood viscosity, resulting in disturbances to the circulation in the vasculature system. HVS is commonly associated with an increase of large plasma proteins and abnormalities in the properties of red blood cells, such as cell interactions, cell stiffness, and increased hematocrit. Here, we perform a systematic study of the effect of each biophysical factor on the viscosity of blood by employing the dissipative particle dynamic method. Our in silico platform enables manipulation of each parameter in isolation, providing a unique scheme to quantify and accurately investigate the role of each factor in increasing the blood viscosity. To study the effect of these four factors independently, each factor was elevated more than its values for a healthy blood while the other factors remained constant, and viscosity measurement was performed for different hematocrits and flow rates. Although all four factors were found to increase the overall blood viscosity, these increases were highly dependent on the hematocrit and the flow rates imposed. The effect of cell aggregation and cell concentration on blood viscosity were predominantly observed at low shear rates, in contrast to the more magnified role of cell rigidity and plasma viscosity at high shear rates. Additionally, cell-related factors increase the whole blood viscosity at high hematocrits compared with the relative role of plasma-related factors at lower hematocrits. Our results, mapped onto the flow rates and hematocrits along the circulatory system, provide a correlation to underpinning mechanisms for HVS findings in different blood vessels.     

Effects of surfactant, salt and solvent on the structure and activity of adenosine deaminase: molecular dynamic and spectrophotometric studies

Effects of sodium dodecyl sulfate, dodecyltrimethylammonium bromide, sodium chloride, sodium sulfate, methanol and ethanol, on the structure and activity of adenosine deaminase (ADA) were investigated by UV-Vis, circular dichroism spectrophotometry and molecular dynamics (MDs) studies. Relative activity, experimental and computational helix content, total accessible surface area (ASA) and exposed charged surface area (ECSA) were obtained. The relative activity of ADA in the absence and the presence of denaturants were compared with structural results. It was shown that an increase in the surface area and a decrease in the amount of helicity are associated with a decrease in the activity of ADA.     

Identification of a seed coat-specific promoter fragment from the Arabidopsis <Emphasis Type="Italic">MUCILAGE-MODIFIED4</Emphasis> gene

Key message

Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation.

Abstract

The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.

     

Plant Symposia and Workshops

Type 2 diabetes (T2D) is a condition with insufficient insulin production or in the setting of insulin resistance with many origins including intestinal microbiota-related molecular mechanism. Insulin-degrading enzyme (IDE) is responsible for insulin breakdown in various tissues and is known as a potential drug target for T2D. Here, we assessed the effects of cell-free supernatant (CFS) and UV-killed Lactobacillus casei IBRC_M10711 on IDE expression, IDE activity, and insulin degradation in Caco-2 cell line. It was found that CFS and UV-killed L. casei IBRC_M10711 led to lower expression of IDE. UV-killed L. casei IBRC_M10711 significantly inhibited IDE activity but CFS did not. Insulin degradation was affected with none of them. In conclusion, L. casei IBRC_M10711 is effective on IDE expression and its activity, but not on insulin degradation. Future studies are recommended to explore the effect of this probiotic on other substrates of IDE.     

Pyrosequencing for microbial typing

Pyrosequencing is a real-time DNA sequencing technique generating short reads rapidly and inexpensively. This technology has the potential advantage of accuracy, ease-of-use, high flexibility and is now emerging as a popular platform for microbial typing. Here, we review the methodology and the use of this technique for viral typing, bacterial typing, and fungal typing. In addition, we describe how to use multiplexing for accurate and rapid typing.     

Rapid synthesis of new block copolyurethanes derived from l-leucine cyclodipeptide in reusable molten ammonium salts: novel and efficient green media for the synthesis of new hydrolysable and biodegradable copolyurethanes

This study concerns the synthesis of novel multi block polyurethane (PU) copolymers containing cyclodipeptide, taking the advantage of ionic liquids (ILs) under microwave irradiation. For this, l-leucine anhydride cyclodipeptide (LACP) was prepared and then a new class of poly(ether-urethane-urea)s (PEUUs) was synthesized in molten ammonium type ILs. ILs were used as reaction media and PUs were prepared via two-step polymerization method. In the first step, 4,4′-methylene-bis-(4-phenylisocyanate) (MDI) was reacted with LACP to produce isocyanate-terminated oligo(imide-urea) as hard segment (NCO-OIU). Chain extension of the aforementioned pre-polymer with polyethyleneglycol (PEG) of molecular weights of 1000 (PEG-1000) was the second step to furnish a series of new PEUUs. These multiblock copolymers are thermally stable, soluble in amide-type solvents, hydrolysable and biodegradable. PEUUs prepared in ILs under microwave irradiation showed more phase separation and crystallinity than PEUUs prepared under conventional method. The protocol presented here has the merits of environmentally benign, simple operation, convenient work-up, short reaction time and good yields without using volatile organic solvents, and catalysts. Ammonium type reaction media were air and water stable, and relatively cheap, which makes them suitable for application. The results demonstrate that they can be easily separated into water and reused without losing activity. Reusability of tetrabutylammonium bromide as reaction media makes the method a cost effective and environmentally benign method under microwave irradiation. Thus, we could prepare environmentally friendly polymers via environmentally benign method.     

Neural differentiation of mouse embryonic stem cells on conductive nanofiber scaffolds

Nerve tissue engineering requires suitable precursor cells as well as the necessary biochemical and physical cues to guide neurite extension and tissue development. An ideal scaffold for neural regeneration would be both fibrous and electrically conductive. We have contrasted the growth and neural differentiation of mouse embryonic stem cells on three different aligned nanofiber scaffolds composed of poly L: -lactic acid supplemented with either single- or multi-walled carbon-nanotubes. The addition of the nanotubes conferred conductivity to the nanofibers and promoted mESC neural differentiation as evidenced by an increased mature neuronal markers expression. We propose that the conductive scaffold could be a useful tool for the generation of neural tissue mimics in vitro and potentially as a scaffold for the repair of neural defects in vivo.     

The effects of nickel oxide nanoparticles on structural changes,heme degradation,aggregation of hemoglobin and expression of apoptotic genes in lymphocytes

Abstract

Nickel oxide nanoparticles (NiO NPs) have received great interests in medical and biotechnological applications. However, their adverse impacts against biological systems have not been well-explored. Herein, the influence of NiO NPs on structural changes, heme degradation and aggregation of hemoglobin (Hb) was evaluated by UV-visible (Vis) spectroscopy, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), and molecular modeling investigations. Also, the morphological changes and expression of Bax/Bcl-2 mRNA in human lymphocyte cell exposed to NiO NPs were assayed by DAPI staining and quantitative real-time PCR (qPCR), respectively. The UV-Vis study depicted that NiO NPs resulted in the displacement of aromatic residues and heme groups and production of the pro-aggregatory species. Intrinsic and Thioflavin T (ThT) fluorescence studies revealed that NiO NPs resulted in heme degradation and amorphous aggregation of Hb, respectively, which the latter result was also confirmed by TEM study. Moreover, far UV-CD study depicted that NiO NPs lead to substantial secondary structural changes of Hb. Furthermore, near UV-CD displayed that NiO NPs cause quaternary conformational changes of Hb as well as heme displacement. Molecular modelling study also approved that NiO NPs resulted in structural alterations of Hb and heme deformation. Moreover, morphological and genotoxicity assays revealed that the DNA fragmentation and expression ratio of Bax/Bcl-2 mRNA increased in lymphocyte cells treated with NiO NPs for 24?hr. In conclusion, this study indicates that NiO NPs may affect the biological media and their applications should be limited.

Communicated by Ramaswamy H. Sarma