Conformational effects on the pro-S hydrogen abstraction reaction in cyclooxygenase-1: an integrated QM/MM and MD study

A key step in the cyclooxygenase reaction cycle of cyclooxygenase 1 (COX-1) is abstraction of the pro-S hydrogen atom of the arachidonic acid by a radical that is formed at the protein residue Tyr-385. Here we investigate this reaction step by a quantum-mechanics/molecular-mechanics approach in combination with molecular-dynamics simulations. The simulations identify the hydrogen abstraction angle as a crucial geometric determinant of the reaction, thus revealing the importance of the cyclooxygenase active site for calculating the potential energy surface of the reaction

In vitro study: binding of 99mTc-DPD to synthetic amyloid fibrils

Abstract This paper is an report of the investigation of the in vitro binding of 99mTc-DPD for synthetic amyloid fibrils used for the diagnosis of cardiac amyloidosis (CA), as compared with the use of 99mTc-HMDP and 99mTc-PPI. It also includes an inquiry into the role played by Ca2+ ions and serum proteins on binding to amyloid like materials, as well as the saturability and specificity of DPD for fibrils versus amorphous precipitates (AP). In the work, synthetic insulin fibrils (SIF) and AP were characterized by Congo red staining and TEM imaging. An equal amount of three radiopharmaceuticals were then added to fibrils in Ca2+ (0-4.2 mmol/L) or human serum (HS) adjoined samples and radiopharmaceutical uptake was assessed. To test the saturability of amyloid binding sites, a displacement assays with cold DPD was performed, while adding 50-1500 nmol of 99mTc-DPD to SIF or AP, saturation binding tests were subsequently carried out for evaluating its specificity for amyloid. Herein, synthetic fibrils and AP showed conformational differences at TEM and polarized microscopy analysis. In our study, 99mTc-DPD fibrils uptake was seen to be the highest and increased with calcium ions concentration. What is more, serum proteins reduced the bound fraction to the amyloid deposits of about 15%, and the Kd values of 90 nM and 114 nM relative to SIF and AP, respectively, did not significantly differ. We saw that 99mTc-DPD is the best seeker for amyloid fibrils in cardiac amyloidosis, and that Ca2+ concentration positively influenced DPD fibrils binding. Furthermore, the radioactivity bound to the serum protein clear up the idea of nuclide exchanging dynamic balance between amyloid and circulating proteins. Moreover, non-labeled DPD did not exert a competition for 99mTc-DPD binding sites, and, finally, DPD cannot be defined a radiopharmaceutical specific for amyloid deposits

N-Acylethanolamine Acid Amidase (NAAA): Structure, Function, and Inhibition

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase primarily found in the endosomal-lysosomal compartment of innate and adaptive immune cells. NAAA catalyzes the hydrolytic deactivation of palmitoylethanolamide (PEA), a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that exerts profound anti-inflammatory effects in animal models. Emerging evidence points to NAAA-regulated PEA signaling at PPAR-α as a critical control point for the induction and the resolution of inflammation and to NAAA itself as a target for anti-inflammatory medicines. The present Perspective discusses three key aspects of this hypothesis: the role of NAAA in controlling the signaling activity of PEA; the structural bases for NAAA function and inhibition by covalent and noncovalent agents; and finally, the potential value of NAAA-targeting drugs in the treatment of human inflammatory disorders

Conjugated polymers mediate intracellular Ca2+ signals in circulating endothelial colony forming cells through the reactive oxygen species-dependent activation of Transient Receptor Potential Vanilloid 1 (TRPV1)

Endothelial colony forming cells (ECFCs) represent the most suitable cellular substrate to induce revascularization of ischemic tissues. Recently, optical excitation of the light-sensitive conjugated polymer, regioregular Poly (3-hexyl-thiophene), rr-P3HT, was found to stimulate ECFC proliferation and tube formation by activating the non-selective cation channel, Transient Receptor Potential Vanilloid 1 (TRPV1). Herein, we adopted a multidisciplinary approach, ranging from intracellular Ca2+ imaging to pharmacological manipulation and genetic suppression of TRPV1 expression, to investigate the effects of photoexcitation on intracellular Ca2+ concentration ([Ca2+](i)) in circulating ECFCs plated on rr-P3HT thin films. Polymer-mediated optical excitation induced a long-lasting increase in [Ca2+](i) that could display an oscillatory pattern at shorter light stimuli. Pharmacological and genetic manipulation revealed that the Ca2+ response to light was triggered by extracellular Ca2+ entry through TRPV1, whose activation required the production of reactive oxygen species at the interface between rr-P3HT and the cell membrane. Light-induced TRPV1-mediated Ca2+ entry was able to evoke intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors, followed by store-operated Ca2+ entry on the plasma membrane. These data show that TRPV1 may serve as a decoder at the interface between rr-P3HT thin films and ECFCs to translate optical excitation in pro-angiogenic Ca2+ signals

Micro- and Nanopatterned Silk Substrates for Antifouling Applications

A major problem of current biomedical implants is the bacterial colonization and subsequent biofilm formation, which seriously affects their functioning and can lead to serious post-surgical complications. Intensive efforts have been directed toward the development of novel technologies that can prevent bacterial colonization while requiring minimal antibiotics doses. To this end, biocompatible materials with intrinsic antifouling capabilities are in high demand. Silk fibroin, widely employed in biotechnology, represents an interesting candidate. Here, we employ a soft-lithography approach to realize micro- and nanostructured silk fibroin substrates, with different geometries. We show that patterned silk film substrates support mammal cells (HEK-293) adhesion and proliferation, and at the same time, they intrinsically display remarkable antifouling properties. We employ Escherichia coli as representative Gram-negative bacteria, and we observe an up to 66% decrease in the number of bacteria that adhere to patterned silk surfaces as compared to control, flat silk samples. The mechanism leading to the inhibition of biofilm formation critically depends on the microstructure geometry, involving both a steric and a hydrophobic effect. We also couple silk fibroin patterned films to a biocompatible, optically responsive organic semiconductor, and we verify that the antifouling properties are very well preserved. The technology described here is of interest for the next generation of biomedical implants, involving the use of materials with enhanced antibacterial capability, easy processability, high biocompatibility, and prompt availability for coupling with photoimaging and photodetection techniques

Calcium as a key player in arrhythmogenic cardiomiopathy : adhesion disorder or intracellular alteration?

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation\u2012contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease

How does conformational flexibility influence key structural features involved in activation of anaplastic lymphoma kinase?

Anaplastic Lymphoma Kinase (ALK) plays a major role in developing tumor processes and therefore has emerged as a validated therapeutic target. Applying atomistic molecular dynamics simulations on the wild type enzyme and the nine most frequently occurring and clinically important activation mutants we revealed important conformational effects on key interactions responsible for the activation of the enzyme

N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) inhibits the angiogenic activity of heparin-binding growth factors.

The peptides N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) and BOC-Met-Leu-Phe (BOC1) are widely used antagonists of formyl peptide receptors (FPRs), BOC2 acting as an FPR1/FPR2 antagonist whereas BOC1 inhibits FPR1 only. Extensive investigations have been performed by using these FPR antagonists as a tool to assess the role of FPRs in physiological and pathological conditions. Based on previous observations from our laboratory, we assessed the possibility that BOC2 may exert also a direct inhibitory effect on the angiogenic activity of vascular endothelial growth factor-A (VEGF-A). Our data demonstrate that BOC2, but not BOC1, inhibits the angiogenic activity of heparin-binding VEGF-A165 with no effect on the activity of the non-heparin-binding VEGF-A121 isoform. Endothelial cell-based bioassays, surface plasmon resonance analysis, and computer modeling indicate that BOC2 may interact with the heparin-binding domain of VEGF-A165, thus competing for heparin interaction and preventing the binding of VEGF-A165 to tyrosine kinase receptor VEGFR2, its phosphorylation and downstream signaling. In addition, BOC2 inhibits the interaction of a variety of heparin-binding angiogenic growth factors with heparin, including fibroblast growth factor 2 (FGF2) whose angiogenic activity is blocked by the compound. Accordingly, BOC2 suppresses the angiogenic potential of human tumor cell lines that co-express VEGF-A and FGF2. Thus, BOC2 appears to act as a novel multi-heparin-binding growth factor antagonist. These findings caution about the interpretation of FPR-focusing experimental data obtained with this compound and set the basis for the design of novel BOC2-derived, FPR independent multi-target angiogenesis inhibitors

Phenotype Screening of an Azole-bisindole Chemical Library Identifies URB1483 as a New Antileishmanial Agent Devoid of Toxicity on Human Cells

We report the evaluation of a small library of azole-bisindoles for their antileishmanial potential, in terms of efficacy on Leishmania infantum promastigotes and intracellular amastigotes. Nine compounds showed good activity on L. infantum MHOM/TN/80/IPT1 promastigotes with IC50 values ranging from 4 to 10 μM. These active compounds were also tested on human (THP-1, HEPG2, HaCaT, and human primary fibroblasts) and canine (DH82) cell lines. URB1483 was selected as the best compound, with no quantifiable cytotoxicity in mammalian cells, to test the efficacy on intracellular amastigotes. URB1483 significantly reduced the infection index of both human and canine macrophages with an effect comparable to the clinically used drug pentamidine. URB1483 emerges as a new anti-infective agent with remarkable antileishmanial activity and no cytotoxic effects on human and canine cells

PixFEL: development of an X-ray diffraction imager for future FEL applications

A readout chip for diffraction imaging applications at new generation X-ray FELs (Free Electron Lasers) has been designed in a 65 nm CMOS technology. It consists of a 32 × 32 matrix, with square pixels and a pixel pitch of 110 µm. Each cell includes a low-noise charge sensitive amplifier (CSA) with dynamic signal compression, covering an input dynamic range from 1 to 104 photons and featuring single photon resolution at small signals at energies from 1 to 10 keV. The CSA output is processed by a time-variant shaper performing gated integration and correlated double sampling. Each pixel includes also a small area, low power 10-bit time-interleaved Successive Approximation Register (SAR) ADC for in-pixel digitization of the amplitude measurement. The channel can be operated at rates up to 4.5 MHz, to be compliant with the rates foreseen for future X-ray FEL machines. The ASIC has been designed in order to be bump bonded to a slim/active edge pixel sensor, in order to build the first demonstrator for the PixFEL (advanced X-ray PIXel cameras at FELs) imager