Lipase-Mediated Synthesis of Oleoyl Ethanolamide Starting from High-Oleic Sunflower Oil Soapstock

This work describes the lipase-mediated synthesis of oleoyl ethanolamide, a dietary supplement for body weight loss recently approved by FDA. The target compound is prepared by conversion of the oleic acid contained in a mixture of fatty acids recovered by enzymatic hydrolysis of soapstock, a side-product of high oleic sunflower oil refinement. The use of a packed-bed reactor (a glass column loaded with the commercial lipase Lipozyme 435) in continuous flow mode improves the space-time yield of the reaction and the catalyst productivity. The nontoxic, bioderived, and renewable solvent limonene is used in the reaction medium. The process has been run for more than 157 h of continuous operation, demonstrating the stability and efficiency of the biocatalyst. Additionally, at the end of the reaction, only oleoyl ethanolamide crystallizes from the reaction mixture, thus, it is collected by simple filtration of the outlet solution in 53% isolation yield, showing 99% chemical purity, while all the byproducts of the reaction are left behind in the mother liquors

Mechanistic insights into the release of doxorubicin from graphene oxide in cancer cells

Liposomal doxorubicin (L-DOX) is a popular drug formulation for the treatment of several cancer types (e.g., recurrent ovarian cancer, metastatic breast cancer, multiple myeloma, etc.), but poor nuclear internalization has hampered its clinical applicability so far. Therefore, novel drug-delivery nanosystems are actively researched in cancer chemotherapy. Here we demonstrate that DOX-loaded graphene oxide (GO), GO-DOX, exhibits much higher anticancer efficacy as compared to its L-DOX counterpart if administered to cellular models of breast cancer. Then, by a combination of live-cell confocal imaging and fluorescence lifetime imaging microscopy (FLIM), we suggest that GO-DOX may realize its superior performances by inducing massive intracellular DOX release (and its subsequent nuclear accumulation) upon binding to the cell plasma membrane. Reported results lay the foundation for future exploitation of these new adducts as high-performance nanochemotherapeutic agents

Immobilization of old yellow enzymes via covalent or coordination bonds

Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziG™ particles (OYE3-EziG). The immobilized OYE3-GA was demonstrated to be active (activity recovery = 52%) and to retain almost 100% of its activity under the enzymatic assay conditions (50 mM phosphate buffer pH 7, 28 °C) for six days, whereas the activity of the non-immobilized enzyme dropped to 50% after two days. In the case of EziG™, the highest activity recovery (54%) was achieved by using the most hydrophilic carrier (EziG™ Opal) that was selected for the full characterization of this type of enzyme preparation (stability, recycling, re-use, enzyme leakage). OYE3-EziG was slightly less stable than OYE3-GA under the same experimental conditions. OYE3-GA could be recycled and re-used for up to 12 reaction cycles in the bioreduction of α-methyl-trans-cinnamaldehyde; after 12 runs, the highest conversion achieved was 40%. In the case of the co-immobilized OYE3/GDH-EziG, the conversion dropped to 56% after two reaction cycles. No enzyme leakage was detected over 48 h for both OYE3-GA and OYE3/GDH-EziG (50 mM phosphate buffer pH 7, 28 °C). These seed results pave the way for a true optimization of the immobilization of OYE3, as well as for the use of immobilized OYE3 for preparative applications both in batch and continuous flow conditions

Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation

A gemini cationic lipid with histidine residues as a novel lipid-based gene nanocarrier: a biophysical and biochemical study

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120–290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies

Ellagic acid inhibits bladder cancer invasiveness and in vivo tumor growth

Ellagic acid (EA) is a polyphenolic compound that can be found as a naturally occurring hydrolysis product of ellagitannins in pomegranates, berries, grapes, green tea and nuts. Previous studies have reported the antitumor properties of EA mainly using in vitro models. No data are available about EA influence on bladder cancer cell invasion of the extracellular matrix triggered by vascular endothelial growth factor-A (VEGF-A), an angiogenic factor associated with disease progression and recurrence, and tumor growth in vivo. In this study, we have investigated EA activity against four different human bladder cancer cell lines (i.e., T24, UM-UC-3, 5637 and HT-1376) by in vitro proliferation tests (measuring metabolic and foci forming activity), invasion and chemotactic assays in response to VEGF-A and in vivo preclinical models in nude mice. Results indicate that EA exerts anti-proliferative effects as a single agent and enhances the antitumor activity of mitomycin C, which is commonly used for the treatment of bladder cancer. EA also inhibits tumor invasion and chemotaxis, specifically induced by VEGF-A, and reduces VEGFR-2 expression. Moreover, EA down-regulates the expression of programmed cell death ligand 1 (PD-L1), an immune checkpoint involved in immune escape. EA in vitro activity was confirmed by the results of in vivo studies showing a significant reduction of the growth rate, infiltrative behavior and tumor-associated angiogenesis of human bladder cancer xenografts. In conclusion, these results suggest that EA may have a potential role as an adjunct therapy for bladder cancer

A phase I study of the safety and tolerability of olaparib (AZD2281, KU0059436) and dacarbazine in patients with advanced solid tumours

BACKGROUND: Poly adenosine diphosphate (ADP)-ribose polymerase (PARP) is essential in cellular processing of DNA damage via the base excision repair pathway (BER). The PARP inhibition can be directly cytotoxic to tumour cells and augments the anti-tumour effects of DNA-damaging agents. This study evaluated the optimally tolerated dose of olaparib (4-(3--4-fluorophenyl) methyl-1(2H)-one; AZD2281, KU0059436), a potent PARP inhibitor, with dacarbazine and assessed safety, toxicity, clinical pharmacokinetics and efficacy of combination treatment. PATIENTS AND METHODS: Patients with advanced cancer received olaparib (20-200 mg PO) on days 1-7 with dacarbazine (600-800 mg m(-2) IV) on day 1 (cycle 2, day 2) of a 21-day cycle. An expansion cohort of chemonaive melanoma patients was treated at an optimally tolerated dose. The BER enzyme, methylpurine-DNA glycosylase and its substrate 7-methylguanine were quantified in peripheral blood mononuclear cells. RESULTS: The optimal combination to proceed to phase II was defined as 100 mg bd olaparib with 600 mg m(-2) dacarbazine. Dose-limiting toxicities were neutropaenia and thrombocytopaenia. There were two partial responses, both in patients with melanoma. CONCLUSION: This study defined a tolerable dose of olaparib in combination with dacarbazine, but there were no responses in chemonaive melanoma patients, demonstrating no clinical advantage over single-agent dacarbazine at these doses

Bioprocess intensification using flow reactors: stereoselective oxidation of achiral 1,3-diols with immobilized Acetobacter Aceti

Enantiomerically enriched 2-hydroxymethylalkanoic acids were prepared by oxidative desymmetrisation of achiral 1,3-diols using immobilized cells of Acetobacter aceti in water at 28 C. The biotransformations were first performed in batch mode with cells immobilized in dry alginate, furnishing the desired products with high molar conversion and reaction times ranging from 2 to 6 h. The biocatalytic process was intensified using a multiphasic flow reactor, where a segmented gas–liquid flow regime was applied for achieving an efficient O2-liquid transfer; the continuous flow systems allowed for high yields and high biocatalyst productivity

Ambient-aware continuous care through semantic context dissemination

Background: The ultimate ambient-intelligent care room contains numerous sensors and devices to monitor the patient, sense and adjust the environment and support the staff. This sensor-based approach results in a large amount of data, which can be processed by current and future applications, e. g., task management and alerting systems. Today, nurses are responsible for coordinating all these applications and supplied information, which reduces the added value and slows down the adoption rate. The aim of the presented research is the design of a pervasive and scalable framework that is able to optimize continuous care processes by intelligently reasoning on the large amount of heterogeneous care data. Methods: The developed Ontology-based Care Platform (OCarePlatform) consists of modular components that perform a specific reasoning task. Consequently, they can easily be replicated and distributed. Complex reasoning is achieved by combining the results of different components. To ensure that the components only receive information, which is of interest to them at that time, they are able to dynamically generate and register filter rules with a Semantic Communication Bus (SCB). This SCB semantically filters all the heterogeneous care data according to the registered rules by using a continuous care ontology. The SCB can be distributed and a cache can be employed to ensure scalability. Results: A prototype implementation is presented consisting of a new-generation nurse call system supported by a localization and a home automation component. The amount of data that is filtered and the performance of the SCB are evaluated by testing the prototype in a living lab. The delay introduced by processing the filter rules is negligible when 10 or fewer rules are registered. Conclusions: The OCarePlatform allows disseminating relevant care data for the different applications and additionally supports composing complex applications from a set of smaller independent components. This way, the platform significantly reduces the amount of information that needs to be processed by the nurses. The delay resulting from processing the filter rules is linear in the amount of rules. Distributed deployment of the SCB and using a cache allows further improvement of these performance results