In vitro Biological Tests as the First Tools To Validate Magnetic Nanotheranostics for Colorectal Cancer Models

Colorectal cancer (CRC) remains a leading cause of cancer death. Nanotechnology has focused on reaching more effective treatments. In this concern, magnetic nanoparticles (MNPs) have been studied for a wide range of biomedical applications related to CRC, such as diagnostic imaging, drug delivery and thermal therapy. However, limited research is currently found in the open literature that refers to nanosystems combining all these mentioned areas (theranostics). When developing nanosystems intended as theranostics applied to CRC, possible variations between patients must be considered. Therefore, multiple in vitro assays are required as guidance for future preclinical and clinical trials. The objective of this contribution is to evaluate the available and recent literature regarding the interactions of MNP and CRC models, aiming to critically analyze the information given by the commonly used assays and evaluate the data provided by each one with a view to implementing this novel technology in CRC diagnostics and therapy.     

Identification of Kukoamine A,Zeaxanthin, and Clexane as New Furin Inhibitors

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.     

The Impact of Membrane Lipid Composition on Macrophage Activation in the Immune Defense against Rhodococcus equi and Pseudomonas aeruginosa

Nutritional fatty acids are known to have an impact on membrane lipid composition of body cells, including cells of the immune system, thus providing a link between dietary fatty acid uptake, inflammation and immunity. In this study we reveal the significance of macrophage membrane lipid composition on gene expression and cytokine synthesis thereby highlighting signal transduction processes, macrophage activation as well as macrophage defense mechanisms. Using RAW264.7 macrophages as a model system, we identified polyunsaturated fatty acids (PUFA) of both the n-3 and the n-6 family to down-regulate the synthesis of: (i) the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α; (ii) the co-stimulatory molecule CD86; as well as (iii) the antimicrobial polypeptide lysozyme. The action of the fatty acids partially depended on the activation status of the macrophages. It is particularly important to note that the anti-inflammatory action of the PUFA could also be seen in case of infection of RAW264.7 with viable microorganisms of the genera R. equi and P. aeruginosa. In summary, our data provide strong evidence that PUFA from both the n-3 and the n-6 family down-regulate inflammation processes in context of chronic infections caused by persistent pathogens.     

Increase in the length of poly(ethylene oxide) blocks in amphiphilic copolymers facilitates their cellular uptake

Amphiphilic nonionic block copolymers induce different biological effects on living cells. In particular, the hydrophobic members of the Pluronics family suppress drug efflux systems in multidrug‐resistant cells, whereas hydrophilic Pluronics support cell viability. However, the relationship between the copolymer structure and its binding to cells is still unclear. Using a tritium‐labeling approach, we analyzed interactions of nine Pluronics and three diblock copolymers with human and murine cells in vitro and revealed that the binding efficiency of the copolymers increased in line with the length of their poly(ethylene oxide) (PEO) blocks. In contrast, partitioning of the same polymers into artificial lipid bilayers determined by Isothermal Titration Calorimetry (ITC) decreased with increasing length of the PEO block. The opposite influence of hydrophilic blocks on the copolymer affinity to living cells and artificial lipid bilayers implied the binding of long PEO blocks to the hydrophilic moieties of cellular membranes. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45492.     

Fatty acid metabolism of the calanoid copepodParacalanus parvus: 2. Palmitate,stearate, oleate and acetate

The de novo biosynthesis of fatty acids in the wild, calanoid copepodParacalanus parvus was studied. The incubation of labeled acetate proved the de novo biosynthesis of saturated and monounsaturated even fatty acids from 14 to 20 carbons and the 22∶1 acid. Saturated and monounsaturated uneven fatty acids from 15 to 21 carbons were also synthesized. The copepod could not synthesize linoleic and α-linolenic acids. By administration of [1-¹⁴C]palmitate, [1-¹⁴C] stearate and [1-¹⁴C]oleate, it was possible to elucidate the general pattern of the de novo biosynthesis of fatty acids in the wildP. parvus.     

CCL2 Inhibition of Pro-Resolving Mediators Potentiates Neuroinflammation in Astrocytes

The chemokine CCL2 participates in multiple neuroinflammatory processes, mainly through the recruitment of glial cells. However, CCL2 has also been proven to exert different types of actions on these cells, including the modification of their response to inflammatory stimuli. In the present study we analyzed the effect of CCL2 on the resolution of inflammation in astrocytes. We observed that genetic removal of CCL2 increases the expression of the enzymes responsible for the synthesis of specialized pro-resolving mediators arachidonate 15-lipoxygenase and arachidonate 5-lipoxygenase in the brain cortex of 5xFAD mice. The expression of FPR2 receptor, known to mediate the activity of pro-resolving mediators was also increased in mice lacking CCL2.The downregulation of these proteins by CCL2 was also observed in cultured astrocytes. This suggests that CCL2 inhibition of the resolution of inflammation could facilitate the progression of neuroinflammatory processes. The production of the pro-inflammatory cytokine IL-1beta by astrocytes was analyzed, and allowed us to confirm that CCL2 potentiates the activation of astrocytes trough the inhibition of pro-resolving pathways mediated by Resolvin D1. In addition, the analysis of the expression of TNFalpha, MIP1alpha and NOS2 further confirmed CCL2 inhibition of inflammation resolution in astrocytes.     

Cancer-Associated Fibroblasts Regulate Kinase Activity in Mesothelioma Cell Lines via Paracrine Signaling and Thereby Dictate Cell Faith and Behavior

Background: Malignant pleural mesothelioma (MPM) has an infaust prognosis due to resistance to systemic treatment with platin-analoga. MPM cells modulate the immune response to their benefit. They release proinflammatory cytokines, such as TGF-ß, awakening resting fibrocytes that switch their phenotype into activated fibroblasts. Signaling interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an integral part in tumor progression. This study aimed to investigate the role CAFs play in MPM progression, analyzing the impact this complex, symbiotic interaction has on kinase-related cell signaling in vitro. Methods: We simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. NCI-H2052, MSTO-211H, and NCI-H2452 cell lines representing the three mayor MPM subtypes, while embryonal myofibroblast cell lines, IMR-90 and MRC-5, provide a CAFs-like phenotype. Subsequently, differences in proliferation rates, migratory behavior, apoptosis, necrosis, and viability were used as covariates for data analysis. Kinase activity of treated samples and corresponding controls were then analyzed using the PamStation12 platform (PamGene); Results: Treatment with myofibroblast-derived CM revealed significant changes in phosphorylation patterns in MPM cell lines. The observed effect differs strongly between the analyzed MPM cell lines and depends on the origin of CM. Overall, a much stronger effect was observed using CM derived from IMR-90 than MRC-5. The phosphorylation changes mainly affected the MAPK signaling pathway.; Conclusions: The factors secreted by myofibroblasts in fibroblasts CM significantly influence the phosphorylation of kinases, mainly affecting the MAPK signaling cascade in tested MPM cell lines. Our in vitro results indicate promising therapeutic effects by the use of MEK or ERK inhibitors and might have synergistic effects in combination with cisplatin-based treatment, improving clinical outcomes for MPM patients.     

BTK and PI3K Inhibitors Reveal Synergistic Inhibitory Anti-Tumoral Effects in Canine Diffuse Large B-Cell Lymphoma Cells

Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3β, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.     

Mass transfer effects in degradation of bismaleimide matrix composite

The role of mass transfer effects in degradation of carbon fiber reinforced bismaleimides was examined utilizing the concepts of Thiele modulus and effectiveness factor. Unidirectional samples were aged at 250, 260, and 270°C. The weight loss rates from the three types of composite surfaces (resin covered surface, 0° surface, and 90° surface) were obtained from the weight loss data of different sample geometries. As observed previously, the weight loss rate from the resin covered surface showed a typical behavior for a reaction that becomes increasingly diffusion limited. The effective reaction and diffusion coeficients were obtained based on the initial and final weight loss rates, and activation energies for reaction and diffusion were also determined. The activation energy for diffusion was an order of magnitude higher than customary, indicating that phenomena other than diffusion (increase in effective surface area due to cracking) also contributed to the weight loss rate. Overall, this work illustrated the applicability of an unreacted core type model to composite degradation and the importance of accounting for the anisotropy as well as mass transfer effects in composite degradation. © 1996 John Wiley & Sons, Inc.