A co-ultramicronized palmitoylethanolamide/luteolin composite mitigates clinical score and disease-relevant molecular markers in a mouse model of experimental autoimmune encephalomyelitis

Background: Persistent and/or recurrent inflammatory processes are the main factor leading to multiple sclerosis (MS) lesions. The composite ultramicronized palmitoylethanolamide, an endogenous N-acylethanolamine, combined with the flavonoid luteolin, PEALut, have been found to exert neuroprotective activities in experimental models of spinal and brain injury and Alzheimer disease, as well as a clinical improvement in human stroke patients. Furthermore, PEALut enhances the expression of different myelin proteins in oligodendrocyte progenitor cells suggesting that this composite might have protective effects in MS experimental models. Methods: The mouse model of experimental autoimmune encephalomyelitis (EAE) based on active immunization with a fragment of myelin oligodendrocyte glycoprotein (MOG35-55) was used. The daily assessment of clinical score and the expression of serum amyloid A (SAA1), proinflammatory cytokines TNF-\u3b1, IL-1\u3b2, IFN-\u3b3, and NLRP3 inflammasome, as well as TLR2, Fpr2, CD137, CD3-\u3b3, and TCR-\u3b6 chain, heterodimers that form T cell surface glycoprotein (TCR), and cannabinoid receptors CB1, CB2, and MBP, were evaluated in the brainstem and cerebellum at different postimmunization days (PIDs). Results: Vehicle-MOG35-55-immunized (MOG35-55) mice developed ascending paralysis which peaked several days later and persisted until the end of the experiment. PEALut, given intraperitoneally daily starting on day 11 post-immunization, dose-dependently improved clinical score over the range 0.1-5 mg/kg. The mRNA expression of SAA1, TNF-\u3b1, IL-1\u3b2, IFN-\u3b3, and NLRP3 were significantly increased in MOG35-55 mice at 14 PID. In MOG35-55 mice treated with 5 mg /kg PEALut, the increase of SAA1, TNF- \u3b1, IL-1\u3b2, and IFN-\u3b3transcripts at 14 PID was statistically downregulated as compared to vehicle-MOG35-55 mice (p < 0.05). The expression of TLR2, Fpr2, CD137, CD3-\u3b3, TCR-\u3b6 chain, and CB2 receptors showed a significant upregulation in vehicle-MOG35-55 mice at 14 PID. Instead, CB1 and MBP transcripts have not changed in expression at any time. In MOG/PEALut-treated mice, TLR2, Fpr2, CD137, CD3-\u3b3, TCR-\u3b6 chain, and CB2 mRNAs were significantly downregulated as compared to vehicle MOG35-55 mice. Conclusions: The present results demonstrate that the intraperitoneal administration of the composite PEALut significantly reduces the development of clinical signs in the MOG35-55 model of EAE. The dose-dependent improvement of clinical score induced by PEALut was associated with a reduction in transcript expression of the acute-phase protein SAA1, TNF-\u3b1, IL-1\u3b2, IFN-\u3b3, and NLRP3 proinflammatory proteins and TLR2, Fpr2, CD137, CD3-\u3b3, TCR-\u3b6 chain, and CB2 receptors

Process Evaluation of Georgia's Integrated Family Support Demonstration Project: Findings from First Year Implementation Efforts

This report summarizes findings from first year implementation efforts of the Georgia Department of Human Resources' Family Support Demonstration Project. The goal of the project was to reduce child abuse and neglect and improve child health by providing support to Georgia's families with infants and young children at highest risk for child maltreatment

The kinesin Eg5 inhibitor K858 induces apoptosis but also survivin-related chemoresistance in breast cancer cells

Inhibitors of kinesin spindle protein Eg5 are characterized by pronounced antitumor activity. Our group has recently synthesized and screened a library of 1,3,4-thiadiazoline analogues with the pharmacophoric structure of K858, an Eg5 inhibitor. We herein report the effects of K858 on four different breast cancer cell lines: MCF7 (luminal A), BT474 (luminal B), SKBR3 (HER2 like) and MDA-MB231 (basal like). We demonstrated that K858 displayed anti-proliferative activity on every analyzed breast cancer cell line by inducing apoptosis. However, at the same time, we showed that K858 up-regulated survivin, an anti-apoptotic molecule. We then performed a negative regulation of survivin expression, with the utilization of wortmannin, an AKT inhibitor, and obtained a significant increase of K858-dependent apoptosis. These data demonstrate that K858 is a potent inhibitor of replication and induces apoptosis in breast tumor cells, independently from the tumor phenotype. This anti-proliferative response of tumor cells to K858 can be limited by the contemporaneous over-expression of survivin; consequently, the reduction of survivin levels, obtained with AKT inhibitors, can sensitize tumor cells to K858-induced apoptosis

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes – NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases

Expression of Fluorescent Genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): Its Application to Parasite-Vector Biology

Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasitesFil: Guevara, Palmira. Universidad Central de Venezuela; VenezuelaFil: Dias, Manuel. Universidad de los Andes; ColombiaFil: Rojas, Agustina. Universidad de los Andes; ColombiaFil: Crisante, Gladys. Universidad de los Andes; ColombiaFil: Abreu Blanco, Maria Teresa. Universidad Central de Venezuela; VenezuelaFil: Umezawa, Eufrozina. Universidade de Sao Paulo; BrasilFil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Añez, Nestor. Universidad de los Andes; ColombiaFil: Ramirez, Jose Luis. Instituto de Estudios Avanzados; Venezuel

The 4q25 variant rs13143308T links risk of atrial fibrillation to defective calcium homoeostasis

Aims: Single nucleotide polymorphisms on chromosome 4q25 have been associated with risk of atrial fibrillation (AF) but the exiguous knowledge of the mechanistic links between these risk variants and underlying electrophysiological alterations hampers their clinical utility. Here, we tested the hypothesis that 4q25 risk variants cause alterations in the intracellular calcium homoeostasis that predispose to spontaneous electrical activity. Methods and results: Western blotting, confocal calcium imaging, and patch-clamp techniques were used to identify mechanisms linking the 4q25 risk variants rs2200733T and rs13143308T to defects in the calcium homoeostasis in human atrial myocytes. Our findings revealed that the rs13143308T variant was more frequent in patients with AF and that myocytes from carriers of this variant had a significantly higher density of calcium sparks (14.1¿±¿4.5 vs. 3.1¿±¿1.3 events/min, P¿=¿0.02), frequency of transient inward currents (ITI) (1.33¿±¿0.24 vs. 0.26¿±¿0.09 events/min, P¿<¿0.001) and incidence of spontaneous membrane depolarizations (1.22¿±¿0.26 vs. 0.56¿±¿0.17 events/min, P¿=¿0.001) than myocytes from patients with the normal rs13143308G variant. These alterations were linked to higher sarcoplasmic reticulum calcium loading (10.2¿±¿1.4 vs. 7.3¿±¿0.5¿amol/pF, P¿=¿0.01), SERCA2 expression (1.37¿±¿0.13 fold, P¿=¿0.03), and RyR2 phosphorylation at ser2808 (0.67¿±¿0.08 vs. 0.47¿±¿0.03, P¿=¿0.01) but not at ser2814 (0.28¿±¿0.14 vs. 0.31¿±¿0.14, P¿=¿0.61) in patients carrying the rs13143308T risk variant. Furthermore, the presence of a risk variant or AF independently increased the ITI frequency and the increase in the ITI frequency observed in carriers of the risk variants was exacerbated in those with AF. By contrast, the presence of a risk variant did not affect the amplitude or properties of the L-type calcium current in patients with or without AF. Conclusions: Here, we identify the 4q25 variant rs13143308T as a genetic risk marker for AF, specifically associated with excessive calcium release and spontaneous electrical activity linked to increased SERCA2 expression and RyR2 phosphorylation.Peer ReviewedPostprint (author's final draft

pH-Sensitive Chitosan–Heparin Nanoparticles for Effective Delivery of Genetic Drugs into Epithelial Cells

Chitosan has been extensively studied as a genetic drug delivery platform. However, its efficiency is limited by the strength of DNA and RNA binding. Expecting a reduced binding strength of cargo with chitosan, we proposed including heparin as a competing polyanion in the polyplexes. We developed chitosan–heparin nanoparticles by a one-step process for the local delivery of oligonucleotides. The size of the polyplexes was dependent on the mass ratio of polycation to polyanion. The mechanism of oligonucleotide release was pH-dependent and associated with polyplex swelling and collapse of the polysaccharide network. Inclusion of heparin enhanced the oligonucleotide release from the chitosan-based polyplexes. Furthermore, heparin reduced the toxicity of polyplexes in the cultured cells. The cell uptake of chitosan–heparin polyplexes was equal to that of chitosan polyplexes, but heparin increased the transfection efficiency of the polyplexes two-fold. The application of chitosan–heparin small interfering RNA (siRNA) targeted to vascular endothelial growth factor (VEGF) silencing of ARPE-19 cells was 25% higher. Overall, chitosan–heparin polyplexes showed a significant improvement of gene release inside the cells, transfection, and gene silencing efficiency in vitro, suggesting that this fundamental strategy can further improve the transfection efficiency with application of non-viral vectors