Comparison of Efficacy of the Disease-Specific LOX1- and Constitutive Cytomegalovirus-Promoters in Expressing Interleukin 10 through Adeno-Associated Virus 2/8 Delivery in Atherosclerotic Mice

The development of gene therapy vectors for treating diseases of the cardiovascular system continues at a steady pace. Moreover, in the field of gene therapy the utility of “disease-specific promoters” has strong appeal. Many therapeutic genes, including transforming growth factor beta 1 or interleukin 10, are associated to adverse effects. The use of a disease-specific promoter might minimize toxicity. The lectin-like oxidized low density lipoprotein receptor 1 is a marker of cardiovascular disease and a potential therapeutic target. The lectin-like oxidized low density lipoprotein receptor 1 is known to be up-regulated early during disease onset in a number of cell types at the sites where the disease will be clinically evident. In this study an adeno-associated virus-2 DNA vector (AAV2) using the AAV8 capsid, and containing the full length The lectin-like oxidized low density lipoprotein receptor 1 promoter, was generated and assayed for its ability to express human interleukin 10 in low density lipoprotein receptor knockout mice on high cholesterol diet. The cytomegalovirus early promoter was used for comparison in a similarly structured vector. The two promoters were found to have equal efficacy in reducing atherogenesis as measured by aortic systolic blood velocity, aortic cross sectional area, and aortic wall thickness. This is the first head-to-head comparison of a constitutive with a disease-specific promoter in a therapeutic context. These data strongly suggest that the use of a disease-specific promoter is appropriate for therapeutic gene delivery.     

Maternal Care and Infanticide by Males in Helvibis longicauda (Araneae: Theridiidae)

Maternal care in spiders varies from just the construction of a protective silken structure for the eggs and the selection of a safe site to place them, to a long period of association between the mother and spiderlings. Such extended care may involve the active protection from predators and parasitoids, food regurgitation, the production of trophic eggs and even matriphagy. In this study, we describe extended maternal care in Helvibis longicauda (Theridiidae) and evaluate the effectiveness of maternal protection against predators of eggs and spiderlings. We conducted experiments comparing the frequency of egg sac destruction and mortality of spiderlings in the presence and absence of mothers. We also observed the behaviour of the mother and spiderlings during prey capture events and interactions with possible predators. Helvibis longicauda females guard their egg sacs until the emergence of the young and guard the spiderlings for several instar stages, fighting possible predators, including conspecifics. We found that aggressive behaviour by females increased the survival of both eggs and spiderlings in our experiments. Intruder males were the main source of mortality in the absence of females. The benefits of maternal care for the young also include the acquisition of prey items that are captured, immobilized and pre‐digested by the mother. Effective maternal protection and the extended period of supplying food to juveniles probably contribute to the late dispersal of offspring in H. longicauda.     

Antimicrobial activity and antibiotic synergy of a biphosphinic ruthenium complex against clinically relevant bacteria

Abstract

The aim of this study was to investigate the antibacterial activity, antibiotic-associated synergy, and anti-biofilm activity of the ruthenium complex, cis-[RuCl₂ (dppb) (bqdi)]²⁺ (RuNN). RuNN exhibited antimicrobial activity against Gram-positive bacteria with minimum inhibitory concentration (MIC) values ranging from 15.6 to 62.5?µg ml^?1 and minimum bactericidal concentration (MBC) values ranging from 62.5 to 125?µg ml^?1. A synergistic effect against Staphylococcus spp. was observed when RuNN was combined with ampicillin, and the range of associated fractional inhibitory concentration index (FICI) values was 0.187 to 0.312. A time-kill curve indicated the bactericidal activity of RuNN in the first 1–5?h. In general, RuNN inhibited biofilm formation and disrupted mature biofilms. Furthermore, RuNN altered the cellular morphology of S. aureus biofilms. Further, RuNN did not cause hemolysis of erythrocytes. The results of this study provide evidence that RuNN is a novel therapeutic candidate to treat bacterial infections caused by Staphylococcus biofilms.     

Role of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> peroxiredoxins in mitochondrial bioenergetics

Trypanosoma cruzi cytosolic (TcCPx) and mitochondrial tryparedoxin peroxidase (TcMPx) play a fundamental role in H₂O₂ detoxification. Herein, mitochondrial bioenergetics was evaluated in cells that overexpressed TcCPx (CPx) and TcMPx (MPx) and in pTEX. In MPx, a higher expression was observed for TcCPx, and the same correlation was true for CPx. Differences in H₂O₂ release among the overexpressing cells were detected when the mitochondrial respiratory chain was inhibited using antimycin A or thenoyltrifluoroacetone. MPx had higher O₂ consumption rates than pTEX and CPx, especially in the presence of oligomycin. In all of the cells, the mitochondrial membrane potential and the ATP levels were similar. Because of the mild uncoupling that was observed in MPx, the presence or induction of a proton transporter in the mitochondrial membrane is suggested when TcMPx is expressed at higher levels. Our results show a possible interplay between the cytosolic and mitochondrial antioxidant systems in a trypanosomatid.     

Induction of β-1,3-glucanase in seeds of maize defective-kernel mutant (827Kpro1)

β-1,3-glucanases are found in organisms as diverse as plants, animals, bacteria and fungi. In plants, such enzymes are not only associated with defense mechanisms against pathogens, but also play critical roles in physiological and developmental processes. Here we identified a new β-1,3-glucanase in maize seeds, and named it ZmGlucA. Sequence analysis revealed that ZmGlucA belongs to the class A of β-1,3-glucanase, a class related to defense and physiological processes in plants. mRNA and protein assays showed that zmGlucA is expressed exclusively in seeds, and it is differentially regulated during seed development. Additionally, zmGlucA expression is strongly induced in seeds of the mutant dek 827Kpro1, which is defective for embryo and endosperm development. Our data support the idea that ZmGlucA protein is relevant to seed development.     

Differential sympathetic and angiotensinergic responses in rats submitted to low- or high-salt diet

The present study was designed to evaluate, in Wistar rats, the effect of high- or low-salt diet on the hemodynamic parameters and on the renal and lumbar sympathetic nerve activity. The renal gene expression of the renin angiotensin system components was also evaluated, aiming to find some correlation between salt intake, sodium homeostasis and blood pressure increase. Male Wistar rats received low (0.06% Na, TD 92141-Harlan Teklad), a normal (0.5% Na, TD 92140), or a high-salt diet (3.12% Na, TD 92142) from weaning to adulthood. Hemodynamic parameters such as cardiac output and total peripheral resistance, and the renal and lumbar sympathetic nerve activity were determined (n=45). Plasma renin activity, plasma and renal content of angiotensin (ANG) I and II, and the renal mRNA expression of angiotensinogen, renin, AT1 and AT2 receptors were also measured (n=24). Compared to normal- and low-salt diet-, high-salt-treated rats were hypertensive and developed an increase (P<0.05) in total peripheral resistance and lumbar sympathetic nerve activity. A decrease in renal renin and angiotensinogen-mRNAs and in plasma ANG II and plasma renin activity was also found in salt overloaded animals. The renal sympathetic nerve activity was higher (P<0.05) in low- compared to high-salt-treated rats, and was associated with an increase (P<0.05) in renal ANG I and II and with a decrease (P<0.05) in AT2 renal mRNA. Plasma ANG I and II and plasma renin activity were higher in low- than in normal-salt rats. Our results show that increased blood pressure is associated with increases in lumbar sympathetic nerve activity and total peripheral resistance in high-salt-treated rats. However, in low-salt-treated rats an increase in the renal sympathetic nerve was correlated with an increase in the renal content of ANG I and II and with a decrease in AT2 renal mRNA. These changes are probably in favor of the antinatriuretic response and the sodium homeostasis in the low-salt group.     

Intensification of high cell-density cultivations of rE. coli for production of S. pneumoniae antigenic surface protein, PspA3, using model-based adaptive control

This work proposes an innovative methodology to control high density fed-batch cultures of E. coli, based on measurements of the concentration of dissolved oxygen and on estimations of the cellular specific growth rate (μ), of the yield of biomass/limiting substrate (Y (xs)) and of the maintenance coefficient (m). The underlying idea is to allow cells to grow according to their metabolic capacity, without the constraints inherent to pre-set growth rates. Cellular concentration was assessed on-line through a capacitance probe. Three configurations of the control system were compared: (1) pre-set value for the three control parameters; (2) continuously updating μ; (3) updating μ, Y (xs) and m. Implementation of an efficient noise filter for the signal of the capacitance probe was essential for a good performance of the control system. The third control strategy, within the framework of an adaptive model-based control, led to the best results, with biomass productivity reaching 9.2?g(DCW)/L/h.     

Reactive oxygen species derived from NAD(P)H oxidase play a role on ethanol-induced hypertension and endothelial dysfunction in rat resistance arteries

Chronic ethanol consumption is a risk factor for cardiovascular diseases. We studied whether NAD(P)H oxidase-derived reactive oxygen species (ROS) play a role in ethanol-induced hypertension, vascular dysfunction, and protein expression in resistance arteries. Male Wistar rats were treated with ethanol (20 %?v/v) for 6 weeks. Ethanol treatment increased blood pressure and decreased acetylcholine-induced relaxation in the rat mesenteric arterial bed (MAB). These responses were attenuated by apocynin (30 mg/kg/day; p.o. gavage). Ethanol consumption increased superoxide anion (O₂ ^?) generation and decreased nitrate/nitrite (NO _x ) concentration in the rat MAB and apocynin prevented these responses. Conversely, ethanol did not affect the concentration of hydrogen peroxide (H₂O₂) and reduced glutathione (GSH) or the activity of superoxide dismutase (SOD) and catalase (CAT) in the rat MAB. Ethanol increased interleukin (IL)-10 levels in the rat MAB but did not affect the levels of tumor necrosis factor (TNF)-α, IL-6, or IL-1β. Ethanol increased the expression of Nox2 and the phosphorylation of SAPK/JNK, but reduced eNOS expression in the rat MAB. Apocynin prevented these responses. However, ethanol treatment did not affect the expression of Nox1, Nox4, p38MAPK, ERK1/2, or SAPK/JNK in the rat MAB. Ethanol increased plasma levels of TBARS, TNF-α, IL-6, IL-1β, and IL-10, whereas it decreased NO _x levels. The major finding of our study is that NAD(P)H oxidase-derived ROS play a role on ethanol-induced hypertension and endothelial dysfunction in resistance arteries. Moreover, ethanol consumption affects the expression and phosphorylation of proteins that regulate vascular function and NAD(P)H oxidase-derived ROS play a role in such responses.