Early IGF-1 Gene Therapy Prevented Oxidative Stress and Cognitive Deficits Induced by Traumatic Brain Injury.

Traumatic Brain Injury (TBI) remains a leading cause of morbidity and mortality in adults under 40 years old. Once primary injury occurs after TBI, neuroinflammation and oxidative stress (OS) are triggered, contributing to the development of many TBI-induced neurological deficits, and reducing the probability of critical trauma patients´ survival. Regardless the research investment on the development of anti-inflammatory and neuroprotective treatments, most pre-clinical studies have failed to report significant effects, probably because of the limited blood brain barrier permeability of no-steroidal or steroidal anti-inflammatory drugs. Lately, neurotrophic factors, such as the insulin-like growth factor 1 (IGF-1), are considered attractive therapeutic alternatives for diverse neurological pathologies, as they are neuromodulators linked to neuroprotection and anti-inflammatory effects. Considering this background, the aim of the present investigation is to test early IGF-1 gene therapy in both OS markers and cognitive deficits induced by TBI. Male Wistar rats were injected via Cisterna Magna with recombinant adenoviral vectors containing the IGF-1 gene cDNA 15 min post-TBI. Animals were sacrificed after 60 min, 24 h or 7 days to study the advanced oxidation protein products (AOPP) and malondialdehyde (MDA) levels, to recognize the protein oxidation damage and lipid peroxidation respectively, in the TBI neighboring brain areas. Cognitive deficits were assessed by evaluating working memory 7 days after TBI. The results reported significant increases of AOPP and MDA levels at 60 min, 24 h, and 7 days after TBI in the prefrontal cortex, motor cortex and hippocampus. In addition, at day 7, TBI also reduced working memory performance. Interestingly, AOPP, and MDA levels in the studied brain areas were significantly reduced after IGF-1 gene therapy that in turn prevented cognitive deficits, restoring TBI-animals working memory performance to similar values regarding control. In conclusion, early IGF-1 gene therapy could be considered a novel therapeutic approach to targeting neuroinflammation as well as to preventing some behavioral deficits related to TBI.

Amphetamine Induces Oxidative Stress, Glial Activation and Transient Angiogenesis in Prefrontal Cortex via AT1-R.

Background: Amphetamine (AMPH) alters neurons, glia and microvessels, which affects neurovascular unit coupling, leading to disruption in brain functions such as attention and working memory. Oxidative stress plays a crucial role in these alterations. The angiotensin type I receptors (AT1-R) mediate deleterious effects, such as oxidative/inflammatory responses, endothelial dysfunction, neuronal oxidative damage, alterations that overlap with those observed from AMPH exposure. Aims: The aim of this study was to evaluate the AT1-R role in AMPH-induced oxidative stress and glial and vascular alterations in the prefrontal cortex (PFC). Furthermore, we aimed to evaluate the involvement of AT1-R in the AMPH-induced short-term memory and working memory deficit. Methods: Male Wistar rats were repeatedly administered with the AT1-R blocker candesartan (CAND) and AMPH. Acute oxidative stress in the PFC was evaluated immediately after the last AMPH administration by determining lipid and protein peroxidation. After 21 off-drug days, long-lasting alterations in the glia, microvessel architecture and to cognitive tasks were evaluated by GFAP, CD11b and von Willebrand immunostaining and by short-term and working memory assessment. Results: AMPH induced acute oxidative stress, long-lasting glial reactivity in the PFC and a working memory deficit that were prevented by AT1-R blockade pretreatment. Moreover, AMPH induces transient angiogenesis in PFC via AT1-R. AMPH did not affect short-term memory. Conclusion: Our results support the protective role of AT1-R blockade in AMPH-induced oxidative stress, transient angiogenesis and long-lasting glial activation, preserving working memory performance.

Facile synthesis of antibiotic-functionalized gold nanoparticles for colorimetric bacterial detection.

The development of quick and efficient methods for the detection of pathogenic bacteria is urgently needed for the diagnosis of infectious diseases and the control of microbiological contamination in global waterways, potable water sources and the food industry. This contribution will describe the synthesis of gold nanoparticles and their conjugation to broad spectrum, polypeptide and ß-lactam antibiotics that function as both reducing agents and surface protectants (ATB@AuNP). Nanoparticle colloids examined using transmission electron microscopy are generally spherical in shape and range from 2-50 nm in size. Dynamic light scattering and infrared spectroscopy were also used to confirm encapsulation of the AuNP surface by antibiotic molecules. ATB@AuNP were then used to detect 3 common pathogenic bacterial species: Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The colour of the AuNP colloid was monitored visually and using UV-visible spectroscopy. A red shift of the UV visible absorbance and a visible colour change following introduction of each pathogen is indicative of ATB binding to the bacteria surface, ascribed to AuNP agglomeration. This work demonstrates that ATB@AuNP may be an efficient and high throughput tool for the rapid detection of common bacterial contaminants.

Oxidative stress response in reference and clinical Staphylococcus aureus strains under Linezolid exposure.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) strains are some of the most widespread pathogens with multi-resistant to antimicrobial agents (AA). AA provoke several changes inside bacteria, which cannot be solely explained by the main mechanisms of action reported. OBJECTIVE: The role of oxidative stress in bacteria exposed to bacteriostatic AA has not been widely studied; hence, the aim of our work was to investigate the effect of linezolid (LZD) on S. aureus strains. METHODS: Oxidative stress markers, such as superoxide dismutase (SOD) enzyme activity, the global antioxidant response, advanced oxidation protein products (AOPP) and basal levels of glutathione in 28 clinical and 2 reference strains were measured. RESULTS AND CONCLUSIONS: We identified 10 of 30 strains showing a slight increase in reactive species under LZD treatment with respect to the untreated control (between 22% and 56%). Higher generation was detected in clinical strains compared with the reference strains; however, the impact on the antioxidant response was not significant, and the oxidized protein levels were almost undetectable. The strains exposed to this oxazolidinone did not suffer acute oxidative stress. This is the first work reporting the behaviour of clinical and reference strains of S. aureus exposed to LZD, showing negligible oxidative stress.

Novel Antibacterial Resin-Based Filling Material Containing Nanoparticles for the Potential One-Step Treatment of Caries.

The aim of this work was to study the application of resin filling containing nanomaterials for the potential treatment of caries. Zinc nanoparticles (ZnO@NP, 50 nm) were chosen for their antimicrobial capacity against aerobic bacteria, and here, they have proved to be bactericidal against anaerobic bacterial strains (Streptococcus mutans, Streptococcus mitis, and Lactobacillus spp.). Potential mechanism of action is proposed based on microbiological assays and seems to be independent of oxidative stress because the nanoparticles are effective in microaerophilic conditions. The loading of nanoparticles on the demineralized dental surface and their infiltration power were significantly improved when ZnO@NP were carried by the resin. Overall, this material seems to have a high potential to become a one-step treatment for caries lesions.

Cooperative Behavior of Fluoroquinolone Combinations against Escherichia coli and Staphylococcus aureus.

The effects of different combinations of ciprofloxacin (CIP) and norfloxacin (NOR) against Escherichia coli and Staphylococcus aureus were studied using checkerboard, fractional inhibitory concentration (FIC) and time-kill analysis methods. Results obtained by the checkerboard method showed that the more effectives combinations against Escherichia coli were 0.0009 µg/mL CIP+0.0312 µg/mL NOR and 0.0037 µg/mL CIP+0.0075 µg/mL NOR with a FIC index of 0.62. For Staphylococcus aureus, the combination of 0.0625 µg/mL CIP+0.2500 µg/mL NOR showed a synergistic effect, with a FIC index of 0.50. The results of the time-kill method demonstrated either indifference or additivity of the combinations 0.0009 µg/mL CIP+0.0312 µg/mL NOR, 0.0018 µg/mL CIP+0.0312 µg/mL NOR, 0.0037 µg/mL CIP+0.0075 µg/mL NOR and 0.0037 µg/mL CIP+0.0156 µg/mL NOR at 24 h against E. coli. The combination 0.0037 µg/mL CIP+0.0312 µg/mL NOR showed synergistic activity. All the analyzed combinations evidenced bactericidal effects at 4 h. The combinations 0.0625 µg/mL CIP+0.2500 µg/mL NOR and 0.0625 µg/mL CIP+0.0625 µg/mL NOR showed indifference or additivity against S. aureus. None of them generated bactericidal effect at 4 h. Moreover, this last equimolecular combination (equivalent to 1/4 minimum inhibitory concentration (MIC) CIP+1/16 MIC NOR) generated higher reduction of nitro blue tetrazolium than drugs alone. By another way, combinations not equimolecular of CIP and NOR assayed, generated less levels of reactive oxygen species (ROS) than the components alone.

Evaluation of Antibacterial Activity and Reactive Species Generation of N-Benzenesulfonyl Derivatives of Heterocycles.

Two N-benzenesulfonyl (BS) derivatives of 1,2,3,4-tetrahydroquinoline (THQ) were designed, prepared, and screened for antibacterial activity. This approach was based on combining the two privileged structures, BS and THQ, which are known to be active. The objective of this study was to evaluate the antibacterial activity of BS-THQ and its analogue 4-NH2BS-THQ, and to investigate the roles of reactive oxygen species and reactive nitrogen species in their lethality. Both showed bactericidal activity against Staphylococcus aureus ATCC 29213 and methicillin-resistant S. aureus (MRSA) ATCC 43300, with transmission electron microscopy revealing a disturbed membrane architecture. Furthermore, an increase of reactive oxygen species (ROS) in strains treated with BS-THQ with respect to the control was detected when fluorescent microscopy and spectrophotometric techniques were used. The analogue 4-NH2BS-THQ demonstrated a broader spectrum of activity than BS-THQ, with a minimum inhibitory concentration of 100 µg/mL against reference strains of S. aureus, Escherichia coli and Pseudomonas aeruginosa. The assayed compounds represent promising structures for the development of new synthetic classes of antimicrobials.

Aspartame-stabilized gold-silver bimetallic biocompatible nanostructures with plasmonic photothermal properties, antibacterial activity, and long-term stability.

Gold-silver core-shell nanoparticles stabilized with a common sweetener, aspartame (AuNP@Ag@Asm), combine the antimicrobial properties of silver with the photoinduced plasmon-mediated photothermal effects of gold. The particles were tested with several bacterial strains, while biocompatibility was verified with human dermal fibroblasts.

Induction of NADPH oxidase activity and reactive oxygen species production by a single Trypanosoma cruzi antigen.

Trypanosoma cruzi is an intracellular protozoan parasite that predominantly invades mononuclear phagocytes and is able to establish a persistent infection. The production of reactive oxygen species (ROS) by phagocytes is an innate defence mechanism against microorganisms. It has been postulated that ROS such as superoxide anion (O(2)), hydrogen peroxide and peroxynitrite, may play a crucial role in the control of pathogen growth. However, information on parasite molecules able to trigger ROS production is scarce. In this work, we investigated whether cruzipain, an immunogenic glycoprotein from T. cruzi, was able to trigger the oxidative burst by murine cells. By employing chemiluminiscense and flow-cytometric analysis, we demonstrated that cruzipain induced ROS production in splenocytes from non-immune and cruzipain immune C57BL/6 mice and in a Raw 264.7 macrophage cell line. We also identified an O(2)(-) molecule as one of the ROS produced after antigen stimulation. Cruzipain stimulation induced NOX2 (gp91(phox)) and p47(phox) expression, as well as the co-localisation of both NADPH oxidase enzyme subunits. In the current study, we provide evidence that cruzipain not only increased ROS production but also promoted IL-6 and IL-1ß cytokine production. Taken together, we believe these results demonstrate for the first time that cruzipain, a single parasite molecule, in the absence of infection, favors oxidative burst in murine cells. This represents an important advance in the knowledge of parasite molecules that interact with the phagocyte defence mechanism.

Antioxidative mechanisms protect resistant strains of Staphylococcus aureus against ciprofloxacin oxidative damage.

The aim of this investigation was to determine whether the antioxidant defences protect resistant strains of Staphylococcus aureus against ciprofloxacin oxidative damage. Reactive oxygen species (ROS) were determined by chemiluminescence and nitric oxide (NO) was assayed by Griess reaction. The accumulation of ciprofloxacin was examined by fluorometry and oxidation of protein, catalase, ferrous reduction antioxidant potency (FRAP), carbonyls and advanced oxidation protein products (AOPP), studied by spectrophotometry. Ciprofloxacin stimulated higher production of ROS and NO in the susceptible strains than in the resistant ones. There was higher accumulation of antibiotic in sensitive strains than in resistant ones, except for the most resistant strain, which accumulated an elevated amount of antibiotic. The FRAP/ciprofloxacin accumulation ratio of the antibiotic was lower in sensitive than in resistant strains. The most resistant strain exhibited the highest FRAP and presented a high catalase activity. There was oxidation of proteins in the presence of ciprofloxacin, with the carbonyl residues increasing in sensitive and resistant S. aureus. The degradation of carbonyls to AOPP in oxidized proteins was higher in the resistant than in sensitive strains. In conclusion, an increase in antioxidant capacity and a rapid oxidation of carbonyls to AOPP contributed to resistance to ciprofloxacin.

Effect of the association of reduced glutathione and ciprofloxacin on the antimicrobial activity in Staphylococcus aureus.

We report the effect of glutathione and the role of reactive oxygen species (ROS), assayed by a nitro blue tetrazolium reaction, on the antibacterial action of ciprofloxacin, gentamicin and chloramphenicol in Staphylococcus aureus 22 resistant to ciprofloxacin and gentamicin, and in S. aureus ATCC 29213 sensitive to the above three antibiotics. The association of glutathione with ciprofloxacin or gentamicin significantly reduced the value of the minimum inhibitory concentration (MIC) in resistant S. aureus 22, measured using the macrodilution method, with a concomitant increase of intracellular ROS and a decrease of extracellular ROS. However, glutathione did not induce modifications in MIC or ROS generated by chloramphenicol. Furthermore, in the sensitive S. aureus ATCC 29213, the association of glutathione with ciprofloxacin, gentamicin or chloramphenicol did not induce any significant variations of MIC or ROS. There was a correlation between the stimulus of intracellular ROS and the decrease of MIC caused by exogenous glutathione. According to the results obtained, it is possible to modify the sensitivity of resistant strains of S. aureus by the addition of exogenous glutathione.

Lipids and DNA oxidation in Staphylococcus aureus as a consequence of oxidative stress generated by ciprofloxacin.

Ciprofloxacin induced an increment of reactive oxygen species in sensitive strains of Staphylococcus aureus leading to oxidative stress detected by chemiluminescence while resistant strains did not suffer such stress. Oxidation of lipids was performed by employing thiobarbituric acid reaction to detect the formation of the amplified intermediate between reactive species oxygen and cytoplasmic macromolecules, namely malondialdehyde (MDA). The sensitive strain presented higher peroxidation of lipids than the resistant strain. The oxidative consequence for DNA was investigated by means of bacteria incubation with ciprofloxacin and posterior extraction of DNA, which was studied by high performance liquid chromatography (HPLC). Sensitive S. aureus ATCC 29213 showed an increase of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) respect controls without antibiotic; there was evident increase of the ratio between 8-oxodG and deoxyguanosine (dG) as a consequence of oxidation of dG to 8-oxodG considered the major DNA marker of oxidative stress. The resistant strain showed low oxidation of DNA and the analysis of 8-oxodG/dG ratio indicated lesser formation of 8-oxodG than S. aureus ATCC 29213.

Light effect and reactive oxygen species in the action of ciprofloxacin on Staphylococcus aureus.

Oxygen consumption by Staphylococcus aureus ATCC 29213 sensitive to ciprofloxacin was determined with an oxygen selective electrode. Increase in the O(2) consumption was observed with 0.45 micromL(-1) ciprofloxacin while higher concentrations gave rise to a reduction of O(2) consumption. Resistant S. aureus strain did not show increase of O(2) consumption in presence of ciprofloxacin. Nitro Blue Tetrazolium assay showed that production of reactive oxygen species (ROS) increased intracellularly in sensitive bacteria incubated with this antibiotic. The exposition to UV light (360 nm) augmented the intracellular oxidative stress of S. aureus and provoked increment of ROS in extracellular media. Generation of singlet oxygen O(2) ((1)Delta(g)) in S. aureus was measured by means of oxidation of methionine. The absorbance of methionine was monitored at 215 nm and a clear decrease was detected when sensitive S. aureus was stressed with ciprofloxacin. Sodium azide and 2,5-dimethylfuran were used to reinforce the evidence of O(2) ((1)Delta(g)) generation during oxidative stress. Assays with methionine and 2,5-dimethylfuran demonstrated that resistant S. aureus did not increase the production of O(2) ((1)Delta(g)) in the presence of antibiotic. DNA oxidation was investigated in presence of O(2) ((1)Delta(g)) generated by laser excitation of perinaphthenone and subsequent energy transfer. Deactivation of O(2) ((1)Delta(g)) by reaction with DNA of sensitive and resistant bacteria was observed. According to the results obtained, the effect of ciprofloxacin in S. aureus led to an increment of O(2) ((1)Delta(g)) generating oxidative stress in the bacteria.