Chitosan-Based Films with 2-Aminothiophene Derivative: Formulation, Characterization and Potential Antifungal Activity.

In this study, films of chitosan and 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (6CN), a 2-aminothiophene derivative with great pharmacological potential, were prepared as a system for a topical formulation. 6CN-chitosan films were characterized by physicochemical analyses, such as Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and scanning electronic microscopy (SEM). Additionally, the antifungal potential of the films was evaluated in vitro against three species of Candida (C. albicans, C. tropicalis, and C. parapsilosis). The results of the FTIR and thermal analysis showed the incorporation of 6CN in the polymer matrix. In the diffractogram, the 6CN-chitosan films exhibited diffraction halos that were characteristic of amorphous structures, while the micrographs showed that 6CN particles were dispersed in the chitosan matrix, exhibiting pores and cracks on the film surface. In addition, the results of antifungal investigation demonstrated that 6CN-chitosan films were effective against Candida species showing potential for application as a new antifungal drug.

Afferent innervation of the ischemic kidney contributes to renal dysfunction in renovascular hypertensive rats.

The ablation of renal nerves, by destroying both the sympathetic and afferent fibers, has been shown to be effective in lowering blood pressure in resistant hypertensive patients. However, experimental studies have reported that the removal of sympathetic fibers may lead to side effects, such as the impairment of compensatory cardiorenal responses during a hemodynamic challenge. In the present study, we evaluated the effects of the selective removal of renal afferent fibers on arterial hypertension, renal sympathetic nerve activity, and renal changes in a model of renovascular hypertension. After 4 weeks of clipping the left renal artery, afferent renal denervation (ARD) was performed by exposing the left renal nerve to a 33 mM capsaicin solution for 15 min. After 2 weeks of ARD, we found reduced MAP (~ 18%) and sympathoexcitation to both the ischemic and contralateral kidneys in the hypertensive group. Moreover, a reduction in reactive oxygen species was observed in the ischemic (76%) and contralateral (27%) kidneys in the 2K1C group. In addition, ARD normalized renal function markers and proteinuria and podocin in the contralateral kidney. Taken altogether, we show that the selective removal of afferent fibers is an effective method to reduce MAP and improve renal changes without compromising the function of renal sympathetic fibers in the 2K1C model. Renal afferent nerves may be a new target in neurogenic hypertension and renal dysfunction.

Theoretical Investigation of Single-Molecule Sensing Using Nanotube-Enhanced Circular Dichroism.

First-principles calculations have been used to investigate the potential use of circular dichroism (CD) spectroscopy in single-molecule sensing. Using a real-space implementation of time-dependent density functional theory (TDDFT), several systems involving single-walled carbon nanotubes (SWCNT) and small molecules have been studied to evaluate their CD response. Large induced CD (ICD) effects, differing for each test molecule, were observed in all SWCNT-molecule complexes. As the SWCNT used in this study shows no intrinsic CD response, the ICD spectra are the result of interaction with the small molecules. This finding is general and independent of the (a)chiral nature of the adsorbed molecule. Our results indicate that it is possible to design a system that uses SWCNT for detection of molecules using the change in CD spectrum of the system induced by adsorption of the molecule onto the SWCNT surface.

Dynamic Influence of pH on Metalloproteinase Activity in Human Coronal and Radicular Dentin.

The aim of this study was to evaluate the effect of pH on the activation of matrix metalloproteinases (MMPs) of human coronal (CD) and radicular dentin (RD). CD and RD were pulverized to powder, and proteins were extracted with 1% phosphoric acid. The extracted proteins and the demineralized powder were separately incubated in the following solutions: 4-aminophenylmercuric acetate (control) or a buffer solution at different pHs (2.5, 4.5, 5.0, 6.0, and 7.0). After incubation, proteins were separated by electrophoresis to measure MMP activities by zymography. To assess the solubilized dentin collagen, the demineralized dentin powder was sustained in incubation buffer, and the amount of hydroxyproline (HYP) released was measured. Zymography revealed MMP-2 gelatinolytic activities for CD and RD in all experimental groups. For both substrates, the lowest pH solutions (2.5, 4.5, and 5.0) yielded higher gelatinolytic activity than those obtained by the highest pH solutions (6.0 and 7.0). For HYP analysis, no detectable absorbance values were observed for pHs of 2.5 and 4.5. The amount of HYP was higher for pH 7.0 than those of all other groups (p < 0.05), except for pH 6.0. No statistical differences were found between pHs 6.0 and 5.0 and control (p > 0.05). The MMP-2 enzyme from human CD and RD is dynamically influenced by pH: at low pH, the extracted enzyme activates this latent form, whereas collagen degradation by the matrix-bound enzyme is only observed when pHs are close to neutral.

Metabolic remodeling triggered by salivation and diabetes in major salivary glands.

The metabolic profile of major salivary glands was evaluated by 13 C nuclear magnetic resonance isotopomer analysis (13 C NMR-IA) following the infusion of [U-13 C]glucose in order to define the true metabolic character of submandibular (SM) and parotid (PA) glands at rest and during salivary stimulation, and to determine the metabolic remodeling driven by diabetes. In healthy conditions, the SM gland is characterized at rest by a glycolytic metabolic profile and extensive pyruvate cycling. On the contrary, the PA gland, although also dominated by glycolysis, also possesses significant Krebs' cycle activity and does not sustain extensive pyruvate cycling. Under stimulation, both glands increase their glycolytic and Krebs' cycle fluxes, but the metabolic coupling between the two pathways is further compromised to account for the much increased biosynthetic anaplerotic fluxes. In diabetes, the responsiveness of the PA gland to a salivary stimulus is seriously hindered, mostly as a result of the incapacity to burst glycolytic activity and also an inability to improve the Krebs' cycle flux to compensate. The Krebs' cycle activity in the SM gland is also consistently compromised, but the glycolytic flux in this gland is more resilient. This diabetes-induced metabolic remodeling in SM and PA salivary glands illustrates the metabolic need to sustain adequate saliva production, and identifies glycolytic and oxidative pathways as key players in the metabolic dynamism of salivary glands.

Development and Validation of HPLC-DAD and UHPLC-DAD Methods for the Simultaneous Determination of Guanylhydrazone Derivatives Employing a Factorial Design.

Guanylhydrazones are molecules with great pharmacological potential in various therapeutic areas, including antitumoral activity. Factorial design is an excellent tool in the optimization of a chromatographic method, because it is possible quickly change factors such as temperature, mobile phase composition, mobile phase pH, column length, among others to establish the optimal conditions of analysis. The aim of the present work was to develop and validate a HPLC and UHPLC methods for the simultaneous determination of guanylhydrazones with anticancer activity employing experimental design. Precise, exact, linear and robust HPLC and UHPLC methods were developed and validated for the simultaneous quantification of the guanylhydrazones LQM10, LQM14, and LQM17. The UHPLC method was more economic, with a four times less solvent consumption, and 20 times less injection volume, what allowed better column performance. Comparing the empirical approach employed in the HPLC method development to the DoE approach employed in the UHPLC method development, we can conclude that the factorial design made the method development faster, more practical and rational. This resulted in methods that can be employed in the analysis, evaluation and quality control of these new synthetic guanylhydrazones.

Taurine supplementation improves economy of movement in the cycle test independently of the detrimental effects of ethanol.

Taurine (TA) ingestion has been touted as blunting the deleterious effects of ethanol (ET) ingestion on motor performance. This study investigated the effects of ingestion of 0.6 mL·kg-1 of ET, 6 grams of TA, and ethanol in combination with taurine (ET+TA) on economy of movement (EM) and heart rate (HR). Nine volunteers, five female (22 ± 3 years) and four male (26 ± 5 years), participated in a study that used a counterbalanced experimental design. EM and HR were measured for 6 min while the subjects were pedalling at a fixed load 10% below the anaerobic threshold. The blood alcohol concentration (BAC) was similar between ET and ET+TA treatments at 30 min after ingestion and after exercise (12.3 mmol·L-1 vs. 13.7 mmol·L-1, and 9.7 mmol • L-1 vs 10.9 mmol·L-1, respectively). EM was significantly different among treatments, with lower mL·W-1 following ingestion of TA (-7.1%, p<0.001) than placebo and ET+TA (-2.45%, p=0.001) compared to ET. HR (bpm) was significantly (p<0.05) higher for ET (137 ± 14 bpm) than the other three treatments (placebo = 129 ± 14 bpm; TA = 127 ± 11 bpm; TA+ET = 133 ± 12 and ET = 137 ± 14 bpm). Taurine improved EM when compared to placebo or ET, and reduced HR when compared to ET. The combination of ET+TA also enhanced EM compared to placebo, and reduced HR in comparison to ET alone. Therefore, these findings indicate that taurine improves EM and counteracts ethanol-induced increases in HR during submaximal exercise.

Metabolic Acidosis Assessment in High-Risk Surgeries: Prognostic Importance.

BACKGROUND: Metabolic acidosis frequently is present in surgical patients; however, different types of metabolic acidosis (hyperlactatemia, hyperchloremia, and others) may have different relationships to perioperative outcomes. We hypothesized that in postoperative surgical patients, distinctive types of metabolic acidosis would correlate differently with the outcomes of high-risk surgeries. METHODS: A prospective, multicenter observational study was performed in 3 different tertiary care hospitals. Patients who required postoperative admission to the intensive care unit (ICU) were included in this study. Patients with a short life expectancy (those with untreated cancer and limited treatment), hepatic failure, renal failure, or a diagnosis of diabetes were excluded. Patients were classified at ICU admission according to the presence and type of metabolic acidosis into 4 groups: those without acidosis, those with a base excess <-4 mmol/L and albumin-corrected anion gap ≤12 mmol/L (hyperchloremic), those with a base excess <-4 mmol/L and increased albumin-corrected anion gap >12 mmol/L, and those with a base excess <-4 mmol/L and hyperlactatemia >2 mmol/L. Furthermore, patients were reclassified 12 hours after admission to the ICU to verify the metabolic acidosis behavior and outcome differences among the groups. RESULTS: The study included 618 patients. The incidence of acidosis at ICU admission was 59.1%; 23.9% presented with hyperchloremia, 21.3% with hyperlactatemia, 13.9% with increased anion gap, and 40.9% of the patients presented without metabolic acidosis. Patients whose metabolic acidosis persisted for 12 hours had an incidence of ICU complications rates in hyperlactatemia group of 68.8%, increased anion gap of 68.6%, hyperchloremic of 65.8%, and those without acidosis over 12 hours of 59.3%. A Cox regression model for postoperative 30-day mortality showed: in hyperlactatemic acidosis, hazard ratio (HR) = 1.74, 95% confidence interval (CI) = 1.02-2.96; increased anion gap acidosis, HR = 1.68, 95% CI = 0.85-3.81; hyperchloremic acidosis, HR = 1.47, 95% CI = 0.75-2.89, and 10.3% of 30-day mortality rate in patients without acidosis. An adjusted survival curve by Cox regression found a worse 30-day survival in the hyperlactatemic group compared with the other groups (P = .03). Furthermore, in multiple comparisons among groups, patients with hyperlactatemic acidosis were more likely to develop renal dysfunction (P < .001) up to the seventh day postoperatively. CONCLUSIONS: We found that among patients with different types of acidosis, patients who developed hyperlactatemic metabolic acidosis postoperatively showed greater rates of renal dysfunction within 7 days and hyperlactatemic acidosis represented an independent factor on 30-day mortality in high-risk surgical patients.

Assessment of Phenolic Compounds and Anti-Inflammatory Activity of Ethyl Acetate Phase of Anacardium occidentale L. Bark.

The bark of A. occidentale L. is rich in tannins. Studies have described various biological activities of the plant, including antimicrobial, antioxidant, antiulcerogenic and antiinflammatory actions. The objective of this study was to assess the activity of the ethyl acetate phase (EtOAc) of A. occidentale on acute inflammation and to identify and quantify its phenolic compounds by HPLC. The method was validated and shown to be linear, precise and accurate for catechin, epicatechin, epigallocatechin and gallic acid. Swiss albino mice (Mus musculus) were treated with saline, Carrageenan (2.5%), Indomethacin (10 mg/kg), Bradykinin (6 nmol) and Prostaglandine E2 (5 µg) at different concentrations of EtOAc - A. occidentale (12.5; 25; 50; and 100 mg/kg/weight p.o.) for the paw edema test. Challenge was performed with carrageenan (500 µg/mL i.p.) for the doses 50 and 100 mg/kg of EtOAc. Levels of cytokines IL-1, TNF-α, IL-6 and IL-10 were also measured. All EtOAc - A. occidentale concentrations reduced the edema. At 50 and 100 mg/kg, an anti-inflammatory response of the EtOAc was observed. Carrageenan stimulus produced a neutrophil count of 28.6% while 50 and 100 mg/kg of the phase reduced this to 14.5% and 9.1%, respectively. The EtOAc extract reduced levels of IL-1 and TNF-α. These results suggest that the EtOAc plays a modulatory role in the inflammatory response. The chromatographic method can be used for the analysis of the phenolic compounds of the EtOAc phase.

Metabolic evaluations of cancer metabolism by NMR-based stable isotope tracer methodologies.

BACKGROUND: Cancer cells are widely recognized for being able to adapt their metabolism towards converting available nutrients into biomass to increase proliferation rates. MATERIALS AND METHODS: We will review a series of nuclear magnetic resonance (NMR)-based stable isotope tracer methodologies for probing cancer metabolism. RESULTS: The monitoring of such adaptations is of the utmost importance to unravel cancer metabolism and tumour growth. Several major metabolic targets have been recognized as promising foci and have been addressed by multiple studies in recent years. In this work are presented strategies to quantify glycolysis, pentose phosphate pathway, Krebs cycle turnover and de novo lipogenesis by NMR isotopomer analysis. CONCLUSIONS: Being able to adequately define the interplay between metabolic pathways allows the monitoring of their prevalence in tissues and such information is critical for an accurate knowledge of the metabolic distinctive nature of tumours towards devising more efficient antitumorigenic strategies. Discussed methodologies are currently available in the literature, but to date, no single review has compiled all their possible uses, particularly in an interdependent perspective.

Insights into colour-tuning of chlorophyll optical response in green plants.

First-principles calculations within the framework of real-space time-dependent density functional theory have been performed for the complete chlorophyll (Chl) network of the light-harvesting complex from green plants, LHC-II. A local-dipole analysis method developed for this work has made possible the studies of the optical response of individual Chl molecules subjected to the influence of the remainder of the chromophore network. The spectra calculated using our real-space TDDFT method agree with previous suggestions that weak interaction with the protein microenvironment should produce only minor changes in the absorption spectrum of Chl chromophores in LHC-II. In addition, relative shifting of Chl absorption energies leads the stromal and lumenal sides of LHC-II to absorb in slightly different parts of the visible spectrum providing greater coverage of the available light frequencies. The site-specific alterations in Chl excitation energies support the existence of intrinsic energy transfer pathways within the LHC-II complex.

Real-space grids and the Octopus code as tools for the development of new simulation approaches for electronic systems.

Real-space grids are a powerful alternative for the simulation of electronic systems. One of the main advantages of the approach is the flexibility and simplicity of working directly in real space where the different fields are discretized on a grid, combined with competitive numerical performance and great potential for parallelization. These properties constitute a great advantage at the time of implementing and testing new physical models. Based on our experience with the Octopus code, in this article we discuss how the real-space approach has allowed for the recent development of new ideas for the simulation of electronic systems. Among these applications are approaches to calculate response properties, modeling of photoemission, optimal control of quantum systems, simulation of plasmonic systems, and the exact solution of the Schrödinger equation for low-dimensionality systems.

Fish oil provides protection against the oxidative stress in pilocarpine model of epilepsy.

Temporal lobe epilepsy (TLE), the most common form of epilepsy is often resistant to pharmacological treatment. Neuronal loss observed in epileptic brain may be result of an overproduction of free radicals (oxidative stress). Oxidative stress is characterized by an imbalance between antioxidant defenses and oxidizing agents (free radicals), which can lead to tissue injury. The n-3 PUFAs are important for the development and maintenance of central nervous system functions. Research by our group has shown that chronic treatment with fish oil, immediately after status epilepticus (SE), exhibits both neuroprotective effects and effects on neuroplasticity. The main purpose of this research was to evaluate if fish oil exhibits a protective effect against oxidative stress. Animals were subjected to TLE model by pilocarpine administration. After 3 h of SE they were randomly divided into the following groups: control animals treated daily with vehicle or with 85 mg/kg of fish oil and animals with epilepsy treated daily with vehicle or with 85 mg/kg of fish oil. After 90 days, superoxide anion production, enzymatic activity of superoxide dismutase (SOD) and catalase (CAT) and protein expression of NAD(P)H oxidase subunits (p47(PHOX) and gp91(PHOX)) were analyzed. Our results showed evidences that reactive oxygen species are increased in animals with epilepsy and that fish oil supplementation could counteract it. Fish oil supplementation promoted protection against oxidative stress by multiple ways, which involved the reduction of activity and expression of NAD(P)H oxidase subunits and increased the activity and expression of antioxidants enzymes, contributing to well-known neuroprotective effect in epilepsy.

Development and Validation of an HPLC Method for Simultaneous Determination of Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol Hydrochloride in Pharmaceutical Formulations.

Tuberculosis treatment consists of a fixed dose combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PYZ), and ethambutol hydrochloride (EMB). The combined treatment using various drugs is necessary for patient curing, without recrudescence, and for prevention of drug-resistant mutants, which may occur during treatment. An HPLC-diode array detector (DAD) method for the simultaneous determination of RIF, INH, PYZ, and EMB in fixed dose combination tablets was developed and validated. Chromatographic experiments were performed on an Agilent 1200 HPLC system, and the separation was carried out on a Purospher STAR RP18e (250×4.6 mm id, 5 µm, Merck) analytical column. Gradient elution was carried out with a mobile phase of 20 mM monobasic sodium phosphate buffer with 0.2% triethylamine (pH 7.0) and acetonitrile at a flow rate of 1.5 mL/min. The total run time was 12 min, and the re-equilibration time was 5 min. EMB detection was performed at 210 nm, and RIF, INH, and PYZ were detected at 238 nm, using a DAD. The method proved to be specific, linear (r2>0.99), precise (RSD<2%), accurate, and robust and may be applied to the QC analysis of pharmaceutical formulations.

On the influence of water on the electronic structure of firefly oxyluciferin anions from absorption spectroscopy of bare and monohydrated ions in vacuo.

A complete understanding of the physics underlying the varied colors of firefly bioluminescence remains elusive because it is difficult to disentangle different enzyme-lumophore interactions. Experiments on isolated ions are useful to establish a proper reference when there are no microenvironmental perturbations. Here, we use action spectroscopy to compare the absorption by the firefly oxyluciferin lumophore isolated in vacuo and complexed with a single water molecule. While the process relevant to bioluminescence within the luciferase cavity is light emission, the absorption data presented here provide a unique insight into how the electronic states of oxyluciferin are altered by microenvironmental perturbations. For the bare ion we observe broad absorption with a maximum at 548 ± 10 nm, and addition of a water molecule is found to blue-shift the absorption by approximately 50 nm (0.23 eV). Test calculations at various levels of theory uniformly predict a blue-shift in absorption caused by a single water molecule, but are only qualitatively in agreement with experiment highlighting limitations in what can be expected from methods commonly used in studies on oxyluciferin. Combined molecular dynamics simulations and time-dependent density functional theory calculations closely reproduce the broad experimental peaks and also indicate that the preferred binding site for the water molecule is the phenolate oxygen of the anion. Predicting the effects of microenvironmental interactions on the electronic structure of the oxyluciferin anion with high accuracy is a nontrivial task for theory, and our experimental results therefore serve as important benchmarks for future calculations.

The effect of excess fluid balance on the mortality rate of surgical patients: a multicenter prospective study.

INTRODUCTION: In some studies including small populations of patients undergoing specific surgery, an intraoperative liberal infusion of fluids was associated with increasing morbidity when compared to restrictive strategies. Therefore, to evaluate the role of excessive fluid infusion in a general population with high-risk surgery is very important. The aim of this study was to evaluate the impact of intraoperative fluid balance on the postoperative organ dysfunction, infection and mortality rate. METHODS: We conducted a prospective cohort study during one year in four ICUs from three tertiary hospitals, which included patients aged 18 years or more who required postoperative ICU after undergoing major surgery. Patients who underwent palliative surgery and whose fluid balance could change in outcome were excluded. The calculation of fluid balance was based on preoperative fasting, insensible losses from surgeries and urine output minus fluid replacement intraoperatively. RESULTS: The study included 479 patients. Mean age was 61.2 ± 17.0 years and 8.8% of patients died at the hospital during the study. The median duration of surgery was 4.0 (3.2 to 5.5) h and the value of the Simplified Acute Physiology Score (SAPS) 3 score was 41.8 ± 14.5. Comparing survivors and non-survivors, the intraoperative fluid balance from non-survivors was higher (1,950 (1,400 to 3,400) mL vs. 1,400 (1,000 to 1,600) mL, P <0.001). Patients with fluid balance above 2,000 mL intraoperatively had a longer ICU stay (4.0 (3.0 to 8.0) vs. 3.0 (2.0 to 6.0), P <0.001) and higher incidence of infectious (41.9% vs. 25.9%, P = 0.001), neurological (46.2% vs. 13.2%, P <0.001), cardiovascular (63.2% vs. 39.6%, P <0.001) and respiratory complications (34.3% vs. 11.6%, P <0.001). In multivariate analysis, the fluid balance was an independent factor for death (OR per 100 mL = 1.024; P = 0.006; 95% CI 1.007 to 1.041). CONCLUSIONS: Patients with excessive intraoperative fluid balance have more ICU complications and higher hospital mortality.

Marine natural products from the deep Pacific as potential non-linear optical chromophores.

Theoretical analysis using quadratic response theory within the time-dependent density functional theory (TDDFT) formalism shows that the dermacozines, a group of phenazine-based compounds isolated from cultures of Dermacoccus abyssi found in the Mariana Trench, possess large first hyperpolarisability (ß) values at common incident laser wavelengths that are highly sensitive to the degree and type of substitution of the core structure. The phenazine moiety is a versatile and tunable chromophore for non-linear optics and this work serves to highlight the potential that (marine) natural products, even those found in the darkest places on the planet, may have for aiding developments in optical materials design.

Laser irradiation affects enzymatic antioxidant system of streptozotocin-induced diabetic rats.

The aim of the present study was to analyze the effect of low-power laser irradiation in the antioxidant enzymatic system of submandibular (SMG) and parotid (PG) salivary glands of streptozotocin-induced diabetic rats. The animals were randomly divided into six groups: three diabetic groups (D0, D5, and D20) and three non-diabetic groups (C0, C5, and C20), according to laser dose received (0, 5, and 20 J/cm(2), respectively). Areas of approximately 1 cm(2) were demarcated in the salivary glands (each parotid and both submandibular glands) and after irradiated according to Simões et.al. (Lasers Med Sci 24:202-208, 2009). A diode laser (660 nm/100 mW) was used, with laser beam spot of 0.0177 cm(2). The group treated with 5 J/cm(2) laser dose was subjected to irradiation for 1 min and 4 s (total irradiation time) and the group treated with 20 J/cm(2) laser dose was subjected to irradiation for 4 min and 16 s. Twenty-four hours after irradiation the animals were euthanized and the salivary glands were removed for biochemical analysis. The total antioxidant values (TA), the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase enzymes were determined. SOD and CAT activities, as well as TA were higher in SMG of irradiated diabetic rats. However, in SMG of non-diabetic rats, laser irradiation decreased TA values and led to an increase in the CAT activity. In addition, there was a decrease in the activity of CAT in PG of diabetic and non-diabetic animals after laser irradiation. According to the results of the present study, low-power laser irradiation can affect the enzymatic antioxidant system of salivary glands of streptozotocin-induced diabetic rats.