Optical fiber laser induced fluorescence spectroscopy as a citrus canker diagnostic

Citrus canker is a serious disease caused by Xanthomonas citri subsp. citri bacteria, which infects citrus plants (Citrus spp.) leading to large economic losses in citrus production worldwide. In this work, laser induced fluorescence spectroscopy (LIF) was investigated as a diagnostic technique for citrus canker disease in citrus trees at an orchard using a portable optical fiber based spectrometer. For comparison we have applied LIF to leaves contaminated with citrus canker, citrus scab, citrus variegates chlorosis, and Huanglongbing (HLB, Greening). In order to reduce the noise in the data, we collected spectra from ten leaves with visual symptoms of diseases and from five healthy leaves per plant. This procedure is carried out in order to minimize the environmental effect on the spectrum (water and nutrient supply) of each plant. Our results show that this method presents a high sensitivity (similar to 90%), however it does present a low specificity (similar to 70%) for citrus canker diagnostic. We believe that such poor performance is due to the fact that the optical fiber collects light from only a small part of the leaf. Such results may be improved using the fluorescence imaging technique on the whole leaf. (C) 2010 Optical Society of America

Development and validation of a fast RP-HPLC method to determine the analogue of the thyroid hormone, 3,5,3 '-triiodothyroacetic acid (TRIAC), in polymeric nanoparticles

The objective of this work was to develop and validate a rapid Reversed-Phase High-Performance Liquid Chromatography method for the quantification of 3,5,3 '-triiodothyroacetic acid (TRIAC) in nanoparticles delivery system prepared in different polymeric matrices. Special attention was given to developing a reliable reproductive technique for the pretreatment of the samples. Chromatographic runs were performed on an Agilent 1200 Series HPLC with a RP Phenomenex (R) Gemini C18 (150 x 4, 6 mm i.d., 5 mu m) column using acetonitrile and triethylamine buffer 0.1% (TEA) (40 : 60 v/v) as a mobile phase in an isocratic elution, pH 5.6 at a flow rate of 1 ml min(-1). TRIAC was detected at a wavelength of 220 nm. The injection volume was 20 mu l and the column temperature was maintained at 35 degrees C. The validation characteristics included accuracy, precision, specificity, linearity, recovery, and robustness. The standard curve was found to have a linear relationship (r(2) - 0.9996) over the analytical range of 5-100 mu g ml(-1) . The detection and quantitation limits were 1.3 and 3.8 mu g ml(-1), respectively. The recovery and loaded TRIAC in colloidal system delivery was nearly 100% and 98%, respectively. The method was successfully applied in polycaprolactone, polyhydroxybutyrate, and polymethylmethacrylate nanoparticles.

Cross-Talk Between Mitochondria and NADPH Oxidase: Effects of Mild Mitochondrial Dysfunction on Angiotensin II-Mediated Increase in Nox Isoform Expression and Activity in Vascular Smooth Muscle Cells

Mitochondria and NADPH oxidase activation are concomitantly involved in pathogenesis of many vascular diseases. However, possible cross-talk between those ROS-generating systems is unclear. We induced mild mitochondrial dysfunction due to mitochondrial DNA damage after 24 h incubation of rabbit aortic smooth muscle (VSMC) with 250 ng/mL ethidium bromide (EtBr). VSMC remained viable and had 29% less oxygen consumption, 16% greater baseline hydrogen peroxide, and unchanged glutathione levels. Serum-stimulated proliferation was unaltered at 24 h. Although PCR amplification of several mtDNA sequences was preserved, D-Loop mtDNA region showed distinct amplification of shorter products after EtBr. Such evidence for DNA damage was further enhanced after angiotensin-II (AngII) incubation. Remarkably, the normally observed increase in VSMC membrane fraction NADPH oxidase activity after AngII was completely abrogated after EtBr, together with failure to upregulate Nox1 mRNA expression. Conversely, basal Nox4 mRNA expression increased 1.6-fold, while being unresponsive to AngII. Similar loss in AngII redox response occurred after 24 h antimycin-A incubation. Enhanced Nox4 expression was unassociated with endoplasmic reticulum stress markers. Protein disulfide isomerase, an NADPH oxidase regulator, exhibited increased expression and inverted pattern of migration to membrane fraction after EtBr. These results unravel functionally relevant cross-talk between mitochondria and NADPH oxidase, which markedly affects redox responses to AngII. Antioxid Redox Signal 11, 1265-1278.

Intermolecular vibrations and fast relaxations in supercooled ionic liquids

Short-time dynamics of ionic liquids has been investigated by low-frequency Raman spectroscopy (4 < omega < 100 cm(-1)) within the supercooled liquid range. Raman spectra are reported for ionic liquids with the same anion, bis(trifluoromethylsulfonyl)imide, and different cations: 1-butyl-3-methylimidazolium, 1-hexyl-3-methylimidazolium, 1-butyl-1-methylpiperidinium, trimethylbutylammonium, and tributylmethylammonium. It is shown that low-frequency Raman spectroscopy provides similar results as optical Kerr effect (OKE) spectroscopy, which has been used to study intermolecular vibrations in ionic liquids. The comparison of ionic liquids containing aromatic and non-aromatic cations identifies the characteristic feature in Raman spectra usually assigned to librational motion of the imidazolium ring. The strength of the fast relaxations (quasi-elastic scattering, QES) and the intermolecular vibrational contribution (boson peak) of ionic liquids with non-aromatic cations are significantly lower than imidazolium ionic liquids. A correlation length assigned to the boson peak vibrations was estimated from the frequency of the maximum of the boson peak and experimental data of sound velocity. The correlation length related to the boson peak (similar to 19 angstrom) does not change with the length of the alkyl chain in imidazolium cations, in contrast to the position of the first-sharp diffraction peak observed in neutron and X-ray scattering measurements of ionic liquids. The rate of change of the QES intensity in the supercooled liquid range is compared with data of excess entropy, free volume, and mean-squared displacement recently reported for ionic liquids. The temperature dependence of the QES intensity in ionic liquids illustrates relationships between short-time dynamics and long-time structural relaxation that have been proposed for glass-forming liquids. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3604533]

Exploiting Mn(III)/EDTA complex in a flow system with solenoid micro-pumps coupled to long pathlength spectrophotometry for fast manganese determination

The formation of the Mn(III)/EDTA complex in a flow system with solenoid micro-pumps was exploited for fast manganese determination in freshwater. Manganese(II) was oxidized in a solid-phase reactor containing lead dioxide immobilized on polyester. Long pathlength spectrophotometry was exploited to increase sensitivity, aiming to reach the threshold limit established by environmental legislation. A linear response was observed from 25 to 1500 mu g L(-1), with a detection limit of 6 mu g L(-1) (99.7% confidence level). Sample throughput and coefficient of variation were 36 samples/h and 2.6% (n = 10), respectively. EDTA consumption and waste generation were estimated as 500 mu g and 3 mL per determination, respectively. The amount of Pb in the residue corresponds to 250 mu g per determination and a solid-phase reactor could be used for up to 1600 determinations. Adsorption in active charcoal avoided interferences caused by organic matter and the developed procedure was successfully applied for determination of manganese in freshwater samples. Results were in agreement with those attained by GFAAS at the 95% confidence level. (C) 2010 Elsevier B.V. All rights reserved.

Effect of different resistance-training regimens on the WNT-signaling pathway

The purpose of the present study was to evaluate the effects of 8 weeks of strength and power training on the expression of genes related to the canonical WNT pathway and beta-catenin protein levels in physically active men. Twenty-five subjects (27.4 +/- A 4.6 years) were balanced based on their relative maximum strength in the squat exercise (squat 1RM/body mass) and randomly assigned to strength training (ST) (n = 10), power training (PT) (n = 10), and control (C) (n = 5) groups. The ST and the PT groups performed high and low intensity squats, respectively, thrice a week, for 8 weeks. Muscle biopsies from the vastus lateralis muscle were collected before and after the training period. Relative strength and power increased similarly in both ST and PT groups (P < 0.001). Fiber cross-sectional area also increased similarly in both ST and PT groups. Gene expression and beta-catenin protein expression levels were assessed by real-time PCR and Western blot. Certain genes were up-regulated in the ST group (WNT1: 6.4-fold, P < 0.0001; SFRP1: 3.3-fold, P < 0.0001 and LEF1: 7.3-fold, P < 0.0001) and also in the PT group (WNT1: 24.9-fold, P < 0.0001; SFRP1: 2.7-fold, P < 0.0001; LEF1: 34.1-fold, P < 0.0001 and Cyclin D1: 7.7-fold, P < 0.001). In addition, the expression of key WNT pathway genes was substantially more responsive to PT than to ST (WNT1: P < 0.0001; LEF1: P < 0.0001 and Cyclin D1: P < 0.001). Finally, the total beta-catenin protein content increased only in the PT group (P < 0.05). Our data indicate that a PT regimen triggers greater responses in key elements of the WNT pathway.

EXPRESSION OF GENES RELATED TO MYOSTATIN SIGNALING DURING RAT SKELETAL MUSCLE LONGITUDINAL GROWTH

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin-like 3 protein (FLST-L3), growth and differentiation factor-associated protein-1 (GASP-1), activin IIB receptor (ActIIB), and SMAD-7 were determined by real-time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12-24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST-L3, and GASP-1 mRNA levels increased significantly over all time-points. ActIIB gene expression decreased quickly at 12-24 hours. SMAD-7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching. Muscle Nerve 40: 992-999, 2009

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. Results. We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4,6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. Conclusions. These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.

Effect of bench press exercise intensity on muscle soreness and inflammatory mediators

This study compared four different intensities of a bench press exercise for muscle soreness, creatine kinase activity, interleukin (IL)-1, IL-6, tumor necrosis factor- (TNF-), and prostaglandin E2 (PGE2) concentrations in the blood. Thirty-five male Brazilian Army soldiers were randomly assigned to one of five groups: 50% one-repetition maximum (1-RM), 75% 1-RM, 90% 1-RM, 110% 1-RM, and a control group that did not perform the exercise. The total volume (setsrepetitionsload) of the exercise was matched among the exercise groups. Muscle soreness and plasma creatine kinase activity increased markedly (P0.05) after exercise, with no significant differences among the groups. Serum PGE2 concentration also increased markedly (P0.05) after exercise, with a significantly (P0.05) greater increase in the 110% 1-RM group compared with the other groups. A weak but significant (P0.05) correlation was found between peak muscle soreness and peak PGE2 concentration, but no significant correlation was evident between peak muscle soreness and peak creatine kinase activity, or peak creatine kinase activity and peak PGE2 concentration. All groups showed no changes in IL-1, IL-6 or TNF-. Our results suggest that the intensity of bench press exercise does not affect the magnitude of muscle soreness and blood markers of muscle damage and inflammation.

LEUCINE ATTENUATES SKELETAL MUSCLE WASTING VIA INHIBITION OF UBIQUITIN LIGASES

The aim of this study was to assess the effect of leucine supplementation on elements of the ubiquitin proteasome system (UPS) in rat skeletal muscle during immobilization. This effect was evaluated by submitting the animals to a leucine supplementation protocol during hindlimb immobilization, after which different parameters were determined, including: muscle mass; cross-sectional area (CSA); gene expression of E3 ligases/deubiquitinating enzymes; content of ubiquitinated proteins; and rate of protein synthesis. Our results show that leucine supplementation attenuates soleus muscle mass loss driven by immobilization. In addition, the marked decrease in the CSA in soleus muscle type I fibers, but not type II fibers, induced by immobilization was minimized by leucine feeding. Interestingly, leucine supplementation severely minimized the early transient increase in E3 ligase [muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1] gene expression observed during immobilization. The reduced peak of E3 ligase gene expression was paralleled by a decreased content of ubiquitinated proteins during leucine feeding. The protein synthesis rate decreased by immobilization and was not affected by leucine supplementation. Our results strongly suggest that leucine supplementation attenuates muscle wasting induced by immobilization via minimizing gene expression of E3 ligases, which consequently could downregulate UPS-driven protein degradation. It is notable that leucine supplementation does not restore decreased protein synthesis driven by immobilization. Muscle Nerve 41: 800-808, 2010

Role of beta-alanine supplementation on muscle carnosine and exercise performance

ARTIOLI, G. G., B. GUALANO, A. SMITH, J. STOUT, and A. H. LANCHA, JR. Role of beta-Alanine Supplementation on Muscle Carnosine and Exercise Performance. Med. Sci. Sports Exerc., Vol. 42, No. 6, pp. 1162-1173, 2010. In this narrative review, we present and discuss the current knowledge available on carnosine and beta-alanine metabolism as well as the effects of beta-alanine supplementation on exercise performance. Intramuscular acidosis has been attributed to be one of the main causes of fatigue during intense exercise. Carnosine has been shown to play a significant role in muscle pH regulation. Carnosine is synthesized in skeletal muscle from the amino acids L-histidine and beta-alanine. The rate-limiting factor of carnosine synthesis is beta-alanine availability. Supplementation with beta-alanine has been shown to increase muscle carnosine content and therefore total muscle buffer capacity, with the potential to elicit improvements in physical performance during high-intensity exercise. Studies on beta-alanine supplementation and exercise performance have demonstrated improvements in performance during multiple bouts of high-intensity exercise and in single bouts of exercise lasting more than 60 s. Similarly, beta-alanine supplementation has been shown to delay the onset of neuromuscular fatigue. Although beta-alanine does not improve maximal strength or (V) over dotO(2max), some aspects of endurance performance, such as anaerobic threshold and time to exhaustion, can be enhanced. Symptoms of paresthesia may be observed if a single dose higher than 800 mg is ingested. The symptoms, however, are transient and related to the increase in plasma concentration. They can be prevented by using controlled release capsules and smaller dosing strategies. No important side effect was related to the use of this amino acid so far. In conclusion, beta-alanine supplementation seems to be a safe nutritional strategy capable of improving high-intensity anaerobic performance.

Experimental chronic low-frequency resistance training produces skeletal muscle hypertrophy in the absence of muscle damage and metabolic stress markers

Volitional animal resistance training constitutes an important approach to modeling human resistance training. However, the lack of standardization protocol poses a frequent impediment to the production of skeletal muscle hypertrophy and the study of related physiological variables (i.e., cellular damage/inflammation or metabolic stress). Therefore, the purposes of the present study were: (1) to test whether a long-term and low frequency experimental resistance training program is capable of producing absolute increases in muscle mass; (2) to examine whether cellular damage/inflammation or metabolic stress is involved in the process of hypertrophy. In order to test this hypothesis, animals were assigned to a sedentary control (C, n = 8) or a resistance trained group (RT, n = 7). Trained rats performed 2 exercise sessions per week (16 repetitions per day) during 12 weeks. Our results demonstrated that the resistance training strategy employed was capable of producing absolute mass gain in both soleus and plantaris muscles (12%, p<0.05). Furthermore, muscle tumor necrosis factor (TNF-alpha) protein expression (soleus muscle) was reduced by 24% (p<0.01) in trained group when compared to sedentary one. Finally, serum creatine kinase (CK) activity and serum lactate concentrations were not affected in either group. Such information may have practical applications if reproduced in situations where skeletal muscle hypertrophy is desired but high mechanical stimuli of skeletal muscle and inflammation are not. Copyright (C) 2010 John Wiley & Sons, Ltd.

The effect of 5 days of aspartate and asparagine supplementation on glucose transport activity in rat muscle

The consumption of protein supplements containing amino acids is increasing around the world Aspartate (Asp) and asparagine (Asn) are amino acids metabolized by skeletal muscle. This metabolism involves biochemical pathways that are involved in increasing Krebs cycle activity via anaplerotic reactions. resulting in higher glutamine concentrations. A connection between amino acid supplementation, glycogen concentration, and glucose uptake has been previously demonstrated. The purpose of this study was to evaluate the effect of asp and Asn Supplementation on glucose uptake in rats using three different glycogen concentrations The results indicate that Asp and Asn supplementation in rats with high glycogen concentrations (fed state) further increased the glycogen concentration in the muscle, and decreased in vitro 2-deoxyglucose (a glucose analog.) uptake by the muscle at maximal insulin concentrations When animals had a medium glycogen concentration (consumed lard for 3 days). glucose uptake was higher in the supplemented group at sub-maximal insulin concentrations. We conclude that supplementation of Asp and Asn reduced glucose transport in rat muscle only at higher levels of glycogen. The ingestion of lard for 3 days changed the responsiveness and sensitivity to insulin, and that group had higher levels of insulin sensivity with Asp and Asn supplementation. Copyright (C) 2009 John Wiley & Sons, Ltd.

SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence of or defective muscular proteins. The murine model for limb-girdle muscular dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4-6 weeks and is nearly complete by 8 months of age. The possibility of restoring the defective muscle protein and improving muscular performance by cell therapy is a promising approach for the treatment of LGMDs or other forms of progressive muscular dystrophies. Here we have injected human adipose stromal cells (hASCs) into the SJL mice, without immunosuppression, aiming to assess their ability to engraft into recipient dystrophic muscle after systemic delivery; form chimeric human/mouse muscle fibers; express human muscle proteins in the dystrophic host and improve muscular performance. We show for the first time that hASCs are not rejected after systemic injection even without immunosuppression, are able to fuse with the host muscle, express a significant amount of human muscle proteins, and improve motor ability of injected animals. These results may have important applications for future therapy in patients with different forms of muscular dystrophies.

Radicicol improves regeneration of skeletal muscle previously damaged by crotoxin in mice

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured Muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom. (C) 2008 Elsevier Ltd. All rights reserved.