Staphylococcus aureus growth delay after exposure to low fluencies of blue light (470 nm) / Atraso no crescimento de Staphylococcus aureus após exposição à baixas fluências de luz azul (470 nm)

Antibiotic resistance is one of the greatest challenges to treat bacterial infections worldwide, leading to increase in medical expenses, prolonged hospital stay and increased mortality. The use of blue light has been suggested as an innovative alternative to overcome this problem. In this study we analyzed the antibacterial effect of blue light using low emission parameters on Staphylococcus aureus cultures. In vitro bacterial cultures were used in two experimental approaches. The first approach included single or fractionated blue light application provided by LED emitters (470 nm), with the following fluencies: 16.29, 27.16 and 54.32 J/cm2. For the second approach a power LED (470 nm) was used to deliver 54.32 J/cm2 fractionated in 3 applications. Our results demonstrated that bacterial cultures exposed to fractionated blue light radiation exhibited significantly smaller sizes colonies than the control group after 24 h incubation, however the affected bacteria were able to adapt and continue to proliferate after prolonged incubation time. We could conclude that the hypothetical clinical use of low fluencies of blue light as an antibacterial treatment is risky, since its action is not definitive and proves to be ineffective at least for the strain used in this study.

A resistência a antibióticos é um dos maiores desafios para o tratamento de infecções bacterianas em todo o mundo, levando ao aumento de despesas médicas, prolongamento da internação hospitalar e aumento da mortalidade. O uso da luz azul tem sido sugerido como uma alternativa inovadora para superar esse problema. Neste estudo, analisamos o efeito antibacteriano da luz azul usando parâmetros de baixa emissão em culturas de Staphylococcus aureus. Culturas bacterianas foram usadas em duas abordagens experimentais in vitro. A primeira abordagem incluiu o uso da aplicação única ou fracionada de luz azul fornecida por emissores de LED (470 nm), com as seguintes fluências: 16,29, 27,16 e 54,32 J/cm2. Para a segunda abordagem, um LED de potência (470 nm) foi usado para fornecer 54,32 J/cm2 fracionado em 3 aplicações. Nossos resultados demonstraram que as culturas bacterianas expostas à radiação de luz azul fracionada exibiram colônias de tamanhos significativamente menores do que o grupo controle após 24 h de incubação, no entanto, as bactérias afetadas foram capazes de se adaptar e continuar a proliferar após um tempo prolongado de incubação. Podemos concluir que o uso clínico hipotético de baixas fluências de luz azul como tratamento antibacteriano é arriscado, pois sua ação não é definitiva e mostra-se ineficaz, pelo menos para a cepa utilizada neste estudo.

Milk yield, milk composition, and hepatic lipid metabolism in transition dairy cows fed flaxseed or linola.

The response of transition dairy cows to dietary supplementation with fat sources of various fatty acid profiles could affect hepatic fat metabolism differently. Twenty-eight Holstein cows were blocked for similar calving date 4wk before expected parturition to compare the effects of feeding sources of n-3 and n-6 fatty acids on milk production and composition, plasma metabolites, and liver parameters. Cows within each block were assigned to 1 of 3 isonitrogenous and isoenergetic diets: control with a source of calcium salts of palm oil (MEG; 1.1 and 2.6% of the dry matter in prepartum and postpartum diets, respectively); n-3 fatty acids supplied as whole flaxseed (WFL; 4.8 and 7.7% of the dry matter in prepartum and postpartum diets, respectively); and n-6 fatty acids supplied as whole linola (WLO; 4.8 and 7.7% of the dry matter in prepartum and postpartum diets, respectively). Diets were fed until wk 14 of lactation. Contrasts of WFL versus WLO and polyunsaturated fatty acids versus MEG were compared. Cows fed polyunsaturated fatty acids increased dry matter intake over time at a greater extent than those fed MEG, which resulted in enhanced energy balance. Cows fed MEG produced more milk compared with those fed polyunsaturated fatty acids, and there was no difference between those fed WFL and WLO. We found no effect on body condition score and body weight. Plasma concentrations of glucose, fatty acids, and BHB were similar among diets. There was no effect of diet on concentration of glycogen and activities of superoxide dismutase and glutathione peroxidase in the liver. We observed higher concentrations of hepatic lipids and triacylglycerol in cows fed MEG compared with those fed polyunsaturated fatty acids, and no difference between WFL and WLO. Hepatic catalase activity tended to be higher on wk 4 after calving for cows supplemented with WFL compared with those fed WLO. Feeding linoleic and linolenic acids as unprotected oilseeds increased dry matter intake over time at a greater extent for cows fed MEG, improved the energy status, and lowered hepatic lipids and triacylglycerol contents, which may contribute to enhance the health status of transition dairy cows.

Qualitative/quantitative strategy for the determination of glufosinate and metabolites in plants.

A simple method for the simultaneous determination of glufosinate and itsmetabolites in plants based on liquid chromatography­ultraviolet (LC­UV) absorption detection after derivatization with fluorenylmethoxycarbonyl chloride (FMOC-Cl) of some analytes to facilitate separation is reported here. Nonavailable standard metabolites were identified by LC­TOF/mass spectrometry (MS), which also confirmed all target analytes. Ultrasound-assisted extraction was used for sample preparation (power of 70 Wand duty cycle of 0.7 s/s for 10 min) with subsequent evaporation of the extractant, reconstitution and filtration as the cleanup/concentration step prior to derivatization, and chromatographic separation and detection at 270 nm for underivatized analytes and 340 nm for those that were derivatized. The chromatographic analysis was completed in 40 min using a Luna® column (C18 phase). The analytical characteristics of the method were linear dynamic range of the calibration curves within 0.047­700 µg/mL with a regression coefficient (rc) of 0.999 for glufosinate, 0.077­700 µg/mL with a rc of 0.998 for N-acetyl-glufosinate, and 0.116­600 µg/mL with a rc of 0.998 for 3-(methylphosphinico)propanoic acid. The precision for the determination of glufosinate (studied at two levels, 0.1 and 5 µg/mL) was 2.7 and 6.0 % for repeatability and 4.7 and 7.2%for within-laboratory reproducibility, respectively. Identification and confirmatory analysis of the presence of glufosinate and metabolites in the extracts from treated plants was carried out by LC­TOF/MS in high-resolution mode for the precursor ion. The method was validated by analyzing wheat (Triticum aestivum) samples (resistant and susceptible biotypes) treated with 300 g of glufosinate/ha following conventional agronomical practices.

Qualitative/quantitative strategy for the determination of glufosinate and metabolites in plants.

A simple method for the simultaneous determination of glufosinate and its metabolites in plants based on liquid chromatography-ultraviolet (LC-UV) absorption detection after derivatization with fluorenylmethoxycarbonyl chloride (FMOC-Cl) of some analytes to facilitate separation is reported here. Nonavailable standard metabolites were identified by LC-TOF/mass spectrometry (MS), which also confirmed all target analytes. Ultrasound-assisted extraction was used for sample preparation (power of 70 W and duty cycle of 0.7 s/s for 10 min) with subsequent evaporation of the extractant, reconstitution and filtration as the cleanup/concentration step prior to derivatization, and chromatographic separation and detection at 270 nm for underivatized analytes and 340 nm for those that were derivatized. The chromatographic analysis was completed in 40 min using a Luna® column (C18 phase). The analytical characteristics of the method were linear dynamic range of the calibration curves within 0.047-700 µg/mL with a regression coefficient (rc) of 0.999 for glufosinate, 0.077-700 µg/mL with a rc of 0.998 for N-acetyl-glufosinate, and 0.116-600 µg/mL with a rc of 0.998 for 3-(methylphosphinico)propanoic acid. The precision for the determination of glufosinate (studied at two levels, 0.1 and 5 µg/mL) was 2.7 and 6.0 % for repeatability and 4.7 and 7.2 % for within-laboratory reproducibility, respectively. Identification and confirmatory analysis of the presence of glufosinate and metabolites in the extracts from treated plants was carried out by LC-TOF/MS in high-resolution mode for the precursor ion. The method was validated by analyzing wheat (Triticum aestivum) samples (resistant and susceptible biotypes) treated with 300 g of glufosinate/ha following conventional agronomical practices.

Efeito da utilização de diferentes diluidores para a produção in vitro de embriões bovinos / Effect of different bovine semen extenders on in vitro development of bovine embryos

Objetivou-se avaliar as características morfológica e funcional do sêmen bovino congelado comparando-se a eficácia de dois diferentes diluidores. O ejaculado de quatro touros foi dividido em duas partes iguais, uma submetida ao diluidor Tris e gema de ovo (A) e outra ao diluidor à base de lecitina de soja (Andromed®) (B). No experimento I, cinco palhetas dos diluidores A e B de cada touro foram descongeladas e avaliadas quanto à motilidade, vigor, concentração, morfologia espermática e teste de termor-resistência lento. Foram feitas, ainda, avaliação da integridade de membranas, por meio da associação das sondas iodeto de propídio, isotiocionato de fluoresceína - Pisum sativum e carbocianina catiônica lipofílica, e avaliação funcional da membrana plasmática com teste hiposmótico. A avaliação da integridade da cromatina foi realizada pelo método de coloração com laranja de acridina. No experimento II, o sêmen com os diferentes diluidores foi utilizado na fecundação in vitro, sendo observadas taxas de clivagem e desenvolvimento embrionário in vitro. Em relação aos resultados obtidos, apenas a porcentagem de espermatozoides no sêmen congelado foi discretamente maior com o diluidor A, concluindo-se que o diluidor composto por lecitina de soja pode substituir o composto por Tris e gema de ovo, respeitando-se as variações individuais de cada touro utilizado no presente experimento.

The goal of this study was to evaluate the protective effect of egg yolk compared with a soybean lecithin-based extender on morphologic and functional characteristics of frozen bovine semen. The ejaculate of four bulls was divided into two equal parts, one diluted with egg-yolk-Tris extender (A) and the other with a soy-lecithin based extender (Andromed®) (B). In experiment I, five straws of extender A and B from each bull were thawed and assessed regarding motility (subjective and computerized analysis), vigor, concentration, sperm morphology and slow thermoresistance (STR), evaluation of membrane integrity through association of propidium iodite probes (PI), fluorescein isotiocianate - Pisum sativum (FITC-PSA) and lipophilic cationic carbocyanine (JC-1) and functional evaluation of the plasmatic membrane through Hyposmotic Swelling Test (HOST). An evaluation of chromatin integrity was performed with the acridine orange staining procedure. In experiment II, the frozen semen with different extenders was used for in vitro fertilization (IVF), analyzing cleavage rates and in vitro embryo development. No statistical difference between extenders was identified, suggesting that the soy lecithin extender may substitute egg-yolk-Tris extender, considering the individual variations of each bull used in this experiment.

Screening and confirmatory analysis of glyoxylate: a biomarker of plants resistance against herbicides.

The evidence that glyoxylate is a biomarker of tolerance or susceptibility to the action of herbicides belonging to the glycine family makes necessary to develop simple methods for the determination of this metabolite. Glyoxylate level allows both to know the presence/absence of members of the glycine family in plants and plant response to these herbicides. With this aim, a colorimetric-screening method has been developed for determination of glyoxylate based on formation of a phenylhydrazone, then oxidised to red coloured 1,5-diphenylformazan. Simultaneous optimization of ultrasound-assisted extraction of glyoxylate from plants and derivatization by a multivariate design has allowed the determination of the target analyte in fresh plants without interferences from pheophytines and compounds with carbonyl groups. Limits of detection and quantification are 0.05 µg ml(-1) and 0.17 µg ml(-1), respectively, with precision, expressed as relative standard deviation, of 3.3% for repeatability and 5.6% for the within-day laboratory reproducibility. Only 50mg of plant is necessary for determination of glyoxylate within 32 min. Confirmatory analysis by capillary electrophoresis-diode array detection in samples of Lolium spp. subjected to treatment with glyphosate shows that the relative error of the proposed method is always lower than 7%.

Studies on medicinal plants of Ivory Coast: investigation of Sida acuta for in vitro antiplasmodial activities and identification of an active constituent.

Sida acuta Burm. (Malvaceae) originating from Ivory Coast was selected after an ethnobotanical survey: traditional healers of malaria commonly used this plant for the treatment. Extracts were tested on two strains of Plasmodium falciparum: FcM29-Cameroon (chloroquine-resistant strain) and a Nigerian chloroquine-sensitive strain. Extracts were obtained by preparing decoction in water of the powdered plant, the technique used by most of the traditional healers. An ethanol extract was then made and tested. The IC50 values obtained for these extracts ranged from 3.9 to -5.4 microg/ml. Purification of this active fraction led to the identification of cryptolepine as the active antiplasmodial constituent of the plant.

Hyperbaric oxygen therapy protects against mitochondrial dysfunction and delays onset of motor neuron disease in Wobbler mice.

The Wobbler mouse is a model of human motor neuron disease. Recently we reported the impairment of mitochondrial complex IV in Wobbler mouse CNS, including motor cortex and spinal cord. The present study was designed to test the effect of hyperbaric oxygen therapy (HBOT) on (1) mitochondrial functions in young Wobbler mice, and (2) the onset and progression of the disease with aging. HBOT was carried out at 2 atmospheres absolute (2 ATA) oxygen for 1 h/day for 30 days. Control groups consisted of both untreated Wobbler mice and non-diseased Wobbler mice. The rate of respiration for complex IV in mitochondria isolated from motor cortex was improved by 40% (P<0.05) after HBOT. The onset and progression of the disease in the Wobbler mice was studied using litters of pups from proven heterozygous breeding pairs, which were treated from birth with 2 ATA HBOT for 1 h/day 6 days a week for the animals' lifetime. A "blinded" observer examined the onset and progression of the Wobbler phenotype, including walking capabilities ranging from normal walking to jaw walking (unable to use forepaws), and the paw condition (from normal to curled wrists and forelimb fixed to the chest). These data indicate that the onset of disease in untreated Wobbler mice averaged 36+/-4.3 days in terms of walking and 40+/-5.7 days in terms of paw condition. HBOT significantly delayed (P<0.001 for both paw condition and walking) the onset of disease to 59+/-8.2 days (in terms of walking) and 63+/-7.6 days (in terms of paw condition). Our data suggest that HBOT significantly ameliorates mitochondrial dysfunction in the motor cortex and spinal cord and greatly delays the onset of the disease in an animal model of motor neuron disease.

Combined transurethral prostatectomy and mesh-based tension-free inguinal hernia repair.

Previous reports have recognized the benefits of combining prostatic resection and inguinal hernia repair. This study reports the surgical management of bladder-outlet obstruction with simultaneous transurethral prostatectomy and mesh-based tension-free inguinal hernia repair. A prospective study was undertaken of 31 consecutive patients seen from January 1993-December 1998 at the Western Medical Center. All surgery was performed electively under epidural anesthesia, and prophylactic antimicrobial agents were given routinely. Two hernia repair techniques were used: the mesh-plug technique and the Lichtenstein repair. Written informed consent was obtained from all patients. Over a 5-year period, in 31 consecutive patients without urinary tract infection, 36 groin hernias were diagnosed. The mean+/-SD age of patients was 65.9+/-6.3 years. Twenty-four (66.7%) hernias were direct, and 12 (33.3%) were indirect; 61.1% (22) were primary hernias, and 38.8% (14) were recurrent. The mesh-plug and Lichtenstein repair techniques were used to treat 22 (61.1%) and 14 (38.8%) hernias, respectively. Wound hematoma developed after three hernioplasties (8.3%) and wound infection in one (2.7%). Hospital stays ranged between 2 and 4 days. The mean follow-up period was 69 months. The recurrence rate was 2.7% (one hernia). Simultaneous mesh-based tension-free herniorrhaphy and transurethral prostatectomy is a reliable and safe alternative for patients with both prostate enlargement and groin hernia. Hospital stay is not affected by the combined procedure, and the infection rate is acceptably low.

In vitro antiplasmodial activity of extracts of Alchornea cordifolia and identification of an active constituent: ellagic acid.

Extracts of leaves of Alchornea cordifolia were studied for their antiplasmodial activities. Chloroformic and ether extracts were found to be inactive while the ethanolic extract exhibited mild in vitro activity against Plasmodium falciparum. Fractionation of this extract led us to isolate ellagic acid as the active constituent of the extract with IC(50) in the range of 0.2-0.5 microM. Cytotoxicity of ethanolic fraction and ellagic acid was also estimated on human fibroblasts cells (IC(50) on Hela cells = 7.3 microM at 24 h for ellagic acid).

The role of nitric oxide in the pathophysiology of thromboembolic stroke in the rat.

Although nitric oxide (NO) has been shown to play an important role in the pathophysiology of cerebral ischemia, its contribution to the pathogenesis of experimentally induced thromboembolic stroke is unknown. In this study, we pharmacologically manipulated NO levels in the acute post-thrombotic stage and determined the effects on behavior and histopathology. The following drugs were used: nitro-L-arginine-methyl ester (L-NAME), a non-specific endothelial and neuronal nitric oxide synthase (eNOS and nNOS) inhibitor, 3-bromo-7-nitroindazole (7-NI), a specific inhibitor for nNOS, the NO precursor, exogenous L-arginine and the NO-donor, 3-morpholino-sydnonimine (SIN-1). Male Wistar rats (n = 76) were randomly assigned to receive vehicle or drug immediately after common carotid artery thrombosis (CCAT). Regional measurements of cortical NOS activity using the [3H]L-arginine to [3H]L-citrulline conversion assay were decreased 1 h after treatment with L-NAME and 7-NI by 50 and 65%, respectively; hippocampal NOS activity was reduced with L-NAME by 35% and with 7-NI by 65%. L-NAME significantly worsened forelimb placing as compared to other groups. 7-NI accelerated sensorimotor recovery. Water maze retention deficits were noted 48 h after CCAT and these were exacerbated by L-NAME treatment. Histopathological protection was conferred in the hippocampus by 7-NI and SIN-1; conversely, L-NAME increased neuronal injury in the contralateral cortex. L-arginine had no effect on these outcomes. In conclusion, both structural and functional consequences of CCAT can be aggravated by limiting endothelial NO production in the acutely post-thrombotic brain. In contrast, inhibition of nNOS and infusion of an NO donor has a beneficial effect on pathology.

Argon laser-induced arterial photothrombosis. Characterization and possible application to therapy of arteriovenous malformations.

The common carotid, femoral, and middle cerebral arteries in the rat have been occluded thrombotically by means of a rose bengal dye-sensitized photochemical reaction initiated in vascular endothelium by the 514.5-nm beam of an argon laser, focused for maximum excitation efficiency of the photosensitizer according to a derived criterion. The total energy required for vessel occlusion was approximately 1 joule (J) for the middle cerebral artery and 140 to 180 J for the femoral and carotid arteries. At energy fluences (energy deposited per unit area) of 3.5 kJ/sq cm for the middle cerebral artery and 35 kJ/sq cm for the larger arteries, occlusion was observed within 3 minutes. The middle cerebral artery thrombus consisted entirely of aggregated platelets; in the larger arteries the thrombi were composed of platelet aggregates and groups of red blood cells interspersed within a matrix of coagulum. Vessels irradiated similarly in the absence of rose bengal dye displayed no morphological or functional damage. Because the photochemical reaction is mediated by electronic-state transitions, the process of photothrombosis (as opposed to photocoagulation) can be initiated in vessels with high flow rates without the requirement of increased temperature. The photothrombotic technique may be useful in the treatment of arteriovenous malformations owing to its significant enhancement of the efficiency and permanency of vessel occlusion.