Determination of post-mortem interval using in situ tissue optical fluorescence

In this study we have used fluorescence spectroscopy to determine the post-mortem interval. Conventional methods in forensic medicine involve tissue or body fluids sampling and laboratory tests, which are often time demanding and may depend on expensive analysis. The presented method consists in using time-dependent variations on the fluorescence spectrum and its correlation with the time elapsed after regular metabolic activity cessation. This new approach addresses unmet needs for post-mortem interval determination in forensic medicine, by providing rapid and in situ measurements that shows improved time resolution relative to existing methods. (C) 2009 Optical Society of America

Antimicrobial photodynamic action on dentin using a light-emitting diode light source

Objective: The aim of this study was the evaluation of two different photosensitizers activated by red light emitted by light-emitting diodes (LEDs) in the decontamination of carious bovine dentin. Materials and Methods: Fifteen bovine incisors were used to obtain dentin samples which were immersed in brain-heart infusion culture medium supplemented with 1% glucose, 2% sucrose, and 1% young primary culture of Lactobacillus acidophilus 108 CFU/mL and Streptococcus mutans 108 CFU/mL for caries induction. Three different concentrations of the Photogem solution, a hematoporphyrin derivative (1, 2, and 3 mg/mL) and two different concentrations of toluidine blue O (TBO), a basic dye (0.025 and 0.1 mg/mL) were used. To activate the photosensitizers two different light exposure times were used: 60 sec and 120 sec, corresponding respectively to the doses of 24 J/cm(2) and 48 J/cm(2). Results: After counting the numbers of CFU per milligram of carious dentin, we observed that the use of LED energy in association with Photogem or TBO was effective for bacterial reduction in carious dentin, and that the greatest effect on S. mutans and L. acidophilus was obtained with TBO at 0.1 mg/mL and a dose of 48 J/cm(2). It was also observed that the overall toxicity of TBO was higher than that of Photogem, and that the phototoxicity of TBO was higher than that of Photogem. Conclusion: Based on our data we propose a mathematical model for the photodynamic effect when different photosensitizer concentrations and light doses are used.

Fluorescence spectroscopy for the detection of tongue carcinoma - validation in an animal model

The efficacy of fluorescence spectroscopy to detect squamous cell carcinoma is evaluated in an animal model following laser excitation at 442 and 532 nm. Lesions are chemically induced with a topical DMBA application at the left lateral tongue of Golden Syrian hamsters. The animals are investigated every 2 weeks after the 4th week of induction until a total of 26 weeks. The right lateral tongue of each animal is considered as a control site (normal contralateral tissue) and the induced lesions are analyzed as a set of points covering the entire clinically detectable area. Based on fluorescence spectral differences, four indices are determined to discriminate normal and carcinoma tissues, based on intraspectral analysis. The spectral data are also analyzed using a multivariate data analysis and the results are compared with histology as the diagnostic gold standard. The best result achieved is for blue excitation using the KNN (K-nearest neighbor, a interspectral analysis) algorithm with a sensitivity of 95.7% and a specificity of 91.6%. These high indices indicate that fluorescence spectroscopy may constitute a fast noninvasive auxiliary tool for diagnostic of cancer within the oral cavity. (C) 2008 Society of Photo-Optical Instrumentation Engineers.

Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida

Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6(1)22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 A resolution.

On the k-restricted structure ratio in planar and outerplanar graphs

A planar k-restricted structure is a simple graph whose blocks are planar and each has at most k vertices. Planar k-restricted structures are used by approximation algorithms for Maximum Weight Planar Subgraph, which motivates this work. The planar k-restricted ratio is the infimum, over simple planar graphs H, of the ratio of the number of edges in a maximum k-restricted structure subgraph of H to the number edges of H. We prove that, as k tends to infinity, the planar k-restricted ratio tends to 1/2. The same result holds for the weighted version. Our results are based on analyzing the analogous ratios for outerplanar and weighted outerplanar graphs. Here both ratios tend to 1 as k goes to infinity, and we provide good estimates of the rates of convergence, showing that they differ in the weighted from the unweighted case.

Prevalence of fibromyalgia in a low socioeconomic status population

Background: The aim of this study was to estimate the prevalence of fibromyalgia, as well as to assess the major symptoms of this syndrome in an adult, low socioeconomic status population assisted by the primary health care system in a city in Brazil. Methods: We cross-sectionally sampled individuals assisted by the public primary health care system (n = 768, 35-60 years old). Participants were interviewed by phone and screened about pain. They were then invited to be clinically assessed (304 accepted). Pain was estimated using a Visual Analogue Scale (VAS). Fibromyalgia was assessed using the Fibromyalgia Impact Questionnaire (FIQ), as well as screening for tender points using dolorimetry. Statistical analyses included Bayesian Statistics and the Kruskal-Wallis Anova test (significance level = 5%). Results: From the phone-interview screening, we divided participants (n = 768) in three groups: No Pain (NP) (n = 185); Regional Pain (RP) (n = 388) and Widespread Pain (WP) (n = 106). Among those participating in the clinical assessments, (304 subjects), the prevalence of fibromyalgia was 4.4% (95% confidence interval [2.6%; 6.3%]). Symptoms of pain (VAS and FIQ), feeling well, job ability, fatigue, morning tiredness, stiffness, anxiety and depression were statically different among the groups. In multivariate analyses we found that individuals with FM and WP had significantly higher impairment than those with RP and NP. FM and WP were similarly disabling. Similarly, RP was no significantly different than NP. Conclusion: Fibromyalgia is prevalent in the low socioeconomic status population assisted by the public primary health care system. Prevalence was similar to other studies (4.4%) in a more diverse socioeconomic population. Individuals with FM and WP have significant impact in their well being.

Antimicrobial mechanisms behind photodynamic effect in the presence of hydrogen peroxide

This study describes the use of methylene blue (MB) plus light (photodynamic inactivation, PDI) in the presence of hydrogen peroxide (H(2)O(2)) to kill Staphylococcus aureus, Escherichia coli, and Candida albicans. When H(2)O(2) was added to MB plus light there was an increased antimicrobial effect, which could be due to a change in the type of ROS generated or increased microbial uptake of MB. To clarify the mechanism, the production of ROS was investigated in the presence and absence of H(2)O(2). It was observed that ROS production was almost inhibited by the presence of H(2)O(2) when cells were not present. In addition, experiments using different sequence combinations of MB and H(2)O(2) were performed and MB optical properties inside the cell were analyzed. Spectroscopy experiments suggested that the amount of MB was higher inside the cells when H(2)O(2) was used before or simultaneously with PDI, and ROS formation inside C. albicans cells confirmed that ROS production is higher in the presence of H(2)O(2). Moreover enzymatic reduction of MB by E. coli during photosensitizer uptake to the photochemically inactive leucoMB could be reversed by the oxidative effects of hydrogen peroxide, increasing ROS formation inside the microorganism. Therefore, the combination of a photosensitizer such as MB and H(2)O(2) is an interesting approach to improve PDI efficiency.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Diversity of free-living marine nematodes (Enoplida) from Baja California assessed by integrative taxonomy

We used morphological and molecular approaches to evaluate the diversity of free-living marine nematodes (order Enoplida) at four coastal sites in the Gulf of California and three on the Pacific coast of Baja California, Mexico. We identified 22 morphological species belonging to six families, of which Thoracostomopsidae and Oncholaimidae were the most diverse. The genus Mesacanthion (Thoracostomopsidae) was the most widespread and diverse. Five allopatric species, genetically and morphologically differentiated, were found in two localities in the Gulf of California (M. sp1 and M. sp2) and three in the Pacific coast (M. sp3, M. sp4 and M. sp5). Overall, we produced 19 and 20 sequences for the 18S and 28S genes, respectively. Neither gene displayed intraspecific polymorphisms, which allowed us to establish that some morphological variation was likely either ontogenetic or due to phenotypic plasticity. Although 18S and 28S phylogenies were topologically congruent (incongruence length difference test, P > 0.05), divergences between species were much higher in the 28S gene. Moreover, this gene possessed a stronger phylogenetic signal to resolve relationships involving Rhabdodemania and Bathylaimus. On the other hand, the close relationship of Pareurystomina (Enchilidiidae) with oncholaimids warrants further study. The 28S sequences (D2D3 domain) may be better suited for DNA barcoding of marine nematodes than those from the 18S rDNA, particularly for differentiating closely related or cryptic species. Finally, our results underline the relevance of adopting an integrative approach encompassing morphological and molecular analyses to improve the assessment of marine nematode diversity and advance their taxonomy.

In vitro organogenesis of Passiflora alata

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO(3) incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA(3) and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.

Evaluation of grinding methods for pellets preparation aiming at the analysis of plant materials by laser induced breakdown spectrometry

It has been demonstrated that laser induced breakdown spectrometry (LIBS) can be used as an alternative method for the determination of macro (P, K. Ca, Mg) and micronutrients (B, Fe, Cu, Mn, Zn) in pellets of plant materials. However, information is required regarding the sample preparation for plant analysis by LIBS. In this work, methods involving cryogenic grinding and planetary ball milling were evaluated for leaves comminution before pellets preparation. The particle sizes were associated to chemical sample properties such as fiber and cellulose contents, as well as to pellets porosity and density. The pellets were ablated at 30 different sites by applying 25 laser pulses per site (Nd:YAG@1064 nm, 5 ns, 10 Hz, 25J cm(-2)). The plasma emission collected by lenses was directed through an optical fiber towards a high resolution echelle spectrometer equipped with an ICCD. Delay time and integration time gate were fixed at 2.0 and 4.5 mu s, respectively. Experiments carried out with pellets of sugarcane, orange tree and soy leaves showed a significant effect of the plant species for choosing the most appropriate grinding conditions. By using ball milling with agate materials, 20 min grinding for orange tree and soy, and 60 min for sugarcane leaves led to particle size distributions generally lower than 75 mu m. Cryogenic grinding yielded similar particle size distributions after 10 min for orange tree, 20 min for soy and 30 min for sugarcane leaves. There was up to 50% emission signal enhancement on LIBS measurements for most elements by improving particle size distribution and consequently the pellet porosity. (C) 2011 Elsevier B.V. All rights reserved.

Exploitation of a single interface flow system for on-line aqueous biphasic extraction

The exploitation of aqueous biphasic extraction is proposed for the first time in flow analysis This extraction strategy stands out for being environmentally attractive since it is based in the utilization of two immiscible phases that are intrinsically aqueous The organic solvents of the traditional liquid-liquid extractions ale no longer used, being replaced by non-toxic, non-flammable and non-volatile ones. A single interface flow analysis (SIFA) system was implemented to carry out the extraction process due to its favourable operational characteristics that include the high automation level and simplicity of operation, the establishment of a dynamic interface where the mass transfer occurred between the two immiscible aqueous phases, and the versatile control over the extraction process namely the extraction time The application selected to demonstrate the feasibility of SIFA to perform this aqueous biphasic extraction was the pre-concentration of lead. After extraction, lead reacted with 8-hydroxyquinoline-5-sulfonic acid and the resulting product was determined by a fluorimetric detector included in the flow manifold. Therefore, the SIFA single interface was used both as extraction (enrichment) and reaction interface. (C) 2010 Elsevier B.V All rights reserved.

Comparison of univariate and multivariate calibration for the determination of micronutrients in pellets of plant materials by laser induced breakdown spectrometry

The application of laser induced breakdown spectrometry (LIBS) aiming the direct analysis of plant materials is a great challenge that still needs efforts for its development and validation. In this way, a series of experimental approaches has been carried out in order to show that LIBS can be used as an alternative method to wet acid digestions based methods for analysis of agricultural and environmental samples. The large amount of information provided by LIBS spectra for these complex samples increases the difficulties for selecting the most appropriated wavelengths for each analyte. Some applications have suggested that improvements in both accuracy and precision can be achieved by the application of multivariate calibration in LIBS data when compared to the univariate regression developed with line emission intensities. In the present work, the performance of univariate and multivariate calibration, based on partial least squares regression (PLSR), was compared for analysis of pellets of plant materials made from an appropriate mixture of cryogenically ground samples with cellulose as the binding agent. The development of a specific PLSR model for each analyte and the selection of spectral regions containing only lines of the analyte of interest were the best conditions for the analysis. In this particular application, these models showed a similar performance. but PLSR seemed to be more robust due to a lower occurrence of outliers in comparison to the univariate method. Data suggests that efforts dealing with sample presentation and fitness of standards for LIBS analysis must be done in order to fulfill the boundary conditions for matrix independent development and validation. (C) 2009 Elsevier B.V. All rights reserved.

Phytomass production and nitrogen accumulation in maize cultivated with different doses of (15)N-urea

This work aimed to study the possible alterations in production, accumulation of the vegetative phytomass and nitrogen efficiency use of the maize crop, in different doses of N applied in the fertilization, by using the technique of isotopic dilution of (15)N. The completely randomized block experimental design was adopted, with 5 treatments and 4 replicates. The following treatments were constituted in the doses in covering: 0, 50, 100, 150 and 200 kg ha(-1) of N, with fertilization of N-urea, respectively. Comparisons among the treatments had been run for crop productivity; nitrogen accumulation for the plant, and use of the nitrogen of the urea-(15)N for the crop. The increase of the dose of N-fertilizer resulted in increase of the dry matter mass, of the dry matter yield crop tax, of the productivity and accumulation of N in the maize plants.

Evaluation of laser induced breakdown spectrometry for the determination of macro and micronutrients in pharmaceutical tablets

The aim of this work is to demonstrate the feasibility of laser induced breakdown spectrometry (LIBS) for the determination of macro and micronutrients in multielement tablets. The experimental setup was designed by using a laser Q-switch (Nd:YAG, 10 Hz, lambda = 1064 nm) and the emission signals were collected by lenses into an optical fiber coupled to an echelle spectrometer equipped with a high-resolution intensified charge coupled device (ICCD). Tablets were cryogenically ground and thereafter pelletized before LIBS analysis. Calibration curves were made by employing samples and mixtures of commercial multielement tablets with binders at different ratios. Best results were achieved by using the following experimental conditions: 29 J cm(-2) laser fluence, 165 mm lens to sample distance (f = 200 mm), 2.0 mu s delay time, 5.0 mu s integration time and 5 accumulated laser pulses. In general, the results obtained by the proposed LIBS procedure were in agreement with those obtained by ICP OES from the corresponding acid digests and coefficients variation of LIBS measurements varied from 2 to 16%. The metrological figures of merit indicate that LIBS fits for the intended purposes, and can be recommended for the analysis of multielement tablets and similar matrices aiming the determination of Ca, Cu, Fe, Mg, Mn, P and Zn.