Detection of citrus canker in citrus plants using laser induced fluorescence spectroscopy

Citrus canker is a serious disease caused by Xanthomonas citri subsp. citri bacteria, which infects citrus plants (Citrus spp.) leading to a large economic loss in citrus production worldwide. In Brazil citrus canker control is done by an official eradication campaign, therefore early detection of such disease is important to prevent greater economic losses. However, detection is difficult and so far it has been done by visual inspection of each tree. Suspicious leaves from citrus plants in the field are sent to the laboratory to confirm the infection by laboratory analysis, which is a time consuming. Our goal was to develop a new optical technique to detect and diagnose citrus canker in citrus plants with a portable field spectrometer unit. In this paper, we review two experiments on laser induced fluorescence spectroscopy (LIF) applied to detect citrus canker. We also present new data to show that the length of time a leaf has been detached is an important variable in our studies. Our results show that LIF has the potential to be applied to citrus plants.

Fluorescence spectroscopy to diagnose hepatic steatosis in a rat model of fatty liver

Steatosis is diagnosed on the basis of the macroscopic aspect of the liver evaluated by the surgeon at the time of organ extraction or by means of a frozen biopsy. In the present study, the applicability of laser-induced fluorescence (LIF) spectroscopy was investigated as a method for the diagnosis of different degrees of steatosis experimentally induced in rats. Rats received a high-lipid diet for different periods of time. The animals were divided into groups according to the degree of induced steatosis diagnosis by histology. The concentration of fat in the liver was correlated with LIF by means of the steatosis fluorescence factor (SFF). The histology classification, according to liver fat concentration was, Severe Steatosis, Moderate Steatosis, Mild Steatosis and Control (no liver steatosis). Fluorescence intensity could be directly correlated with fat content. It was possible to estimate an average of fluorescence intensity variable by means of different confidence intervals (P=95%) for each steatosis group. SFF was significantly higher in the Severe Steatosis group (P < 0.001) compared with the Moderate Steatosis, Mild Steatosis and Control groups. The various degrees of steatosis could be directly correlated with SFF. LIF spectroscopy proved to be a method capable of identifying the degree of hepatic steatosis in this animal model, and has the potential of clinical application for non-invasive evaluation of the degree of steatosis.

Temperature dependence of molecular dynamics and supramolecular aggregation in MEH-PPV films: A solid-state NMR, X-ray and fluorescence spectroscopy study

This article presents an investigation of the temperature induced modification in the microstructure and dynamics of poly[2-methoxy-5-(2`-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) cast films using Wide-Angle X-ray Scattering (WAXS), solid-state Nuclear Magnetic Resonance (NMR), and Fluorescence Spectroscopy (PL). MEH-PPV chain motions were characterized as a function of temperature by NMR. The results indicated that the solvent used to cast the films influences the activation energy of the side-chain motions. This was concluded from the comparison of the activation energy of the toluene cast film, E(a) = (54 +/- 8) kJ/mol, and chloroform cast film, E(a) = (69 +/- 5) kJ/mol, and could be attributed to the higher side-chain packing provided by chloroform, that preferentially solvates the side chain in contrast to toluene that solvates mainly the backbone. Concerning the backbone mobility, it was observed that the torsional motions in the MEH-PPV have average amplitude of similar to 10 degrees at 300 K, which was found to be independent of the solvent used to cast the films. In order to correlate the molecular dynamics processes with the changes in the microstructure of the polymer, in situ WAXS experiments as a function of temperature were performed and revealed that the interchain spacing in the MEH-PPV molecular aggregates increases as a function of temperature, particularly at temperatures where molecular relaxations occur. It was also observed that the WAXS peak associated with the bilayer spacing becomes narrower and its intensity increases whereas the peak associated with the inter-backbone planes reduces its intensity for higher temperatures. This last result Could be interpreted as a decrease in the number of aggregates and the reduction of the interchain species during the MEH-PPV relaxation processes. These WAXS results were correlated with PL spectra modifications observed upon temperature treatments. (C) 2008 Elsevier Ltd. All rights reserved.

Noninvasive Evaluation of Oral Lesions Using Depth-sensitive Optical Spectroscopy

BACKGROUND: Optical spectroscopy is a noninvasive technique with potential applications for diagnosis of oral dysplasia and early cancer. In this study, we evaluated the diagnostic performance of a depth-sensitive optical spectroscopy (DSOS) system for distinguishing dysplasia and carcinoma from non-neoplastic oral mucosa. METHODS: Patients with oral lesions and volunteers without any oral abnormalities were recruited to participate. Autofluorescence and diffuse reflectance spectra of selected oral sites were measured using the DSOS system. A total of 424 oral sites in 124 subjects were measured and analyzed, including 154 sites in 60 patients with oral lesions and 270 sites in 64 normal volunteers. Measured optical spectra were used to develop computer-based algorithms to identify the presence of dysplasia or cancer. Sensitivity and specificity were calculated using a gold standard of histopathology for patient sites and clinical impression for normal volunteer sites. RESULTS: Differences in oral spectra were observed in: (1) neoplastic versus nonneoplastic sites, (2) keratinized versus nonkeratinized tissue, and (3) shallow versus deep depths within oral tissue. Algorithms based on spectra from 310 nonkeratinized anatomic sites (buccal, tongue, floor of mouth, and lip) yielded an area under the receiver operating characteristic curve of 0.96 in the training set and 0.93 in the validation set. CONCLUSIONS: The ability to selectively target epithelial and shallow stromal depth regions appeared to be diagnostically useful. For nonkeratinized oral sites, the sensitivity and specificity of this objective diagnostic technique were comparable to that of clinical diagnosis by expert observers. Thus, DSOS has potential to augment oral cancer screening efforts in community settings. Cancer 2009;115:1669-79. (C) 2009 American Cancer Society.

Autofluorescence spectroscopy in liver transplantation: Preliminary results from a pilot clinical study

The evaluation of graft function at various stages after transplantation is relevant, particularly at the moment of organ harvest, when a decision must be made whether to use the organ. Autofluorescence spectroscopy is noninvasive technique to monitor the metabolic condition of a liver graft throughout its course, from an initial evaluation in the donor, through cold ischemia transportation, to reperfusion and reoxygenation in the recipient. Preliminary results are presented in six liver transplantations spanning the periods from liver harvest to implant. The laser-induced fluorescence spectrum at 532-mn excitation was investigated before cold perfusion (autofluorescence), during cold ischemia, at the back table procedure, as well as 5 and 60 minutes after reperfusion. The results showed that the fluorescence analysis was sensitive to changes during the transplantation procedure. Fluorescence spectroscopy potentially provides a real-time, noninvasive technique to monitor liver graft function. The information could potentially be valuable for surgical decisions and transplant success.

Study of Blood Porphyrin Spectral Profile for Diagnosis of Chronic Renal Failure

The progression to end-stage renal failure is independent of the initial pathogenic mechanism. Metabolic acidosis is a common consequence of chronic renal failure that results from inadequate ammonium excretion and decreased tubular bicarbonate reabsorption. Protoporphyrin IX (PpIX) is the immediate metabolic precursor of the heme molecule. The purpose of this study was to evaluate the levels of erythrocytes protoporphyrin IX at an animal model during progressive renal disease. A total of 36 eight-week-old male Wistar rats were divided into six groups: Normal, 4 and 8 weeks after 5/6 nephrectomy (NX). Renal function was evaluated by creatinine clearance and plasma creatinine levels. The autofluorescence of erythrocytes porphyrin of healthy and NX rats was analyzed using fluorescence spectroscopy. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences between normal and NX rats autofluorescence shape occurred in the 600-700 nm spectral region. A correlation was observed between emission band intensity at 635 nm and progression of renal disease.

Synthesis and characterization of phosphate glass microspheres for radiotherapy applications

Class microspheres containing the radioisotope (32)P, a beta(-) particle emitter, and half-life of 14.3 days, can be easily introduced in specific human organs such as liver, pancreas. and uterus to kill cancer cells. In the present work phosphate glass microspheres were produced with different compositions and particle size distribution in the range of 20- 30 mu m. Two different thermal processes were used to spherodize glass particles originally with irregular shapes. Samples were characterized by X-rays diffraction to check the amorphous structure, energy dispersive X-rays fluorescence spectroscopy to determine the final glass composition, and Fourier transformed infrared spectroscopy to determine the structural groups in the glass structure. The dissolution rate of glass samples in water was determined at 90 degrees C, and in simulated body fluid (SBF) at 37 degrees C. Classes with dissolution rates close to 10(-5) g/(cm(2) day) were obtained, which make them suitable for the present application. Scanning electron microscopy was used to evaluate the shape of the microspheres before and after the dissolution tests. The cytotoxicity tests showed that these microspheres can be used for biological applications. (C) 2008 Elsevier B.V. All rights reserved.

Phototransformation of hematoporphyrin in aqueous solution: Anomalous behavior at low oxygen concentration

The photoactivation of a photosensitizer is the initial step in photodynamic therapy (PDT) where photochemical reactions result in the production of reactive oxygen species and eventually cell death. In addition to oxidizing biomolecules, some of these photochemical reactions lead to photosensitizer degradation at a rate dependent on the oxygen concentration among other factors. We investigated photodegradation of Photogem A (R) (28 mu M), a hematoporphyrin derivative, at different oxygen concentrations (9.4 to 625.0 mu M) in aqueous solution. The degradation was monitored by fluorescence spectroscopy. The degradation rate (M/s) increases as the oxygen concentration increases when the molar ratio of oxygen to PhotogemA (R) is greater than 1. At lower oxygen concentrations (< 25 mu M) an inversion of this behavior was observed. The data do not fit a simple kinetic model of first-order dependence on oxygen concentration. This inversion of the degradation rate at low oxygen concentration has not previously been demonstrated and highlights the relationship between photosensitizer and oxygen concentrations in determining the photobleaching mechanism(s). The findings demonstrate that current models for photobleaching are insufficient to explain completely the effects at low oxygen concentration.

Histopathology and laser autofluorescence of ischemic kidneys of rats

The purpose of this research was to evaluate the severity of renal ischemia/reperfusion injury as determined by histology and by laser-induced fluorescence (LIF) with excitation wavelengths of 442 nm and 532 nm. Wistar rats (four groups of six animals) were subjected to left renal warm ischemia for 20, 40, 60 and 80 min followed by 10 min of reperfusion. Autofluorescence was determined before ischemia (control) and then every 5-10 min thereafter. Tissue samples for histology were harvested from the right kidney (control) and from the left kidney after reperfusion. LIF and ischemia time showed a significant correlation (p < 0.0001 and r (2)=0.47, and p=0.006 and r (2)=0.25, respectively, for the excitation wavelengths of 442 nm and 532 nm). Histological scores showed a good correlation with ischemia time (p < 0.0001). The correlations between optical spectroscopy values and histological damage were: LIF at 442 nm p < 0.0001, LIF at 532 nm p=0.001; IFF (peak of back scattered light/LIF) at 442 nm p > 0.05, and IFF at 532 nm p > 0.05. After reperfusion LIF tended to return to preischemic basal levels which occurred in the presence of histological damage. This suggests that factors other than morphological alterations may have a more relevant effect on changes observed in LIF. In conclusion, renal ischemia/reperfusion changed tissue fluorescence induced by laser. The excitation light of 442 nm showed a better correlation with the ischemia time and with the severity of tissue injury.

Immobilization of Alcohol Dehydrogenase in Phospholipid Langmuir-Blodgett Films To Detect Ethanol

Enzyme immobilization in nanostructured films may be useful for a number of biomimetic systems, particularly if suitable matrixes are identified. Here we show that alcohol dehydrogenase (ADH) has high affinity toward a negatively charged phospholipid, dimyristoylphosphatidic acid (DMPA), which forms a Langmuir monolayer at an air-water interface. Incorporation of ADH into the DMPA monolayer was monitored with Surface pressure measurements; and polarization-modulation infrared reflection absorption spectroscopy, with the alpha-helices from ADH being mainly oriented parallel to the water surface. ADH remained at the interface even at high surface pressures, thus allowing deposition of Langmuir-Blodgett (LB) films from the DMPA-ADH film. Indeed, interaction with DMPA enhances the transfer of ADH, where the mass transferred onto a solid support increased from 134 ng for ADH on a Gibbs monolayer to 178 ng for an LB film with DMPA. With fluorescence spectroscopy it was possible to confirm that the ADH structure was preserved even after one month of the LB deposition. ADH-containing films deposited onto gold-interdigitated electrodes were employed in a sensor array capable of detecting ethanol at concentrations down to 10 ppb (in volume), using impedance spectroscopy as the method of detection.

Chitosan as a removing agent of beta-lactoglobulin from membrane models

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

Non-homogeneous liver distribution of photosensitizer and its consequence for photodynamic therapy outcome

Background: Photodynamic therapy is mainly used for treatment of malignant lesions, and is based on selective location of a photosensitizer in the tumor tissue, followed by light at wavelengths matching the photosensitizer absorption spectrum. In molecular oxygen presence, reactive oxygen species are generated, inducing cells to die. One of the limitations of photodynamic therapy is the variability of photosensitizer concentration observed in systemically photosensitized tissues, mainly due to differences of the tissue architecture, cell lines, and pharmacokinetics. This study aim was to demonstrate the spatial distribution of a hematoporphyrin derivative, Photogem(R), in the healthy liver tissue of Wistar rats via fluorescence spectroscopy, and to understand its implications on photodynamic response. Methods: Fifteen male Wistar rats were intravenously photosensitized with 1.5 mg/kg body weight of Photogem(R). Laser-induced fluorescence spectroscopy at 532nm-excitation was performed on ex vivo liver slices. The influence of photosensitizer surface distribution detected by fluorescence and the induced depth of necrosis were investigated in five animals. Results: Photosensitizer distribution on rat liver showed to be greatly non-homogeneous. This may affect photodynamic therapy response as shown in the results of depth of necrosis. Conclusions: As a consequence of these results, this study suggests that photosensitizer surface spatial distribution should be taken into account in photodynamic therapy dosimetry, as this will help to better predict clinical results. (C) 2010 Elsevier B.V. All rights reserved.

Mixing alternating copolymers containing fluorenyl groups with phospholipids to obtain Langmuir and Langmuir-Blodgett films

The control of molecular architectures may be essential to optimize materials properties for producing luminescent devices from polymers, especially in the blue region of the spectrum. In this Article, we report on the fabrication of Langmuir-Blodgett (LB) films of polyfluorene copolymers mixed with the phospholipid dimyristoyl phosphatidic acid (DMPA). The copolymers poly(9.9-dioetylfluorene)-co-phenylene (copolymer I) and poly(9,9-dioctylfluorene)-co-quaterphenylene) (copolymer 2) were synthesized via Suzuki reaction. Copolymer I could not form a monolayer on its own, but it yielded stable films when mixed with DMPA. In contrast, Langmuir monolayers could be formed from either the neat copolymer 2 or when mixed with DMPA. The surface pressure and surface potential measurements, in addition to Brewster angle microscopy, indicated that DMPA provided a suitable matrix for copolymer I to form a stable Langmuir film, amenable to transfer as LB films, while enhancing the ability of copolymer 2 to form LB films with enhanced emission, as indicated by fluorescence spectroscopy. Because a high emission was obtained with the mixed LB films and since the molecular-level interactions between the film components can be tuned by changing the experimental conditions to allow For further optimization, one may envisage applications of these films in optical devices such as organic light-emitting diodes (OLEDs).

Isolation of a lectin and a galactoxyloglucan from Mucuna sloanei seeds

A lectin and a galactoxyloglucan were characterized from Mucuna sloanei seed cotyledons. The galactoxyloglucan, isolated by water extraction and ethanol precipitation, had Glc:Xyl:Gal proportions in a molar ratio of 1.8:1.7:1.0 and a molar mass (M(w)) of 1.6 x 10(6) g mol(-1). The lectin (sloanin), isolated from the same seed by affinity chromatography on cross-linked Adenanthera pavonina galactomannan, gave two protein bands by SDS-PAGE (36 and 34 kDa) and one peak by gel filtration (63.6 kDa). Its N-terminal sequence indicated similar to 69% identity with soybean agglutinin to leguminous lectins. Circular dichroism (CD) spectra established that sloanin predominantly contains beta-sheet structures. Sloanin has similar to 5.5% carbohydrate and displayed hemagglutinating activity against rabbit and enzyme treated human erythrocytes, inhibited only by D-Gal containing sugars. The interaction between sloanin and storage cell-wall galactoxyloglucan was tested by affinity chromatography and fluorescence spectroscopy. (C) 2009 Elsevier Ltd. All rights reserved.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.