Phytoplasma closely related to the pigeon pea witches`-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of Sao Paulo, Brazil

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of Sao Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fDI/rPI was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the Pigeon pea witches`-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these result.,;. With two primers D7f2/D7r2 designed based oil the 16S rDNA Sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and `Candidatus Liberibacter asiaticus`. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results Show that a phytoplasma of group IX is associated with citrus HLB symptoms ill northern, central, and Southern SPs. This phytoplasma has very probably been transmitted to citrus from an external Source of inoculum, but the Putative insect vector is not yet known.

Effects of natural antioxidants on the lipid profile of electron beam-irradiated beef burgers

The purpose of this study was to assess the efficacy of rosemary and oregano extracts in avoiding oxidative changes in beef burgers, and to evaluate the fatty acid profile of these products after electron beam exposition. Extracts, individually or in combination, were added to beef burgers and compared to synthetic antioxidants commonly used in food (butylated hydroxytoluene, butylated hydroxyanisole). The ground beef were submitted to electron beam irradiation at doses of 0, 3.5 and 7 kGy, and stored for 90 days. At regular time intervals, lipid oxidation and fatty acid composition were evaluated through measurement of thiobarbituric acid-reactive substances (TBARS) and gas chromatography, respectively. The results indicate that, although the irradiation process triggers an increase in the lipid oxidation ratio expressed by TBARS values, great changes in the fatty acid profiles were not observed; instead, they continued to present characteristics very similar to that of non-irradiated beef. Thus, as irradiation doses of up to 7 kGy for frozen meat can make foods safe from foodborne pathogens, natural antioxidants derived from spices are able to reduce and avoid lipid changes that may cause a deterioration of the sensory quality of these foods, and these natural extracts offer a good choice for replacing synthetic additives.

Interference of raw milk autochthonous microbiota on the performance of conventional methodologies for Listeria monocytogenes and Salmonella spp. detection

Pathogen detection in foods by reliable methodologies is very important to guarantee microbilogical safety. However, peculiar characteristics of certain foods, such as autochthonous microbiota, can directly influence pathogen development and detection. With the objective of verifying the performance of the official analytical methodologies for the isolation of Listeria monocytogenes and Salmonella in milk, different concentrations of these pathogens were inoculated in raw milk treatments with different levels of mesophilic aerobes, and then submitted to the traditional isolation procedures for the inoculated pathogens. Listeria monocytogenes was inoculated at the range of 0.2-5.2 log CFU/mL in treatments with 1.8-8.2 log CFU/mL. Salmonella Enteritidis was inoculated at 0.9-3.9 log CFU/mL in treatments with 3.0-8.2 log CFU/mL. The results indicated that recovery was not possible or was more difficult in the treatments with high counts of mesophilic aerobes and low levels of the pathogens, indicating interference of raw milk autochthonous microbiota. This interference was more evident for L. monocytogenes, once the pathogen recovery was not possible in treatments with mesophilic aerobes up to 4.0 log CFU/mL and inoculum under 2.0 log CFU/mL. For S. Enteritidis the interference appeared to be more non-specific. (C) 2007 Elsevier GmbH. All rights reserved.

Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay

Objectives: The resazurin microtitre plate assay (REMA) was evaluated to determine the susceptibility of Mycobacterium tuberculosis to pyrazinamide, and was compared with the broth microdilution method (BMM), the absolute concentration method (ACM) and pyrazinamidase (PZase) determination. Methods: Thirty-four M. tuberculosis clinical isolates (26 susceptible and 8 resistant to pyrazinamide) and reference strains M. tuberculosis H37Rv ATCC 27294 and Mycobacterium bovis AN5 were tested. Results: REMA and BMM showed 100% specificity and sensitivity when compared with ACM; BMM, however, demanded more reading time. The PZase determination assay showed 87.50% and 100% sensitivity and specificity, respectively. Conclusions: All tested methods in this preliminary study showed excellent sensitivity and specificity for the determination of pyrazinamide susceptibility of M. tuberculosis, but REMA was faster, low-cost and easy to perform and interpret. Additional studies evaluating REMA for differentiating pyrazinamide-resistant and-susceptible M. tuberculosis should be conducted on an extended panel of clinical isolates.

Methods for detection of anatoxin-a(s) by liquid chromatography coupled to electrospray ionization-tandem mass spectrometry

Anatoxin-a(s) is a potent irreversible inhibitor of the enzyme acetylcholinesterase with a unique N-hydroxyguanidine methylphosphate ester chemical structure. Determination of this toxin in environmental samples is hampered by the lack of specific methods for its detection. Using the toxic strain of Anabaena lemmermani PH-160 B as positive control, the fragmentation characteristics of anatoxin-a(s) under collision-induced dissociation conditions have been investigated and new LC-MS/MS methods proposed. Recommended ion transitions for correct detection of this toxin are 253 > 58, 253 > 159, 235 > 98 and 235 > 96. Chromatographic separation is better achieved under HILIC conditions employing a ZIC-HILIC column. This method was used to confirm for the first time the production of anatoxin-a(s) by strains of Anabaena oumiana ITEP-025 and ITEP-026. Considering no standard solutions are commercially available, our results will be of significant use for the correct identification of this toxin by LC-MS/MS. (C) 2009 Elsevier Ltd. All rights reserved.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

P>Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Parana, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff`s method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 mu l DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Detection of the TLR4 1196C > T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

Background: Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. Results: Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C> T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK-plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 mu L PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 mL PCR product. In standardized conditions, TLR4 1196C> T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C> T.

Micromethod for Quantification of Carbamazepine, Phenobartital and Phenytoin in Human Plasma by HPLC-UV Detection for Therapeutic Drug Monitoring Application

A simple, rapid, selective and sensitive analytical method by HPLC with UV detection was developed for the quantification of carbamazepine, phenobarbital and phenytoin in only 0.2 mL of plasma. A C18 column (150 x 3.9 mm, 4 micra) using a binary mobile phase consisting of water and acetonitrile (70:30, v/v) at a flow rate of 0.5 mL/min were proposed. Validation of the analytical method showed a good linearity (0.3 to 20.0 mg/L for CBZ, 0.9 to 60.0 mg/L for PB and 0.6 to 40.0 mg/L for PHT), high sensitivity (LOQ: 0.3, 0.9 and 0.6 mg/L respectively). The method was applied for drug monitoring of antiepileptic drugs (AED) in 27 patients with epilepsy under polytherapy.

Electron beam irradiation of Matricaria chamomilla L. for microbial decontamination

Wild chamomile (Matricaria chamomilla L.) is one of the most popular herbal materials with both internal and external use to cure different health disturbances. As a consequence of its origin, chamomile could carry various microbial contaminants which offer different hazards to the final consumer. Reduction of the microbial load to the in force regulation limits represents an important phase in the technological process of vegetal materials, and the electron beam treatment might be an efficient alternative to the classical methods of hygienic quality assurance. The purpose of the study was to analyze the potential application of the electron beam treatment in order to assure the microbial safety of the wild chamomile. Samples of chamomile dry inflorescences were treated in electron beam (e-beam) of 6 MeV mean energy, at room temperature and ambient pressure. Some loss of the chemical compounds with bioactive role could be noticed, but the number of microorganisms decreased as a function on the absorbed dose. Consequently, the microbial quality of studied vegetal material inflorescences was improved by e-beam. irradiation. (C) 2008 Elsevier B.V. All rights reserved.

In vitro safety assessment of papain on human skin: A qualitative Light and Transmission Electron Microscopy (TEM) study

Papain is a thiol proteolytic enzyme widely used in dermatology that found applications in wound treatment. Recently, papain was also used as absorption enhancer which can modify the peptide/ protein material in the bilayer domain. We investigated papain safety using human skin that was exposed to papain in vitro at different times: 4, 24 and 48 hours. The samples were examined using Light and Transmission Electron Microscopy (TEM) to study of the mechanisms involved in enhancer-skin interaction. After 24 hours, changes occurred in corneosomes. However, samples of 48 hours did not show major changes in agreement with the control. These findings indicated that papain could be used safely onto the skin.

Characterization of the stimulus for reactive oxygen species generation in calcium-overloaded mitochondria

We have used two different probes with distinct detection properties, dichlorodihydrofluorescein diacetate and Amplex Red/horseradish peroxidase, as well as different respiratory substrates and electron transport chain inhibitors, to characterize the reactive oxygen species (ROS) generation by the respiratory chain in calcium-overloaded mitochondria. Regardless of the respiratory substrate, calcium stimulated the mitochondrial generation of ROS, which were released at both the mitochondrial-matrix side and the extramitochondrial space, in a way insensitive to the mitochondrial permeability transition pores inhibitor cyclosporine A. In glutamate/malate-energized mitochondria, inhibition at complex I or complex III (ubiquinone cycle) similarly modulated ROS generation at either mitochondrial-matrix side or extramitochondrial space; this also occurred when the backflow of electrons to complex I in succinate-energized mitochondria was inhibited. On the other hand, in succinate-energized mitochondria the modulation of ROS generation at mitochondrial-matrix side or extra-mitochondrial space depends on the site of complex III which was inhibited. These results allow a straight comparison between the effects of different respiratory substrates and electron transport chain inhibitors on ROS generation at either mitochondrial-matrix side or extra-mitochondrial space in calcium-overloaded mitochondria.

Photoinduced Nitric Oxide and Singlet Oxygen Release from ZnPC Liposome Vehicle Associated with the Nitrosyl Ruthenium Complex: Synergistic Effects in Photodynamic Therapy Application

Under continuous photolysis at 675 nm, liposomal zinc phthalocyanine associated with nitrosyl ruthenium complex [Ru(NH.NHq)(tpy)NO](3+) showed the detection and quantification of nitric oxide (NO) and singlet oxygen ((1)O(2)) release. Photophysical and photochemical results demonstrated that the interaction between the nitrosyl ruthenium complex and the photosensitizer can enable an electron transfer process from the photosensitizer to the nitrosyl ruthenium complex which leads to NO release. Synergistic action of both photosensitizers and the nitrosyl ruthenium complex results in the production of reactive oxygen species and reactive nitrogen species, which is a potent oxidizing agent to many biological tissues, in particular neoplastic cells.

A method to lower the detection limit for optical beat signals from a frequency-dithered stabilized laser

A narrow absorption feature in an atomic or molecular gas (such as iodine or methane) is used as the frequency reference in many stabilized lasers. As part of the stabilization scheme an optical frequency dither is applied to the laser. In optical heterodyne experiments, this dither is transferred to the RF beat signal, reducing the spectral power density and hence the signal to noise ratio over that in the absence of dither. We removed the dither by mixing the raw beat signal with a dithered local oscillator signal. When the dither waveform is matched to that of the reference laser the output signal from the mixer is rendered dither free. Application of this method to a Winters iodine-stabilized helium-neon laser reduced the bandwidth of the beat signal from 6 MHz to 390 kHz, thereby lowering the detection threshold from 5 pW of laser power to 3 pW. In addition, a simple signal detection model is developed which predicts similar threshold reductions.

Development and evaluation of neural network freeway incident detection models using field data

This paper discusses a multi-layer feedforward (MLF) neural network incident detection model that was developed and evaluated using field data. In contrast to published neural network incident detection models which relied on simulated or limited field data for model development and testing, the model described in this paper was trained and tested on a real-world data set of 100 incidents. The model uses speed, flow and occupancy data measured at dual stations, averaged across all lanes and only from time interval t. The off-line performance of the model is reported under both incident and non-incident conditions. The incident detection performance of the model is reported based on a validation-test data set of 40 incidents that were independent of the 60 incidents used for training. The false alarm rates of the model are evaluated based on non-incident data that were collected from a freeway section which was video-taped for a period of 33 days. A comparative evaluation between the neural network model and the incident detection model in operation on Melbourne's freeways is also presented. The results of the comparative performance evaluation clearly demonstrate the substantial improvement in incident detection performance obtained by the neural network model. The paper also presents additional results that demonstrate how improvements in model performance can be achieved using variable decision thresholds. Finally, the model's fault-tolerance under conditions of corrupt or missing data is investigated and the impact of loop detector failure/malfunction on the performance of the trained model is evaluated and discussed. The results presented in this paper provide a comprehensive evaluation of the developed model and confirm that neural network models can provide fast and reliable incident detection on freeways. (C) 1997 Elsevier Science Ltd. All rights reserved.