Quantitative determination of cytotoxic friedo-nor-oleanane derivatives from five morphological types of Maytenus ilicifolia (Celastraceae) by reverse-phase high-performance liquid chromatography

Five different morphological types of Maytenus ilicifolia of the same age and harvested under the same conditions showed distinct accumulations of some friedo-nor-oleananes. A rapid, sensitive and reliable reverse-phase HPLC method (employing an external standard) was used for the determination of the cytotoxic triterpenoids, 20alpha-hydroxymaytenin, 22beta-hydroxymaytenin, maytenin, celastrol and pristimerin in each of the five types. Well resolved peaks with good detection response and linearity in the range 1.0-100 mug/mL were obtained. Copyright (C) 2002 John Wiley Sons, Ltd.

An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations

The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota-LS-(vaccine strain) and Sao Joao do Meriti-SJM-strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity. © 2012 Springer Science+Business Media B.V.

Using the Analytic Network Process to evaluate the relation between Reverse Logistics and corporate performance in Brazilian companies

Reverse Logistics activities are practiced by most Brazilian companies. However, a relevant problem is to identify how different Reverse Logistics programs can affect corporate performance indicators. Analytic Network Process is one of the analytical tools, which can be used to handle a multi-criteria decision-making problem and it is the only one that can capture the interdependencies between the criteria under consideration. This method was adopted here to study the influence of Reverse Logistics practices in corporate performance. Preliminary results indicated that the method can be used, reaching a result compatible to the reality of the Brazilian companies.

Validation of a stability-indicating RP-LC method for the determination of tigecycline in lyophilized powder

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of tigecycline in lyophilized powder. The LC method was conducted on a Luna C18 column (250 × 4.6 mm i.d.), maintained at room temperature. The mobile phase consisted of buffer containing sodium phosphate monobasic (0.015M) and oxalic acid (0.015M) (pH 7.0)-acetonitrile (75:25, v/v), run at a flow rate of 1.0 mL/min and using ultraviolet detection at 280 nm. The chromatographic separation was obtained with a retention time of 8.6 min, and was linear in the range of 40-100 μg/mL (r2 = 0.9997). The specificity and stability-indicating capability of the method was proven through forced degradation studies, which also showed no interference of the excipients. The accuracy was 99.01% with a bias lower than 1.81%. The limits of detection and quantitation were 1.67 and 5.05 μg/mL, respectively. Moreover, method validation demonstrated satisfactory results for precision and robustness. The proposed method was applied for the analysis of the lyophilized powder formulation, contributing to improve the quality control and to assure the therapeutic efficacy. © The Author [2012]. Published by Oxford University Press. All rights reserved.

An emergy-based evaluation of a reverse logistics network for steel recycling

Efficient implementation of recycling networks requires appropriate logistical structures for managing the reverse flow of materials from users to producers. The steel sheet distributor studied had established a protocol for scrap recovery with the steel plant and its customers. The company invested in producing containers, hiring a specialized labor force and in purchasing trucks for container transportation to implement the logistics network for recycling. That network interconnected the company with two kinds of customers: the ones returning scrap and the ones who preferred to continue business-as-usual. The logistical network was analyzed using emergy synthesis, and the data obtained were used to evaluate and compare the system's environmental costs and benefits from the perspective of the distributor and the steel plant operator. The use of emergy ternary diagrams provided a way to assess recycle strategies to compare the relative economic and environmental benefits of the logistical network implemented. The minimum quantity of scrap that the distributor must recover to improve environmental benefits was determined allowing decision on whether it is worth keeping the system running. The new assessment method proposed also may help policy-makers to create strategies to reward or incentive users of reverse logistics, and help to establish regulations, by decreasing taxes or stimulating innovation, for effectively implement the National Policy on Solid Waste. (C) 2013 Elsevier Ltd. All rights reserved.

Application of reverse Monte Carlo simulations to diatomic molecules. Part 1. Noncomplete radial distribution functions

The reverse Monte Carlo (RMC) method generates sets of points in space which yield radial distribution functions (RDFS) that approximate those of the system of interest. Such sets of configurations should, in principle, be sufficient to determine the structural properties of the system. In this work we apply the RMC technique to fluids of hard diatomic molecules. The experimental RDFs of the hard-dimer fluid were generated by the conventional MC method and used as input in the RMC simulations. Our results indicate that the RMC method is only satisfactory in determining the local structure of the fluid studied by means of only mono-variable RDF. Also we suggest that the use of multi-variable RDFs would improve the technique significantly. However, the accuracy of the method turned out to be very sensitive to the variance of the input experimental RDF. © 1995.

Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts.

Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48-72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.

A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNIA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADM), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type I virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

Method for detection of respiratory viruses in the sputa of patients with cystic fibrosis

Since the role of respiratory viruses in lung exacerbations of patients with cystic fibrosis has been hampered by the difficulty of detecting viruses in viscous sputum specimens, a multiplex reverse transcriptase PCR (RT-PCR) assay combined with colorimetric amplicon detection was tested for the identification of seven common respiratory viruses in the sputa of cystic fibrosis patients. Of 52 sputa from 38 patients, 12 (23%) samples from 12 patients were positive for a respiratory virus (4 for influenza B, 3 for parainfluenza 1, 3 for influenza A and 2 for respiratory syncytial virus). These results suggest that the RT-PCR method carried out on sputum may provide a convenient means of investigating the role of virus infection in lung exacerbations of cystic fibrosis patients.

A preform design method for sheet superplastic bulging with finite element modeling

Superplastic bulging is the most successful application of superplastic forming (SPF) in industry, but the non-uniform wall thickness distribution of parts formed by it is a common technical problem yet to be overcome. Based on a rigid-viscoplastic finite element program developed by the authors, for simulation of the sheet superplastic forming process combined with the prediction of microstructure variations (such as grain growth and cavity growth), a simple and efficient preform design method is proposed and applied to the design of preform mould for manufacturing parts with uniform wall thickness. Examples of formed parts are presented here to demonstrate that the technology can be used to improve the uniformity of wall thickness to meet practical requirements. (C) 2004 Elsevier B.V. All rights reserved.

Optimization of flocculation process and continuous filtration for sea water primary treatment filtration to reduce SDI for entree to reverse osmosis system

Today , Providing drinking water and process water is one of the major problems in most countries ; the surface water often need to be treated to achieve necessary quality, and in this way, technological and also financial difficulties cause great restrictions in operating the treatment units. Although water supply by simple and cheap systems has been one of the important objectives in most scientific and research centers in the world, still a great percent of population in developing countries, especially in rural areas, don't benefit well quality water. One of the big and available sources for providing acceptable water is sea water. There are two ways to treat sea water first evaporation and second reverse osmosis system. Nowadays R.O system has been used for desalination because of low budget price and easily to operate and maintenance. The sea water should be pretreated before R.O plants, because there is some difficulties in raw sea water that can decrease yield point of membranes in R.O system. The subject of this research may be useful in this way, and we hope to be able to achieve complete success in design and construction of useful pretreatment systems for R.O plant. One of the most important units in the sea water pretreatment plant is filtration, the conventional method for filtration is pressurized sand filters, and the subject of this research is about new filtration which is called continuous back wash sand filtration (CBWSF). The CBWSF designed and tested in this research may be used more economically with less difficulty. It consists two main parts first shell body and second central part comprising of airlift pump, raw water feeding pipe, air supply hose, backwash chamber and sand washer as well as inlet and outlet connections. The CBWSF is a continuously operating filter, i.e. the filter does not have to be taken out of operation for backwashing or cleaning. Inlet water is fed through the sand bed while the sand bed is moving downwards. The water gets filtered while the sand becomes dirty. Simultaneously, the dirty sand is cleaned in the sand washer and the suspended solids are discharged in backwash water. We analyze the behavior of CBWSF in pretreatment of sea water instead of pressurized sand filter. There is one important factor which is not suitable for R.O membranes, it is bio-fouling. This factor is defined by Silt Density Index (SDI).measured by SDI. In this research has been focused on decreasing of SDI and NTU. Based on this goal, the prototype of pretreatment had been designed and manufactured to test. The system design was done mainly by using the design fundamentals of CBWSF. The automatic backwash sand filter can be used in small and also big water supply schemes. In big water treatment plants, the units of filters perform the filtration and backwash stages separately, and in small treatment plants, the unit is usually compacted to achieve less energy consumption. The analysis of the system showed that it may be used feasibly for water treating, especially for limited population. The construction is rapid, simple and economic, and its performance is high enough because no mobile mechanical part is used in it, so it may be proposed as an effective method to improve the water quality and consequently the hygiene level in the remote places of the country.

An approximate method for the solution of an influence function foil rolling model

Fleck and Johnson (Int. J. Mech. Sci. 29 (1987) 507) and Fleck et al. (Proc. Inst. Mech. Eng. 206 (1992) 119) have developed foil rolling models which allow for large deformations in the roll profile, including the possibility that the rolls flatten completely. However, these models require computationally expensive iterative solution techniques. A new approach to the approximate solution of the Fleck et al. (1992) Influence Function Model has been developed using both analytic and approximation techniques. The numerical difficulties arising from solving an integral equation in the flattened region have been reduced by applying an Inverse Hilbert Transform to get an analytic expression for the pressure. The method described in this paper is applicable to cases where there is or there is not a flat region.