Development of a DNA-based method for detection and identification of Phytophthora species

Phytophthora diseases cause major losses to agricultural and horticultural production in Australia and worldwide. Most Phytophthora diseases are soilborne and difficult to control, making disease prevention an important component of many disease management strategies. Detection and identification of the causal agent, therefore, is an essential part of effective disease management. This paper describes the development and validation of a DNA-based diagnostic assay that can detect and identify 27 different Phytophthora species. We have designed PCR primers that are specific to the genus Phytophthora. The resulting amplicon after PCR is subjected to digestion by restriction enzymes to yield a specific restriction pattern or fingerprint unique to each species. The restriction patterns are compared with a key comprising restriction patterns of type specimens or representative isolates of 27 different Phytophthora species. A number of fundamental issues, such as genetic diversity within and among species which underpin the development and validation of DNA-based diagnostic assays, are addressed in this paper.

A solvent-free matrix application method for matrix-assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI-MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules.

Novel sol–gel preparation of (P2O5)0.4–(CaO)0.25–(Na2O)X–(TiO2)(0.35−X) bioresorbable glasses (X = 0.05, 0.1, and 0.15)

Quaternary phosphate-based glasses in the P2O5–CaO–Na2O–TiO2 system with a fixed P2O5 and CaO content of 40 and 25 mol% respectively have been successfully synthesised via sol–gel method and bulk, transparent samples were obtained. The structure, elemental proportion, and thermal properties of stabilised sol–gel glasses have been characterised using X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), 31P nuclear magnetic resonance (31P NMR), titanium K-edge X-ray absorption near-edge structure (XANES), fourier transform infrared (FTIR) spectroscopy, and differential thermal analysis (DTA). The XRD results confirmed the amorphous nature for all stabilized sol–gel derived glasses. The EDX result shows the relatively low loss of phosphorus during the sol–gel process and Ti K-edge XANES confirmed titanium in the glass structure is in mainly six-fold coordination environment. The 31P NMR and FTIR results revealed that the glass structure consist of mainly Q1 and Q2 phosphate units and the Ti4+ cation was acting as a cross-linking between phosphate units. In addition DTA results confirmed a decrease in the glass transition and crystallisation temperature with increasing Na2O content. Ion release studies also demonstrated a decrease in degradation rates with increasing TiO2 content therefore supporting the use of these glasses for biomedical applications that require a degree of control over glass degradation. These sol–gel glasses also offer the potential to incorporate proactive molecules for drug delivery application due to the low synthesis temperature employed.

The use of sodium carboxymethylcellulose in the preparation of spray-dried proteins for pulmonary drug delivery

The use of sodium carboxymethylcellulose (NaCMC) as a spray-drying excipient in the preparation of inhalable formulations of proteins was investigated, using alkaline phosphatase as a model functional protein. Two spray-dried powders were investigated: a control powder comprising 100% (w/w) alkaline phosphatase and a test powder comprising 67% (w/w) NaCMC and 33% (w/w) alkaline phosphatase. Following physicochemical characterisation, the powders were prepared as both dry powder inhaler (DPI) and pressurised metered dose inhaler (pMDI) formulations. The aerosolisation performance of the formulations was assessed using a Multi-Stage Liquid Impinger, both immediately after preparation and over a 16-week storage period. Formulating the control powder as a DPI resulted in a poor fine particle fraction (FPF: 10%), whereas the FPF of the NaCMC-modified DPI formulation was significantly greater (47%). When the powders were formulated as pMDI systems, the control and NaCMC-modified powders demonstrated FPFs of 52% and 55%, respectively. Following storage, reduced FPF was observed for all formulations except the NaCMC-modified pMDI system; the performance of this formulation following storage was statistically equivalent to that immediately following preparation. Co-spray-drying proteins and peptides with NaCMC may therefore offer an alternative method for the preparation of stable and respirable pMDI formulations for pulmonary delivery. © 2010 Elsevier B.V.

Development of thresholds for surface preparation in repair systems with wet lay -up fiber reinforced polymers

Bonded repair of concrete structures with fiber reinforced polymer (FRP) systems is increasingly being accepted as a cost-efficient and structurally viable method of rapid rehabilitation of concrete structures. However, the relationships between long-term performance attributes, service-life, and details of the installation process are not easy to quantify. Accordingly, there is currently a lack of generally accepted construction specifications, making it difficult for the field engineer to certify the adequacy of the construction process. ^ The objective of the present study, as part of the National Cooperative Highway Research Program (NCHRP) Project 10-59B, was to investigate the effect of surface preparation on the behavior of wet lay-up FRP repair systems and consequently develop rational thresholds that provide sufficient performance. ^ The research program was comprised of both experimental and analytical work for wet lay-up FRP applications. The experimental work included flexure testing of sixty-seven (67) reinforced concrete beams and bond testing of ten (10) reinforced concrete blocks. Four different parameters were studied: surface roughness, surface flatness, surface voids and bug holes, and surface cracks/cuts. The findings were analyzed from various aspects and compared with the data available in the literature. As part of the analytical work, finite element models of the flexural specimens with surface flaws were developed using ANSYS. The purpose of this part was to extend the parametric study on the effects of concrete surface flaws and verify the experimental results based on nonlinear finite element analysis. ^ Test results showed that surface roughness does not appear to have a significant influence on the overall performance of the wet lay-up FRP systems with or without adequate anchorage, and whether failure was by debonding or rupture of FRP. Both experimental and analytical results for surface flatness proved that peaks on concrete surface, in the range studied, do not have a significant effect on the performance of wet lay-up FRP systems. However, valleys of particular size could reduce the strength of wet lay-up FRP systems. Test results regarding surface voids and surface cracks/cuts revealed that previously suggested thresholds for these flaws appear to be conservative, as also confirmed by analytical study. ^

Online solid phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of sucralose in reclaimed and drinking waters and its photo degradation in natural waters from South Florida

Background Sucralose has gained popularity as a low calorie artificial sweetener worldwide. Due to its high stability and persistence, sucralose has shown widespread occurrence in environmental waters, at concentrations that could reach up to several μg/L. Previous studies have used time consuming sample preparation methods (offline solid phase extraction/derivatization) or methods with rather high detection limits (direct injection) for sucralose analysis. This study described a faster and sensitive analytical method for the determination of sucralose in environmental samples. Results An online SPE-LC–MS/MS method was developed, being capable to quantify sucralose in 12 minutes using only 10 mL of sample, with method detection limits (MDLs) of 4.5 ng/L, 8.5 ng/L and 45 ng/L for deionized water, drinking and reclaimed waters (1:10 diluted with deionized water), respectively. Sucralose was detected in 82% of the reclaimed water samples at concentrations reaching up to 18 μg/L. The monthly average for a period of one year was 9.1 ± 2.9 μg/L. The calculated mass loads per capita of sucralose discharged through WWTP effluents based on the concentrations detected in wastewaters in the U. S. is 5.0 mg/day/person. As expected, the concentrations observed in drinking water were much lower but still relevant reaching as high as 465 ng/L. In order to evaluate the stability of sucralose, photodegradation experiments were performed in natural waters. Significant photodegradation of sucralose was observed only in freshwater at 254 nm. Minimal degradation (<20%) was observed for all matrices under more natural conditions (350 nm or solar simulator). The only photolysis product of sucralose identified by high resolution mass spectrometry was a de-chlorinated molecule at m/z 362.0535, with molecular formula C12H20Cl2O8. Conclusions Online SPE LC-APCI/MS/MS developed in the study was applied to more than 100 environmental samples. Sucralose was frequently detected (>80%) indicating that the conventional treatment process employed in the sewage treatment plants is not efficient for its removal. Detection of sucralose in drinking waters suggests potential contamination of surface and ground waters sources with anthropogenic wastewater streams. Its high resistance to photodegradation, minimal sorption and high solubility indicate that sucralose could be a good tracer of anthropogenic wastewater intrusion into the environment.

A novel method for rapid and selective extraction of male DNA from rape kits using alkaline lysis and pressure cycling technology (PCT)

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. ^ Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. ^ Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.^

The use of quantitative polymerase chain reaction amplification method to determine sex ratio of human spermatozoa in semen and to determine maternal contamination of amniotic fluid samples

We have modified a technique which uses a single pair of primer sets directed against homologous but distinct genes on the X and Y chromosomes, all of which are coamplified in the same reaction tube with trace amounts of radioactivity. The resulting bands are equal in length, yet distinguishable by restriction enzyme sites generating two independent bands, a 364 bp X-specific band and a 280 bp Y-specific band. A standard curve was generated to show the linear relationship between X/Y ratio average vs. %Y or %X chromosomal content. Of the 51 purified amniocyte DNA samples analyzed, 16 samples showed evidence of high % X contamination while 2 samples demonstrated higher % Y than the expected 50% X and 50% Y chromosomal content. With regards to the 25 processed sperm samples analyzed, X-sperm enrichment was evident when compared to the primary sex ratio whereas Y-sperm was enriched when we compared before and after selection samples.

Selective Preparation of Semiconducting Single-Walled Carbon Nanotubes: From Fundamentals to Applications

Aligned single-walled carbon nanotubes (SWNTs) synthesized by the chemical vapor deposition (CVD) method have exceptional potential for next-generation nanoelectronics. However, there are considerable challenges in the preparation of semiconducting (s-) SWNTs with controlled properties (e.g., density, selectivity, and diameter) for their application in solving real-world problems. This dissertation describes research that aims to overcome the limitations by novel synthesis strategies and post-growth treatment. The application of as-prepared SWNTs as functional devices is also demonstrated. The dissertation includes the following parts: 1) decoupling the conflict between density and selectivity of s-SWNTs in CVD growth; 2) investigating the importance of diameter control for the selective synthesis of s-SWNTs; 3) synthesizing highly conductive SWNT thin film by thiophene-assisted CVD method; 4) eliminating metallic pathways in SWNT crossbars by gate-free electrical breakdown method; 5) enhancing the density of SWNT arrays by strain-release method; 6) studying the sensing mechanism of SWNT crossbar chemical sensors.

A Novel Method for Rapid and Selective Extraction of Male DNA from Rape Kits using Alkaline Lysis and Pressure Cycling Technology (PCT)

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.

Preparation and characterization of Pr0.6Sr0.4FeO3-δ-Ce0.9Pr0.1O2-δ nanofiber structured composite cathode for IT-SOFCs

In this work, Pr_0.6Sr_0.4FeO_3-δ -Ce_0.9Pr_0.1O_2-δ (PSFO-CPO) nanofibers were synthesized by a one-step electrospin technique for use in intermediate-temperature solid oxide fuel cell (IT-SOFC) applications. PSFO-CPO nanofibers were produced with a diameter of about 100nm and lengths exceeding tens of microns. The thermal expansion coefficient (TEC) matches with standard GDC electrolytes and the resulting conductivity also satisfies the needs of IT-SOFCs cathodes. EIS analysis of the nanofiber structured electrode gives a polarization resistance of 0.072Ωcm² at 800°C, smaller than that from the powdered cathode with the same composition. The excellent electrochemical performance can be attributed to the well-constructed microstructure of the nanofiber structured cathode, which promotes surface oxygen diffusion and charge transfer processes. All the results imply that the one-step electrospin method is a facile and practical way of improving the cathode properties and that PSFO-CPO is a promising cathode material for IT-SOFCs.

Factors affecting fertility in frozen-thawed boar sperm

Frozen-thawed boar sperm holds the potential to have an impact on the future of the swine industry. Utilization of this technology could improve a swine producer’s ability to access top-tier genetics from around the world, to improve efficiency, profitability, and the quality of product to meet consumer demands. Effective application of frozen-thawed sperm can help reduce the potential risk associated with devastating economic loss due to the spread of disease. Frozen storage of boar sperm also provides a safeguard in the event of disease outbreaks, as genetic material from paternal lines can be preserved and banked for repopulation purposes. Historically these benefits have been masked by reduction in fertility measures such as litter size. The reduced fertility results from the damage sustained by the sperm cell during cryopreservation. However, increased understanding of this damage has lead to improved cryopreservation methods, ultimately increasing post-thaw viability and fertility. Enhancements in breeding technology have also resulted in a better understanding of the AI methods required to achieve acceptable farrowing rates and litter size. Fertility following AI with frozen-thawed sperm is approaching that of liquid stored sperm, and producers may soon reap the benefits of this technology. This thesis will outline the current swine industry, opportunities for utilizing frozen-thawed sperm, the main components of sperm, why they are susceptible to damage, and current freezing and breeding practices. Objective 1 was to develop a cryopreservation protocol for our lab that resulted in consistent post-thaw motility ( ≥ 40%) that would eventually be used by Illinois boar studs for domestic and international sale of frozen sperm. Evaluation with both manual microscopy and CASA methods were conducted to verify quality. A preliminary breeding trial was then conducted to test the fertility of sperm frozen with this method. There were 41 ejaculates from 23 boars used for freezing. Sperm were frozen at 1.4x109 sperm/mL, averaging 55.61.1% (meanSE) motility, following thaw. The samples assessed were not different (P>0.05) in motility when compared with manual or CASA systems, and results were most reliable at a 1:40 sperm dilution. In the preliminary breeding trial, gilts (n=14) were inseminated with either a single (n=10) or double (n=4) AI using 1, 2, or 4x109 motile, frozen-thawed sperm. Overall, the resulting pregnancy rates averaged 71.4% and numbers of normal fetuses per litter averaged 15.51.3 per litter. A feasibility study for freezing cost per ejaculate was estimated at $275/ejaculate or $11/dose of frozen-thawed semen at standard doses of 5x109 total frozen-thawed sperm. This cost estimate did not include genetic value, fixed equipment costs, depreciation, or variable lab space fees. Objective 2 focused on the proper methods for breeding with frozen-thawed boar sperm to achieve fertility. Our hypothesis was that increased numbers of inseminations and increased numbers of motile frozen-thawed sperm would improve pregnancy rate and litter size. Results showed acceptable fertility at high sperm numbers, but also the optimal method for insemination with the lowest dose tested. Gilts (n=111) responded to synchronization methods and were bred with 1, 2, or 4x109 motile frozen-thawed sperm from six boars using a single AI at 32 h, or a double AI, with the first AI at 24 and 32 h following estrus. Ultrasound was conducted at 12 h intervals to estimate the time of ovulation. On day 32 of gestation, overall pregnancy rate (73%) and number of normal fetuses per litter (10.80.5) across all treatments did not differ, and were not affected by number of motile sperm, or the interaction of number of motile sperm and number of inseminations. However, the number of inseminations tended to affect (P=0.14) the number of normal fetuses. Litter size increased with a double AI compared to single AI. Multiple inseminations helped to allow insemination to occur close to ovulation in response to variation in the time of ovulation. Both pregnancy rate and number of normal fetuses were greater when the time of the AI at 32 h occurred closer to the estimated time of ovulation (P<0.05). In addition, other factors such as presence of an abnormal ovary at day 30 decreased (P<0.001) pregnancy rate, while boar affected number of normal fetuses (P<0.01). Analysis of our data using a fertility index revealed doses of 2x109 motile sperm with multiple AI can achieve acceptable fertility with use of less sperm, when compared to AI using 4x109 motile sperm. The methods described here will investigate the potential for improved fertility when using frozen-thawed sperm, while accounting for variation in time of ovulation.

Preparation of environmental monitoring plan pattern for surface water quality in Iran (Case study: Gharehsou River)

Using water quality management programs is a necessary and inevitable way for preservation and sustainable use of water resources. One of the important issues in determining the quality of water in rivers is designing effective quality control networks, so that the measured quality variables in these stations are, as far as possible, indicative of overall changes in water quality. One of the methods to achieve this goal is increasing the number of quality monitoring stations and sampling instances. Since this will dramatically increase the annual cost of monitoring, deciding on which stations and parameters are the most important ones, along with increasing the instances of sampling, in a way that shows maximum change in the system under study can affect the future decision-making processes for optimizing the efficacy of extant monitoring network, removing or adding new stations or parameters and decreasing or increasing sampling instances. This end, the efficiency of multivariate statistical procedures was studied in this thesis. Multivariate statistical procedure, with regard to its features, can be used as a practical and useful method in recognizing and analyzing rivers’ pollution and consequently in understanding, reasoning, controlling, and correct decision-making in water quality management. This research was carried out using multivariate statistical techniques for analyzing the quality of water and monitoring the variables affecting its quality in Gharasou river, in Ardabil province in northwest of Iran. During a year, 28 physical and chemical parameters were sampled in 11 stations. The results of these measurements were analyzed by multivariate procedures such as: Cluster Analysis (CA), Principal Component Analysis (PCA), Factor Analysis (FA), and Discriminant Analysis (DA). Based on the findings from cluster analysis, principal component analysis, and factor analysis the stations were divided into three groups of highly polluted (HP), moderately polluted (MP), and less polluted (LP) stations Thus, this study illustrates the usefulness of multivariate statistical techniques for analysis and interpretation of complex data sets, and in water quality assessment, identification of pollution sources/factors and understanding spatial variations in water quality for effective river water quality management. This study also shows the effectiveness of these techniques for getting better information about the water quality and design of monitoring network for effective management of water resources. Therefore, based on the results, Gharasou river water quality monitoring program was developed and presented.

Host-guest system of 4-nerolidylcatechol in 2-hydroxypropyl-beta-cyclodextrin: preparation, characterization and molecular modeling

The interaction of 4-nerolidylcatechol (4-NRC), a potent antioxidant agent, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was investigated by the solubility method using Fourier transform infrared (FTIR) methods in addition to UV-Vis, (1)H-nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. The inclusion complexes were prepared using grinding, kneading and freeze-drying methods. According to phase solubility studies in water a B(S)-type diagram was found, displaying a stoichiometry complexation of 2:1 (drug:host) and stability constant of 6494 +/- A 837 M(-1). Stoichiometry was established by the UV spectrophotometer using Job's plot method and, also confirmed by molecular modeling. Data from (1)H-NMR, and FTIR, experiments also provided formation evidence of an inclusion complex between 4-NRC and HP-beta-CD. 4-NRC complexation indeed led to higher drug solubility and stability which could probably be useful to improve its biological properties and make it available to oral administration and topical formulations.

Polymeric implant of methylprednisolone for spinal injury: preparation and characterization

Purpose: To improve the effectiveness and reduce the systemic side effects of methylprednisolone in traumatic spinal injuries, its polymeric implants were prepared using chitosan and sodium alginate as the biocompatible polymers. Methods: Implants of methylprednisolone sodium succinate (MPSS) were prepared by molding the drug-loaded polymeric mass obtained after ionotropic gelation method. The prepared implants were evaluated for drug loading, in vitro drug release and in vivo performance in traumatic spinal-injury rat model with paraplegia. Results: All the implant formulations were light pale solid matrix with smooth texture. Implants showed 86.56 ± 2.07 % drug loading. Drug release was 89.29 ± 1.25 % at the end of 7 days. Motor function was evaluated in traumatic spinal injury-induced rats in terms of its movement on the horizontal bar. At the end of 7 days, the test group showed the activity score (4.75 ± 0.02) slightly higher than that of standard (4.62 ± 0.25), but the difference was not statistically different (p > 0.05). Conclusion: MPSS-loaded implants produces good recovery in traumatic spinal-injury rats.