Uric acid biorhythm, a feature of long-term variation in a clinical laboratory database

Background: The biorhythm of serum uric acid was evaluated in a large sample of a clinical laboratory database by spectral analysis and the influence of the gender and age on uric acid variability. Methods: Serum uric acid values were extracted from a large database of a clinical laboratory from May 2000 to August 2006. Outlier values were excluded from the analysis and the remaining data (n = 73,925) were grouped by gender and age ranges. Rhythm components were obtained by the Lomb Scargle method and Cosinor analysis. Results: Serum uric acid was higher in men than in women older than 13 years (p<0.05). Compared with 0-12 year group, uric acid increased in men but not in women older than 13 years (p<0.05). Circannual (12 months) and transyear (17 months) rhythm components were detected, but they were significant only in adult individuals (>26 years, p<0.05). Cosinor analysis showed that midline estimating statistic of rhythm (MESOR) values were higher in men (range: 353-368 mu mol/L) than in women (range: 240-278 mu mol/L; p<0.05), independent of the age and rhythm component. The extent of predictable change within a cycle, approximated by the double amplitude, represented up to 20% of the corresponding MESOR. Conclusions: Serum uric acid biorhythm is dependent on gender and age and it may have relevant influence on preanalytical variability of clinical laboratory results.

Reliable identification of mycobacterial species by PCR-restriction enzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRA-hsp65 patterns

Abstract Background Identification of nontuberculous mycobacteria (NTM) based on phenotypic tests is time-consuming, labor-intensive, expensive and often provides erroneous or inconclusive results. In the molecular method referred to as PRA-hsp65, a fragment of the hsp65 gene is amplified by PCR and then analyzed by restriction digest; this rapid approach offers the promise of accurate, cost-effective species identification. The aim of this study was to determine whether species identification of NTM using PRA-hsp65 is sufficiently reliable to serve as the routine methodology in a reference laboratory. Results A total of 434 NTM isolates were obtained from 5019 cultures submitted to the Institute Adolpho Lutz, Sao Paulo Brazil, between January 2000 and January 2001. Species identification was performed for all isolates using conventional phenotypic methods and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing a 441 bp fragment of hsp65. Phenotypic evaluation and PRA-hsp65 were concordant for 321 (74%) isolates. These assignments were presumed to be correct. For the remaining 113 discordant isolates, definitive identification was based on sequencing a 441 bp fragment of hsp65. PRA-hsp65 identified 30 isolates with hsp65 alleles representing 13 previously unreported PRA-hsp65 patterns. Overall, species identification by PRA-hsp65 was significantly more accurate than by phenotype methods (392 (90.3%) vs. 338 (77.9%), respectively; p < .0001, Fisher's test). Among the 333 isolates representing the most common pathogenic species, PRA-hsp65 provided an incorrect result for only 1.2%. Conclusion PRA-hsp65 is a rapid and highly reliable method and deserves consideration by any clinical microbiology laboratory charged with performing species identification of NTM.

Accuracy, precision and robustness of different methods to obtain samples from silages in fermentation studies

The objective of this study was to evaluate accuracy, precision and robustness of two methods to obtain silage samples, in comparison with extraction of liquor by manual screw-press. Wet brewery residue alone or combined with soybean hulls and citrus pulp were ensiled in laboratory silos. Liquor was extracted by a manual screw-press and a 2-mL aliquot was fixed with 0.4 mL formic acid. Two 10-g silage samples from each silo were diluted in 20 mL deionized water or 17% formic acid solution (alternative methods). Aliquots obtained by the three methods were used to determine the silage contents of fermentation end-products. The accuracy of the alternative methods was evaluated by comparing mean bias of estimates obtained by manual screw-press and by alternative methods, whereas precision was assessed by the root mean square prediction error and the residual error. Robustness was determined by studying the interaction between bias and chemical components, pH, in vitro dry matter digestibility (IVDMD) and buffer capacity. The 17% formic acid method was more accurate for estimating acetic, butyric and lactic acids, although it resulted in low overestimates of propionic acid and underestimates of ethanol. The deionized water method overestimated acetic and propionic acids and slightly underestimated ethanol. The 17% formic acid method was more precise than deionized water for estimating all organic acids and ethanol. The robustness of each method with respect to variation in the silage chemical composition, IVDMD and pH is dependent on the fermentation end-product at evaluation. The robustness of the alternative methods seems to be critical at the determination of lactic acid and ethanol contents.

Safety factors in sheet pile wall design: considerations by using traditional methods and FEM analysis

The purpose of the work is: define and calculate a factor of collapse related to traditional method to design sheet pile walls. Furthermore, we tried to find the parameters that most influence a finite element model representative of this problem. The text is structured in this way: from chapter 1 to 5, we analyzed a series of arguments which are usefull to understanding the problem, while the considerations mainly related to the purpose of the text are reported in the chapters from 6 to 10. In the first part of the document the following arguments are shown: what is a sheet pile wall, what are the codes to be followed for the design of these structures and what they say, how can be formulated a mathematical model of the soil, some fundamentals of finite element analysis, and finally, what are the traditional methods that support the design of sheet pile walls. In the chapter 6 we performed a parametric analysis, giving an answer to the second part of the purpose of the work. Comparing the results from a laboratory test for a cantilever sheet pile wall in a sandy soil, with those provided by a finite element model of the same problem, we concluded that:in modelling a sandy soil we should pay attention to the value of cohesion that we insert in the model (some programs, like Abaqus, don’t accept a null value for this parameter), friction angle and elastic modulus of the soil, they influence significantly the behavior of the system (structure-soil), others parameters, like the dilatancy angle or the Poisson’s ratio, they don’t seem influence it. The logical path that we followed in the second part of the text is reported here. We analyzed two different structures, the first is able to support an excavation of 4 m, while the second an excavation of 7 m. Both structures are first designed by using the traditional method, then these structures are implemented in a finite element program (Abaqus), and they are pushed to collapse by decreasing the friction angle of the soil. The factor of collapse is the ratio between tangents of the initial friction angle and of the friction angle at collapse. At the end, we performed a more detailed analysis of the first structure, observing that, the value of the factor of collapse is influenced by a wide range of parameters including: the value of the coefficients assumed in the traditional method and by the relative stiffness of the structure-soil system. In the majority of cases, we found that the value of the factor of collapse is between and 1.25 and 2. With some considerations, reported in the text, we can compare the values so far found, with the value of the safety factor proposed by the code (linked to the friction angle of the soil).

Biofilms on exposed monumental stones: mechanism of formation and development of new control methods

Within the stone monumental artefacts artistic fountains are extremely favorable to formation of biofilms, giving rise to biodegradation processes related with physical-chemical and visual aspect alterations, because of their particular exposure conditions. Microbial diversity of five fountains (two from Spain and three from Italy) was investigated. It was observed an ample similarity between the biodiversity of monumental stones reported in literature and that one found in studied fountains. Mechanical procedures and toxic chemical products are usually employed to remove such phototrophic patinas. Alternative methods based on natural antifouling substances are recently experimented in the marine sector, due to their very low environmental impact and for the bio settlement prevention on partially immersed structures of ships. In the present work groups of antibiofouling agents (ABAs) were selected from literature for their ability to interfere, at molecular level, with the microbial communication system “quorum sensing”, inhibiting the initial phase of biofilm formation. The efficacy of some natural antibiofoulants agents (ABAs) with terrestrial (Capsaicine - CS, Cinnamaldehyde - CI) and marine origin (Zosteric Acid - ZA, poly-Alkyl Pyridinium Salts – pAPS and Ceramium botryocarpum extract - CBE), incorporated into two commercial coatings (Silres BS OH 100 - S and Wacker Silres BS 290 - W) commonly used in stone conservation procedures were evaluated. The formation of phototrophic biofilms in laboratory conditions (on Carrara marble specimens and Sierra Elvira stone) and on two monumental fountains (Tacca’s Fountain 2 - Florence, Italy and Fountain from Patio de la Lindaraja - Alhambra Palace, Granada, Spain) has been investigated in the presence or absence of these natural antifouling agents. The natural antibiofouling agents, at tested concentrations, demonstrated a certain inhibitory effect. The silane-siloxane based silicone coating (W) mixing with ABAs was more suitable with respect to ethyl silicate coating (S) and proved efficacy against biofilm formation only when incompletely cured. The laboratory results indicated a positive action in inhibiting the patina formation, especially for poly-alkyl pyridinium salts, zosteric acid and cinnamaldehyde, while on site tests revealed a good effect for zosteric acid.

Development of muliplexed analytical methods based on bioluminescent whole-cell biosensors

The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.

Development of instrumental and sensory analytical methods of food obtained by traditional and emerging technologies

The consumer demand for natural, minimally processed, fresh like and functional food has lead to an increasing interest in emerging technologies. The aim of this PhD project was to study three innovative food processing technologies currently used in the food sector. Ultrasound-assisted freezing, vacuum impregnation and pulsed electric field have been investigated through laboratory scale systems and semi-industrial pilot plants. Furthermore, analytical and sensory techniques have been developed to evaluate the quality of food and vegetable matrix obtained by traditional and emerging processes. Ultrasound was found to be a valuable technique to improve the freezing process of potatoes, anticipating the beginning of the nucleation process, mainly when applied during the supercooling phase. A study of the effects of pulsed electric fields on phenol and enzymatic profile of melon juice has been realized and the statistical treatment of data was carried out through a response surface method. Next, flavour enrichment of apple sticks has been realized applying different techniques, as atmospheric, vacuum, ultrasound technologies and their combinations. The second section of the thesis deals with the development of analytical methods for the discrimination and quantification of phenol compounds in vegetable matrix, as chestnut bark extracts and olive mill waste water. The management of waste disposal in mill sector has been approached with the aim of reducing the amount of waste, and at the same time recovering valuable by-products, to be used in different industrial sectors. Finally, the sensory analysis of boiled potatoes has been carried out through the development of a quantitative descriptive procedure for the study of Italian and Mexican potato varieties. An update on flavour development in fresh and cooked potatoes has been realized and a sensory glossary, including general and specific definitions related to organic products, used in the European project Ecropolis, has been drafted.

Perfluoroalkylated substances in food matrices: development of mass spectrometry based analytical methods and preliminary monitoring

Perfluoroalkylated substances are a group of chemicals that have been largely employed during the last 60 years in several applications, widely spreading and accumulating in the environment due to their extreme resistance to degradation. As a consequence, they have been found also in various types of food as well as in drinking water, proving that they can easily reach humans through the diet. The available information concerning their adverse effects on health has recently increased the interest towards these contaminants and highlighted the importance of investigating all the potential sources of human exposure, among which diet was proved to be the most relevant. This need has been underlined by the European Union through Recommendation 2010/161/EU: in this document, Member States were called to monitor their presence of in food, producing accurate estimations of human exposure. The purpose of the research presented in this thesis, which is the result of a partnership between an Italian and a French laboratory, was to develop reliable tools for the analysis of these pollutants in food, to be used for generating data on potentially contaminated matrices. An efficient method based on liquid chromatography-mass spectrometry for the detection of 16 different perfluorinated compounds in milk has been validated in accordance with current European regulation guidelines (2002/657/EC) and is currently under evaluation for ISO 17025 accreditation. The proposed technique was applied to cow, powder and human breast milk samples from Italy and France to produce a preliminary monitoring on the presence of these contaminants. In accordance with the above mentioned European Recommendation, this project led also to the development of a promising technique for the quantification of some precursors of these substances in fish. This method showed extremely satisfying performances in terms of linearity and limits of detection, and will be useful for future surveys.

Development of Measurement Methods for Testing of Hydrokinetic Devices to Evaluate the Environmental Effect on Local Substrate

Modifications and upgrades to the hydraulic flume facility in the Environmental Fluid Mechanics and Hydraulics Laboratory (EFM&H) at Bucknell University are described. These changes enable small-scale testing of model marine hydrokinetic(MHK) devices. The design of the experimental platform provides a controlled environment for testing of model MHK devices to determine their effect on localsubstrate. Specifically, the effects being studied are scour and erosion around a cylindrical support structure and deposition of sediment downstream from the device.

Proposing new approaches for dating young volcanic eruptions by luminescence methods

The application of luminescence dating to young volcanic sediments has been first investigated over three decades ago, but it was only with the technical innovations of the last decade that such analyses became viable. While current analytical procedures show promise for dating late Quaternary volcanic events, most efforts have been aimed at unconsolidated volcanic tephra. Investigations into direct dating of lava flows or of non-heated volcanoclastics like phreatic explosion layers, however, remain scarce. These volcanic deposits are of common occurrence and represent important chrono- and volcanostratigraphic markers. Their age determination is therefore of great importance in volcanologic, tectonic, geomorphological and climate studies. In this article, we propose the use of phreatic explosion deposits and xenolithic inclusions in lava flows as target materials for luminescence dating applications. The main focus is on the crucial criterion whether it is probable that such materials experience complete luminescence signal resetting during the volcanic event to be dated. This is argued based on the findings from existing literature, model calculations and laboratory tests.

A technique to detect and to quantify fasciocutaneous blood vessels in small laboratory animals ex vivo

PURPOSE: A microangiographical technique is described, which allows visualization of small and capillary blood vessels and quantification of fasciocutaneous blood vessels by means of digital computer analysis in very small laboratory animals. MATERIALS AND METHODS: The left carotid artery of 20 nu/nu mice was cannulated (26 gauge) and a mixture of gelatin, bariumsulfate, and green ink was injected according to standardized protocol. Fasciocutaneous blood vessels were visualized by digital mammography and analyzed for vessel length and vessel surface area as standardized units [SU] by computer program. RESULTS: With the described microangiography method, fasciocutaneous blood vessels down to capillary size level can be clearly visualized. Regions of interest (ROIs) can be defined and the containing vascular network quantified. Comparable results may be obtained by calculating the microvascular area index (MAI) and the microvascular length index (MLI), related to the ROIs size. Identical ROIs showed a high reproducibility for measured [SU] < 0.01 +/- 0.0012%. CONCLUSION: Combining microsurgical techniques, pharmacological knowledge, and modern digital image technology, we were able to visualize small and capillary blood vessels even in small laboratory animals. By using our own computer analytical program, quantification of vessels was reliable, highly reproducible, and fast.

Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0-3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.

Discrete element methods for asphalt concrete : development and application of user-defined microstructural models and a viscoelastic micromechanical model

As an important Civil Engineering material, asphalt concrete (AC) is commonly used to build road surfaces, airports, and parking lots. With traditional laboratory tests and theoretical equations, it is a challenge to fully understand such a random composite material. Based on the discrete element method (DEM), this research seeks to develop and implement computer models as research approaches for improving understandings of AC microstructure-based mechanics. In this research, three categories of approaches were developed or employed to simulate microstructures of AC materials, namely the randomly-generated models, the idealized models, and image-based models. The image-based models were recommended for accurately predicting AC performance, while the other models were recommended as research tools to obtain deep insight into the AC microstructure-based mechanics. A viscoelastic micromechanical model was developed to capture viscoelastic interactions within the AC microstructure. Four types of constitutive models were built to address the four categories of interactions within an AC specimen. Each of the constitutive models consists of three parts which represent three different interaction behaviors: a stiffness model (force-displace relation), a bonding model (shear and tensile strengths), and a slip model (frictional property). Three techniques were developed to reduce the computational time for AC viscoelastic simulations. It was found that the computational time was significantly reduced to days or hours from years or months for typical three-dimensional models. Dynamic modulus and creep stiffness tests were simulated and methodologies were developed to determine the viscoelastic parameters. It was found that the DE models could successfully predict dynamic modulus, phase angles, and creep stiffness in a wide range of frequencies, temperatures, and time spans. Mineral aggregate morphology characteristics (sphericity, orientation, and angularity) were studied to investigate their impacts on AC creep stiffness. It was found that aggregate characteristics significantly impact creep stiffness. Pavement responses and pavement-vehicle interactions were investigated by simulating pavement sections under a rolling wheel. It was found that wheel acceleration, steadily moving, and deceleration significantly impact contact forces. Additionally, summary and recommendations were provided in the last chapter and part of computer programming codes wree provided in the appendixes.

Imported diphyllobothriasis in Switzerland: molecular methods to define a clinical case of Diphyllobothrium infection as Diphyllobothrium dendriticum, August 2010

Following a first clinical case of infection by Diphyllobothrium dendriticum in Switzerland in 2006, we report a second case in the country. The species was identified by molecular methods. In the Swiss, French and Italian subalpine regions, human diphyllobothriasis has seen a comeback since the late 1980's, and Diphyllobothrium latum is usually considered the causative agent of the disease. In addition, several locally acquired and imported clinical infections due to allochthonous Diphyllobothrium species have been documented in the last years. Due to the colonisation potential of these parasites and their probably underestimated presence in the human population, there is a need for discriminating them at the medical laboratory level. Because the morphological characters are very similar among the different taxa, a correct identification requires the use of molecular methods. Molecular identification would improve diagnosis and help monitor the distribution of Diphyllobothrium species in Europe.