Use of a solvent-free dry matrix coating for quantitative matrix-assisted laser desorption ionization imaging of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in rat brain and quantitative analysis of the drug from laser microdissected tissue regions

A dry matrix application for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was used to profile the distribution of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate, monohydrochloride (BDNC, SSR180711) in rat brain tissue sections. Matrix application involved applying layers of finely ground dry alpha-cyano-4-hydroxycinnamic acid (CHCA) to the surface of tissue sections thaw mounted onto MALDI targets. It was not possible to detect the drug when applying matrix in a standard aqueous-organic solvent solution. The drug was detected at higher concentrations in specific regions of the brain, particularly the white matter of the cerebellum. Pseudomultiple reaction monitoring imaging was used to validate that the observed distribution was the target compound. The semiquantitative data obtained from signal intensities in the imaging was confirmed by laser microdissection of specific regions of the brain directed by the imaging, followed by hydrophilic interaction chromatography in combination with a quantitative high-resolution mass spectrometry method. This study illustrates that a dry matrix coating is a valuable and complementary matrix application method for analysis of small polar drugs and metabolites that can be used for semiquantitative analysis.

Analytical techniques for the study of soft contact lens spoilation

Soft contact lens wear has become a common phenomenon in recent times. The contact lens when placed in the eye rapidly undergoes change. A film of biological material builds up on and in the lens matrix. The long term wear characteristics of the lens ultimately depend on this process. With time distinct structures made up of biological material have been found to build up on the lens. A fuller understanding of this process and how it relates to the lens chemistry could lead to contact lenses that are better tolerated by the eye. The tear film is a complex biological fluid, it is this fluid that bathes the lens during wear. It is reasonable to suppose that it is material derived from this source that accumulates on the lens. To understand this phenomenon it was decided to investigate the make up and conformation of the protein species that are found on and in the lens. As inter individual variations in tear fluid composition have been found it is important to be able to study the proteins on a single lens. Many of the analytical techniques used in bio research are not suitable for this study because of the lack of sensitivity. Work with poly acrylamide electrophoresis showed the possibility of analyzing the proteins extracted from a single lens. The development of a biotin avidin electro-blot and an enzyme linked aniibody electro-blot, lead to the high sensitivity detection and identification of the proteins present. The extraction of proteins from a lens is always incomplete. A method that analyses the proteins in situ would be a great advancement. Fourier transform infra red microscopy was developed to a point where a thin section of a contact lens could yield information about the proteins present and their conformation. The three dimensional structure of the gross macroscopic structures termed white spots was investigated using confocal laser microscopy.

Analytical model for active metamaterials with quantum ingredients

We present an analytical model for describing complex dynamics of a hybrid system consisting of resonantly coupled classical resonator and quantum structures. Classical resonators in our model correspond to plasmonic metamaterials of various geometries, as well as other types of nano- and microstructure, the optical responses of which can be described classically. Quantum resonators are represented by atoms or molecules, or their aggregates (for example, quantum dots, carbon nanotubes, dye molecules, polymer or bio-molecules etc), which can be accurately modelled only with the use of the quantum mechanical approach. Our model is based on the set of equations that combines well established density matrix formalism appropriate for quantum systems, coupled with harmonic-oscillator equations ideal for modelling sub-wavelength plasmonic and optical resonators. As a particular example of application of our model, we show that the saturation nonlinearity of carbon nanotubes increases multifold in the resonantly enhanced near field of a metamaterial. In the framework of our model, we discuss the effect of inhomogeneity of the carbon-nanotube layer (bandgap value distribution) on the nonlinearity enhancement. © 2012 IOP Publishing Ltd.

Wave Operators for Defocusing Matrix Zakharov-Shabat Systems with Pnonvanishing at Infinity

2000 Mathematics Subject Classification: Primary: 34L25; secondary: 47A40, 81Q10.

Improved estimation of postmortem interval with Cardiac Troponin T and improved analytical methods

Accurate knowledge of the time since death, or postmortem interval (PMI), has enormous legal, criminological, and psychological impact. In this study, an investigation was made to determine whether the relationship between the degradation of the human cardiac structure protein Cardiac Troponin T and PMI could be used as an indicator of time since death, thus providing a rapid, high resolution, sensitive, and automated methodology for the determination of PMI. ^ The use of Cardiac Troponin T (cTnT), a protein found in heart tissue, as a selective marker for cardiac muscle damage has shown great promise in the determination of PMI. An optimized conventional immunoassay method was developed to quantify intact and fragmented cTnT. A small sample of cardiac tissue, which is less affected than other tissues by external factors, was taken, homogenized, extracted with magnetic microparticles, separated by SDS-PAGE, and visualized with Western blot by probing with monoclonal antibody against cTnT. This step was followed by labeling and available scanners. This conventional immunoassay provides a proper detection and quantitation of cTnT protein in cardiac tissue as a complex matrix; however, this method does not provide the analyst with immediate results. Therefore, a competitive separation method using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was developed to study the interaction between human cTnT protein and monoclonal anti-TroponinT antibody. ^ Analysis of the results revealed a linear relationship between the percent of degraded cTnT and the log of the PMI, indicating that intact cTnT could be detected in human heart tissue up to 10 days postmortem at room temperature and beyond two weeks at 4C. The data presented demonstrates that this technique can provide an extended time range during which PMI can be more accurately estimated as compared to currently used methods. The data demonstrates that this technique represents a major advance in time of death determination through a fast and reliable, semi-quantitative measurement of a biochemical marker from an organ protected from outside factors. ^

The Effect of Sample and Sample Matrix on DNA Processing: Mechanisms for the Detection and Management of Inhibition in Forensic Samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

Origin authentication of distillers' dried grains and solubles (DDGS) - Application and comparison of different analytical strategies

In the context of products from certain regions or countries being banned because of an identified or non-identified hazard, proof of geographical origin is essential with regard to feed and food safety issues. Usually, the product labeling of an affected feed lot shows origin, and the paper documentation shows traceability. Incorrect product labeling is common in embargo situations, however, and alternative analytical strategies for controlling feed authenticity are therefore needed. In this study, distillers' dried grains and solubles (DDGS) were chosen as the product on which to base a comparison of analytical strategies aimed at identifying the most appropriate one. Various analytical techniques were investigated for their ability to authenticate DDGS, including spectroscopic and spectrometric techniques combined with multivariate data analysis, as well as proven techniques for authenticating food, such as DNA analysis and stable isotope ratio analysis. An external validation procedure (called the system challenge) was used to analyze sample sets blind and to compare analytical techniques. All the techniques were adapted so as to be applicable to the DDGS matrix. They produced positive results in determining the botanical origin of DDGS (corn vs. wheat), and several of them were able to determine the geographical origin of the DDGS in the sample set. The maintenance and extension of the databanks generated in this study through the analysis of new authentic samples from a single location are essential in order to monitor developments and processing that could affect authentication.

The effect of sample and sample matrix on DNA processing: Mechanisms for the detection and management of inhibition in forensic samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.^

The study of thermal stresses in a single long elastic fiber embedded in an infinite matrix

This work considered the micro-mechanical behavior of a long fiber embedded in an infinite matrix. Using the theory of elasticity, the idea of boundary layer and some simplifying assumptions, an approximate analytical solution was obtained for the normal and shear stresses along the fiber. The analytical solution to the problem was found for the case when the length of the embedded fiber is much greater than its radius, and the Young's modulus of the matrix was much less than that of the fiber. The analytical solution was then compared with a numerical solution based on Finite Element Analysis (FEA) using ANSYS. The numerical results showed the same qualitative behavior of the analytical solution, serving as a validation tool against lack of experimental results. In general this work provides a simple method to determine the thermal stresses along the fiber embedded in a matrix, which is the foundation for a better understanding of the interaction between the fiber and matrix in the case of the classical problem of thermal-stresses.

Phosphorus quantification in fertilizers using laser induced breakdown spectroscopy (LIBS): a methodology of analysis to correct physical matrix effcts.

2016

Mechanical behaviour and durability assessment of Fibre Reinforced Cementitious Matrix (FRCM) strengthening systems.

In recent years, the seismic vulnerability of existing masonry buildings has been underscored by the destructive impacts of earthquakes. Therefore, Fibre Reinforced Cementitious Matrix (FRCM) retrofitting systems have gained prominence due to their high strength-to-weight ratio, compatibility with substrates, and potential reversibility. However, concerns linger regarding the durability of these systems when subjected to long-term environmental conditions. This doctoral dissertation addressed these concerns by studying the effects of mild temperature variations on three FRCM systems, featuring basalt, glass, and aramid fibre textiles with lime-based mortar matrices. The study subjected various specimens, including mortar triplets, bare textile specimens, FRCM coupons, and single-lap direct shear wallets, to thermal exposure. A novel approach utilizing embedded thermocouple sensors facilitated efficient monitoring and active control of the conditioning process. A shift in the failure modes was obtained in the single lap-direct shear tests, alongside a significant impact on tensile capacity for both textiles and FRCM coupons. Subsequently, bond tests results were used to indirectly calibrate an analytical approach based on mode-II fracture mechanics. A comparison between Cohesive Material Law (CML) functions at various temperatures was conducted for each of the three systems, demonstrating a good agreement between the analytical model and experimental curves. Furthermore, the durability in alkaline environment of two additional FRCM systems, characterized by basalt and glass fibre textiles with lime-based mortars, was studied through an extensive experimental campaign. Tests conducted on single yarn and textile specimens after exposure at different durations and temperatures revealed a significant impact on tensile capacity. Additionally, FRCM coupons manufactured with conditioned textile were tested to understand the influence of aged textile and curing environment on the final tensile behavior. These results contributed significantly to the existing knowledge on FRCM systems and could be used to develop a standardized alkaline testing protocol, still lacking in the scientific literature.

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Matrix Metalloproteinases And Left Ventricular Function And Structure In Spinal Cord Injured Subjects.

Subjects with spinal cord injury (SCI) exhibit impaired left ventricular (LV) diastolic function, which has been reported to be attenuated by regular physical activity. This study investigated the relationship between circulating matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) and echocardiographic parameters in SCI subjects and the role of physical activity in this regard. Forty-two men with SCI [19 sedentary (S-SCI) and 23 physically-active (PA-SCI)] were evaluated by clinical, anthropometric, laboratory, and echocardiographic analysis. Plasmatic pro-MMP-2, MMP-2, MMP-8, pro-MMP-9, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay and zymography. PA-SCI subjects presented lower pro-MMP-2 and pro-MMP-2/TIMP-2 levels and improved markers of LV diastolic function (lower E/Em and higher Em and E/A values) than S-SCI ones. Bivariate analysis showed that pro-MMP-2 correlated inversely with Em and directly with E/Em, while MMP-9 correlated directly with LV mass index and LV end-diastolic diameter in the whole sample. Following multiple regression analysis, pro-MMP-2, but not physical activity, remained associated with Em, while MMP-9 was associated with LV mass index in the whole sample. These findings suggest differing roles for MMPs in LV structure and function regulation and an interaction among pro-MMP-2, diastolic function and physical activity in SCI subjects.

Identification Of Corynebacterium Spp. Isolated From Bovine Intramammary Infections By Matrix-assisted Laser Desorption Ionization-time Of Flight Mass Spectrometry.

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Inactivation Of Ampk Mediates High Phosphate-induced Extracellular Matrix Accumulation Via Nox4/tgfß-1 Signaling In Human Mesangial Cells.

High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs). iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively. In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression. AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.