Development of a novel in vitro system for nasal drug delivery development

There is currently, no ideal system for studying nasal drug delivery in vitro. The existing techniques such as the Ussing chamber and cell culture all have major disadvantages. Most importantly, none of the existing techniques accurately represent the interior of the nasal cavity, with its airflow and humidity; neither do they allow the investigation of solid dosage forms.The work in this thesis represents the development of an in vitro model system in which the interior characteristics of the nasal cavity are closely represented, and solid or minimal volume dosage forms can be investigated. The complete nasal chamber consists of two sections: a lower tissue, viability chamber and an upper nasal chamber. The lower tissue viability chamber has been shown, using existing tissue viability monitoring techniques, to maintain the viability of a number of epithelial tissues, including porcine and rabbit nasal tissue, and rat ileal and Payers' patch tissue. The complete chamber including the upper nasal chamber has been shown to provide tissue viability for porcine and rabbit nasal tissue above that available using the existing Ussing chamber techniques. Adaptation of the complete system, and the development of the necessary experimental protocols that allow aerosol particle-sizing, together with videography, has shown that the new factors investigated, humidity and airflow, have a measurable effect on the delivered dose from a typical nasal pump. Similarly, adaptation of the chamber to fit under a confocal microscope, and the development of the necessary protocols has shown the effect of surface and size on the penetration of microparticulate materials into nasal epithelial tissues. The system developed in this thesis has been shown to be flexible, in allowing the development of the confocal and particle-sizing systems. For future nasal drug delivery studies, the ability to measure such factors as the size of the delivered system in the nasal cavity, the depth of penetration of the formulation into the tissue are essential. Additionally, to have access to other data such as that obtained from drug transport in the same system, and to have the tissue available for histological examination represents a significant advance in the usefulness of such an in vitro technique for nasal delivery.

Development and deployment of an autonomous micro-drilling system for cochleostomy

This thesis describes the design and development of an autonomous micro-drilling system capable of accurately controlling the penetration of complaint tissues and its application to the drilling of the cochleostomy; a key stage in the cochlea implant procedure. The drilling of the cochleostomy is a precision micro-surgical task in which the control of the burr penetration through the outer bone tissue of the cochlea is vital to prevent damage to the structures within and requires a high degree of skill to perform successfully. The micro-drilling system demonstrates that the penetration of the cochlea can be achieved consistently and accurately. Breakthrough can be detected and controlled to within 20µm of the distal surface and the hole completed without perforation of the underlying endosteal membrane, leaving the membranous cochlea intact. This device is the first autonomous surgical tool successfully deployed in the operating theatre. The system is unique due to the way in which it uses real-time data from the cutting tool to derive the state of the tool-tissue interaction. Being a smart tool it uses this state information to actively control the way in which the drilling process progresses. This sensor guided strategy enables the tool to self-reference to the deforming tissue and navigate without the need for pre-operative scan data. It is this capability that enables the system to operate in circumstances where the tissue properties and boundary conditions are unknown, without the need to restrain the patient.

Spray dried combinations of lactoferrin with antibiotics appear superior to monotherapy for reducing biofilm formation by pseudomonas aeruginosa

SD Apo Lactoferrin-Tobramycin/Gentamicin Combinations are superior to monotherapy in the eradication of Pseudomonas aeruginosa Biofilm in the lungs Wilson Oguejiofor1, Lindsay J. Marshall1, Andrew J. Ingham1, Robert Price2, Jag. Shur2 1School of Life and Health Sciences, Aston University, Birmingham, UK. 2School of Pharmacy and Pharmacology, University of Bath, Bath, UK. KEYWORDS: lactoferrin, apo lactoferrin, spray drying, biofilm, cystic fibrosis Introduction Chronic lung infections from the opportunistic pathogeen Pseudomonas aeruginosa has been recognised as a major contributor to the incidences of high morbidity and mortality amongst cystic fibrosis (CF) patients (1,2). Currently, strategies for managing lung infections in CF patients involves the aggressive use of aerosolised antibiotics (3), however, increasing evidence suggests that the biofilm component of P. aeruginosa in the lower airway remains unperturbed and is associated with the development of antibiotic resistance. If this is so then, there is an urgent need to suitably adjust the current treatment strategy so that it includes compounds that prevent biofilm formation or disrupt established biofilms. It is well understood that biofilm formation is strongly dependent on iron (Fe3+) availability (4), therefore aerosolised anti-infective formulations which has the ability to chelate iron may essentially be a well suited therapy for eliminating P. aeruginosa biofilms on CF airway epithelial cells (5). In this study, we report the use of combination therapy; an aminoglycosides (tobramycin and gentamicin) and an antimicrobial peptide (lactoferrin) to significantly deplete P. aeruginosa biofilms. We demonstrate that lactoferrin-tobramycin and lactoferrin-gentamicin combinations are superior to the single antibiotic regime currently being employed to combat P. aeruginosa biofilms. MATERIALS AND METHOD Antibiotics: The antibiotics used in this study included gentamicin and tobramycin supplied by Fagron, UK. Bacterial strain and growth conditions: Pseudomonas aeruginosa strain PAO1 was provided by Prof. Peter Lambert of Aston University, Birmingham UK. The Strains were routinely grown from storage in a medium supplemented with magnesium chloride, glucose and casamino acids. Dialysis of lactoferrin: Apo lactoferrin was prepared by dialyzing a suspension of lactoferrin for 24 hrs at 4 °C against 20 mmol/L sodium dihydrogen phosphate, 20 mmol/L sodium acetate and 40 mmol/L EDTA (pH 3.5). Ferric ion (Fe3+) removal was verified by atomic absorption spectroscopy measurements. Spray drying of combinations of lactoferrin and apo lactoferrin with the different aminoglycosides: Combinations of tobramycin and gentamicin with the different preparations of lactoferrin were spray dried (SD) as a 2% (w/v) aqueous suspension. The spray drying parameters utilized for the production of suitable micron-sized particles includes: Inlet temperature, 180°C, spray flow rate, 606 L/hr; pump setting, 10%; aspirator setting, 85% (34m3/hr) to produce various outlet temperatures ranging from 99 - 106°C. Viability assay: To test the bactericidal activity of the various combinations, a viability assay was performed as previously described by Xu, Xiong et al. (6) with some modifications. Briefly, 10µL of ~ c. 6.6 x 107 CFU mL-1 P. aeruginosa strain PAO1 suspension were incubated (37°C, 60 mins) with 90 µL of a 2 µg/mL concentration of the various combinations and sampled every 10 mins. After incubation, the cells were diluted in deionised water and plated in Mueller hinton agar plates. Following 24 h incubation of the plates at 37°C, the percentage of viable cells was determined relative to incubation without added antibiotics. Biofilm assay: To test the susceptibility of the P. aeruginosa strain to various antibiotics in the biofilms mode of growth, overnight cultures of P. aeruginosa were diluted 1:100 into fresh medium supplemented with magnesium chloride, glucose and casamino acids. Aliquots of the dilution were dispensed into a 96 well dish and incubated (37°C, 24 h). Excess broth was removed and the number of colony forming units per milliliter (CFU/mL) of the planktonic bacteria was quantified. The biofilms were then washed and stained with 0.1% (w/v) crystal violet for 15 mins at room temperature. Following vigorous washing with water, the stained biofilms were solubilized in 30% acetic acid and the absorbance at 550nm of a 125 µL aliquot was determined in a microplate reader (Multiskan spectrum, Thermo Scientific) using 30% acetic acid in water as the blank. Aliquots of the broth prior to staining were used as an indicator of the level of planktonic growth. RESULTS AND DISCUSSION Following spray drying, the mean yield, volume weighted mean diameter and moisture content of lactoferrin powder were measured and were as follows (Table 1 and table 2); Table 1: Spray drying parameters FormulationInlet temp (°C)Outlet temp (°C)Airflow rate (L/hr)Mean yield (%)Moisture content (%) SD Lactoferrin18099 - 10060645.2 ±2.75.9 ±0.4 SD Apo Lactoferrin180100 - 10260657.8 ±1.85.7 ±0.2 Tobramycin180102 - 10460682.1 ±2.23.2 ±0.4 Lactoferrin + Tobramycin180104 - 10660687.5 ±1.43.7 ±0.2 Apo Lactoferrin + Tobramycin180103 - 10460676.3 ±2.43.3 ±0.5 Gentamicin18099 - 10260685.4 ±1.34.0 ±0.2 Lactoferrin + Gentamicin180102 - 10460687.3 ±2.13.9 ±0.3 Apo Lactoferrin + Gentamicin18099 -10360680.1±1.93.4 ±0.4 Table 2: Particle size distribution d10 d50d90 SD Lactoferrin1.384.9111.08 SD Apo Lactoferrin1.284.7911.04 SD Tobramycin1.254.9011.29 SD Lactoferrin + Tobramycin1.175.2715.23 SD Apo Lactoferrin + Tobramycin1.115.0614.31 SD Gentamicin1.406.0614.38 SD Lactoferrin + Gentamicin1.476.2314.41 SD Apo Lactoferrin + Gentamicin1.465.1511.53 The bactericidal activity of the various combinations were tested against P. aeruginosa PAO1 following a 60 minute incubation period (Figure 1 and Figure 2). While 2 µg/mL of a 1:1 combination of spray dried apo lactoferrin and Gentamicin was able to completely kill all bacterial cells within 40 mins, the same concentration was not as effective for the other antibiotic combinations. However, there was an overall reduction of bacterial cells by over 3 log units by the other combinations within 60 mins. Figure 1: Logarithmic plot of bacterial cell viability of various combinations of tobramycin and lactoferrin preparations at 2µg/mL (n = 3). Figure 2: Logarithmic plot of bacterial cell viability of various combinations of gentamicin and lactoferrin preparations at 2µg/mL (n = 3). Crystal violet staining showed that biofilm formation by P. aeruginosa PAO1 was significantly (ANOVA, p < 0.05) inhibited in the presence of the different lactoferrin preparations. Interestingly, apo lactoferrin and spray dried lactoferrin exhibited greater inhibition of both biofilm formation and biofilm persistence (Figure 2). Figure 2: Crystal violet staining of residual biofilms of P. aeruginosa following a 24hr incubation with the various combinations of antibiotics and an exposure to 48 hr formed biofilms. CONCLUSION In conclusion, combination therapy comprising of an antimicrobial peptide (lactoferrin) and an aminoglycosides (tobramycin or gentamicin) provides a feasible and alternative approach to monotherapy since the various combinations are more efficient than the respective monotherapy in the eradication of both planktonic and biofilms of P. aeruginosa. ACKNOWLEDGEMENT The authors would like to thank Mr. John Swarbrick and Friesland Campina for their generous donation of the Lactoferrin. REFERENCES 1.Hassett, D.J., Sutton, M.D., Schurr, M.J., Herr, A.B., Caldwell, C.C. and Matu, J.O. (2009), "Pseudomonas aeruginosa hypoxic or anaerobic biofilm infections within cystic fibrosis airways". Trends in Microbiology, 17, 130-138. 2.Trust, C.F. (2009), "Antibiotic treatment for cystic fibrosis". Report of the UK Cystic Fibrosis Trust Antibiotic Working Group. Consensus document. London: Cystic Fibrosis Trust. 3.Garcia-Contreras, L. and Hickey, A.J. (2002), "Pharmaceutical and biotechnological aerosols for cystic fibrosis therapy". Advanced Drug Delivery Reviews, 54, 1491-1504. 4.O'May, C.Y., Sanderson, K., Roddam, L.F., Kirov, S.M. and Reid, D.W. (2009), "Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions". J Med Microbiol, 58, 765-773. 5.Reid, D.W., Carroll, V., O'May, C., Champion, A. and Kirov, S.M. (2007), "Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis". European Respiratory Journal, 30, 286-292. 6.Xu, G., Xiong, W., Hu, Q., Zuo, P., Shao, B., Lan, F., Lu, X., Xu, Y. and Xiong, S. (2010), "Lactoferrin-derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa". J Appl Microbiol, 109, 1311-1318.

The spatial patterns of beta/A4 deposit subtypes in Alzheimer's disease

The spatial patterns of diffuse, primitive, classic (cored) and compact (burnt-out) subtypes of beta/A4 deposits were studied in coronal sections of the frontal lobe and hippocampus, including the adjacent gyri, in nine cases of Alzheimer's disease (AD). If the more mature deposits were derived from the diffuse deposits then there should be a close association between their spatial patterns in a brain region. In the majority of tissues examined, all deposit subtypes occurred in clusters which varied in dimension from 200 to 6400 microns. In many tissues, the clusters appeared to be regularly spaced parallel to the pia or alveus. The mean dimension of the primitive deposit clusters was greater than those of the diffuse, classic and compact types. In about 60% of cortical tissues examined, the clusters of primitive and diffuse deposits were not in phase, i.e. they alternated along the cortical strip. Clusters of classic deposits appeared to be distributed independently of the diffuse deposit clusters. Cluster size of the primitive deposits was positively correlated with the density of the primitive deposits in a tissue but no such relationship could be detected for the diffuse deposits. This study suggested that there was a complex relationship between the clusters of the different subtypes of beta/A4 deposits. If the diffuse deposits do give rise to the primitive and classic varieties then factors unrelated to the initial deposition of beta/A4 in the form of diffuse plaques were important in the formation of the mature deposits.

Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition

Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest. © 2011 Elsevier Inc.

Method of Elimination Data Sources Conflicts in Information System

Use of modern object-oriented methods of designing of information systems (IS) both descriptions of interrelations IS and automated with its help business-processes of the enterprises leads to necessity of construction uniform complete IS on the basis of set of local models of such system. As a result of use of such approach there are the contradictions caused by inconsistency of actions of separate developers IS with each other and that is much more important, inconsistency of the points of view of separate users IS. Besides similar contradictions arise while in service IS at the enterprise because of constant change separate business- processes of the enterprise. It is necessary to note also, that now overwhelming majority IS is developed and maintained as set of separate functional modules. Each of such modules can function as independent IS. However the problem of integration of separate functional modules in uniform system can lead to a lot of problems. Among these problems it is possible to specify, for example, presence in modules of functions which are not used by the enterprise to destination, to complexity of information and program integration of modules of various manufacturers, etc. In most cases these contradictions and the reasons, their caused, are consequence of primary representation IS as equilibrium steady system. In work [1] representation IS as dynamic multistable system which is capable to carry out following actions has been considered:

Optimal Control of Heterogeneous Systems

Цветомир Цачев - В настоящия доклад се прави преглед на някои резултати от областта на оптималното управление на непрекъснатите хетерогенни системи, публикувани в периодичната научна литература в последните години. Една динамична система се нарича хетерогенна, ако всеки от нейните елементи има собствена динамиката. Тук разглеждаме оптимално управление на системи, чиято хетерогенност се описва с едномерен или двумерен параметър – на всяка стойност на параметъра отговаря съответен елемент на системата. Хетерогенните динамични системи се използват за моделиране на процеси в икономиката, епидемиологията, биологията, опазване на обществената сигурност (ограничаване на използването на наркотици) и др. Тук разглеждаме модел на оптимално инвестиране в образование на макроикономическо ниво [11], на ограничаване на последствията от разпространението на СПИН [9], на пазар на права за въглеродни емисии [3, 4] и на оптимален макроикономически растеж при повишаване на нивото на върховите технологии [1]. Ключови думи: оптимално управление, непрекъснати хетерогенни динамични системи, приложения в икономиката и епидемиолегията

The development of attenuation compensation models of fluorescence spectroscopy signals

This study examines the effect of blood absorption on the endogenous fluorescence signal intensity of biological tissues. Experimental studies were conducted to identify these effects. To register the fluorescence intensity, the fluorescence spectroscopy method was employed. The intensity of the blood flow was measured by laser Doppler flowmetry. We proposed one possible implementation of the Monte Carlo method for the theoretical analysis of the effect of blood on the fluorescence signals. The simulation is constructed as a four-layer skin optical model based on the known optical parameters of the skin with different levels of blood supply. With the help of the simulation, we demonstrate how the level of blood supply can affect the appearance of the fluorescence spectra. In addition, to describe the properties of biological tissue, which may affect the fluorescence spectra, we turned to the method of diffuse reflectance spectroscopy (DRS). Using the spectral data provided by the DRS, the tissue attenuation effect can be extracted and used to correct the fluorescence spectra.

Kameralizmus, történelmi iskola, osztrák gazdaságtan. Három vázlat a német és osztrák közgazdasági diskurzus történetéből (Cameralism, the historical school and Austrian economics. Three outlines from the history of German and Austrian economic debate)

A kameralizmus a 16-18. századi német gyakorlati államtudomány és egyetemi tantárgy volt, amely összefoglalta a hivatalnokok képzéséhez szükséges elméleti és gyakorlati gazdasági ismereteket. Felfogása szerint az alattvalók jóléte és boldogsága feltétele az uralkodó gazdagságának, de az alattvalók önmaguktól nem képesek utat találni ehhez a boldogsághoz, szükség van az állandó külső irányításra. Igazgatásközpontúsága és egyetemi intézményesülése miatt nem tekinthető a merkantilizmus helyi változatának. Az 1840 és 1945 közötti német történeti iskola hagyományos ábrázolása több mint egy évszázadon át a német és a neoklasszikus tradíció szembenállását hangsúlyozta, kiemelve az organicizmus, a fejlődésgondolat és az egyediség jelentőségét, a szociális kérdés fontosságát, illetve a deduktív módszer és a gazdaság változatlan törvényeinek tagadását. A modern rekonstrukciók a történeti iskolát a posztklasszikus válságra adott egyik európai válaszként fogják fel, amely a történelemből levont, empirikusan megalapozott induktív alternatívát kínált. Az 1871-től kialakult osztrák iskola a neoklasszikus paradigmának egyszerre volt alkotórésze és versenytársa. A módszertani individualizmus, a szubjektivizmus, az idő fontossága, a tudás szerepe, az alternatív költségek elmélete stb. ugyan beépültek a mainstream közgazdaságtanba, de hangsúlyos kiemelésük lehetővé tette, hogy a társadalomelméleti magyarázat igényét őrző osztrák iskola megtartson valamit önálló beszédmódjából. / === / Cameralism was a practical political science and university subject in 16th–18th century Germany, summarizing the theoretical and practical economic knowledge required in the training of officials. The assumption was that the prosperity of the ruler depended on the welfare and happiness of the subjects, but the subjects themselves were not capable of achieving this happiness without permanent directions from above. Cameralism’s emphasis on administration and university institutionalization means that this approach cannot be seen as a local variant of mercantilism. The traditional account of the German historical school from 1840 to 1945 emphasized for over a century the contrast between the German and the Neoclassical traditions. It underlined the significance of the organic approach, the concept of development and individuality, the importance of the social question, and the denial of the deductive method and unalterable laws of the economy. Modern reconstructions see the historical school as one European response to the post-Classical crisis, offering an inductive alternative grounded empirically on history. The Austrian school formed in 1871 was at once a constituent of the Neoclassical paradigm and a rival to it. Methodological individualism, subjectivism, the importance of time, the role of knowledge, the theory of alternative costs etc. were absorbed into mainstream economics, but the focusing on these issues allowed the Austrian school, in keeping alive its demand for a social-theoretical explanation, to preserve something of an alternative discourse.

Versenyképesség haloptikával – Helyi gazdaságfejlesztési problémák fejlődő városokban = Competitiveness at short range - Local problems of economic development in medium-sized towns

The aim of our research in the first half of 2011 was to find out what were the administrative, regulative and other problems that were specific obstacles to local economic development and what best practices can be found in local economies. During our research we carried out interviews with leaders and local professionals in five medium size towns of Hungary. We stated that the most obstructive factors were the imperfection of vocational training, the excessively bureaucratic administrative proceedings (supplying of data, acquisition of authority permits, the attitude of the authorities, etc.) and the system of application and finding sources of funds. We think that the most innovative solutions are the good examples of the institutionalized co-operation between local governments and local businesses. We've come to the conclusion that there is a need for reducing administrative burdens for the sake of local economic development.

Biogeochemical effects of iron availability on primary producers in a shallow marine carbonate environment

We completed a synoptic survey of iron, phosphorus, and sulfur concentrations in shallow marine carbonate sediments from south Florida. Total extracted iron concentrations typically were 50 μmol g-1 dry weight (DW) and tended to decrease away from the Florida mainland, whereas total extracted phosphorus concentrations mostly were 10 μmol g-1 DW and tended to decrease from west to east across Florida Bay. Concentrations of reduced sulfur compounds, up to 40 μmol g-1 DW, tended to covary with sediment iron concentrations, suggesting that sulfide mineral formation was iron-limited. An index of iron availability derived from sediment data was negatively correlated with chlorophyll a concentrations in surface waters, demonstrating the close coupling of sediment-water column processes. Eight months after applying a surface layer of iron oxide granules to experimental plots, sediment iron, phosphorus, and sulfur were elevated to a depth of 10 cm relative to control plots. Biomass of the seagrass Thalassia testudinum was not different between control and iron addition plots, but individual shoot growth rates were significantly higher in experimental plots after 8 months. Although the iron content of leaf tissues was significantly higher from iron addition plots, no difference in phosphorus content of T. testudinum leaves was observed. Iron addition altered plant exposure to free sulfide, documented by a significantly higher δ34S of leaf tissue from experimental plots relative to controls. Iron as a buffer to toxic sulfides may promote individual shoot growth, but phosphorus availability to plants still appears to limit production in carbonate sediments.

Arsenic transport by zebrafish aquaglyceroporins

Background Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized. Results In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were namedaqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic. Conclusion This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

Size-based variation in intertissue comparisons of stable carbon and nitrogen isotopic signatures of bull sharks (Carcharhinus leucas) and tiger sharks (Galeocerdo cuvier)

Stable isotopes are important tools for understanding the trophic roles of elasmobranchs. However, whether different tissues provide consistent stable isotope values within an individual are largely unknown. To address this, the relationships among carbon and nitrogen isotope values were quantified for blood, muscle, and fin from juvenile bull sharks (Carcharhinus leucas) and blood and fin from large tiger sharks (Galeocerdo cuvier) collected in two different ecosystems. We also investigated the relationship between shark size and the magnitude of differences in isotopic values between tissues. Isotope values were significantly positively correlated for all paired tissue comparisons, but R2 values were much higher for δ13C than for δ15N. Paired differences between isotopic values of tissues were relatively small but varied significantly with shark total length, suggesting that shark size can be an important factor influencing the magnitude of differences in isotope values of different tissues. For studies of juvenile sharks, care should be taken in using slow turnover tissues like muscle and fin, because they may retain a maternal signature for an extended time. Although correlations were relatively strong, results suggest that correction factors should be generated for the desired study species and may only allow coarse-scale comparisons between studies using different tissue types.

Probing Tissue Microstructure Using Susceptibility Contrast Magnetic Resonance Imaging

Magnetic resonance imaging is a research and clinical tool that has been applied in a wide variety of sciences. One area of magnetic resonance imaging that has exhibited terrific promise and growth in the past decade is magnetic susceptibility imaging. Imaging tissue susceptibility provides insight into the microstructural organization and chemical properties of biological tissues, but this image contrast is not well understood. The purpose of this work is to develop effective approaches to image, assess, and model the mechanisms that generate both isotropic and anisotropic magnetic susceptibility contrast in biological tissues, including myocardium and central nervous system white matter.

This document contains the first report of MRI-measured susceptibility anisotropy in myocardium. Intact mouse heart specimens were scanned using MRI at 9.4 T to ascertain both the magnetic susceptibility and myofiber orientation of the tissue. The susceptibility anisotropy of myocardium was observed and measured by relating the apparent tissue susceptibility as a function of the myofiber angle with respect to the applied magnetic field. A multi-filament model of myocardial tissue revealed that the diamagnetically anisotropy α-helix peptide bonds in myofilament proteins are capable of producing bulk susceptibility anisotropy on a scale measurable by MRI, and are potentially the chief sources of the experimentally observed anisotropy.

The growing use of paramagnetic contrast agents in magnetic susceptibility imaging motivated a series of investigations regarding the effect of these exogenous agents on susceptibility imaging in the brain, heart, and kidney. In each of these organs, gadolinium increases susceptibility contrast and anisotropy, though the enhancements depend on the tissue type, compartmentalization of contrast agent, and complex multi-pool relaxation. In the brain, the introduction of paramagnetic contrast agents actually makes white matter tissue regions appear more diamagnetic relative to the reference susceptibility. Gadolinium-enhanced MRI yields tensor-valued susceptibility images with eigenvectors that more accurately reflect the underlying tissue orientation.

Despite the boost gadolinium provides, tensor-valued susceptibility image reconstruction is prone to image artifacts. A novel algorithm was developed to mitigate these artifacts by incorporating orientation-dependent tissue relaxation information into susceptibility tensor estimation. The technique was verified using a numerical phantom simulation, and improves susceptibility-based tractography in the brain, kidney, and heart. This work represents the first successful application of susceptibility-based tractography to a whole, intact heart.

The knowledge and tools developed throughout the course of this research were then applied to studying mouse models of Alzheimer’s disease in vivo, and studying hypertrophic human myocardium specimens ex vivo. Though a preliminary study using contrast-enhanced quantitative susceptibility mapping has revealed diamagnetic amyloid plaques associated with Alzheimer’s disease in the mouse brain ex vivo, non-contrast susceptibility imaging was unable to precisely identify these plaques in vivo. Susceptibility tensor imaging of human myocardium specimens at 9.4 T shows that susceptibility anisotropy is larger and mean susceptibility is more diamagnetic in hypertrophic tissue than in normal tissue. These findings support the hypothesis that myofilament proteins are a source of susceptibility contrast and anisotropy in myocardium. This collection of preclinical studies provides new tools and context for analyzing tissue structure, chemistry, and health in a variety of organs throughout the body.

Unraveling the function of bacterial-type phosphoenolpyruvate carboxylase in vascular plants

Two distinct phosphoenolpyruvate carboxylase (PEPC) isozymes occur in vascular plants and green algae: plant-type PEPC (PTPC) and bacterial-type PEPC (BTPC). PTPC polypeptides typically form a tightly regulated cytosolic Class-1 PEPC homotetramer. BTPCs, however, appear to be less widely expressed and to exist only as catalytic and regulatory subunits that physically interact with co-expressed PTPC subunits to form hetero-octameric Class-2 PEPC complexes that are highly desensitized to Class-1 PEPC allosteric effectors. Yeast two-hybrid studies indicated that castor plant BTPC (RcPPC4) interacts with all three Arabidopsis thaliana PTPC isozymes, and that it forms stronger interactions with AtPPC2 and AtPPC3, suggesting that specific PTPCs are preferred for Class-2 PEPC formation. In contrast, Arabidopsis BTPC (AtPPC4) appeared to interact very weakly with AtPPC2 and AtPPC3, suggesting that BTPCs from different species may have different physical properties, hypothesized to be due to sequence dissimilarities within their ~10 kDa intrinsically disordered region. Recent RNA-seq and microarray data were analyzed to obtain a better understanding of BTPC expression patterns in different tissues of various monocot and dicot species. High levels of BTPC transcripts, polypeptides and Class-2 PEPC complexes were originally discovered in developing castor seeds, but the analysis revealed a broad range of diverse tissues where abundant BTPC transcripts are also expressed, such as the developing fruits of cucumber, grape, and tomato. Marked BTPC expression correlated well with the presence of ~116 kDa immunoreactive BTPC polypeptides, as well as Class-2 PEPC complexes in the immature fruit of cucumbers and tomatoes. It is therefore hypothesized that in vascular plants BTPC and thus Class-2 PEPC complexes maintain anaplerotic PEP flux in tissues with elevated malate levels that would potently inhibit ‘housekeeping’ Class-1 PEPCs. Elevated levels of malate can be used by biosynthetically active sink tissues such as immature tomatoes and cucumbers for rapid cell expansion, drought or salt stressed roots for osmoregulation, and developing seeds and pollen as a precursor for storage lipid and protein biosynthesis.