Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen''s (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)²; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction² and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations³. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.     

The possible role of soluble salts in chemical evolution

Summary A model is proposed for a prebiotic environment in which concentration, condensation, and chemical evolution of biomolecules could have taken place. The main reactions expected of proteins, nucleic acids, lipids, and some of their precursors in this environment are examined.The model is based on our previously developed concept of a fluctuating system in which hydration and dehydration processes take place in a cyclic manner. In the present model, however, high concentrations of soluble salts, such as chlorides and sulfates, are taken into account, whereas previously a more or less salt-free system had been assumed. Thus the preponderance of surfaces of soluble salts is implied, even though sparingly soluble minerals, such as clay minerals or quartz, are also present.During the dehydration stage biomolecules tend to leave the solution and concentrate at certain microenvironments, such as in micelles and aggregates, at the liquid-gas surface and, possibly, at the emerging solid surfaces. Moreover, in these brines, and especially during the last stages of dehydration, high temperatures are attainable, which may enhance certain reactions between the organic molecules, and result in a net increase of condensation over degradation.In the dehydrated state, solid-state condensation and synthesis reactions are possible in which the surface of soluble salts may serve as a catalyst. Several reports in the literature support this hypothesis. Hydration brings about dissolution of the minerals and redistribution of the biomolecules. In such a system, evolutionary processes like those postulated by White (1980) and by Lahav and White (1980) are possible. Moreover, since several soluble salts of known geological occurrence are optically active in their crystalline state, the involvement of the model system in the selection and evolution of chiral organic compounds should also be considered. In addition, organic molecules in the above microenvironments are also expected to undergo selective interactions based on factors such as molecular pattern and chiral recognition and hydrophobicity. The proposed system emphasizes the need to develop the theoretical background and experimental methods for the study of interactions among biomolecules in the presence of high salt concentrations and solid surfaces of soluble salts, as well as interactions between the biomolecules and these surfaces.     

Imaging growth dynamics of tumour spheroids using optical coherence tomography

Optical coherence tomography (OCT) was used to monitor the dynamics of tumour spheroid formation by the hanging drop method. In contrast to microscopy, the estimates obtained using OCT for the volume of the spheroid, were consistent with the measured changes in cell number as a function of time. The OCT images also revealed heterogeneous structures in the spheroids of ∼200 μm diameter. These corresponded to the necrotic regions identified by fluorescence of propidium iodide stained cells.     

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein–surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein–surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein–surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein–surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

A facile HPLC method for optical purity and quantitative measurements of phenylalanine from the hydrolyzed aspartame under different pH and temperature after its derivatization with a fluorescent reagent

Summary. In this paper, the artificial sweetener aspartame is deliberately hydrolyzed under different pH and temperature in the matrix, and time period for the hydrolysis. The HPLC analysis is then performed to quantitatively measure the amount and the optical purity of phenylalanine produced as a result of hydrolysis in the matrix after its functionalization with a fluorescent reagent. The results show that the amount of phenylalanine in the matrix is affected by the pH variation during the hydrolysis and found increased in low pH conditions. High temperature or long time periods for the decomposition also increases the amount, which indicates that beverages and foods containing aspartame as a sweetener may not be safe for phenylketonuria patients to consume if they are stored under these conditions. Conversely, the optical purity of phenylalanine, expressed as the percentage of d-enantiomer, is not affected by pH variations. However, it decreases as the length of time elapsed is increased or surrounding temperature is elevated during the decomposition.     

NEKTAR,SPICE and Vortonics: using federated grids for large scale scientific applications

In response to a joint call from US’s NSF and UK’s EPSRC for applications that aim to utilize the combined computational resources of the US and UK, three computational science groups from UCL, Tufts and Brown Universities teamed up with a middleware team from NIU/Argonne to meet the challenge. Although the groups had three distinct codes and aims, the projects had the underlying common feature that they were comprised of large-scale distributed applications which required high-end networking and advanced middleware in order to be effectively deployed. For example, cross-site runs were found to be a very effective strategy to overcome the limitations of a single resource. The seamless federation of a grid-of-grids remains difficult. Even if interoperability at the middleware and software stack levels were to exist, it would not guarantee that the federated grids can be utilized for large scale distributed applications. There are important additional requirements for example, compatible and consistent usage policy, automated advanced reservations and most important of all co-scheduling. This paper outlines the scientific motivation and describes why distributed resources are critical for all three projects. It documents the challenges encountered in using a grid-of-grids and some of the solutions devised in response.     

Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells

A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO.     

Nanotechnologies in proteomics

Progress in proteomic researches is largely determined by development and implementation of new methods for the revelation and identification of proteins in biological material in a wide concentration range (from 10(-3) M to single molecules). The most perspective approaches to address this problem involve (i) nanotechnological physicochemical procedures for the separation of multicomponent protein mixtures; among these of particular interest are biospecific nanotechnological procedures for selection of proteins from multicomponent protein mixtures with their subsequent concentration on solid support; (ii) identification and counting of single molecules by use of molecular detectors. The prototypes of biospecific nanotechnological procedures, based on the capture of ligand biomolecules by biomolecules of immobilized ligate and the concentration of the captured ligands on appropriate surfaces, are well known; these are affinity chromatography, magnetic biobeads technology, different biosensor methods, etc. Here, we review the most promising nanotechnological approaches for selection of proteins and kinetic characterization of their complexes based on these biospecific methods with subsequent MS/MS identification of proteins and protein complexes. Two major groups of methods for the analysis and identification of individual molecules and their complexes by use of molecular detectors will be reviewed: scanning probe microscopy (SPM) (including atomic-force microscopy) and cryomassdetector technology.     

Coumarin derived chromophores in the donor-acceptor-donor format that gives fluorescence enhancement and large two-photon activity in presence of specific metal ions

Two coumarin derived dyes (compounds L_a and L) have been synthesized in high yields by Schiff base condensation. On probing with a femtosecond laser, none of these compounds show any two-photon activity in the wavelength range, 760-860 nm. However, L_a in presence of Zn(II) and L in presence of Mg(II), exhibit large two-photon absorption as well as emission in the same wavelength range. Theoretical calculations at the B3LYP functional with 6-31G∗ and LanL2DZ mixed basis set under DFT formalism support experimental results.     

The anatomy of organogenesis: novel solutions to old problems

Understanding anatomical aspects of mammalian organ development, in both normal and mutant animals, is important for basic biology and also for regenerative medicine and tissue engineering. The size and complexity of developing organs, together with variations in their detailed anatomy, has made the obtaining of high-resolution time-courses of anatomical change difficult to obtain. The fact that organ development tends to use the same genes as early embryogenesis also makes genetic manipulation difficult, as so many mutant embryos die before organogenesis begins. These problems have seriously hampered the study of organogenesis. Here, we describe three significant advances that promise solutions: (1) the production of GFP-reporter mice that can be used for high-resolution live-imaging of small tissues as they grow, (2) RNA interference, which allows the manipulation of specific genes at any stage of organ development, and (3) optical projection tomography, which allows medium-resolution three-dimensional images of complete embryos to be obtained easily. We finish by looking ahead to the prospects of uniting these three technologies to allow accurate, high-throughput screening of mutants and automated comparison of biological data with computer predictions.