Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

Children’s exposure to sustainability practices during the transition from preschool into school and their learning and socioemotional development

Evidence that the learning gains of preschool fade as children transition into elementary school has led to increased efforts to sustain preschool advantages during this key transitional period. This study explores whether the observed benefits of sustainability practices for a range of child outcomes are explained and/or moderated by family and school mechanisms selecting children into experiencing these practices. Analyses of the Early Childhood Longitudinal Study-Birth Cohort revealed that both family and school factors predicted children’s exposure to several PK-3 sustainability practices. PK-3 sustainability practices were associated with reading (but not math) gains and better interpersonal skills (but not fewer externalizing behaviors) following the transition into kindergarten. These links were not conditioned by the selection mechanisms. The findings highlight who is more likely to seek out (at the family level) or offer (at the school level) sustainability practices and how relevant they are to fighting preschool fadeout.     

TheisfA mutation inhibits mutator activity and processing of UmuD protein inEscherichia coli recA730 strains

Further studies on theisfA mutation responsible for anti-SOS and antimutagenic activities inEscherichia coli are described. We have previously shown that theisfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. TheisfA mutation has now been demonstrated also to suppress mutator activity inE. coli recA730 andrecA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of theisfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in therecA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

An Ecteola-cellulose chromatography assay for 3′-phosphoadenosine 5′-phosphosulfate: Phenol sulfotransferase

A new assay procedure for phenol sulfotransferase which employs [35S]-3'-phosphoadenosine-5'-phosphosulfate as a sulfate donor and a variety of phenols as sulfate acceptors was developed. The appearance of the 35S-sulfated products or the disappearance of the [35S]-3'-phosphoadenosine-5'-phosphosulfate are determined simultaneously by chromatography of the assay incubation mixtures on Ecteola-cellulose columns, eluting with an NH4HCO3 step gradient. Various acidic, neutral, and basic phenols can be employed as substrates for phenol sulfotransferase using this procedure.     

Trends in mortality statistics in England and Wales with particular reference to AIDS from 1984 to April 1987

Death certification should be able to provide accurate data on the number of deaths due to AIDS as a basis for predicting future deaths from the syndrome. Trends in deaths from other causes may identify conditions that have not been recognised to be associated with HIV infection. Mortality statistics with reference to AIDS in England and Wales were completed from death certificates. Increases in deaths from selected causes likely to be associated with AIDS or HIV infection suggested that in some patients with HIV infection, AIDS was not stated on the death certificate or subsequently notified by the doctor who signed the certificate. From calculations of excess deaths between the beginning of 1985 and the end of April 1987, compared with 1984 at least 495 deaths possibly associated with HIV infection were estimated to have occurred among men aged 15-54 during that period. In 261 AIDS or HIV infection was stated on the original or amended death entry as the cause of death, and of these 198 were included in the estimated number of excess deaths.Accurate notification of the underlying cause of death and associated diseases is required for the precise monitoring of trends in mortality from AIDS and possible identification of unrecognised conditions associated with HIV infection.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Starch Binding Domain-containing Protein 1/Genethonin 1 Is a Novel Participant in Glycogen Metabolism

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.     

Simple models including energy and spike constraints reproduce complex activity patterns and metabolic disruptions

In this work, we introduce new phenomenological neuronal models (eLIF and mAdExp) that account for energy supply and demand in the cell as well as the inactivation of spike generation how these interact with subthreshold and spiking dynamics. Including these constraints, the new models reproduce a broad range of biologically-relevant behaviors that are identified to be crucial in many neurological disorders, but were not captured by commonly used phenomenological models. Because of their low dimensionality eLIF and mAdExp open the possibility of future large-scale simulations for more realistic studies of brain circuits involved in neuronal disorders. The new models enable both more accurate modeling and the possibility to study energy-associated disorders over the whole time-course of disease progression instead of only comparing the initially healthy status with the final diseased state. These models, therefore, provide new theoretical and computational methods to assess the opportunities of early diagnostics and the potential of energy-centered approaches to improve therapies.