MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings.     

Effects of data structure on the estimation of covariance functions to describe genotype by environment interactions in a reaction norm model

Covariance functions have been proposed to predict breeding values and genetic (co)variances as a function of phenotypic within herd-year averages (environmental parameters) to include genotype by environment interaction. The objective of this paper was to investigate the influence of definition of environmental parameters and non-random use of sires on expected breeding values and estimated genetic variances across environments. Breeding values were simulated as a linear function of simulated herd effects. The definition of environmental parameters hardly influenced the results. In situations with random use of sires, estimated genetic correlations between the trait expressed in different environments were 0.93, 0.93 and 0.97 while simulated at 0.89 and estimated genetic variances deviated up to 30% from the simulated values. Non random use of sires, poor genetic connectedness and small herd size had a large impact on the estimated covariance functions, expected breeding values and calculated environmental parameters. Estimated genetic correlations between a trait expressed in different environments were biased upwards and breeding values were more biased when genetic connectedness became poorer and herd composition more diverse. The best possible solution at this stage is to use environmental parameters combining large numbers of animals per herd, while losing some information on genotype by environment interaction in the data.     

Inflammatory neurodegeneration induced by lipoteichoic acid from Staphylococcus aureus is mediated by glia activation, nitrosative and oxidative stress, and caspase activation

In this study we investigated the mechanisms of neuronal cell death induced by lipoteichoic acid (LTA) and muramyl dipeptide (MDP) from Gram-positive bacterial cell walls using primary cultures of rat cerebellum granule cells (CGCs) and rat cortical glial cells (astrocytes and microglia). LTA (+/- MDP) from Staphylococcus aureus induced a strong inflammatory response of both types of glial cells (release of interleukin-1beta, tumour necrosis factor-alpha and nitric oxide). The death of CGCs was caused by activated glia because in the absence of glia (treatment with 7.5 microm cytosine-d-arabinoside to inhibit non-neuronal cell proliferation) LTA + MDP did not cause significant cell death (less than 20%). In addition, staining with rhodamine-labelled LTA confirmed that LTA was bound only to microglia and astrocytes (not neurones). Neuronal cell death induced by LTA (+/- MDP)-activated glia was partially blocked by an inducible nitric oxide synthase inhibitor (1400 W; 100 microm), and completely blocked by a superoxide dismutase mimetic [manganese (III) tetrakis (4-benzoic acid)porphyrin chloride; 50 microm] and a peroxynitrite scavenger [5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III); 100 microm] suggesting that nitric oxide and peroxynitrite contributed to LTA-induced cell death. Moreover, neuronal cell death was inhibited by selective inhibitors of caspase-3 (z-DEVD-fmk; 50 microm) and caspase-8 (z-Ile-Glu(O-Me)-Thr-Asp(O-Me) fluoromethyl ketone; 50 microm) indicating that they were involved in LTA-induced neuronal cell death.     

Release of regulators of angiogenesis following Hypocrellin-A and -B photodynamic therapy of human brain tumor cells

Photodynamic therapy (PDT) is an innovative strategy for the treatment of solid neoplasms of the brain. Aside from inducing cell death in tumor cells, PDT induces endothelial cell death and promotes formation of blood clots; however, exact mechanisms that trigger these phenomena remain largely unknown. We now used Western blotting to analyze secretion of regulators of angiogenesis to the supernatants of one glioma, one macrophage, and one endothelial cell line following Hypocrellin-A and -B photodynamic therapy. We observed induction of proangiogenic VEGF (vascular endothelial growth factor) and of antiangiogenic sFlt-1, angiostatin, p43, allograft inflammatory factor-1, and connective tissue growth factor. Release of thrombospondin-1 was diminished in a glioma cell line supernatant. Endostatin release was induced in glioma cells and reduced in macrophages and endothelial cells. These data show that a wide range of antiangiogenic factors are secreted by brain tumor cells following Hypocrellin photochemotherapy. However, VEGF release is also induced thus suggesting both favorable and deleterious effects on tumor outgrowth.     

Alu repeats and human genomic diversity

During the past 65 million years, Alu elements have propagated to more than one million copies in primate genomes, which has resulted in the generation of a series of Alu subfamilies of different ages. Alu elements affect the genome in several ways, causing insertion mutations, recombination between elements, gene conversion and alterations in gene expression. Alu-insertion polymorphisms are a boon for the study of human population genetics and primate comparative genomics because they are neutral genetic markers of identical descent with known ancestral states.     

Potential for retroposition by old Alu subfamilies

Alu elements sharing sequence characteristics of the old subfamilies are thought to currently be retrotranspositionally inactive. We analyzed one of these old subfamilies of Alu elements, Sx, for sequence conservation relative to the consensus and the length of the A-tail as parameters to define the presence of potential Alu Sx source genes in the human genome. Sequence identity to the left half or the right half of the Alu Sx consensus sequence was evaluated for 4424 complete elements obtained from the human genome draft sequence. A small subset of Alu Sx left halves were found to be more conserved than any of the Alu Sx right halves. Selection for promoter function in active elements may explain the slightly higher conservation of the left half. In order to determine whether this sequence identity was the result of recent activity, or simply sequence conservation for older elements, PCR amplification of some of the loci containing Sx elements with conserved left/right halves from different primate genomes was carried out. Several of these Sx Alus were found to have amplified at a later evolutionary period (<35 mya) than expected based on previous studies of Sx elements. Analysis of A-tail length, a feature correlated with current retroposition activity, varied between Alu Sx element loci in different primates, where the length increased in specific Alu elements in the human genome. The presence of few conserved Alu Sx elements and the dynamic expansion/contraction of the A-tail suggests that some of these older subfamilies may still be active at very low levels or in a few individuals. Present address: (Claudina Alemán Stevenson) Laboratory of Cell Biology, NCI/NIH Building 37/Rm 1A09, Bethesda, MD 20892, USA     

Glycosylphosphatidyl inositol-anchored proteins and fyn kinase assemble in noncaveolar plasma membrane microdomains defined by reggie-1 and -2

Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in < or =0.1-microm plasma membrane microdomains of neurons and astrocytes. In astrocytes, reggie-1 and -2 do not occur in caveolae but clearly outside these structures. Microscopy and coimmunoprecipitation show that reggie-1 and -2 are associated with fyn kinase and with the glycosylphosphatidyl inositol-anchored proteins Thy-1 and F3 that, when activated by antibody cross-linking, selectively copatch with reggie. Jurkat cells, after cross-linking of Thy-1 or GM1 (with the use of cholera toxin), exhibit substantial colocalization of reggie-1 and -2 with Thy-1, GM1, the T-cell receptor complex and fyn. This, and the accumulation of reggie proteins in detergent-resistant membrane fractions containing F3, Thy-1, and fyn imparts to reggie-1 and -2 properties of raft-associated proteins. It also suggests that reggie-1 and -2 participate in the formation of signal transduction centers. In addition, we find reggie-1 and -2 in endolysosomes. In Jurkat cells, reggie-1 and -2 together with fyn and Thy-1 increase in endolysosomes concurrent with a decrease at the plasma membrane. Thus, reggie-1 and -2 define raft-related microdomain signaling centers in neurons and T cells, and the protein complex involved in signaling becomes subject to degradation.