Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts ^{1 2}. Approximately 250 consecutive sections (4 μm thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (www.proteinatlas.org) (Figure 1) ^{3 4}. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.     

The relationship between the diversity of arbuscular mycorrhizal fungi and grazing in a meadow steppe

Aims

To study the relationship between changes in soil properties and plant community characters produced by grazing in a meadow steppe grassland and the composition and diversity of spore-producing arbuscular mycorrhizal fungi (AMF).

Methods

A field survey was carried out in a meadow steppe area with a gradient of grazing pressures (a site with four grazing intensities and a reserve closed to grazing). The AMF community composition (characterized by spore abundance) and diversity, the vegetation characters and soil properties were measured, and root colonization by AMF was assessed.

Results

AMF diversity (richness and evenness) was higher under light to moderate grazing pressure and declined under intense grazing pressures. Results of multiple regressions indicated that soil electrical conductivity was highly associated with AMF diversity. The variation in AMF diversity was partially associated to the density of tillers of the dominant grass (Leymus chinensis), the above and below-ground biomass and the richness of the plant community.

Conclusions

We propose that the relationship between plants and AMF is altered by environmental stress (salinity) which is in turn influenced by animal grazing. Direct and indirect interactions between vegetation, soil properties, and AMF community need to be elucidated to improve our ability to manage these communities.     

Cardiovascular risk factors and cognitive function in middle aged and elderly Lithuanian urban population: results from the HAPIEE study

Background

The purpose of this study was to examine associations between cardiovascular risk factors and cognitive ability in middle aged and elderly Lithuanian urban population.

Methods

Data from the survey performed in the framework of the HAPIEE (Health, Alcohol, Psychosocial Factors in Eastern Europe) study were presented. A random sample of 7,087 individuals aged 45–72 years was screened in 2006–2008.

Results

The scores of immediate recall and delayed verbal recall, cognitive speed and attention were significantly lower in men than in women; yet numerical ability scores were higher in men. Significant associations between lowered cognitive functions and previous stroke (in male OR?=?2.52; 95% CI?=?1.75-3.64; in female OR?=?2.45; 95% CI?=?1.75, 3.64) as well as ischemic heart disease history (among male OR?=?1.28; 95% CI?=?1.03-1.60) have been determined. Higher level of physical activity in leisure time (among female OR?=?1.32; 95% CI?=?1.03-1.69), poor self-rated health (among male OR?=?1.57; 95% CI?=?1.15-2.14) and poor quality of life (in male OR?=?1.67; 95% CI?=?1.07-2.61; in female OR?=?2.81; 95% CI?=?1.92-4.11) were related to lowered cognitive function.

Conclusions

The findings of the study suggest that associations between cardiovascular risk factors and lowered cognitive function among healthy middle-aged and elderly adults strongly depend on gender.

     

The Lipopolysaccharide from Capnocytophaga canimorsus Reveals an Unexpected Role of the Core-Oligosaccharide in MD-2 Binding

Capnocytophaga canimorsus is a usual member of dog''s mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a “hybrid backbone” lacking the 4′ phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 – lipid A complex in case the 4′ phosphate is not present.     

Epigenetic modulators for brain cancer stem cells: Implications for anticancer treatment

Primary malignant brain tumors are a major cause of morbidity and mortality in both adults and children, with a dismal prognosis despite multimodal therapeutic approaches. In the last years, a specific subpopulation of cells within the tumor bulk, named cancer stem cells(CSCs) or tumor-initiating cells, have been identified in brain tumors as responsible for cancer growth and disease progression. Stemness features of tumor cells strongly affect treatment response, leading to the escape from conventional therapeutic approaches and subsequently causing tumor relapse. Recent research efforts have focused at identifying new therapeutic strategies capable of specifically targeting CSCs in cancers by taking into consideration their complex nature. Aberrant epigenetic machinery plays a key role in the genesis and progression of brain tumors as well as inducing CSC reprogramming and preserving CSC characteristics. Thus, reverting the cancer epigenome can be considered a promising therapeutic strategy. Three main epigenetic mechanisms have been described: DNA methylation, histone modifications, and non-coding RNA, particularly micro RNAs. Each of these mechanisms has been proven to be targetable by chemical compounds, known as epigeneticbased drugs or epidrugs, that specifically target epigenetic marks. We review here recent advances in the study of epigenetic modulators promoting and sustaining brain tumor stem-like cells. We focus on their potential role in cancer therapy.     

Structure of the O-polysaccharides of the lipopolysaccharides of Mesorhizobium loti HAMBI 1148 and Mesorhizobium amorphae ATCC 19655 containing two O-methylated monosaccharides

The O-polysaccharide of Mesorhizobium loti HAMBI 1148 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses, Smith degradation, and ¹H and ¹³C NMR spectroscopies, including 2D ¹H/¹H COSY, TOCSY, ROESY, and H-detected ¹H/¹³C HSQC experiments. The O-polysaccharide was found to have a branched hexasaccharide-repeating unit of the following structure:where 2-acetamido-2-deoxy-4-O-methyl-d-glucose (d-GlcNAc4Me) and methyl group on 2-substituted d-rhamnose (Me) shown in italics are present in ∼80% and ∼40% repeating units, respectively. Similar studies of the O-polysaccharide from Mesorhizobium amorphae ATCC 19655 by sugar analysis and NMR spectroscopy revealed essentially the same structure but a higher content of 3-O-methyl-d-rhamnose (∼70%).     

Role of oxidative stress in the antitumoral action of a new vanadyl(IV) complex with the flavonoid chrysin in two osteoblast cell lines: relationship with the radical scavenger activity

The new complex [VO(chrysin)₂EtOH]₂ (VOchrys) has been synthesized and thoroughly characterized. Fourier transform IR, UV–vis, diffuse reflectance, and EPR spectroscopies as well as elemental analysis and thermal measurements were performed. In solution, different species could be detected by EPR spectroscopy as a function of the ligand-to-metal ratio. The stoichiometry of the chelate complex formed at pH 5 was also determined by spectrophotometric titrations. Since flavonoids are natural antioxidant compounds, the antioxidant capacity of chrysin and its vanadyl(IV) complex was investigated using different radicals. Chrysin and its complex were not able to diminish the level of superoxide and 1,1-diphenyl-2-picrylhydrazyl radicals to a great extent. In contrast, they were strong scavengers for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt radical cations and OH· radicals with a greater potency for VOchrys. Taking into account their selective antioxidant properties, we investigated the bioactivity of these compounds in two osteoblast-like cells in culture. Chrysin and VOchrys caused an inhibition of cell proliferation in MC3T3E1 normal osteoblasts and UMR106 tumor cells in a dose-response manner, with a greater effect in the latter cell line. The generation of reactive oxygen species (ROS) was evaluated in both cell lines and a correlation could be established between the antiproliferative effects of chrysin and the increase in the ROS levels. The complex did not generate types of ROS that can be detected by the dihydrorhodamine 123 technique so the antiproliferative effect may be attributed to the formation of other radicals such as superoxide, which is not detected by this probe. The morphological alterations were in agreement with these changes.     

Reactive oxygen species are required for maintenance and differentiation of primary lung fibroblasts in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms.

Methodology/Principal Findings

We first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger.

Conclusions/Significance

Robust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.     

Superoxide dismutase activity and electrochemical study of the binuclear [Cu(TSA)2py]2 complex.

The superoxide dismutase-mimetic activity and the electrochemical behavior of the binuclear complex [Cu(TSA)2py]2 is reported. The complex exhibits a marked SOD activity in the nitroblue tetrazolium assay. Its redox response is in agrement with two copper atoms partially coupled through the carboxylate moieties. The electrode reactions lie in the range of the superoxide/peroxide and oxygen/superoxide couples. This fact is indicative that this complex can act as catalyst for the SOD reaction.     

Understanding the cell biology of antigen presentation: the dendritic cell contribution

The study of the cell biology of antigen processing and presentation has greatly contributed to our understanding of the immune response. The work of many immunologically inclined cell biologists has also permitted us to gain new insights on cellular mechanisms shared by many cell types. Dendritic cells are master regulators of the immune system and consequently have received a lot of attention in recent years. With the aim of controlling antigen processing and presentation, the solutions used by dendritic cells to respond to environmental changes are numerous and surprising. In the presence of pathogens, dendritic cells regulate strongly their endocytic pathway by interfering with uptake, proteolysis, membrane dynamics and transport in and out of the lysosome to become the most potent antigen-presenting cells known.