Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes

The mechanisms driving bacterial chromosome segregation remain poorly characterized. While a number of factors influencing chromosome segregation have been described in recent years, none of them appeared to play an essential role in the process comparable to the eukaryotic centromere/spindle complex. The research community involved in bacterial chromosome was becoming familiar with the fact that bacteria have selected multiple redundant systems to ensure correct chromosome segregation. Over the past few years a new perspective came out that entropic forces generated by the confinement of the chromosome in the crowded nucleoid shell could be sufficient to segregate the chromosome. The segregating factors would only be required to create adequate conditions for entropy to do its job. In the article by Yazdi et al. ( 2012 ) in this issue of Molecular Microbiology, this model was challenged experimentally in live Escherichia coli cells. A Fis–GFP fusion was used to follow nucleoid choreography and analyse it from a polymer physics perspective. Their results suggest strongly that E. coli nucleoids behave as self‐adherent polymers. Such a structuring and the specific segregation patterns observed do not support an entropic like segregation model. Are we back to the pre‐entropic era?     

Exposure classification of MRI workers in epidemiological studies

We estimate that there are about 100,000 workers from different disciplines, such as radiographers, nurses, anesthetists, technicians, engineers, etc., who can be exposed to substantial electromagnetic fields (compared to normal background levels) around magnetic resonance imaging (MRI) scanners. There is a need for well‐designed epidemiological studies of MRI workers but since the exposure from MRI equipment is a very complex mixture of static magnetic fields, switched gradient magnetic fields, and radiofrequency electromagnetic fields (RF EMF), it is necessary to discuss how to assess the exposure in epidemiological studies. As an alternative to the use of job title as a proxy of exposure, we propose an exposure categorization for the different professions working with MRI equipment. Specifically, we propose defining exposure in three categories, depending on whether people are exposed to only the static field, to the static plus switched gradient fields or to the static plus switched gradient plus RF fields, as a basis for exposure assessment in epidemiological studies. Bioelectromagnetics 34:81–84, 2013. © 2012 Wiley Periodicals, Inc.     

Community and species responses to water level fluctuations with reference to soil layers in different habitats of mid-boreal mire complexes

The present paper discusses water level fluctuations in different parts of boreal mire complexes (eleven localities), mainly aapa mire complexes, on the basis of measurements performed by means of shallow observation wells and a few deeper observation tubes (piezometers) in the coastal half of the southern aapa mire zone in Finland. The sites represented intact vegetation from 12 different habitat types (communities), which were divided a priori into habitats with a stable surface moisture status (stable habitats) and into habitats with an unstable surface moisture status (unstable habitats). In stable habitats water level fluctuations took place according to the acrotelm–catotelm model, but the unstable habitats clearly deviated from the general pattern: water level fluctuations in them were not at all concentrated to the surficial, porous peat layer. Direct gradient analysis was used for arranging the communities along the water level fluctuation gradient. Variability of the water table, using 80% amplitude of water table residence, was used for the arrangement. The gradient was split into three groups: (1) habitats with a slightly fluctuating water table, (2) habitats with a considerably fluctuating water table and (3) habitats with an extremely fluctuating water table. The last group nearly corresponded to aro wetlands, and represented a very special habitat type. Indirect gradient analysis (NMDS ordination) also revealed the water level fluctuation gradient along with the gradient of traditional water level categories. According to the results of direct and indirect gradient analysis, the water level fluctuation seems to be an independent and important vegetation gradient. In peatlands, it occurs alongside with the traditional gradient of water level categories reflecting the mean water table. The responses of species to the range of water level fluctuations seem to reflect their tolerance to disturbances and evidently to seasonal drought. Most Sphagnum species are absent or in poor condition in habitats with extremely fluctuating water table. Vascular plant species that experienced most extreme water level fluctuations (Carex nigra, Juncus filiformis) have earlier been regarded as disturbance indicators. In addition, the difference between the piezometric water level and simultaneously measured water table depth reached the highest values within the habitats of those species (i.e., within Polytrichum commune aro wetlands) showing the downward direction of water movement in sandy mineral soil.     

Pathways affected by asbestos exposure in normal and tumour tissue of lung cancer patients

Background:

The early detection of prostate cancer has resulted in an increase in the number of patients with localized prostate cancer and has paralleled the reported reduction in prostate cancer mortality. The increased rate of detection of patients with localized prostate cancer may also increase the risk of potentially morbid therapy in a patient with indolent cancer. Defining the biomarker correlates of prostate cancer virulence will facilitate the appropriate application and development of therapy for patients with early disease.

Methods:

A 255 core prostate cancer tissue microarray (TMA) from 47 prostatectomy specimens with organ confined tumor was constructed. Prostate cancer foci of transition and peripheral zone origin were represented on the TMA. Further, replicate cores of the two Gleason grades comprising the Gleason score, representative of Gleason scores 5-9, were arrayed from each prostatectomy specimen. Standard immunohistochemical techniques were used to assess expression of nine, cell death and cell cycle regulatory proteins implicated in the pathogenesis of prostate cancer (bax, bcl-2, bcl-x_L, bin1, CD95, mdm2, p21, p53, and NFkB).

Results:

The Spearman correlation coefficient revealed a strong correlation of bax, bin1, FAS, p65 and p21 expression with Gleason grade. Spearman correlation coefficients showed that expression of, bax and bin1, bax and MDM2, Bax and p21, and bax and p65 NFkB was highly associated. Other significant associations were identified between bin1 and p21, bin1 and MDM2, bin1 and p65 NFkB and between p21 and p65 NFκB. A model for predicting the biological potential of Gleason score 7 prostate cancer using multivariable logistic regression methods was developed. The findings also indicate that the profile of specific markers for Gleason grade 3 prostate cancer correlates with the overall context of the Gleason score.

Conclusion:

These data support the view that important molecular differences exist among and between the Gleason scores. Furthermore, there is significant molecular heterogeneity among prostatectomy specimens containing Gleason grade 3 cancer. This observation may have broader implications regarding the determination of risk among patients with prostate cancer that is currently considered to be of either good prognosis or unclear prognosis, i.e. Gleason score 7 tumors.     

Targeting of the AE2 anion exchanger to the Golgi apparatus is cell type-dependent and correlates with the expression of Ank(195), a Golgi membrane skeletal protein

Sodium-independent anion exchangers (AE1-4) show remarkable variability in their tissue-specific expression and subcellular localization. Currently, isoform-specific targeting mechanisms are considered to be responsible for this variable localization. Here, we report that targeting can also be cell type-specific. We show that the full-length AE2 protein and its green fluorescent protein- or DsRed-tagged variants localize predominantly either to the Golgi apparatus in COS-7 cells, or to the plasma membrane in HeLa cells. This alternative targeting did not seem to result from either translational or post-translational differences, but rather from differential expression of at least one of the Golgi membrane skeletal proteins, ankyrin(195) (Ank(195)), between the two cell types. Comparative studies with several different cell lines revealed that the Golgi localization of the AE2 protein correlated strictly with the expression of Ank(195) in the cells. The two Golgi-associated proteins also co-localized well and similarly resisted detergent extraction in the cold, whereas the plasma membrane-localized AE2 in Ank(195)-deficient cells was mostly detergent-soluble. Collectively, our results suggest that Ank(195) expression is a key determinant for the variable and cell type-dependent localization of the AE2 protein in the Golgi apparatus in mammalian cells.     

OligoDesign: Optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling

We report the development of new software, OligoDesign, which provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. LNAs constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Furthermore it includes routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. We have successfully used the OligoDesign tool to design a Caenorhabditis elegans LNA oligonucleotide microarray, which allows monitoring of the expression of a set of 120 potential marker genes for a variety of stress and toxicological processes and toxicologically relevant pathways. The OligoDesign program is freely accessible at http://lnatools.com/.     

The complex effects of the invasive polychaetes Marenzelleria spp. on benthic nutrient dynamics

The effects of the polychaetes Marenzelleria sp. (Polychaeta, Spionidae), nonindigenous, rapidly increasing species in the Baltic Sea, on benthic nutrient fluxes, denitrification and sediment pore water nutrient concentration were studied in laboratory experiments using a flow-through setup with muddy sediment from coastal regions of the Gulf of Finland. In addition, different forms of sediment phosphorus (P), separated by chemical fractionation, were studied in three sediment layers. At a population density corresponding to about half the highest measured in the northern Baltic Sea, Marenzelleria sp. increased the fluxes of P and ammonium to the water column. No effect could be recorded for denitrification. Since the previously dominant species of the area, Monoporeia affinis, can enhance denitrification and lower the amount of dissolved P in the pore water, the replacement of M. affinis with Marenzelleria spp. may lead to increased P flux to the water column and decreased denitrification, further increasing the ammonium flux to the water column. However, sediment reworking by Marenzelleria spp. also oxidizes the surface sediment in the long run, improving its ability to retain P and support nitrification. Therefore, the impact of Marenzelleria spp. on sediment nutrient release may not be as drastic as the initial reactions seen in our experiments suggest.     

N-linked (N-) Glycoproteomics of Urimary Exosomes*

Epithelial cells lining the urinary tract secrete urinary exosomes (40–100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk.Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS.We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.Urine is a combination of plasma filtrate and the secretion profile of cells lining the urino-genital tract. This secretion profile, in addition to proteins and metabolites, also contains exosomes and larger microvesicles that have glycoproteins on their surface. In healthy individuals, ∼70% of the urinary proteome originates from kidneys and the rest represents plasma filtered by glomeruli (1). Proteins present in urine are a collection of proteins secreted by a number of tissues, which changes in disease states (2). Therefore, the urinary proteome may serve as a rich source of biomarkers for urogenital and systemic diseases, which have been reviewed previously (3). Moreover, urine collection is a noninvasive procedure, which makes it an ideal candidate for discovery of novel biomarkers. Only a few large scale studies on urinary proteome and glycoproteome have been published (4, 5). However, most of them have not focused on the glycopeptide characterization.Microvesicles, including exosomes are secreted by many cell types and involved in functions including antigen-presentation, cell-to-cell communication, and immunomodulation (6). These are specialized compartments of cells and they mirror the physiological state of cells secreting them while also providing information about the environment into which they are secreted. For instance, the immunosuppressive and pro-angiogenic environment of cancer may be mediated partially by exosomes (6). Exosomes and other types of microvesicles are abundantly found in urine and thought to be mainly secreted by epithelial cells lining the urinary system (7). These vesicles contain DNA, RNA, and proteins.Glycosylation is an important post-translational modification of proteins and lipids and appears to play many roles, e.g. in cell adhesion, cell-to-cell communication, and immune response (8). Glycosylation is also very important for targeting of proteins to various compartments of the cells. Accordingly, glycans of glycoproteins have important roles in protein sorting to membrane microdomains and furthermore in influencing their intracellular trafficking (9). Microvesicles have a glycan signature that is distinct from the parent cell, suggesting that they originate from specialized membrane microdomains implying a role of glycosylation in microvesicle protein sorting (10, 11). Changes in N-glycans of exosomes from expressed prostatic secretions correlate with disease severity (12). HIV-1 particles were found to have a glycome (the comprehensive glycan profile of a protein, cell, or tissue) largely shared with microvesicles, which is taken to imply that the virus hijacks the glycomachinery of infected cells and uses it systematically to infect additional cells or to deceive the normal immunodefence (13). Thus, the specific glycoproteins of exosomes may be of major impact in targeting exosomes to distinct cells and tissues.Exosome uptake in various cell types has been shown to occur through the mechanisms of clathrin-mediated endocytosis, phagocytosis, and micropinocytosis (14, 15). The uptake of exosomes by dendritic cells and macrophages has been shown to be inhibited by mannose, N-acetylglucosamine, and lactose residues, respectively. This uptake is mediated by a C-type lectin in dendritic cells and galectin-5 in macrophages (14, 15). All this points toward a system in which the exosome glycosylation pattern is kept specific by the cells secreting them to suit the target cell makeup and uptake pathways, and further downstream functions. Taken together, these findings suggest that better understanding of surface glycosylation patterns as well as the glycomics and glycoproteomics of exosomes might help in establishing the specificity of exosome uptake by target cells and activated downstream pathways. This information about exosome uptake might be utilized in therapeutics involving exosomes. Glycome and glycoproteome of urinary microvesicles will provide information not only about the functional state of constituent proteins, but it will also highlight the similarities and differences among proteins that are specifically targeted to exosomes.An N-glycopeptide analysis using collision induced dissociation (CID)-Tandem MS has been reported previously for different sample types (16, 17). This approach provides information about the composition of glycan, partial structure, glycosylation site, and peptide sequence from the same molecule compared with approaches where released glycans or peptides of N-glycopeptides are analyzed separately.We have published an algorithm for an automated analysis of N-glycopeptides (18, 19). A public, web-based software with some changes to the original was developed and named as GlycopeptideId (www.appliednumerics.com, GlycopeptideID version 28–02-28 0.91 beta). The major change was to analyze glycan structures against a database and not with an iterative de novo glycan structure analysis. The proposed structures were further manually validated by the presence of diagnostic ions, when they were available for the given structure.This software was utilized in this study and a comprehensive glycopeptide characterization of urinary exosomal glycoproteins was carried out. We report here the glycan structure determination of urinary exosomal glycoproteins. We have characterized 126 N-glycopeptides representing 51 N-glycosylation sites that belong to 37 glycoproteins. Additionally, glycomic analysis of released N-glycans was also performed and 66 unique modified and 13 sulfated and/or phosphorylated glycans were found. A third of total glycan compositions were common to both the analysis, whereas approximately a third were unique to both the analysis.     

Minor Role of Plasminogen in Complement Activation on Cell Surfaces

Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation.