Effect of biofouling on anodized and sol-gel treated titanium surfaces: a comparative study

Anodization and sol-gel treatments of titanium (Ti) were evaluated as biofilm control measures on surfaces exposed to seawater exposed to ultraviolet light. Anodized and sol-gel treated specimens were characterized using Raman spectroscopy to confirm the presence of TiO(2). The single anatase phase was observed at the anodized surfaces whereas the anatase/rutile mixed phase was detected on the sol-gel coated surfaces. After exposure of the specimens to seawater, biofilms were characterized by total viable counts, and epifluorescence and Raman microscopy. These techniques confirmed the reduction in biofilm formation on both the anodized and sol-gel coated Ti specimens compared to the untreated specimens. Biofilm control by anodization was found to be more effective than by sol-gel treatment of the specimens. The higher particle size and the inhomogeneity at the sol-gel coated surfaces produced less effective biofilm control.     

In Situ trans Ligands of CD22 Identified by Glycan-Protein Photocross-linking-enabled Proteomics

CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAcα2–6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell.Glycan-binding proteins (GBPs)¹ mediate diverse aspects of cell communication through their interactions with their counter-receptors comprising glycan ligands carried on cell surface glycoproteins and glycolipids. Identification of the in situ counter-receptors of glycan-binding proteins is problematic due to the fact that the vast majority of the glycoproteins of a cell will carry highly related glycan structures because they share the same secretory pathway that elaborates their glycans post-translationally en route to the cell surface. Thus, although many glycoproteins will carry the glycan structure recognized by a GBP, the challenge is to determine whether one, several, or all of these cell surface glycoproteins (and glycolipids) are recognized in situ as physiologically relevant counter-receptors (1–4). Standard in vitro methods, such as co-precipitation from cell lysates or Western blotting using binding protein probes, are useful for identifying glycoproteins that contain the glycan structure recognized by the GBP. However, these may not be relevant ligands in situ due to constraints imposed by their microdomain localization and the geometric arrangement of their glycans relative to the GBP presented on the apposing cell.In this report, we examine the in situ ligands of CD22 (Siglec-2), a member of the siglec family and a regulator of B-cell receptor (BCR) signaling that recognizes glycans containing the sequence NeuAcα2–6Gal as ligands (2, 5, 6). Regulation of BCR signaling by CD22 is effected by its proximity to the BCR through recruitment of a tyrosine phosphatase, SHP-1, which is in turn influenced by CD22 binding to its glycan ligands (6). Glycoproteins bearing CD22 ligands are abundantly expressed on B-cells and bind to CD22 in cis (on the same cell) (7), regulating BCR signaling (2, 5, 6). Although binding to cis ligands has been shown to “mask” CD22 from binding low avidity synthetic sialoside probes (2, 7), CD22 can also interact with ligands on apposing immune cells in trans (8–10). Interactions of CD22 with trans ligands influence T-cell signaling in vitro (11, 12), mediate B-cell homing via binding to sinusoidal endothelial cells in the bone marrow (13), and aid in “self”-recognition (14). Thus, interactions with both cis and trans ligands modulate CD22 function in immune homeostasis.Several groups have demonstrated that recombinant CD22-Fc chimera is capable of binding and precipitating the majority of glycoproteins from B- and T-cell lysates whose glycans contain the sequence NeuAcα2–6Gal (15–18). Among them, CD45, IgM, and CD22 itself were identified as specific B-cell binding partners and were postulated to have functional significance as in situ cis ligands of CD22 in regulation of BCR signaling (11, 16, 18–20). Several reports have also documented in situ interactions of CD22 with IgM and CD45, but these interactions were found to be of low stoichiometry and sialic acid-independent (19–21), leaving open the question of which glycoproteins served as in situ cis ligands of CD22 on B-cells that masked the glycan ligand binding site of CD22 (7). Subsequently, using metabolically labeled B-cells with sialic acids containing a photoactivatable 9-aryl azide moiety, we demonstrated that CD22 could be photocross-linked to its cis ligands, effectively tagging the in situ cis ligands with CD22 (15). Notably, there was no cross-linking observed to IgM or CD45, demonstrating that they are not significant in situ cis ligands of CD22 (15). Instead, only glycans of neighboring CD22 molecules interacted significantly with CD22, resulting in photocross-linking of homomultimeric complexes of CD22. Thus, despite the fact that most B-cell glycoproteins are recognized in vitro, CD22 selectively recognizes glycans of neighboring CD22 molecules as cis ligands in situ.With the perspective gained from analysis of cis ligands, we wished to determine whether CD22 was also selective in recognition of trans ligands upon cell contact. We have previously demonstrated that CD22 is redistributed to sites of cell contact of interacting B-cells and T-cells and that redistribution is mediated by the interaction of CD22 with sialic acid-containing trans ligands on the apposing cell (8). Stamenkovic et al. (22) had previously demonstrated that binding of T-cells to CD22-expressing COS cells was blocked by an anti-CD45RO antibody, suggesting that CD45 was a functional trans ligand of CD22 on T-cells. However, we found that redistribution of CD22 to sites of cell contact was also observed with CD45-deficient B-cells (8), indicating that, at a minimum, other glycoproteins must also serve as trans ligands of CD22 on B-cells.To assess whether CD22 recognizes all or a subset of glycoproteins as trans ligands on an apposing cell, we initiated an unbiased analysis of the trans ligands of CD22 on apposing B-cells using our protein-glycan cross-linking strategy (15). By cross-linking CD22-Fc to intact B-cells, we identified 27 candidate trans ligands of CD22 by quantitative mass spectrometry-based proteomics. We then looked at the in situ trans interactions of CD22 in the physiologically relevant cellular context by cross-linking CD22 expressed on one cell to the trans ligands with photoreactive sialic acids on the apposing cell. Our results indicate that only a subset of cell surface glycoproteins, including IgM and, to a lesser extent, CD45 and Basigin, are selectively recognized in trans by CD22. Indeed, IgM in particular is a preferred trans ligand that is selectively redistributed to the sites of cell contact on apposing B-cells in a CD22- and sialic acid-dependent manner despite a vast excess of cell surface glycoproteins that carry a glycan recognized by CD22. The results support the view that factors other than glycan sequence are critical for the in situ engagement of glycan-binding proteins with glycan ligand bearing counter-receptors on the same cell (in cis) or apposing cell (in trans).     

A Novel 3-D Mineralized Tumor Model to Study Breast Cancer Bone Metastasis

Background

Metastatic bone disease is a frequent cause of morbidity in patients with advanced breast cancer, but the role of the bone mineral hydroxyapatite (HA) in this process remains unclear. We have developed a novel mineralized 3-D tumor model and have employed this culture system to systematically investigate the pro-metastatic role of HA under physiologically relevant conditions in vitro.

Methodology/Principal Findings

MDA-MB231 breast cancer cells were cultured within non-mineralized or mineralized polymeric scaffolds fabricated by a gas foaming-particulate leaching technique. Tumor cell adhesion, proliferation, and secretion of pro-osteoclastic interleukin-8 (IL-8) was increased in mineralized tumor models as compared to non-mineralized tumor models, and IL-8 secretion was more pronounced for bone-specific MDA-MB231 subpopulations relative to lung-specific breast cancer cells. These differences were pathologically significant as conditioned media collected from mineralized tumor models promoted osteoclastogenesis in an IL-8 dependent manner. Finally, drug testing and signaling studies with transforming growth factor beta (TGFβ) confirmed the clinical relevance of our culture system and revealed that breast cancer cell behavior is broadly affected by HA.

Conclusions/Significance

Our results indicate that HA promotes features associated with the neoplastic and metastatic growth of breast carcinoma cells in bone and that IL-8 may play an important role in this process. The developed mineralized tumor models may help to reveal the underlying cellular and molecular mechanisms that may ultimately enable more efficacious therapy of patients with advanced breast cancer.     

A mosquito-specific protein family includes candidate receptors for malaria sporozoite invasion of salivary glands

We describe a previously unrecognized protein family from Aedes and Anopheles mosquitoes, here named SGS proteins. There are no SGS homologues in Drosophila or other eukaryotes, but SGS presence in two mosquito genera suggests that the protein family is widespread among mosquitoes. Ae. aegypti aaSGS1 mRNA and protein are salivary gland specific, and protein is localized in the basal lamina covering the anatomical regions that are preferentially invaded by malaria sporozoites. Anti-aaSGS1 antibodies inhibited sporozoite invasion into the salivary glands in vivo, confirming aaSGS1 as a candidate sporozoite receptor. By homology to aaSGS1 we identified the complete complement of four SGS genes in An. gambiae, which were not recognized in the genome annotation. Two An. gambiae SGS genes display salivary gland specific expression like aaSGS1. Bioinformatic analysis predicts that SGS proteins possess heparin-binding domains, and have among the highest density of tyrosine sulphation sites of all An. gambiae proteins. The major sporozoite surface proteins (CS and TRAP) also bind heparin, and interact with sulphoconjugates during liver cell invasion. Thus, we speculate that sporozoite invasion of mosquito salivary glands and subsequently the vertebrate liver may share similar mechanisms based on sulphation. Phylogenomic analysis suggests that an SGS ancestor was involved in a lateral gene transfer.     

Knockout of the Erythromycin Biosynthetic Cluster Gene, eryBI, Blocks Isoflavone Glucoside Bioconversion during Erythromycin Fermentations in Aeromicrobium erythreum but Not in Saccharopolyspora erythraea

Isoflavone glucosides are valuable nutraceutical compounds and are present in commercial fermentations, such as the erythromycin fermentation, as constituents of the soy flour in the growth medium. The purpose of this study was to develop a method for recovery of the isoflavone glucosides as value-added coproducts at the end of either Saccharopolyspora erythraea or Aeromicrobium erythreum fermentation. Because the first step in isoflavone metabolism was known to be the conversion of isoflavone glucosides to aglycones by a β-glucosidase, we chose to knock out the only β-glucosidase gene known at the start of the study, eryBI, to see what effect this had on metabolism of isoflavone glucosides in each organism. In the unicellular erythromycin producer A. erythreum, knockout of eryBI was sufficient to block the conversion of isoflavone glucosides to aglycones. In S. erythraea, knockout of eryBI had no effect on this reaction, suggesting that other β-glucosidases are present. Erythromycin production was not significantly affected in either strain as a result of the eryBI knockout. This study showed that isoflavone metabolism could be blocked in A. erythreum by eryBI knockout but that eryBI knockout was not sufficient to block isoflavone metabolism in S. erythraea.     

The Profile and Treatment Outcomes of the Older (Aged 60 Years and Above) Tuberculosis Patients in Tamilnadu,South India

Background

With changing demographic patterns in the context of a high tuberculosis (TB) burden country, like India, there is very little information on the clinical and demographic factors associated with poor treatment outcome in the sub-group of older TB patients. The study aimed to assess the proportion of older TB patients (60 years of age and more), to compare the type of TB and treatment outcomes between older TB patients and other TB patients (less than 60 years of age) and to describe the demographic and clinical characteristics of older TB patients and assess any associations with TB treatment outcomes.

Methods

A retrospective cohort study involving a review of records from April to June 2011 in the 12 selected districts of Tamilnadu, India. Demographic, clinical and WHO defined disease classifications and treatment outcomes of all TB patients aged 60 years and above were extracted from TB registers maintained routinely by Revised National TB Control Program (RNTCP).

Results

Older TB patients accounted for 14% of all TB patients, of whom 47% were new sputum positive. They had 38% higher risk of unfavourable treatment outcomes as compared to all other TB patients (Relative risk (RR)-1.4, 95% CI 1.2–1.6). Among older TB patients, the risk for unfavourable treatment outcomes was higher for those aged 70 years and more (RR 1.5, 95% CI 1.2–1.9), males (RR 1.5, 95% CI 1.0–2.1), re-treatment patients (RR 2.5, 95% CI 1.9–3.2) and those who received community-based Direct Observed Treatment (RR 1.4, 95% CI 1.1–1.9).

Conclusion

Treatment outcomes were poor in older TB patients warranting special attention to this group – including routine assessment and recording of co-morbidities, a dedicated recording, reporting and monitoring of outcomes for this age-group and collaboration with National programme of non-communicable diseases for comprehensive management of co-morbidities.