Identification of Chondroitin Sulfate Linkage Region Glycopeptides Reveals Prohormones as a Novel Class of Proteoglycans

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.Chondroitin sulfates (CS)¹ are complex polysaccharides present at cell surfaces and in extracellular matrices. The polysaccharides belong to a subclass of glycosaminoglycans (GAGs) and are covalently linked to various core proteins to form CS-proteoglycans (CSPGs), each with differences in the protein structures and/or numbers of CS side chains. Apart from their structural role in cartilage, CSPGs contribute to the regulation of a diverse set of biological processes such as neurogenesis, growth factor signaling, angiogenesis, and morphogenesis (1–5). Although the molecular basis of CSPGs functions remains elusive, accumulating evidence suggests that the underlying activities relate to selective ligand binding to discrete structural variants of the polysaccharides. Thus, the current strategy for understanding the biological role of CSPGs aims to identify selective CS polysaccharide–ligand interactions. However, information on the number of CS-chains and their specific attachment site(s) on any given core protein is often scarce which limits our functional understanding of CSPGs.The biosynthesis of GAGs occurs in the endoplasmic reticulum and Golgi compartments and is initiated by the enzymatic addition of a beta-linked xylose (Xyl) to a Ser residue of the core protein. The sequential addition of two galactose residues (Gal) and a glucuronic acid (GlcA) onto the growing saccharide chain completes the formation of a tetrasaccharide linkage region (GlcAβ3Galβ3Galβ4XylβSer). This part of the biosynthesis is the same for CS and heparan sulfate (HS). However, for CS the biosynthesis continues with the addition of an N-acetylgalactosamine (GalNAcβ3), whereas HS biosynthesis continues with the addition of an N-acetylglucosamine (GlcNAcα4) (6). The CS-chains are thereafter elongated through the addition of repeating units of GlcA and GalNAc and are further modified by the addition of specifically positioned sulfate groups (7). Certain features of the core protein seem to influence if a certain Ser residue is selected for GAG attachment and whether CS or HS will be synthesized, but the selection mechanism is largely unknown. Sequence analysis of previously known GAG-substituted core proteins reveals that the glycosylated serine residues are usually flanked by a glycine residue (-SG-), and are associated with a cluster of acidic residues in close proximity (8). This motif may assist in the prediction of potential GAG-sites of core proteins; however, the use of such strategy is ambiguous because proteoglycans may also contain unoccupied motifs or motifs that are occasionally occupied (9).Glycoproteomics strategies have recently appeared that provide site-specific information of N- and O-glycans. Such strategies are typically based on a specific enrichment of glycopeptides and a subsequent analysis with nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) (10). By further developing this concept for proteoglycans (11), we have now analyzed CSPG linkage region glycopeptides of human samples, which enabled us to identify 13 novel human CSPGs in addition to 13 already established CSPGs. Urine and cerebrospinal fluid (CSF) samples were trypsinized and CS glycopeptides were enriched using strong anion exchange (SAX) chromatography. The CS chains were depolymerized with chondroitinase ABC, generating free disaccharides and a residual hexameric structure composed of the linkage region and a GlcA-GalNAc disaccharide dehydrated on the terminal GlcA residue (12). MS/MS analysis provided the combined sequencing of the residual hexasaccharide and of the core peptide.     

Three New Species of Phytotelm-Breeding Melanophryniscus from the Atlantic Rainforest of Southern Brazil (Anura: Bufonidae)

Three new species of Melanophryniscus are described from the Serra do Mar mountain range of the state of Santa Catarina, southern Brazil. All species are found at intermediate to high altitudes and share phytotelm-breeding as their reproductive strategy. The new species are distinguished from other phytotelm-breeding Melanophryniscus based on different combinations of the following traits: snout-vent length, presence of white and/or yellow spots on forearms, mouth, belly and cloaca, pattern and arrangement of warts, and presence and number of corneous spines. The discovery of these species in a rather restricted geographical area suggests that the diversity of phytotelm-breeding species of Melanophryniscus might be severely underestimated. The conservation status of these species is of particular concern, given that one of them is at risk of extinction not only due to its restricted habitat, but also because of anthropogenic disturbances.     

Ecological Risk Assessment of a Metal-Contaminated Area in the Tropics. Tier II: Detailed Assessment

This study presents data on the detailed evaluation (tier 2) of a site-specific ecological risk assessment (ssERA) in a former smelter area contaminated with metals (Santo Amaro, Bahia, Brazil). Combining information from three lines of evidence (LoE), chemical (ChemLoE), ecotoxicological (EcotoxLoE) and ecological (EcoLoE), in the Triad approach, integrated risk values were calculated to rank sites and confirm the potential risk disclosed with tier 1. Risk values were calculated for the habitat and for the retention functions in each sampling point. Habitat function included the ChemLoE calculated from total metal concentrations. The EcotoxLoE was based on reproduction tests with terrestrial invertebrates (Folsomia candida, Enchytraeus crypticus, Eisenia andrei), shoot length and plant biomass (Avena sativa, Brassica rapa). For the EcoLoE, ecological parameters (microbial parameters, soil invertebrate community, litter breakdown) were used to derive risk values. Retention function included the ChemLoE, calculated from extractable metal concentrations, and the EcotoxLoE based on eluate tests with aquatic organisms (Daphnia magna reproduction and Pseudokirchneriella subcapitata growth). Results related to the habitat function indicated that the metal residues are sufficient to cause risk to biota, while the low metal levels in extracts and the general lack of toxicity in aquatic tests indicated a high soil retention capacity in most sampling points. Integrated risk of tier 2 showed the same trend of tier 1, suggesting the need to proceed with remediation actions. The high risk levels were related to direct toxicity to organisms and indirect effects, such as failure in the establishment of vegetation and the consequent loss of habitat quality for microorganisms and soil fauna. This study shed some light on the selection of tools for the tier 2 of an ssERA in tropical metal-contaminated sites, focusing on ecological receptors at risk and using available chemical methods, ecological surveys and ecotoxicity tests.     

Does Severe Maternal Morbidity Affect Female Sexual Activity and Function? Evidence from a Brazilian Cohort Study

Objective

to assess Female Sexual Function Index (FSFI) scores and delay to resume sexual activity associated with a previous severe maternal morbidity.

Method

This was a multidimensional retrospective cohort study. Women who gave birth at a Brazilian tertiary maternity between 2008 and 2012 were included, with data extraction from the hospital information system. Those with potentially life-threatening conditions and maternal near miss episodes (severe maternal morbidity) were considered the exposed group. The control group was a random sample of women who had had uncomplicated pregnancy. Female sexual function was evaluated through FSFI questionnaire, and general and reproductive aspects were addressed through specific questions. Statistical analyses were performed using Mann-Whitney and Pearson´s Chi-square for bivariate analyses. Logistic regression was used to identify variables independently associated with lower FSFI scores.

Results

638 women were included (315 at exposed and 323 at not exposed groups). The majority of women were under 30 years-old in the control group and between 30 and 46 years-old in the exposed group (p = 0.003). Women who experienced severe maternal morbidity (SMM) had statistically significant differences regarding cesarean section (82.4% versus 47.1% among deliveries without complications, p<0.001), and some previous pathological conditions. FSFI mean scores were similar among groups ranging from 24.39 to 24.42. It took longer for exposed women to resume sexual activity after index pregnancy (mean 84 days after SMM and 65 days for control group, p = 0.01). Multiple analyses showed no significant association of FSFI below cut-off value with any predictor.

Conclusion

FSFI scores were not different in both groups. However, they were lower than expected. SMM delayed resumption of sexual activity after delivery, beyond postpartum period. However, the proportion of women in both groups having sex at 3 months after delivery was similar. Altered sexual response may be evaluated as one of possible long-term consequences after SMM episodes. Further studies on the growing population of women surviving severe maternal conditions might be worth for improvement of care for women.     

Maturation of Oral Microbiota in Children with or without Dental Caries

Background

The aim of this longitudinal study was to evaluate the oral microbiota in children from age 3 months to 3 years, and to determine the association of the presence of caries at 3 years of age.

Methods and findings

Oral biofilms and saliva were sampled from children at 3 months (n = 207) and 3 years (n = 155) of age, and dental caries was scored at 3 years of age. Oral microbiota was assessed by culturing of total lactobacilli and mutans streptococci, PCR detection of Streptococcus mutans and Streptococcus sobrinus, 454 pyrosequencing and HOMIM (Human Oral Microbe Identification Microarray) microarray detection of more then 300 species/ phylotypes. Species richness and taxa diversity significantly increased from 3 months to 3 years. Three bacterial genera, present in all the 3-month-old infants, persisted at 3 years of age, whereas three other genera had disappeared by this age. A large number of new taxa were also observed in the 3-year-olds. The microbiota at 3 months of age, except for lactobacilli, was unrelated to caries development at a later age. In contrast, several taxa in the oral biofilms of the 3-year-olds were linked with the presence or absence of caries. The main species/phylotypes associated with caries in 3-year-olds belonged to the Actinobaculum, Atopobium, Aggregatibacter, and Streptococcus genera, whereas those influencing the absence of caries belonged to the Actinomyces, Bergeyella, Campylobacter, Granulicatella, Kingella, Leptotrichia, and Streptococcus genera.

Conclusions

Thus, during the first years of life, species richness and taxa diversity in the mouth increase significantly. Besides the more prevalent colonization of lactobacilli, the composition of the overall microbiota at 3 months of age was unrelated to caries development at a later age. Several taxa within the oral biofilms of the 3-year-olds could be linked to the presence or absence of caries.     

MET Gene Copy Number Alterations and Expression of MET and Hepatocyte Growth Factor Are Potential Biomarkers in Angiosarcomas and Undifferentiated Pleomorphic Sarcomas

Soft tissue sarcomas are a heterogeneous group of tumors with many different subtypes. In 2014 an estimated 12,020 newly diagnosed cases and 4,740 soft tissue sarcoma related deaths can be expected in the United States. Many soft tissue sarcomas are associated with poor prognosis and therapeutic options are often limited. The evolution of precision medicine has not yet fully reached the clinical treatment of sarcomas since therapeutically tractable genetic changes have not been comprehensively studied so far. We analyzed a total of 484 adult-type malignant mesenchymal tumors by MET fluorescence in situ hybridization and MET and hepatocyte growth factor immunohistochemistry. Eleven different entities were included, among them the most common and clinically relevant subtypes and tumors with specific translocations or complex genetic changes. MET protein expression was observed in 2.6% of the cases, all of which were either undifferentiated pleomorphic sarcomas or angiosarcomas, showing positivity rates of 14% and 17%, respectively. 6% of the tumors showed hepatocyte growth factor overexpression, mainly seen in undifferentiated pleomorphic sarcomas and angiosarcomas, but also in clear cell sarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas and gastrointestinal stromal tumors. MET and hepatocyte growth factor overexpression were significantly correlated and may suggest an autocrine activation in these tumors. MET FISH amplification and copy number gain were present in 4% of the tumors (15/413). Two samples, both undifferentiated pleomorphic sarcomas, fulfilled the criteria for high level amplification of MET, one undifferentiated pleomorphic sarcoma reached an intermediate level copy number gain, and 12 samples of different subtypes were categorized as low level copy number gains for MET. Our findings indicate that angiosarcomas and undifferentiated pleomorphic sarcomas rather than other frequent adult-type sarcomas should be enrolled in screening programs for clinical trials with MET inhibitors. The screening methods should include both in situ hybridization and immunohistochemistry.     

Thiessen polygons as a model for animal territory estimation

Thiessen polygons are often used to model territory characteristics. However, information about the quality of Thiessen polygon‐based estimates is currently lacking. We used published data to investigate the match between Thiessen polygons and mapped bird territories regarding territory size, shape and neighbourhood. Although territory sizes and the number of neighbours were strongly correlated between these two methods, both parameters were overestimated by the Thiessen polygons. Therefore, caution is required when Thiessen polygons are used as a model for absolute values and when the assumptions of Thiessen polygons, such as formation of discrete territories and a contiguous study area, are not met.     

In vitro assessment of the antimicrobial activity of new N-acyl-thiourea derivatives

The qualitative screening of the susceptibility spectra of different microbial strains to the newly synthesized substances complexes was performed by adapted disk diffusion techniques, while the quantitative assay of the minimal inhibitory concentration (M.I.C., microg/cm3) value was based on liquid medium serial microdilutions. The compounds were solubilized in dimethylsulfoxide (DMSO). The in vitro biological screening effects were tested against a microbial inoculum of approximately 1.5 x 10(8) UFC/cm3, corresponding to 0.5 McFarland standard density, obtained from Gram positive (Staphylococcus aureus, Bacillus subtilis), Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and fungal strains (Candida albicans). In order to investigate the influence of the subinhibitory concentration of the tested substances on the expression of different virulence features, the strains were incubated overnight in the presence of the newly synthesized thiourea derivatives (vol:vol) and different virulence features were investigated, i.e: adherence capacity to the cellular substrate represented by HeLa cells and to inert substrata quantified by slime test and soluble enzymatic virulence factors (haemolysins and other pore-forming toxins, proteases activity, DN-ase and siderophores production). The cytotoxicity was assessed microscopically, by observing the effect of the tested compounds on the cellular substratum integrity.