Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.     

Central giant cell lesion of the jaws: study of CCND1 gene amplification and p16INK4a protein levels

Central giant cell lesions (CGCLs) are uncommon benign jaw lesions with uncertain etiology and a variable clinical behavior. In neoplasms, alterations in molecules involved in the G1/S checkpoint are frequently found. Loss of p16^INK4a expression or overexpression of cyclin D1 may stimulate cell proliferation. The purpose of this study was to analyze CCND1 gene amplification and the expression of p16^INK4a in CGCLs. Structural analysis of the CCND1 was performed using chromogenic in situ hybridization. Immmunohistochemistry was used to identify p16^INK4a protein levels. Statistical analysis correlated the two biomarkers with clinical behavior and between each other. Twenty-four lesions were included, being 11 aggressive and 13 non-aggressive. Moderate/high-level CCND1 amplification was found in 12 lesions. Also, immunoreactivity for p16^INK4a was present in 12 cases, mainly in mononuclear cells. There was a significantly higher level of p16^INK4a expression in mononuclear cells of non-aggressive lesions and lesions with moderate/high-level CCND1 amplification in mononuclear cells. It could be speculated that some CGCLs may develop as a true benign neoplasm. The higher expression of p16^INK4a in non-aggressive lesions and in cases with moderate/high-level CCND1 amplification may show that these molecules have a role in CGCLs.     

Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe,Protective Vaccine Antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.     

Field Enhancement by Shaping Nanocavities in a Gold Film

We report on 2D plasmonic crystals composed of a hexagonal lattice of polymeric nanopillars embedded in an optically thick gold film on a glass substrate. A tapered shape of the polymeric pillars is proved to localize the electric field distribution close to the free surface of the device and to determine a significant increase in the electric field intensity particularly when the incident light comes from the glass side. These effects significantly improve the sample sensitivity to a refractive index change occurring at the free surface of the device.     

Expression,surface immobilization,and characterization of functional recombinant cannabinoid receptor CB2

Human peripheral cannabinoid receptor CB₂, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB₂ may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB₂ and immobilization at solid interfaces. Expression of CB₂ as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB₂ was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB₂ was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB₂ from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB₂ and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB₂.     

Dual Role of G-runs and hnRNP F in the Regulation of a Mutation-Activated Pseudoexon in the Fibrinogen Gamma-Chain Transcript

Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting elements and trans-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (FGG) pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T). This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H. To explore the effect of these proteins on FGG pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other trans-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for FGG pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating FGG pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.     

Prevalence of HIV,STIs, and Risk Behaviors in a Cross-Sectional Community- and Clinic-Based Sample of Men Who Have Sex with Men (MSM) in Lima,Peru

Background

Further research is necessary to understand the factors contributing to the high prevalence of HIV/STIs among men who have sex with men (MSM) in Peru. We compared HIV/STI prevalence and risk factors between two non-probability samples of MSM, one passively enrolled from an STI clinic and the other actively enrolled from community venues surrounding the clinic in Lima, Peru.

Methods

A total of 560 self-identified MSM were enrolled between May-December, 2007. 438 subjects enrolled from a municipal STI clinic and 122 subjects enrolled during community outreach visits. All participants underwent screening for HIV, syphilis, HSV-2, gonorrhoea, and chlamydia and completed a survey assessing their history of HIV/STIs, prior HIV testing, and sexual behavior.

Results

HIV prevalence was significantly higher among MSM enrolled from the clinic, with previously undiagnosed HIV identified in 9.1% compared with 2.6% of community participants. 15.4 % of all MSM screened were infected with ≥1 curable STI, 7.4% with early syphilis (RPR≥1∶16) and 5.5% with urethral gonorrhoea and/or chlamydia. No significant differences between populations were reported in prevalence of STIs, number of male sex partners, history of unprotected anal intercourse, or alcohol and/or drug use prior to sex. Exchange of sex for money or goods was reported by 33.5% of MSM enrolled from the clinic and 21.2% of MSM from the community (p = 0.01).

Conclusions

Our data demonstrate that the prevalence of HIV and STIs, including syphilis, gonorrhoea, and chlamydia are extremely high among MSM enrolled from both clinic and community venues in urban Peru. New strategies are needed to address differences in HIV/STI epidemiology between clinic- and community-enrolled samples of MSM.     

Is It Possible to Improve Memory Function by Upregulation of the Cholesterol 24S-Hydroxylase (CYP46A1) in the Brain?

We previously described a heterozygous mouse model overexpressing human HA-tagged 24S-hydroxylase (CYP46A1) utilizing a ubiquitous expression vector. In this study, we generated homozygotes of these mice with circulating levels of 24OH 30–60% higher than the heterozygotes. Female homozygous CYP46A1 transgenic mice, aged 15 months, showed an improvement in spatial memory in the Morris water maze test as compared to the wild type mice. The levels of N-Methyl-D-Aspartate receptor 1, phosphorylated-N-Methyl-D-Aspartate receptor 2A, postsynaptic density 95, synapsin-1 and synapthophysin were significantly increased in the hippocampus of the CYP46A1 transgenic mice as compared to the controls. The levels of lanosterol in the brain of the CYP46A1 transgenic mice were significantly increased, consistent with a higher synthesis of cholesterol. Our results are discussed in relation to the hypothesis that the flux in the mevalonate pathway in the brain is of importance in cognitive functions.     

Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest Streptococcus pneumoniae Clonal Complex in the MLST Database

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.     

BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

TGF-β family members play a relevant role in tumorigenic processes, including hepatocellular carcinoma (HCC), but a specific implication of the Bone Morphogenetic Protein (BMP) subfamily is still unknown. Although originally isolated from fetal liver, little is known about BMP9, a BMP family member, and its role in liver physiology and pathology. Our results show that BMP9 promotes growth in HCC cells, but not in immortalized human hepatocytes. In the liver cancer cell line HepG2, BMP9 triggers Smad1,5,8 phosphorylation and inhibitor of DNA binding 1 (Id1) expression up- regulation. Importantly, by using chemical inhibitors, ligand trap and gene silencing approaches we demonstrate that HepG2 cells autocrinely produce BMP9 that supports their proliferation and anchorage independent growth. Additionally, our data reveal that in HepG2 cells BMP9 triggers cell cycle progression, and strikingly, completely abolishes the increase in the percentage of apoptotic cells induced by long-term incubation in low serum. Collectively, our data unveil a dual role for BMP9, both promoting a proliferative response and exerting a remarkable anti-apoptotic function in HepG2 cells, which result in a robust BMP9 effect on liver cancer cell growth. Finally, we show that BMP9 expression is increased in 40% of human HCC tissues compared with normal human liver as revealed by immunohistochemistry analysis, suggesting that BMP9 signaling may be relevant during hepatocarcinogenesis in vivo. Our findings provide new clues for a better understanding of BMPs contribution, and in particular BMP9, in HCC pathogenesis that may result in the development of effective and targeted therapeutic interventions.