Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data

The Photo-Activatable Ribonucleoside-enhanced CrossLinking and ImmunoPrecipitation (PAR-CLIP) method was recently developed for global identification of RNAs interacting with proteins. The strength of this versatile method results from induction of specific T to C transitions at sites of interaction. However, current analytical tools do not distinguish between non-experimentally and experimentally induced transitions. Furthermore, geometric properties at potential binding sites are not taken into account. To surmount these shortcomings, we developed a two-step algorithm consisting of a non-parametric two-component mixture model and a wavelet-based peak calling procedure. Our algorithm can reduce the number of false positives up to 24% thereby identifying high confidence interaction sites. We successfully employed this approach in conjunction with a modified PAR-CLIP protocol to study the functional role of nuclear Moloney leukemia virus 10, a putative RNA helicase interacting with Argonaute2 and Polycomb. Our method, available as the R package wavClusteR, is generally applicable to any substitution-based inference problem in genomics.     

An invasive tree alters the structure of seed dispersal networks between birds and plants in French Polynesia

Aim We studied how the abundance of the highly invasive fruit‐bearing tree Miconia calvescens DC. influences seed dispersal networks and the foraging patterns of three avian frugivores. Location Tahiti and Moorea, French Polynesia. Methods Our study was conducted at six sites which vary in the abundance of M. calvescens. We used dietary data from three frugivores (two introduced, one endemic) to determine whether patterns of fruit consumption are related to invasive tree abundance. We constructed seed dispersal networks for each island to evaluate how patterns of interaction between frugivores and plants shift at highly invaded sites. Results Two frugivores increased consumption of M. calvescens fruit at highly invaded sites and decreased consumption of other dietary items. The endemic fruit dove, Ptilinopus purpuratus, consumed more native fruit than either of the two introduced frugivores (the red‐vented bulbul, Pycnonotus cafer, and the silvereye, Zosterops lateralis), and introduced frugivores showed a low potential to act as dispersers of native plants. Network patterns on the highly invaded island of Tahiti were dominated by introduced plants and birds, which were responsible for the majority of plant–frugivore interactions. Main conclusions Shifts in the diet of introduced birds, coupled with reduced populations of endemic frugivores, caused differences in properties of the seed dispersal network on the island of Tahiti compared to the less invaded island of Moorea. These results demonstrate that the presence of invasive fruit‐bearing plants and introduced frugivores can alter seed dispersal networks, and that the patterns of alteration depend both on the frugivore community and on the relative abundance of available fruit.     

Soluble expression and purification of Brucella cell surface protein (BCSP31) of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis.     

Y-SNP haplogroups related to the Yqh+ heteromorphism in the Mexican northwestern population

Morphological variation of the Y chromosome has been observed in different populations. This variation is mostly related to the heteromorphic Yq12 band, which is composed of a variable block of constitutive heterochromatin. The Yqh+ heteromorphism has a worldwide frequency of 2.85% and is considered clinically innocuous. The aim of this study was to identify the ancestry of the Yqh+ heteromorphism present in individuals from western Mexico. For this purpose, 17 Y-chromosome single nucleotide polymorphisms were analysed by multiplex polymerase chain reaction and SNaPshot assays. In 28 Yqh+ males, only five haplogroups were observed; with a haplogroup diversity of 0.4841 ± 0.1094, which was less than that observed in a study of unselected Mexican mestizo population. Differences were specifically conferred by the high frequencies of haplogroups R1b1 and P*(xQ,R), and by the absence of the Amerindian haplogroup Q (Q*(xQ1a3a) plus Q1a3a) from the Yqh+ group. This study suggests a post-1492 incorporation for Yqh+ chromosomes into the Mexican northwestern population.     

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.     

In vitro antigen ELISA for quality control of tetanus vaccines

Consistency of production is recognised as an important aspect of vaccine manufacture and suitably validated in vitro assays are required for quality control testing of these products. For the manufacture and batch release of tetanus vaccines, antigen content and integrity, and degree of adsorption of antigen to the adjuvant are critical parameters that should be monitored for consistency. Here we describe the development and use of an Enzyme Linked Immunosorbent Assay (ELISA) to quantify tetanus antigen in combined vaccine products and to measure the degree of adsorption of antigen to adjuvant. Whilst the antigen assay cannot be assumed to predict potency for different products, it can be used as part of a panel of in vitro methods to provide a more informative product profile and to monitor trends in production. The antigen assay is particularly valuable for providing quantitative information on every final lot when modifications of in vivo potency tests, such as single dilution assays, are used.     

Cumulus cell layers as a critical factor in meiotic competence and cumulus expansion of ovine oocytes

A critical stage in the optimization of in vitro maturation (IVM) is the selection of good quality oocytes. There exists a relationship between the size of the cumulus investment and the in vitro developmental ability of the cumulus–oocyte complex (COC), which provides a basis for the selection of the COCs. This study was designed to evaluate the effect of the number of cumulus cell layers which enclose the oocytes, on the in vitro maturation, cytoplasm quality and cumulus expansion of the ovine oocytes. Ovaries were obtained from an abattoir and transported to the laboratory within 1–2 h, at 37 °C. Oocytes (n = 535) were recovered by means of an aspiration pump (set at a flow rate of 10 mL H₂O/min), with a disposable 20 G needle attached. Oocytes were divided into four classes (classes I to IV – with more than 5, 3–4, 1–2 and no cumulus cell layers, respectively) and separately cultured in a TCM199 medium for 24 h. The morphology of oocytes was evaluated following in vitro culture (IVC) to assess cumulus expansion, cytoplasm quality (score I with a homogenous cytoplasm and II with granulated cytoplasm) and nuclear maturation stage. The percentage of maximum cumulus expansion for classes I to III oocytes were 53.0 ± 1.0, 36.3 ± 2.2 and 16.3 ± 1.8% respectively. The rate of meiotic resumption of oocytes in classes I to IV were 77.0 ± 2.7, 77.2 ± 1.9, 53.0 ± 2.1 and 2.7 ± 1.1% respectively. The proportion of oocytes with a cytoplasm quality I in oocyte classes I to IV were 62.8 ± 1.5, 59.4 ± 1.2, 36.4 ± 2.1 and 0.5 ± 1.1%, respectively. Results showed that the presence of ≥3 cumulus cell layers in the COC prior to IVM led to a better (p < 0.05) cumulus expansion, meiotic resumption and cytoplasmic maturation rate. Thus the morphological grading of immature ovine oocytes may be an appropriate selection criterion regarding their developmental ability.