Rapid radiation of ant parasitic butterflies during the Miocene aridification of Africa.

Africa has undergone a progressive aridification during the last 20 My that presumably impacted organisms and fostered the evolution of life history adaptations. We test the hypothesis that shift to living in ant nests and feeding on ant brood by larvae of phyto-predaceous Lepidochrysops butterflies was an adaptive response to the aridification of Africa that facilitated the subsequent radiation of butterflies in this genus. Using anchored hybrid enrichment we constructed a time-calibrated phylogeny for Lepidochrysops and its closest, non-parasitic relatives in the Euchrysops section (Poloyommatini). We estimated ancestral areas across the phylogeny with process-based biogeographical models and diversification rates relying on time-variable and clade-heterogeneous birth-death models. The Euchrysops section originated with the emerging Miombo woodlands about 22 million years ago (Mya) and spread to drier biomes as they became available in the late Miocene. The diversification of the non-parasitic lineages decreased as aridification intensified around 10 Mya, culminating in diversity decline. In contrast, the diversification of the phyto-predaceous Lepidochrysops lineage proceeded rapidly from about 6.5 Mya when this unusual life history likely first evolved. The Miombo woodlands were the cradle for diversification of the Euchrysops section, and our findings are consistent with the hypothesis that aridification during the Miocene selected for a phyto-predaceous life history in species of Lepidochrysops, with ant nests likely providing caterpillars a safe refuge from fire and a source of food when vegetation was scarce.

A global phylogeny of butterflies reveals their evolutionary history, ancestral hosts and biogeographic origins.

Butterflies are a diverse and charismatic insect group that are thought to have evolved with plants and dispersed throughout the world in response to key geological events. However, these hypotheses have not been extensively tested because a comprehensive phylogenetic framework and datasets for butterfly larval hosts and global distributions are lacking. We sequenced 391 genes from nearly 2,300 butterfly species, sampled from 90 countries and 28 specimen collections, to reconstruct a new phylogenomic tree of butterflies representing 92% of all genera. Our phylogeny has strong support for nearly all nodes and demonstrates that at least 36 butterfly tribes require reclassification. Divergence time analyses imply an origin ~100 million years ago for butterflies and indicate that all but one family were present before the K/Pg extinction event. We aggregated larval host datasets and global distribution records and found that butterflies are likely to have first fed on Fabaceae and originated in what is now the Americas. Soon after the Cretaceous Thermal Maximum, butterflies crossed Beringia and diversified in the Palaeotropics. Our results also reveal that most butterfly species are specialists that feed on only one larval host plant family. However, generalist butterflies that consume two or more plant families usually feed on closely related plants.

Peroxisomes and pancreatic beta-cell lipo-dysfunction.

AIMS: Pancreatic beta-cell lipo-dysfunction decreases insulin secretion and predisposes to the development of type 2 diabetes. Through targeted Pex11ß knockdown and peroxisome depletion, our aim was to investigate the specific contribution of peroxisomes to palmitate mediated pancreatic beta-cell dysfunction. METHODS: MIN6 cells were transfected with probes targeted against Pex11ß, a regulator of peroxisome abundance, or with scrambled control probes. Peroxisome abundance was measured by PMP-70 protein expression. 48 h post transfection, cells were incubated with 250 µM palmitate or BSA control for a further 48 h before measurement of glucose stimulated insulin secretion and of reactive oxygen species. RESULTS: Pex11ß knockdown decreased target gene expression by >80% compared with the scrambled control (P<0.001). This led to decreased PMP-70 expression (p<0.01) and a 22% decrease in peroxisome number (p<0.05). At 25 mM glucose, palmitate treatment decreased insulin secretion by 64% in the scrambled control cells (2.54±0.25 vs 7.07±0.83 [mean±SEM] ng/h/µg protein; Palmitate vs BSA P<0.001), but by just 37% in the Pex11ß knockdown cells. Comparing responses in the presence of palmitate, insulin secretion at 25 mM glucose was significantly greater in the Pex11ß knockdown cells compared with the scrambled controls (4.04±0.46 vs 2.54±0.25 ng/h/µg protein; p<0.05). Reactive oxygen species generation with palmitate was lower in the Pex11ß knockdown cells compared with the scrambled controls (P<0.001). CONCLUSION: Pex11ß knockdown decreased peroxisome abundance, decreased palmitate mediated reactive oxygen species generation, and reversed the inhibitory effect of palmitate on insulin secretion. These findings reveal a distinct role of peroxisomes in palmitate mediated beta-cell dysfunction.

Repeat UVA exposure of human skin fibroblasts induces both a transitionary and recovery DNA methylation response.

Aim: UVA radiation drives skin photoaging in the dermis, plausibly via persistent changes to DNA methylation in dermal fibroblasts. Methods: Genome-wide DNA methylation changes after five repeated daily UVA doses were determined at 48 h (transitionary) and 1 week (recovery) post final irradiation. Results: Differential methylation was found at the transitionary time point in active chromatin states near genes that are highly expressed in fibroblasts and are involved in cellular defensive mechanisms; the majority of these methylation differences were restored to control levels after 7 day recovery. At the recovery time point, new differential methylation occurred at repressed regions near developmental genes, normally weakly expressed in fibroblasts. Conclusion: UVA irradiation induces transitionary and recovery-associated DNA methylation responses in fibroblasts with contrasting functional characteristics.

Recent diversification of Chrysoritis butterflies in the South African Cape (Lepidoptera: Lycaenidae).

Although best known for its extraordinary radiations of endemic plant species, the South African fynbos is home to a great diversity of phytophagous insects, including butterflies in the genus Chrysoritis (Lepidoptera: Lycaenidae). These butterflies are remarkably uniform morphologically; nevertheless, they comprise 43 currently accepted species and 68 currently valid taxonomic names. While many species have highly restricted, dot-like distributions, others are widespread. Here, we investigate the phylogenetic and biogeographic history underlying their diversification by analyzing molecular markers from 406 representatives of all described species throughout their respective ranges. We recover monophyletic clades for both C. chrysaor and C. thysbe species-groups, and identify a set of lineages that fall between them. The estimated age of divergence for the genus is 32 Mya, and we document significantly rapid diversification of the thysbe species-group in the Pleistocene (~2 Mya). Using ancestral geographic range reconstruction, we show that West Fynbos is the most likely region of origin for the radiation of the thysbe species-group. The colonization of this region occurred 9 Mya and appears to have been followed by a long period of relative stasis before a recent increase in diversification. Thus, the thysbe radiation does not appear to have resulted from the colonization of new biogeographic areas. Rather, the impact of species interactions (with ants and plants), the appearance of key innovations, and/or the opening of new ecological niche space in the region might explain the sudden burst of speciation that occurred in this group 2 Mya. The biogeographic model suggests two different diversification processes with few historical cross-colonisations, one in eastern South Africa for the C. chrysaor group and the other in western South Africa for the remaining taxa. Distributional range assessments and ecological niche models for each species show important niche overlap, and in a few cases, complete overlap. However, these shared traits are not explained by phylogenetic history. Chrysoritis taxa frequently fly in sympatry and gene tree reticulation appears to be widespread at the species level, suggesting that several episodes of range shifts might have led to secondary sympatries, allowing limited gene flow that challenges species delimitation efforts. In addition, the unusually high diversification rate for the thysbe clade of 1.35 [0.91-1.81] lineages per million years also suggests the possibility of taxonomic oversplitting. The phylogeny presented here provides a framework for a taxonomic revision of the genus. We highlight cases of potential synonymy both in allopatry and sympatry, and stress the importance of dedicated studies to assess potential pre- and post-zygotic barriers giving rise to species delimitations of the thysbe group.

Development and validation of a monocyte activation test for the control/safety testing of an OMV-based meningococcal B vaccine.

It is imperative to ensure biological products are free of contaminating pyrogenic material prior to administration to patients. Historically the rabbit pyrogen test (RPT) was used to screen for such contamination in medicines for intravenous delivery. This test was adapted for use to screen vaccines. However, some, including meningococcal vaccines containing outer membrane vesicles, are intrinsically pyrogenic. Indeed, this is the case for Bexsero which contains relatively high levels of endotoxin and other potential pyrogens such as lipoproteins and porins. The RPT proved a difficult method for measuring the pyrogenic content of Bexsero and differences between laboratories in different countries made repeat testing at the control laboratories problematic resulting in batches being wrongly identified as unsafe. At NIBSC a monocyte activation test (MAT) was adapted and validated as an alternative. This required setting of a specification in-house and deciding on a decisional procedure using multiple donors, allowing batches equally pyrogenic or less, than those batches shown to be safe in a clinical trial, to be certified as safe. The resulting format was a reference comparison method with an upper limit of 1.8 relative pyrogen units (RPU). The batch passed if an initial four donors had a response equal to or less than 1.8 RPU, if one donor is above this limit the batch was tested in a further four donors and seven of the eight must be equal to or below 1.8 RPU. If two donors have a response greater than 1.8 the batch failed.

A collaborative study to establish the 3rd WHO International Standard for hepatitis B virus for nucleic acid amplification techniques.

Nucleic acid amplification techniques (NAT) are routinely used for clinical diagnostics and monitoring hepatitis B virus (HBV) infections, and are implemented on a voluntary basis for blood screening. A collaborative study was performed to evaluate a replacement WHO International Standard for HBV for the standardization of NAT. Two lyophilised HBV candidates were evaluated by 16 laboratories worldwide, alongside the existing HBV International Standard. The overall mean potency estimates for the candidate samples 1 and 2, relative to sample 3 (2nd HBV International Standard), from quantitative assays, were 5.93 and 5.98 log10 International Units (IU)/mL respectively. The variability in individual laboratory mean estimates for samples 1-3 for quantitative assays was ∼0.3 log10 IU/mL. The inter-laboratory variability for qualitative assays was higher. Accelerated thermal degradation studies indicate that both lyophilised candidates are stable and suitable for long-term use. Overall, the results suggested that both candidates were suitable as replacement International Standards. Sample 1 (NIBSC code 10/264) was established as the 3rd WHO International Standard for HBV for NAT with an assigned potency of 850,000 IU/mL (∼5.93 log10 IU/mL), when reconstituted in 0.5 mL of nuclease-free water. It is intended for the calibration (in IU) of secondary reference materials used in HBV NAT.

A collaborative study to establish the 1st WHO International Standard for Epstein-Barr virus for nucleic acid amplification techniques.

Variability in viral load measurements using nucleic acid amplification techniques (NAT) has a significant impact on the management of Epstein-Barr virus (EBV)-associated diseases, and has highlighted a need for standardisation of these measurements. The aim of this collaborative study was to evaluate the suitability of a range of candidate reference materials to harmonise EBV viral load measurements in a wide range of NAT assays. Candidate materials included lyophilised and liquid whole virus preparations of the EBV B95-8 strain, and preparations of Namalwa and Raji cells. Variability between the individual laboratory mean estimates for each candidate was 2.5 log10 copies/mL. The agreement between laboratories was improved when the potency of each candidate was expressed relative to the lyophilised B95-8 preparation. The results of the study indicate the suitability of this candidate as the 1st WHO International Standard for EBV for NAT. It was established in October 2011 by the WHO's Expert Committee on Biological Standardisation with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/260). It is intended to be used for the calibration of secondary reference materials, used in EBV NAT assays, in IU, thereby improving the comparability of patient viral load measurements.

Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples.

BACKGROUND: Interassay harmonization of cytomegalovirus (CMV) DNA measurement is important for infection management. Uncertainty exists regarding the result harmonization achievable in patient plasma samples using quantitative polymerase chain reaction (qPCR) assays with calibrators now traceable to the First World Health Organization International Standard (IS) for CMV DNA. METHOD: Serial dilutions of the IS and a blinded panel of 40 genotyped CMV DNA-positive pooled plasma samples and 10 negative plasma samples were tested by 6 laboratories using 10 qPCR assays calibrated to the IS. Each clinical sample was constructed using plasma from a single unique transplant recipient. RESULTS: The variance for individual CMV DNA-positive samples was greater for clinical samples (median, 1.50 [range, 1.22-2.82] log10 IU/mL) than for IS dilutions (median, 0.94 [range, 0.69-1.35] log10 IU/mL) (P < .001); 58.9% of all clinical sample results and 93.6% of IS dilution results fell within ±0.5 log10 IU/mL of the mean viral load of each sample. Result variability was not impacted by either genotype or quantitative levels of CMV DNA. Testing procedure differences can significantly influence results, even when analyte-specific reagents are identical. For clinical samples, all assays demonstrated result bias (P < .008). Assays with amplicon sizes ≤86 bp had significantly higher results compared to assays with larger amplicon sizes (≥105 bp) (P < .001). CONCLUSIONS: The variability in CMV DNA results reported on individual samples has been reduced by the IS, but ongoing clinically relevant variability persists, preventing meaningful interassay result comparison.

International collaborative study on the 3rd WHO International Standard for hepatitis B surface antigen.

BACKGROUND: The WHO International Standard (IS) for hepatitis B surface antigen (HBsAg) is used to standardize HBsAg assays. Stocks of the 2nd IS for HBsAg are depleted. The proposal to establish its replacement was endorsed by WHO in 2012. OBJECTIVE: Preparation of a freeze-dried candidate 3rd IS (NIBSC 12/226); evaluation of its suitability in a WHO international collaborative study; calibration of its potency in International Units (IU). STUDY DESIGN: The 3rd IS is based on plasma-derived, purified, inactivated HBsAg from Vietnam. Qualitative and quantitative HBsAg assays were used to evaluate 12/226 alongside the 2nd IS and 1st IS. Blinded study samples included a duplicate of 12/226, a negative control and two diluted plasma samples representing hepatitis B virus (HBV) genotypes A and B. RESULTS: Twelve laboratories from 9 countries returned 22 data sets from 15 methods. The overall geometric mean potency of 12/226 is 47.3IU/mL (±13% CV) when compared to the 2nd IS with HBV subgenotype A2. The 3rd IS has HBV subgenotype B4 with a heterogeneous HBsAg subtype population of ayw1 and adw2. Some genotype-dependent effects on the inter-laboratory variability were observed but overall mean potencies were virtually identical irrespective of the IS used for calibration. Stability studies indicate that the candidate is stable for long-term use. CONCLUSIONS: 12/226 was established in October 2014 by the WHO Expert Committee on Biological Standardization as the 3rd IS for HBsAg with a potency of 47.3IU per ampoule maintaining the continuity in the standardization of HBsAg assays.

A collaborative study to establish the 1st WHO International Standard for human cytomegalovirus for nucleic acid amplification technology.

Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU.

International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE.

The microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future.

Standardization of human IL-29 (IFN-λ1): establishment of a World Health Organization international reference reagent for IL-29 (IFN-λ1).

Human interleukin-29 (IL-29), a helical cytokine with interferon-like activities, is currently being developed as a clinical biotherapeutic to treat chronic hepatitis C infection and some cancers. As such, the World Health Organization (WHO) has recognized a need for biological standardization of IL-29 and the establishment of an internationally available reference reagent of IL-29. In order to accomplish this, an international collaborative study that evaluates WHO candidate reference reagents of IL-29 was instigated by the National Institute for Biological Standards and Control (NIBSC) in 2010 and was carried out in the succeeding year. Two preparations of human sequence recombinant IL-29, one expressed in murine NS0 cells and the other in Escherichia coli, were formulated and lyophilized at NIBSC before evaluation in the collaborative study for their suitability to serve as a reference reagent. The preparations were tested by 6 laboratories from 4 countries using in vitro bioassays and also evaluated for thermal stability within the NIBSC laboratory. On the basis of the results of the collaborative study, both preparations, 07/212 (NS0-derived) and 10/176 (E. coli-derived) were judged sufficiently active and stable to serve as a reference reagent. However, since IL-29 produced in E. coli is in development for clinical applications, it was recommended that the preparation coded 10/176 be established as the WHO international reference reagent for human IL-29. This recommendation was accepted, and the IL-29 preparation coded 10/176 was formally established by the WHO ECBS at its meeting in October 2012 as the WHO international reference reagent for IL-29 with an assigned unitage of 5,000 reference units per ampoule.

The 3rd International Standard for serum IgE: international collaborative study to evaluate a candidate preparation.

BACKGROUND: The measurement of serum IgE aids in the diagnosis and management of atopic allergic disease and hyper-IgE immunodeficiency syndromes. The 2nd World Health Organization (WHO) International Reference Reagent (IRR) for serum IgE (75/502; 5000 IU/ampoule), is widely used to calibrate assays for serum IgE. Exhaustion of stocks of the 2nd IRR necessitated the production of a replacement preparation and its evaluation in an international collaborative study to determine its suitability to serve as the 3rd International Standard (IS) for serum IgE. METHODS: Sera and defibrinated plasma with elevated IgE levels were pooled and lyophilised in ampoules. This preparation, coded 11/234, was assayed by 18 laboratories in 11 countries using commercial assay methodology for IgE, along with the 2nd IRR, 75/502, and two lyophilised serum samples. RESULTS: Overall, there were no consistent differences in the way that the candidate IS (11/234), the IRR (75/502), and the two serum samples behaved in the assays with respect to linearity and parallelism. The mean IgE value of the candidate IS, 11/234, relative to the IRR, 75/502, was 13,411 IU/mL based on parallel line analysis of raw assay data at NIBSC, and 13,551 IU/mL based on the laboratories' own estimates after correcting for the values obtained for 75/502. CONCLUSIONS: The use of 11/234 will ensure that assays for serum IgE continue to be well standardised. The preparation was established by the WHO Expert Committee on Biological Standardization as the 3rd IS for serum IgE with an assigned value of 13,500 IU/mL, corresponding to 6750 IU/ampoule.

The 2nd International Standard for Interleukin-2 (IL-2). Report of a collaborative study.

Two candidate preparations of human sequence recombinant Interleukin-2 (IL-2) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement international standard. The preparations were tested by eight laboratories using in vitro bioassays and immunoassays. The candidate preparation 86/500 was judged suitable to serve as a replacement international standard based on the data obtained for activity and stability. On the basis of the results reported here, the preparation coded 86/500 was established by the WHO Expert Committee on Biological Standardisation (ECBS) in 2012 as the WHO 2nd IS for human IL-2 with an assigned value for IL-2 activity of 210IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies exclusively on the estimates calculated relative to the WHO 1st IS for continuity of the IU.