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The impact of preserved museum specimens is transforming and increasing by three-dimensional (3D) imaging that creates high-fidelity online digital specimens. Through examples from the openVertebrate (oVert) Thematic Collections Network, we describe how we created a digitization community dedicated to the shared vision of making 3D data of specimens available and the impact of these data on a broad audience of scientists, students, teachers, artists, and more. High-fidelity digital 3D models allow people from multiple communities to simultaneously access and use scientific specimens. Based on our multiyear, multi-institution project, we identify significant technological and social hurdles that remain for fully realizing the potential impact of digital 3D specimens., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Institute of Biological Sciences.)

Museum specimens collected prior to cryogenic tissue storage are increasingly being used as genetic resources, and though high-throughput sequencing is becoming more cost-efficient, whole genome sequencing (WGS) of historical DNA (hDNA) remains inefficient and costly due to its short fragment sizes and high loads of exogenous DNA, among other factors. It is also unclear how sequencing efficiency is influenced by DNA sources. We aimed to identify the most efficient method and DNA source for collecting WGS data from avian museum specimens. We analyzed low-coverage WGS from 60 DNA libraries prepared from four American Robin ( Turdus migratorius ) and four Abyssinian Thrush ( Turdus abyssinicus ) specimens collected in the 1920s. We compared DNA source (toepad versus incision-line skin clip) and three library preparation methods: (1) double-stranded DNA (dsDNA), single tube (KAPA); (2) single-stranded DNA (ssDNA), multi-tube (IDT); and (3) ssDNA, single tube (Claret Bioscience). We found that the ssDNA, multi-tube method resulted in significantly greater endogenous DNA content, average read length, and sequencing efficiency than the other tested methods. We also tested whether a predigestion step reduced exogenous DNA in libraries from one specimen per species and found promising results that warrant further study. The ~10% increase in average sequencing efficiency of the best-performing method over a commonly implemented dsDNA library preparation method has the potential to significantly increase WGS coverage of hDNA from bird specimens. Future work should evaluate the threshold for specimen age at which these results hold and how the combination of library preparation method and DNA source influence WGS in other taxa., Competing Interests: IDT has been a corporate sponsor of the Field Museum of Natural History as recently as 2020, but no funds from IDT were used to directly support the research that is the subject of the publication., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Unlike most bird species, individual kingfisher species (Aves: Alcedinidae) are typically parasitized by only a single genus of louse (Alcedoffula, Alcedoecus, or Emersoniella). These louse genera are typically specific to a particular kingfisher subfamily. Specifically, Alcedoecus and Emersoniella parasitize Halcyoninae, whereas Alcedoffula parasitizes Alcedininae and Cerylinae. Although Emersoniella is geographically restricted to the Indo-Pacific region, Alcedoecus and Alcedoffula are geographically widespread. We used DNA sequences from two genes, the mitochondrial COI and nuclear EF-1α genes, to infer phylogenies for the two geographically widespread genera of kingfisher lice, Alcedoffula and Alcedoecus. These phylogenies included 47 kingfisher lice sampled from 11 of the 19 currently recognized genera of kingfishers. We compared louse phylogenies to host phylogenies to reconstruct their cophylogenetic history. Two distinct clades occur within Alcedoffula, one that infests Alcedininae and a second that infests Cerylinae. All species of Alcedoecus were found only on host species of the subfamily Halcyoninae. Cophylogenetic analysis indicated that Alcedoecus, as well as the clade of Alcedoffula occurring on Alcedininae, do not show evidence of cospeciation. In contrast, the clade of Alcedoffula occurring on Cerylinae showed strong evidence of cospeciation.

Phylogenomics, the use of large-scale data matrices in phylogenetic analyses, has been viewed as the ultimate solution to the problem of resolving difficult nodes in the tree of life. However, it has become clear that analyses of these large genomic data sets can also result in conflicting estimates of phylogeny. Here, we use the early divergences in Neoaves, the largest clade of extant birds, as a "model system" to understand the basis for incongruence among phylogenomic trees. We were motivated by the observation that trees from two recent avian phylogenomic studies exhibit conflicts. Those studies used different strategies: 1) collecting many characters [$\sim$ 42 mega base pairs (Mbp) of sequence data] from 48 birds, sometimes including only one taxon for each major clade; and 2) collecting fewer characters ($\sim$ 0.4 Mbp) from 198 birds, selected to subdivide long branches. However, the studies also used different data types: the taxon-poor data matrix comprised 68% non-coding sequences whereas coding exons dominated the taxon-rich data matrix. This difference raises the question of whether the primary reason for incongruence is the number of sites, the number of taxa, or the data type. To test among these alternative hypotheses we assembled a novel, large-scale data matrix comprising 90% non-coding sequences from 235 bird species. Although increased taxon sampling appeared to have a positive impact on phylogenetic analyses the most important variable was data type. Indeed, by analyzing different subsets of the taxa in our data matrix we found that increased taxon sampling actually resulted in increased congruence with the tree from the previous taxon-poor study (which had a majority of non-coding data) instead of the taxon-rich study (which largely used coding data). We suggest that the observed differences in the estimates of topology for these studies reflect data-type effects due to violations of the models used in phylogenetic analyses, some of which may be difficult to detect. If incongruence among trees estimated using phylogenomic methods largely reflects problems with model fit developing more "biologically-realistic" models is likely to be critical for efforts to reconstruct the tree of life. [Birds; coding exons; GTR model; model fit; Neoaves; non-coding DNA; phylogenomics; taxon sampling.]., (© The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: The Buff-spotted Woodpecker (Campethera nivosa) is a resident bird species that is distributed in lowland rainforest habitats from western to eastern Africa. We developed species-specific microsatellite markers to examine the population genetics of this species., Findings: Twenty-one microsatellite loci were isolated from C. nivosa. Of these, 15 were found to amplify consistently. These loci were then tested for variability in 15 individuals from different lowland forest localities. The number of alleles ranged from 3 to 13 per locus, with observed and expected heterozygosity ranging from 0.100 to 0.917 and 0.485 to 0.901, respectively. Four loci exhibited significant heterozygote deficiency while one had an excess of heterozygotes. None of the loci exhibited linkage disequilibrium., Conclusion: These polymorphic microsatellite markers will be used to study genetic variability in populations of C. nivosa across either sides of the Congo River to evaluate the effect of the river as a barrier to gene flow.

The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies., (©2013 AACR.)

The Riverine Barriers Hypothesis (RBH) posits that tropical rivers can be effective barriers to gene flow, based on observations that range boundaries often coincide with river barriers. Over the last 160 years, the RBH has received attention from various perspectives, with a particular focus on vertebrates in the Amazon Basin. To our knowledge, no molecular assessment of the RBH has been conducted on birds in the Afrotropics, despite its rich avifauna and many Afrotropical bird species being widely distributed across numerous watersheds and basins. Here, we provide the first genetic evidence that an Afrotropical river has served as a barrier for birds and for their lice, based on four understory bird species collected from sites north and south of the Congo River. Our results indicate near-contemporaneous, Pleistocene lineage diversification across the Congo River in these species. Our results further indicate differing levels of genetic variation in bird lice; the extent of this variation appears linked to the life-history of both the host and the louse. Extensive cryptic diversity likely is being harbored in Afrotropical forests, in both understory birds and their lice. Therefore, these forests may not be "museums" of old lineages. Rather, substantial evolutionary diversification may have occurred in Afrotropical forests throughout the Pleistocene, supporting the Pleistocene Forest Refuge Hypothesis. Strong genetic variation in birds and their lice within a small part of the Congo Basin forest indicates that we may have grossly underestimated diversity in the Afrotropics, making these forests home of substantial biodiversity in need of conservation.

Developing molecularly targeted therapeutics with minimal off-target effects is facilitated by an understanding of compound selectivity. However, for HDAC inhibitors, a clear understanding of specificity has been challenging. In particular, it has been suggested that use of nonspecific substrates and the presence of multiple HDAC activities in enzyme preparations may complicate interpretation of inhibitor experiments. To overcome these and other potential limitations of activity-based HDAC assays, the authors have developed an assay format based on measurement of the binding affinity of inhibitors rather than measurement of enzyme activity. One advantage of this format is that it does not require use of a substrate and thus ameliorates concerns about lack of specificity of existing substrates. This assay is based on an Alexa Fluor® 647-labeled HDAC inhibitor or "tracer," which binds with a high affinity to Class I and Class IIb HDACs. Unlike activity assays, which can be affected by the presence of residual untagged endogenous HDACs from the host expression system, the signal in this format is dependent on the presence of an epitope tag on the specific HDAC of interest. The authors demonstrate the utility of this method by determining the potencies of commonly used inhibitors for six human HDACs.

Background: Microinversions are cytologically undetectable inversions of DNA sequences that accumulate slowly in genomes. Like many other rare genomic changes (RGCs), microinversions are thought to be virtually homoplasy-free evolutionary characters, suggesting that they may be very useful for difficult phylogenetic problems such as the avian tree of life. However, few detailed surveys of these genomic rearrangements have been conducted, making it difficult to assess this hypothesis or understand the impact of microinversions upon genome evolution., Results: We surveyed non-coding sequence data from a recent avian phylogenetic study and found substantially more microinversions than expected based upon prior information about vertebrate inversion rates, although this is likely due to underestimation of these rates in previous studies. Most microinversions were lineage-specific or united well-accepted groups. However, some homoplastic microinversions were evident among the informative characters. Hemiplasy, which reflects differences between gene trees and the species tree, did not explain the observed homoplasy. Two specific loci were microinversion hotspots, with high numbers of inversions that included both the homoplastic as well as some overlapping microinversions. Neither stem-loop structures nor detectable sequence motifs were associated with microinversions in the hotspots., Conclusions: Microinversions can provide valuable phylogenetic information, although power analysis indicates that large amounts of sequence data will be necessary to identify enough inversions (and similar RGCs) to resolve short branches in the tree of life. Moreover, microinversions are not perfect characters and should be interpreted with caution, just as with any other character type. Independent of their use for phylogenetic analyses, microinversions are important because they have the potential to complicate alignment of non-coding sequences. Despite their low rate of accumulation, they have clearly contributed to genome evolution, suggesting that active identification of microinversions will prove useful in future phylogenomic studies.

A recent trend in the literature highlights the special role that tropical montane regions and habitat transitions peripheral to large blocks of lowland rainforest play in the diversification process. The emerging view is one of lowland rainforests as evolutionary 'museums'; where biotic diversity is maintained over evolutionary time, and additional diversity is accrued from peripheral areas, but where there has been little recent diversification. This leads to the prediction of genetic diversity without geographic structure in widespread taxa. Here, I assess the notion of the lowland rainforest 'museum' with a phylogeographic study of the green hylia (Aves: Sylviidae: Hylia prasina) using 1132 bp of mtDNA sequence data. The distribution of genetic diversity within the mainland subspecies of Hylia reveals five highly divergent haplotype groups distributed in accordance with broad-scale areas of endemism in the Afrotropics. This pattern of genetic diversity within a currently described subspecies refutes the characterization of lowland forests as evolutionary museums. If the pattern of geographic variation in Hylia occurs broadly in widespread rainforest species, conservation policy makers may need to rethink their priorities for conservation in the Afrotropics., ((c) 2009 Elsevier Inc. All rights reserved.)

The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor 647 conjugate of staurosporine (a "tracer") from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.

The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug-drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z'-factors of >or=0.5. Seven- to 15-point concentration-response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format.

Ratites (ostriches, emus, rheas, cassowaries, and kiwis) are large, flightless birds that have long fascinated biologists. Their current distribution on isolated southern land masses is believed to reflect the breakup of the paleocontinent of Gondwana. The prevailing view is that ratites are monophyletic, with the flighted tinamous as their sister group, suggesting a single loss of flight in the common ancestry of ratites. However, phylogenetic analyses of 20 unlinked nuclear genes reveal a genome-wide signal that unequivocally places tinamous within ratites, making ratites polyphyletic and suggesting multiple losses of flight. Phenomena that can mislead phylogenetic analyses, including long branch attraction, base compositional bias, discordance between gene trees and species trees, and sequence alignment errors, have been eliminated as explanations for this result. The most plausible hypothesis requires at least three losses of flight and explains the many morphological and behavioral similarities among ratites by parallel or convergent evolution. Finally, this phylogeny demands fundamental reconsideration of proposals that relate ratite evolution to continental drift.

Deep avian evolutionary relationships have been difficult to resolve as a result of a putative explosive radiation. Our study examined approximately 32 kilobases of aligned nuclear DNA sequences from 19 independent loci for 169 species, representing all major extant groups, and recovered a robust phylogeny from a genome-wide signal supported by multiple analytical methods. We documented well-supported, previously unrecognized interordinal relationships (such as a sister relationship between passerines and parrots) and corroborated previously contentious groupings (such as flamingos and grebes). Our conclusions challenge current classifications and alter our understanding of trait evolution; for example, some diurnal birds evolved from nocturnal ancestors. Our results provide a valuable resource for phylogenetic and comparative studies in birds.

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.

The bee-eaters (family Meropidae) comprise a group of brightly colored, but morphologically homogeneous, birds with a wide variety of life history characteristics. A phylogeny of bee-eaters was reconstructed using nuclear and mitochondrial DNA sequence data from 23 of the 25 named bee-eater species. Analysis of the combined data set provided a well-supported phylogenetic hypothesis for the family. Nyctiornis is the sister taxon to all other bee-eaters. Within the genus Merops, we recovered two well-supported clades that can be broadly separated into two groups along geographic and ecological lines, one clade with mostly African resident species and the other clade containing a mixture of African and Asian taxa that are mostly migratory species. The clade containing resident African species can be further split into two groups along ecological lines by habitat preference into lowland forest specialists and montane forest and forest edge species. Intraspecific sampling in several of the taxa revealed moderate to high (3.7-6.5%, ND2) levels of divergence in the resident taxa, whereas the lone migratory taxon showed negligible levels of intraspecific divergence. This robust molecular phylogeny provides the phylogenetic framework for future comparative tests of hypotheses about the evolution of plumage patterns, sociality, migration, and delayed breeding strategies.

The Grey-cheeked Fulvetta, Alcippe morrisonia, is a polytypic species of Quaker babbler (Timaliidae) occurring mainly in highlands from Burma across southern China to Taiwan. To examine gene flow among populations, we sequenced the mitochondrial ND2 gene of 39 individuals of six of the seven subspecies, plus multiple individuals of three outgroup Alcippe species. A lack of shared haplotypes and high FST values suggested no gene flow among populations. The nucleotide divergence between geographically juxtaposed subspecies ranged from 0.8% between Guangdong and Hainan to 9.4% between Yunnan and Vietnam. Phylogenetic analysis of the populations yielded a well resolved tree with two major clades. One clade consisted of the geographically central subspecies schaefferi and davidi, which are located largely in the "Mid-central" zoogeographic region of China's "Oriental" realm. The other clade, the geographically peripheral group, consisted of all other A. morrisonia subspecies, as well as an erstwhile outgroup, the Mountain Fulvetta (Alcippe peracensis annamensis) from central Vietnam. This peripheral group was further divided into two clades, one consisting of taxa occurring in China's "Southwest" zoogeographic region (fratercula and A. p. annamensis), and one occurring in China's "Southern" region (morrisonia, rufescentior, and hueti). These three geographic and phylogenetic groups represent at least four different species based on plumage differences and genetic differentiation. The phylogeny provides the first avian molecular evidence of area relationships among China's zoogeographic zones. It also highlights a remarkable and unexpected amount of genetic divergence and structure in a Sino-Indian "species". If such diversity occurs in other groups of birds with similar distribution, the ramifications are important for conservation planning.

The interactions of the ligand binding domain (LBD) of androgen receptor (AR) and the AR T877A mutant, found in prostate cancer, with peptides from coactivator and corepressor proteins or random phage display peptides were investigated using in vitro time-resolved fluorescence resonance energy transfer (TR-FRET). Interaction of wild-type AR LBD with the random phage display peptide D11FxxLF was observed with dihydrotestosterone (DHT), testosterone, R1881, estradiol, spironolactone, progesterone, and cortisol resulting in distinct dose dependency (EC50) values for each ligand and correlating well with the reported rank order potency of these agonists. Increasing concentrations of cyproterone acetate and mifepristone resulted in more complete disruption of the DHT-mediated AR-D11FxxLF peptide interaction, while flutamide, hydroxyflutamide, and bicalutamide caused only partial disruption of the complex. The mutant AR T877A LBD exhibited increased binding affinities for all ligands tested except for bicalutamide, mifepristone, DHT, and R1881 in a competitive binding assay as compared to wild-type AR LBD. This mutation was also characterized by increased ligand potency for agonist-induced peptide recruitment. Although usually an antagonist, hydroxyflutamide was more potent in the recruitment of D11FxxLF or an SRC3-1 LXXLL motif to AR T877A LBD than AR LBD. The antagonist cyproterone acetate behaved as a full antagonist of D11FxxLF recruitment to AR LBD and AR T877A LBD but as a more potent agonist in the recruitment of SRC3-1 to AR T877A LBD. These results suggest that the AR T877A mutation affects both ligand affinity and ligand dose dependency for peptide recruitment and may explain in part the altered responses of antagonists and increased transcriptional activation reported in androgen-independent prostate cancers.

The estrogen-related receptor-gamma (ERRgamma) is a constitutively active orphan receptor that belongs to the nuclear receptor superfamily and is most closely related to the estrogen receptors. Although its physiological ligand is unknown, ERRgamma has been shown to interact with synthetic estrogenic compounds such as 4-hydroxytamoxifen (4-OHT), tamoxifen, and diethylstilbestrol (DES). To assess how coregulator proteins interact with ERRgamma in response to ligand, an in vitro interaction methodology using time-resolved fluorescence resonance energy transfer (TR-FRET) was developed using glutathione S-transferase (GST)-tagged ERRgamma ligand-binding domain (LBD), a terbium-labeled anti-GST antibody, a fluorescein-labeled peptide containing sequences derived from coregulator proteins, and various ligands. An initial screen of these coregulator peptides bearing the coactivator LXXLL motif, the corepressor LXXI/HIXXXI/L motif, or other interaction motifs from natural coactivator sequences or random phage display peptides indicated that the peptides PGC1alpha, D22, and SRC1-4, known as class III coregulators, interacted most strongly with ERRgamma in the absence of ligand. Given its assay window and biological relevance in energy metabolism and obesity, further studies were conducted with PGC1alpha. Fluorescein-labeled PGC1alpha peptide was displaced from the ERRgamma LBD in the presence of increasing concentrations of 4-OHT and tamoxifen, but DES was less effective in PGC1alpha displacement. The statistical parameter Z' factor that measures the robustness of the assay was greater than 0.8 for displacement of PGC1alpha from ERRgamma LBD in the presence of saturating 4-OHT over an assay incubation time of 1-6 h, indicating an excellent assay. These findings also suggest that binding of 4-OHT, tamoxifen, or DES to ERRgamma results in differential affinity of coregulators for ERRgamma due to unique ligand-induced conformations.

Bulbuls (Aves: Pycnonotidae) are a fairly speciose (ca. 130 sp.) bird family restricted to the Old World. Family limits and taxonomy have been revised substantially over the past decade, but a comprehensive molecular phylogeny for the family has not been undertaken. Using nuclear and mitochondrial DNA sequences, we reconstructed a well-supported phylogenetic hypothesis for the bulbuls. Three basal lineages were identified: a large African clade, a large Asian clade that also included African Pycnonotus species, and the monotypic African genus Calyptocichla. The African clade was sister to the other two lineages, but this placement did not have high branch support. The genus Pycnonotus was not monophyletic because three species (eutilotus, melanoleucos, and atriceps) were highly diverged from the other species and sister to all other Asian taxa. Additional taxon sampling is needed to further resolve relationships and taxonomy within the large and variable Hypsipetes complex.

Direct measurement of the fluorescence lifetime (FLT) of a fluorescent label is an emerging method for high-throughput screening. Changes in the fluorescence lifetime can be correlated to changes in the non-radiative relaxation pathway(s) for the excited state of the label. These pathways can be environmentally sensitive, such as when a labeled analyte is free in solution versus bound to a receptor. Because lifetime is an intrinsic property of a fluorophore, it is not concentration dependent, and therefore has advantages similar to those of ratiometric fluorescent techniques such as fluorescence resonance energy transfer or fluorescence polarization. We have applied the FLT measurement technique to a screen of a small compound library in order to identify compounds that bind to the progesterone receptor, and compared the results to those obtained by performing the assay in fluorescence polarization mode. Each readout modality showed excellent Z'; values, with the FLT readout performing slightly better in this respect. Interfering compounds could be rapidly identified for either assay format by comparing the results between the two formats.

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.

CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid(R) CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid(R) CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent K(m), V(max), and V(max)/K(m) ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid(R) CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid(R) CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.

CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo- and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme's involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (K(i), K(d), or K(m) values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.

Large-scale screening of multiple compound libraries and combinatorial libraries for pharmacological activity is one of the novel approaches of the modern drug discovery process. The application of isozyme-specific high-throughput screening (HTS) assays for characterizing the interactions of potential drug candidates with major human drug-metabolizing cytochrome p450 enzymes (p450s) is newly becoming an essential part of this process. Fluorescence-based HTS assays have been successfully employed for in vitro assessment of drug-drug interactions and enzyme inhibition with several p450 isoforms, including CYP3A4, CYP2D6, CYP2C9, and CYP2C19. Here we describe a fluorescence-based HTS assay for detecting drug metabolism and inhibition with human CYP2E1. CYP2E1 plays an important role in the metabolism of several drugs, many solvents, and toxins and therefore has been repeatedly linked to numerous pathologies, including cancer, liver and kidney toxicity, diabetes, and alcoholism. The assay is based on the ability of a drug to compete with the fluorogenic Vivid CYP2E1 Blue Substrate for CYP2E1 metabolism and thus enables rapid screening of lead molecules for their inhibitory potential. We have used this assay to screen a panel of drugs and compounds for their effects on CYP2E1 metabolism and inhibition. Our results demonstrate the assay's usefulness in identifying CYP2E1 substrates and inhibitors and in enabling in-depth characterization of their interactions with the CYP2E1 isozyme. We also present detailed characteristics of the assay, including its dynamic range and Z'-factor values, which indicate that this robust assay is well suited for kinetic and inhibition studies in HTS formats.

Studies of the distribution of South American taxa have identified several areas of endemism that may have contributed to the historical diversification of the region. We constructed a phylogeny of Glyphorynchus spirurus (Aves: Dendrocolaptidae) populations using mtDNA sequence data from portions of cytochrome b, NADH dehydrogenase subunit II (ND2), and complete NADH dehydrogenase subunit III (ND3). Using this phylogeny we evaluate five previous hypotheses of area-relationships, two based on phylogenetic studies of morphological characters in birds and three based on parsimony analysis of endemism in birds and primates. Maximum likelihood and maximum parsimony analyses recovered two phylogenetic hypotheses that differed in the placement of one of the areas. Within each of the areas of endemism, the two analyses support the same clades. Neither of the phylogenetic hypotheses for Glyphorynchus exactly matches any of the five previous hypotheses of area-relationships, although ambiguous support exists for one of them. Five areas-Central America, Inambari, Napo, Pará, and Rondônia-are supported as composites with component taxa having phylogenetic affinities with more than one area. Data reported here also indicate high levels of sequence divergence within Glyphorynchus. Genetic breaks within Glyphorynchus are only partially congruent with subspecific taxonomy. The regional sampling design used makes this study the largest scale genetic assay of a widespread Neotropical avian taxon published to date.