Unexpected Species Research Articles

Data Quality in Citizen Science Projects: Challenges and Solutions

Data Quality in Citizen Science Projects: Challenges and Solutions

Active and passive environmental DNA surveillance of aquatic invasive species

Environmental DNA (eDNA) is useful for delimiting species ranges in aquatic systems, whereby water samples are screened for the presence of DNA from a single species. However, DNA from many species is collected in every sample, and high-throughput sequencing approaches allow for more passive surveillance where a community of species is identified. In this study, we use active (targeted) and passive molecular surveillance approaches to detect species in the Muskingum River Watershed in Ohio, USA. The presence of bighead carp (Hypophthalmichthys nobilis) eDNA in the Muskingum River Watershed was confirmed with active surveillance using digital droplet polymerase chain reaction (ddPCR). The passive surveillance method detected the presence of eDNA from northern snakehead (Channa argus), which was further confirmed with active ddPCR. Whereas active surveillance may be more sensitive to detecting rare DNA, passive surveillance has the capability of detecting unexpected invasive species. Deploying both active and passive surveillance approaches with the same eDNA samples is beneficial for invasive species management.

English

Abstract As part of a study of aquatic faunal community changes along riverine-lacustrine transition zones upstream of Lewis Smith Reservoir in northwest Alabama, USA, we collected crayfish from 60 sites in the Sipsey Fork, Brushy Creek, and selected tributaries (Black Warrior River system). After finding two unexpected and possibly-introduced crayfish species, we expanded our investigation of crayfish distributions to include crayfish obtained from stomachs of black bass (Micropterus spp.) caught at seven sites in the reservoir. To explore what crayfish species were in the drainage historically, we examined museum databases as well as stomach and intestinal contents of a variety of preserved fishes that were caught in the Sipsey Fork and Brushy Creek drainages upstream of the reservoir in the early 1990’s. Of the seven crayfish species collected, one, Orconectes (Procericambarus) sp. nr ronaldi, was not previously reported from Alabama, and another, O. lancifer, was not reported from the Black Warrior River system prior to the study. Three are known or possibly introduced species. Upstream of the reservoir, the native species Cambarus obstipus, C. striatus, and O. validus were common. The same three species were found in fish collected in the 1990’s. Orconectes perfectus was found only in the reservoir but may be native to the drainage. Orconectes lancifer was in the reservoir and in stream reaches influenced by the reservoir. Evidence points to O. lancifer being introduced in the drainage, but this is uncertain. Orconectes sp. nr ronaldi was found in a relatively small portion of Brushy Creek and its tributaries, in both flowing and impounded habitats, and may be introduced. Orconectes virilis is introduced in Alabama and was found only in stomachs of fish collected in the reservoir.

A new subfossil bulbul (Aves: Passerines: Pycnonotidae) from Rodrigues Island, Mascarenes, south-western Indian Ocean

The Hypsipetes bulbuls (Pycnonotidae) occur on Madagascar and oceanic islands throughout the Indian Ocean, as well as continental Asia. Endemic species are found on the Mascarene Islands of Mauritius and Réunion, but the genus was purportedly never reported from Rodrigues, the smallest and most isolated of the Mascarene group, which was somewhat anomalous considering present avian biogeography. Based on recently discovered subfossil remains, this paper describes a new Hypsipetes bulbul from Rodrigues Island, which presumably became extinct historically, thus filling in the biogeographical gap. This discovery further emphasises the need for palaeontological surveys in situations in which unexpected bird species distribution patterns occur, before robust biogeographical conclusions can be ascertained.http://zoobank.org/urn:lsid:zoobank.org:pub:D979AC29-B662-4A39-B4CA-984E04EB6B65

Crystal structure of potassium sodium hepta-hydrogen hexa-molybdocobaltate(III) octa-hydrate: an extra-protonated B-series Anderson-type heteropolyoxidometalate.

The title compound, KNa[Co(III)(OH)7{Mo6O17}]·8H2O, was obtained by the ion-exchange technique from K3[Co(μ3-OH)6Mo6O18]·7H2O. Six μ3-O atoms and one O atom of the bridging μ2-O atom are protonated. This novel polyanion protonated by an extra H atom is an unexpected polyanion species among the B-series Anderson-type polyoxidometalates (POMs), [X(n) (+)(μ3-OH)6Mo6O18]((6-n)-)] (X = heteroatom). The extra H atom (seventh H atom) in the polyanion does not lie on a crystallographic centre of symmetry, but is located at the mid-point between two μ2-O atoms of adjacent polyanions, and forms a very short hydrogen bond [2.430 (5) Å]. The present structure is considered as particularly significant in understanding noncentrosymmetric strong hydrogen bonding.

Differences in exploration behaviour in common ravens and carrion crows during development and across social context.

Exploration is particularly important for young animals, as it enables them to learn to exploit their surroundings. It is likely to be affected by species ecology and social context, though there are few comparative, longitudinal studies that control for effects of early experience. Here, we investigated group level exploration behaviour in two closely related and identically reared, generalist corvid species: common ravens (Corvus corax) and carrion crows (C. corone, C. cornix), during development and across social context. Subjects were repeatedly presented with a range of novel items, whilst alone and in a dyad/ subgroup, at the fledging (1–2 months old), juvenile (3–8 months old) and sub-adult (14–18 months old) stages. Whilst alone, they were also presented with a novel and familiar person, at the fledging and juvenile stages. We expected developmental differences and a facilitating influence of social context on exploration. Developmental differences were present, with both species interacting most frequently with novel items as juveniles, which may relate to major developmental steps, such as dispersal and a neophobia increase as sub-adults. When a conspecific(s) was present, subjects generally interacted more frequently, though took longer to interact, with novel items. Additionally, we found unexpected species differences, with the most striking difference being the crows’ significantly lower rate of interaction with the novel person, though not the familiar person; a species difference that was present from fledging. We discuss these findings by relating to potential differences in the two species ecology and behaviour, such as habitat use and caching proficiency.

Strange little flies in the big city: exotic flower-breeding drosophilidae (Diptera) in urban Los Angeles.

Urban landscapes are commonly considered too mundane and corrupted to be biotically interesting. Recent insect surveys employing 29 Malaise traps throughout Los Angeles, California, however, have uncovered breeding populations of two unexpected species of one of the most studied and familiar groups of organisms, Drosophila “fruit” flies. Unlike most introduced species of drosophilids, which breed in fresh or decaying fruits, these are specialized flower-breeders. A common species in the survey was Drosophila (Drosophila) gentica Wheeler and Takada, previously collected only once, in El Salvador. It belongs to the flavopilosa species group, all species of which have been known until now from central Chile, Argentina and Uruguay, to Veracruz, Mexico and the Caribbean, breeding in flowers of Cestrum (“jessamine”) and Sessea (Solanaceae). The Los Angeles populations are probably breeding in a native and/or introduced Cestrum; in addition, populations in San Luis Obispo County were visiting ornamental Cestrum. Drosophila gentica occurs as far north as San Francisco, where it was found breeding in Cestrum aurantiacum. D. gentica is redescribed and figured in detail for diagnostic and identification purposes. Specimens from Jamaica previously identified as D. gentica are a distinct species but are not formally described in lieu of complete male specimens. Rare in the Malaise traps was Drosophila (Sophophora) flavohirta Malloch, a common species in Australia on the blossoms of native Myrtaceae, found on introduced Eucalyptus in South Africa and both Eucalyptus and Syzygium in Madagascar; adults feed on myrtaceous pollen and nectar, larvae breed in the flowers. It is also redescribed in detail, including its unusual egg. This is the first New World report of this species; DNA sequences confirm it is a morphologically highly aberrant member of the D. melanogaster species group. This study reveals how intensive field sampling can uncover remarkable biodiversity in even the most urbanized areas.

Cornuvesica: A little known mycophilic genus with a unique biology and unexpected new species

Little is known about the biology of the monotypic genus Cornuvesica (Microascales), apart from that isolates are notoriously difficult to culture on artificial media. A recent collection of material resembling this genus from freshly made wounds on Gmelina arborea in Indonesia, provided an opportunity to reconsider all available material of Cornuvesica falcata, type species of the genus. In addition to morphological comparisons, multigene phylogenetic analyses were made using sequences of the SSU, ITS, LSU and TEF-1α genes. Our results showed that the holotype of Cor. falcata from pine in Canada differed from all other material previously considered to represent this species and also from the new Indonesian collections. The collections considered represented three additional species that we describe here as new. Three New Zealand isolates and an isolate from UK were respectively described as Cor. acuminata and Cor. crypta, while the Indonesian isolates were described as Cor. magnispora. Phylogenies based on the SSU and LSU data sets showed that Cornuvesica spp. do not belong in the Ceratocystidaceae as previously suggested, but represent a distinct lineage in the Microascales that has yet to be named. Results showed that culture filtrates from other fungi or ferric chloride markedly stimulated the growth of Cor. magnispora.

Identification and Characterization of a Selective Allosteric Antagonist of Human P2X4 Receptor Channels

P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 µM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional.

Diverse and widespread contamination evident in the unmapped depths of high throughput sequencing data.

Trace quantities of contaminating DNA are widespread in the laboratory environment, but their presence has received little attention in the context of high throughput sequencing. This issue is highlighted by recent works that have rested controversial claims upon sequencing data that appear to support the presence of unexpected exogenous species. I used reads that preferentially aligned to alternate genomes to infer the distribution of potential contaminant species in a set of independent sequencing experiments. I confirmed that dilute samples are more exposed to contaminating DNA, and, focusing on four single-cell sequencing experiments, found that these contaminants appear to originate from a wide diversity of clades. Although negative control libraries prepared from ‘blank’ samples recovered the highest-frequency contaminants, low-frequency contaminants, which appeared to make heterogeneous contributions to samples prepared in parallel within a single experiment, were not well controlled for. I used these results to show that, despite heavy replication and plausible controls, contamination can explain all of the observations used to support a recent claim that complete genes pass from food to human blood. Contamination must be considered a potential source of signals of exogenous species in sequencing data, even if these signals are replicated in independent experiments, vary across conditions, or indicate a species which seems a priori unlikely to contaminate. Negative control libraries processed in parallel are essential to control for contaminant DNAs, but their limited ability to recover low-frequency contaminants must be recognized.

Symposium on the “Magnitude of Molluscan Diversity — the Known and the Unknown”

The symposium formed part of the 78 Annual Meeting of the American Malacological Society in Cherry Hill, New Jersey, and was held on 19 and 20 June 2012. While everyone talks about biodiversity we asked the basic question: What do we know about molluscan diversity? Or even more simply: How many species of molluscs exist today? A simple question to ask but diffi cult to answer. Of course, this question is not a new one, but ever rising threats to our environment and thus, habitats of molluscs render it more urgent than ever to know what lives on our earth and what will we lose. Land and freshwater molluscs are among the most endangered groups worldwide (Lydeard et al. 2004, Regnier et al. 2009). More recent comprehensive estimates of molluscan diversity suggest the number of described species at 31,000 non-marine and 52,500 marine, but the number of yet unknown diversity may be roughly one third to more than an equal amount (Lydeard et al. 2004, Bouchet 2006). The symposium title refl ects these two aspects while the “known” refers to described species rather than implies that their number is known considering the often unknown share of synonyms among available names. The goal of the symposium was to gather new data to narrow down a realistic estimate of the diversity of this second largest phylum in the animal kingdom and to reveal the progress made during the last few years. The 22 talks presented in Cherry Hill encompassed studies on special taxonomic groups, geographic areas as well as theoretical approaches like analyzing saturation curves, the portion of valid species versus synonyms, correlation to rarity, sampling methods, the amount of undescribed species in collections versus discovery by new fi eldwork, and last but not least elucidated the potential of new methods. The “unknown” stood for what remains to be discovered in terms of species not yet found in underexplored habitats and regions, species not yet recognized due to the lack of taxonomic expertise, revisionary works, and cryptic species. If the title was mirrored in two talks, Gary Rosenberg’s analyses of databases reducing the estimated diversity of described species to 70,000–76,000 represented the “known“ (this issue) while Philippe Bouchet certainly took the audience to the frontier of the “unknown“. Based on his tremendous exploration efforts in the tropical Indo-Pacifi c Bouchet assumed that only 30% of the molluscan diversity of this area is known. This already anticipates that despite some detailed approaches the latter aspect remains not only as undescribed diversity but still unknown in its extent. In terms of marine species, Rudiger Bieler questioned our knowledge of the molluscan fauna in the well known Florida Keys. Philippe Bouchet, as mentioned above, characterized the tropical Indo-Pacifi c as a treasure trove for new species and highlighted the phenomenon of a high percentage of species occurring in low abundances. Janet Voight evaluated the exploration of hydrothermal vent habitats (this issue). John Taylor, co-authored by Emily Glover, gave insight into the enormous diversifi cation of Lucinidae triggered by their obligate chemosymbiosis. Continuing with bivalves, Paul Valentich-Scott dug deep into the hyperdiverse group of Galeommatoidea which not only amaze us with their previously unexpected species diversity but even more by their range of unusual lifestyles. Patrick LaFollette described the attempt to catalogue the published taxa of the megadiverse family Pyramidellidae resulting in an estimated 10,000 names regardless of status. James McLean’s review of the Liotiidae and Areneidae more than doubles the known diversity and sampling still seems far from complete. Exposing the most cryptic groups, Timea Neusser and colleagues gave insights into the mesopsammic fauna and their underestimated diversity and ability to colonize unusual habitats (this issue, Jorger et al.). Moving from the least known to the best known, Fabio Moretzsohn reviewed the progress of cataloging the Cypraeidae (this issue). An equally attractive group but less popular among shell collectors, Terrence Gosliner described nudibranch exploration. Greatly supported by citizen scientists like scuba divers, Gosliner feels their role is not only in accelerating the discovery of new species but also in helping to monitor environmental changes. The contributions focusing on advances by the application of molecular techniques identifi ed signifi cant numbers of so-called cryptic species: Ellen Strong estimated the diversity of Cerithiidae two to three times higher than currently recognized. John Slapcinsky deduced equal ranges from his analyses of the land snail genera Tropidophora and Daedolochila (this issue, on Tropidophora). To the relief of less wellequipped biologists both pointed out that after recognizing the hidden species differentiating morphological characters Stuttgart State Museum of Natural History, Rosenstein 1, 70191 Stuttgart, Germany

All-Food-Seq (AFS): a quantifiable screen for species in biological samples by deep DNA sequencing.

BackgroundDNA-based methods like PCR efficiently identify and quantify the taxon composition of complex biological materials, but are limited to detecting species targeted by the choice of the primer assay. We show here how untargeted deep sequencing of foodstuff total genomic DNA, followed by bioinformatic analysis of sequence reads, facilitates highly accurate identification of species from all kingdoms of life, at the same time enabling quantitative measurement of the main ingredients and detection of unanticipated food components.ResultsSequence data simulation and real-case Illumina sequencing of DNA from reference sausages composed of mammalian (pig, cow, horse, sheep) and avian (chicken, turkey) species are able to quantify material correctly at the 1% discrimination level via a read counting approach. An additional metagenomic step facilitates identification of traces from animal, plant and microbial DNA including unexpected species, which is prospectively important for the detection of allergens and pathogens.ConclusionsOur data suggest that deep sequencing of total genomic DNA from samples of heterogeneous taxon composition promises to be a valuable screening tool for reference species identification and quantification in biosurveillance applications like food testing, potentially alleviating some of the problems in taxon representation and quantification associated with targeted PCR-based approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-639) contains supplementary material, which is available to authorized users.

A genome signature derived from the interplay of word frequencies and symbol correlations

Genome signatures are statistical properties of DNA sequences that provide information on the underlying species. It is not understood, how such species-discriminating statistical properties arise from processes of genome evolution and from functional properties of the DNA. Investigating the interplay of different genome signatures can contribute to this understanding. Here we analyze the statistical dependences of two such genome signatures: word frequencies and symbol correlations at short and intermediate distances.We formulate a statistical model of word frequencies in DNA sequences based on the observed symbol correlations and show that deviations of word counts from this correlation-based null model serve as a new genome signature. This signature (i) performs better in sorting DNA sequence segments according to their species origin and (ii) reveals unexpected species differences in the composition of microsatellites, an important class of repetitive DNA.While the first observation is a typical task in metagenomics projects and therefore an important benchmark for a genome signature, the latter suggests strong species differences in the biological mechanisms of genome evolution.On a more general level, our results highlight that the choice of null model (here: word abundances computed via symbol correlations rather than shorter word counts) substantially affects the interpretation of such statistical signals.

Elemental ratios for characterization of quantum-dots populations in complex mixtures by asymmetrical flow field-flow fractionation on-line coupled to fluorescence and inductively coupled plasma mass spectrometry

Separation and identification of nanoparticles of different composition, with similar particle diameter, coexisting in heterogeneous suspensions of polymer-coated CdSe/ZnS quantum dots (QDs) have been thoroughly assessed by asymmetric flow field-flow fractionation (AF4) coupled on-line to fluorescence and inductively coupled plasma mass spectrometry (ICPMS) detectors. Chemical characterization of any previously on-line separated nanosized species was achieved by the measurement of the elemental molar ratios of every element involved in the synthesis of the QDs, using inorganic standards and external calibration by flow injection analysis (FIA). Such elemental molar ratios, strongly limited so far to pure single nanoparticles suspensions, have been achieved with adequate accuracy by coupling for the first time an ICP-QQQ instrument to an AF4 system. This hyphenation turned out to be instrumental to assess the chemical composition of the different populations of nanoparticles coexisting in the relatively complex mixtures, due to its capabilities to detect the hardly detectable elements involved in the synthesis. Interestingly such information, complementary to that obtained by fluorescence, was very valuable to detect and identify unexpected nanosized species, present at significant level, produced during QDs synthesis and hardly detectable by standard approaches.

Ammonia-Borane Dehydrogenation by Means of an Unexpected Pentacoordinate Boron Species: Insights from Density Functional and Molecular Dynamics Studies

Liberating H2: Static and dynamic density-functional studies reveal that a metal-free Brønsted acid triggered dehydrocoupling of ammonia–borane initiates through the protonation of a BH bond. This protonation leads to the formation of an intermediate containing a pentacoordinate boron centre and results in the loss of H2 from the hypervalent boron centre (see figure).

Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement

Flavoproteins catalyze a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyzes the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics coupled with isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unanticipated stable flavin oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization.

Multiple Population-Period Transient Spectroscopy (MUPPETS) of CdSe/ZnS Nanoparticles. II. Effects of High Fluence and Solvent Heating

Multiple population-period transient spectroscopy (MUPPETS) is a six-pulse experiment with two time dimensions that is capable of adding information about systems with complicated kinetics. The core theory for MUPPETS focuses on the χ(5) response of the chromophores. This theory was used to analyze the dynamics of excitons and biexcitons in CdSe/ZnS core-shell nanoparticles in part I of this paper [J. Phys. Chem. B 2013, DOI:10.1021/jp405785a]. In real experiments, the potential role of additional processes must also be considered, in particular, the χ(7), "saturation" of the MUPPETS signal and nonresonant signals from heating of the solvent. A pathway method for calculating fluence effects in MUPPETS is developed. The fluence dependence of the biexciton signal and its sign reversal, as found in part I, are explained without invoking higher excitons or unexpected species. A method is presented for quantitatively predicting the magnitude of signals from solvent heating using an external standard. Thermal effects in this system are found to be too small to affect the conclusions in part I. Their small size, combined with small, systematic errors in the data, also makes it difficult to measure the yield of solvent heat in these experiments.

Antibiotic Resistance of Bacteria Isolated from the Internal Organs of Edible Snow Crabs

Antibiotic resistance and microbiota within edible snow crabs are important for the Chionoecetes (snow crab) fishing industry. We investigated these parameters using culture methods and antibiotic susceptibility tests with six internal organs from three species of Chionoecetes. Each sample revealed many unexpected microbial species within Chionoecetes internal organs. On the basis of 16S rRNA sequence analysis of 381 isolates, the most abundant genera identified in Chionoecetes opilio were Acinetobacter spp. (24%), Bacillus spp. (4%), Pseudomonas spp. (34%), Stenotrophomonas spp. (28%), and Agreia spp. (11%). In Chionoecetes sp. crabs, Acinetobacter spp. (23%), Bacillus spp. (12%), and Psychrobacter spp. (20%) were most prevalent, while Agreia spp. (11%), Bacillus spp. (31%), Microbacterium spp. (10%), Rhodococcus spp. (12%), and Agrococcus spp. (6%) were most abundant in C. japonicus. Our antibiotic resistance test found resistance to all nine antibiotics tested in 19, 14, and two of the isolates from C. opilio, Chionoecetes sp., and, C. japonicus respectively. Our results are the first to show that microbes with antibiotic resistance are widely distributed throughout the internal organs of natural snow crabs.

New Phosphine-Functionalized NHC Ligands: Discovery of an Effective Catalyst for the Room-Temperature Amination of Aryl Chlorides with Primary and Secondary Amines

We report convenient and high-yielding syntheses of new phosphine-functionalized dihydroimidazolium salts and demonstrate their utility as ligand precursors for Buchwald–Hartwig amination. Several examples of the general formula [1-Mes-3-{2-(PR2)phenyl}imidazolidin-2-ylium][BF4] have been prepared, where phosphines of varying steric and electronic properties (R = Ph (9), Cy (10), 1-Ad (11)) are tethered by an o-phenylene group. The synthesis was not adaptable to N-aryl groups other than mesityl, giving unexpected phosphonium salt species instead. The synthesis was adapted to flexible benzyl-linked variants of the formula [1-Ar-3-{2-(PCy2)benzyl}imidazolidin-2-ylium][BF4], which allowed more steric variation of the dihydroimidazolium N-aryl group (Ar = Mes (21), Dipp (22)). A preliminary study of these hybrid NHC/P ligands in Buchwald–Hartwig amination catalysis (in situ precatalyst formation) revealed 11 to be the most active of the series. Premixing the isolated free NHC ligand 1-Mes-3-{2-(PAd2)phenyl}imidazolidin-2-ylidene (23) with [Pd(cinnamyl)Cl]2 provided a highly active precatalyst that performed well at room temperature and 1 mol % catalyst loading. The system was shown to have an unprecedented ability to arylate both primary alkylamines (monoarylation) and secondary dialkylamines with aryl chlorides at room temperature. Electron-rich and -poor aryl and heteroaryl halides, as well as those featuring ortho substitution, were well tolerated, while substrates featuring both primary and secondary amine groups were selectively arylated at the NH2 position. Furthermore, a preliminary examination of performance in ammonia arylation and acetone α-arylation showed promising results, giving good conversion and high selectivity for monoarylation in both cases.

THREE-DIMENSIONAL MAPPING OF DIFFERENTIAL AMINO ACIDS OF HUMAN, MURINE, CANINE AND EQUINE TLR4/MD-2 RECEPTOR COMPLEXES CONFERRING ENDOTOXIC ACTIVATION BY LIPID A, ANTAGONISM BY ERITORAN AND SPECIES-DEPENDENT ACTIVITIES OF LIPID IVA IN THE MAMMALIAN LPS SENSOR SYSTEM

A literature review concerning the unexpected species differences of the vertebrate innate immune response to lipid IVA was published in CSBJ prior to the present computational study to address the unpaired activity-sequence correlation of prototypic E. coli -type lipid A and its precursor lipid IVA regarding human, murine, equine and canine species. To this end, their sequences and structures of hitherto known Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) complexes were aligned and their differential side chain patterns studied. If required due to the lack of the corresponding X-ray crystallographic data, three-dimensional models of TLR4/MD-2/ligand complexes were generated using mono and dimeric crystal structures as templates and in silico docking of the prototypic ligands lipid A, lipid IVA and Eritoran. All differential amino acids were mapped to pinpoint species dependency on an atomic scale, i.e. the possible concert of mechanistically relevant side chains. In its most abstract and general form the three-dimensional (3D-) models devise a triangular interface or “wedge” where molecular interactions between TLR4, MD-2 and ligand itself take place. This study identifies two areas in the wedge related to either agonism or antagonism reflecting why ligands like lipid IVA can possess a species dependent dual activity. Lipid IVA represents an imperfect (underacylated and backbone-flipped), low affinity ligand of mammalian TLR4/MD-2 complexes. Its specific but weak antagonistic activity in the human system is in particular due to the loss of phosphate attraction in the wedge-shaped region conferred by nonhomologous residue changes when compared to crystal and modeled structures of the corresponding murine and equine TLR4/MD-2 complexes. The counter-TLR4/MD-2 unit was also taken into account since agonist-mediated dimerization in a defined m-shaped complex composed of two TLR4/MD-2/agonist subunits triggers intracellular signaling during the innate immune response to bacterial endotoxin exposure.