Serological Evidence of Influenza A Viruses in Frugivorous Bats from Africa

Bats are likely natural hosts for a range of zoonotic viruses such as Marburg, Ebola, Rabies, as well as for various Corona- and Paramyxoviruses. In 2009/10, researchers discovered RNA of two novel influenza virus subtypes – H17N10 and H18N11 – in Central and South American fruit bats. The identification of bats as possible additional reservoir for influenza A viruses raises questions about the role of this mammalian taxon in influenza A virus ecology and possible public health relevance. As molecular testing can be limited by a short time window in which the virus is present, serological testing provides information about past infections and virus spread in populations after the virus has been cleared. This study aimed at screening available sera from 100 free-ranging, frugivorous bats (Eidolon helvum) sampled in 2009/10 in Ghana, for the presence of antibodies against the complete panel of influenza A haemagglutinin (HA) types ranging from H1 to H18 by means of a protein microarray platform. This technique enables simultaneous serological testing against multiple recombinant HA-types in 5μl of serum. Preliminary results indicate serological evidence against avian influenza subtype H9 in about 30% of the animals screened, with low-level cross-reactivity to phylogenetically closely related subtypes H8 and H12. To our knowledge, this is the first report of serological evidence of influenza A viruses other than H17 and H18 in bats. As avian influenza subtype H9 is associated with human infections, the implications of our findings from a public health context remain to be investigated.     

The complete cDNA and amino acid sequence of human apolipoprotein B-100

We have determined the complete sequence of apolipoprotein (apo) B-100 cDNA. It is 14.1 kilobases in length and codes for a 4563-amino acid protein, including a 27-amino acid signal peptide and a 4536-amino acid mature protein. Further, we identified 2366 residues of apoB-100 by direct sequence analysis of apoB-100 tryptic peptides. The mature peptide is characterized by high hydrophobicity (0.916 kcal/residue) and predicted beta-sheet content (21%). Dot matrix analysis revealed the presence of many long internal repeats in apoB-100. The mature peptide contains 25 cysteine residues, 12 of which are in the N-terminal 500 residues. Twenty potential N-linked glycosylation sites were identified, of which 13 were proven to be glycosylated, and 4 were found not to be glycosylated by direct analysis of tryptic peptides. Our findings on apoB structure provide a basis for future experimentation on the role of apoB-100-containing lipoproteins in atherosclerosis.     

Assessing developmental hazard: the reliability of the A/D ratio.

The quantitative relationship between developmentally toxic exposure levels and adult toxic exposure levels has been used as an index of developmental hazard and has figured prominently in discussions of legal regulation of developmentally toxic agents. Perhaps the most frequently cited index of developmental hazard is the A/D ratio. This index, a ratio of marginally toxic adult and developmental dose levels (e.g., NOAELs or LOAELs), is attractive because it is easily calculated from published toxicity assays and because it has been argued that A/D is relatively constant across species for a given agent. We explored some quantitative aspects of the A/D ratio and of the concept of developmental hazard by simulating 661,500 mammalian developmental toxicity assays on 441 hypothetical compounds. In our simulations, A/D often varied substantially among replicate assays: the median ratio of the upper and lower limits of the distribution of A/D values that include about 95% of the observed A/D values is 16. In addition, A/D did a poor job of predicting the relative developmental and adult responses at dosages lower than those used to calculate the index: among simulated compounds with A/Ds of about 1.0, the developmental response at 1/100th of the NOAEL ranged from about 0.1% to 20,000% of the adult response. Finally, we measured the concordance between pairs of four different indices of developmental hazard, including A/D. Concordance was greatest when the indices were based on the same portion of the dose response, and was much weaker between indices that examined different portions of the dose response. Therefore, it seems likely that no single index can quantify "developmental hazard," as defined by relative adult and developmental susceptibility, and more effort needs to be expended in refining the concept if it is to be useful for hazard assessment.     

A simple vector modification to facilitate oligonucleotide-directed mutagenesis.

We describe a simple modification of commonly used single-stranded cloning vectors that permits the efficient recovery of mutant DNA molecules in oligonucleotide-directed mutagenesis experiments, even when the absolute efficiency of mutagenesis is very low. The modification consists of the insertion of a short synthetic DNA fragment into the vector's polylinker and permits the identification of mutant clones based on a standard chromogenic plate assay for bacterial colonies or phage plaques producing functional beta-galactosidase. Other useful properties of the original vector are retained in the modified version. In vitro mutagenesis reactions are carried out with two oligonucleotides, one to introduce the mutation of interest, and the second to correct a frameshift mutation introduced into the beta-galactosidase gene of the modified vector. We have found that these two sequence changes are closely linked following transformation of an appropriate E. coli strain with the products of the in vitro mutagenesis reaction, and have thereby recovered desired mutations at a frequency of about 50% even when the overall mutagenesis efficiency is less than 1%. By alternately correcting and re-introducing the beta-galactosidase frameshift mutation, we have shown that multiple rounds of mutagenesis can be carried out on the same template with a high efficiency of mutant recovery in each step. Modifications similar or identical to those we describe here should be feasible for most commonly used single-stranded cloning vectors and should increase the usefulness of these vectors by providing an additional option for oligonucleotide-directed mutagenesis to be used in conjunction with or in lieu of other commonly used approaches.     

A landscape ecology approach identifies important drivers of urban biodiversity

Cities are growing rapidly worldwide, yet a mechanistic understanding of the impact of urbanization on biodiversity is lacking. We assessed the impact of urbanization on arthropod diversity (species richness and evenness) and abundance in a study of six cities and nearby intensively managed agricultural areas. Within the urban ecosystem, we disentangled the relative importance of two key landscape factors affecting biodiversity, namely the amount of vegetated area and patch isolation. To do so, we a priori selected sites that independently varied in the amount of vegetated area in the surrounding landscape at the 500‐m scale and patch isolation at the 100‐m scale, and we hold local patch characteristics constant. As indicator groups, we used bugs, beetles, leafhoppers, and spiders. Compared to intensively managed agricultural ecosystems, urban ecosystems supported a higher abundance of most indicator groups, a higher number of bug species, and a lower evenness of bug and beetle species. Within cities, a high amount of vegetated area increased species richness and abundance of most arthropod groups, whereas evenness showed no clear pattern. Patch isolation played only a limited role in urban ecosystems, which contrasts findings from agro‐ecological studies. Our results show that urban areas can harbor a similar arthropod diversity and abundance compared to intensively managed agricultural ecosystems. Further, negative consequences of urbanization on arthropod diversity can be mitigated by providing sufficient vegetated space in the urban area, while patch connectivity is less important in an urban context. This highlights the need for applying a landscape ecological approach to understand the mechanisms shaping urban biodiversity and underlines the potential of appropriate urban planning for mitigating biodiversity loss.     

ADP ribosylation of Escherichia coli RNA polymerase is nonessential for bacteriophage T4 development.

The bacteriophage T4-induced alt and mod gene products covalently add ADP-ribose to the Escherichia coli RNA polymerase alpha polypeptides; phage carrying either an alt or a mod mutation are viable. A genetic cross between T4alt and T4mod phages yielded alt mod recombinant progeny which could not ADP ribosylate RNA polymerase at all, yet grew apparently normally. Thus, ADP ribosylation of RNA polymerase appeared to be nonessential for T4 development (at least in E. coli B/r and E. coli CR63), even though the phage has evolved two distinct enzymes to catalyze this reaction.     

Exposure-disease continuum for 2-chloro-2'-deoxyadenosine, a prototype ocular teratogen. 1. Dose-response analysis

BACKGROUND: Treatment of pregnant mice with 2-chloro-2'-deoxyadenosine (2CdA) on day 8 of gestation induces microphthalmia through a mechanism coupled to the p53 tumor suppressor gene. The present study defines 2CdA dosimetry with respect to exposure (pharmacokinetics), p53 protein induction, and disease (microphthalmia). METHODS: Pregnant CD-1 mice dosed with 0.5-10.0 mg/kg 2CdA on day 8 provided fetuses for teratological evaluation; 2CdA was measured by HPLC in the antimesometrium through 180 min postexposure, and p53 was assessed with immunostaining of the embryo through 270 min. 5'-/3'-RACE was used to sequence the candidate gene for 2CdA bioactivation from target cells. RESULTS: Microphthalmia appeared first in the dose-response curve. The highest 2CdA dose having no observable adverse effect (NOAEL) was 1.5 mg/kg; the benchmark dose that produced an extra 5% risk of microphthalmia (BMD(5)) was 2.5 mg/kg, and the lower confidence limit (BMDL) was 2.0 mg/kg. Pharmacokinetic parameters for doses encompassing the threshold (1.5-2.5 mg/kg) were modeled at 1.0-1.8 microM (C(max)) and 30-80 microM-min (AUC). The p53 response was not detected below the BMDL; however, a low-grade response appeared 4.5 hr after a teratogenic dose (5.0 mg/kg), and high-grade induction followed an embryolethal dose (10.0 mg/kg). RACE identified a novel splice variant of mitochondrial deoxyguanosine kinase, dGK-3, as the likely candidate for 2CdA bioactivation in the embryo. CONCLUSIONS: Microphthalmia represented the critical effect malformation of 2CdA. The findings suggest a mitochondrial mechanism for 2CdA bioactivation, leading to an embryonic p53 response only after 2CdA elimination and implying pharmacodynamic coupling to the exposure-disease continuum. Published 2001 Wiley-Liss, Inc.     

Ab initio analysis of the Cope rearrangement of germacrane sesquiterpenoids

The energetics of the Cope rearrangement of 17 germacrane sesquiterpenoids to their respective elemane forms have been calculated using both density functional theory (B3LYP/6-31G*) and post Hartee-Fock (MP2/6-31G**) ab initio methods. The calculations are in qualitative agreement with experimentally observed Cope rearrangements, but the two methods give slightly different results. MP2 calculations generally show more favorable elemene energies compared to the respective germacrenes (by around 3–4 kcal mol⁻¹) and smaller activation energies (by 2–3 kcal mol⁻¹). Additionally, neither method is accurate enough to consistently reproduce the germacrene/elemene equilibrium. Apparently, the generally small energy differences between the two forms in these sesquiterpenoids cannot be adequately reproduced at these levels of calculation. Figure The Cope rearrangement of the germacrane sesquiterpenoid bacchascandon to the elemane shyobunone     

Incomplete Lineage Sorting and Hybridization Statistics for Large-Scale Retroposon Insertion Data

Ancient retroposon insertions can be used as virtually homoplasy-free markers to reconstruct the phylogenetic history of species. Inherited, orthologous insertions in related species offer reliable signals of a common origin of the given species. One prerequisite for such a phylogenetically informative insertion is that the inserted element was fixed in the ancestral population before speciation; if not, polymorphically inserted elements may lead to random distributions of presence/absence states during speciation and possibly to apparently conflicting reconstructions of their ancestry. Fortunately, such misleading fixed cases are relatively rare but nevertheless, need to be considered. Here, we present novel, comprehensive statistical models applicable for (1) analyzing any pattern of rare genomic changes, (2) testing and differentiating conflicting phylogenetic reconstructions based on rare genomic changes caused by incomplete lineage sorting or/and ancestral hybridization, and (3) differentiating between search strategies involving genome information from one or several lineages. When the new statistics are applied, in non-conflicting cases a minimum of three elements present in both of two species and absent in a third group are considered significant support (p<0.05) for the branching of the third from the other two, if all three of the given species are screened equally for genome or experimental data. Five elements are necessary for significant support (p<0.05) if a diagnostic locus derived from only one of three species is screened, and no conflicting markers are detected. Most potentially conflicting patterns can be evaluated for their significance and ancestral hybridization can be distinguished from incomplete lineage sorting by considering symmetric or asymmetric distribution of rare genomic changes among possible tree configurations. Additionally, we provide an R-application to make the new KKSC insertion significance test available for the scientific community at http://retrogenomics.uni-muenster.de:3838/KKSC_significance_test/.     

A family of repeated DNA sequences, one of which resides in the second intervening sequence of the mouse dihydrofolate reductase gene

One member of a repeated sequence family has been found in the second intron of the mouse dihydrofolate reductase gene. The family consists of approximately 20 members/haploid genome, two of which have been sequenced and found to contain a 480-base pair region of homology with a significant amount of sequence divergence. Rat, Chinese hamster, Syrian hamster, and human DNAs contain homologous repetitious elements, and the four rodent species have a member of this family associated with the dihydrofolate reductase gene.