Systematics within Gyps vultures: a clade at risk

Background  

Populations of the Oriental White-backed Vulture (Gyps bengalensis) have declined by over 95% within the past decade. This decline is largely due to incidental consumption of the non-steroidal anti-inflammatory veterinary pharmaceutical diclofenac, commonly used to treat domestic livestock. The conservation status of other Gyps vultures in southern Asia is also of immediate concern, given the lack of knowledge regarding status of their populations and the continuing existence of taxonomic uncertainties. In this study, we assess phylogenetic relationships for all recognized species and the majority of subspecies within the genus Gyps. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits to Gyps bengalensis. Therefore, an accurate assessment of the phylogenetic relationships among Gyps vultures should aid in their conservation by clarifying taxonomic uncertainties, and enabling inference of their respective relatedness to susceptible G. bengalensis.     

A double helix B-type geometry based on high-resolution proton NMR of single-helical DNA fragments: d(TA)5 x d(TA)5.

A single-helical B-type geometry is presented based on 1H NMR observations on d(TATA) and several other small single-helical DNA fragments. The structure is extended to one complete turn of double-helical DNA and its characteristics are compared with other known B-type structures.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.     

Effect of culture filtrates of black-pigmented Bacteroides species on the matrix production of chick embryo chondrocytes in vitro

Antonie van Leeuwenhoek -     

Optimal Triage Test Characteristics to Improve the Cost-Effectiveness of the Xpert MTB/RIF Assay for TB Diagnosis: A Decision Analysis

Background

High costs are a limitation to scaling up the Xpert MTB/RIF assay (Xpert) for the diagnosis of tuberculosis in resource-constrained settings. A triaging strategy in which a sensitive but not necessarily highly specific rapid test is used to select patients for Xpert may result in a more affordable diagnostic algorithm. To inform the selection and development of particular diagnostics as a triage test we explored combinations of sensitivity, specificity and cost at which a hypothetical triage test will improve affordability of the Xpert assay.

Methods

In a decision analytical model parameterized for Uganda, India and South Africa, we compared a diagnostic algorithm in which a cohort of patients with presumptive TB received Xpert to a triage algorithm whereby only those with a positive triage test were tested by Xpert.

Findings

A triage test with sensitivity equal to Xpert, 75% specificity, and costs of US$5 per patient tested reduced total diagnostic costs by 42% in the Uganda setting, and by 34% and 39% respectively in the India and South Africa settings. When exploring triage algorithms with lower sensitivity, the use of an example triage test with 95% sensitivity relative to Xpert, 75% specificity and test costs $5 resulted in similar cost reduction, and was cost-effective by the WHO willingness-to-pay threshold compared to Xpert for all in Uganda, but not in India and South Africa. The gain in affordability of the examined triage algorithms increased with decreasing prevalence of tuberculosis among the cohort.

Conclusions

A triage test strategy could potentially improve the affordability of Xpert for TB diagnosis, particularly in low-income countries and with enhanced case-finding. Tests and markers with lower accuracy than desired of a diagnostic test may fall within the ranges of sensitivity, specificity and cost required for triage tests and be developed as such.     

Validation of an HIV-1 inactivation protocol that is compatible with intracellular drug analysis by mass spectrometry

Mass spectrometry is a powerful tool for studying the intracellular pharmacokinetics of antiretroviral drugs. However, the biohazard of HIV-1 calls for a safety protocol for such analyses. To this end, we extracted HIV-1 producing cells with methanol or ethanol at 4 degrees C. After extraction, no viral infectivity was detected, as shown by a reduction in infectious titers of more than 6log. In addition, this protocol is compatible with the quantitative analysis of antiretroviral drugs in cell extracts using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Thus, using this protocol, infectious HIV-1 is inactivated and antiretroviral drugs are extracted from cells in a single step.