Transmembrane molecules for phylogenetic analyses of pathogenic protists: Leishmania-specific informative sites in hydrophilic loops of trans- endoplasmic reticulum N-acetylglucosamine-1-phosphate transferase

A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.     

Auditory spatial acuity approximates the resolving power of space-specific neurons

The relationship between neuronal acuity and behavioral performance was assessed in the barn owl (Tyto alba), a nocturnal raptor renowned for its ability to localize sounds and for the topographic representation of auditory space found in the midbrain. We measured discrimination of sound-source separation using a newly developed procedure involving the habituation and recovery of the pupillary dilation response. The smallest discriminable change of source location was found to be about two times finer in azimuth than in elevation. Recordings from neurons in its midbrain space map revealed that their spatial tuning, like the spatial discrimination behavior, was also better in azimuth than in elevation by a factor of about two. Because the PDR behavioral assay is mediated by the same circuitry whether discrimination is assessed in azimuth or in elevation, this difference in vertical and horizontal acuity is likely to reflect a true difference in sensory resolution, without additional confounding effects of differences in motor performance in the two dimensions. Our results, therefore, are consistent with the hypothesis that the acuity of the midbrain space map determines auditory spatial discrimination.     

Four-body scoring function for mutagenesis

MOTIVATION: There is a need for an efficient and accurate computational method to identify the effects of single- and multiple-residue mutations on the stability and reactivity of proteins. Such a method should ideally be consistent and yet applicable in a widespread manner, i.e. it should be applied to various proteins under the same parameter settings, and have good predictive power for all of them. RESULTS: We develop a Delaunay tessellation-based four-body scoring function to predict the effects of single- and multiple-residue mutations on the stability and reactivity of proteins. We test our scoring function on sets of single-point mutations used by several previous studies. We also assemble a new, diverse set of 237 single- and multiple-residue mutations, from over 24 different publications. The four-body scoring function correctly predicted the changes to the stability of 169 out of 210 mutants (80.5%), and the changes to the reactivity of 17 out of 27 mutants (63%). For the mutants that had the changes in stability/reactivity quantified (using reaction rates, temperatures, etc.), an average Spearman rank correlation coefficient of 0.67 was achieved with the four-body scores. We also develop an efficient method for screening huge numbers of mutants of a protein, called combinatorial mutagenesis. In one study, 64 million mutants of a cold-shock nucleus binding domain protein 1CSQ, with six of its residues being changed to all possible (20) amino acids, were screened within a few hours on a PC, and all five stabilizing mutants reported were correctly identified as stabilizing by combinatorial mutagenesis.     

Neck muscle paths and moment arms are significantly affected by wrapping surface parameters

In this paper, we studied the effects of wrapping surfaces on muscle paths and moment arms of the neck muscle, semispinalis capitis. Sensitivities to wrapping surface size and the kinematic linkage to vertebral segments were evaluated. Kinematic linkage, but not radius, significantly affected the accuracy of model muscle paths compared to centroid paths from images. Both radius and linkage affected the moment arm significantly. Wrapping surfaces that provided the best match to centroid paths over a range of postures had consistent moment arms. For some wrapping surfaces with poor matches to the centroid path, a kinematic method (tendon excursion) predicted flexion moment arms in certain postures, whereas geometric method (distance to instant centre) predicted extension. This occurred because the muscle lengthened as it wrapped around the surface. This study highlights the sensitivity of moment arms to wrapping surface parameters and the importance of including multiple postures when evaluating muscle paths and moment arm.     

DNA topoisomerase I from parasitic protozoa: a potential target for chemotherapy

The growing occurrence of drug resistant strains of unicellular prokaryotic parasites, along with insecticide-resistant vectors, are the factors contributing to the increased prevalence of tropical diseases in underdeveloped and developing countries, where they are endemic. Malaria, cryptosporidiosis, African and American trypanosomiasis and leishmaniasis threaten human beings, both for the high mortality rates involved and the economic loss resulting from morbidity. Due to the fact that effective immunoprophylaxis is not available at present; preventive sanitary measures and pharmacological approaches are the only sources to control the undesirable effects of such diseases. Current anti-parasitic chemotherapy is expensive, has undesirable side effects or, in many patients, is only marginally effective. Under this point of view molecular biology techniques and drug discovery must walk together in order to find new targets for chemotherapy intervention. The identification of DNA topoisomerases as a promising drug target is based on the clinical success of camptothecin derivatives as anticancer agents. The recent detection of substantial differences between trypanosome and leishmania DNA topoisomerase IB with respect to their homologues in mammals has provided a new lead in the study of the structural determinants that can be effectively targeted. The present report is an up to date review of the new findings on type IB DNA topoisomerase in unicellular parasites and the role of these enzymes as targets for therapeutic agents.     

Reliability at the Lower Limits of HIV-1 RNA Quantification in Clinical Samples: A Comparison of RT-PCR versus bDNA Assays

Introduction

To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods.

Methodology/Principal Findings

We selected patients with ≥1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable CD4 counts, excluding patients with viral loads ≥1,000 copies/ml or any significant changes in therapy. Out of 4500 clinic patients, 419 patients (1588 viral loads) were included. 39% of viral loads were reported as detectable by RT-PCR vs. 5% reported as detectable by bDNA. The mean coefficient of variation was higher before vs. after assay change. We found an odds'' ratio of 16.7 for having a viral load >75 copies/ml during the RT-PCR vs. bDNA periods.

Discussion

These data support previous reports, suggesting that bDNA may more reliably discriminate between viral suppression and low level viremia in stable patients on therapy. Low-level viremia, noted more with RT-PCR, may promote unneeded testing, while differences in viral load reliability may impact antiretroviral trial and quality assurance endpoints. Commonly used plasma separator tubes may differentially affect RT-PCR and bDNA results.     

Maximizing power production in a stack of microbial fuel cells using multiunit optimization method

This study demonstrates real‐time maximization of power production in a stack of two continuous flow microbial fuel cells (MFCs). To maximize power output, external resistances of two air–cathode membraneless MFCs were controlled by a multiunit optimization algorithm. Multiunit optimization is a recently proposed method that uses multiple similar units to optimize process performance. The experiment demonstrated fast convergence toward optimal external resistance and algorithm stability during external perturbations (e.g., temperature variations). Rate of the algorithm convergence was much faster than in traditional maximum power point tracking algorithms (MPPT), which are based on temporal perturbations. A power output of 81–84 mW/L_A (A = anode volume) was achieved in each MFC. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009