Fecal steroid analysis of ovarian cycles in free-ranging baboons

This paper reports field and laboratory tests of serial sampling, solid phase extraction, and microradioimmunoassay methods for the collection, preservation, and analysis of fecal steroids. The field study was conducted in a troop of 87 yellow baboons (Papio cynocephalus) in the Tana River Primate Reserve, Kenya. Serial samples of four focal females and opportunistic sampling of 18 additional females over 22 days of sampling yielded a total of 62 samples, X = 3.1 ± 0.4/day, demonstrating the feasibility of regular field collection and extraction. Estradiol and progesterone concentrations in the field-extracted samples exhibited high recovery and statistically significant correlations (P < 0.05) with concentrations in the lab-extracted samples, suggesting that solid phase extraction could provide a useful alternative to freezing in sites where electricity or liquid nitrogen is not available. Tests of microradioimmunoassays demonstrated that these assays were sensitive, accurate, and precise when applied to the assay of fecal extracts, providing estimates of ovarian steroids that varied significantly with reproductive state. The demonstration that testosterone could be accurately and reliably assayed in fecal extracts suggests that these techniques also could be applied to the study of male reproductive function. Parallels between fecal profiles of cycling and pregnant baboons with patterns reported for serum steroids in baboons suggest that fecal steroids might be useful in distinguishing amenorrhea from early pregnancy in free-ranging baboons as well as in species lacking external indices of reproductive state. © 1995 Wiley-Liss, Inc.     

Chromosomal polymorphism in an Australian leafhopper (Homoptera: Cicadellidae)

Variation in chromosome number from 2n=9 to 2n=7 was found in a species of leafhopper, Alodeltocephalus draba. It is argued that this variation is due to chromosome fusion and that A. draba is presently evolving to a lower number. The process of reduction is more advanced in some populations than in others.Seconded from: Department of Biology, University of Oregon, Eugene, Oregon, U.S.A.     

Molecular phylogenetics and the evolution of fruit and leaf morphology of Dichaea (Orchidaceae: Zygopetalinae)

Background and Aims

The orchid genus Dichaea, with over 100 species found throughout the neotropics, is easily recognized by distichous leaves on long stems without pseudobulbs and flowers with infrastigmatic ligules. The genus has previously been divided into four sections based primarily on presence of ovary bristles and a foliar abscission layer. The aim of this work is to use DNA sequence data to estimate phylogenetic relationships within Dichaea and map the distribution of major morphological characters that have been used to delimit subgenera/sections.

Methods

Sequence data for the nuclear ribosomal internal transcribed spacers and plastid matK, trnL intron, trnL-F spacer and ycf1 for 67 ingroup and seven outgroup operational taxonomic units were used to estimate phylogenetic relationships within Dichaea. Taxa from each of the four sections were sampled, with the greatest representation from section Dichaea, the most diverse and taxonomically puzzling group.

Key Results

Molecular data and morphology support monophyly of Dichaea. Results indicate that section Dichaeopsis is polyphyletic and based on symplesiomorphies, including deciduous leaves and smooth ovaries that are widespread in Zygopetalinae. There are at least three well-supported clades within section Dichaeopsis. Section Pseudodichaea is monophyletic and defined by setose ovaries and leaves with an abscission layer. Sections Dichaea and Dichaeastrum are monophyletic and defined by pendent habit and persistent leaves. Section Dichaeastrum, distinguished from section Dichaea primarily by a glabrous ovary, is potentially polyphyletic.

Conclusions

The leaf abscission layer was lost once, occurring only in the derived sections Dichaea and Dichaeastrum. The setose fruit is a more homoplasious character with several losses and gains within the genus. We propose an informal division of the genus based upon five well-supported clades.Key words: Dichaea, matK, nrITS, Orchidaceae, trnL intron, trnL-F spacer, ycf1, Zygopetalinae     

Multiplexed DNA sequence capture of mitochondrial genomes using PCR products

Background

To utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length.

Methodology/Principal Findings

We describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GA_II instrument.

Conclusions/Significance

This method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA.     

Remission in psoriatic arthritis: is it possible and how can it be predicted?

Introduction

Since remission is now possible in psoriatic arthritis (PsA) we wished to examine remission rates in PsA patients following anti tumour necrosis factor alpha (TNFα) therapy and to examine possible predictors of response.

Methods

Analysis of a prospective patient cohort attending a biologic clinic, between November 2004 and March 2008, was performed prior to commencing therapy and at regular intervals. Baseline clinical characteristics including demographics, previous disease-modifying antirheumatic drug (DMARD) response, tender and swollen joint counts, early morning stiffness, pain visual analogue score, patient global assessment, C reactive protein (CRP) and health assessment questionnaire (HAQ) were collected.

Results

A total of 473 patients (152 PsA; 321 rheumatoid arthritis (RA)) were analyzed. At 12 months remission, defined according to the disease activity score using 28 joint count and CRP (DAS28-CRP), was achieved in 58% of PsA patients compared to 44% of RA patients, significant improvement in outcome measures were noted in both groups (P < 0.05). Analysis of a subgroup of PsA and RA patients matched for DAS28-CRP at baseline also showed higher numbers of PsA patients achieving remission. Linear regression analysis identified the HAQ at baseline as the best predictor of remission in PsA patients (P < 0.001).

Conclusions

DAS28 remission is possible in PsA patients at one year following anti-TNF therapy, at higher rates than in RA patients and is predicted by baseline HAQ.     

Supramolecular photochemical self-assemblies for fluorescence "turn on" and "turn off" assays for chem-bio-helices.

We describe the development of an optical sensing system for the high-throughput screening (HTS) of a broad range of biological molecules, whole cells, organisms and pathogens, and illustrate the technology applications by a hyaluronidase enzyme activity assay as a specific example. At the core of the technology described in this paper, is the exciton concept that is relevant to molecular aggregation. J-aggregates of cyanine dyes have a narrower, red-shifted absorption band compared to monomer. We demonstrate that self-assembly may be driven by the helicogenic nature of the cyanine dye, converting the linear polymers of hyaluronic acid or carboxymethyl cellulose into supramolecular helical assemblies. This self-assembly is accompanied by an intense, sharp, red-shifted J-aggregate fluorescence. We utilized this property to develop an assay for the enzyme hyaluronidase, based upon the concept of "scaffold destruction," whereby the disruption/destruction of the hyaluronic acid polymer by hyaluronidase is accompanied by an attenuation of light emission from the J-aggregate. The extent of light attenuation provides an index of hyaluronidase activity. Other polymers of carbohydrates, proteins, nucleic acids and chemical polymers (such as the carbon nanotube) might provide a similar scaffold for helicogenic dyes upon which molecular aggregation can occur. A key feature of these assays is that they are label-free.     

Accurate determination of phenotypic information from historic thoroughbred horses by single base extension

Historic DNA data have the potential to identify phenotypic information otherwise invisible in the historical, archaeological and palaeontological record. In order to determine whether a single nucleotide polymorphism typing protocol based on single based extension (SNaPshot™) could produce reliable phenotypic data from historic samples, we genotyped three coat colour markers for a sample of historic Thoroughbred horses for which both phenotypic and correct genotypic information were known from pedigree information in the General Stud Book. Experimental results were consistent with the pedigrees in all cases. Thus we demonstrate that historic DNA techniques can produce reliable phenotypic information from museum specimens.     

Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures

Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1–2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.     

The role of protein conformational fluctuations in allostery, function, and evolution

It is now well-known that proteins exist at equilibrium as ensembles of conformational states rather than as unique static structures. Here we review from an ensemble perspective important biological effects of such spontaneous fluctuations on protein allostery, function, and evolution. However, rather than present a thorough literature review on each subject, we focus instead on connecting these phenomena through the ensemble-based experimental, theoretical, and computational investigations from our laboratory over the past decade. Special emphasis is given to insights that run counter to some of the prevailing ideas that have emerged over the past 40 years of structural biology research. For instance, when proteins are viewed as conformational ensembles rather than as single structures, the commonly held notion of an allosteric pathway as an obligate series of individual structural distortions loses its meaning. Instead, allostery can result from energetic linkage between distal sites as one Boltzmann distribution of states transitions to another. Additionally, the emerging principles from this ensemble view of proteins have proven surprisingly useful in describing the role of intrinsic disorder in inter-domain communication, functional adaptation mediated by mutational control of fluctuations, and evolutionary conservation of the energetics of protein stability.