Giant taxon-character matrices: the future of morphological systematics.

Simões et al. () argued that large matrices are linked to the construction of "problematic" characters, and that those characters negatively affect tree topology. In their re-evaluation of two squamate datasets, however, Simões et al. () simply eliminated what they termed "problematic" characters, rather than recode them. This practice ignores potential sources of phylogenetic information and, if it were to be more widely followed, would inhibit the advancement of the field of systematics. Here, we defend the necessity and inevitability of large morphological (phenomic) datasets and discuss best practices for morphological data collection in contemporary phylogenetics.

Effective methods to improve recruitment and retention in school-based substance use prevention studies.

BACKGROUND: Poor recruitment and high attrition may invalidate results of research studies. This paper describes successful recruitment and retention strategies in a school-based substance use prevention trial and explores factors associated with intervention attendance and retention. METHODS: A total of 384 parent-child dyads from 15 schools in the New York Metropolitan area participated in a control trial, testing the efficacy of parent-training to prevent youth substance use. Assessments were completed immediately post-intervention and 6-, 12-, and 24-month postintervention. Logistic regression analyses were used to determine which familial and study characteristics predicted attendance in the intervention and retention by parents and youth. RESULTS: 84% of intervention parents attended 4 of the 5 workshops; 83% of control parents attended their single workshop. Intervention attendance was predicted by parent job status, but this was not significant after controlling for other family factors. Retention rates ranged from 87% to 91% over the 2 years. No family characteristics predicted retention, but time since baseline and attendance at treatment workshops and the control workshop did. For children, age at baseline and ethnicity predicted retention, but this did not remain significant in the adjusted model. CONCLUSION: Intervention attendance was high and retention rates far exceeded the minimum standard of 70% retention in behavioral studies. Recruitment and retention strategies were effective for different family constellations. Efforts to maximize participation in both treatment and control interventions are critical to retention in longitudinal trials.

Screening for the expression of soluble recombinant protein in Escherichia coli.

Protein expression and purification have traditionally been time-consuming, case-specific endeavors, and are considered to be the greatest bottlenecks in most proteomics pipelines. Escherichia coli (E. coli) is the most convenient and cost-effective host, although optimal conditions for the expression of different proteins vary widely. Proteins vary in their structural stability, solubility, and toxicity in this environment, resulting in differing rates of protein degradation, formation into insoluble inclusion bodies, and cell death, thus affecting the amount of soluble protein that can be obtained from E. coli grown in culture. To take full advantage of a variety of strategies developed to improve the expression of soluble protein in E. coli, an easy, rapid means to test many growth parameters is necessary. This chapter describes a dot-blot expression screen to test the effects of growth and induction parameters on the yield of soluble protein. The expression screen is used to detect hexahistidine-tagged proteins expressed in E. coli; however, it is adaptable for the detection of other affinity tags or fusion partners that have suitable antibodies available. In this example, induction time and temperature are tested; however, it can be used to test additional parameters, such as affinity tag type and placement, E. coli host type, and growth medium formulations.

High-throughput purification of hexahistidine-tagged proteins expressed in E. coli.

This chapter describes a method for efficient high-throughput purification of hexahistidine-tagged proteins that are expressed in Escherichia coli (E. coli) using immobilized metal affinity chromatography (IMAC) in a 96-well format. This approach is particularly suitable for proteomic applications that require modest amounts of highly purified proteins to be generated very efficiently. This approach is also very useful for identifying protein targets that are most amenable to scaled-up production for use in structural studies. The typical yield of proteins purified using this system is 50-150 microg, which is generally greater than that of many in vitro expression systems and much less costly. The method as described has been optimized for purifying approx 150 microg of hexahistidine-tagged protein, but the method is flexible, so that the amount of affinity matrix and culture volumes can be adjusted for optimal binding capacity and consequently highest purity. Although the method detailed here uses IMAC to purify hexahistidine-tagged proteins, this basic platform can be used with many other tags and affinity resins.

High-throughput cloning for proteomics research.

Ligation-independent cloning (LIC) is a simple, rapid, and efficient method for high-throughput cloning. In this system, linear plasmid vector and insert DNA are treated to generate complementary single-stranded overhangs that anneal during a short incubation. The LIC system is adaptable for use with any vector following an alteration of the vector sequence. This chapter describes the creation of an LIC-compatible vector, with tips on how to make any vector LIC-enabled. It also includes a protocol for generating high-quality linearized vector template for the LIC reaction. Lastly, a step-by-step protocol of the LIC reaction is outlined, with useful tips and tricks for optimization and screening.

Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein.

Protein interactions critical to DNA repair and cell cycle control systems are often coordinated by modules that belong to a superfamily of structurally conserved BRCT domains. Because the mechanisms of BRCT interactions and their significance are not well understood, we sought to define the affinity and specificity of those BRCT modules that orchestrate base excision repair and single-strand break repair. Common to these pathways is the essential XRCC1 DNA repair protein, which interacts with at least nine other proteins and DNA. Here, we characterized the interactions of four purified BRCT domains, two from XRCC1 and their two partners from DNA ligase IIIalpha and poly(ADP-ribosyl) polymerase 1. A monoclonal antibody was selected that recognizes the ligase IIIalpha BRCT domain, but not the other BRCT domains, and was used to capture the relevant ligase IIIalpha BRCT complex. To examine the assembly states of isolated BRCT domains and pairwise domain complexes, we used size-exclusion chromatography coupled with on-line light scattering. This analysis indicated that isolated BRCT domains form homo-oligomers and that the BRCT complex between the C-terminal XRCC1 domain and the ligase IIIalpha domain is a heterotetramer with 2:2 stoichiometry. Using affinity capture and surface plasmon resonance methods, we determined that specific heteromeric interactions with high nanomolar dissociation constants occur between pairs of cognate BRCT domains. A structural model for a XRCC1 x DNA ligase IIIalpha heterotetramer is proposed as a core base excision repair complex, which constitutes a scaffold for higher order complexes to which other repair proteins and DNA are brought into proximity.

Fluffy, the major regulator of conidiation in Neurospora crassa, directly activates a developmentally regulated hydrophobin gene.

The fluffy (fl) gene of Neurospora crassa is required for asexual sporulation and encodes an 88 kDa polypeptide containing a typical fungal Zn2Cys6 DNA-binding motif. Identification of genes regulated by fl will provide insight into how fungi regulate growth during morphogenesis. As a step towards identifying the target genes on which FL may act, we sought to define target sequences to which the FL protein binds. The DNA binding domain of FL was expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified using glutathione-sepharose affinity chromatography. The DNA binding sites were selected and amplified by means of a polymerase chain reaction (PCR)-mediated random-site selection method involving affinity bead-binding and gel mobility shift analysis. Sequencing and comparison of the selected clones suggested that FL binds to the motif 5'-CGG(N)9CCG-3'. A potential binding site was found in the promoter region of the eas (ccg-2) gene, which encodes a fungal hydrophobin. In vitro competitive binding assays revealed a preferred binding site for FL in the eas promoter, 5'-CGGAAGTTTC CTCCG-3', which is located 1498 bp upstream of the eas translation initiation codon. In vivo experiments using a foreign DNA sequence tag also confirmed that this sequence resides in a region required for FL regulation. In addition, yeast one hybrid experiments demonstrated that the C-terminal portion of FL functions in transcriptional activation. Transcriptional profiling was used to identify additional potential targets for regulation by fl.

A saturation screen for cis-acting regulatory DNA in the Hox genes of Ciona intestinalis.

A screen for the systematic identification of cis-regulatory elements within large (>100 kb) genomic domains containing Hox genes was performed by using the basal chordate Ciona intestinalis. Randomly generated DNA fragments from bacterial artificial chromosomes containing two clusters of Hox genes were inserted into a vector upstream of a minimal promoter and lacZ reporter gene. A total of 222 resultant fusion genes were separately electroporated into fertilized eggs, and their regulatory activities were monitored in larvae. In sum, 21 separable cis-regulatory elements were found. These include eight Hox linked domains that drive expression in nested anterior-posterior domains of ectodermally derived tissues. In addition to vertebrate-like CNS regulation, the discovery of cis-regulatory domains that drive epidermal transcription suggests that C. intestinalis has arthropod-like Hox patterning in the epidermis.

A quality mix: using evidence and experience to evaluate new technologies.

This article describes BlueCross BlueShield of Tennessee's (BCBST) success in blending evidence with experience. This process has provided a winning solution for members, providers, and the health plan. Provider input has proved to be an invaluable tool in assessing both new and established technologies. BCBST uses a variety of methods, from formal panels to a modified Delphi approach, to obtain provider input into the creation of clinical decision tools. Provider experience fills the gap in the assessment of technologies that are widely used but do not have sufficient evidence to support them. This approach has significant implications for health plan delivery. It also meets the Institute of Medicine's concern that every healthcare organization, purchaser, regulator and educational institution encourage the delivery of services based upon the best available scientific knowledge.

An improved method for the in vitro evolution of aptamers and applications in protein detection and purification.

One of the key components of proteomics initiatives is the production of high affinity ligands or probes that specifically recognize protein targets in assays that detect and capture proteins of interest. Particularly versatile probes with tremendous potential for use as affinity molecules are aptamers. Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on affinity for a target molecule. Aptamers offer advantages over traditional antibody-based affinity molecules in their ease of production, regeneration and stability, largely due to the chemical properties of nucleic acids versus amino acids. We describe an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. We demonstrate the ability of aptamers that recognize thyroid transcription factor 1 (TTF1) to bind their target protein with high affinity and specificity, and detail their uses in a number of assays. The TTF1 aptamers were characterized using surface plasmon resonance, and shown to be useful for enzyme-linked assays, western blots and affinity purification.

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

High-throughput proteomics: a flexible and efficient pipeline for protein production.

Many studies that aim to characterize the proteome require the production of pure protein in a high-throughput format. We have developed a system for high-throughput subcloning, protein expression and purification that is simple, fast, and inexpensive. We utilized ligation-independent cloning with a custom-designed vector and developed an expression screen to test multiple parameters for optimal protein production in E. coli. A 96-well format purification protocol that produced microgram quantities of pure protein was also developed.