Netrin-1 Peptide Is a Chemorepellent in Tetrahymena thermophila.

Netrin-1 is a highly conserved, pleiotropic signaling molecule that can serve as a neuronal chemorepellent during vertebrate development. In vertebrates, chemorepellent signaling is mediated through the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2. Tetrahymena thermophila has been used as a model system for chemorepellent signaling because its avoidance response is easily characterized under a light microscope. Our experiments showed that netrin-1 peptide is a chemorepellent in T. thermophila at micromolar concentrations. T. thermophila adapts to netrin-1 over a time course of about 10 minutes. Netrin-adapted cells still avoid GTP, PACAP-38, and nociceptin, suggesting that netrin does not use the same signaling machinery as any of these other repellents. Avoidance of netrin-1 peptide was effectively eliminated by the addition of the tyrosine kinase inhibitor, genistein, to the assay buffer; however, immunostaining using an anti-phosphotyrosine antibody showed similar fluorescence levels in control and netrin-1 exposed cells, suggesting that tyrosine phosphorylation is not required for signaling to occur. In addition, ELISA indicates that a netrin-like peptide is present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Further study will be required in order to fully elucidate the signaling mechanism of netrin-1 peptide in this organism.

FtsHi1/ARC1 is an essential gene in Arabidopsis that links chloroplast biogenesis and division.

The Arabidopsis arc1 (accumulation and replication of chloroplasts 1) mutant has pale seedlings and smaller, more numerous chloroplasts than the wild type. Previous work has suggested that arc1 affects the timing of chloroplast division but does not function directly in the division process. We isolated ARC1 by map-based cloning and discovered it encodes FtsHi1 (At4g23940), one of several FtsHi proteins in Arabidopsis. These poorly studied proteins resemble FtsH metalloproteases important for organelle biogenesis and protein quality control but are presumed to be proteolytically inactive. FtsHi1 bears a predicted chloroplast transit peptide and localizes to the chloroplast envelope membrane. Phenotypic studies showed that arc1 (hereafter ftsHi1-1), which bears a missense mutation, is a weak allele of FtsHi1 that disrupts thylakoid development and reduces de-etiolation efficiency in seedlings, suggesting that FtsHi1 is important for chloroplast biogenesis. Consistent with this finding, transgenic plants suppressed for accumulation of an FtsHi1 fusion protein were often variegated. A strong T-DNA insertion allele, ftsHi1-2, caused embryo-lethality, indicating that FtsHi1 is an essential gene product. A wild-type FtsHi1 transgene rescued both the chloroplast division and pale phenotypes of ftsHi1-1 and the embryo-lethal phenotype of ftsHi1-2. FtsHi1 overexpression produced a subtle increase in chloroplast size and decrease in chloroplast number in wild-type plants while suppression led to increased numbers of small chloroplasts, providing new evidence that FtsHi1 negatively influences chloroplast division. Taken together, our analyses reveal that FtsHi1 functions in an essential, envelope-associated process that may couple plastid development with division.

Plastid chaperonin proteins Cpn60 alpha and Cpn60 beta are required for plastid division in Arabidopsis thaliana.

BACKGROUND: Plastids arose from a free-living cyanobacterial endosymbiont and multiply by binary division as do cyanobacteria. Plastid division involves nucleus-encoded homologs of cyanobacterial division proteins such as FtsZ, MinD, MinE, and ARC6. However, homologs of many other cyanobacterial division genes are missing in plant genomes and proteins of host eukaryotic origin, such as a dynamin-related protein, PDV1 and PDV2 are involved in the division process. Recent identification of plastid division proteins has started to elucidate the similarities and differences between plastid division and cyanobacterial cell division. To further identify new proteins that are required for plastid division, we characterized previously and newly isolated plastid division mutants of Arabidopsis thaliana. RESULTS: Leaf cells of two mutants, br04 and arc2, contain fewer, larger chloroplasts than those of wild type. We found that ARC2 and BR04 are identical to nuclear genes encoding the plastid chaperonin 60 alpha (ptCpn60alpha) and chaperonin 60 beta (ptCpn60beta) proteins, respectively. In both mutants, plastid division FtsZ ring formation was partially perturbed though the level of FtsZ2-1 protein in plastids of ptcpn60beta mutants was similar to that in wild type. Phylogenetic analyses showed that both ptCpn60 proteins are derived from ancestral cyanobacterial proteins. The A. thaliana genome encodes two members of ptCpn60alpha family and four members of ptCpn60beta family respectively. We found that a null mutation in ptCpn60alpha abolished greening of plastids and resulted in an albino phenotype while a weaker mutation impairs plastid division and reduced chlorophyll levels. The functions of at least two ptCpn60beta proteins are redundant and the appearance of chloroplast division defects is dependent on the number of mutant alleles. CONCLUSION: Our results suggest that both ptCpn60alpha and ptCpn60beta are required for the formation of a normal plastid division apparatus, as the prokaryotic counterparts are required for assembly of the cell division apparatus. Since moderate reduction of ptCpn60 levels impaired normal FtsZ ring formation but not import of FtsZ into plastids, it is suggested that the proper levels of ptCpn60 are required for folding of stromal plastid division proteins and/or regulation of FtsZ polymer dynamics.

Automatic word recognition: the validity of a universally accessible assessment task.

In the current study, the validity of a task designed to assess the automatic word recognition skills of persons with complex communication needs was investigated. A total of 78 students without communication impairments in kindergarten through second grade completed a standard automatic word recognition task requiring oral reading of words presented for less than 0.25 s. The same students completed an experimental word recognition task that did not require a spoken response. Results support the validity of the experimental task. For example, the mean performance scores on both tasks decreased in the expected direction, and there was a significant correlation between the standard and experimental tasks. Other results suggest that the same trait was being measured by both tasks. The data highlight directions for future research and development of the experimental task, while leaving us enthusiastic about the future of the experimental task as a valid means of assessing automatic word recognition for persons with complex communication needs.

New connections across pathways and cellular processes: industrialized mutant screening reveals novel associations between diverse phenotypes in Arabidopsis.

In traditional mutant screening approaches, genetic variants are tested for one or a small number of phenotypes. Once bona fide variants are identified, they are typically subjected to a limited number of secondary phenotypic screens. Although this approach is excellent at finding genes involved in specific biological processes, the lack of wide and systematic interrogation of phenotype limits the ability to detect broader syndromes and connections between genes and phenotypes. It could also prevent detection of the primary phenotype of a mutant. As part of a systems biology approach to understand plastid function, large numbers of Arabidopsis thaliana homozygous T-DNA lines are being screened with parallel morphological, physiological, and chemical phenotypic assays (www.plastid.msu.edu). To refine our approaches and validate the use of this high-throughput screening approach for understanding gene function and functional networks, approximately 100 wild-type plants and 13 known mutants representing a variety of phenotypes were analyzed by a broad range of assays including metabolite profiling, morphological analysis, and chlorophyll fluorescence kinetics. Data analysis using a variety of statistical approaches showed that such industrial approaches can reliably identify plant mutant phenotypes. More significantly, the study uncovered previously unreported phenotypes for these well-characterized mutants and unexpected associations between different physiological processes, demonstrating that this approach has strong advantages over traditional mutant screening approaches. Analysis of wild-type plants revealed hundreds of statistically robust phenotypic correlations, including metabolites that are not known to share direct biosynthetic origins, raising the possibility that these metabolic pathways have closer relationships than is commonly suspected.

Effects of mutations in Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and positioning, and FtsZ filament morphology in vivo.

In plants, chloroplast division FtsZ proteins have diverged into two families, FtsZ1 and FtsZ2. FtsZ1 is more divergent from its bacterial counterparts and lacks a C-terminal motif conserved in most other FtsZs. To begin investigating FtsZ1 structure-function relationships, we first identified a T-DNA insertion mutation in the single FtsZ1 gene in Arabidopsis thaliana, AtFtsZ1-1. Homozygotes null for FtsZ1, though impaired in chloroplast division, could be isolated and set seed normally, indicating that FtsZ1 is not essential for viability. We then mapped five additional atftsZ1-1 alleles onto an FtsZ1 structural model and characterized chloroplast morphologies, FtsZ protein levels and FtsZ filament morphologies in young and mature leaves of the corresponding mutants. atftsZ1-1(G267R), atftsZ1-1(R298Q) and atftsZ1-1(Delta404-433) exhibit reduced FtsZ1 accumulation but wild-type FtsZ2 levels. The semi-dominant atftsZ1-1(G267R) mutation caused the most severe phenotype, altering a conserved residue in the predicted T7 loop. atftsZ1-1(G267R) protein accumulates normally in young leaves but is not detected in rings or filaments. atftsZ1-1(R298Q) has midplastid FtsZ1-containing rings in young leaves, indicating that R298 is not critical for ring formation or positioning despite its conservation. atftsZ1-1(D159N) and atftsZ1-1(G366A) both have overly long, sometimes spiral-like FtsZ filaments, suggesting that FtsZ dynamics are altered in these mutants. However, atftsZ1-1(D159N) exhibits loss of proper midplastid FtsZ positioning while atftsZ1-1(G366A) does not. Finally, truncation of the FtsZ1 C-terminus in atftsZ1-1(Delta404-433) impairs chloroplast division somewhat but does not prevent midplastid Z ring formation. These alleles will facilitate understanding of how the in vitro biochemical properties of FtsZ1 are related to its in vivo function.

Chloroplast division.

Chloroplasts are descendants of cyanobacteria and divide by binary fission. Several components of the division apparatus have been identified in the past several years and we are beginning to appreciate the plastid division process at a mechanistic level. In this review, we attempt to summarize the most recent developments in the field and assemble these observations into a working model of plastid division in plants.

What happens to reading between first and third grade? Implications for students who use AAC.

School-age students who use AAC need access to communication, reading, and writing tools that can support them to actively engage in literacy learning. They also require access to core literacy learning opportunities across grade levels that foster development of conventional literacy skills. The importance of the acquisition of conventional literacy skills for students who use AAC cannot be overemphasized. And yet, one of the critical challenges in supporting the literacy learning of students who use AAC has been a lack of knowledge about literacy curricula and supports to literacy learning for these students. Most students who use AAC do not become conventionally literate and few of those who do achieve literacy skills beyond the second grade level. This article will provide an overview of the most frequent reading instructional activities in first and third grade classrooms. To better understand the foundational experiences important to literacy learning, the results of a survey project that examined the reading activities of general education students and teachers during primary grade instruction are presented, and critical shifts in instruction that occurred between first and third grade are highlighted. The primary instructional focus of core reading activities is also examined, along with adaptations for students who use AAC.

Threshold denial rates in prior authorization prescription programs.

In this special report, the economics of prior authorization is examined with a focus on the break-even rate that requests are denied. Using a simple theoretical model, comparative static results are derived and used to consider the cost-effectiveness implications of future changes in prior authorization policies.

Impact of the fourth hurdle on the international pharmaceutical industry.

Traditionally, pharmaceutical companies were required to provide evidence to demonstrate their product's safety, efficacy and quality for purposes of registration and reimbursement. Increasingly, a fourth hurdle has been added which requires that companies demonstrate the economic value of a product to be reimbursed. This paper provides empirical observations and anecdotes, as well as economic theory to explain the impact of the fourth hurdle on the pharmaceutical industry. Specifically, the paper discusses the impact on drug development, clinical trials, sales, launches, corporate value, human resources and access to new medicines by patients. Using game theory, the potential for suboptimal investment in pharmacoeconomics by the pharmaceutical industry will be explored through a theoretical application of the prisoner's dilemma model. The paper concludes with a hypothetical example for calculating the monetary impact of the fourth hurdle on society when making formulary decisions related to three drugs within a therapeutic class.

Enhancing literacy development through AAC technologies.

There is a critical need to understand teaching and technology supports that enable students who use augmentative and alternative communication (AAC) systems to engage in meaningful literacy experiences and foster conventional literacy skills. To thrive in classroom environments, they must have access to tools that can support them in active and independent literacy learning. These students need technology that allows them to move seamlessly between reading, writing, and communicating. They require technology that takes into account access needs, individual learning needs, the learning demands of technology, and literacy development across grades. Families and school teams need information that will assist them in providing the best tools and the most appropriate content within these tools throughout the school day. Teachers need information that supports them in providing exemplary literacy instruction to students who use AAC systems. This article explores and summarizes factors impacting literacy learning, including literacy capabilities of school-age students who use AAC, communication in literacy learning and use, reading and writing instruction in general education classrooms, and technology to support literacy learning. It is important that future technology tools provide a platform for levels of literacy learning: The power of technology will be reflected in its ability to provide access to and display the right content at the right time for students who use AAC. This article summarizes current factors thought to influence literacy learning and discusses priorities for future research and technology development.