Organ-Specific and Development Regulation of the Nopaline Synthase Promoter in Transgenic Tobacco Plants

Control regions of the nopaline synthase (nos) gene have been widely used to express foreign genes in plants since the promoter is active in a wide variety of plant tissues. We report here the characteristics of the nos promoter activity in transgenic tobacco (Nicotiana tabacum) plants at various developmental stages. The promoter was highly active in the lower parts of a plant and gradually decreased in the upper parts. This vertical gradient was maintained throughout plant growth until the flowering stage when the overall promoter strength decreased significantly in the vegetative organs. However, in various flower organs, the nos promoter activities increased dramatically. Higher activity was observed in calyx, corolla, and stamens although the maximum promoter activity in each organ was found at different stages of flower development. The promoter activity in pistils was low and gradually increased in the ovaries after anthesis. In developing fruits, the nos promoter activity was strongly induced during the mid-stage of embryogenesis. These results indicate that the expression of the nos promoter is developmentally regulated and organ specific in transgenic tobacco plants

The Dawn of Open Access to Phylogenetic Data

The scientific enterprise depends critically on the preservation of and open access to published data. This basic tenet applies acutely to phylogenies (estimates of evolutionary relationships among species). Increasingly, phylogenies are estimated from increasingly large, genome-scale datasets using increasingly complex statistical methods that require increasing levels of expertise and computational investment. Moreover, the resulting phylogenetic data provide an explicit historical perspective that critically informs research in a vast and growing number of scientific disciplines. One such use is the study of changes in rates of lineage diversification (speciation - extinction) through time. As part of a meta-analysis in this area, we sought to collect phylogenetic data (comprising nucleotide sequence alignment and tree files) from 217 studies published in 46 journals over a 13-year period. We document our attempts to procure those data (from online archives and by direct request to corresponding authors), and report results of analyses (using Bayesian logistic regression) to assess the impact of various factors on the success of our efforts. Overall, complete phylogenetic data for ~60% of these studies are effectively lost to science. Our study indicates that phylogenetic data are more likely to be deposited in online archives and/or shared upon request when: (1) the publishing journal has a strong data-sharing policy; (2) the publishing journal has a higher impact factor, and; (3) the data are requested from faculty rather than students. Although the situation appears dire, our analyses suggest that it is far from hopeless: recent initiatives by the scientific community -- including policy changes by journals and funding agencies -- are improving the state of affairs

RAC1 P29S regulates PD-L1 expression in melanoma.

Whole exome sequencing of cutaneous melanoma has led to the detection of P29 mutations in RAC1 in 5-9% of samples, but the role of RAC1 P29 mutations in melanoma biology remains unclear. Using reverse phase protein array analysis to examine the changes in protein/phospho-protein expression, we identified cyclin B1, PD-L1, Ets-1, and Syk as being selectively upregulated with RAC1 P29S expression and downregulated with RAC1 P29S depletion. Using the melanoma patient samples in TCGA, we found PD-L1 expression to be significantly increased in RAC1 P29S patients compared to RAC1 WT as well as other RAC1 mutants. The finding that PD-L1 is upregulated suggests that oncogenic RAC1 P29S may promote suppression of the antitumor immune response. This is a new insight into the biological function of RAC1 P29S mutations with potential clinical implications as PD-L1 is a candidate biomarker for increased benefit from treatment with anti-PD1 or anti-PD-L1 antibodies

α8β1 integrin regulates nutrient absorption through an Mfge8-PTEN dependent mechanism.

Coordinated gastrointestinal smooth muscle contraction is critical for proper nutrient absorption and is altered in a number of medical disorders. In this work, we demonstrate a critical role for the RGD-binding integrin α8β1 in promoting nutrient absorption through regulation of gastrointestinal motility. Smooth muscle-specific deletion and antibody blockade of α8 in mice result in enhanced gastric antral smooth muscle contraction, more rapid gastric emptying, and more rapid transit of food through the small intestine leading to malabsorption of dietary fats and carbohydrates as well as protection from weight gain in a diet-induced model of obesity. Mechanistically, ligation of α8β1 by the milk protein Mfge8 reduces antral smooth muscle contractile force by preventing RhoA activation through a PTEN-dependent mechanism. Collectively, our results identify a role for α8β1 in regulating gastrointestinal motility and identify α8 as a potential target for disorders characterized by hypo- or hyper-motility

The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.</jats:p

Serendipitous discoveries in microarray analysis

Background Scientists are capable of performing very large scale gene expression experiments with current microarray technologies. In order to find significance in the expression data, it is common to use clustering algorithms to group genes with similar expression patterns. Clusters will often contain related genes, such as co-regulated genes or genes in the same biological pathway. It is too expensive and time consuming to test all of the relationships found in large scale microarray experiments. There are many bioinformatics tools that can be used to infer the significance of microarray experiments and cluster analysis. Materials and methods In this project we review several existing tools and used a combination of them to narrow down the number of significant clusters from a microarray experiment. Microarray data was obtained through the Cerebellar Gene Regulation in Time and Space (Cb GRiTS) database [2]. The data was clustered using paraclique, a graph-based clustering algorithm. Each cluster was evaluated using Gene-Set Cohesion Analysis Tool (GCAT) [3], ONTO-Pathway Analysis [4], and Allen Brain Atlas data [1]. The clusters with the lowest p-values in each of the three analysis methods were researched to determine good candidate clusters for further experimental confirmation of gene relationships. Results and conclusion While looking for genes important to cerebellar development, we serendipitously came across interesting clusters related to neural diseases. For example, we found two clusters that contain genes known to be associated with Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease pathways. Both clusters scored low in all three analyses and have very similar expression patterns but at different expression levels. Such unexpected discoveries help unlock the real power of high throughput data analysis

Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen-the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains. IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen-the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies

Actuation of Micro-Optomechanical Systems Via Cavity-Enhanced Optical Dipole Forces

We demonstrate a new type of optomechanical system employing a movable, micron-scale waveguide evanescently-coupled to a high-Q optical microresonator. Micron-scale displacements of the waveguide are observed for milliwatt(mW)-level optical input powers. Measurement of the spatial variation of the force on the waveguide indicates that it arises from a cavity-enhanced optical dipole force due to the stored optical field of the resonator. This force is used to realize an all-optical tunable filter operating with sub-mW control power. A theoretical model of the system shows the maximum achievable force to be independent of the intrinsic Q of the optical resonator and to scale inversely with the cavity mode volume, suggesting that such forces may become even more effective as devices approach the nanoscale.Comment: 4 pages, 5 figures. High resolution version available at (http://copilot.caltech.edu/publications/CEODF_hires.pdf). For associated movie, see (http://copilot.caltech.edu/research/optical_forces/index.htm

Adaptor Protein 2 (Ap-2) Complex is Essential for Functional Axogenesis in Hippocampal Neurons

The complexity and diversity of a neural network requires regulated elongation and branching of axons, as well as the formation of synapses between neurons. In the present study we explore the role of AP-2, a key endocytic adaptor protein complex, in the development of rat hippocampal neurons. We found that the loss of AP-2 during the early stage of development resulted in impaired axon extension and failed maturation of the axon initial segment (AIS). Normally the AIS performs two tasks in concert, stabilizing neural polarity and generating action potentials. In AP-2 silenced axons polarity is established, however there is a failure to establish action potential firing. Consequently, this impairs activity-driven Ca2+ influx and exocytosis at nerve terminals. In contrast, removal of AP-2 from older neurons does not impair axonal growth or signaling and synaptic function. Our data reveal that AP-2 has important roles in functional axogenesis by proper extension of axon as well as the formation of AIS during the early step of neurodevelopment