A universal primer for isolation of fragments of a gene encoding phytoene desaturase for use in virus-induced gene silencing (VIGS) studies

We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae

Patterns of periodic holes created by increased cell motility

The reaction and diffusion of morphogens is a mechanism widely used to explain many spatial patterns in physics, chemistry and developmental biology. However, because experimental control is limited in most biological systems, it is often unclear what mechanisms account for the biological patterns that arise. Here, we study a biological model of cultured vascular mesenchymal cells (VMCs), which normally self-organize into aggregates that form into labyrinthine configurations. We use an experimental control and a mathematical model that includes reacting and diffusing morphogens and a third variable reflecting local cell density. With direct measurements showing that cell motility was increased ninefold and threefold by inhibiting either Rho kinase or non-muscle myosin-II, respectively, our experimental results and mathematical modelling demonstrate that increased motility alters the multicellular pattern of the VMC cultures, from labyrinthine to a pattern of periodic holes. These results suggest implications for the tissue engineering of functional replacements for trabecular or spongy tissue such as endocardium and bone

MUC2 polymorphisms are associated with endometriosis development and infertility: a case-control study

<p>Abstract</p> <p>Background</p> <p>Mucins are highly glycosylated proteins protecting and lubricating epithelial surface of respiratory, gastrointestinal and reproductive tracts. Members of the mucin protein family have been suggested to play an important role in development of endometriosis and infertility. This study investigates genetic association of mucin2 (<it>MUC2</it>) with the risk of endometriosis and endometriosis-related infertility.</p> <p>Methods</p> <p>This case-control study was conducted at China Medical University Hospital, with 195 endometriosis patients and 196 healthy controls enrolled. Genotyping of six SNPs (rs2856111, rs11245936, rs10794288, rs10902088, rs7103978 and rs11245954) within <it>MUC2 </it>gene were performed by using <it>Taqman </it>genotyping assay; individual SNP and haplotype associations with endometriosis and endometriosis-related infertility were assessed by <it>χ</it>²test.</p> <p>Results</p> <p>Endometriosis patients exhibit significantly lower frequency of the rs10794288 C allele, the rs10902088 T allele and the rs7103978 G allele (<it>P </it>= 0.030, 0.013 and 0.040, respectively). In addition, the rs10794288 C allele and the rs10902088 T allele were also less abundant in patients with infertility versus fertile ones (<it>P </it>= 0.015 and 0.024, respectively). Haplotype analysis of the endometriosis associated SNPs in <it>MUC2 </it>also showed significantly association between the most common haplotypes and endometriosis or endometriosis-related infertility.</p> <p>Conclusions</p> <p><it>MUC2 </it>polymorphisms, especially rs10794288 and rs10902088, are associated with endometriosis as well as endometriosis-related infertility. Our data present MUC2 as a new candidate involved in development of endometriosis and related infertility in Taiwanese Han women.</p

Hairpin ribozyme-antisense RNA constructs can act as molecular lassos

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5′- and 3′-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-α (TNF-α) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns

Running away experience and psychoactive substance use among adolescents in Taiwan: multi-city street outreach survey

<p>Abstract</p> <p>Background</p> <p>This study aimed to examine: 1) the relationship between being a runaway and the time since the first absconding event and adolescent substance use; 2) whether different kinds of psychoactive substances have a different temporal relationship to the first absconding event; and 3) whether the various reasons for the first absconding event are associated with different risks of substance use.</p> <p>Methods</p> <p>Participants were drawn from the 2004-2006 nationwide outreach programs across 26 cities/towns in Taiwan. A total of 17,133 participants, age 12-18 years, who completed an anonymous questionnaire on their experience of running away and substances use and who were now living with their families, were included in the analysis.</p> <p>Results</p> <p>The lifetime risk of tobacco, alcohol, betel nut, and illegal drug/inhalant use increased steadily from adolescents who had experienced a trial runaway episode (one time lasting ≤ 1 day), to those with extended runaway experience (≥ 2 times or lasting > 1 day), when compared to those who had never ran away. Adolescents who had their first running away experience > 6 months previously had a greater risk of betel nut or illegal drug/inhalant use over the past 6-months than those with a similar experience within the last 6 months. Both alcohol and tobacco use were most frequently initiated before the first running away, whereas both betel nut and illegal drug/inhalant use were most frequently initiated after this event. When adolescents who were fleeing an unsatisfactory home life were compared to those who ran away for excitement, the risk of alcohol use was similar but the former tended to have a higher risk of tobacco, betel nut, and illegal drug/inhalant use.</p> <p>Conclusions</p> <p>More significant running away and a longer time since the first absconding experience were associated with more advanced substance involvement among adolescents now living in a family setting. Once adolescents had left home, they developed additional psychoactive substance problems, regardless of their reasons for running away. These findings have implications for caregivers, teachers, and healthcare workers when trying to prevent and/or intervening in adolescent substance use.</p

TNFα-Induced Apoptosis Enabled by CCN1/CYR61: Pathways of Reactive Oxygen Species Generation and Cytochrome c Release

Although TNFα is a strong inducer of apoptosis, its cytotoxicity in most normal cells in vitro requires blockade of NFκB signaling or inhibition of de novo protein synthesis, typically by the addition of cycloheximide. However, several members of CCN (CYR61/CTGF/NOV) family of extracellular matrix proteins enable TNFα-dependent apoptosis in vitro without inhibiting NFκB or de novo protein synthesis, and CCN1 (CYR61) is essential for optimal TNFα cytotoxicity in vivo. Previous studies showed that CCN1 unmasks the cytotoxicity of TNFα by binding integrins αvβ5, α6β1, and the cell surface heparan sulfate proteoglycan syndecan 4 to induce the accumulation of a high level of reactive oxygen species (ROS), leading to a biphasic activation of JNK necessary for apoptosis. Here we show for the first time that CCN1 interacts with the low density lipoprotein receptor-related protein 1 (LRP1) in a protein complex, and that binding to LRP1 is critical for CCN1-induced ROS generation and apoptotic synergism with TNFα. We also found that neutral sphingomyelinase 1 (nSMase1), which contributes to CCN1-induced ROS generation, is required for CCN1/TNFα-induced apoptosis. Furthermore, CCN1 promotes the activation of p53 and p38 MAPK, which mediate enhanced cytochrome c release to amplify the cytotoxicity of TNFα. By contrast, LRP1, nSMase1, p53, and p38 MAPK are not required when TNFα-dependent apoptosis is facilitated by the presence of cycloheximide, indicating that they function in the CCN1 signaling pathway that converges with TNFα-induced signaling events. Since CCN1/CYR61 is a physiological regulator of TNFα cytotoxicity at least in some contexts, these findings may reveal important mediators of TNFα-induced apoptosis in vivo and identify potential therapeutic targets for thwarting TNFα-dependent tissue damage

Mesoscale phenomena and their contribution to the global response: a focus on the magnetotail transition region and magnetosphere-ionosphere coupling

An important question that is being increasingly studied across subdisciplines of Heliophysics is “how do mesoscale phenomena contribute to the global response of the system?” This review paper focuses on this question within two specific but interlinked regions in Near-Earth space: the magnetotail’s transition region to the inner magnetosphere and the ionosphere. There is a concerted effort within the Geospace Environment Modeling (GEM) community to understand the degree to which mesoscale transport in the magnetotail contributes to the global dynamics of magnetic flux transport and dipolarization, particle transport and injections contributing to the storm-time ring current development, and the substorm current wedge. Because the magnetosphere-ionosphere is a tightly coupled system, it is also important to understand how mesoscale transport in the magnetotail impacts auroral precipitation and the global ionospheric system response. Groups within the Coupling, Energetics and Dynamics of Atmospheric Regions Program (CEDAR) community have also been studying how the ionosphere-thermosphere responds to these mesoscale drivers. These specific open questions are part of a larger need to better characterize and quantify mesoscale “messengers” or “conduits” of information—magnetic flux, particle flux, current, and energy—which are key to understanding the global system. After reviewing recent progress and open questions, we suggest datasets that, if developed in the future, will help answer these questions

A Genome-Wide Linkage Scan for Distinct Subsets of Schizophrenia Characterized by Age at Onset and Neurocognitive Deficits

As schizophrenia is genetically and phenotypically heterogeneous, targeting genetically informative phenotypes may help identify greater linkage signals. The aim of the study is to evaluate the genetic linkage evidence for schizophrenia in subsets of families with earlier age at onset or greater neurocognitive deficits.Patients with schizophrenia (n  =  1,207) and their first-degree relatives (n  =  1,035) from 557 families with schizophrenia were recruited from six data collection field research centers throughout Taiwan. Subjects completed a face-to-face semi-structured interview, the Continuous Performance Test (CPT), the Wisconsin Card Sorting Test, and were genotyped with 386 microsatellite markers across the genome.A maximum nonparametric logarithm of odds (LOD) score of 4.17 at 2q22.1 was found in 295 families ranked by increasing age at onset, which had significant increases in the maximum LOD score compared with those obtained in initial linkage analyses using all available families. Based on this subset, a further subsetting by false alarm rate on the undegraded and degraded CPT obtained further increase in the nested subset-based LOD on 2q22.1, with a score of 7.36 in 228 families and 7.71 in 243 families, respectively.We found possible evidence of linkage on chromosome 2q22.1 in families of schizophrenia patients with more CPT false alarm rates nested within the families with younger age at onset. These results highlight the importance of incorporating genetically informative phenotypes in unraveling the complex genetics of schizophrenia