North Carolina State University

About: North Carolina State University is a education organization based out in Raleigh, North Carolina, United States. It is known for research contribution in the topics: Population & Thin film. The organization has 44161 authors who have published 101744 publications receiving 3456774 citations. The organization is also known as: NCSU & North Carolina State University at Raleigh.

Higher‐order dimensions of the big five personality traits and the big six vocational interest types

Abstract: The purpose of this study was to identify higher-order dimensions that explain the relationships among the Big 6 interest types and the Big 5 personality traits. Meta-analyses were conducted to identify an 11 × 11 true score correlation matrix of interest and personality attributes. Cluster analysis and nonmetric multidimensional scaling were used to identify 3 dimensions that explained relations among the 11 attributes: (a) Interests versus Personality Traits; (b) Striving for Accomplishment Versus Striving for Personal Growth, and (c) Interacting with People Versus Interacting with Things. Overall, results clarified the relationships among interests and personality traits by showing that 3 rather than 2 dimensions best explain the relationships among interests and personality traits. Personality traits and vocational interests are two major, noncognitive individual difference domains in the field of psychology. Both sets of dispositional attributes are important because they influence numerous outcomes associated with work and life success. One common thread that links personality traits and vocational interests is that they influence behavior through motivational processes. That is, they influence choices individuals make about which tasks and activities to engage in, how much effort to exert on those tasks, and how long to persist with those tasks. Although psychologists have conducted hundreds of studies that investigate one or both topics, the precise nature of the linkages between the two domains remains ambiguous and controversial.

Small-Molecule Inhibitors of MicroRNA miR-21 Function

Abstract: MicroRNAs (miRNAs) have recently emerged as an important class of gene regulators, and their misregulation has been linked to a variety of cancers. Small molecule inhibitors of miRNAs would be important tools to elucidate the detailed mechanisms of miRNA function and provide lead structures for the development of new therapeutics. We are reporting a cellular screen for miRNA pathway inhibitors and the first small molecule modifiers of miRNA function. miRNAs are single-stranded noncoding RNAs of 21-23 nucleotides. They are a novel class of gene regulators that function by binding to the 3’ untranslated region of target messenger RNAs leading to either suppression of their translation or acceleration of their degradation.[1] The majority of miRNAs are initially transcribed as primary miRNAs (primiRNAs),[2] which are further processed in the nucleus by the enzyme Drosha, thus transforming pri-miRNAs into shorter stem-loop-structured, double-stranded RNAs called precursor miRNAs (pre-miRNAs).[3] Pre-miRNAs are then transported from the nucleus to the cytoplasm and are processed by Dicer into mature miRNAs.[4] Mature miRNAs enter the effector complex, called the RNA-induced silencing complex (RISC), to then target single-stranded complementary mRNAs (Supporting Figure 1).[5] It is estimated that miRNAs are involved in the regulation of about 30% of all genes and almost every genetic pathway.[6] Moreover, recent evidence suggests that they can function as oncogenes and tumor suppressors.[7, 8] Thus, small molecule regulation of misregulated miRNAs has the potential to provide a new area of therapeutics. So far, specific miRNA inhibition has been only achieved by antisense nucleic acids.[9] We developed an assay for small molecule inhibitors of miRNA function and discovered potentially specific miRNA pathway inhibitors. Although inhibitors of the siRNA pathway have been identified,[10] to our knowledge no small molecule inhibitors of the miRNA pathway have been reported. We selected miR-21 as a target miRNA due to its documented function as an anti-apoptotic factor in cancer cells and its elevated levels in various cancers such as breast, ovarian, and lung cancer as well as glioblastomas.[7, 11] Lentiviral reporter constructs for miRNA activity were assembled by introducing the complementary sequences of mature miR-21, the specificity control miR-30, or a negative control linker sequence (a site with no detectable recognition by natural miRNAs) downstream of a luciferase reporter gene (Supporting Figure 2). These plasmids serve as sensors to detect the presence of specific mature miRNAs (Scheme 1). Scheme 1 Luciferase expression under control of a miRNA binding sequence in the 3’ untranslated region (3’ UTR) provides an efficient miRNA assay. Endogenous miR-21 (HeLa cells) or exogenous miR-30 downregulate luciferase activity when paired with ... The reporter constructs were stably introduced into HeLa cells which express high levels of miR-21, but relatively low levels of miR-30.[12] In order to test the miRNA specificity of the reporter system, cells that contained both the Luc-miR-30A reporter construct and a construct expressing exogenous primary miR-30 were assayed. These cells displayed a greatly diminished luciferase signal compared to cells with a mismatched Luc-miR-30A reporter/miR-21 combination (Supporting Figure 3), demonstrating that the Luc-miR-21 and Luc-miR-30A reporters are specific and react only to miR-21 and miR-30, respectively. The ability to detect endogenous miR-21 was proven by the fact that the Luc-miR-21 reporter, when introduced into HeLa cells, led to a 90% decreased luciferase signal in comparison to the control luciferase-linker construct, visualizing the high level of endogenous miR-21 expression in HeLa cells (Supporting Figure 4). As expected, the miR-30A reporter displayed only a modest decrease since HeLa cells express relatively low levels of endogenous miR-30. Subsequently, a primary screen of >1000 compounds from our own compound collection and the Library of Pharmacologically Active Compounds (Sigma-Aldrich) was conducted at a 10 μM compound concentration and an initial hit compound 1 was discovered. This diazobenzene led to an increase of the luciferase signal by 251% compared to untreated cells (the DMSO control had no effect on the luciferase signal; Supporting Figure 5). Through several rounds of screening and structural modification a preliminary structure-activity relationship was developed (Supporting Figure 6). Acylation and alkylation of the amino group in 1 led to diminished activities. However, the screening of a wide range of molecules structurally related to the azobenzene core delivered the highly active compound 2 (5-fold increase of the luciferase signal at 10 μM, Figure 1 and ​and2A).2A). Other molecules derived from 2 through introduction of an amino or nitro group in the 4’ position led to 12% or 64% reduced activity, respectively. Other amide substituents displayed a substantial loss of activity (24-53%), with the exception of allyl and propyl groups, which showed 11% and 16% lower activity, respectively. An exchange of the amide for a sulfonamide delivered compounds with no activity and, interestingly, the styrene analog of 2 had a 40% lower activity. Thus far, 2 is the most effective small molecule inhibitor of microRNA miR-21 function inducing a 485% increase in the luciferase reporter signal at 10 μM. The diazobenzene 2 does not display cytotoxic effects at this concentration as determined by an MTT assay (data not shown). Dose response studies from 0-10 μM revealed a concentration dependence of the luciferase signal with an EC50 of 2 μM (Figure 1). Figure 1 Dose-response curve of 2, revealing an EC50 of 2 μM and a luciferase signal increase of ~500% at 10 μM. The error bars represent the standard deviation from three independent measurements. Figure 2 A) Change in luciferase signal of cells treated with 2 (10 μM) relative to a DMSO control. B) Mature or primary miRNA levels in cells treated with 2 (10 μM) relative to a DMSO control, as determined by quantitative RT-PCR. The error bars ... Several experiments were conducted in order to investigate the mode of action of the inhibitor 2. The compound does not affect the luciferase signal in HeLa cells expressing the Luc-linker control sequence (Figure 2A), thus indicating that it does not increase the luciferase signal through means other than inhibiting the miRNA pathway. Furthermore, HeLa cells expressing both the miR-30 luciferase reporter construct and miR-30 were treated with 2. In this case, no increase of the luciferase signal was detected (Figure 2A), demonstrating that 2 is potentially specific towards miR-21 and does not have a general effect on the common miRNA pathway. The specificity of 2 for the inhibition of miR-21 function was further validated by measuring intracellular miRNA levels via quantitative RT-PCR (Figure 2B). We found that levels of the stably expressed, exogenous mature miR-30, endogenous mature miR-93 and endogenous non-miRNA genes such as E-chaderin, ID1, RAP1A, Fibronectin are not reduced by treatment with 2 (Figure 2B, Supporting Figure 7). Gratifyingly, miR-21 expression is reduced by 78% compared to the DMSO control in HeLa cells. Quantitative RT-PCR experiments with primers specific for the primary miR-21 (pri-miR-21) sequence but not mature or precursor miR-21 revealed that the pri-miR-21 levels in cells treated with 2 were reduced by 87% (Figure 2B). Similar observations were also made in three other cell lines, MCF-7, A172, and MDA-MB-231, which endogenously express miR-21 (Supporting Figure 7 & 8). These results strongly suggest that compound 2 is an inhibitor that targets the transcription of the miR-21 gene into pri-miR-21, but not downstream processes of the common miRNA pathway. In summary, we developed a method to identify inhibitors of the miRNA pathway in live cells, specifically of miR-21 an important anti-apoptotic factor in several cancers. A screening of >1000 small organic molecules followed by a structure activity relationship analysis of an initial hit delivered the azobenzene 2 as a specific and efficient inhibitor of miR-21 expression. Research on miRNAs is still in its infancy and the biogenesis of many miRNAs (including miR-21) is not fully understood, thus specific inhibitors of the miRNA pathway (like 2) will be unique tools for the investigation of miRNAs and their involvement in various types of diseases.

Identification and Functional Analysis of in Vivo Phosphorylation Sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 Receptor Kinase

Abstract: Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography–tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.

Agile software development: it's about feedback and change

Abstract: Currently, the focus is on determining how to blend agile methodologies with plan-driven approaches to software development.