In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Background: Adenine nucleotide/phosphate carriers (APCs) from mammals and yeast are commonly known to adapt the mitochondrial adenine nucleotide pool in accordance to cellular demands. They catalyze adenine nucleotide - particularly ATP-Mg - and phosphate exchange and their activity is regulated by calcium. Our current knowledge about corresponding proteins from plants is comparably limited. Recently, the three putative APCs from Arabidopsis thaliana were shown to restore the specific growth phenotype of APC yeast loss-of-function mutants and to interact with calcium via their N-terminal EF-hand motifs in vitro. In this study, we performed biochemical characterization of all three APC isoforms from A. thaliana to gain further insights into their functional properties. Results: Recombinant plant APCs were functionally reconstituted into liposomes and their biochemical characteristics were determined by transport measurements using radiolabeled substrates. All three plant APCs were capable of ATP, ADP and phosphate exchange, however, high preference for ATP-Mg, as shown for orthologous carriers, was not detectable. By contrast, the obtained data suggest that in the liposomal system the plant APCs rather favor ATP-Ca as substrate. Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Ca2+-binding to the EF-hand motifs in the N-terminal domain of the carrier. Conclusion: Biochemical characteristics suggest that plant APCs can mediate net transport of adenine nucleotides and hence, like their pendants from animals and yeast, might be involved in the alteration of the mitochondrial adenine nucleotide pool. Although, ATP-Ca was identified as an apparent import substrate of plant APCs in vitro it is arguable whether ATP-Ca formation and thus the corresponding transport can take place in vivo

Looking ahead: forecasting and planning for the longer-range future, April 1, 2, and 3, 2005

This repository item contains a single issue of the Pardee Conference Series, a publication series that began publishing in 2006 by the Boston University Frederick S. Pardee Center for the Study of the Longer-Range Future. This was the Center's spring Conference that took place during April 1, 2, and 3, 2005.The conference allowed for many highly esteemed scholars and professionals from a broad range of fields to come together to discuss strategies designed for the 21st century and beyond. The speakers and discussants covered a broad range of subjects including: long-term policy analysis, forecasting for business and investment, the National Intelligence Council Global Trends 2020 report, Europe’s transition from the Marshal plan to the EU, forecasting global transitions, foreign policy planning, and forecasting for defense

Immersive multi-user decision training games with ARLearn

Serious gaming approaches so far focus mainly on skill development, motivational aspects or providing immersive learning situations. Little work has been reported to foster awareness and decision competencies in complex deci-sion situations involving incomplete information and multiple stakeholders. We address this issue exploring the technical requirements and possibilities to de-sign games for such situations in three case studies: a hostage taking situation, a multi-stakeholder logistics case, and a health-care related emergency case. To implement the games, we use a multi-user enabled mobile game development platform (ARLearn). We describe the underlying real world situations and edu-cational challenges and analyse how these are reflected in the ARLearn games realized. Based on these cases we propose a way to increase the immersiveness of mobile learning games.SALOM

Tapping the nucleotide pool of the host: novel nucleotide carrier proteins of Protochlamydia amoebophila

Protochlamydia amoebophila UWE25 is related to the Chlamydiaceae comprising major pathogens of humans, but thrives as obligate intracellular symbiont in the protozoan host Acanthamoeba sp. The genome of P. amoebophila encodes five paralogous carrier proteins belonging to the nucleotide transporter (NTT) family. Here we report on three P. amoebophila NTT isoforms, PamNTT2, PamNTT3 and PamNTT5, which possess several conserved amino acid residues known to be critical for nucleotide transport. We demonstrated that these carrier proteins are able to transport nucleotides, although substrate specificities and mode of transport differ in an unexpected manner and are unique among known NTTs. PamNTT2 is a counter exchange transporter exhibiting submillimolar apparent affinities for all four RNA nucleotides, PamNTT3 catalyses an unidirectional proton-coupled transport confined to UTP, whereas PamNTT5 mediates a proton-energized GTP and ATP import. All NTT genes of P. amoebophila are transcribed during intracellular multiplication in acanthamoebae. The biochemical characterization of all five NTT proteins from P. amoebophila in this and previous studies uncovered that these metabolically impaired bacteria are intimately connected with their host cell’s metabolism in a surprisingly complex manner

Mechanisms behind temsirolimus resistance causing reactivated growth and invasive behavior of bladder cancer cells in vitro

Background: Although mechanistic target of rapamycin (mTOR) inhibitors, such as temsirolimus, show promise in treating bladder cancer, acquired resistance often hampers efficacy. This study evaluates mechanisms leading to resistance. Methods: Cell growth, proliferation, cell cycle phases, and cell cycle regulating proteins were compared in temsirolimus resistant (res) and sensitive (parental—par) RT112 and UMUC3 bladder cancer cells. To evaluate invasive behavior, adhesion to vascular endothelium or to immobilized extracellular matrix proteins and chemotactic activity were examined. Integrin α and β subtypes were analyzed and blocking was done to evaluate physiologic integrin relevance. Results: Growth of RT112res could no longer be restrained by temsirolimus and was even enhanced in UMUC3res, accompanied by accumulation in the S- and G2/M-phase. Proteins of the cdk-cyclin and Akt-mTOR axis increased, whereas p19, p27, p53, and p73 decreased in resistant cells treated with low-dosed temsirolimus. Chemotactic activity of RT112res/UMUC3res was elevated following temsirolimus re-exposure, along with significant integrin α2, α3, and β1 alterations. Blocking revealed a functional switch of the integrins, driving the resistant cells from being adhesive to being highly motile. Conclusion: Temsirolimus resistance is associated with reactivation of bladder cancer growth and invasive behavior. The α2, α3, and β1 integrins could be attractive treatment targets to hinder temsirolimus resistance

High-Level Expression of Wild-Type p53 in Melanoma Cells is Frequently Associated with Inactivity in p53 Reporter Gene Assays

Background: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date. Methodology/Principal Findings: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5–8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53. Conclusions/Significance: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level

Assessment of a novel smartglass-based point-of-care fusion approach for mixed reality-assisted targeted prostate biopsy: A pilot proof-of-concept study

PurposeWhile several biopsy techniques and platforms for magnetic resonance imaging (MRI)-guided targeted biopsy of the prostate have been established, none of them has proven definite superiority. Augmented and virtual reality (mixed reality) smartglasses have emerged as an innovative technology to support image-guidance and optimize accuracy during medical interventions. We aimed to investigate the benefits of smartglasses for MRI-guided mixed reality-assisted cognitive targeted biopsy of the prostate.MethodsFor prospectively collected patients with suspect prostate PIRADS lesions, multiparametric MRI was uploaded to a smartglass (Microsoft® Hololens I), and smartglass-assisted targeted biopsy (SMART TB) of the prostate was executed by generation of a cognitive fusion technology at the point-of-care. Detection rates of prostate cancer (PCA) were compared between SMART TB and 12-core systematic biopsy. Assessment of SMART-TB was executed by the two performing surgeons based on 10 domains on a 10-point scale ranging from bad (1) to excellent (10).ResultsSMART TB and systematic biopsy of the prostate were performed for 10 patients with a total of 17 suspect PIRADS lesions (PIRADS 3, n = 6; PIRADS 4, n = 6; PIRADS 5, n = 5). PCA detection rate per core was significant (p < 0.05) higher for SMART TB (47%) than for systematic biopsy (19%). Likelihood for PCA according to each core of a PIRADS lesion (17%, PIRADS 3; 58%, PIRADS 4; 67%, PIRADS 5) demonstrated convenient accuracy. Feasibility scores for SMART TB were high for practicality (10), multitasking (10), execution speed (9), comfort (8), improvement of surgery (8) and image quality (8), medium for physical stress (6) and device handling (6) and low for device weight (5) and battery autonomy (4).ConclusionSMART TB has the potential to increase accuracy for PCA detection and might enhance cognitive MRI-guided targeted prostate biopsy in the future