Coulomb crystal mass spectrometry in a digital ion trap

We present a mass spectrometric technique for identifying the masses and relative abundances of Coulomb-crystallized ions held in a linear Paul trap. A digital radio-frequency wave form is employed to generate the trapping potential, as this can be cleanly switched off, and static dipolar fields are subsequently applied to the trap electrodes for ion ejection. Close to 100% detection efficiency is demonstrated for Ca+ and CaF+ ions from bicomponent Ca+ − CaF+ Coulomb crystals prepared by the reaction of Ca+ with CH3F. A quantitative linear relationship is observed between ion number and the corresponding integrated time-of-flight (TOF) peak, independent of the ionic species. The technique is applicable to a diverse range of multicomponent Coulomb crystals—demonstrated here for Ca+ − NH3+ − NH4+ and Ca+ − CaOH+ − CaOD+ crystals—and will facilitate the measurement of ion-molecule reaction rates and branching ratios in complicated reaction systems

Peripheral halo-functionalization in [Cu(N^N)(P^P)]+ emitters: influence on the performances of light-emitting electrochemical cells

A series of heteroleptic [Cu(N^N)(P^P)][PF6] complexes is described in which P^P = bis(2-(diphenylphosphino)phenyl)ether (POP) or 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (xantphos) and N^N = 4,4′-diphenyl-6,6′-dimethyl-2,2′-bipyridine substituted in the 4-position of the phenyl groups with atom X (N^N = 1 has X = F, 2 has X = Cl, 3 has X = Br, 4 has X = I; the benchmark N^N ligand with X = H is 5). These complexes have been characterized by multinuclear NMR spectroscopy, mass spectrometry, elemental analyses and cyclic voltammetry; representative single crystal structures are also reported. The solution absorption spectra are characterized by high energy bands (arising from ligand-centred transitions) which are red-shifted on going from X = H to X = I, and a broad metal-to-ligand charge transfer band with λmax in the range 387–395 nm. The ten complexes are yellow emitters in solution and yellow or yellow-orange emitters in the solid-state. For a given N^N ligand, the solution photoluminescence (PL) spectra show no significant change on going from [Cu(N^N)(POP)]+ to [Cu(N^N)(xantphos)]+; introducing the iodo-functionality into the N^N domain leads to a red-shift in λmaxem compared to the complexes with the benchmark N^N ligand 5. In the solid state, [Cu(1)(POP)][PF6] and [Cu(1)(xantphos)][PF6] (fluoro-substituent) exhibit the highest PL quantum yields (74 and 25%, respectively) with values of τ1/2 = 11.1 and 5.8 μs, respectively. Light-emitting electrochemical cells (LECs) with [Cu(N^N)(P^P)][PF6] complexes in the emissive layer have been tested. Using a block-wave pulsed current driving mode, the best performing device employed [Cu(1)(xantphos)]+ and this showed a maximum luminance (Lummax) of 129 cd m−2 and a device lifetime (t1/2) of 54 h; however, the turn-on time (time to reach Lummax) was 4.1 h. Trends in performance data reveal that the introduction of fluoro-groups is beneficial, but that the incorporation of heavier halo-substituents leads to poor devices, probably due to a detrimental effect on charge transport; LECs with the iodo-functionalized N^N ligand 4 failed to show any electroluminescence after 50 h

Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

Background The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system. Results The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci. Conclusion This archived set of Gateway® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome

Wheat sprout extract-induced apoptosis in human cancer cells by proteasomes modulation.

Natural occurring modulators of proteasome functionality are extensively investigated for their implication in cancer therapy. On the basis of our previous evidences both on proteasomal inhibition by monomeric polyphenols, and on the characterization of wheat sprout hydroalcoholic extract, herein we thoroughly report on a comparative study of the effect of wheat sprout extract on both normal and tumour cells. Treatment of isolated 20S proteasomes with wheat sprout extracts induced a gradual inhibition of all proteasome activities. Next, two wheat sprout extract components were separated: a polyphenol and a protein fraction. Both components exerted an in vitro inhibitory effect on proteasome activity. HeLa tumour cells and FHs 74 Int normal cells were exposed to both fractions, resulting in different rates of proteasome inhibition, with tumour cells showing a significantly higher degree of proteasome impairment and apoptosis induction. Furthermore, a decrease in proteasome activities and in cell survival of the human plasmacytoma RPMI 8226 cell line, upon the same treatments, was observed. Collectively, our results provide additional evidences supporting the possible use of natural extracts as coadjuvants in cancer treatments

CoAIMs: A Cost-Effective Panel of Ancestry Informative Markers for Determining Continental Origins

Genetic ancestry is known to impact outcomes of genotype-phenotype studies that are designed to identify risk for common diseases in human populations. Failure to control for population stratification due to genetic ancestry can significantly confound results of disease association studies. Moreover, ancestry is a critical factor in assessing lifetime risk of disease, and can play an important role in optimizing treatment. As modern medicine moves towards using personal genetic information for clinical applications, it is important to determine genetic ancestry in an accurate, cost-effective and efficient manner. Self-identified race is a common method used to track and control for population stratification; however, social constructs of race are not necessarily informative for genetic applications. The use of ancestry informative markers (AIMs) is a more accurate method for determining genetic ancestry for the purposes of population stratification.Here we introduce a novel panel of 36 microsatellite (MSAT) AIMs that determines continental admixture proportions. This panel, which we have named Continental Ancestry Informative Markers or CoAIMs, consists of MSAT AIMs that were chosen based upon their measure of genetic variance (F(st)), allele frequencies and their suitability for efficient genotyping. Genotype analysis using CoAIMs along with a Bayesian clustering method (STRUCTURE) is able to discern continental origins including Europe/Middle East (Caucasians), East Asia, Africa, Native America, and Oceania. In addition to determining continental ancestry for individuals without significant admixture, we applied CoAIMs to ascertain admixture proportions of individuals of self declared race.CoAIMs can be used to efficiently and effectively determine continental admixture proportions in a sample set. The CoAIMs panel is a valuable resource for genetic researchers performing case-control genetic association studies, as it can control for the confounding effects of population stratification. The MSAT-based approach used here has potential for broad applicability as a cost effective tool toward determining admixture proportions

Clinical prediction models for treatment outcomes in newly diagnosed epilepsy: A systematic review.

Up to 35% of individuals diagnosed with epilepsy continue to have seizures despite treatment, commonly referred to as drug-resistant epilepsy. Uncontrolled seizures can directly, or indirectly, negatively impact an individual's quality of life. To inform clinical management and life decisions, it is important to be able to predict the likelihood of seizure control. Those likely to achieve seizure control will be able to return sooner to their usual work and leisure activities and require less follow-up, whereas those with a poor prognosis will need more frequent clinical attendance and earlier consideration of epilepsy surgery. This is a systematic review aimed at identifying demographic, clinical, physiological (e.g., electroencephalographic), and imaging (e.g., magnetic resonance imaging) factors that may be predictive of treatment outcomes in patients with newly diagnosed epilepsy (NDE). MEDLINE and Embase were searched for prediction models of treatment outcomes in patients with NDE. Study characteristics were extracted and subjected to assessment of risk of bias (and applicability concerns) using the PROBAST (Prediction Model Risk of Bias Assessment Tool) tool. Baseline variables associated with treatment outcomes are reported as prognostic factors. After screening, 48 models were identified in 32 studies, which generally scored low for concerns of applicability, but universally scored high for susceptibility to bias. Outcomes reported fit broadly into four categories: drug resistance, short-term treatment response, seizure remission, and mortality. Prognostic factors were also heterogenous, but the predictors that were commonly significantly associated with outcomes were those related to seizure characteristics/types, epilepsy history, and age at onset. Antiseizure medication response was often included as a baseline variable, potentially obscuring other factor relationships at baseline. Currently, outcome prediction models for NDE demonstrate a high risk of bias. Model development could be improved with a stronger adherence to recommended TRIPOD (Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis) practices. Furthermore, we outline actionable changes to common practices that are intended to improve the overall quality of prediction model development in NDE

Re-structuring of marine communities exposed to environmental change

Species richness is the most commonly used but controversial biodiversity metric in studies on aspects of community stability such as structural composition or productivity. The apparent ambiguity of theoretical and experimental findings may in part be due to experimental shortcomings and/or heterogeneity of scales and methods in earlier studies. This has led to an urgent call for improved and more realistic experiments. In a series of experiments replicated at a global scale we translocated several hundred marine hard bottom communities to new environments simulating a rapid but moderate environmental change. Subsequently, we measured their rate of compositional change (re-structuring) which in the great majority of cases represented a compositional convergence towards local communities. Re-structuring is driven by mortality of community components (original species) and establishment of new species in the changed environmental context. The rate of this re-structuring was then related to various system properties. We show that availability of free substratum relates negatively while taxon richness relates positively to structural persistence (i.e., no or slow re-structuring). Thus, when faced with environmental change, taxon-rich communities retain their original composition longer than taxon-poor communities. The effect of taxon richness, however, interacts with another aspect of diversity, functional richness. Indeed, taxon richness relates positively to persistence in functionally depauperate communities, but not in functionally diverse communities. The interaction between taxonomic and functional diversity with regard to the behaviour of communities exposed to environmental stress may help understand some of the seemingly contrasting findings of past research

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication

Preclinical Testing of Erlotinib in a Transgenic Alveolar Rhabdomyosarcoma Mouse Model

Rhabdomyosarcoma is an aggressive childhood malignancy, accounting for more than 50% of all soft-tissue sarcomas in children. Even with extensive therapy, the survival rate among alveolar rhabdomyosarcoma patients with advanced disease is only 20%. The receptor tyrosine kinase Epidermal Growth Factor Receptor (EGFR) has been found to be expressed and activated in human rhabdomyosarcomas. In this study we have used a genetically engineered mouse model for alveolar rhabdomyosarcoma (ARMS) which faithfully recapitulates the human disease by activating the pathognomic Pax3:Fkhr fusion gene and inactivating p53 in the maturing myoblasts. We have demonstrated that tumors from our mouse model of alveolar rhabdomyosarcoma express EGFR at both the mRNA and protein levels. We then tested the EGFR inhibitor, Erlotinib, for its efficacy in this mouse model of alveolar rhabdomyosarcoma. Surprisingly, Erlotinib had no effect on tumor progression, yet mice treated with Erlotinib showed 10–20% loss of body weight. These results suggest that EGFR might not be an a priori monotherapy target in alveolar rhabdomyosarcoma