Evaluation of a Commercial Enzyme Linked Immunosorbent Assay (ELISA) for the Determination of the Neurotoxin BMAA in Surface Waters

The neurotoxin ß-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA

Association between the c.*229C>T polymorphism of the topoisomerase IIb binding protein 1 (TopBP1) gene and breast cancer

Topoisomerase IIb binding protein 1 (TopBP1) is involved in cell survival, DNA replication, DNA damage repair and cell cycle checkpoint control. The biological function of TopBP1 and its close relation with BRCA1 prompted us to investigate whether alterations in the TopBP1 gene can influence the risk of breast cancer. The aim of this study was to examine the association between five polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 30UTR region of the TopBP1 gene and breast cancer risk as well as allele-specific gene expression. Five hundred thirty-four breast cancer patients and 556 population controls were genotyped for these SNPs. Allele-specific Top- BP1 mRNA and protein expressions were determined by using real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with breast cancer. Compared to homozygous common allele carriers, heterozygous and homozygous for the T variant had significantly increased risk of breast cancer (adjusted odds ratio = 3.81, 95 % confidence interval: 1.63–8.34, p = 0.001). Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT or TT genotype. There was a significant association between the rs115160714 and tumor grade and stage. Most carriers of minor allele had a high grade (G3) tumors classified as T2-T4N1M0. Our study raises a possibility that a genetic variation of TopBP1 may be implicated in the etiology of breast cancer

Identifying component modules

A computer-based system for modelling component dependencies and identifying component modules is presented. A variation of the Dependency Structure Matrix (DSM) representation was used to model component dependencies. The system utilises a two-stage approach towards facilitating the identification of a hierarchical modular structure. The first stage calculates a value for a clustering criterion that may be used to group component dependencies together. A Genetic Algorithm is described to optimise the order of the components within the DSM with the focus of minimising the value of the clustering criterion to identify the most significant component groupings (modules) within the product structure. The second stage utilises a 'Module Strength Indicator' (MSI) function to determine a value representative of the degree of modularity of the component groupings. The application of this function to the DSM produces a 'Module Structure Matrix' (MSM) depicting the relative modularity of available component groupings within it. The approach enabled the identification of hierarchical modularity in the product structure without the requirement for any additional domain specific knowledge within the system. The system supports design by providing mechanisms to explicitly represent and utilise component and dependency knowledge to facilitate the nontrivial task of determining near-optimal component modules and representing product modularity

Inducible high-efficiency CRISPR-Cas9-targeted gene editing and precision base editing in African trypanosomes

The Cas9 endonuclease can be programmed by guide RNA to introduce sequence-specific breaks in genomic DNA. Thus, Cas9-based approaches present a range of novel options for genome manipulation and precision editing. African trypanosomes are parasites that cause lethal human and animal diseases. They also serve as models for studies on eukaryotic biology, including 'divergent' biology. Genome modification, exploiting the native homologous recombination machinery, has been important for studies on trypanosomes but often requires multiple rounds of transfection using selectable markers that integrate at low efficiency. We report a system for delivering tetracycline inducible Cas9 and guide RNA to Trypanosoma brucei. In these cells, targeted DNA cleavage and gene disruption can be achieved at close to 100% efficiency without further selection. Disruption of aquaglyceroporin (AQP2) or amino acid transporter genes confers resistance to the clinical drugs pentamidine or eflornithine, respectively, providing simple and robust assays for editing efficiency. We also use the new system for homology-directed, precision base editing; a single-stranded oligodeoxyribonucleotide repair template was delivered to introduce a single AQP2 - T 791G/L 264R mutation in this case. The technology we describe now enables a range of novel programmed genome-editing approaches in T. brucei that would benefit from temporal control, high-efficiency and precision. </p

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter

Radio continuum emission in the northern Galactic plane: Sources and spectral indices from the THOR survey

Radio continuum surveys of the Galactic plane can find and characterize HII regions, supernova remnants (SNRs), planetary nebulae (PNe), and extragalactic sources. A number of surveys at high angular resolution (<25") at different wavelengths exist to study the interstellar medium (ISM), but no comparable high-resolution and high-sensitivity survey exists at long radio wavelengths around 21cm. We observed a large fraction of the Galactic plane in the first quadrant of the Milky Way (l=14.0-67.4deg and |b| < 1.25deg) with the Karl G. Jansky Very Large Array (VLA) in the C-configuration covering six continuum spectral windows. These data provide a detailed view on the compact as well as extended radio emission of our Galaxy and thousands of extragalactic background sources. We used the BLOBCAT software and extracted 10916 sources. After removing spurious source detections caused by the sidelobes of the synthesised beam, we classified 10387 sources as reliable detections. We smoothed the images to a common resolution of 25" and extracted the peak flux density of each source in each spectral window (SPW) to determine the spectral indices $\alpha$ (assuming $I(\nu)\propto\nu^\alpha$). By cross-matching with catalogs of HII regions, SNRs, PNe, and pulsars, we found radio counterparts for 840 HII regions, 52 SNRs, 164 PNe, and 38 pulsars. We found 79 continuum sources that are associated with X-ray sources. We identified 699 ultra-steep spectral sources ($\alpha < -1.3$) that could be high-redshift galaxies. Around 9000 of the sources we extracted are not classified specifically, but based on their spatial and spectral distribution, a large fraction of them is likely to be extragalactic background sources. More than 7750 sources do not have counterparts in the SIMBAD database, and more than 3760 sources do not have counterparts in the NED database

Modifiable predictors of depression following childhood maltreatment: a systematic review and meta-analysis

Although maltreatment experiences in childhood increase the risk for depression, not all maltreated children become depressed. This review aims to systematically examine the existing literature to identify modifiable factors that increase vulnerability to, or act as a buffer against, depression, and could therefore inform the development of targeted interventions. Thirteen databases (including Medline, PsychINFO, SCOPUS) were searched (between 1984 and 2014) for prospective, longitudinal studies published in English that included at least 300 participants and assessed associations between childhood maltreatment and later depression. The study quality was assessed using an adapted Newcastle-Ottawa Scale checklist. Meta-analyses (random effects models) were performed on combined data to estimate the effect size of the association between maltreatment and depression. Meta-regressions were used to explore effects of study size and quality. We identified 22 eligible articles (N=12 210 participants), of which 6 examined potential modifiable predictors of depression following maltreatment. No more than two studies examined the same modifiable predictor; therefore, it was not possible to examine combined effects of modifiable predictors with meta-regression. It is thus difficult to draw firm conclusions from this study, but initial findings indicate that interpersonal relationships, cognitive vulnerabilities and behavioral difficulties may be modifiable predictors of depression following maltreatment. There is a lack of well-designed, prospective studies on modifiable predictors of depression following maltreatment. A small amount of initial research suggests that modifiable predictors of depression may be specific to maltreatment subtypes and gender. Corroboration and further investigation of causal mechanisms is required to identify novel targets for intervention, and to inform guidelines for the effective treatment of maltreated children

Identification and functional characterisation of CRK12:CYC9, a novel cyclin-dependent kinase (CDK)-cyclin complex in Trypanosoma brucei

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively

Continuum sources from the THOR survey between 1 and 2 GHz

We carried out a large program with the Karl G. Jansky Very Large Array (VLA): "THOR: The HI, OH, Recombination line survey of the Milky Way". We observed a significant portion of the Galactic plane in the first quadrant of the Milky Way in the 21cm HI line, 4 OH transitions, 19 radio recombination lines, and continuum from 1 to 2 GHz. In this paper we present a catalog of the continuum sources in the first half of the survey (l=14.0-37.9deg and l=47.1-51.2deg, |b|<1.1deg) at a spatial resolution of 10-25", with a spatially varying noise level of ~0.3-1 mJy/beam. The catalog contains ~4400 sources. Around 1200 of these are spatially resolved, and ~1000 are possible artifacts, given their low signal-to-noise ratios. Since the spatial distribution of the unresolved objects is evenly distributed and not confined to the Galactic plane, most of them are extragalactic. Thanks to the broad bandwidth of the observations from 1 to 2 GHz, we are able to determine a reliable spectral index for ~1800 sources. The spectral index distribution reveals a double-peaked profile with maxima at spectral indices of alpha = -1 and alpha = 0 , corresponding to steep declining and flat spectra, respectively. This allows us to distinguish between thermal and non-thermal emission, which can be used to determine the nature of each source. We examine the spectral index of ~300 known HII regions, for which we find thermal emission with spectral indices around alpha = 0. In contrast, supernova remnants (SNR) show non-thermal emission with alpha = -0.5 and extragalactic objects generally have a steeper spectral index of alpha = -1. Using the spectral index information of the THOR survey, we investigate potential SNR candidates. We classify the radiation of four SNR candidates as non-thermal, and for the first time, we provide strong evidence for the SNR origin of these candidates

The HI/OH/Recombination line survey of the inner Milky Way (THOR)

Context: The past decade has witnessed a large number of Galactic plane surveys at angular resolutions below 20". However, no comparable high-resolution survey exists at long radio wavelengths around 21cm in line and continuum emission. Methods: Employing the Very Large Array (VLA) in the C-array configuration and a large program, we observe the HI 21cm line, four OH lines, nineteen Halpha radio recombination lines as well as the continuum emission from 1 to 2GHz in full polarization over a large part of the first Galactic quadrant. Results: Covering Galactic longitudes from 14.5 to 67.4deg and latitudes between +-1.25deg, we image all of these lines and the continuum at ~20" resolution. These data allow us to study the various components of the interstellar medium (ISM): from the atomic phase, traced by the HI line, to the molecular phase, observed by the OH transitions, to the ionized medium, revealed by the cm continuum and the Halpha radio recombination lines. Furthermore, the polarized continuum emission enables magnetic field studies. In this overview paper, we discuss the survey outline and present the first data release as well as early results from the different datasets. We now release the first half of the survey; the second half will follow later after the ongoing data processing has been completed. The data in fits format (continuum images and line data cubes) can be accessed through the project web-page http://www.mpia.de/thor. Conclusions: The HI/OH/Recombination line survey of the Milky Way (THOR) opens a new window to the different parts of the ISM. It enables detailed studies of molecular cloud formation, conversion of atomic to molecular gas, and feedback from HII regions as well as the magnetic field in the Milky Way. It is highly complementary to other surveys of our Galaxy, and comparing different datasets allows us to address many open questions