Evaluating the performance of an updated high-sensitivity troponin T assay with increased tolerance to biotin.

OBJECTIVES: Biotin >20 ng/mL may interfere with the Elecsys® Troponin T-high sensitive assay (cTnT-hs; Roche Diagnostics International Ltd). We evaluated the performance of an updated assay, cTnT-hs*, which was designed to reduce biotin interference. METHODS: cTnT-hs* assay performance was assessed using up to two applications (18 min/9 min) on three analyzers (cobas e 411/cobas e 601/cobas e 801). Biotin interference was determined by measuring recovery in an 11-sample series dilution with biotin ranging from 0-3600 ng/mL. Repeatability/reproducibility were evaluated in five serum sample pools (n=75 each). Method comparisons tested: cTnT-hs* vs. cTnT-hs (18 min/cobas e 601); cTnT-hs* assay 18 vs. 9 min (cobas e 601); cTnT-hs* (18 min) on cobas e 601 vs. cobas e 411 and cobas e 601 vs. cobas e 801. Concordance at the 99th percentile decision limit between cTnT-hs* and cTnT-hs (9 min/cobas e 601) was calculated using 300 lithium-heparin plasma samples and a 14 ng/L assay cutoff. RESULTS: cTnT-hs* assay (18 min/cobas e 601) recovery was ≥96% for biotin ≤1250 ng/mL. Across all applications/analyzers, coefficients of variation for repeatability/reproducibility with the cTnT-hs* assay were <5% in most serum sample pools (mean cardiac troponin T: 8.528-9484 ng/L). High correlation (Pearson's r=1.000) was demonstrated for all method comparisons. Concordance at the 99th percentile decision limit was high between the cTnT-hs* and cTnT-hs assays. CONCLUSIONS: The updated cTnT-hs* assay may provide greater tolerance to biotin interference, and shows good analytical and clinical agreement/concordance with the previous cTnT-hs assay.

Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila.

RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site - contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

Sex-specific programming effects of parental obesity in pre-implantation embryonic development.

BACKGROUND: Obesity is a global rising problem with epidemiological dimension. Obese parents can have programming effects on their offspring leading to obesity and associated diseases in later life. This constitutes a vicious circle. Epidemiological data and studies in rodents demonstrated differential programming effects in male and female offspring, but the timing of their developmental origin is not known. METHODS: This study investigated if sex-specific programming effects of parental obesity can already be detected in the pre-implantation period. Diet-induced obese male or female mice were mated with normal-weight partners and blastocysts were recovered. RESULTS: Gene expression profiling revealed sex-specific responses of the blastocyst transcriptome to maternal and paternal obesity. The changes in the transcriptome of male blastocysts were more pronounced than those of female blastocysts, with a stronger impact of paternal than of maternal obesity. The sperm of obese mice revealed an increased abundance of several miRNAs compared with lean mice. CONCLUSIONS: Our study indicates that sex-specific programming effects of parental obesity already start in the pre-implantation period and reveals specific alterations of the sperm miRNA profile as mechanistic link to programming effects of paternal obesity.

Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference.

RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC.

Reversible perturbations of gene regulation after genome editing in Drosophila cells.

The prokaryotic phage defense CRISPR/cas-system has developed into a versatile toolbox for genome engineering and genetic studies in many organisms. While many efforts were spent on analyzing the consequences of off-target effects, only few studies addressed side-effects that occur due to the targeted manipulation of the genome. Here, we show that the CRISPR/cas9-mediated integration of an epitope tag in combination with a selection cassette can trigger an siRNA-mediated, epigenetic genome surveillance pathway in Drosophila melanogaster cells. After homology-directed insertion of the sequence coding for the epitope tag and the selection marker, a moderate level of siRNAs covering the inserted sequence and extending into the targeted locus was detected. This response affected protein levels less than two-fold and it persisted even after single cell cloning. However, removal of the selection cassette abolished the siRNA generation, demonstrating that this response is reversible. Consistently, marker-free genome engineering did not trigger the same surveillance mechanism. These two observations indicate that the selection cassette we employed induces an aberrant transcriptional arrangement and ultimately sets off the siRNA production. There have been prior concerns about undesirable effects induced by selection markers, but fortunately we were able to show that at least one of the epigenetic changes reverts as the marker gene is excised. Although the effects observed were rather weak (less than twofold de-repression upon ago2 or dcr-2 knock-down), we recommend that when selection markers are used during genome editing, a strategy for their subsequent removal should always be included.

A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells.

Custom genome editing has become an essential element of molecular biology. In particular, the generation of fusion constructs with epitope tags or fluorescent proteins at the genomic locus facilitates the analysis of protein expression, localization, and interaction partners at physiologic levels. Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. While our first PCR-based editing approach offered only blasticidin-resistance for selection, we now introduce puromycin-resistance as a second, independent selection marker; it is thus possible to edit two loci (e.g., for coimmunoprecipitation) without marker removal. Finally, we describe a modified FLP recombinase expression plasmid that improves the efficiency of marker cassette FLP-out. In summary, our technique and reagents enable a flexible, robust, and cloning-free genome editing approach that can be parallelized for scale-up.