Variation in point-of-care testing of HbA1c in diabetes care in general practice

Background: Point-of-care testing (POCT) of HbA1c may result in improved diabetic control, better patient outcomes, and enhanced clinical efficiency with fewer patient visits and subsequent reductions in costs. In 2008, the Danish regulators created a framework agreement regarding a new fee-for-service fee for the remuneration of POCT of HbA1c in general practice. According to secondary research, only the Capital Region of Denmark has allowed GPs to use this new incentive for POCT. The aim of this study is to use patient data to characterize patients with diabetes who have received POCT of HbA1c and analyze the variation in the use of POCT of HbA1c among patients with diabetes in Danish general practice. Methods: We use register data from the Danish Drug Register, the Danish Health Service Register and the National Patient Register from the year 2011 to define a population of 44,981 patients with diabetes (type 1 and type 2 but not patients with gestational diabetes) from the Capital Region. The POCT fee is used to measure the amount of POCT of HbA1c among patients with diabetes. Next, we apply descriptive statistics and multilevel logistic regression to analyze variation in the prevalence of POCT at the patient and clinic level. We include patient characteristics such as gender, age, socioeconomic markers, health care utilization, case mix markers, and municipality classifications. Results: The proportion of patients who received POCT was 14.1% and the proportion of clinics which were “POCT clinics” was 26.9%. There were variations in the use of POCT across clinics and patients. A part of the described variation can be explained by patient characteristics. Male gender, age differences (older age), short education, and other ethnicity imply significantly higher odds for POCT. High patient costs in general practice and other parts of primary care also imply higher odds for POCT. In contrast, high patient costs for drugs and/or morbidity in terms of the Charlson Comorbidity index mean lower odds for POCT. The frequency of patients with diabetes per 1000 patients was larger in POCT clinics than Non-POCT clinics. A total of 22.5% of the unexplained variability was related to GP clinics. Conclusions: This study demonstrates variation in the use of POCT which can be explained by patient characteristics such as demographic, socioeconomic, and case mix markers. However, it appears relevant to reassess the system for POCT. Further studies are warranted in order to assess the impacts of POCT of HbA1c on health care outcomes

Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online

Assembly of custom TALE-type DNA binding domains by modular cloning

Transcription activator-like effector (TALE) DNA binding proteins show tremendous potential as molecular tools for targeted binding to any desired DNA sequence. Their DNA binding domain consists of tandem arranged repeats, and due to this repetitive structure it is challenging to generate designer TALEs (dTALEs) with user-defined specificity. We present a cloning approach that facilitates the assembly of multiple repeat-encoding DNA fragments that translate into dTALEs with pre-defined DNA binding specificity. This method makes use of type IIS restriction enzymes in two sequential cut-ligase reactions to build dTALE repeat arrays. We employed this modular approach for generation of a dTALE that differentiates between two highly similar DNA sequences that are both targeted by the Xanthomonas TALE, AvrBs3. These data show that this modular assembly system allows rapid generation of highly specific TALE-type DNA binding domains that target binding sites of predefined length and sequence. This approach enables the rapid and flexible production of dTALEs for gene regulation and genome editing in routine and high-throughput applications

Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a ‘modular assembly’ method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry

Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator–like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.Harvard University. Society of FellowsNational Human Genome Research Institute (U.S.) (Center for Excellence in Genomics Science P50 HG003170)United States. Dept. of Energy (Genomes to Life DE-FG02-02ER63445)United States. Defense Advanced Research Projects Agency (W911NF-08-1-0254, G.M.C.)Wyss Institute of Biologically Inspired EngineeringNational Institutes of Health (U.S.) (Transformative R01 (R01 NS073124-01))European School of Molecular Medicine (predoctoral fellowship

GrassPlot v. 2.00 – first update on the database of multi-scale plant diversity in Palaearctic grasslands

Abstract: GrassPlot is a collaborative vegetation-plot database organised by the Eurasian Dry Grassland Group (EDGG) and listed in the Global Index of Vegetation-Plot Databases (GIVD ID EU-00-003). Following a previous Long Database Report (Dengler et al. 2018, Phyto- coenologia 48, 331–347), we provide here the first update on content and functionality of GrassPlot. The current version (GrassPlot v. 2.00) contains a total of 190,673 plots of different grain sizes across 28,171 independent plots, with 4,654 nested-plot series including at least four grain sizes. The database has improved its content as well as its functionality, including addition and harmonization of header data (land use, information on nestedness, structure and ecology) and preparation of species composition data. Currently, GrassPlot data are intensively used for broad-scale analyses of different aspects of alpha and beta diversity in grassland ecosystems

Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases

Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease

Context dependence between subdomains in the DNA binding interface of the I-CreI homing endonuclease

Homing endonucleases (HE) have emerged as precise tools for achieving gene targeting events. Redesigned HEs with tailored specificities can be used to cleave new sequences, thereby considerably expanding the number of targetable genes and loci. With HEs, as well as with other protein scaffolds, context dependence of DNA/protein interaction patterns remains one of the major limitations for rational engineering of new DNA binders. Previous studies have shown strong crosstalk between different residues and regions of the DNA binding interface. To investigate this phenomenon, we systematically combined mutations from three groups of amino acids in the DNA binding regions of the I-CreI HE. Our results confirm that important crosstalk occurs throughout this interface in I-CreI. Detailed analysis of success rates identified a nearest-neighbour effect, with a more pronounced level of dependence between adjacent regions. Taken together, these data suggest that combinatorial engineering does not necessarily require the identification of separable functional or structural regions, and that groups of amino acids provide acceptable building blocks that can be assembled, overcoming the context dependency of the DNA binding interface. Furthermore, the present work describes a sequential method to engineer tailored HEs, wherein three contiguous regions are individually mutated and assembled to create HEs with engineered specificity