Efficient preparation of internally modified single-molecule constructs using nicking enzymes

Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required

Functional KV10.1 Channels Localize to the Inner Nuclear Membrane

Ectopically expressed human KV10.1 channels are relevant players in tumor biology. However, their function as ion channels at the plasma membrane does not totally explain their crucial role in tumors. Both in native and heterologous systems, it has been observed that a majority of KV10.1 channels remain at intracellular locations. In this study we investigated the localization and possible roles of perinuclear KV10.1. We show that KV10.1 is expressed at the inner nuclear membrane in both human and rat models; it co-purifies with established inner nuclear membrane markers, shows resistance to detergent extraction and restricted mobility, all of them typical features of proteins at the inner nuclear membrane. KV10.1 channels at the inner nuclear membrane are not all transported directly from the ER but rather have been exposed to the extracellular milieu. Patch clamp experiments on nuclei devoid of external nuclear membrane reveal the existence of channel activity compatible with KV10.1. We hypothesize that KV10.1 channels at the nuclear envelope might participate in the homeostasis of nuclear K+, or indirectly interact with heterochromatin, both factors known to affect gene expression

MyD88 signalling in myeloid cells is sufficient to prevent chronic mycobacterial infection

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that is responsible for almost 1.5 million deaths per year. Sensing of mycobacteria by the host's immune system relies on different families of receptors present on innate immune cells. Amongst them, several members of the TLR family are involved in the activation of immune cells by mycobacteria, yet the in vivo contribution of individual TLRs to the protective immune response remains controversial. On the contrary, MyD88, the adaptor molecule for most TLRs, plays a non-redundant role in the protection against tuberculosis and mice with a complete germline deletion of MyD88 succumb very early to infection. MyD88 is expressed in both immune and non-immune cells, but it is not clear whether control of mycobacteria requires ubiquitous or cell-type specific MyD88 expression. Therefore, using novel conditional switch-on mouse models, we aimed to investigate the importance of MyD88 signalling in DCs and macrophages for the induction of protective effector mechanisms against mycobacterial infection. We conclude that specific reactivation of MyD88 signalling in CD11c- or lysozyme M-expressing myeloid cells during Mycobacterium bovis Bacille Calmette-Guerin infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden.Fil: Berod, Luciana. Helmholtz Centre for Infection Research; Alemania. Medical School Hanover; AlemaniaFil: Stüve, Philipp. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Swallow, Maxine. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Arnold Schrauf, Catharina. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Kruse, Friederike. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Gentilini, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Helmholtz Centre for Infection Research; Alemania. Medical School Hanover; AlemaniaFil: Freitag, Jenny. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Holzmann, Bernhard. Universitat Technical Zu Munich; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Sparwasser, Tim. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; Alemani

Calcium modulates force sensing by the von Willebrand factor A2 domain

von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. VWF potency critically depends on multimer size, which is regulated by a feedback mechanism involving shear-induced unfolding of the VWF-A2 domain and cleavage by the metalloprotease ADAMTS-13. Here we report crystallographic and single-molecule optical tweezers data on VWF-A2 providing mechanistic insight into calcium-mediated stabilization of the native conformation that protects A2 from cleavage by ADAMTS-13. Unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate with non-native calcium coordination. Calcium further accelerates refolding markedly, in particular, under applied load. We propose that calcium improves force sensing by allowing reversible force switching under physiologically relevant hydrodynamic conditions. Our data show for the first time the relevance of metal coordination for mechanical properties of a protein involved in mechanosensing

Genomic tools development for Aquilegia: construction of a BAC-based physical map

<p>Abstract</p> <p>Background</p> <p>The genus <it>Aquilegia</it>, consisting of approximately 70 taxa, is a member of the basal eudicot lineage, Ranuculales, which is evolutionarily intermediate between monocots and core eudicots, and represents a relatively unstudied clade in the angiosperm phylogenetic tree that bridges the gap between these two major plant groups. <it>Aquilegia </it>species are closely related and their distribution covers highly diverse habitats. These provide rich resources to better understand the genetic basis of adaptation to different pollinators and habitats that in turn leads to rapid speciation. To gain insights into the genome structure and facilitate gene identification, comparative genomics and whole-genome shotgun sequencing assembly, BAC-based genomics resources are of crucial importance.</p> <p>Results</p> <p>BAC-based genomic resources, including two BAC libraries, a physical map with anchored markers and BAC end sequences, were established from <it>A. formosa</it>. The physical map was composed of a total of 50,155 BAC clones in 832 contigs and 3939 singletons, covering 21X genome equivalents. These contigs spanned a physical length of 689.8 Mb (~2.3X of the genome) suggesting the complex heterozygosity of the genome. A set of 197 markers was developed from ESTs induced by drought-stress, or involved in anthocyanin biosynthesis or floral development, and was integrated into the physical map. Among these were 87 genetically mapped markers that anchored 54 contigs, spanning 76.4 Mb (25.5%) across the genome. Analysis of a selection of 12,086 BAC end sequences (BESs) from the minimal tiling path (MTP) allowed a preview of the <it>Aquilegia </it>genome organization, including identification of transposable elements, simple sequence repeats and gene content. Common repetitive elements previously reported in both monocots and core eudicots were identified in <it>Aquilegia </it>suggesting the value of this genome in connecting the two major plant clades. Comparison with sequenced plant genomes indicated a higher similarity to grapevine (<it>Vitis vinifera</it>) than to rice and <it>Arabidopsis </it>in the transcriptomes.</p> <p>Conclusions</p> <p>The <it>A. formosa </it>BAC-based genomic resources provide valuable tools to study <it>Aquilegia </it>genome. Further integration of other existing genomics resources, such as ESTs, into the physical map should enable better understanding of the molecular mechanisms underlying adaptive radiation and elaboration of floral morphology.</p

The Baker's Yeast Diploid Genome Is Remarkably Stable in Vegetative Growth and Meiosis

Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1–2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms

Survival of atraumatic restorative treatment (ART) sealants and restorations: a meta-analysis

The purpose of this study is to perform a systematic investigation plus meta-analysis into survival of atraumatic restorative treatment (ART) sealants and restorations using high-viscosity glass ionomers and to compare the results with those from the 2005 ART meta-analysis. Until February 2010, four databases were searched. Two hundred four publications were found, and 66 reported on ART restorations or sealant survival. Based on five exclusion criteria, two independent reviewers selected the 29 publications that accounted for the meta-analysis. Confidence intervals (CI) and or standard errors were calculated and the heterogeneity variance of the survival rates was estimated. Location (school/clinic) was an independent variable. The survival rates of single-surface and multiple-surface ART restorations in primary teeth over the first 2 years were 93% (CI, 91–94%) and 62% (CI, 51–73%), respectively; for single-surface ART restorations in permanent teeth over the first 3 and 5 years it was 85% (CI, 77–91%) and 80% (CI, 76–83%), respectively and for multiple-surface ART restorations in permanent teeth over 1 year it was 86% (CI, 59–98%). The mean annual dentine lesion incidence rate, in pits and fissures previously sealed using ART, over the first 3 years was 1%. No location effect and no differences between the 2005 and 2010 survival rates of ART restorations and sealants were observed. The short-term survival rates of single-surface ART restorations in primary and permanent teeth, and the caries-preventive effect of ART sealants were high. Clinical relevance: ART can safely be used in single-surface cavities in both primary and permanent teeth. ART sealants have a high caries preventive effect

Localization and function of the renal calcium-sensing receptor

The ability to monitor changes in the ionic composition of the extracellular environment is a crucial feature that has evolved in all living organisms. The cloning and characterization of the extracellular calcium-sensing receptor (CaSR) from the mammalian parathyroid gland in the early 1990s provided the first description of a cellular, ion-sensing mechanism. This finding demonstrated how cells can detect small, physiological variations in free ionized calcium (Ca 2+) in the extracellular fluid and subsequently evoke an appropriate biological response by altering the secretion of parathyroid hormone (PTH) that acts on PTH receptors expressed in target tissues, including the kidney, intestine, and bone. Aberrant Ca 2+ sensing by the parathyroid glands, as a result of altered CaSR expression or function, is associated with impaired divalent cation homeostasis. CaSR activators that mimic the effects of Ca 2+ (calcimimetics) have been designed to treat hyperparathyroidism, and CaSR antagonists (calcilytics) are in development for the treatment of hypercalciuric disorders. The kidney expresses a CaSR that might directly contribute to the regulation of many aspects of renal function in a PTH-independent manner. This Review discusses the roles of the renal CaSR and the potential impact of pharmacological modulation of the CaSR on renal function