Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis. Keywords: embryonic stem cells; human genome engineering; shelterin; telomerase telomere maintenanceNational Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022

Designing and implementing a COPD discharge care bundle

National surveys have revealed significant differences in patient outcomes following admission to hospital with acute exacerbation of COPD which are likely to be due to variations in care. We developed a care bundle, comprising a short list of evidence-based practices to be implemented prior to discharge for all patients admitted with this condition, based on a review of national guidelines and other relevant literature, expert opinion and patient consultation. Implementation was then piloted using action research methodologies with patient input. Actively involving staff was vital to ensure that the changes introduced were understood and the process followed. Implementation of a care bundle has the potential to produce a dramatic improvement in compliance with optimum health care practice

miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer.

MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management

“Dogged” Search of Fresh Nakhla Surfaces Reveals New Alteration Textures

Special Issue: 74th Annual Meeting of the Meteoritical Society, August 8-12, 2011, London, U.K.International audienceCarbonaceous chondrites are considered as amongst the most primitive Solar System samples available. One of their primitive characteristics is their enrichment in volatile elements.This includes hydrogen, which is present in hydrated and hydroxylated minerals. More precisely, the mineralogy is expected to be dominated by phyllosilicates in the case of CM chondrites, and by Montmorillonite type clays in the case of CI. Here, in order to characterize and quantify the abundance of lowtemperature minerals in carbonaceous chondrites, we performed thermogravimetric analysis of matrix fragments of Tagish Lake, Murchison and Orgueil

Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice

Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery–Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations

Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders.

International audienceSHANK3 (also known as ProSAP2) regulates the structural organization of dendritic spines and is a binding partner of neuroligins; genes encoding neuroligins are mutated in autism and Asperger syndrome. Here, we report that a mutation of a single copy of SHANK3 on chromosome 22q13 can result in language and/or social communication disorders. These mutations concern only a small number of individuals, but they shed light on one gene dosage-sensitive synaptic pathway that is involved in autism spectrum disorders

An investigation of causes of false positive single nucleotide polymorphisms using simulated reads from a small eukaryote genome

Background: Single Nucleotide Polymorphisms (SNPs) are widely used molecular markers, and their use has increased massively since the inception of Next Generation Sequencing (NGS) technologies, which allow detection of large numbers of SNPs at low cost. However, both NGS data and their analysis are error-prone, which can lead to the generation of false positive (FP) SNPs. We explored the relationship between FP SNPs and seven factors involved in mapping-based variant calling - quality of the reference sequence, read length, choice of mapper and variant caller, mapping stringency and filtering of SNPs by read mapping quality and read depth. This resulted in 576 possible factor level combinations. We used error- and variant-free simulated reads to ensure that every SNP found was indeed a false positive. Results: The variation in the number of FP SNPs generated ranged from 0 to 36,621 for the 120 million base pairs (Mbp) genome. All of the experimental factors tested had statistically significant effects on the number of FP SNPs generated and there was a considerable amount of interaction between the different factors. Using a fragmented reference sequence led to a dramatic increase in the number of FP SNPs generated, as did relaxed read mapping and a lack of SNP filtering. The choice of reference assembler, mapper and variant caller also significantly affected the outcome. The effect of read length was more complex and suggests a possible interaction between mapping specificity and the potential for contributing more false positives as read length increases. Conclusions: The choice of tools and parameters involved in variant calling can have a dramatic effect on the number of FP SNPs produced, with particularly poor combinations of software and/or parameter settings yielding tens of thousands in this experiment. Between-factor interactions make simple recommendations difficult for a SNP discovery pipeline but the quality of the reference sequence is clearly of paramount importance. Our findings are also a stark reminder that it can be unwise to use the relaxed mismatch settings provided as defaults by some read mappers when reads are being mapped to a relatively unfinished reference sequence from e.g. a non-model organism in its early stages of genomic exploration

Genetic engineering of human ES and iPS cells using TALE nucleases

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022)Howard Hughes Medical Institut