Microtubule detyrosination guides chromosomes during mitosis

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.We thank F. I. Ataullakhanov for help with the laser trap and data analysis; A. Kiyatkin, V. Mustyatsa, M. Molodtsov, A. Gautreau, G. Lakisic, and M. Barisic for technical assistance; and members of our laboratories for stimulating discussions. This work was supported by National Institutes of Health grant R01-GM098389 and RSG-14-018-01-CCG from the American Cancer Society to E.L.G.; by the Institut Curie, the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the L'Agence Nationale de la Recherche (ANR) award ANR-12-BSV2-0007, INCA_6517, ANR-10-LBX-0038, part of the IDEX Idex PSL, ANR-10-IDEX-0001-02 PSL to C.J.; and Fundacao Luso-Americana para o Desenvolvimento (FLAD) Life Science 2020 and PRECISE grant from the European Research Council to H.M. A.V.Z. is supported by the RAS Presidium Grants "Mechanisms of the Molecular Systems Integration," " Molecular and Cell Biology programs," and Russian Fund for Basic Research Grant 12-04-00111-a and 13-00-40188. R.S.S. is supported by a fellowship from the Programa Graduado em Areas da Biologia Basica e Aplicada (GABBA) PhD program from the University of Porto. A.L.P. is supported by fellowship SFRH/BPD/66707/2009 from Fundacao para a Ciencia e a Tecnologia of Portugal. M.B., R.S.S., S.K.T., M.M.M., C.J., E.L.G., and H.M. designed the experiments; M.B. performed all experiments in cells; M. M. M. established and performed the tubulin purification protocol from HeLa cells; R.S.S. performed single-molecule experiments; S.K.T. performed force measurements; A.L.P. provided reagents; all authors analyzed data; H.M., E.L.G., and M.B. wrote the paper, with contributions from all authors; H.M. conceived and coordinated the project. Data described can be found in the main figures and supplementary materials. The authors declare no conflict of interests

Magnetic resonance spectroscopy as a non-invasive method to quantify muscle carnosine in humans: a comprehensive validity assessment

Carnosine is a dipeptide abundantly found in human skeletal muscle, cardiac muscle and neuronal cells having numerous properties that confers performance enhancing effects, as well as a wide-range of potential therapeutic applications. A reliable and valid method for tissue carnosine quantification is crucial for advancing the knowledge on biological processes involved with carnosine metabolism. In this regard, proton magnetic resonance spectroscopy (1H-MRS) has been used as a non-invasive alternative to quantify carnosine in human skeletal muscle. However, carnosine quantification by 1H-MRS has some potential limitations that warrant a thorough experimental examination of its validity. The present investigation examined the reliability, accuracy and sensitivity for the determination of muscle carnosine in humans using in vitro and in vivo experiments and comparing it to reference method for carnosine quantification (high-performance liquid chromatography – HPLC). We used in vitro 1H-MRS to verify signal linearity and possible noise sources. Carnosine was determined in the m. gastrocnemius by 1H-MRS and HPLC to compare signal quality and convergent validity. 1H-MRS showed adequate discriminant validity, but limited reliability and poor agreement with a reference method. Low signal amplitude, low signal-to-noise ratio, and voxel repositioning are major sources of error

Health promoting potential of herbal teas and tinctures from Artemisia campestris subsp maritima: from traditional remedies to prospective products

This work explored the biotechnological potential of the medicinal halophyte Artemisia campestris subsp. maritima (dune wormwood) as a source of health promoting commodities. For that purpose, infusions, decoctions and tinctures were prepared from roots and aerial-organs and evaluated for in vitro antioxidant, anti-diabetic and tyrosinase-inhibitory potential, and also for polyphenolic and mineral contents and toxicity. The dune wormwood extracts had high polyphenolic content and several phenolics were identified by ultra-high performance liquid chromatography-photodiode array-mass-spectrometry (UHPLC-PDA-MS). The main compounds were quinic, chlorogenic and caffeic acids, coumarin sulfates and dicaffeoylquinic acids; several of the identified phytoconstituents are here firstly reported in this A. campestris subspecies. Results obtained with this plant's extracts point to nutritional applications as mineral supplementary source, safe for human consumption, as suggested by the moderate to low toxicity of the extracts towards mammalian cell lines. The dune wormwood extracts had in general high antioxidant activity and also the capacity to inhibit a-glucosidase and tyrosinase. In summary, dune wormwood extracts are a significant source of polyphenolic and mineral constituents, antioxidants and a-glucosidase and tyrosinase inhibitors, and thus, relevant for different commercial segments like the pharmaceutical, cosmetic and/or food industries.FCT - Foundation for Science and Technology [CCMAR/Multi/04326/2013]; Portuguese National Budget; FCT [IF/00049/2012, SFRH/BD/94407/2013]; Research Foundation - Flanders (FWO) [12M8315N]info:eu-repo/semantics/publishedVersio

Time spent with cats is never wasted: Lessons learned from feline acromegalic cardiomyopathy, a naturally occurring animal model of the human disease

<div><p>Background</p><p>In humans, acromegaly due to a pituitary somatotrophic adenoma is a recognized cause of increased left ventricular (LV) mass. Acromegalic cardiomyopathy is incompletely understood, and represents a major cause of morbidity and mortality. We describe the clinical, echocardiographic and histopathologic features of naturally occurring feline acromegalic cardiomyopathy, an emerging disease among domestic cats.</p><p>Methods</p><p>Cats with confirmed hypersomatotropism (IGF-1>1000ng/ml and pituitary mass; n = 67) were prospectively recruited, as were two control groups: diabetics (IGF-1<800ng/ml; n = 24) and healthy cats without known endocrinopathy or cardiovascular disease (n = 16). Echocardiography was performed in all cases, including after hypersomatotropism treatment where applicable. Additionally, tissue samples from deceased cats with hypersomatotropism, hypertrophic cardiomyopathy and age-matched controls (n = 21 each) were collected and systematically histopathologically reviewed and compared.</p><p>Results</p><p>By echocardiography, cats with hypersomatotropism had a greater maximum LV wall thickness (6.5mm, 4.1–10.1mm) than diabetic (5.9mm, 4.2–9.1mm; Mann Whitney, p<0.001) or control cats (5.2mm, 4.1–6.5mm; Mann Whitney, p<0.001). Left atrial diameter was also greater in cats with hypersomatotropism (16.6mm, 13.0–29.5mm) than in diabetic (15.4mm, 11.2–20.3mm; Mann Whitney, p<0.001) and control cats (14.0mm, 12.6–17.4mm; Mann Whitney, p<0.001). After hypophysectomy and normalization of IGF-1 concentration (n = 20), echocardiographic changes proved mostly reversible. As in humans, histopathology of the feline acromegalic heart was dominated by myocyte hypertrophy with interstitial fibrosis and minimal myofiber disarray.</p><p>Conclusions</p><p>These results demonstrate cats could be considered a naturally occurring model of acromegalic cardiomyopathy, and as such help elucidate mechanisms driving cardiovascular remodeling in this disease.</p></div

Training in neurology: lessons learnt.

There is no consensus on how to structure and deliver neurology training. The General Medical Council's annual National Training Survey indicates that the quality of UK neurology training is very variable, but does not explain this variation. We used the survey data to identify the four highest and lowest performing sites for neurology training across the UK. We conducted semistructured interviews with groups of local trainees and, separately, local trainers in an exploratory qualitative study, and identified common themes across a range of aspects of neurology training. Here we present our findings, share case studies from top-performing sites and make recommendations on how best to train a neurologist

Molecular identification of adenoviruses associated with respiratory infection in Egypt from 2003 to 2010.

BACKGROUND: Human adenoviruses of species B, C, and E (HAdV-B, -C, -E) are frequent causative agents of acute respiratory infections worldwide. As part of a surveillance program aimed at identifying the etiology of influenza-like illness (ILI) in Egypt, we characterized 105 adenovirus isolates from clinical samples collected between 2003 and 2010. METHODS: Identification of the isolates as HAdV was accomplished by an immunofluorescence assay (IFA) and confirmed by a set of species and type specific polymerase chain reactions (PCR). RESULTS: Of the 105 isolates, 42% were identified as belonging to HAdV-B, 60% as HAdV-C, and 1% as HAdV-E. We identified a total of six co-infections by PCR, of which five were HAdV-B/HAdV-C co-infections, and one was a co-infection of two HAdV-C types: HAdV-5/HAdV-6. Molecular typing by PCR enabled the identification of eight genotypes of human adenoviruses; HAdV-3 (n = 22), HAdV-7 (n = 14), HAdV-11 (n = 8), HAdV-1 (n = 22), HAdV-2 (20), HAdV-5 (n = 15), HAdV-6 (n = 3) and HAdV-4 (n = 1). The most abundant species in the characterized collection of isolates was HAdV-C, which is concordant with existing data for worldwide epidemiology of HAdV respiratory infections. CONCLUSIONS: We identified three species, HAdV-B, -C and -E, among patients with ILI over the course of 7 years in Egypt, with at least eight diverse types circulating

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements

The secreted triose phosphate isomerase of Brugia malayi is required to sustain microfilaria production in vivo

Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60–70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4+ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections