Using selenomethionyl derivatives to assign sequence in low-resolution structures of the AP2 clathrin adaptor.

Selenomethionine incorporation is a powerful technique for assigning sequence to regions of electron density at low resolution. Genetic introduction of methionine point mutations and the subsequent preparation and crystallization of selenomethionyl derivatives permits unambiguous sequence assignment by enabling the placement of the anomalous scatterers (Se atoms) thus introduced. Here, the use of this approach in the assignment of sequence in a part of the AP2 clathrin adaptor complex that is responsible for clathrin binding is described. AP2 plays a pivotal role in clathrin-mediated endocytosis, a tightly regulated process in which cell-surface transmembrane proteins are internalized from the plasma membrane by incorporation into lipid-enclosed transport vesicles. AP2 binds cargo destined for internalization and recruits clathrin, a large trimeric protein that helps to deform the membrane to produce the transport vesicle. By selenomethionine labelling of point mutants, it was shown that the clathrin-binding site is buried within a deep cleft of the AP2 complex. A membrane-stimulated conformational change in AP2 releases the clathrin-binding site from autoinhibition, thereby linking clathrin recruitment to membrane localization.We would like to thank the I02, I03 and I04-1 beamline staff at the Diamond Light Source (mx6641) and Chris Oubridge for advice and assistance with SeMet mapping of the hinge residues. D.J.O. and B.T.K. are supported by Wellcome Trust Principal Research Fellowship (090909/Z/09/Z). S.C.G. is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (098406/Z/12/Z). CIMR is supported by a Wellcome Trust Strategic Award (079895).This is the final version of the article. It first appeared from the International Union of Crystallography via http://dx.doi.org/10.1107/S205979831502158

Osteosarcopenic Obesity Syndrome: What Is It and How Can It Be Identified and Diagnosed?

Conditions related to body composition and aging, such as osteopenic obesity, sarcopenia/sarcopenic obesity, and the newly termed osteosarcopenic obesity (triad of bone muscle and adipose tissue impairment), are beginning to gain recognition. However there is still a lack of definitive diagnostic criteria for these conditions. Little is known about the long-term impact of these combined conditions of osteoporosis, sarcopenia, and obesity in older adults. Many may go undiagnosed and progress untreated. Therefore, the objective of this research is to create diagnostic criteria for osteosarcopenic obesity in older women. The proposed diagnostic criteria are based on two types of assessments: physical, via body composition measurements, and functional, via physical performance measures. Body composition measurements such as T-scores for bone mineral density, appendicular lean mass for sarcopenia, and percent body fat could all be obtained via dual energy X-ray absorptiometry. Physical performance tests: handgrip strength, one-leg stance, walking speed, and sit-to-stand could be assessed with minimal equipment. A score could then be obtained to measure functional decline in the older adult. For diagnosing osteosarcopenic obesity and other conditions related to bone loss and muscle loss combined with obesity, a combination of measures may more adequately improve the assessment process

Stalna kronična upala niskoga stupnja zbog suvremenog načina prehrane potiče pretilost i osteoporozu

Some of the universal characteristics of pre-agricultural hominin diets are strikingly different from the modern human diet. Hominin dietary choices were limited to wild plant and wild animal foods, while the modern diet includes more than 70 % of energy consumed from refined sugars, refined vegetable oils, and highly processed cereals and dairy products. The modern diet, with higher intake of fat has also resulted in a higher ratio of omega-6 (n-6) to omega-3 (n-3) polyunsaturated fatty acids (PUFA), contributing to low-grade chronic inflammation (LGCI) and thus promoting the development of many chronic diseases, including obesity and osteoporosis. In this review, we describe the changes in modern diet, focusing on the kind and amount of consumed fat; explain the shortcomings of the modern diet with regard to inflammatory processes; and delineate the reciprocity between adiposity and inflammatory processes, with inflammation being a common link between obesity and osteoporosis. We present the evidence that overconsumption of n-6 PUFA coupled with under-consumption of n-3 PUFA results in LGCI and, along with the increased presence of reactive oxygen species, leads to a shift in mesenchymal stem cells (precursors for both osteoblasts and adipocytes) lineage commitment toward increased adipogenesis and suppressed osteoblastogenesis. In turn, high n-6 to n-3 PUFA ratios in the modern diet, coupled with increased synthesis of pro-inflammatory cytokines due to adiposity, propagate obesity and osteoporosis by increasing or maintaining LGCI.Pojedina univerzalna svojstva prehrane hominina prije pojave poljodjelstva izrazito se razlikuju od suvremene prehrane. Prehrambene navike hominina vjerojatno su bile ograničene na plodove divljeg bilja i lovinu; više od 70 % moderne prehrane odnosi se na konzumaciju rafiniranih šećera, biljnih ulja, žitarica i mliječnih prerađevina. Moderna je prehrana s višim unosom masti također dovela do nepovoljnijeg omjera omega-6 i omega-3 višestruko nezasićenih masnih kiselina (engl. krat. PUFA), koji pridonosi održavanju kronične upale niskoga stupnja, a time i nastanku mnogih kroničnih bolesti, uključujući pretilost i osteoporozu. U ovom se preglednom članku opisuju promjene uslijed modernog načina prehrane, s posebnim osvrtom na vrste i količine konzumirane masti. Također se objašnjavaju nedostatci moderne prehrane s obzirom na upalne procese te međusobna povezanost između pretilosti i upalnih procesa, koji su usto i poveznica između pretilosti i osteoporoze. U članku se iznose saznanja o tome da pretjerana konzumacija omega-6 masnih kiselina uz nedostatnu konzumaciju omega-3 masnih kiselina dovodi do kronične upale niskoga stupnja i povišenih vrijednosti reaktivnih kisikovih čestica (ROS), a time i do pomaka u mezenhimskih matičnih stanica (prekursora osteoblasta i adipocita) prema povećanoj adipogenezi i smanjenoj osteoblastogenezi. Uz povećanu sintezu upalnih citokina zbog pretilosti, moderna prehrana s nepovoljnim omjerom omega-6 i omega-3 kiselina u korist prvih nastavlja poticati pretilost i osteoporozu jer održava i pogoršava kroničnu upalu

Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

AbstractClathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.</jats:p

Thorium Energy Futures

The potential for thorium as an alternative or supplement to uranium in fission power generation has long been recognised, and several reactors, of various types, have already operated using thorium-based fuels. Accelerator Driven Subcritical (ADS) systems have benefits and drawbacks when compared to conventional critical thorium reactors, for both solid and molten salt fuels. None of the four options – liquid or solid, with or without an accelerator – can yet be rated as better or worse than the other three, given today's knowledge. We outline the research that will be necessary to lead to an informed choice

The impact of routine open nonsuction drainage on fluid accumulation after thyroid surgery: a prospective randomised clinical trial.

Background: Thyroid drains following thyroid surgery are routinely used despite minimal supportive evidence. Our aim in this study is to determine the impact of routine open drainage of the thyroid bed postoperatively on ultrasound-determined fluid accumulation at 24 hours. Methods: We conducted a prospective randomised clinical trial on patients undergoing thyroid surgery. Patients were randomly assigned to a drain group (n = 49) or a no-drain group (n = 44) immediately prior to wound closure. Patients underwent a neck ultrasound on day 1 and day 2 postoperatively. After surgery, we evaluated visual analogue scale pain scores, postoperative analgesic requirements, self-reported scar satisfaction at 6 weeks and complications. Results: There was significantly less mean fluid accumulated in the drain group on both day 1, 16.4 versus 25.1 ml (P-value = 0.005), and day 2, 18.4 versus 25.7 ml (P-value = 0.026), following surgery. We found no significant differences between the groups with regard to length of stay, scar satisfaction, visual analogue scale pain score and analgesic requirements. There were four versus one wound infections in the drain versus no-drain groups. This finding was not statistically significant (P = 0.154). No life-threatening bleeds occurred in either group. Conclusions: Fluid accumulation after thyroid surgery was significantly lessened by drainage. However, this study did not show any clinical benefit associated with this finding in the non-emergent setting. Drains themselves showed a trend indicating that they may augment infection rates. The results of this study suggest that the frequency of acute life-threatening bleeds remains extremely low following abandoning drains. We advocate abandoning routine use of thyroid drains. Trial registration: ISRCTN94715414

CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.S.E.M. and D.J.O. are funded by a Wellcome Trust Fellowship (to D.J.O. no. 090909/Z). N.A.B. is funded by MRC grant MR/M010007/1, and S.H. is funded by a grant from the German Science Foundation (SFB 635, TP A3). D.S. and S.M. acknowledge financial support from the Lundbeck Foundation and the Danish Councils for Independent and Strategic Research. C.J.M. and F.P. were funded by the Fondation pour la Recherche Medicale.This is the final published version. It first appeared at http://www.cell.com/developmental-cell/fulltext/S1534-5807%2815%2900144-6

Deep Chandra X-ray Imaging of a Nearby Radio Galaxy 4C+29.30: X-ray/Radio Connection

We report results from our deep Chandra X-ray observations of a nearby radio galaxy, 4C+29.30 (z=0.0647). The Chandra image resolves structures on sub-arcsec to arcsec scales, revealing complex X-ray morphology and detecting the main radio features: the nucleus, a jet, hotspots, and lobes. The nucleus is absorbed (N(H)=3.95 (+0.27/-0.33)x10^23 atoms/cm^2) with an unabsorbed luminosity of L(2-10 keV) ~ (5.08 +/-0.52) 10^43 erg/s characteristic of Type 2 AGN. Regions of soft (<2 keV) X-ray emission that trace the hot interstellar medium (ISM) are correlated with radio structures along the main radio axis indicating a strong relation between the two. The X-ray emission beyond the radio source correlates with the morphology of optical line-emitting regions. We measured the ISM temperature in several regions across the galaxy to be kT ~ 0.5 with slightly higher temperatures (of a few keV) in the center and in the vicinity of the radio hotspots. Assuming these regions were heated by weak shocks driven by the expanding radio source, we estimated the corresponding Mach number of 1.6 in the southern regions. The thermal pressure of the X-ray emitting gas in the outermost regions suggest the hot ISM is slightly under-pressured with respect to the cold optical-line emitting gas and radio-emitting plasma, which both seem to be in a rough pressure equilibrium. We conclude that 4C+29.30 displays a complex view of interactions between the jet-driven radio outflow and host galaxy environment, signaling feedback processes closely associated with the central active nucleus.Comment: ApJ in pres

Enrichment of CpG island shore region hypermethylation in epigenetic breast field cancerization

While changes in DNA methylation are known to occur early in breast carcinogenesis and the landscape of breast tumour DNA methylation is profoundly altered compared with normal tissue, there have been limited efforts to identify DNA methylation field cancerization effects in histologically normal breast tissue adjacent to tumour. Matched tumour, histologically normal tissue of the ipsilateral breast (ipsilateral-normal), and histologically normal tissue of the contralateral breast (contralateral-normal) were obtained from nine women undergoing bilateral mastectomy. Laser capture microdissection was used to select epithelial cells from normal tissue, and neoplastic cells from tumour for genome-scale measures of DNA methylation with the Illumina HumanMethylationEPIC array. We identified substantially more CpG loci that were differentially methylated between contralateral-normal and tumour (63,271 CpG loci q \u3c 0.01), than between ipsilateral-normal and tumour (38,346 CpG loci q \u3c 0.01). We identified differential methylation in ipsilateral-normal relative to contralateral-normal tissue (9,562 CpG loci p \u3c 0.01). In this comparison, hypomethylated loci were significantly enriched for breast cancer-relevant transcription factor binding sites including those for ESR1, FoxA1, and GATA3 and hypermethylated loci were significantly enriched for CpG island shore regions. In addition, progression of shore hypermethylation was observed in tumours compared to matched ipsilateral normal tissue, and these alterations tracked to several well-established tumour suppressor genes. Our results indicate an epigenetic field effect in surrounding histologically normal tissue. This work offers an opportunity to focus investigations of early DNA methylation alterations in breast carcinogenesis and potentially develop epigenetic biomarkers of disease risk