ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathy gene.

OBJECTIVES: We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. BACKGROUND: CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. METHODS: In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. CONCLUSIONS: ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

The physical map of {\it Escherichia coli\/} K-12 strain MG1655: Construction, comparison with other strains and implications for mechanisms controlling oligonucleotide frequency

Complete NotI, SfiI, XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 were constructed. Techniques used included: CHEF pulsed field gel electrophoresis; transposon mutagenesis; fragment hybridization to the ordered $\lambda$ library of Kohara et al.; fragment and cosmid hybridization to Southern blots; correlation of fragments and cleavage sites with EcoMap, a sequence-modified version of the genomic restriction map of Kohara et al.; and correlation of cleavage sites with DNA sequence databases. In all, 105 restriction sites were mapped and correlated with the EcoMap coordinate system.^ NotI, SfiI, XbaI and BlnI restriction patterns of five commonly used E. coli K-12 strains were compared to those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the difference between the strains on the basis of simple insertions, deletions, and in one case an inversion. Insertions and deletions ranged in size from 1 kb to 86 kb. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains.^ Some aspects of the frequency and distribution of NotI, SfiI, XbaI and BlnI cleavage sites were analyzed using a method based on Markov chain theory. Overlaps of Dam and Dcm methylase sites with XbaI and SfiI cleavage sites were examined. The one XbaI-Dam overlap in the database is in accord with the expected frequency of this overlap. The occurrence of certain types of SfiI-Dcm overlaps are overrepresented. Of the four subtypes of SfiI-Dcm overlap, only one has a partial inhibitory effect on the activity of SfiI. Recognition sites for all four enzymes are rarer than expected based on oligonucleotide frequency data, with this effect being much stronger for XbaI and BlnI than for NotI and SfiI. The latter two enzyme sites are rare mainly due to apparent negative selection against GGCC (both) and CGGCCG (NotI). The former two enzyme sites are rare mainly due to effects of the VSP repair system on certain di-tri- and tetranucleotides, most notably CTAG. Models are proposed to explain several of the anomalies of oligonucleotide distribution in E. coli, and the biological significance of the systems that produce these anomalies is discussed. ^

Regulation of Phytoplankton Carbon to Chlorophyll Ratio By Light, Nutrients and Temperature in the Equatorial Pacific Ocean: A Basin-Scale Model

The complex effects of light, nutrients and temperature lead to a variable carbon to chlorophyll (C:Chl) ratio in phytoplankton cells. Using field data collected in the Equatorial Pacific, we derived a new dynamic model with a non-steady C:Chl ratio as a function of irradiance, nitrate, iron, and temperature. The dynamic model is implemented into a basin-scale ocean circulation-biogeochemistry model and tested in the Equatorial Pacific Ocean. The model reproduces well the general features of phytoplankton dynamics in this region. For instance, the simulated deep chlorophyll maximum (DCM) is much deeper in the western warm pool (similar to 100 m) than in the Eastern Equatorial Pacific (similar to 50 m). The model also shows the ability to reproduce chlorophyll, including not only the zonal, meridional and vertical variations, but also the interannual variability. This modeling study demonstrates that combination of nitrate and iron regulates the spatial and temporal variations in the phytoplankton C:Chl ratio in the Equatorial Pacific. Sensitivity simulations suggest that nitrate is mainly responsible for the high C:Chl ratio in the western warm pool while iron is responsible for the frontal features in the C:Chl ratio between the warm pool and the upwelling region. In addition, iron plays a dominant role in regulating the spatial and temporal variations of the C:Chl ratio in the Central and Eastern Equatorial Pacific. While temperature has a relatively small effect on the C:Chl ratio, light is primarily responsible for the vertical decrease of phytoplankton C:Chl ratio in the euphotic zone.

Conditioned medium from fresh and demineralized bone enhances osteoclastogenesis in murine bone marrow cultures

OBJECTIVES Osteoclasts rapidly form on the surface of bone chips at augmentation sites. The underlying molecular mechanism, however, is unclear. Soluble factors released from bone chips in vitro have a robust impact on mesenchymal cell differentiation. Whether these soluble factors change the differentiation of hematopoietic cells into osteoclasts remains unknown. METHODS Osteoclastogenesis, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells, was studied with murine bone marrow cultures exposed to RANKL and M-CSF, and conditioned medium from fresh (BCM) and demineralized bone matrix (DCM). Histochemical staining, gene and protein expression, as well as viability assays were performed. RESULTS This study shows that BCM had no impact on osteoclastogenesis. However, when BCM was heated to 85°C (BCMh), the number of tartrate-resistant acid phosphatase-positive multinucleated cells that developed in the presence of RANKL and M-CSF approximately doubled. In line with the histochemical observations, there was a trend that BCMh increased expression of osteoclast marker genes, in particular the transcription factor c-fos. The expression of c-fos was significantly reduced by the TGF-β receptor I antagonist SB431542. DCM significantly stimulated osteoclastogenesis, independent of thermal processing. CONCLUSIONS These data demonstrate that activated BCM by heat and DBM are able to stimulate osteoclastogenesis in vitro. These in vitro results support the notion that the resorption of autografts may be supported by as yet less defined paracrine mechanisms.

Myocardial expression profiles of candidate molecules in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia compared to those with dilated cardiomyopathy and healthy controls.

BACKGROUND Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is mainly an autosomal dominant disease characterized by fibrofatty infiltration of the right ventricle, leading to ventricular arrhythmias. Mutations in desmosomal proteins can be identified in about half of the patients. The pathogenic mechanisms leading to disease expression remain unclear. OBJECTIVE The purpose of this study was to investigate myocardial expression profiles of candidate molecules involved in the pathogenesis of ARVC/D. METHODS Myocardial messenger RNA (mRNA) expression of 62 junctional molecules, 5 cardiac ion channel molecules, 8 structural molecules, 4 apoptotic molecules, and 6 adipogenic molecules was studied. The averaged expression of candidate mRNAs was compared between ARVC/D samples (n = 10), nonfamilial dilated cardiomyopathy (DCM) samples (n = 10), and healthy control samples (n = 8). Immunohistochemistry and quantitative protein expression analysis were performed. Genetic analysis using next generation sequencing was performed in all patients with ARVC/D. RESULTS Following mRNA levels were significantly increased in patients with ARVC/D compared to those with DCM and healthy controls: phospholamban (P ≤ .001 vs DCM; P ≤ .001 vs controls), healthy tumor protein 53 apoptosis effector (P = .001 vs DCM; P ≤ .001 vs controls), and carnitine palmitoyltransferase 1β (P ≤ .001 vs DCM; P = 0.008 vs controls). Plakophillin-2 (PKP-2) mRNA was downregulated in patients with ARVC/D with PKP-2 mutations compared with patients with ARVC/D without PKP-2 mutations (P = .04). Immunohistochemistry revealed significantly increased protein expression of phospholamban, tumor protein 53 apoptosis effector, and carnitine palmitoyltransferase 1β in patients with ARVC/D and decreased PKP-2 expression in patients with ARVC/D carrying a PKP-2 mutation. CONCLUSION Changes in the expression profiles of sarcolemmal calcium channel regulation, apoptosis, and adipogenesis suggest that these molecular pathways may play a critical role in the pathogenesis of ARVC/D, independent of the underlying genetic mutations.

Phytol and phytyldiol concentrations at DYFAMED time series station and sediment trap

Particulate samples from the water column were collected monthly from depths of 5-150 m, between May 1996 and March 1997, in the northwestern Mediterranean Sea (Ligurian Sea) as part of the DYFAMED project within the French JGOFS program. These samples were analyzed by gas chromatography-electron impact mass spectrometry for their phytol and 3-methylidene-3,7,11-trimethylhexadecan-1,2-diol (phytyldiol) content. The corresponding Chlorophyll Phytyl side chain Photodegradation Index, molar ratio of phytyldiol to phytol, was calculated and the mean amount of chlorophyll photodegraded within the euphotic zone estimated. Seasonal differences in the chlorophyll photodegradation process appear in the one-year study. The chlorophyll appeared more photodegraded in the surface water (generally more than 40% photodegraded at 5-10 m) than at the deep chlorophyll maximum (DCM) (40-50 m) observed in the summer stratified waters (about 20% photodegraded). This difference was attributed to the healthy state of the phytoplankton community (coincidence with the highest primary production levels) and to the lower intensity of irradiance at the DCM level. On the other hand, the bulk of the detrital chlorophyll (chlorophyll associated with phytodetritus, phaeopigments) undergoes photodegradation before it sinks out of the photic zone. However, in January (winter mixed water) the pigments exported towards the sea floor were less photodegraded. This is thought to result from a shorter period of residence of the pigments in the photic zone due to vertical convection and grazing activity of macrozooplankton (salps), which are producers of rapid sinking fecal pellets.

(Table 3) Stable oxagen isotope record of planktonic foraminifera from Central Walvish Ridge

Above the Walvis Ridge, in the SE Atlantic Ocean, we collected living plantkic foraminifera from the upper water column using depth stratified plantkon tows. The oxygen isotope composition (d18Oc) in shells of foraminifera and shell concentration profiles show seasonal and depth habitats of individual species. The tow results are compared with the average annual deposition d18Oc from sediment traps and the interannual average d18Oc of fossil specimens in top sediments at the same site. The species Globigerinita glutinata best reflects the austral winter/spring sea surface temperature (SST). Its d18Oc signal in top sediments remains pristine. In contrast, tow results also show that Globigerinoides ruber continues to calcify below the surface mixed layer (SML), i.e., down to the deep chlorophyll maximum (DCM); hence its d18Oc signature of exported specimens reflects the SST only when SML incorporates the DCM. Deep tow and sediment trap results show that both Globorotalia truncatulinoides and Globorotalia inflata record the temperature between 150 and 350 m, depending on the season and the shell size. However, for all fossil taxa in sediments apart from Globigerinita glutinata, we observe a positive d18Oc shift with respect to the sediment trap and plankton tow values, likely related to the interannual flux changes and deep encrustation.

Planktonic assemblages during sapropel S5 deposits in the Urania Basin

A micropaleontological study of planktonic assemblages on the partially laminated sapropel S5 (late Pleistocene, marine isotope stage (MIS) 5e) was performed in two piston cores from Urania Basin area (eastern Mediterranean, west of Crete): UM94PC16 and UM94PC31 recovered during a PALEOFLUX Project Cruise. The abundance of Florisphaera profunda indicates the development of a deep chlorophyll maximum (DCM) before the anoxic condition at bottom were established, whereas patterns of upper photic zone coccoliths suggest extreme oligotrophy in surface water. The short appearance of Globorotalia scitula and the presence of Globigerinoides ruber in the lower part of sapropel testify to a thermal stratification, also recorded by changes in primary producers. During G. scitula occurrence, diatoms, mainly represented by Pseudosolenia calcar-avis, appear and bloom because of their capability in using nutrients from DCM. Scanning electron microscope analyses performed on selected intervals from UM94PC16 show that the sapropel is organized in microlaminae mostly composed by siliceous microfossils. In particular, sapropel S5 could be related to an enhanced nutrient availability in the lower-middle part of the photic zone, stratified conditions, and a higher continental input.

Coccolithophora and geochemistry of sapropel S1 in ODP Hole 160-964B

The cyclic development of anoxic conditions in the eastern Mediterranean deep sea waters is one of the most fascinating research topics in paleoceanographic studies. In combination with bottom water stagnation, enhanced primary production is a common explanation for the deposition of organic-rich layers (sapropels). This is supported by extensive evidence from both geochemical and micropaleontological studies. The correspondence of recent sapropel layers with peaks of the lower photic zone coccolithophore species Florisphaera profunda has been interpreted as a proxy for the development of a deep chlorophyll maximum (DCM), due to the pycnocline/nutricline shallowing into the lower part of the photic zone. We present millennial-scale data for coccolithophore assemblages from sediments across the most recent sapropel (S1), in the ODP Hole 964B drilled in the Ionian Sea. Relative and absolute abundances of taxa are compared with selected elemental composition of the bulk sediments. The Mn/Al and Ba/Al profiles are used to determine the original thickness of the S1 interval, and show that the upper part of S1 was affected by post-depositional oxidation of organic matter. The Nannofossil Accumulation Rate, defined by the number of coccoliths/cm**2/kyr, suggests that there is no evidence of increased productivity within most of the sapropel layer. In fact, coccolithophore production was at its minimum in the lower part. Minimum coccolith concentrations are reached despite the increase in F. profunda in both relative and absolute abundance. We suggest that the DCM deduced from the increased productivity of this species did not significantly contribute to the putative overall increased primary productivity during the deposition of most of the sapropel layer. Within the upper oxidized part of S1, coccolith accumulation was at least five times higher than in the lower part. This period of high coccolith productivity finds a counterpart in the increase of the Ba/Al ratio. The total concentration of coccoliths is again controlled by the amount of E. huxleyi, but it is also supported by concomitant increases in all the other groups, suggesting that coccolithophore productivity increased throughout the year and through the total vertical extent of the photic zone. At site 964, this is apparently the only moment when coccolithophores contributed substantially to the increased primary productivity generally assumed for the S1 layer.

Relative abundance of prokaryotic picoplankton populations in the Atlantic Ocean

Members of the prokaryotic picoplankton are the main drivers of the biogeochemical cycles over large areas of the world's oceans. In order to ascertain changes in picoplankton composition in the euphotic and twilight zones at an ocean basin scale we determined the distribution of 11 marine bacterial and archaeal phyla in three different water layers along a transect across the Atlantic Ocean from South Africa (32.9°S) to the UK (46.4°N) during boreal spring. Depth profiles down to 500 m at 65 stations were analysed by catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and automated epifluorescence microscopy. There was no obvious overall difference in microbial community composition between the surface water layer and the deep chlorophyll maximum (DCM) layer. There were, however, significant differences between the two photic water layers and the mesopelagic zone. SAR11 (35 ± 9%) and Prochlorococcus (12 ± 8%) together dominated the surface waters, whereas SAR11 and Crenarchaeota of the marine group I formed equal proportions of the picoplankton community below the DCM (both ~15%). However, due to their small cell sizes Crenarchaeota contributed distinctly less to total microbial biomass than SAR11 in this mesopelagic water layer. Bacteria from the uncultured Chloroflexi-related clade SAR202 occurred preferentially below the DCM (4-6%). Distinct latitudinal distribution patterns were found both in the photic zone and in the mesopelagic waters: in the photic zone, SAR11 was more abundant in the Northern Atlantic Ocean (up to 45%) than in the Southern Atlantic gyre (~25%), the biomass of Prochlorococcus peaked in the tropical Atlantic Ocean, and Bacteroidetes and Gammaproteobacteria bloomed in the nutrient-rich northern temperate waters and in the Benguela upwelling. In mesopelagic waters, higher proportions of SAR202 were present in both central gyre regions, whereas Crenarchaeota were clearly more abundant in the upwelling regions and in higher latitudes. Other phylogenetic groups such as the Planctomycetes, marine group II Euryarchaeota and the uncultured clades SAR406, SAR324 and SAR86 rarely exceeded more than 5% of relative abundance.

Distribution of Florisphaera profunda and Helicosphaera carteri in down core and surface sediments

A detailed assessment of the respective roles of production, export, and subsequent preservation of organic carbon (Corg) in the eastern Mediterranean (EMED) sediments during the formation of sapropels remains elusive. Here we present new micropaleontological results for both surface samples taken at several locations in the EMED and last interglacial sapropel S5 from core LC21 in the southeastern Aegean Sea. A strong exponential anticorrelation between relative abundances of the lower photic zone coccolithophore Florisphaera profundain the surface sediments and modern concentrations of chlorophyll a (Chl-a) at the sea surface suggests thatF. profunda percentages can be used to track past productivity changes in the EMED. Prior to S5 deposition, an abrupt and large increase of F. profunda percentages in LC21 coincided (within the multidecadal resolution of the records) with the marked freshening of EMED surface waters. This suggests a strong coupling between freshwater-bound surface to intermediate water (density) stratification and enhanced upward advection of nutrients to the base of the photic zone, fuelling a productive deep chlorophyll maximum (DCM) underneath a nutrient-starved surface layer. Our findings imply that (at least) at the onset of sapropel formation physical and biogeochemical processes likely operated in tandem, enabling high Corg accumulation at the seafloor.

Seawater samples along several inshore-offshore transects off NW Africa and SE Africa

For the optimal use in palaeoceanographic studies of the stable oxygen isotopic signal and elemental composition of the calcareous photosynthetic dinoflagellate Thoracosphaera heimii, it is essential to gain detailed information about its calcification depth and spatial distribution. We therefore studied the vertical and horizontal distribution patterns of T. heimii in the upper water column (0-200 m) along three transects: an inshore-offshore gradient off Cape Blanc (CB), a south-north transect from CB to the Portuguese coast and a north-south transect off Tanzania. We compared concentrations of living cysts (cells with cell content) with chlorophyll-a, salinity and temperature measurements at the sampling depth. In order to explore the seasonal variability in cyst production, three transect off CB were sampled at three different times of the year. Living T. heimii cysts were found in the upper 160 m of the water column with highest concentrations in the photic zone indicating that the calcification of T. heimii occurs in the upper part of the water column. Maximal abundances of living cysts were found relatively often in or just above the deep chlorophyll maximum (DCM), the depth of which varies regionally from about 20-40 m off CB to about 80 m off Tanzania and along the transect from CB to the Portuguese Coast. However, there was no significant correlation at the 95% confidence level between the cyst concentrations and temperature, salinity and chlorophyll-a concentrations at the sampling depths observed. In both the Atlantic and Indian Oceans, the highest abundances of T. heimii were observed in regions where the upper water masses contained relatively low nutrient concentrations that are influenced only sporadically, or not at all, by enhanced photic zone mixing related to the presence of upwelling cells or river outflow plumes at or close to the sampling sites. The seasonal production of cysts by T. heimii appears to be negatively related to the presence of upwelling filaments across the sampling sites. Our study suggests that turbulence of the upper water masses is a major environmental factor influencing T. heimii production.

Shaking table tests of structures with hysteretic dampers: experimental results vs. prediction using non-linear static methods

The use of seismic hysteretic dampers for passive control is increasing exponentially in recent years for both new and existing buildings. In order to utilize hysteretic dampers within a structural system, it is of paramount importance to have simplified design procedures based upon knowledge gained from theoretical studies and validated with experimental results. Non-linear Static Procedures (NSPs) are presented as an alternative to the force-based methods more common nowadays. The application of NSPs to conventional structures has been well established; yet there is a lack of experimental information on how NSPs apply to systems with hysteretic dampers. In this research, several shaking table tests were conducted on two single bay and single story 1:2 scale structures with and without hysteretic dampers. The maximum response of the structure with dampers in terms of lateral displacement and base shear obtained from the tests was compared with the prediction provided by three well-known NSPs: (1) the improved version of the Capacity Spectrum Method (CSM) from FEMA 440; (2) the improved version of the Displacement Coefficient Method (DCM) from FEMA 440; and (3) the N2 Method implemented in Eurocode 8. In general, the improved version of the DCM and N2 methods are found to provide acceptable accuracy in prediction, but the CSM tends to underestimate the response.

A general framework to testing the effect of transport policy measures to achieve a modal shift: a sequental hybrid model

In order to achieve to minimize car-based trips, transport planners have been particularly interested in understanding the factors that explain modal choices. In the transport modelling literature there has been an increasing awareness that socioeconomic attributes and quantitative variables are not sufficient to characterize travelers and forecast their travel behavior. Recent studies have also recognized that users? social interactions and land use patterns influence travel behavior, especially when changes to transport systems are introduced, but links between international and Spanish perspectives are rarely deal. In this paper, factorial and path analyses through a Multiple-Indicator Multiple-Cause (MIMIC) model are used to understand and describe the relationship between the different psychological and environmental constructs with social influence and socioeconomic variables. The MIMIC model generates Latent Variables (LVs) to be incorporated sequentially into Discrete Choice Models (DCM) where the levels of service and cost attributes of travel modes are also included directly to measure the effect of the transport policies that have been introduced in Madrid during the last three years in the context of the economic crisis. The data used for this paper are collected from a two panel smartphone-based survey (n=255 and 190 respondents, respectively) of Madrid.

Win-win transport strategies: searching for sinergies

In order to achieve to minimize car-based trips, transport planners have been particularly interested in understanding the factors that explain modal choices. In the transport modelling literature there has been an increasing awareness that socioeconomic attributes and quantitative variables are not sufficient to characterize travelers and forecast their travel behavior. Recent studies have also recognized that users? social interactions and land use patterns influence travel behavior, especially when changes to transport systems are introduced, but links between international and Spanish perspectives are rarely deal. In this paper, factorial and path analyses through a Multiple-Indicator Multiple-Cause (MIMIC) model are used to understand and describe the relationship between the different psychological and environmental constructs with social influence and socioeconomic variables. The MIMIC model generates Latent Variables (LVs) to be incorporated sequentially into Discrete Choice Models (DCM) where the levels of service and cost attributes of travel modes are also included directly to measure the effect of the transport policies that have been introduced in Madrid during the last three years in the context of the economic crisis. The data used for this paper are collected from a two panel smartphone-based survey (n=255 and 190 respondents, respectively) of Madrid.