Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation

Synchronous dynamics of zooplankton competitors prevail in temperate lake ecosystems

Although competing species are expected to exhibit compensatory dynamics (negative temporal covariation), empirical work has demonstrated that competitive communities often exhibit synchronous dynamics (positive temporal covariation). This has led to the suggestion that environmental forcing dominates species dynamics; however, synchronous and compensatory dynamics may appear at different length scales and/or at different times, making it challenging to identify their relative importance. We compiled 58 long-term datasets of zooplankton abundance in north-temperate and sub-tropical lakes and used wavelet analysis to quantify general patterns in the times and scales at which synchronous/compensatory dynamics dominated zooplankton communities in different regions and across the entire dataset. Synchronous dynamics were far more prevalent at all scales and times and were ubiquitous at the annual scale. Although we found compensatory dynamics in approximately 14% of all combinations of time period/scale/lake, there were no consistent scales or time periods during which compensatory dynamics were apparent across different regions. Our results suggest that the processes driving compensatory dynamics may be local in their extent, while those generating synchronous dynamics operate at much larger scales. This highlights an important gap in our understanding of the interaction between environmental and biotic forces that structure communities

State Behavioral Scale (SBS) A Sedation Assessment Instrument for Infants and Young Children Supported on Mechanical Ventilation

Objective: To develop and test the reliability and validity of the State Behavioral Scale for use in describing sedation/agitation levels in young intubated patients supported on mechanical ventilation. Design: In this prospective, psychometric evaluation, pairs of trained pediatric critical care nurse evaluators simultaneously and independently assessed a convenience sample of pediatric intensive care unit patients along eight state/behavioral dimensions and a numeric rating scale (NRS) of 0 (extremely sedated) to 10 (extremely agitated). The eight dimensions were derived from the sedation/agitation literature and expert opinion and included respiratory drive, response to ventilation, coughing, best response to stimulation, attentiveness to careprovider, tolerance to care, consolability, and movement after consoled, each with 3–5 levels. Setting: An 18-bed pediatric medical–surgical intensive care unit and 26-bed pediatric cardiovascular intensive care unit in a university-affiliated academic children’s hospital. Patients: A total of 91 intubated mechanically ventilated patients 6 wks to 6 yrs of age provided a median of two observations (interquartile range, 1–3) for a total of 198 sets of observations. Excluded were postoperative patients or those receiving neuromuscular blockade. Interventions: Patients were observed for 1 min, and then incremental levels of stimulation were applied until patient response. After 2 mins of consoling, the state behavioral assessment and NRS were completed. Measurements: Weighted kappa and intraclass coefficients were generated to assess interrater reliability of the eight dimension and NRS ratings. Distinct state behavior profiles were empirically identified from the dimension ratings using hierarchical cluster analysis using a squared Euclidean distance measure and between-groups linkage. Construct validity of these profiles was assessed by comparing group mean NRS scores using one-way analysis of variance. Main Results: Weighted kappa scores for all 198 dimension ratings ranged from .44 to .76, indicating moderate to good interrater reliability. The intraclass coefficient of .79 was high for NRS ratings. Cluster analysis revealed five distinct state profiles, with mean NRS ratings of 1.1, 2.5, 4.0, 5.3, and 7.6, all of which differed significantly from each other (F = 75.8, p \u3c .001), supporting the profiles’ construct validity. Conclusions: Based on empirically derived state behavior profiles, we have constructed the State Behavioral Scale to allow systematic description of the sedation–agitation continuum in young pediatric patients supported on mechanical ventilation. Further studies including prospective validation and describing the effect of State Behavioral Scale implementation on clinical outcomes, including the quality of sedation and length of mechanical ventilation, are warranted

Insulin doseresponse curves for stimulation of splanchnic glucose uptake and suppression of endogenous glucose production differ in nondiabetic humans and are abnormal in people with type 2 diabetes.

To determine whether the insulin dose-response curves for suppression of endogenous glucose production (EGP) and stimulation of splanchnic glucose uptake (SGU) differ in nondiabetic humans and are abnormal in type 2 diabetes, 14 nondiabetic and 12 diabetic subjects were studied. Glucose was clamped at ϳ9.5 mmol/l and endogenous hormone secretion inhibited by somatostatin, while glucagon and growth hormone were replaced by an exogenous infusion. Insulin was progressively increased from ϳ150 to ϳ350 and ϳ700 pmol/l by means of an exogenous insulin infusion, while EGP, SGU, and leg glucose uptake (LGU) were measured using the splanchnic and leg catheterization methods, combined with a [3-3 H]glucose infusion. In nondiabetic subjects, an increase in insulin from ϳ150 to ϳ350 pmol/l resulted in maximal suppression of EGP, whereas SGU continued to increase (P < 0.001) when insulin was increased to ϳ700 pmol/l. In contrast, EGP progressively decreased (P < 0.001) and SGU progressively increased (P < 0.001) in the diabetic subjects as insulin increased from ϳ150 to ϳ700 pmol/l. Although EGP was higher (P < 0.01) in the diabetic than nondiabetic subjects only at the lowest insulin concentration, SGU was lower (P < 0.01) in the diabetic subjects at all insulin concentrations tested. On the other hand, in contrast to LGU and overall glucose disposal, the increment in SGU in response to both increments in insulin did not differ in the diabetic and nondiabetic subjects, implying a right shifted but parallel dose-response curve. These data indicate that the dose-response curves for suppression of glucose production and stimulation of glucose uptake differ in nondiabetic subjects and are abnormal in people with type 2 diabetes. Taken together, these data also suggest that agents that enhance SGU in diabetic patients (e.g. glucokinase activators) are likely to improve glucose tolerance. Diabete

Essential and checkpoint functions of budding yeast ATM and ATR during meiotic prophase are facilitated by differential phosphorylation of a meiotic adaptor protein, Hop1

A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis

Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery

An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks

Unconventional human T cells accumulate at the site of infection in response to microbial ligands and induce local tissue remodeling

The antimicrobial responsiveness and function of unconventional human T cells are poorly understood, with only limited access to relevant specimens from sites of infection. Peritonitis is a common and serious complication in individuals with end-stage kidney disease receiving peritoneal dialysis. By analyzing local and systemic immune responses in peritoneal dialysis patients presenting with acute bacterial peritonitis and monitoring individuals before and during defined infectious episodes, our data show that Vg9/ Vd2+ gd T cells and mucosal-associated invariant T cells accumulate at the site of infection with organisms producing (E)-4- hydroxy-3-methyl-but-2-enyl pyrophosphate and vitamin B2, respectively. Such unconventional human T cells are major producers of IFN-g and TNF-a in response to these ligands that are shared by many microbial pathogens and affect the cells lining the peritoneal cavity by triggering local inflammation and inducing tissue remodeling with consequences for peritoneal membrane integrity. Our data uncover a crucial role for Vg9/Vd2 T cells and mucosal-associated invariant T cells in bacterial infection and suggest that they represent a useful predictive marker for important clinical outcomes, which may inform future stratification and patient management. These findings are likely to be applicable to other acute infections where local activation of unconventional T cells contributes to the antimicrobial inflammatory response

Visually assessed breast density, breast cancer risk and the importance of the craniocaudal view

Contains fulltext : 69403.pdf (publisher's version ) (Open Access)INTRODUCTION: Mammographic density is known to be a strong risk factor for breast cancer. A particularly strong association with risk has been observed when density is measured using interactive threshold software. This, however, is a labour-intensive process for large-scale studies. METHODS: Our aim was to determine the performance of visually assessed percent breast density as an indicator of breast cancer risk. We compared the effect on risk of density as measured with the mediolateral oblique view only versus that estimated as the average density from the mediolateral oblique view and the craniocaudal view. Density was assessed using a visual analogue scale in 10,048 screening mammograms, including 311 breast cancer cases diagnosed at that screening episode or within the following 6 years. RESULTS: Where only the mediolateral oblique view was available, there was a modest effect of breast density on risk with an odds ratio for the 76% to 100% density relative to 0% to 25% of 1.51 (95% confidence interval 0.71 to 3.18). When two views were available, there was a considerably stronger association, with the corresponding odds ratio being 6.77 (95% confidence interval 2.75 to 16.67). CONCLUSION: This indicates that a substantial amount of information on risk from percentage breast density is contained in the second view. It also suggests that visually assessed breast density has predictive potential for breast cancer risk comparable to that of density measured using the interactive threshold software when two views are available. This observation needs to be confirmed by studies applying the different measurement methods to the same individuals

Ecology of \u3ci\u3eVibrio parahaemolyticus\u3c/i\u3e and \u3ci\u3eVibrio vulnificus\u3c/i\u3e in the Coastal and Estuarine Waters of Louisiana, Maryland, Mississippi, and Washington (United States)

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaetnolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST

The Ninth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the SDSS-III Baryon Oscillation Spectroscopic Survey

The Sloan Digital Sky Survey III (SDSS-III) presents the first spectroscopic data from the Baryon Oscillation Spectroscopic Survey (BOSS). This ninth data release (DR9) of the SDSS project includes 535,995 new galaxy spectra (median z=0.52), 102,100 new quasar spectra (median z=2.32), and 90,897 new stellar spectra, along with the data presented in previous data releases. These spectra were obtained with the new BOSS spectrograph and were taken between 2009 December and 2011 July. In addition, the stellar parameters pipeline, which determines radial velocities, surface temperatures, surface gravities, and metallicities of stars, has been updated and refined with improvements in temperature estimates for stars with T_eff<5000 K and in metallicity estimates for stars with [Fe/H]>-0.5. DR9 includes new stellar parameters for all stars presented in DR8, including stars from SDSS-I and II, as well as those observed as part of the SDSS-III Sloan Extension for Galactic Understanding and Exploration-2 (SEGUE-2). The astrometry error introduced in the DR8 imaging catalogs has been corrected in the DR9 data products. The next data release for SDSS-III will be in Summer 2013, which will present the first data from the Apache Point Observatory Galactic Evolution Experiment (APOGEE) along with another year of data from BOSS, followed by the final SDSS-III data release in December 2014.Comment: 9 figures; 2 tables. Submitted to ApJS. DR9 is available at http://www.sdss3.org/dr