Binding of the Cytosolic P200 Protein to Golgi Membranes Is Regulated by Heterotrimeric G-Proteins

The formation of vesicles for protein trafficking requires the dynamic binding of cytosolic coat proteins onto Golgi membranes and this binding is regulated by a variety of GTPases, including heterotrimeric G proteins. We have previously shown the presence of the pertussis toxin-sensitive Galpha(i-3) protein on Golgi membranes and demonstrated a functional role for Galpha(i-3) in the trafficking of secretory proteins through the Golgi complex. We have also described a brefeldin A-sensitive phosphoprotein, p200, which is found in the cytoplasm and on Golgi membranes. The present study investigations the role of heterotrimeric G proteins in the regulation of p200 binding to Golgi membranes. An in vitro binding assay was used to measure the binding of cytosolic p200 to LLC-PK1 cell microsomal membranes and to purified rat liver Golgi membranes in the presence of specific activators of G proteins. The binding of p200 to Golgi membranes was compared to that of the coatomer protein beta-COP, for which G protein-dependent membrane binding has previously been established. Membrane binding of both p200 and beta-COP was induced maximally by activation of all G proteins in the presence of GTPgammaS. More selective activation of the heterotrimeric G proteins, with AlFn or mastoparan, also induced membrane binding of p200 and beta-COP. Pertussis toxin pretreatment of Golgi membranes, to selectively inactivate Galpha(i-3), reduced the AlFn and mastoparan-induced binding of p200 to Golgi membranes, whereas no significant effect of pertussis toxin on beta-COP binding was found in this assay. The effect of pertussis toxin thus implicates Galpha(i-3), as one component of a regulatory pathway, in the binding of cytosolic p200 to Golgi membranes. The effects of AlFn and pertussis toxin on p200 membrane binding were also shown in intact cells by immunofluorescence staining. AlFn treatment of cells induced translocation of p200 from the cytoplasm onto the Golgi complex, resulting in a conformational change in some Golgi membranes. The translocation of p200 was blocked by pretreatment of intact NRK cells with pertussis toxin. The data presented here support the conclusion that the binding of the p200 protein to Golgi membranes involves regulation by the pertussis toxin-sensitive heterotrimeric G proteins, specifically the Galpha(i-3) protein

Targeting of Chimeric G-Alpha(I) Proteins to Specific Membrane Domains

Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous Galpha(i-2) is localized on the basolateral plasma membrane, whereas Galpha(i-3) is localized on Golgi membranes. The targeting of these highly homologous Galpha(i) proteins to distinct membrane domains was studied by the transfection and expression of chimeric Galpha(i) proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for Galpha(i-3) and Galpha(i-2) and introduced into a pMXX eukaryotic expression vector containing a mouse metaltothionein-I promotor. Stably transfected cell lines were produced that expressed either Galpha(i-2/3) or Galpha(i-3/2) chimeric proteins. Chimeric and endogenous Galpha(i) proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric Galpha(i) proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous Galpha(i) proteins. Using confocal microscopy and image analysis we localized Galpha(i-2) to a specific sub-domain of the lateral membrane of polarized cells, the chimeric Galpha(i-3/2) protein was then shown to colocalize with endognenous Galpha(i-2) in the same lateral plasma membrane domain. The chimeric Galpha(i-2/3) protein colocalized with endogenous Galpha(i-3) on Golgi membranes in LLC-PK1 cells. These results show that chimeric Galpha(i) proteins were targeted to the same membrane domains as endogenous Galpha(i) proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of Galpha(i) proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins

Lizards Cooperatively Tunnel to Construct a Long-Term Home for Family Members

Constructing a home to protect offspring while they mature is common in many vertebrate groups, but has not previously been reported in lizards. Here we provide the first example of a lizard that constructs a long-term home for family members, and a rare case of lizards behaving cooperatively. The great desert skink, Liopholis kintorei from Central Australia, constructs an elaborate multi-tunnelled burrow that can be continuously occupied for up to 7 years. Multiple generations participate in construction and maintenance of burrows. Parental assignments based on DNA analysis show that immature individuals within the same burrow were mostly full siblings, even when several age cohorts were present. Parents were always captured at burrows containing their offspring, and females were only detected breeding with the same male both within- and across seasons. Consequently, the individual investments made to construct or maintain a burrow system benefit their own offspring, or siblings, over several breeding seasons

Genome-wide association study of thyroid-stimulating hormone highlights new genes, pathways and associations with thyroid disease.

Thyroid hormones play a critical role in regulation of multiple physiological functions and thyroid dysfunction is associated with substantial morbidity. Here, we use electronic health records to undertake a genome-wide association study of thyroid-stimulating hormone (TSH) levels, with a total sample size of 247,107. We identify 158 novel genetic associations, more than doubling the number of known associations with TSH, and implicate 112 putative causal genes, of which 76 are not previously implicated. A polygenic score for TSH is associated with TSH levels in African, South Asian, East Asian, Middle Eastern and admixed American ancestries, and associated with hypothyroidism and other thyroid disease in South Asians. In Europeans, the TSH polygenic score is associated with thyroid disease, including thyroid cancer and age-of-onset of hypothyroidism and hyperthyroidism. We develop pathway-specific genetic risk scores for TSH levels and use these in phenome-wide association studies to identify potential consequences of pathway perturbation. Together, these findings demonstrate the potential utility of genetic associations to inform future therapeutics and risk prediction for thyroid diseases

Statistical process control of mortality series in the Australian and New Zealand Intensive Care Society (ANZICS) adult patient database: implications of the data generating process

for the ANZICS Centre for Outcome and Resource Evaluation (CORE) of the Australian and New Zealand Intensive Care Society (ANZICS)BACKGROUND Statistical process control (SPC), an industrial sphere initiative, has recently been applied in health care and public health surveillance. SPC methods assume independent observations and process autocorrelation has been associated with increase in false alarm frequency. METHODS Monthly mean raw mortality (at hospital discharge) time series, 1995–2009, at the individual Intensive Care unit (ICU) level, were generated from the Australia and New Zealand Intensive Care Society adult patient database. Evidence for series (i) autocorrelation and seasonality was demonstrated using (partial)-autocorrelation ((P)ACF) function displays and classical series decomposition and (ii) “in-control” status was sought using risk-adjusted (RA) exponentially weighted moving average (EWMA) control limits (3 sigma). Risk adjustment was achieved using a random coefficient (intercept as ICU site and slope as APACHE III score) logistic regression model, generating an expected mortality series. Application of time-series to an exemplar complete ICU series (1995-(end)2009) was via Box-Jenkins methodology: autoregressive moving average (ARMA) and (G)ARCH ((Generalised) Autoregressive Conditional Heteroscedasticity) models, the latter addressing volatility of the series variance. RESULTS The overall data set, 1995-2009, consisted of 491324 records from 137 ICU sites; average raw mortality was 14.07%; average(SD) raw and expected mortalities ranged from 0.012(0.113) and 0.013(0.045) to 0.296(0.457) and 0.278(0.247) respectively. For the raw mortality series: 71 sites had continuous data for assessment up to or beyond lag ₄₀ and 35% had autocorrelation through to lag ₄₀; and of 36 sites with continuous data for ≥ 72 months, all demonstrated marked seasonality. Similar numbers and percentages were seen with the expected series. Out-of-control signalling was evident for the raw mortality series with respect to RA-EWMA control limits; a seasonal ARMA model, with GARCH effects, displayed white-noise residuals which were in-control with respect to EWMA control limits and one-step prediction error limits (3SE). The expected series was modelled with a multiplicative seasonal autoregressive model. CONCLUSIONS The data generating process of monthly raw mortality series at the ICU level displayed autocorrelation, seasonality and volatility. False-positive signalling of the raw mortality series was evident with respect to RA-EWMA control limits. A time series approach using residual control charts resolved these issues.John L Moran, Patricia J Solomo

Integrating Multiple Biomarkers of Fish Health: A Case Study of Fish Health in Ports

Biomarkers of fish health are recognised as valuable biomonitoring tools that inform on the impact of pollution on biota. The integration of a suite of biomarkers in a statistical analysis that better illustrates the effects of exposure to xenobiotics on living organisms is most informative; however, most published ecotoxicological studies base the interpretation of results on individual biomarkers rather than on the information they carry as a set. To compare the interpretation of results from individual biomarkers with an interpretation based on multivariate analysis, a case study was selected where fish health was examined in two species of fish captured in two ports located in Western Australia. The suite of variables selected included chemical analysis of white muscle, body condition index, liver somatic index (LSI), hepatic ethoxyresorufin-O-deethylase activity, serum sorbitol dehydrogenase activity, biliary polycyclic aromatic hydrocarbon metabolites, oxidative DNA damage as measured by serum 8-oxo-dG, and stress protein HSP70 measured on gill tissue. Statistical analysis of individual biomarkers suggested little consistent evidence of the effects of contaminants on fish health. However, when biomarkers were integrated as a set by principal component analysis, there was evidence that the health status of fish in Fremantle port was compromised mainly due to increased LSI and greater oxidative DNA damage in fish captured within the port area relative to fish captured at a remote site. The conclusions achieved using the integrated set of biomarkers show the importance of viewing biomarkers of fish health as a set of variables rather than as isolated biomarkers of fish health

Translocation of Threatened New Zealand Falcons to Vineyards Increases Nest Attendance, Brooding and Feeding Rates

Anthropogenic landscapes can be rich in resources, and may in some cases provide potential habitat for species whose natural habitat has declined. We used remote videography to assess whether reintroducing individuals of the threatened New Zealand falcon Falco novaeseelandiae into a highly modified agricultural habitat affected the feeding rates of breeding falcons or related breeding behavior such as nest attendance and brooding rates. Over 2,800 recording hours of footage were used to compare the behavior of falcons living in six natural nests (in unmanaged, hilly terrain between 4 km and 20 km from the nearest vineyard), with that of four breeding falcon pairs that had been transported into vineyards and nested within 500 m of the nearest vineyard. Falcons in vineyard nests had higher feeding rates, higher nest attendance, and higher brooding rates. As chick age increased, parents in vineyard nests fed chicks a greater amount of total prey and larger prey items on average than did parents in hill nests. Parents with larger broods brought in larger prey items and a greater total sum of prey biomass. Nevertheless, chicks in nests containing siblings received less daily biomass per individual than single chicks. Some of these results can be attributed to the supplementary feeding of falcons in vineyards. However, even after removing supplementary food from our analysis, falcons in vineyards still fed larger prey items to chicks than did parents in hill nests, suggesting that the anthropogenic habitat may be a viable source of quality food. Although agricultural regions globally are rarely associated with raptor conservation, these results suggest that translocating New Zealand falcons into vineyards has potential for the conservation of this species

Increasing the Depth of Current Understanding: Sensitivity Testing of Deep-Sea Larval Dispersal Models for Ecologists

Larval dispersal is an important ecological process of great interest to conservation and the establishment of marine protected areas. Increasing numbers of studies are turning to biophysical models to simulate dispersal patterns, including in the deep-sea, but for many ecologists unassisted by a physical oceanographer, a model can present as a black box. Sensitivity testing offers a means to test the models' abilities and limitations and is a starting point for all modelling efforts. The aim of this study is to illustrate a sensitivity testing process for the unassisted ecologist, through a deep-sea case study example, and demonstrate how sensitivity testing can be used to determine optimal model settings, assess model adequacy, and inform ecological interpretation of model outputs. Five input parameters are tested (timestep of particle simulator (TS), horizontal (HS) and vertical separation (VS) of release points, release frequency (RF), and temporal range (TR) of simulations) using a commonly employed pairing of models. The procedures used are relevant to all marine larval dispersal models. It is shown how the results of these tests can inform the future set up and interpretation of ecological studies in this area. For example, an optimal arrangement of release locations spanning a release area could be deduced; the increased depth range spanned in deep-sea studies may necessitate the stratification of dispersal simulations with different numbers of release locations at different depths; no fewer than 52 releases per year should be used unless biologically informed; three years of simulations chosen based on climatic extremes may provide results with 90% similarity to five years of simulation; and this model setup is not appropriate for simulating rare dispersal events. A step-by-step process, summarising advice on the sensitivity testing procedure, is provided to inform all future unassisted ecologists looking to run a larval dispersal simulation

Determinants of Habitat Selection by Hatchling Australian Freshwater Crocodiles

Animals almost always use habitats non-randomly, but the costs and benefits of using specific habitat types remain unknown for many types of organisms. In a large lake in northwestern Australia (Lake Argyle), most hatchling (<12-month-old) freshwater crocodiles (Crocodylus johnstoni) are found in floating vegetation mats or grassy banks rather than the more widely available open banks. Mean body sizes of young crocodiles did not differ among the three habitat types. We tested four potential explanations for non-random habitat selection: proximity to nesting sites, thermal conditions, food availability, and exposure to predation. The three alternative habitat types did not differ in proximity to nesting sites, or in thermal conditions. Habitats with higher food availability harboured more hatchlings, and feeding rates (obtained by stomach-flushing of recently-captured crocodiles) were highest in such areas. Predation risk may also differ among habitats: we were twice as likely to capture a crocodile after seeing it in open-bank sites than in the other two habitat types. Thus, habitat selection of hatchling crocodiles in this system may be driven both by prey availability and by predation risk

Evaluating the effectiveness of a tailored multifaceted performance feedback intervention to improve the quality of care: protocol for a cluster randomized trial in intensive care

Contains fulltext : 95871.pdf (publisher's version ) (Open Access)ABSTRACT: BACKGROUND: Feedback is potentially effective in improving the quality of care. However, merely sending reports is no guarantee that performance data are used as input for systematic quality improvement (QI). Therefore, we developed a multifaceted intervention tailored to prospectively analyzed barriers to using indicators: the Information Feedback on Quality Indicators (InFoQI) program. This program aims to promote the use of performance indicator data as input for local systematic QI. We will conduct a study to assess the impact of the InFoQI program on patient outcome and organizational process measures of care, and to gain insight into barriers and success factors that affected the program's impact. The study will be executed in the context of intensive care. This paper presents the study's protocol. METHODS/DESIGN: We will conduct a cluster randomized controlled trial with intensive care units (ICUs) in the Netherlands. We will include ICUs that submit indicator data to the Dutch National Intensive Care Evaluation (NICE) quality registry and that agree to allocate at least one intensivist and one ICU nurse for implementation of the intervention. Eligible ICUs (clusters) will be randomized to receive basic NICE registry feedback (control arm) or to participate in the InFoQI program (intervention arm). The InFoQI program consists of comprehensive feedback, establishing a local, multidisciplinary QI team, and educational outreach visits. The primary outcome measures will be length of ICU stay and the proportion of shifts with a bed occupancy rate above 80%. We will also conduct a process evaluation involving ICUs in the intervention arm to investigate their actual exposure to and experiences with the InFoQI program. DISCUSSION: The results of this study will inform those involved in providing ICU care on the feasibility of a tailored multifaceted performance feedback intervention and its ability to accelerate systematic and local quality improvement. Although our study will be conducted within the domain of intensive care, we believe our conclusions will be generalizable to other settings that have a quality registry including an indicator set available. TRIAL REGISTRATION: Current Controlled Trials ISRCTN50542146