Nucleotide-dependent shape changes in the reverse direction motor, myosin VI

We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca²⁺). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO₄, which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix.     

Systematics of genus Gnomoniopsis (Gnomoniaceae, Diaporthales) based on a three gene phylogeny, host associations and morphology

Species of Gnomoniopsis are leaf- and stem-inhabiting pyrenomycetes that infect plants in Fagaceae, Onagraceae and Rosaceae. Morphology and analyses of DNA sequences from three ribosomal DNA and protein coding regions, namely β-tubulin, translation elongation factor 1α (tef-1α) and the ITS region including ITS1, 5.8S rDNA and ITS2, were used to define species in Gnomoniopsis. Secondary structural alignment of the ITS region across four genera in Gnomoniaceae was used to increase the potential number of homologous positions in the ITS alignment. Ascospore isolates were grown from newly collected specimens. Type specimens were compared with these specimens to determine their identity. In this paper a recent concept of Gnomoniopsis is confirmed with phylogenetic resolution of additional species. Four new combinations and one new species are proposed. Nine species are described and illustrated, and a key is provided to the 13 species currently recognized in Gnomoniopsis.     

Fenoldopam use in a burn intensive care unit: a retrospective study

Background

We sought to evaluate agreement between a new and widely implemented method of temperature measurement in critical care, temporal artery thermometry and an established method of core temperature measurement, bladder thermometry as performed in clinical practice.

Methods

Temperatures were simultaneously recorded hourly (n = 736 observations) using both devices as part of routine clinical monitoring in 14 critically ill adult patients with temperatures ranging ≥1°C prior to consent.

Results

The mean difference between temporal artery and bladder temperatures measured was -0.44°C (95% confidence interval, -0.47°C to -0.41°C), with temporal artery readings lower than bladder temperatures. Agreement between the two devices was greatest for normothermia (36.0°C to < 38.3°C) (mean difference -0.35°C [95% confidence interval, -0.37°C to -0.33°C]). The temporal artery thermometer recorded higher temperatures during hypothermia (< 36°C) (mean difference 0.66°C [95% confidence interval, 0.53°C to 0.79°C]) and lower temperatures during hyperthermia (≥38.3°C) (mean difference -0.90°C [95% confidence interval, -0.99°C to -0.81°C]). The sensitivity for detecting fever (core temperature ≥38.3°C) using the temporal artery thermometer was 0.26 (95% confidence interval, 0.20 to 0.33), and the specificity was 0.99 (95% confidence interval, 0.98 to 0.99). The positive likelihood ratio for fever was 24.6 (95% confidence interval, 10.7 to 56.8); the negative likelihood ratio was 0.75 (95% confidence interval, 0.68 to 0.82).

Conclusions

Temporal artery thermometry produces somewhat surprising disagreement with an established method of core temperature measurement and should not to be used in situations where body temperature needs to be measured with accuracy.     

Study protocol of the YOU CALL - WE CALL TRIAL: impact of a multimodal support intervention after a "mild" stroke

Background

More than 60% of new strokes each year are "mild" in severity and this proportion is expected to rise in the years to come. Within our current health care system those with "mild" stroke are typically discharged home within days, without further referral to health or rehabilitation services other than advice to see their family physician. Those with mild stroke often have limited access to support from health professionals with stroke-specific knowledge who would typically provide critical information on topics such as secondary stroke prevention, community reintegration, medication counselling and problem solving with regard to specific concerns that arise. Isolation and lack of knowledge may lead to a worsening of health problems including stroke recurrence and unnecessary and costly health care utilization. The purpose of this study is to assess the effectiveness, for individuals who experience a first "mild" stroke, of a sustainable, low cost, multimodal support intervention (comprising information, education and telephone support) - "WE CALL" compared to a passive intervention (providing the name and phone number of a resource person available if they feel the need to) - "YOU CALL", on two primary outcomes: unplanned-use of health services for negative events and quality of life.

Method/Design

We will recruit 384 adults who meet inclusion criteria for a first mild stroke across six Canadian sites. Baseline measures will be taken within the first month after stroke onset. Participants will be stratified according to comorbidity level and randomised to one of two groups: YOU CALL or WE CALL. Both interventions will be offered over a six months period. Primary outcomes include unplanned use of heath services for negative event (frequency calendar) and quality of life (EQ-5D and Quality of Life Index). Secondary outcomes include participation level (LIFE-H), depression (Beck Depression Inventory II) and use of health services for health promotion or prevention (frequency calendar). Blind assessors will gather data at mid-intervention, end of intervention and one year follow up.

Discussion

If effective, this multimodal intervention could be delivered in both urban and rural environments. For example, existing infrastructure such as regional stroke centers and existing secondary stroke prevention clinics, make this intervention, if effective, deliverable and sustainable.

Trial Registration

ISRCTN95662526     

Role of Purα in the cellular response to ultraviolet-C radiation

Purα is a nucleic acid-binding protein with DNA-unwinding activity, which has recently been shown to have a role in the cellular response to DNA damage. We have investigated the function of Purα in Ultraviolet-C (UVC) radiation-induced DNA damage and nucleotide excision repair (NER). Mouse embryo fibroblasts from PURA^-/- knockout mice, which lack Purα, showed enhanced sensitivity to UVC irradiation as assessed by assays for cell viability and clonogenicity compared to Purα positive control cultures. In reporter plasmid reactivation assays to measure the removal of DNA adducts induced in vitro by UVC, the Purα-negative cells were less efficient in DNA damage repair. Purα-negative cells were also more sensitive to UVC-induced DNA damage measured by Comet assay and showed a decreased ability to remove UVC-induced cyclobutane pyrimidine dimers. In wild-type mouse fibroblasts, expression of Purα is induced following S-phase checkpoint activation by UVC in a similar manner to the NER factor TFIIH. Moreover, co-immunoprecipitation experiments showed that Purα physically associates with TFIIH. Thus, Purα has a role in NER and the repair of UVC-induced DNA damage.Key words: purα, ultraviolet radiation, DNA damage, DNA repair, nucleotide excision repair, TFIIH     

Use of Data-Biased Random Walks on Graphs for the Retrieval of Context-Specific Networks from Genomic Data

Extracting network-based functional relationships within genomic datasets is an important challenge in the computational analysis of large-scale data. Although many methods, both public and commercial, have been developed, the problem of identifying networks of interactions that are most relevant to the given input data still remains an open issue. Here, we have leveraged the method of random walks on graphs as a powerful platform for scoring network components based on simultaneous assessment of the experimental data as well as local network connectivity. Using this method, NetWalk, we can calculate distribution of Edge Flux values associated with each interaction in the network, which reflects the relevance of interactions based on the experimental data. We show that network-based analyses of genomic data are simpler and more accurate using NetWalk than with some of the currently employed methods. We also present NetWalk analysis of microarray gene expression data from MCF7 cells exposed to different doses of doxorubicin, which reveals a switch-like pattern in the p53 regulated network in cell cycle arrest and apoptosis. Our analyses demonstrate the use of NetWalk as a valuable tool in generating high-confidence hypotheses from high-content genomic data.     

Oxygen consumption is maintained in fetal sheep during prolonged hypoxaemia.

Experiments were conducted in 12 chronically-catheterized pregnant sheep to examine the effect of prolonged hypoxaemia secondary to the restriction of uterine blood flow on fetal oxygen consumption. Surgery was performed at 115 days gestation to place a teflon vascular occluder around the maternal common internal iliac artery and for insertion of vascular catheters. Following a 5-day recovery period, uterine blood flow was reduced in 6 animals for 24 hours and in 6 animals, the occluder was not adjusted. Fetal arterial PO2 decreased from 19.9 +/- 2.0 mmHg to 12.8 +/- 2.0 mmHg and 11.0 +/- 2.0 mmHg at 1 and 24 hours respectively in the experimental group and did not change the control group. Fetal pH decreased from 7.34 +/- 0.01 to 7.25 +/- 0.03 and 7.29 +/- 0.02 at 1 and 24 hours of hypoxaemia respectively. Fetal arterial lactate concentrations remained elevated throughout the experimental period with maximum concentrations of 6.6 +/- 2.1 mmol/l being present at 4 hours compared to 1.3 +/- 0.2 mmol/l during the control period. Umbilical blood flow increased from 186 +/- 19 ml/min/kg to 251 +/- 39 ml/min/kg at 1 h of hypoxaemia and returned to 191 +/- 21 ml/min/kg at 24 h. In association with the progressive fall in oxygen delivery to the fetus, oxygen extraction increased from 0.33 +/- 0.04 to 0.43 +/- 0.04 and 0.54 +/- 0.05 at 1 and 24 hours, respectively. Overall oxygen consumption by the fetus remained unchanged from control values.(ABSTRACT TRUNCATED AT 250 WORDS)