Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

Modeling interactions between voltage-gated Ca2+ channels and KCa1.1 channels

High voltage-activated (HVA) Cav channels form complexes with KCa1.1 channels, allowing reliable activation of KCa1.1 current through a nanodomain interaction. We recently found that low voltage-activated Cav3 calcium channels also create KCa1.1-Cav3 complexes. While coimmunoprecipitation studies again supported a nanodomain interaction, the sensitivity to calcium chelating agents was instead consistent with a microdomain interaction. A computational model of the KCa1.1-Cav3 complex suggested that multiple Cav3 channels were necessary to activate KCa1.1 channels, potentially causing the KCa1.1-Cav3 complex to be more susceptible to calcium chelators. Here, we expanded the model and compared it to a KCa1.1-Cav2.2 model to examine the role of Cav channel conductance and kinetics on KCa1.1 activation. As found for direct recordings, the voltage-dependent and kinetic properties of Cav3 channels were reflected in the activation of KCa1.1 current, including transient activation from lower voltages than other KCa1.1-Cav complexes. Substantial activation of KCa1.1 channels required the concerted activity of several Cav3.2 channels. Combined with the effect of EGTA, these results suggest that the Ca²⁺ domains of several KCa1.1-Cav3 complexes need to cooperate to generate sufficient [Ca²⁺]_i, despite the physical association between KCa1.1 and Cav3 channels. By comparison, Cav2.2 channels were twice as effective at activating KCa1.1 channels and a single KCa1.1-Cav2.2 complex would be self-sufficient. However, even though Cav3 channels generate small, transient currents, the regulation of KCa1.1 activity by Cav3 channels is possible if multiple complexes cooperate through microdomain interactions.     

New parasites and predators follow the introduction of two fish species to a subarctic lake: implications for food-web structure and functioning

Introduced species can alter the topology of food webs. For instance, an introduction can aid the arrival of free-living consumers using the new species as a resource, while new parasites may also arrive with the introduced species. Food-web responses to species additions can thus be far more complex than anticipated. In a subarctic pelagic food web with free-living and parasitic species, two fish species (arctic charr Salvelinus alpinus and three-spined stickleback Gasterosteus aculeatus) have known histories as deliberate introductions. The effects of these introductions on the food web were explored by comparing the current pelagic web with a heuristic reconstruction of the pre-introduction web. Extinctions caused by these introductions could not be evaluated by this approach. The introduced fish species have become important hubs in the trophic network, interacting with numerous parasites, predators and prey. In particular, five parasite species and four predatory bird species depend on the two introduced species as obligate trophic resources in the pelagic web and could therefore not have been present in the pre-introduction network. The presence of the two introduced fish species and the arrival of their associated parasites and predators increased biodiversity, mean trophic level, linkage density, and nestedness; altering both the network structure and functioning of the pelagic web. Parasites, in particular trophically transmitted species, had a prominent role in the network alterations that followed the introductions.     

Simultaneous determination of 6 beta-blockers, 3 calcium-channel antagonists, 4 angiotensin-II antagonists and 1 antiarrhythmic drug in post-mortem whole blood by automated solid phase extraction and liquid chromatography mass spectrometry. Method development and robustness testing by experimental design

A method for the simultaneous determination of the beta-blockers atenolol, sotalol, metoprolol, bisoprolol, propranolol and carvedilol, the calcium-channel antagonists diltiazem, amlodipine and verapamil, the angiotensin-II antagonists losartan, irbesartan, valsartan and telmisartan, and the antiarrhythmic drug flecainide, in whole blood samples from forensic autopsies was developed. Sample clean-up was achieved by precipitation and solid phase extraction (SPE) with a mixed-mode column. Quantification was performed by reversed phase high performance liquid chromatography with positive electrospray ionization mass spectrometric detection (HPLC-MS). The method has been developed and robustness tested by systematically searching for satisfactory conditions using experimental designs including factorial and response surface designs. With the exception of amlodipine, the concentration limit of quantification (cLOQ) covered low therapeutic concentration levels for all the compounds. Within assay precisions and accuracies (bias) were 3.4-21% RSD and from -24 to 21% for the concentration range 1.00-5.00 microM, respectively. Between assay precisions were 4.4-28% RSD for the concentration range from 0.1 to 5 microM and recoveries varied from 9 to 103%. The method is used for determination of cardiovascular drugs in post-mortem whole blood samples from forensic autopsy cases.     

Calcium dynamics during fertilization in C. elegans

Background

Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism, we have characterized fertilization-induced calcium dynamics in C. elegans.

Results

Owing to the transparency of the nematode, we have been able to study the calcium signal in C. elegans fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that is initiated soon after oocyte entry into the spermatheca, the compartment that contains sperm. Therefore, it is likely that the calcium transient is initiated by contact with sperm. This calcium elevation spreads throughout the oocyte, and decays monotonically after which the cytosolic calcium concentration returns to that preceding fertilization. Only this single calcium transient is observed.

Conclusion

Development of a technique to study fertilization induced calcium transients opens several experimental possibilities, e.g., identification of the signaling events intervening sperm binding and calcium elevation, identifying the possible roles of the calcium elevation such as the completion of meiosis, the formation of the eggshell, and the establishing of the embryo's axis of symmetry.     

Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function. Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea. We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA. The purified AfalkA protein differs from E. coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) > 3-methylguanine approximately 7-methyladenine > 7-methylguanine. Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A. fulgidus. At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates. The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures. This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.     

Developmental regulation of the maize Zm-p60.1 gene encoding a β-glucosidase located to plastids

A β-glucosidase that cleaves the biologically inactive hormone conjugates cytokinin-O- and kinetin-N3-glucosides is encoded by the maize Zm-p60.1 gene. The expression of the Zm-p60.1 gene was analyzed by Northern blot analysis and in-situ hybridization. It was found that the expression levels of the Zm-p60.1-specific mRNA changed after pollination of carpellate inflorescences. The Zm-p60.1 cDNA was expressed in E. coli and antibodies were raised against this protein. An antibody was used to determine the tissue-specific localization of this protein. By in situ immunolocalization experiments, this protein was found to be located in cell layers below the epidermis and around the vascular bundles of the coleoptile. In the primary leaf, the Zm-p60.1 protein was detected in cells of the outermost cell layer and around the vascular tissue. In floral tissue, Zm-p60.1 was present in the glumes, the carpels and in the outer cell layer of the style. In coleoptiles, as determined by immuno-electronmicroscopy, the Zm-p60.1 protein was located exclusively in the plastids. Received: 11 August 1998 / Accepted: 30 December 1998     

30-Day Survival Probabilities as a Quality Indicator for Norwegian Hospitals: Data Management and Analysis

Background

The Norwegian Knowledge Centre for the Health Services (NOKC) reports 30-day survival as a quality indicator for Norwegian hospitals. The indicators have been published annually since 2011 on the website of the Norwegian Directorate of Health (www.helsenorge.no), as part of the Norwegian Quality Indicator System authorized by the Ministry of Health. Openness regarding calculation of quality indicators is important, as it provides the opportunity to critically review and discuss the method. The purpose of this article is to describe the data collection, data pre-processing, and data analyses, as carried out by NOKC, for the calculation of 30-day risk-adjusted survival probability as a quality indicator.

Methods and Findings

Three diagnosis-specific 30-day survival indicators (first time acute myocardial infarction (AMI), stroke and hip fracture) are estimated based on all-cause deaths, occurring in-hospital or out-of-hospital, within 30 days counting from the first day of hospitalization. Furthermore, a hospital-wide (i.e. overall) 30-day survival indicator is calculated. Patient administrative data from all Norwegian hospitals and information from the Norwegian Population Register are retrieved annually, and linked to datasets for previous years. The outcome (alive/death within 30 days) is attributed to every hospital by the fraction of time spent in each hospital. A logistic regression followed by a hierarchical Bayesian analysis is used for the estimation of risk-adjusted survival probabilities. A multiple testing procedure with a false discovery rate of 5% is used to identify hospitals, hospital trusts and regional health authorities with significantly higher/lower survival than the reference. In addition, estimated risk-adjusted survival probabilities are published per hospital, hospital trust and regional health authority. The variation in risk-adjusted survival probabilities across hospitals for AMI shows a decreasing trend over time: estimated survival probabilities for AMI in 2011 varied from 80.6% (in the hospital with lowest estimated survival) to 91.7% (in the hospital with highest estimated survival), whereas it ranged from 83.8% to 91.2% in 2013.

Conclusions

Since 2011, several hospitals and hospital trusts have initiated quality improvement projects, and some of the hospitals have improved the survival over these years. Public reporting of survival/mortality indicators are increasingly being used as quality measures of health care systems. Openness regarding the methods used to calculate the indicators are important, as it provides the opportunity of critically reviewing and discussing the methods in the literature. In this way, the methods employed for establishing the indicators may be improved.     

Carrot psyllid (Trioza apicalis) winter habitats – insights in shelter plant preference and migratory capacity

Abstract:  The carrot psyllid ( Trioza apicalis ) is a serious pest in carrot-growing areas in northern and parts of central Europe. The psyllids overwinter as adults on conifers and during summer feed and lay eggs on carrot plants ( Daucus carota ssp. sativus ), thereby destroying the crop. To investigate the migratory capabilities and preferences for different shelter plant species of the carrot psyllid, we made an inventory study of its winter habitats in three carrot-growing regions in southern Sweden. Norway spruce ( Picea abies ) was the preferred conifer over Scots pine ( Pinus sylvestris ) and juniper ( Juniperus communis ). We found psyllids on trees up to 1 km from the carrot fields, which was the largest distance sampled. The regression of catch numbers over distance was non-significant, however all samples containing more than seven psyllids were collected within 250m distance from the fields. There was no obvious pattern between catch directions and the prevailing wind directions of the preceding migratory period. Our study did not show any differences between males and females with respect to migration or shelter species preferences.