Future trends in measuring physiology in free-living animals.

Thus far, ecophysiology research has predominantly been conducted within controlled laboratory-based environments, owing to a mismatch between the recording technologies available for physiological monitoring in wild animals and the suite of behaviours and environments they need to withstand, without unduly affecting subjects. While it is possible to record some physiological variables for free-living animals using animal-attached logging devices, including inertial-measurement, heart-rate and temperature loggers, the field is still in its infancy. In this opinion piece, we review the most important future research directions for advancing the field of 'physiologging' in wild animals, including the technological development that we anticipate will be required, and the fiscal and ethical challenges that must be overcome. Non-invasive, multi-sensor miniature devices are ubiquitous in the world of human health and fitness monitoring, creating invaluable opportunities for animal and human physiologging to drive synergistic advances. We argue that by capitalizing on the research efforts and advancements made in the development of human wearables, it will be possible to design the non-invasive loggers needed by ecophysiologists to collect accurate physiological data from free-ranging animals ethically and with an absolute minimum of impact. In turn, findings have the capacity to foster transformative advances in human health monitoring. Thus, we invite biomedical engineers and researchers to collaborate with the animal-tagging community to drive forward the advancements necessary to realize the full potential of both fields. This article is part of the theme issue 'Measuring physiology in free-living animals (Part II)'.

Restricted gene flow and fine-scale population structuring in tool using New Caledonian crows.

New Caledonian crows Corvus moneduloides are the most prolific avian tool users. It has been suggested that some aspects of their complex tool use behaviour are under the influence of cultural processes, involving the social transmission-and perhaps even progressive refinement-of tool designs. Using microsatellite and mt-haplotype profiling of crows from three distinct habitats (dry forest, farmland and beachside habitat), we show that New Caledonian crow populations can exhibit significant fine-scale genetic structuring. Our finding that some sites of <10 km apart were highly differentiated demonstrates considerable potential for genetic and/or cultural isolation of crow groups. Restricted movement of birds between local populations at such small spatial scales, especially across habitat boundaries, illustrates how specific tool designs could be preserved over time, and how tool technologies of different crow groups could diverge due to drift and local selection pressures. Young New Caledonian crows have an unusually long juvenile dependency period, during which they acquire complex tool-related foraging skills. We suggest that the resulting delayed natal dispersal drives population-divergence patterns in this species. Our work provides essential context for future studies that examine the genetic makeup of crow populations across larger geographic areas, including localities with suspected cultural differences in crow tool technologies.

Identifying governance strategies that effectively support ecosystem services, resource sustainability, and biodiversity.

Conservation scientists, national governments, and international conservation groups seek to devise, and implement, governance strategies that mitigate human impact on the environment. However, few studies to date have systematically investigated the performance of different systems of governance in achieving successful conservation outcomes. Here, we use a newly-developed analytic framework to conduct analyses of a suite of case studies, linking different governance strategies to standardized scores for delivering ecosystem services, achieving sustainable use of natural resources, and conserving biodiversity, at both local and international levels. Our results: (i) confirm the benefits of adaptive management; and (ii) reveal strong associations for the role of leadership. Our work provides a critical step toward implementing empirically justified governance strategies that are capable of improving the management of human-altered environments, with benefits for both biodiversity and people.

[Avian influenza: wildbird monitoring in Switzerland between 2003-2006]. / Aviäre Influenza: Wildvogelmonitoring in der Schweiz zwischen 2003-2006.

During various surveillance programs more than 3500 cloacal swabs and organ samples from songbirds, waterbirds and poultry have been tested for avian influenza using real time RT-PCR. Switzerland carried out the first wildbird monitoring between autumn 2003 and spring 2005. 1053 samples, mostly from songbirds, were tested. LPAI-strains were found in two cases. A second intensified surveillance program started in October 2005 along with the first ban on free range poultry farming. Until the end of April 2006 2455 cloacal swabs from dead wildbirds have been analysed. By the end of february H5N1 was for the first time detected in Switzerland and by the end of march 32 waterbirds have been found positive for H5N1. 146 poultry flocks with a special permission for free range management proved to be AI negative.

Molecular and conformational features of a transport-relevant domain in the C-terminal tail of the vasopressin V(2) receptor.

We have previously shown a conserved glutamate/dileucine motif ((335)ELRSLL(340)) in the intracellular C terminus of the vasopressin V(2) receptor (V(2) receptor) to be essential for receptor transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The motif may represent a transport signal that is recognized by a component of ER to Golgi vesicles. Alternatively, it may be necessary for transport-competent receptor folding to pass the quality-control system of the ER. To assess these two possibilities, we constructed a receptor fragment that allows transport studies independent of full-length receptor folding. Transmembrane domains II-VII were deleted, thereby fusing the intracellular C terminus to the first cytoplasmic loop. The mutations that impaired transport of the full-length receptor were introduced, and receptor fragments were localized in transiently transfected HEK 293 cells. All mutant receptor fragments were detectable at the plasma membrane, demonstrating that the glutamate/dileucine motif does not function as a small, linear vesicular transport signal. Instead, our data strongly suggest that this motif is required for transport-competent folding of the full-length receptor. To assess the underlying conformational features, a three-dimensional homology model of the V(2) receptor was computed. Our model predicts that the glutamate/dileucine motif contributes to a U-like loop within the intracellular C terminus. Residue Leu(339) may be required for folding back the intracellular C terminus to residue Leu(62) of the first cytoplasmic loop. We characterized the naturally occurring L62P and DeltaL62-R64 mutations in the first cytoplasmic loop and show that they lead to transport-defective full-length V(2) receptors that are retained in the ER, consistent with the structure model.

A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain.

The orientation of membrane proteins is determined by the asymmetric distribution of charged residues in the sequences flanking the transmembrane domains. For the inner membrane of Escherichia coli, numerous studies have shown that an excess of positively charged residues defines a cytoplasmic domain of a membrane protein ("positive inside" rule). The role of negatively charged residues in establishing membrane protein topology, however, is not completely understood. To investigate the influence of negatively charged residues on this process in detail, we have constructed a single spanning chimeric receptor fragment comprising the N terminus and first transmembrane domain of the heptahelical G protein-coupled vasopressin V(2) receptor and the first cytoplasmic loop of the beta(2)-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane of E. coli with its N terminus facing the cytoplasm (N(in)-C(out) orientation), although both membrane-flanking domains had rather similar topogenic determinants. The orientation of the receptor fragment was changed after the introduction of single glutamate residues into the N terminus. Orientation inversion, however, was found to be dependent on the location of the glutamate substitutions, which had to lie within a narrow window up to 6 residues distant from the transmembrane domain. These results demonstrate that a single negatively charged residue can play an active role as a topogenic determinant of membrane proteins in the inner membrane of E. coli, but only if it is located adjacent to a transmembrane domain.

Polarized expression of the vasopressin V2 receptor in Madin-Darby canine kidney cells.

BACKGROUND: The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented. METHODS: Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [3H]-arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti-c-myc antibody. RESULTS: The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell. CONCLUSION: The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor.

Topology of eukaryotic multispanning transmembrane proteins: use of LacZ fusions for the localization of cytoplasmic domains in COS.M6 cells.

In Escherichia coli, the topology of inner membrane proteins can be studied conveniently with the alkaline phosphatase/beta-galactosidase (PhoA/LacZ) gene fusion system. PhoA is enzymatically active only when fused to external domains, LacZ when fused to cytoplasmic domains. In eukaryotic cells, only time consuming methods exist to study the topology of membrane proteins. We have extended in the first systematic study the PhoA/LacZ gene fusion system originally developed for E.coli for use in eukaryotic COS.M6 cells. We have fused PhoA and LacZ to the putative external and cytoplasmic loops of rat aquaporin 2 (AQP2), for which a model with six transmembrane domains was proposed previously. The fusion proteins were expressed in E.coli and COS.M6 cells and immunoblot analyses and enzyme activity assays were performed to localize the protein domains in both cell types. The data obtained in E.coli correlated mostly with the predictions of the six transmembrane domain model. However, two fusions were found to exhibit both high PhoA and high LacZ activity, thereby complicating the construction of a complete AQP2 model. In COS.M6 cells, the PhoA fusions were inactive. In contrast, the LacZ fusions succeeded and showed an activity pattern in complete agreement with the predictions of the six transmembrane domain model. Therefore, LacZ fusions can localize cytoplasmic loops in COS.M6 cells by means of a simple enzymatic assay with high reliability and may be used in future studies to develop topological models of other eukaryotic membrane proteins in their authentic cell systems.

A team approach to managing an information security program.

How can HIM professionals step into the spotlight as facilities establish information security policies and procedures? At Hartford Hospital, HIM practitioners collaborated with other departments and formed a team that creatively implements and manages the security process.

Membrane targeting and determination of transmembrane topology of the human vasopressin V2 receptor.

The human vasopressin V2 receptor belongs to the large family of G-protein-coupled receptors, which possess seven transmembrane helices, an extracellular N terminus and an intracellular C terminus. We have determined the sequence requirements of the V2 receptor for membrane insertion and correct topology for the inner membrane of Escherichia coli with the PhoA/LacZ gene fusion system. In addition, we have studied the signals for its membrane insertion and correct topology for the membrane of the endoplasmic reticulum of the authentic eucaryotic transport system. To this end, we have extended the PhoA/LacZ gene fusion system for the first time to eucaryotic cells, i.e. transiently transfected COS.M6 cells. Truncated V2 receptor sequences were fused to PhoA and LacZ and expressed in both E. coli and COS.M6 cells. Cells were fractionated, and LacZ/PhoA activity assays and immunoblots were performed. We show here that a V2 receptor fragment consisting of the N terminus, the first transmembrane segment and the first cytoplasmic loop (71 amino acids) provided sufficient information for membrane insertion and correct orientation (extracellular N terminus) in both procaryotic and eucaryotic cells. Our data differ substantially from those obtained for the human beta2-adrenergic receptor expressed in E. coli (Lacatena, R. M., Cellini, A., Scavizzi, F., and Tocchini-Valentini, G. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10521-10525). To establish correct topology, the beta2-adrenergic receptor requires a larger receptor portion, including the three N-terminal transmembrane segments and/or parts of the second cytoplasmic loop. The present data show that the observations made for the beta2-adrenergic receptor cannot be applied to G-protein-coupled receptors generally.

Vasopressin V2 receptor mutants that cause X-linked nephrogenic diabetes insipidus: analysis of expression, processing, and function.

We investigated the biochemical and functional properties of five vasopressin V2 receptor mutants (L44F, L44P, W164S, S167L, and S167T) that were recently described in families with a history of X-linked nephrogenic diabetes insipidus. COS.M6 cells transfected with cDNA encoding these mutants acquired < 4% specific [3H]arginine vasopressin (AVP) binding sites on the cell surface in comparison with cells transfected with cDNA coding for the wild-type receptor. Membrane preparations from COS.M6 cells or human embryonic kidney 293 cells expressing these mutants did not respond with an increase in adenylyl cyclase activity in response to AVP, which is in contrast to membranes from cells expressing the wild-type. By analyzing fusion proteins of the V2 receptor and Escherichia coli alkaline phosphatase attached to the carboxyl terminus of the receptor moiety, we found that the mutants L44P, W164S, S167L, and S167T lacked complex glycosylation and were expressed at low levels. The data suggest that the mutants L44P, W164S, S167T, and S167L are misfolded and therefore retained within the endoplasmic reticulum and degraded. In contrast, the fusion proteins carrying the mutant L44F and the in vitro mutant S167A were expressed in their mature form at wild-type levels; however, only the mutant S167A was functionally active. Site-directed mutagenesis of S167 revealed that elimination of the endogenous hydroxyl group (S167A) yielded a protein with properties identical to those of the wild-type receptor, whereas both the introduction of a methyl group (S167T) and the replacement of the hydroxyl group by an isopropyl group (S167L) profoundly disturbed receptor processing. The data show that minute changes at codon 167 nearly abolish expression of a mature protein, thus defining structural requirements of this codon.

Cost effectiveness of quality control in bacteriology.

The cost effectiveness of quality control in bacteriology stipulated by regulators is not established. The authors evaluated 111 surveillance procedures applied to 54 different operations; 100 had been performed in the authors' lab between 1965 and 1980, 91 of which had been performed 50 times. Forty-six conformed to CLIA requirements (CLIA-P). Thirty-seven others were CLIA-P, which had been modified (CLIA-PM) by reducing frequency and extent because few or no deficiencies had been observed. Eight others were devised by the authors (HH-P). The number detecting deficiencies, the per cent, and the mean per cent of deficiencies detected were: CLIA-P, 31, 67%, 3.5%; CLIA-PM, 8, 22%, 2.1%; HH-P, 8, 100%, 8.8%. Compliance with CLIA would cost HH $20,700/year (3.4% of total bacteriology laboratory cost). HH-P would cost an additional $9000/year. Discontinuation of CLIA-P not detecting deficiencies would reduce HH costs by $2900/year. Application of other low yield CLIA-P only to new lots of selected dehydrated media and fresh batches of selected reagents would reduce cost further by $2000/year.

Processing control and cost in bacteriology.

Controlled processing was applied to lower respiratory, wound, and cervicovaginal exudates, and urine. The extent of processing was determined by assessment of quality in direct smears and/or by limits placed on complete identification and antimicrobial susceptibility testing of isolates in mixed cultures. Some specimens were not cultured; others were cultured, but certain isolates were identified by colonial morphologic features only. The time in minutes for performing 22 processing operations was determined. The average number of operations performed with processing control was established along with the total cost per minute of labor expended. A 19% reduction was observed in the time expended with controlled processing relative to projected time expended without processing controls. Urine specimens yielded the greatest saving. Processing control speeds recollection of poor-quality specimens, provides reporting of results of examination of all Gram-stained direct smears, minimizes reporting of information potentially misleading to physicians, and may reduce other health-care costs through improved patient care.

Comparative costs of microbial identification employing conventional and prepackaged commercial systems.

The accuracy of commercially prepackaged kits for microbial identification has become well established. Laboratory workers may encounter increasing demands for production of objective data to justify replacement of systems using individual biochemical tests in tubes. The authors present a system of cost analysis in which materials and labor costs are separately computed, and to which are added the effects of (1) fringe benefits, (2) decreased productivity resulting from administration, quality control, education and development, and (3) the additional expense of indirect costs that are allocated to laboratory procedures by accepted and standardized hospital accounting methods. Labor costs should be based on time-engineered studies conducted in individual laboratories. Alternatively, various published "unit values" may be used. The result may present several alternative differences in cost, depending on which unit values are accepted as applicable to the individual laboratory. Despite these uncertainties, the method of analysis provides a more objective means of justifying the cost of introduction of prepackaged kits where accuracy and speed of identification have already been proven to have advantages over biochemical tests in tubes.