[Dyspnea]. / Dyspnoe.

Dyspnea represents one of the most frequent cardinal symptoms in general practice and interdisciplinary emergency care across all sectors. Due to its subjective character, dyspnea is described by patients in many different ways, including "shortness of breath, difficulty of breathing, feeling of chest tightness, etc". The spectrum of differential diagnoses is broad, including in particular pulmonary and cardiovascular diseases. In addition to an evaluation of severity and an assessment of temporal, situation-related, and causal classification criteria, a structured process of multiple diagnostic steps in both primary and emergency care is a prerequisite for fast and correct diagnosis. In this context, it is of crucial importance to identify life-threatening diseases according to defined criteria and thus initiate adequate emergency measures. Further treatment options at the interface between primary and clinical care can be based on the German Appropriate Evaluation Protocol (G-AEP) criteria.

From species divergence to population structure: a multimarker approach on the most basal lineage of Salamandridae, the spectacled salamanders (genus Salamandrina) from Italy.

The Apennine Peninsula is one of Europe's main glacial refugial areas and harbors a large number of lineages and species. Here, a pattern of higher genetic diversity in the south compared to that of the north is characteristic of most vertebrates; however, most studies that have produced these results have relied only on inferences based on mitochondrial DNA. The spectacled salamanders (genus Salamandrina) are endemic to the Apennine Peninsula and have diverged into two sibling species: S. terdigitata (in the south) and S. perspicillata (in the north), presumably in the late Miocene or early Pliocene. By sequencing one mitochondrial (cytb) and two nuclear genes (RAG1 and POMC) and genotyping 10 microsatellite loci, we traced the evolution of these sibling species from their divergence to their contemporary population structure at a fine scale. Using a multilocus coalescent-based approach, we estimated the temporal divergence of both species at approximately 2.25 mya (million years ago), which, hence, is much younger than previous estimates. The classical pattern of high genetic diversity in the south and lower diversity in the north was confirmed only for some markers, and the demographic histories of the two species differed substantially. Whereas S. perspicillata (north) expanded from a single major refugium in the center of the Apennine Peninsula, populations of S. terdigitata (south) persisted through cooler periods in multiple refugia. Further, the fine-scale population genetic structure of 16 S. perspicillata populations revealed significant genetic differentiation, even across short geographic distances. The results of our study stress that for a better understanding of phylogeographic patterns and past demographic processes, both mitochondrial and multiple nuclear loci should be analyzed to avoid gene-specific, and possibly biased results.

Nuclear and mitochondrial multilocus phylogeny and survey of alkaloid content in true salamanders of the genus Salamandra (Salamandridae).

The genus Salamandra represents a clade of six species of Palearctic salamanders of either contrasted black-yellow, or uniformly black coloration, known to contain steroidal alkaloid toxins in high concentrations in their skin secretions. This study reconstructs the phylogeny of the genus Salamandra based on DNA sequences of segments of 10 mitochondrial and 13 nuclear genes from 31 individual samples representing all Salamandra species and most of the commonly recognized subspecies. The concatenated analysis of the complete dataset produced a fully resolved tree with most nodes strongly supported, suggesting that a clade composed of the Alpine salamander (S. atra) and the Corsican fire salamander (S. corsica) is the sister taxon to a clade containing the remaining species, among which S. algira and S. salamandra are sister species. Separate analyses of mitochondrial and nuclear data partitions disagreed regarding basal nodes and in the position of the root but concordantly recovered the S. atra/S. corsica as well as the S. salamandra/S. algira relationship. A species-tree analysis suggested almost simultaneous temporal splits between these pairs of species, which we hypothesize was caused by vicariance events after the Messinian salinity crisis (from late Miocene to early Pliocene). A survey of toxins with combined gas chromatography/mass spectroscopy confirmed the presence of samandarine and/or samandarone steroidal alkaloids in all species of Salamandra as well as in representatives of their sister group, Lyciasalamandra. Samandarone was also detected in lower concentrations in other salamandrids including Calotriton, Euproctus, Lissotriton, and Triturus, suggesting that the presence and possible biosynthesis of this alkaloid is plesiomorphic within the Salamandridae.

Radically different phylogeographies and patterns of genetic variation in two European brown frogs, genus Rana.

We reconstruct range-wide phylogeographies of two widespread and largely co-occurring Western Palearctic frogs, Rana temporaria and R. dalmatina. Based on tissue or saliva samples of over 1000 individuals, we compare a variety of genetic marker systems, including mitochondrial DNA, single-copy protein-coding nuclear genes, microsatellite loci, and single nucleotide polymorphisms (SNPs) of transcriptomes of both species. The two focal species differ radically in their phylogeographic structure, with R. temporaria being strongly variable among and within populations, and R. dalmatina homogeneous across Europe with a single strongly differentiated population in southern Italy. These differences were observed across the various markers studied, including microsatellites and SNP density, but especially in protein-coding nuclear genes where R. dalmatina had extremely low heterozygosity values across its range, including potential refugial areas. On the contrary, R. temporaria had comparably high range-wide values, including many areas of probable postglacial colonization. A phylogeny of R. temporaria based on various concatenated mtDNA genes revealed that two haplotype clades endemic to Iberia form a paraphyletic group at the base of the cladogram, and all other haplotypes form a monophyletic group, in agreement with an Iberian origin of the species. Demographic analysis suggests that R. temporaria and R. dalmatina have genealogies of roughly the same time to coalescence (TMRCA ~3.5 mya for both species), but R. temporaria might have been characterized by larger ancestral and current effective population sizes than R. dalmatina. The high genetic variation in R. temporaria can therefore be explained by its early range expansion out of Iberia, with subsequent cycles of differentiation in cryptic glacial refugial areas followed by admixture, while the range expansion of R. dalmatina into central Europe is a probably more recent event.

A multigene species tree for Western Mediterranean painted frogs (Discoglossus).

Painted frogs (Discoglossus) are an anuran clade that originated in the Upper Miocene. Extant species are morphologically similar and have a circum-Mediterranean distribution. We assembled a multilocus dataset from seven nuclear and four mitochondrial genes for several individuals of all but one of the extant species and reconstructed a robust phylogeny by applying a coalescent-based species-tree method and a concatenation approach, both of which gave congruent results. The earliest phylogenetic split within Discoglossus separates D. montalentii from a clade comprising all other species. Discoglossus montalentii is monophyletic for haplotype variation at all loci and has distinct morphological, bioacoustic and karyotypic characters. We find moderate support for a sister-group relationship between the Iberian taxa and the Moroccan D. scovazzi, and high support for a D. pictus -D. sardus clade distributed around the Tyrrhenian basin. Topological discordance among gene trees during the speciation of D. galganoi, D. scovazzi, D. pictus and D. sardus is interpreted as the consequence of nearly simultaneous, vicariant diversification. The timing of these events is unclear, but possibly coincided with the final geotectonic rearrangement of the Western Mediterranean in the Middle Miocene or later during the Messinian salinity crisis. The Iberian taxa D. galganoi galganoi and D. g. jeanneae are reciprocally monophyletic in mitochondrial DNA but not in nuclear gene trees, and are therefore treated as subspecies of D. galganoi.

[Modeling the requirements on routine data of general practitioners from the health-care researcher's point of view with the help of unified modeling langauge (UML)]. / Modellierung von Anforderungen an hausärztliche Routinedaten aus Sicht der Versorgungsforschung mithilfe der Unified Modeling Language (UML).

BACKGROUND AND AIMS: Health-care research is, besides primary acquired study data, based on data from widely differing secondary sources. In order to link, compare and analyze data sources uniform models and methods are needed. This could be facilitated by a more structured description of requirements, models and methods of health-care research than those currently used. Suitable methods of presentation were sought in an approach to this target and the unified modeling language (UML) identified as a possible alternative. METHODS: Using different tools 3 UML diagrams were created to represent some individual aspects of a scientific use file (SUF): A use case diagram as well as an activity and a class diagram. In the use case diagram we attempted to represent the general use cases of an SUF based on general practitioners routine data. Secondly a class diagram was constructed to visualize the contents and structure of a SUF. Thirdly an activity diagram was developed to graphically represent the concept of a general practitioner's episode of care. RESULTS: The creation of the UML diagrams was possible without any technical difficulties. Regarding the content the 3 diagrams must still be considered as prototypes. The use case diagram shows possible uses and users of an SUF, e. g. a research worker, industry but also the general practitioner who supplies the data. The class diagram reveals a general data structure that can serve information processes in practice and research. Besides aggregation, possibilities for specialization and generalization are essential elements of the class diagram that can be used meaningfully. The activity diagram for the schematic representation of a general practitioner's episode of care reveals the existence of multiple endpoints of an episode and the possibility to form relationships by means of episodes (diagnosis>therapy). CONCLUSION: The constructed diagrams are preliminary results and should be refined in future steps. Use case diagrams enable a rapid overview of the meaning and purpose of a system, in this case an SUF. Class diagrams can help at a professional level to describe relationships between entities (classes/objects) more clearly than with the existing methods of representation. Activity diagrams are successors to classic flow charts. They are complemented appropriately by status diagrams. UML is suitable to uniformly and graphically describe a system (here an SUF) from various points of view. In future, validated UML models will help us to present scientific concepts and results in a more structured form than before and to promote the technological use of these concepts in practice.

[Influenza vaccination by Lower Saxonian general practitioners - a secondary analysis of physicians' data from 1995/1996, 2002/2003, and 2005/2006]. / Influenza-Impfungen durch Niedersächsische Hausärzte - eine Sekundäranalyse vertragsärztlicher Versorgungsdaten aus 1995/1996, 2002/2003 und 2005/2006.

Vaccination against infection with human influenza virus is considered to be one of the most effective preventive measures available, especially when complications such as hospitalisation or death and indirect costs from off-work are considered. General practice is the preferable place for annual influenza vaccination because here the elderly and those endangered from bad health conditions are cared for frequently and regularly. The aim of this study was to find out the frequencies of influenza vaccination by Lower Saxonian contract physicians during three time periods with special respect to patients of older age or at risk. Data from two sources of health-care service have been analysed, namely from the contract physicians' union of Lower Saxony with all physicians vaccinating against influenza in the winter seasons of 1995/1996, 2002/2003, and 2005/2006, and from direct access to the electronic practice record system of 79 general practices via the BDT software data interface. Contract physicians, of whom more than 90 % are general practitioners, from Lower Saxony, vaccinate patients of the statutory health insurance against influenza in markedly increasing numbers, since 1995/1996 and 2002/2003 up to 2005/2006. Those over sixty years old or at high risk from bad health conditions are vaccinated up to seven-fold more frequently, compared to other patients. Influenza vaccination coverage rates (VCRs) are significantly higher in small and in medium-sized practices, compared to those with many patients. Nevertheless, influenza VCRs in Lower Saxony are not yet as high as would be necessary or desirable when compared internationally. Secondary analysis of aggregated health service data revealed inconsistencies in the primary material on cross-checking and validating, probably being caused during the external process of data aggregation and anonymisation. Also major systematic obstacles were found in the subsequent process of analysis. Nevertheless, fundamental results have been produced and are valid for all statutory health insured Lower Saxonians. Data from direct access to electronic practice records allowed for a deeper and multi-faceted insight into 101 928 patients of the same population, limited by the possibility of selection bias ('convenience sample'). Secondary analysis of health service data from different sources and their cross-check comparison is possible and successful. It is important to inform and involve the holder of the primary data extensively, following the guide lines of "good practice secondary data analysis".

[Routine data from general practitioner's software systems - Export, analysis and preparation for research]. / Routinedaten aus hausärztlichen Arztinformationssystemen - Export, Analyse und Aufbereitung für die Versorgungsforschung.

An advanced and integrative information technology (IT)-landscape is needed for optimal support of future processes in health-care, including health services research. Most researches in the primary care sector are based on data collected for reimbursement. The aim of this study is to show the limits and options of secondary analysis based on data that was exported via the "Behandlungsdatentransfer" (treatment data transport) BDT-interface in the software systems of German general practitioners and afterwards prepared for further research in SPSS. From the middle of 2005 to the end of 2007 all 168 teaching practices of the Hannover Medical School (MHH) were invited to join the study. Finally routine data from 28 practices could be collected successfully. The data from 139 other practices which had been collected for the project "Health Care in Practice" ("Medizinische Versorgung in der Praxis" - MedViP) was also added to the pool. The process of data preparation included a complete cycle from data collection, merging the data in a relational database system, via statistics and analysis to publishing and generating a feedback report for the participating practices. During the whole study the limits and options of this method were systematically identified. Of the 168 practices, 68 (40.5%) were interested to participate. From 28 (16.7%) physicians the data could be exported from their software systems. In 15 (8.9%) cases no collection was possible due to technical and in 26 (15.5%) to administrative reasons. The method of data extraction varied, as the BDT-interface was differently implemented by the software companies. Together with the MedViP data, the database at the MHH now consists of 167 practices with 974 304 patients and 12 555 943 treatments. For 44.1% of the 11 497 899 prescription entries an anatomic therapeutic chemical (ATC) code could be applied, by matching the entries to the master data from the Scientific Institute of Local Health-Care Funds ("Wissenschaftliches Instituts der Ortskrankenkassen" - WIdO). Periodically consistent sets of SPSS files could successfully be created for further research and feedback reports for the participating practices were generated as portable document format (PDF) files. The BDT-interface seems quite out of date, but can still reveal interesting information, especially on data about medical treatments and findings. Much of the data is contained in fields based on free text, which makes analysis difficult. Coded information, like agents, as ATC, could partially be extracted from the data, which afterwards was easy to prepare for further research. Quality and content of the data depend mainly on the data enterer, the physicians and their practice staff. Future research could be improved by more classified and coded data, which would better be transported through an interface more advanced than BDT.

Mitochondrial Introgression, Color Pattern Variation, and Severe Demographic Bottlenecks in Three Species of Malagasy Poison Frogs, Genus Mantella.

Madagascar is a biodiversity hotspot particularly rich in amphibian diversity and only a few charismatic Malagasy amphibians have been investigated for their population-level differentiation. The Mantellamadagascariensis group is composed of two rainforest and three swamp forest species of poison frogs. We first confirm the monophyly of this clade using DNA sequences of three nuclear and four mitochondrial genes, and subsequently investigate the population genetic differentiation and demography of the swamp forest species using one mitochondrial, two nuclear and a set of nine microsatellite markers. Our results confirm the occurrence of two main mitochondrial lineages, one dominated by Mantellaaurantiaca (a grouping supported also by our microsatellite-based tree) and the other by Mantellacrocea + Mantellamilotympanum. These two main lineages probably reflect an older divergence in swamp Mantella. Widespread mitochondrial introgression suggests a fairly common occurrence of inter-lineage gene flow. However, nuclear admixture seems to play only a limited role in this group, and the analyses of the RAG-1 marker points to a predominant incomplete lineage sorting scenario between all five species of the group, which probably diverged relatively recently. Our demographic analyses show a common, severe and recent demographic contraction, inferred to be in temporal coincidence with the massive deforestation events that took place in the past 1000 years. Current data do not allow to conclusively delimit independent evolutionary units in these frogs, and we therefore refrain to suggest any taxonomic changes.

Turtle soup, Prohibition, and the population genetic structure of Diamondback Terrapins (Malaclemys terrapin).

Diamondback terrapins (Malaclemys terrapin) were a popular food item in early twentieth century America, and were consumed in soup with sherry. Intense market demand for terrapin meat resulted in population declines, notably along the Atlantic seaboard. Efforts to supply terrapins to markets resulted in translocation events, as individuals were moved about to stock terrapin farms. However, in 1920 the market for turtle soup buckled with the enactment of the eighteenth amendment to the United States' Constitution-which initiated the prohibition of alcoholic drinks-and many terrapin fisheries dumped their stocks into local waters. We used microsatellite data to show that patterns of genetic diversity along the terrapin's coastal range are consistent with historical accounts of translocation and cultivation activities. We identified possible instances of human-mediated dispersal by estimating gene flow over historical and contemporary timescales, Bayesian model testing, and bottleneck tests. We recovered six genotypic clusters along the Gulf and Atlantic coasts with varying degrees of admixture, including increased contemporary gene flow from Texas to South Carolina, from North Carolina to Maryland, and from North Carolina to New York. In addition, Bayesian models incorporating translocation events outperformed stepping-stone models. Finally, we were unable to detect population bottlenecks, possibly due to translocation reintroducing genetic diversity into bottlenecked populations. Our data suggest that current patterns of genetic diversity in the terrapin were altered by the demand for turtle soup followed by the enactment of alcohol prohibition. In addition, our study shows that population genetic tools can elucidate metapopulation dynamics in taxa with complex genetic histories impacted by anthropogenic activities.

A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax).

Western Palearctic water frogs in the genus Pelophylax are a set of morphologically similar anuran species that form hybridogenetic complexes. Fully reliable identification of species and especially of hybrid ploidy depends on karyological and molecular methods. In central Europe, native water frog populations consist of the Pelophylax esculentus complex, that is, P. lessonae (LL), P. ridibundus (RR) and the hybrid form P. esculentus that can have different karyotypes (RL, LLR and RRL). We developed existing molecular methods further and propose a simple PCR method based on size-differences in the length of the serum albumin intron-1 and the RanaCR1, a non-LTR retrotransposon of the chicken repeat (CR) family. This PCR yields taxon-specific banding patterns that can easily be screened by standard agarose gel electrophoresis and correctly identify species in all of the 160 samples that had been identified to karyotype with other methods. To distinguish ploidy levels in LR, LLR and RRL specimens, we used the ratio of the peak heights of the larger (ridibundus specific) to the smaller (lessonae specific) bands of fluorescently labelled PCR products resolved on a capillary DNA sequencer and obtained a correct assignment of the karyotype in 93% of cases. Our new method will cut down time and expenses drastically for a reliable identification of water frogs of the P. esculentus complex and potentially for identification of other hybridogenetic complexes and/or taxa, and it even serves as a good indicator of the ploidy status of hybrid individuals.

Strong correlation between cross-amplification success and genetic distance across all members of 'True Salamanders' (Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite loci.

The unpredictable and low cross-amplification success of microsatellite loci tested for congeneric amphibian species has mainly been explained by the size and complexity of amphibian genomes, but also by taxonomy that is inconsistent with phylogenetic relationships among taxa. Here, we tested whether the cross-amplification success of nine new and 11 published microsatellite loci cloned for an amphibian source species, the fire salamander (Salamandra salamandra), correlated with the genetic distance across all members of True Salamanders (genera Chioglossa, Lyciasalamandra, Mertensiella and Salamandra that form a monophyletic clade within the family of Salamandridae) serving as target species. Cross-amplification success varied strongly among the species and showed a highly significant negative relationship with genetic distance and amplification success. Even though lineages of S. salamandra and Lyciasalamndra have separated more than 30 Ma, a within genus amplification success rate of 65% was achieved for species of Lyciasalamandra thus demonstrating that an efficient cross-species amplification of microsatellite loci in amphibians is feasible even across large evolutionary distances. A decrease in genome size, on the other hand, paralleled also a decrease in amplified loci and therefore contradicted previous results and expectations that amplification success should increase with a decrease in genome size. However, in line with other studies, our comprehensive dataset clearly shows that cross-amplification success of microsatellite loci is well explained by phylogenetic divergence between species. As taxonomic classifications on the species and genus level do not necessarily mirror phylogenetic divergence between species, the pure belonging of species to the same taxonomic units (i.e. species or genus) might be less useful to predict cross-amplification success of microsatellite loci between such species.

Eight new tetranucleotide microsatellite loci for the agile frog (Rana dalmatina).

We describe eight new polymorphic tetranucleotide microsatellite loci isolated from the agile frog (Rana dalmatina). In 25 individuals from the Nature Reserve Lüneburger Heide (Lower Saxony, Germany), the number of alleles per locus ranged from four to nine and average observed heterozygosities from 69.1% to 80.7%. No evidence for linkage disequilibrium was found and none of the loci showed significant deviation from Hardy-Weinberg expectations. These microsatellite DNA markers are suitable tools for addressing population genetics issues in this endangered species.

Molecular characterization of MHC class II in a nonmodel anuran species, the fire-bellied toad Bombina bombina.

While the anuran Xenopus comprises one of the best characterized nonmammalian taxa regarding the major histocompatibility complex (MHC), the organization of this gene complex has never been studied in other anurans, and information on amphibian MHC (other than Xenopus) is generally very scarce. Here, we describe the characterization of the first MHC class II B cDNA sequences from a nonmodel anuran species, the European fire-bellied toad (Bombina bombina). We isolated two transcript sequences differing substantially in amino acid composition and length within the beta2 domain. To investigate the variability of the peptide binding region in this species, we sequenced a 158-bp large fragment from wild B. bombina (n = 20) and identified eight distinct alleles. All substitutions but one were nonsynonymous, and many of the highly polymorphic sites corresponded with amino acid positions known to be involved in antigen binding. The level of variation we found in B. bombina was similar compared to that previously found in a comparable sample of a wild urodelan species, Ambystoma tigrinum, and to that found in Xenopus laevis. Based on the cDNA data and the individual's allelic diversity, we conclude that Bombina possesses at least two class II B loci. With our new beta1 primers, we were able to generate sequences in other species of anurans. We provide here a first phylogenetic analysis of this gene in amphibians.

Local adaptation and pronounced genetic differentiation in an extremophile fish, Poecilia mexicana, inhabiting a Mexican cave with toxic hydrogen sulphide.

We investigated genetic differentiation and migration patterns in a small livebearing fish, Poecilia mexicana, inhabiting a sulfidic Mexican limestone cave (Cueva del Azufre). We examined fish from three different cave chambers, the sulfidic surface creek draining the cave (El Azufre) and a nearby surface creek without the toxic hydrogen sulphide (Arroyo Cristal). Using microsatellite analysis of 10 unlinked loci, we found pronounced genetic differentiation among the three major habitats: Arroyo Cristal, El Azufre and the cave. Genetic differentiation was also found within the cave between different pools. An estimation of first-generation migrants suggests that (i) migration is unidirectional, out of the cave, and (ii) migration among different cave chambers occurs to some extent. We investigated if the pattern of genetic differentiation is also reflected in a morphological trait, eye size. Relatively large eyes were found in surface habitats, small eyes in the anterior cave chambers, and the smallest eyes were detected in the innermost cave chamber (XIII). This pattern shows some congruence with a previously proposed morphocline in eye size. However, our data do not support the proposed mechanism for this morphocline, namely that it would be maintained by migration from both directions into the middle cave chambers. This would have led to an increased variance in eye size in the middle cave chambers, which we did not find. Restricted gene flow between the cave and the surface can be explained by local adaptations to extreme environmental conditions, namely H2S and absence of light. Within the cave system, habitat properties are patchy, and genetic differentiation between cave chambers despite migration could indicate local adaptation at an even smaller scale.

Population genetics of the diamondback terrapin (Malaclemys terrapin).

We examined the population genetic structure of the diamondback terrapins (Malaclemys terrapin), within and among estuaries. Based on mark-recapture studies, these estuarine turtles have high site fidelity that is likely to make them vulnerable to local extinctions. We tested if observed site fidelity of adults would be reflected in intraestuarine population genetic structure of six highly polymorphic microsatellite loci (five tetranucleotide and one dinucleotide). No evidence was found for population structuring within the Charleston estuary nor among three different estuaries in South Carolina. We then examined four other terrapin populations from North Carolina to New York, as well as from the Florida Keys and from Texas. With increasing geographical distance, genetic differentiation increased from South Carolina through New York, but overall values were low. The dinucleotide locus contributed significantly more to the genetic differentiation of some population comparisons than any of the other loci. Interestingly, terrapins from South Carolina to New York were much more genetically similar to those from Texas (rho = 0.154) than to those from Florida (rho = 0.357). We attribute this pattern to extensive translocations of terrapins during the early 20th century to replenish diminished populations and to provide turtle farms with stocks. Terrapins collected in Texas were especially sought for shipment to the northeastern US because of their larger size. Our study indicates no population structure within or among adjacent estuaries. Thus, the mark-recapture information from adult and subadult feeding locations is a poor predictor of population genetic structure. Additionally, it appears that past human activities may have drastically altered the genetics of current populations. Finally, our data suggest that translocation of eggs or head starting of terrapins within estuaries or among adjacent estuaries is acceptable from a genetic standpoint.