Multi-omics for studying and understanding polar life.

Polar ecosystems are experiencing amongst the most rapid rates of regional warming on Earth. Here, we discuss 'omics' approaches to investigate polar biodiversity, including the current state of the art, future perspectives and recommendations. We propose a community road map to generate and more fully exploit multi-omics data from polar organisms. These data are needed for the comprehensive evaluation of polar biodiversity and to reveal how life evolved and adapted to permanently cold environments with extreme seasonality. We argue that concerted action is required to mitigate the impact of warming on polar ecosystems via conservation efforts, to sustainably manage these unique habitats and their ecosystem services, and for the sustainable bioprospecting of novel genes and compounds for societal gain.

Genetic analysis of Boletus edulis suggests that intra-specific competition may reduce local genetic diversity as a woodland ages.

Ectomycorrhizal fungi are key players in terrestrial ecosystems yet their mating systems and population dynamics remain poorly understood. We investigated the fine-scale relatedness structure and genetic diversity of Boletus edulis, one of the world's most commercially important wild mushrooms. Microsatellite genotyping of fruiting bodies from 14 different sites around Bielefeld in Germany revealed little in the way of population structure over a geographic scale of several kilometres. However, on a more local scale we found evidence for elevated relatedness as well as inbreeding. We also observed a significant negative association between the genetic diversity of fruit and the age of the trees under which they were sampled. Taken together, our results suggest that as genets mature, they compete and potentially create conditions under which further spores struggle to become established. By implication, even though this species is widely picked, propagules remain common enough to create strong competition when new habitats become available.

Rapid evolution in native plants cultivated for ecological restoration: not a general pattern.

The growing number of restoration projects worldwide increases the demand for seed material of native species. To meet this demand, seeds are often produced through large-scale cultivation on specialised farms, using wild-collected seeds as the original sources. However, during cultivation, plants experience novel environmental conditions compared to those in natural populations, and there is a danger that the plants in cultivation are subject to unintended selection and lose their adaptation to natural habitats. Although the propagation methods are usually designed to maintain as much natural genetic diversity as possible, the effectiveness of these measures have never been tested. We obtained seed of five common grassland species from one of the largest native seed producers in Germany. For each species, the seeds were from multiple generations of seed production. We used AFLP markers and a common garden experiment to test for genetic and phenotypic changes during cultivation of these plants. The molecular markers detected significant evolutionary changes in three out of the five species and we found significant phenotypic changes in two species. The only species that showed substantial genetic and phenotypic changes was the short-lived and predominantly selfing Medicago lupulina, while in the other, mostly perennial and outcrossing species, the observed changes were mostly minor. Agricultural propagation of native seed material for restoration can cause evolutionary changes, at least in some species. We recommend caution, particularly in selfing and short-lived species, where evolution may be more rapid and effects may thus be more severe.

Muscles and fascial elements of the antebrachium and manus of the African elephant (Loxodonta africana, Blumenbach 1797): starring comparative and functional considerations.

The structure of the limbs of elephants is unusual among mammals. In African elephants (Loxodonta africana, Blumenbach 1797), the front limbs serve to support the greatest part of the body mass of the largest land animal. In this study, we present new and detailed anatomical data regarding muscular and specific fascial structures of the lower front limb which were examined by means of standard anatomical and histological techniques. The muscles and tendons of the forearm (antebrachium) and hand (manus) are tightly surrounded by thick, highly elastic fascial layers which fuse with the lacertus (lac.) fibrosus and the so-called ligamentum (lig.) humeroulnare. A well-developed musculus (m.) brachioradialis occupies the proximolateral aspect of the forearm and its tendon inserts together with the lac. fibrosus on the os carpi intermedium. The lac. fibrosus, the lig. humeroulnare and the m. flexor carpi radialis reveal a large proportion of elastic fibres. These three structures may play an important role in storing strain energy thus promoting energy-saving locomotion. On the palmar aspect of the carpus, metacarpus and digits, short flexor, abductor, adductor, lumbricales and interossei muscles are present, whereas supinator muscles are absent in all specimens. The short muscles of the hand together with specific dorsal tendons enable precise movements of specific toes.

Deleterious acute and chronic effects of bradycardic right ventricular apex pacing: consequences for arrhythmic outcome.

In the chronic complete atrioventricular (AV) block dog (CAVB) model, both bradycardia and altered ventricular activation due to the uncontrolled idioventricular rhythm contribute to ventricular remodeling and the enhanced susceptibility to Torsade de Pointes (TdP) arrhythmias. We investigated the effect of permanent bradycardic right ventricular apex (RVA) pacing on mechanical and electrical remodeling and TdP. In 23 anesthetized dogs, serial experiments were performed at sinus rhythm (SR), acutely after AV block (AAVB) and 3 weeks of remodeling CAVB at a fixed pacing rate of 60/min. ECG, and left (LV) and right ventricular (RV) monophasic action potentials durations (MAPD) were recorded; activation time (AT) and activation recovery interval (ARI) were determined from ten distinct LV electrograms; interventricular mechanical delay (IVMD) and time-to-peak strain (TTP) of the LV septal and lateral wall (ΔTTP: lateral wall minus septal wall) were obtained echocardiographically. Dofetilide (25 µg/kg/5 min) was infused to study TdP inducibility. In baseline AAVB, in comparison to SR, RVA bradypacing acutely increased QT interval, LV, and RVMAPD. Echocardiographic IVMD and ΔTTP were initially increased, which was partially corrected after 3 weeks of RVA pacing (IVMD: 22 ± 13 vs. 42 ± 11 vs. 31 ± 6 ms; ΔTTP: -2 ± 47 vs. -114 ± 38 vs. -36 ± 22 ms). QT interval (362 ± 23 vs. 373 ± 29 ms), LVMAPD (245 ± 18 vs. 253 ± 22 ms), RVMAPD (226 ± 26 vs. 238 ± 31 ms), and mean LV-ARI (268 ± 5 vs. 267 ± 6 ms) were not significantly changed after 3 weeks of RVA pacing. During AAVB, dofetilide increased mean LV-ARI (381 ± 11 ms) with largest increases in the later activated basal areas (slope AT-ARI: +0.96). In contrast with acute RVA pacing, 3 week pacing increased TdP inducibility (0/13 vs. 11/21) and mean LV-ARI (484 ± 18 ms), while the slope of AT-ARI responded differently on dofetilide (-2.37), with larger APD increases in the early region. The latter was supported at the molecular level: reduced RNA expressions of three repolarization-related ion channel genes in early (KCNQ1, KCNH2, and KCNJ2) versus two in late regions (KNCQ1 and KCNJ2). In conclusion, bradycardic RVA pacing acutely induced LV intra- and interventricular mechanical dyssynchrony, which was partially reversed after 3 weeks of pacing (remodeling). The latter occurred without apparent baseline electrical effects. However, dofetilide clearly unmasked (region-specific) arrhythmic consequences of remodeling.

Engineering of Tomato Glandular Trichomes for the Production of Specialized Metabolites.

Glandular trichomes are specialized tissues on the epidermis of many plant species. On tomato they synthesize, store, and emit a variety of metabolites such as terpenoids, which play a role in the interaction with insects. Glandular trichomes are excellent tissues for studying the biosynthesis of specialized plant metabolites and are especially suitable targets for metabolic engineering. Here we describe the strategy for engineering tomato glandular trichomes, first with a transient expression system to provide proof of trichome specificity of selected promoters. Using microparticle bombardment, the trichome specificity of a terpene-synthase promoter could be validated in a relatively fast way. Second, we describe a method for stable expression of genes of interest in trichomes. Trichome-specific expression of another terpene-synthase promoter driving the yellow-fluorescence protein-gene is presented. Finally, we describe a case of the overexpression of farnesyl diphosphate synthase (FPS), specifically in tomato glandular trichomes, providing an important precursor in the biosynthetic pathway of sesquiterpenoids. FPS was targeted to the plastid aiming to engineer sesquiterpenoid production, but interestingly leading to a loss of monoterpenoid production in the transgenic tomato trichomes. With this example we show that trichomes are amenable to engineering though, even with knowledge of a biochemical pathway, the result of such engineering can be unexpected.

The electromechanical window is no better than QT prolongation to assess risk of Torsade de Pointes in the complete atrioventricular block model in dogs.

BACKGROUND AND PURPOSE: The electromechanical window (EMW), the interval between the end of the T-wave and the end of the left ventricular pressure (LVP) curve, has recently been proposed as a predictor of risk of Torsade de Pointes (TdP) in healthy animals, whereby a negative EMW (mechanical relaxation earlier than repolarization) after drug administration indicates an increased TdP risk. The aims of this study were to assess (i) the effect of the ventricular remodelling in the canine chronic, complete atrioventricular block (CAVB) model on EMW; (ii) the effect of the I(Kr) -blocker dofetilide on EMW; and (iii) the correlation of EMW with TdP inducibility. EXPERIMENTAL APPROACH: Our 11 year database of experiments of CAVB in dogs under general anaesthesia was reviewed and experiments included if ECG and LVP were recorded simultaneously at spontaneous rhythm. In total, 89 experiments in 44 dogs were appropriate and were analysed. KEY RESULTS: During normally conducted sinus rhythm or acute atrioventricular block, EMW was positive. During CAVB, EMW was decreased to negative values. Dofetilide further reduced EMW before inducing repetitive TdP in 82% of the experiments. However, subclassification into inducible and non-inducible dogs revealed no difference in EMW. Analysis of the components of EMW revealed that the observed changes in EMW were solely caused by QT prolongation. CONCLUSIONS AND IMPLICATIONS: In the canine CAVB model, ventricular remodelling and I(Kr) block by dofetilide are associated with negative EMW values, but this reflects QT prolongation, and implies that the EMW lacks specificity to predict dofetilide-induced TdP.

Blastocystis subtypes in symptomatic and asymptomatic family members and pets and response to therapy.

BACKGROUND: Blastocystis is a common, enteric parasite. The pathogenicity of the organism is uncertain, but subtypes (ST) 1 and 3 have been reported more likely to cause irritable bowel-like symptoms. AIMS: We treated symptomatic patients positive for Blastocystis with conventional therapy and analysed 16 small-subunit (SSU) rDNA to assess clearance and carriage rates and ST prevalence of the parasite in the asymptomatic household members. METHODS: In a longitudinal, prospective case study, 11 symptomatic patients positive for Blastocystis underwent outpatient clinical assessment to exclude other diagnoses before 14 days of either metronidazole 400 mg three times daily or trimethoprim/sulfamethoxazole 160/800 mg twice-daily therapy. Faecal specimens were collected from patients at baseline, day 15, 28 and 56 after therapy and from 17 family members and eight pets at day 15. Specimens were analysed using faecal smear, culture and polymerase chain reaction analysis of 16SSU rDNA. RESULTS: No patient cleared the organism following therapy. ST 1 (45%), 3 (36%), 4 (36%) and 6 (9%) were found in the symptomatic Blastocystis patients, and ST identified before and after therapy were identical in each individual. All household contacts were positive for Blastocystis and 16/17 (94%) contacts showed identical Blastocystis ST to the symptomatic family member. All pets were positive for Blastocystis with polymerase chain reaction testing, 7/8 (88%) demonstrating ST concordance with the symptomatic Blastocystis patients. CONCLUSIONS: Conventional therapy is ineffective for symptomatic Blastocystis infection. The high prevalence of Blastocystis infection within households suggested transmission between humans and their pets. Subtyping analysis of SSU rDNA alone in Blastocystis does not appear to predict pathogenicity.

Comparison of the IKr blockers moxifloxacin, dofetilide and E-4031 in five screening models of pro-arrhythmia reveals lack of specificity of isolated cardiomyocytes.

BACKGROUND AND PURPOSE: Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell-derived cardiomyocytes (hESC-CM) could be used to identify pro-arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro-arrhythmic properties of I(Kr) blockers, using moxifloxacin (safe compound) and dofetilide or E-4031 (unsafe compounds). EXPERIMENTAL APPROACH: Assays included the anaesthetized remodelled chronic complete AV block (CAVB) dog, the anaesthetized methoxamine-sensitized unremodelled rabbit, multi-cellular hESC-CM clusters, isolated CM obtained from CAVB dogs and isolated CM obtained from the normal rabbit. Arrhythmic outcome was defined as Torsade de Pointes (TdP) in the animal models and early afterdepolarizations (EADs) in the cell models. KEY RESULTS: At clinically relevant concentrations (5-12 µM), moxifloxacin was free of pro-arrhythmic properties in all assays with the exception of the isolated CM, in which 10 µM induced EADs in 35% of the CAVB CM and in 23% of the rabbit CM. At supra-therapeutic concentrations (≥100 µM), moxifloxacin was pro-arrhythmic in the isolated rabbit CM (33%), in the hESC-CM clusters (18%), and in the methoxamine rabbit (17%). Dofetilide and E-4031 induced EADs or TdP in all assays (50-83%), and the induction correlated with a significant increase in beat-to-beat variability of repolarization. CONCLUSION AND IMPLICATIONS: Isolated cardiomyocytes lack specificity to discriminate between TdP liability of the I(Kr) blocking drugs moxifloxacin and dofetilide or E4031.

Fabrication and electrical characterization of a pore-cavity-pore device.

We present a solid state nanopore device structure comprising two nanopores which are stacked above each other and connected via a pyramidal cavity of 10 fl volume. The process of fabrication of the pore-cavity-pore device (PCP) relies on the formation of one pore in a Si(3)N(4) membrane by electron beam lithography, while the other pore is chemically etched into the Si carrier by a feedback controlled process. The dimensions of the two nanopores as well as the cavity can be adjusted independently, which is confirmed by transmission electron microscopy. The PCP device is characterized with respect to its electrical properties, including noise analysis and impedance spectroscopy. An equivalent circuit model is identified and resistance, capacitance, and dielectric loss factors are obtained. Potential and electric field distributions inside the electrically biased device are simulated by finite element methods. The low noise characteristics of the PCP device (comparable to a single solid state nanopore) make it suitable for the stochastic sensing of single molecules; moreover, the pore-cavity-pore architecture allows for novel kinds of experiments including the trapping of single nano-objects and single molecule time-of-flight measurements.

Bioconcentration, biomagnification and metabolism of 14C-terbutryn and 14C-benzo[a]pyrene in Gammarus fossarum and Asellus aquaticus.

The contribution of bioconcentration and biomagnification of 14C-terbutryn and 14C-benzo[a]pyrene via food and water to the bioaccumulation in Gammarus fossarum and Asellus aquaticus was investigated in single species-tests. In this investigation the uptake of 14C-terbutryn and 14C-benzo[a]pyrene via food and water by G. fossarum (L.) and A. aquaticus (L.) was examined. Bioconcentration factors (BCFs) and biomagnification factors (BMFs) were determined. Calculated BCFs were clearly higher than BMFs, indicating that water is the primary route of uptake. The uptake of terbutryn and benzo[a]pyren via water in G. fossarum and A. aquaticus is faster than uptake via food. The elimination and metabolism of the two chemicals by G. fossarum and A. aquaticus were studied. Terbutryn was eliminated almost completely in both species. In general, the elimination of terbutryn from G. fossarum and A. aquaticus was fast with half-life of 5 h. The elimination of terbutryn by G. fossarum after biomagnification is slower than after bioconcentration. No difference was found in elimination of terbutryn by A. aquaticus after biomagnification and after bioconcentration. Metabolites of terbutryn in G. fossarum and A. aquaticus were analyzed by HPLC. After the bioconcentration experiment a higher percentage of metabolites was found in G. fossarum than in A. aquaticus. This was confirmed for the experiment with uptake via food. The spectrum of metabolites was similar in both species, with hydroxyterbutryn being the major metabolism product in water. 14C-B[a]P could nearly completely be eliminated by G. fossarum (rest of activity 2%) after uptake via water. 14C-B[a]P could not be eliminated by G. fossarum and A. aquaticus after uptake via food. The metabolite could not be identified.

Population structure and dynamics of Gammarus fossarum (Amphipoda) upstream and downstream from effluents of sewage treatment plants.

Two streams in Germany (Körsch and Lockwitzbach), each with two sampling sites above and below a sewage treatment plant (STP) discharging effluent, were investigated. Sampling sites were characterized, and exposure monitoring for chemicals with known or assumed endocrine disrupting potential was carried out. Both the population structure and the population dynamics of Gammarus fossarum were examined. The physicochemical parameters measured at the sampling sites of the Lockwitzbach and Körsch streams were found not to reach levels having an acute toxic impact on the development of gammarids. The calculated estrogenic potential in the stream water was 22- to 35-fold higher at the downstream site of the Körsch compared with the other sampling sites, mainly because of the concentrations of 17alpha-ethinylestradiol on two sampling dates. At both streams, an influence of the respective STP effluent on the sex ratio of G. fossarum was not observed. Moreover, intersexuality was not induced by these effluents. Differences in the structure and dynamics of G. fossarum populations were more pronounced at the Körsch than at the Lockwitzbach. At the downstream sampling site at the Körsch, gammarids reached their highest abundances. Particularly at the downstream sampling site of the Körsch, the proportion of breeding female gammarids and the proportion of juvenile gammarids in the smallest body length class were decreased compared with upstream. Adult gammarids were larger from the Lockwitzbach downstream site, but they were smaller from the Körsch downstream site compared with the respective upstream site. At the Körsch, the earlier onset of the autumnal reproductive resting period could be caused by the influence of the STP effluent.

Endocrine effects in Gammarus fossarum (Amphipoda): influence of wastewater effluents, temporal variability, and spatial aspects on natural populations.

In a field study, individuals of autochthounous populations of the amphipod Gammarus fossarum were examined for their maturity status, oocyte development, and biochemical parameters associated with their reproductive cycle. Variability in these parameters was related to abiotic exposure parameters varying in accordance to wastewater discharges, stream identity, and time. Patterns of all investigated parameters showed predominantly seasonal rather than spatial influence for both exposure and biologic effects. Single selected-effect parameters, however--such as the maturity index, late vitellogenic oocyte size and atresia, and the hsp90 level--responded to a sewage treatment plant discharge showing an estrogenic potential and also correlated significantly with the concentration of potential xenoestrogens at the different locations.

Beta-globin-gene haplotypes, mitochondrial DNA, the Y-chromosome: their impact on the genetic epidemiology of the major structural hemoglobinopathies.

The history of the discovery of the globin beta-like globin-gene haplotypes and their importance in the understanding of hemoglobinopathies has been reviewed recently. We will add in this review more recent findings and other molecular genetic tools that can help in the understanding of the genetic epidemiology of structural hemoglobinopathies, leaving the thalassemias for a later effort.

Direct costs of microsurgical management of radiosurgically amenable intracranial pathology in Germany: an analysis of meningiomas, acoustic neuromas, metastases and arteriovenous malformations of less than 3 cm in diameter.

BACKGROUND: The purpose of the analysis was to appreciate and compare the effective direct costs of microsurgical treatment of intracranial pathology potentially amenable to radiosurgery as they arose in 1998-99. METHOD: Treatment costs of 127 microsurgically treated patients harbouring an arteriovenous malformation (AVM), acoustic nEuroma, meningioma or brain metastasis potentially amenable to radiosurgery were reviewed. Costs for the surgical procedure, ICU care, medical and nursing care on the ward, interclinical bills (ICB) for services provided by other departments and the overhead for basic hotel service were added. For comparison Gamma Knife costs were calculated by dividing the global operating cost of the Gamma Knife centre by the number of patients treated in 1999. FINDINGS: Average hospitalisation time for the entire microsurgical patients was 15,4+/-8,6 days. The patients spent an average of 1,2+/-2,8 days on ICU. Average operating time for all patients, including preparation, was 393+/-118 minutes. Average costs for the microsurgical therapy were Euro10.814+/-6.108. These consisted of Euro1417+/-426 for the surgical procedure, Euro1.188+/-2.658 for ICU care, Euro2.333+/-1.582 for medical and nursing care on the ward, Euro1.671+/-1.433 for interclinical bills and Euro 4.204+/-2.338 for basic hotel service (overhead, Euro273/day). 70% of the microsurgically treated patients needed ancillary inpatient rehabilitation or radiotherapy resulting in an average additional cost for all patients of Euro2.744. Furthermore 20% of the microsurgically treated patients required an unplanned readmission after discharge, resulting in an average additional costs for all patients of Euro1.684. Average overall costs per patient including ancillary therapy and unplanned readmissions amounted to Euro15.242. For comparison, Gamma Knife treatment costs per patient amounted to Euro7.920 in 1999. INTERPRETATION: The current analysis showed that for established radiosurgical indications the primary costs of microsurgery exceeded the costs of radiosurgery. Differences with regard to additional expenses as a consequence of disability were not addressed in this study. Microsurgical management as well as Gamma Knife radiosurgery have potential for economic improvement.