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1.  Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function 
The EMBO Journal  2015;34(10):1417-1433.
Key transcription factors control the gene expression program in mature pancreatic β-cells, but their integration into regulatory networks is little understood. Here, we show that Insm1, Neurod1 and Foxa2 directly interact and together bind regulatory sequences in the genome of mature pancreatic β-cells. We used Insm1 ablation in mature β-cells in mice and found pronounced deficits in insulin secretion and gene expression. Insm1-dependent genes identified previously in developing β-cells markedly differ from the ones identified in the adult. In particular, adult mutant β-cells resemble immature β-cells of newborn mice in gene expression and functional properties. We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant β-cells. Remarkably, combinatorial binding of Insm1, Neurod1 and Foxa2 but not binding of Insm1 alone explained a significant fraction of gene expression changes. Human genomic sequences corresponding to the murine sites occupied by Insm1/Neurod1/Foxa2 were enriched in single nucleotide polymorphisms associated with glycolytic traits. Thus, our data explain part of the mechanisms by which β-cells maintain maturity: Combinatorial Insm1/Neurod1/Foxa2 binding identifies regulatory sequences that maintain the mature gene expression program in β-cells, and disruption of this network results in functional failure.
doi:10.15252/embj.201490819
PMCID: PMC4492000  PMID: 25828096
development; differentiation; Insm1; maturation; metabolisms; pancreatic beta cells
2.  Towards understanding microvillus inclusion disease 
Microvillus inclusion disease (MVID) is characterised by onset of intractable life-threatening watery diarrhoea during infancy. Transmission electron microscopy demonstrates shortening or absence of apical microvilli, pathognomonic microvillus inclusions in mature enterocytes and subapical accumulation of periodic acid-Schiff-positive granules or vesicles confirming diagnosis. Mutations in MYO5B have been found to cause MVID. In two patients with MVID, whole-exome sequencing of DNA revealed homozygous truncating mutations in STX3. Mutations in these genes disrupt trafficking between apical cargo vesicles and the apical plasma membrane. Thus, disturbed delivery of certain brush border membrane proteins is a common defect in MVID.
doi:10.1186/s40348-016-0031-0
PMCID: PMC4733813  PMID: 26830108
MVID; Microvillus inclusion disease; Enteropathy; MYO5B; STX3
3.  Technical-Induced Hemolysis in Patients with Respiratory Failure Supported with Veno-Venous ECMO – Prevalence and Risk Factors 
PLoS ONE  2015;10(11):e0143527.
The aim of the study was to explore the prevalence and risk factors for technical-induced hemolysis in adults supported with veno-venous extracorporeal membrane oxygenation (vvECMO) and to analyze the effect of hemolytic episodes on outcome. This was a retrospective, single-center study that included 318 adult patients (Regensburg ECMO Registry, 2009–2014) with acute respiratory failure treated with different modern miniaturized ECMO systems. Free plasma hemoglobin (fHb) was used as indicator for hemolysis. Throughout a cumulative support duration of 4,142 days on ECMO only 1.7% of the fHb levels were above a critical value of 500 mg/l. A grave rise in fHb indicated pumphead thrombosis (n = 8), while acute oxygenator thrombosis (n = 15) did not affect fHb. Replacement of the pumphead normalized fHb within two days. Neither pump or cannula type nor duration on the first system was associated with hemolysis. Multiple trauma, need for kidney replacement therapy, increased daily red blood cell transfusion requirements, and high blood flow (3.0–4.5 L/min) through small-sized cannulas significantly resulted in augmented blood cell trauma. Survivors were characterized by lower peak levels of fHb [90 (60, 142) mg/l] in comparison to non-survivors [148 (91, 256) mg/l, p≤0.001]. In conclusion, marked hemolysis is not common in vvECMO with modern devices. Clinically obvious hemolysis often is caused by pumphead thrombosis. High flow velocity through small cannulas may also cause technical-induced hemolysis. In patients who developed lung failure due to trauma, fHb was elevated independantly of ECMO. In our cohort, the occurance of hemolysis was associated with increased mortality.
doi:10.1371/journal.pone.0143527
PMCID: PMC4659553  PMID: 26606144
4.  The Glass Slide Extraction System Snap Card Improves Non-Invasive Prenatal Genotyping in Pregnancies with Antibodies 
Summary
Background
Determination of fetal blood groups in maternal plasma samples critically depends on adequate pre-analytical steps for optimal amplification of fetal DNA. We compared the extraction of cell-free DNA by binding on a glass surface (BCSI SNAP™ Card) with an automated system based on bead technology (MagnaPure compact™).
Methods
Maternal blood samples from 281 pregnancies (7th-39th week of gestation) with known antibodies were evaluated in this study. Both the SNAP card and the MagnaPure method were applied to isolate DNA in order to directly compare the amplification in a single base extension assay and/or real-time PCR.
Results
The mean concentration of total DNA obtained by the SNAP card (33.8 ng/µl) exceeded more than twofold that of MagnaPure extraction (15.7 ng/µl). SNAP card-extracted samples allowed to detect 3.7 single nucleotide polymorphisms (SNPs) versus 2.5 SNPs in MagnaPure extracts to control for traces of fetal DNA. This difference is highest for samples from 7th-13th week of gestation.
Conclusion
The SNAP card system improves DNA extraction efficacy for prenatal diagnosis in maternal blood samples and provides an at least eightfold higher total amount of DNA for the ensuing analysis. Its advantage is most evident for samples from early stages of pregnancy and thus especially valuable for pregnancies with antibodies.
doi:10.1159/000441542
PMCID: PMC4698643  PMID: 26733769
Cell-free DNA; Non-invasive prenatal genotyping; DNA extraction
5.  Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function 
The EMBO Journal  2015;34(10):1417-1433.
Key transcription factors control the gene expression program in mature pancreatic β-cells, but their integration into regulatory networks is little understood. Here, we show that Insm1, Neurod1 and Foxa2 directly interact and together bind regulatory sequences in the genome of mature pancreatic β-cells. We used Insm1 ablation in mature β-cells in mice and found pronounced deficits in insulin secretion and gene expression. Insm1-dependent genes identified previously in developing β-cells markedly differ from the ones identified in the adult. In particular, adult mutant β-cells resemble immature β-cells of newborn mice in gene expression and functional properties. We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant β-cells. Remarkably, combinatorial binding of Insm1, Neurod1 and Foxa2 but not binding of Insm1 alone explained a significant fraction of gene expression changes. Human genomic sequences corresponding to the murine sites occupied by Insm1/Neurod1/Foxa2 were enriched in single nucleotide polymorphisms associated with glycolytic traits. Thus, our data explain part of the mechanisms by which β-cells maintain maturity: Combinatorial Insm1/Neurod1/Foxa2 binding identifies regulatory sequences that maintain the mature gene expression program in β-cells, and disruption of this network results in functional failure.
doi:10.15252/embj.201490819
PMCID: PMC4492000  PMID: 25828096
development; differentiation; Insm1; maturation; metabolisms; pancreatic beta cells
6.  Nosema Tolerant Honeybees (Apis mellifera) Escape Parasitic Manipulation of Apoptosis 
PLoS ONE  2015;10(10):e0140174.
Apoptosis is not only pivotal for development, but also for pathogen defence in multicellular organisms. Although numerous intracellular pathogens are known to interfere with the host’s apoptotic machinery to overcome this defence, its importance for host-parasite coevolution has been neglected. We conducted three inoculation experiments to investigate in the apoptotic respond during infection with the intracellular gut pathogen Nosema ceranae, which is considered as potential global threat to the honeybee (Apis mellifera) and other bee pollinators, in sensitive and tolerant honeybees. To explore apoptotic processes in the gut epithelium, we visualised apoptotic cells using TUNEL assays and measured the relative expression levels of subset of candidate genes involved in the apoptotic machinery using qPCR. Our results suggest that N. ceranae reduces apoptosis in sensitive honeybees by enhancing inhibitor of apoptosis protein-(iap)-2 gene transcription. Interestingly, this seems not be the case in Nosema tolerant honeybees. We propose that these tolerant honeybees are able to escape the manipulation of apoptosis by N. ceranae, which may have evolved a mechanism to regulate an anti-apoptotic gene as key adaptation for improved host invasion.
doi:10.1371/journal.pone.0140174
PMCID: PMC4596554  PMID: 26445372
7.  Molecular and cellular characteristics of human and non-human primate multipotent stromal cells from the amnion and bone marrow during long term culture 
Introduction
Multipotent stromal cells (MSCs) are among the key candidates in regenerative medicine. However variety of MSC sources and general heterogeneity lead to controversial data in functional characterization. Furthermore, despite intensive usage as preclinical animal model, little is known about MSCs of the common marmoset monkey.
Methods
MSCs derived from placental amnion and bone marrow samples from human and common marmoset were characterized in parallel over 12 passages to monitor similarities and significant differences (p ≤ 0.05, Student’s t-test) in MSC markers and major histocompatibility complex (MHC) class I expression by immunohistochemistry, flow cytometry, real-time PCR, metabolic activity test, with special focus on pluripotency associated genes.
Results
Human and non-human primate MSCs were characterized for expression of MSC markers and capability of differentiation into mesenchymal lineages. MSCs could be cultured more than 100 days (26 passages), but metabolic activity was significantly enhanced in amnion vs. bone marrow MSCs. Interestingly, MHC class I expression is significantly reduced in amnion MSCs until passage 6 in human and marmoset, but not in bone marrow cells. For MSC markers, CD73 and CD105 levels remain unchanged in amnion MSCs and slightly decline in bone marrow at late passages; CD166 is significantly higher expressed in human MSCs, CD106 significantly lower vs. marmoset. All cultured MSCs showed pluripotency marker expression like Oct-4A at passage 3 significantly decreasing over time (passages 6–12) while Nanog expression was highest in human bone marrow MSCs. Furthermore, human MSCs demonstrated the highest Sox2 levels vs. marmoset, whereas the marmoset exhibited significantly higher Lin28A values. Bisulfite sequencing of the Oct-4 promoter region displayed fewer methylations of CpG islands in the marmoset vs. human.
Conclusions
Little is known about MSC characteristics from the preclinical animal model common marmoset vs. human during long term culture. Studied human and common marmoset samples share many similar features such as most MSC markers and reduced MHC class I expression in amnion cells vs. bone marrow. Furthermore, pluripotency markers indicate in both species a subpopulation of MSCs with true ‘stemness’, which could explain their high proliferation capacity, though possessing differences between human and marmoset in Lin28A and Sox2 expression.
doi:10.1186/s13287-015-0146-6
PMCID: PMC4546288  PMID: 26297012
8.  Spatio-temporal Use of Oral Rabies Vaccines in Fox Rabies Elimination Programmes in Europe 
PLoS Neglected Tropical Diseases  2015;9(8):e0003953.
In Europe, the elimination of wildlife rabies using oral rabies vaccination [ORV] of foxes for more than 30 years has been a success story. Since a comprehensive review on the scope of the different oral rabies vaccine baits distributed across Europe has not been available yet, we evaluated the use of different vaccine baits over the entire period of ORV [1978–2014]. Our findings provide valuable insights into the complexity of ORV programs in terms of vaccine related issues. More than 10 oral vaccines against rabies were used over the past four decades. Depending on many factors, the extent to which oral rabies virus vaccines were used varied considerably resulting in huge differences in the number of vaccine doses disseminated in ORV campaigns as well as in large spatial and temporal overlaps. Although vaccine virus strains derived from the SAD rabies virus isolate were the most widely used, the success of ORV campaigns in Europe cannot be assigned to a single oral rabies virus vaccine alone. Rather, the successful elimination of fox rabies is the result of an interaction of different key components of ORV campaigns, i.e. vaccine strain, vaccine bait and strategy of distribution.
Author Summary
Oral rabies vaccination [ORV] is the pre-eminent example of successful vaccination of wildlife populations which has resulted in virtual elimination of fox-mediated rabies from large parts of Western and Central Europe. This achievement is unprecedented in history. Attractive species-specific baits, efficient vaccination strategy, and highly potent and safe oral rabies vaccines have been instrumental for the success. Numerous oral rabies vaccines for wildlife have been developed over the past four decades. However, information on the use of those vaccines in operational ORV programs in Europe that exists has until now not been fully publicly accessible. Here, we provide the first comprehensive review of the spatio-temporal use of oral rabies vaccines over the entire period of ORV [1978–2014]. Although ‘first generation’ vaccine virus strains derived from the SAD rabies isolate are the most widely used, the results do not support significant differences in efficacy of these strains under field conditions nor can success of ORV be attributed solely to one vaccine type. In contrast, vaccination strategy is of major importance. Our analyses could help in the development of adequate rabies control strategies by documenting the importance of vaccination strategy, in which oral rabies vaccines are only one contributing component among other parameters.
doi:10.1371/journal.pntd.0003953
PMCID: PMC4539187  PMID: 26280895
9.  Wagner–Meerwein-Type Rearrangements of Germapolysilanes - A Stable Ion Study 
Organometallics  2015;34(15):3756-3763.
The rearrangement of tris(trimethylsilyl)silyltrimethylgermane 1 to give tetrakis(trimethylsilyl)germane 2 was investigated as a typical example for Lewis acid catalyzed Wagner–Meerwein-type rearrangements of polysilanes and polygermasilanes. Direct 29Si NMR spectroscopic evidence is provided for several cationic intermediates during the reaction. The identity of these species was verified by independent synthesis and NMR characterization, and their transformation was followed by NMR spectroscopy.
doi:10.1021/acs.organomet.5b00431
PMCID: PMC4534834  PMID: 26294805
10.  Stereo- and Regioselective Phyllobilane Oxidation in Leaf Homogenates of the Peace Lily (Spathiphyllum wallisii): Hypothetical Endogenous Path to Yellow Chlorophyll Catabolites 
In senescent leaves, chlorophyll typically is broken down to colorless and essentially photo-inactive phyllobilanes, which are linear tetrapyrroles classified as “nonfluorescent” chlorophyll catabolites (NCCs) and dioxobilane-type NCCs (DNCCs). In homogenates of senescent leaves of the tropical evergreen Spathiphyllum wallisii, when left at room temperature and extracted with methanol, the major endogenous, naturally formed NCC was regio- and stereoselectively oxidized (in part) to a mixture of its 15-hydroxy and 15-methoxy derivative. In the absence of methanol in the extract, only the 15-OH-NCC was observed. The endogenous oxidation process depended upon molecular oxygen. It was inhibited by carbon monoxide, as well as by keeping the leaf homogenate and extract at low temperatures. The remarkable “oxidative activity” was inactivated by heating the homogenate for 10 min at 70 °C. Upon addition of a natural epimeric NCC (epiNCC) to the homogenate of senescent or green Sp. wallisii leaves at room temperature, the exogenous epiNCC was oxidized regio- and stereoselectively to 15-OH-epiNCC and 15-OMe-epiNCC. The identical two oxidized epiNCCs were also obtained as products of the oxidation of epiNCC with dicyanodichlorobenzoquinone (DDQ). Water elimination from 15-OH-epiNCC occurred readily and gave a known “yellow” chlorophyll catabolite (YCC). The endogenous oxidation process, described here, may represent the elusive natural path from the colorless NCCs to yellow and pink coloured phyllobilins, which were found in (extracts of) some senescent leaves.
doi:10.1002/chem.201404783
PMCID: PMC4517098  PMID: 25382809
chlorophyll; metallo-enzyme; oxidation reaction; senescence; tetrapyrrol
11.  Formation and Properties of a Bicyclic Silylated Digermene 
In the presence of PMe3 or N-heterocyclic carbenes, the reaction of oligosilanylene dianions with GeCl2⋅dioxane gives germylene–base adducts. After base abstraction, the free germylenes can dimerize by formation of a digermene. An electrochemical and theoretical study of a bicyclic tetrasilylated digermene revealed formation of a comparably stable radical anion and a more reactive radical cation, which were characterized further by UV/Vis and ESR spectroscopy.
doi:10.1002/chem.201402785
PMCID: PMC4506559  PMID: 24981992
digermenes; electrochemistry; germylene; radicals; silicon
12.  A High Power-Density, Mediator-Free, Microfluidic Biophotovoltaic Device for Cyanobacterial Cells 
Advanced Energy Materials  2014;5(2):1-6.
Biophotovoltaics has emerged as a promising technology for generating renewable energy because it relies on living organisms as inexpensive, self-repairing, and readily available catalysts to produce electricity from an abundant resource: sunlight. The efficiency of biophotovoltaic cells, however, has remained significantly lower than that achievable through synthetic materials. Here, a platform is devised to harness the large power densities afforded by miniaturized geometries. To this effect, a soft-lithography approach is developed for the fabrication of microfluidic biophotovoltaic devices that do not require membranes or mediators. Synechocystis sp. PCC 6803 cells are injected and allowed to settle on the anode, permitting the physical proximity between cells and electrode required for mediator-free operation. Power densities of above 100 mW m-2 are demonstrated for a chlorophyll concentration of 100 μM under white light, which is a high value for biophotovoltaic devices without extrinsic supply of additional energy.
doi:10.1002/aenm.201401299
PMCID: PMC4503997  PMID: 26190957
biophotovoltaic devices; microfluidics; bioenergy; cyanobacteria
14.  Oligosilanylated Antimony Compounds 
Organometallics  2015;34(8):1419-1430.
By reactions of magnesium oligosilanides with SbCl3, a number of oligosilanylated antimony compounds were obtained. When oligosilanyl dianions were used, either the expected cyclic disilylated halostibine was obtained or alternatively the formation of a distibine was observed. Deliberate formation of the distibine from the disilylated halostibine was achieved by reductive coupling with C8K. Computational studies of Sb–Sb bond energies, barriers of pyramidal inversion at Sb, and the conformational behavior of distibines provided insight for the understanding of the spectroscopic properties.
doi:10.1021/om501075v
PMCID: PMC4413694  PMID: 25937691
15.  Estimating the Global Burden of Endemic Canine Rabies 
PLoS Neglected Tropical Diseases  2015;9(4):e0003709.
Background
Rabies is a notoriously underreported and neglected disease of low-income countries. This study aims to estimate the public health and economic burden of rabies circulating in domestic dog populations, globally and on a country-by-country basis, allowing an objective assessment of how much this preventable disease costs endemic countries.
Methodology/Principal Findings
We established relationships between rabies mortality and rabies prevention and control measures, which we incorporated into a model framework. We used data derived from extensive literature searches and questionnaires on disease incidence, control interventions and preventative measures within this framework to estimate the disease burden. The burden of rabies impacts on public health sector budgets, local communities and livestock economies, with the highest risk of rabies in the poorest regions of the world. This study estimates that globally canine rabies causes approximately 59,000 (95% Confidence Intervals: 25-159,000) human deaths, over 3.7 million (95% CIs: 1.6-10.4 million) disability-adjusted life years (DALYs) and 8.6 billion USD (95% CIs: 2.9-21.5 billion) economic losses annually. The largest component of the economic burden is due to premature death (55%), followed by direct costs of post-exposure prophylaxis (PEP, 20%) and lost income whilst seeking PEP (15.5%), with only limited costs to the veterinary sector due to dog vaccination (1.5%), and additional costs to communities from livestock losses (6%).
Conclusions/Significance
This study demonstrates that investment in dog vaccination, the single most effective way of reducing the disease burden, has been inadequate and that the availability and affordability of PEP needs improving. Collaborative investments by medical and veterinary sectors could dramatically reduce the current large, and unnecessary, burden of rabies on affected communities. Improved surveillance is needed to reduce uncertainty in burden estimates and to monitor the impacts of control efforts.
Author Summary
Rabies is a fatal viral disease largely transmitted to humans from bites by infected animals—predominantly from domestic dogs. The disease is entirely preventable through prompt administration of post-exposure prophylaxis (PEP) to bite victims and can be controlled through mass vaccination of domestic dogs. Yet, rabies is still very prevalent in developing countries, affecting populations with limited access to health care. The disease is also grossly underreported in these areas because most victims die at home. This leads to insufficient prioritization of rabies prevention in public health agendas. To address this lack of information on the impacts of rabies, in this study, we compiled available data to provide a robust estimate of the health and economic implications of dog rabies globally. The most important impacts included: loss of human lives (approximately 59,000 annually) and productivity due to premature death from rabies, and costs of obtaining PEP once an exposure has occurred. The greatest risk of developing rabies fell upon the poorest regions of the world, where domestic dog vaccination is not widely implemented and access to PEP is most limited. A greater focus on mass dog vaccination could eliminate the disease at source, reducing the need for costly PEP and preventing the large and unnecessary burden of mortality on at-risk communities.
doi:10.1371/journal.pntd.0003709
PMCID: PMC4400070  PMID: 25881058
17.  Colorless Chlorophyll Catabolites in Senescent Florets of Broccoli (Brassica oleracea var. italica) 
Typical postharvest storage of broccoli (Brassica oleracea var. italica) causes degreening of this common vegetable with visible loss of chlorophyll (Chl). As shown here, colorless Chl-catabolites are generated. In fresh extracts of degreening florets of broccoli, three colorless tetrapyrrolic Chl-catabolites accumulated and were detected by high performance liquid chromatography (HPLC): two “nonfluorescent” Chl-catabolites (NCCs), provisionally named Bo-NCC-1 and Bo-NCC-2, and a colorless 1,19-dioxobilin-type “nonfluorescent” Chl-catabolite (DNCC), named Bo-DNCC. Analysis by nuclear magnetic resonance spectroscopy and mass spectrometry of these three linear tetrapyrroles revealed their structures. In combination with a comparison of their HPL-chromatographic properties, this allowed their identification with three known catabolites from two other brassicacea, namely two NCCs from oil seed rape (Brassica napus) and a DNCC from degreened leaves of Arabidopsis thaliana.
doi:10.1021/jf5055326
PMCID: PMC4329831  PMID: 25620234
broccoli; chlorophyll catabolites; structure elucidation; natural product; nutrition; vegetable
18.  The Longitudinal Course of Gross Motor Activity in Schizophrenia – Within and between Episodes 
Schizophrenia is associated with heterogeneous course of positive and negative symptoms. In addition, reduced motor activity as measured by wrist actigraphy has been reported. However, longitudinal studies of spontaneous motor activity are missing. We aimed to explore whether activity levels were stable within and between psychotic episodes. Furthermore, we investigated the association with the course of negative symptoms. In 45 medicated patients, we investigated motor behavior within a psychotic episode. In addition, we followed 18 medicated patients across 2 episodes. Wrist actigraphy and psychopathological ratings were applied. Within an episode symptoms changed but activity levels did not vary systematically. Activity at baseline predicted the course of negative symptoms. Between two episodes activity recordings were much more stable. Again, activity at the index episode predicted the outcome of negative symptoms. In sum, spontaneous motor activity shares trait and state characteristics, the latter are associated with negative symptom course. Actigraphy may therefore become an important ambulatory instrument to monitor negative symptoms and treatment outcome in schizophrenia.
doi:10.3389/fpsyt.2015.00010
PMCID: PMC4318415  PMID: 25698981
actigraphy; psychosis; negative symptoms; PANSS; avolition
19.  Physical Activity in Schizophrenia is Higher in the First Episode than in Subsequent Ones 
Schizophrenia is frequently associated with abnormal motor behavior, particularly hypokinesia. The course of the illness tends to deteriorate in the first years. We aimed to assess gross motor activity in patients with a first episode (n = 33) and multiple episodes (n = 115) of schizophrenia spectrum disorders using wrist actigraphy. First episode patients were younger, had higher motor activity and reduced negative symptom severity. Covarying for age, chlorpromazine equivalents, and negative symptoms, first episode patients still had higher motor activity. This was also true after excluding patients with schizophreniform disorder from the analyses. In first episode patients, but not in patients with multiple episodes, motor activity was correlated with antipsychotic dosage. In conclusion, after controlling for variables related to disorder chronicity, patients with first episodes were still more active than patients with multiple episodes. Thus, reduced motor activity is a marker of deterioration in the course of schizophrenia spectrum disorders.
doi:10.3389/fpsyt.2014.00191
PMCID: PMC4283447  PMID: 25601842
actigraphy; antipsychotic; negative symptoms; hypokinesia; psychosis
20.  First-line therapy in atypical hemolytic uremic syndrome: consideration on infants with a poor prognosis 
Background
Atypical hemolytic uremic syndrome (aHUS) is a rare and heterogeneous disorder. The first line treatment of aHUS is plasma therapy, but in the past few years, the recommendations have changed greatly with the advent of eculizumab, a humanized monoclonal anti C5-antibody. Although recent recommendations suggest using it as a primary treatment for aHUS, important questions have arisen about the necessity of immediate use of eculizumab in all cases. We aimed to draw attention to a specific subgroup of aHUS patients with rapid disease progression and high mortality, in whom plasma therapy may not be feasible.
Methods
We present three pediatric patients of acute complement-mediated HUS with a fatal outcome. Classical and alternative complement pathway activity, levels of complement factors C3, C4, H, B and I, as well as of anti-factor H autoantibody and of ADAMTS13 activity were determined. The coding regions of CFH, CFI, CD46, THBD, CFB and C3 genes were sequenced and the copy number of CFI, CD46, CFH and related genes were analyzed.
Results
We found severe activation and consumption of complement components in these patients, furthermore, in one patient we identified a previously not reported mutation in CFH (Ser722Stop), supporting the diagnosis of complement-mediated HUS. These patients were not responsive to the FFP therapy, and all cases had fatal outcome.
Conclusion
Taking the heterogeneity and the variable prognosis of atypical HUS into account, we suggest that the immediate use of eculizumab should be considered as first-line therapy in certain small children with complement dysregulation.
doi:10.1186/s13052-014-0101-7
PMCID: PMC4295478  PMID: 25496981
Atypical hemolytic uremic syndrome; Infant; Eculizumab; Plasma therapy
21.  Technical Complications during Veno-Venous Extracorporeal Membrane Oxygenation and Their Relevance Predicting a System-Exchange – Retrospective Analysis of 265 Cases 
PLoS ONE  2014;9(12):e112316.
Objectives
Technical complications are a known hazard in veno-venous extracorporeal membrane oxygenation (vvECMO). Identifying these complications and predictive factors indicating a developing system-exchange was the goal of the study.
Methods
Retrospective study on prospectively collected data of technical complications including 265 adult patients (Regensburg ECMO Registry, 2009-2013) with acute respiratory failure treated with vvECMO. Alterations in blood flow resistance, gas transfer capability, hemolysis, coagulation and hemostasis parameters were evaluated in conjunction with a system-exchange in all patients with at least one exchange (n = 83).
Results
Values presented as median (interquartile range). Patient age was 50(36–60) years, the SOFA score 11(8–14.3) and the Murray lung injury Score 3.33(3.3–3.7). Cumulative ECMO support time 3411 days, 9(6–15) days per patient. Mechanical failure of the blood pump (n = 5), MO (n = 2) or cannula (n = 1) accounted for 10% of the exchanges. Acute clot formation within the pump head (visible clots, increase in plasma free hemoglobin (frHb), serum lactate dehydrogenase (LDH), n = 13) and MO (increase in pressure drop across the MO, n = 16) required an urgent system-exchange, of which nearly 50% could be foreseen by measuring the parameters mentioned below. Reasons for an elective system-exchange were worsening of gas transfer capability (n = 10) and device-related coagulation disorders (n = 32), either local fibrinolysis in the MO due to clot formation (increased D-dimers [DD]), decreased platelet count; n = 24), or device-induced hyperfibrinolysis (increased DD, decreased fibrinogen [FG], decreased platelet count, diffuse bleeding tendency; n = 8), which could be reversed after system-exchange. Four MOs were exchanged due to suspicion of infection.
Conclusions
The majority of ECMO system-exchanges could be predicted by regular inspection of the complete ECMO circuit, evaluation of gas exchange, pressure drop across the MO and laboratory parameters (DD, FG, platelets, LDH, frHb). These parameters should be monitored in the daily routine to reduce the risk of unexpected ECMO failure.
doi:10.1371/journal.pone.0112316
PMCID: PMC4251903  PMID: 25464516
22.  Identification of rhabdoviral sequences in oropharyngeal swabs from German and Danish bats 
Virology Journal  2014;11:196.
Background
In the frame of active lyssavirus surveillance in bats, oropharyngeal swabs from German (N = 2297) and Danish (N = 134) insectivorous bats were investigated using a newly developed generic pan-lyssavirus real-time reverse transcriptase PCR (RT-qPCR).
Findings
In total, 15 RT-qPCR positive swabs were detected. Remarkably, sequencing of positive samples did not confirm the presence of bat associated lyssaviruses but revealed nine distinct novel rhabdovirus-related sequences.
Conclusions
Several novel rhabdovirus-related sequences were detected both in German and Danish insectivorous bats. The results also prove that the novel generic pan-lyssavirus RT-qPCR offers a very broad detection range that allows the collection of further valuable data concerning the broad and complex diversity within the family Rhabdoviridae.
doi:10.1186/s12985-014-0196-x
PMCID: PMC4247638  PMID: 25420461
Pan-lyssavirus PCR; Rhabdoviridae; Dimarhabdovirus supergroup
23.  Transcriptome sequencing of two wild barley (Hordeum spontaneum L.) ecotypes differentially adapted to drought stress reveals ecotype-specific transcripts 
BMC Genomics  2014;15(1):995.
Background
Wild barley is adapted to highly diverse environments throughout its geographical distribution range. Transcriptome sequencing of differentially adapted wild barley ecotypes from contrasting environments contributes to the identification of genes and genetic variation involved in abiotic stress tolerance and adaptation.
Results
Two differentially adapted wild barley ecotypes from desert (B1K2) and Mediterranean (B1K30) environments were analyzed for drought stress response under controlled conditions. The desert ecotype lost more water under both irrigation and drought, but exhibited higher relative water content (RWC) and better water use efficiency (WUE) than the coastal ecotype. We sequenced normalized cDNA libraries from drought-stressed leaves of both ecotypes with the 454 platform to identify drought-related transcripts. Over half million reads per ecotype were de novo assembled into 20,439 putative unique transcripts (PUTs) for B1K2, 21,494 for B1K30 and 28,720 for the joint assembly. Over 50% of PUTs of each ecotype were not shared with the other ecotype. Furthermore, 16% (3,245) of B1K2 and 17% (3,674) of B1K30 transcripts did not show orthologous sequence hits in the other wild barley ecotype and cultivated barley, and are candidates of ecotype-specific transcripts. Over 800 unique transcripts from each ecotype homologous to over 30 different stress-related genes were identified. We extracted 1,017 high quality SNPs that differentiated the two ecotypes. The genetic distance between the desert ecotype and cultivated barley was 1.9-fold higher than between the Mediterranean ecotype and cultivated barley. Moreover, the desert ecotype harbored a larger proportion of non-synonymous SNPs than the Mediterranean ecotype suggesting different demographic histories of these ecotypes.
Conclusions
The results indicate a strong physiological and genomic differentiation between the desert and Mediterranean wild barley ecotypes and a closer relationship of the Mediterranean to cultivated barley. A significant number of novel transcripts specific to wild barley were identified. The higher SNP density and larger proportion of SNPs with functional effects in the desert ecotype suggest different demographic histories and effects of natural selection in Mediterranean and desert wild barley. The data are a valuable genomic resource for an improved genome annotation, transcriptome studies of drought adaptation and a source of new genetic markers for future barley improvement.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-995) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-995
PMCID: PMC4251939  PMID: 25408241
Hordeum spontaneum; Transcriptome; Drought tolerance; Genetic diversity
24.  Anti-Lyssaviral Activity of Interferons κ and ω from the Serotine Bat, Eptesicus serotinus 
Journal of Virology  2014;88(10):5444-5454.
ABSTRACT
Interferons (IFNs) are cytokines produced by host cells in response to the infection with pathogens. By binding to the corresponding receptors, IFNs trigger different pathways to block intracellular replication and growth of pathogens and to impede the infection of surrounding cells. Due to their key role in host defense against viral infections, as well as for clinical therapies, the IFN responses and regulation mechanisms are well studied. However, studies of type I IFNs have mainly focused on alpha interferon (IFN-α) and IFN-β subtypes. Knowledge of IFN-κ and IFN-ω is limited. Moreover, most studies are performed in humans or mouse models but not in the original host of zoonotic pathogens. Bats are important reservoirs and transmitters of zoonotic viruses such as lyssaviruses. A few studies have shown an antiviral activity of IFNs in fruit bats. However, the function of type I IFNs against lyssaviruses in bats has not been studied yet. Here, IFN-κ and IFN-ω genes from the European serotine bat, Eptesicus serotinus, were cloned and functionally characterized. E. serotinus IFN-κ and IFN-ω genes are intronless and well conserved between microchiropteran species. The promoter regions of both genes contain essential regulatory elements for transcription factors. In vitro studies indicated a strong activation of IFN signaling by recombinant IFN-ω, whereas IFN-κ displayed weaker activation. Noticeably, both IFNs inhibit to different extents the replication of different lyssaviruses in susceptible bat cell lines. The present study provides functional data on the innate host defense against lyssaviruses in endangered European bats.
IMPORTANCE We describe here for the first time the molecular and functional characterization of two type I interferons (IFN-κ and -ω) from European serotine bat (Eptesicus serotinus). The importance of this study is mainly based on the fact that very limited information about the early innate immune response against bat lyssaviruses in their natural host serotine bats is yet available. Generally, whereas the antiviral activity of other type I interferons is well studied, the functional involvement of IFN-κ and -ω has not yet been investigated.
doi:10.1128/JVI.03403-13
PMCID: PMC4019090  PMID: 24574413
25.  Comparative studies on the genetic, antigenic and pathogenic characteristics of Bokeloh bat lyssavirus 
The Journal of General Virology  2014;95(Pt 8):1647-1653.
Bokeloh bat lyssavirus (BBLV), a novel lyssavirus, was isolated from a Natterer’s bat (Myotis nattererii), a chiropteran species with a widespread and abundant distribution across Europe. As a novel lyssavirus, the risks of BBLV to animal and human health are unknown and as such characterization both in vitro and in vivo was required to assess pathogenicity and vaccine protection. Full genome sequence analysis and antigenic cartography demonstrated that the German BBLV isolates are most closely related to European bat lyssavirus type 2 (EBLV-2) and Khujand virus and can be characterized within phylogroup I. In vivo characterization demonstrated that BBLV was pathogenic in mice when inoculated peripherally causing clinical signs typical for rabies encephalitis, with higher pathogenicity observed in juvenile mice. A limited vaccination-challenge experiment in mice was conducted and suggested that current vaccines would afford some protection against BBLV although further studies are warranted to determine a serological cut-off for protection.
doi:10.1099/vir.0.065953-0
PMCID: PMC4103065  PMID: 24828330

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