Biodegradation of polyethylene by the marine fungus Parengyodontium album.

Plastic pollution in the marine realm is a severe environmental problem. Nevertheless, plastic may also serve as a potential carbon and energy source for microbes, yet the contribution of marine microbes, especially marine fungi to plastic degradation is not well constrained. We isolated the fungus Parengyodontium album from floating plastic debris in the North Pacific Subtropical Gyre and measured fungal-mediated mineralization rates (conversion to CO2) of polyethylene (PE) by applying stable isotope probing assays with 13C-PE over 9 days of incubation. When the PE was pretreated with UV light, the biodegradation rate of the initially added PE was 0.044 %/day. Furthermore, we traced the incorporation of PE-derived 13C carbon into P. album biomass using nanoSIMS and fatty acid analysis. Despite the high mineralization rate of the UV-treated 13C-PE, incorporation of PE-derived 13C into fungal cells was minor, and 13C incorporation was not detectable for the non-treated PE. Together, our results reveal the potential of P. album to degrade PE in the marine environment and to mineralize it to CO2. However, the initial photodegradation of PE is crucial for P. album to metabolize the PE-derived carbon.

Discovery and quantification of a widespread methane ebullition event in a coastal inlet (Baltic Sea) using a novel sonar strategy.

How much of the greenhouse gas methane is transported from the seafloor to the atmosphere is unclear. Here, we present data describing an extensive ebullition event that occurred in Eckernförde Bay, a shallow gas-hosting coastal inlet in the Baltic Sea, in the fall of 2014. A weak storm induced hydrostatic pressure fluctuations that in turn stimulated gas ebullition from the seabed. In a finely tuned sonar survey of the bay, we obtained a hydroacoustic dataset with exceptionally high sensitivity for bubble detection. This allowed us to identify 2849 bubble seeps rising within 28 h from the seafloor across the 90 km² study site. Based on our calculations, the estimated bubble-driven episodic methane flux from the seafloor across the bay is 1,900 µMol m-2 d-1. Our study demonstrates that storm-associated fluctuations of hydrostatic pressure induce bulk gas-driven ebullitions. Given the extensive occurrence of shallow gas-hosting sediments in coastal seas, similar ebullition events probably take place in many parts of the Western Baltic Sea. However, these are likely to be missed during field investigations, due to the lack of high-quality data acquisition during storms, such that atmospheric inputs of marine-derived methane will be highly underestimated.

First Observation of a Stable Highly Dissipative Divertor Plasma Regime on the Wendelstein 7-X Stellarator.

For the first time, the optimized stellarator Wendelstein 7-X has operated with an island divertor. An operation regime in hydrogen was found in which the total plasma radiation approached the absorbed heating power without noticeable loss of stored energy. The divertor thermography recorded simultaneously a strong reduction of the heat load on all divertor targets, indicating almost complete power detachment. This operation regime was stably sustained over several energy confinement times until the preprogrammed end of the discharge. The plasma radiation is mainly due to oxygen and is located at the plasma edge. This plasma scenario is reproducible and robust at various heating powers, plasma densities, and gas fueling locations. These experimental results show that the island divertor concept actually works and displays good power dissipation potential, producing a promising exhaust concept for the stellarator reactor line.

[Ability to drive with cerebrovascular diseases : Position paper of the German societies DGNB, DGN, DGNC, DGNR, DSG and GNP]. / Fahreignung bei Hirngefäßerkrankungen : Positionspapier der DGNB, DGN, DGNC, DGNR, DSG und GNP.

The regulations for ability to drive with cerebrovascular diseases in the German Driving License Regulations (Fahrerlaubnisverordnung, FeV) and German Guidelines for the Evaluation of Driving Ability of the Federal Highway Research Institute (BASt) are not up to date with the current medical knowledge and are not consistent with comparable regulations regarding cardiovascular diseases. This is particularly true for the assessment of future risks for a sudden loss of control during driving. The present position paper of six medical and neuropsychological societies in Germany presents the current conditions for the assessment of driving ability of patients a cerebrovascular diesease and recommends an estimation of the ability to drive founded on the current state of scientific knowledge. It addresses the following: 1. Physical and mental functional limitations and the possibilities for compensation, which if necessary enable a fitness to drive under conditions or within limits, including the importance of behavioral or personality changes and cognitive deficiencies that interfere with safety. 2. The potential danger due to a sudden loss of control as a result of a transient ischemic attack (TIA) new stroke event, or another cardiovascular event while driving. A summary in the form of a table provides physicians and expert assessors with assistance for the most important cerebrovascular diseases.

A divertor scraper observation system for the Wendelstein 7-X stellarator.

Two graphite divertor elements called scrapers have been installed on the Wendelstein 7-X stellarator in the throat of the magnetic island divertor. To diagnose one, we have designed, built, calibrated, and installed a new infrared/visible imaging endoscope system to enable detailed observations of the plasma interactions and heat loads at one of the scrapers and the nearby divertor surfaces. The new system uses a shuttered pinhole-protected pair of 90° off-axis 228 mm focal length aluminum parabolic mirrors, and two flat turning metal mirrors, to send light to a sapphire vacuum window 1.6 meters away, beyond which we have co-located telephoto lens-based infrared and visible cameras. The back-to-back off-axis parabolas serve to cancel out most aberrations, enabling the use of off-the-shelf commercial optics outside of the vessel. For the infrared, we use a 3-5 µm 1-megapixel FLIR SC8303HD camera and for the visible, a 5-megapixel CMOS PCO 5.5 edge camera. A short 1-m quartz pickoff fiber is used to send 200-1100 nm light to a compact spectrometer, also located in the same iron shield box as the cameras. The camera field of view covers the 700 mm length of the scraper, and includes locations monitored by thermocouples and Langmuir probes embedded in some of the scraper tiles. Predicted and actual optical test performances of the overall system are compared.

Successful knock-in of Hypertrophic Cardiomyopathy-mutation R723G into the MYH7 gene mimics HCM pathology in pigs.

Familial Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiac disease. About 30% of the patients are heterozygous for mutations in the MYH7 gene encoding the ß-myosin heavy chain (MyHC). Hallmarks of HCM are cardiomyocyte disarray and hypertrophy of the left ventricle, the symptoms range from slight arrhythmias to sudden cardiac death or heart failure. To gain insight into the underlying mechanisms of the diseases' etiology we aimed to generate genome edited pigs with an HCM-mutation. We used TALEN-mediated genome editing and successfully introduced the HCM-point mutation R723G into the MYH7 gene of porcine fibroblasts and subsequently cloned pigs that were heterozygous for the HCM-mutation R723G. No off-target effects were determined in the R723G-pigs. Surprisingly, the animals died within 24 h post partem, probably due to heart failure as indicated by a shift in the a/ß-MyHC ratio in the left ventricle. Most interestingly, the neonatal pigs displayed features of HCM, including mild myocyte disarray, malformed nuclei, and MYH7-overexpression. The finding of HCM-specific pathology in neonatal R723G-piglets suggests a very early onset of the disease and highlights the importance of novel large animal models for studying causative mechanisms and long-term progression of human cardiac diseases.

Towards a new image processing system at Wendelstein 7-X: From spatial calibration to characterization of thermal events.

Wendelstein 7-X (W7-X) is the most advanced fusion experiment in the stellarator line and is aimed at proving that the stellarator concept is suitable for a fusion reactor. One of the most important issues for fusion reactors is the monitoring of plasma facing components when exposed to very high heat loads, through the use of visible and infrared (IR) cameras. In this paper, a new image processing system for the analysis of the strike lines on the inboard limiters from the first W7-X experimental campaign is presented. This system builds a model of the IR cameras through the use of spatial calibration techniques, helping to characterize the strike lines by using the information given by real spatial coordinates of each pixel. The characterization of the strike lines is made in terms of position, size, and shape, after projecting the camera image in a 2D grid which tries to preserve the curvilinear surface distances between points. The description of the strike-line shape is made by means of the Fourier Descriptors.

Methane- and dissolved organic carbon-fueled microbial loop supports a tropical subterranean estuary ecosystem.

Subterranean estuaries extend inland into density-stratified coastal carbonate aquifers containing a surprising diversity of endemic animals (mostly crustaceans) within a highly oligotrophic habitat. How complex ecosystems (termed anchialine) thrive in this globally distributed, cryptic environment is poorly understood. Here, we demonstrate that a microbial loop shuttles methane and dissolved organic carbon (DOC) to higher trophic levels of the anchialine food web in the Yucatan Peninsula (Mexico). Methane and DOC production and consumption within the coastal groundwater correspond with a microbial community capable of methanotrophy, heterotrophy, and chemoautotrophy, based on characterization by 16S rRNA gene amplicon sequencing and respiratory quinone composition. Fatty acid and bulk stable carbon isotope values of cave-adapted shrimp suggest that carbon from methanotrophic bacteria comprises 21% of their diet, on average. These findings reveal a heretofore unrecognized subterranean methane sink and contribute to our understanding of the carbon cycle and ecosystem function of karst subterranean estuaries.

Gene-specific profiling of DNA methylation and mRNA expression in bovine oocytes derived from follicles of different size categories.

Epigenetic changes, such as DNA methylation, play an essential role in the acquisition of full developmental competence by mammalian oocytes during the late follicular growth phase. Here we used the bovine model to investigate the DNA methylation profiles of seven candidate genes (imprinted: bH19, bSNRPN; non-imprinted: bZAR1, bDNMT3A, bOCT4, bDNMT3 Lo and bDNMT3 Ls) and the mRNA expression of nine candidate genes (imprinted: bSNRPN, bPEG3, bIGF2R; non-imprinted: bPRDX1, bDNMT1B, bDNMT3A, bZAR1, bHSF1 and bNLRP9) in oocytes from antral follicles of three different size classes (≤2mm, 3-5mm, ≥6mm) to unravel the epigenetic contribution to this process. We observed an increased number of aberrantly methylated alleles in bH19, bSNRPN and bDNMT3 Lo of oocytes from small antral follicles (≤2mm), correlating with lower developmental competence. Furthermore, we detected an increased frequency of CpG sites with an unclear methylation status for DNMT3 Ls, specifically in oocytes from follicles ≥6mm, predominantly at three CpG positions (CpG2, CpG7 and CpG8), of which CpG7 is a potential regulatory site. No major differences in mRNA expression were observed, indicating that the transcriptional machinery may not yet be active during the follicular growth phase. Our results support the notion that a follicle diameter of ~2mm is a critical stage for establishing DNA methylation profiles and indicate a link between DNA methylation and the acquisition of oocyte developmental competence.

Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Effects of cryopreservation on the epigenetic profile of cells.

Effective cryopreservation protocols are essential for long-term storage of cells and their subsequent clinical application. Freezing protocols are generally considered as safe; however, putative effects on epigenetic marks have not yet been studied in detail. While post-thaw cell survival rates have been used to evaluate the success of cryopreservation protocols, increasing evidence suggests that freezing may be associated with deviations from the physiological epigenetic marks with putative long-term effects on the cells and/or their derivatives. A better understanding of the underlying mechanisms would be beneficial for improving safety and effectiveness of freezing protocols. The purpose of this review is to provide current information regarding epigenetic alterations (DNA methylation and histone modification patterns) associated with cryopreservation.

Synthetic plasma edge diagnostics for EMC3-EIRENE, highlighted for Wendelstein 7-X.

Interpretation of spectroscopic measurements in the edge region of high-temperature plasmas can be a challenge since line of sight integration effects make direct interpretation in terms of quantitative, local emission strengths often impossible. The EMC3-EIRENE code-a 3D fluid edge plasma and kinetic neutral gas transport code-is a suitable tool for full 3D reconstruction of such signals. A versatile synthetic diagnostic module has been developed recently which allows the realistic 3D setup of various plasma edge diagnostics to be captured. We highlight these capabilities with two examples for Wendelstein 7-X (W7-X): a visible camera for the analysis of recycling, and a coherent-imaging system for velocity measurements.

Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

Xenotransplantation of porcine islet cells as a potential option for the treatment of type 1 diabetes in the future.

Solid organ and cell transplantation, including pancreatic islets constitute the treatment of choice for chronic terminal diseases. However, the clinical use of allogeneic transplantation is limited by the growing shortage of human organs. This has prompted us to initiate a unique multi-center and multi-team effort to promote translational research in xenotransplantation to bring xenotransplantation to the clinical setting. Supported by the German Research Foundation, an interdisciplinary group of surgeons, internal medicine doctors, diabetologists, material sciences experts, immunologists, cell biologists, virologists, veterinarians, and geneticists have established a collaborative research center (CRC) focusing on the biology of xenogeneic cell, tissue, and organ transplantation. A major strength of this consortium is the inclusion of members of the regulatory bodies, including the Paul-Ehrlich Institute (PEI), infection specialists from the Robert Koch Institute and PEI, veterinarians from the German Primate Center, and representatives of influential ethical and religious institutions. A major goal of this consortium is to promote islet xenotransplantation, based on the extensive expertise and experience of the existing clinical islet transplantation program. Besides comprehensive approaches to understand and prevent inflammation-mediated islet xenotransplant dysfunction [immediate blood-mediated inflammatory reaction (IBMIR)], we also take advantage of the availability of and experience with islet macroencapsulation, with the goal to improve graft survival and function. This consortium harbors a unique group of scientists with complementary expertise under a cohesive program aiming at developing new therapeutic approaches for islet replacement and solid organ xenotransplantation.

Residential traffic and incidence of Type 2 diabetes: the German Health Interview and Examination Surveys.

AIMS: To investigate whether an indicator of overall traffic intensity is related to the risk of Type 2 diabetes in a nationwide cohort. METHODS: The study population comprised 3604 adults aged 18-79 years and without diabetes from the German National Health Interview and Examination Survey (GNHIES98, 1997-1999) who participated again in a follow-up survey (DEGS1, 2008-2011). The association between the participants' reported traffic intensity at their residential address and Type 2 diabetes incidence was examined using logistic regression models. RESULTS: During a mean of 12.1 years of follow-up, 252 of the participants included in the study developed Type 2 diabetes. Compared with people living in traffic-calmed areas, odds ratios were 1.15 (95% CI 0.80-1.67) for people living on moderately busy side streets, 1.11 (95% CI 0.69-1.80) for people living on considerably busy side streets, 1.41 (95% CI 0.96-2.08) for people living on heavily busy roads, and 1.97 (95% CI 1.07-3.64) for people living on extremely busy roads, after adjusting for age, sex, active and passive smoking, type of heating, education, BMI, waist circumference, sport activity and parental diabetes history. CONCLUSIONS: The twofold higher risk of Type 2 diabetes observed for people exposed to intense traffic in this nationwide cohort extends the limited evidence from previous selected populations. Although the underlying traffic-related components and their biological mechanisms still need to be unravelled, traffic exposure control should be considered in public health strategies to reduce the global burden of diabetes.

Temporal constraints on hydrate-controlled methane seepage off Svalbard.

Methane hydrate is an icelike substance that is stable at high pressure and low temperature in continental margin sediments. Since the discovery of a large number of gas flares at the landward termination of the gas hydrate stability zone off Svalbard, there has been concern that warming bottom waters have started to dissociate large amounts of gas hydrate and that the resulting methane release may possibly accelerate global warming. Here, we corroborate that hydrates play a role in the observed seepage of gas, but we present evidence that seepage off Svalbard has been ongoing for at least 3000 years and that seasonal fluctuations of 1° to 2°C in the bottom-water temperature cause periodic gas hydrate formation and dissociation, which focus seepage at the observed sites.

Wound healing potential of a dimeric InlB variant analyzed by in vitro experiments on re-epithelialization of human skin models.

A constitutively dimeric truncated variant of internalin B (InlB321-CD), acting as stimulator of the receptor tyrosine kinase MET, was tested for dermal wound-healing potential. Due to a lack of the endogenous MET agonist HGF/SF in chronic wounds, HGF/SF substitution by an InlB321-CD-loaded hydrogel might be beneficial in chronic wound therapy. In this study, InlB321-CD in solution and incorporated in a hydrogel was tested for mitogenic effects on immortalized human dermal keratinocytes (HaCaT) with an MTT assay. Cell migration was investigated with a scratch assay on primary keratinocytes (PHK) and on HaCaT. For the latter, scratching needed to be mitomycin C-controlled. InlB321-CD effects on a model of human skin were analyzed histologically with respect to viability. InlB321-CD led to dose-dependent proliferative effects on HaCaT cells whereas the equimolar dose of monomeric InlB321 did not. Upon hydrogel incorporation of InlB321-CD its mitogenic activity for HaCaT cells was maintained thus confirming the hydrogel as a promising drug delivery system. Motogenic effects were shown on both HaCaT and PHK cells. InlB321-CD neither possesses cytotoxic effects on the viability of a human skin model nor alters its organotypic cell morphology.

Protracted induction of parturition enhances placental maturation, but does not influence incidence of placental retention in cows.

As the etiopathology of retained placenta is still not resolved in cattle, we compared the effects of protracted induction of parturition (PIP) and conventional induction of parturition (SIP) on placental maturation and the occurrence of retained placenta. PIP was induced in 13 cows by administration of 1.3 mg dexamethasone im twice daily between Days 268 and 273 of gestation and 40 mg dexamethasone im on Day 274 of gestation. For SIP, 10 cows received a single injection of 40 mg dexamethasone on Day 274 of gestation. A third group (SPON, n = 11) served as a nontreated control group. Within 2 hours after birth, two placentomes were extracted from the uterus and used for assessment of placental maturation by histology and immunohistochemistry. Incidence of retained placenta was lower (P < 0.05) in group SPON (9%) compared with groups PIP (54%) and SIP (70%). Staining with Masson's trichrome and pan-cytokeratin indicated a higher degree of atrophy and flatness of the maternal crypt epithelium in cows with physiological release of fetal membranes (REL) compared with cows with retained placentae (RET). Staining with anti-caspase-3 ratified the observations as more apoptotic cells were detected in groups SPON and PIP compared with group SIP; however, data were not statistically significant. Additionally, the expressions of the potent vasodilators endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were evaluated. Both eNOS and iNOS were only expressed in chorionic tissue. Endothelin-1 (ET-1), a major vasoconstrictor, showed positive staining in maternal crypt epithelium and in chorionic epithelium. No differences were found for iNOS and eNOS and ET-1, neither among the experimental groups nor between RET and REL cows. These findings indicate that a PIP results in increased placental maturation, but does not influence the incidence of placental retention in cows. The expression of vasoactive substances does not seem to be related to the placental separation process.