Membrane Topology of Pestiviral Non-Structural Protein 2 and determination of the minimal autoprotease domain.

Pestiviruses like bovine viral diarrhea virus (BVDV) belong to the family Flaviviridae A distinctive feature of the Flaviviridae is the importance of non-structural (NS) proteins for RNA genome replication and virus morphogenesis. For pestiviruses, the NS2 protease-mediated release of NS3 is essential for RNA replication, whereas uncleaved NS2-3 is indispensable for producing viral progeny. Accordingly, in the pestiviral life cycle the switch from RNA replication to virion morphogenesis is temporally regulated by the extent of NS2-3 cleavage, which is catalyzed by the NS2 autoprotease. A detailed knowledge of the structural and functional properties of pestiviral NS2 and NS2-3 is mandatory for a better understanding of these processes.In the present study, we experimentally determined the membrane topology of NS2 of BVDV-1 strain NCP7 by the Substituted Cysteine Accessibility Method (SCAM) assay. According to the resulting model, the N terminus of NS2 resides in the ER lumen and is followed by three transmembrane segments (TM) and a cytoplasmic C-terminal protease domain. We used the resulting model for fine mapping of the minimal autoprotease domain. Only one TM segment was found to be essential for maintaining residual autoprotease activity. While the topology of pestiviral NS2 is overall comparable to the one of hepatitis C virus (HCV) NS2, our data also reveal potentially important differences between the two molecules. The improved knowledge about structural and functional properties of this protein will support future functional and structural studies on pestiviral NS2.ImportancePestiviral NS2 is central to the regulation of RNA replication and virion morphogenesis via its autoprotease activity. This activity is temporally regulated by the cellular DNAJC14 as a cofactor: while free NS3 is required for RNA replication as a component of the viral replicase, only uncleaved NS2-3 supports virion morphogenesis. For a better understanding of the underlying molecular interactions, topological and structural data are required. The topology-based determination of the minimal NS2-protease domain in the present study will facilitate future attempts to determine the structure of this unusual protease cofactor complex. In the hepatitis C virus system, NS2 functions as a hub in virion morphogenesis by interacting with structural as well as non-structural proteins. Our knowledge of the membrane topology will significantly support future detailed interaction studies for pestiviral NS2.

[Innovative laser technologies in the treatment of urolithiasis : A change to more gentle methods with increased patient safety]. / Innovative Lasertechnologien in der Harnsteintherapie : Ein Wandel zu schonenderen Methoden mit hoher Patientensicherheit.

Management of urolithiasis has undergone fundamental changes with the introduction of extracorporeal shock wave lithotripsy (ESWL) and percutaneous and ureterorenoscopic techniques in the 1980s. Since then, these minimally invasive techniques have been continuously optimized and specific laser techniques for stone disintegration have emerged. Besides the established holmium laser, other types of lasers are also emerging. Especially the thulium fiber laser is the subject of promising research due to its variable adjustment options. In terms of patient safety, both holmium and thulium techniques seem to be similar . While serious direct physical lesions are rare, there is increasing evidence of clinically relevant secondary thermal injury due to increased temperatures in the upper urinary tract during treatment. Our research group has recently demonstrated in both in vitro and in vivo (porcine animal model) experiments that monitoring the fluorescence spectra of calculi allows precise target differentiation between stone, tissue, and endoscope components. Consequently, pulse emissions were only emitted when stone material was detected. We believe that target monitoring will minimize the risk of laser-induced urothelial damage and decrease energy release into the upper urinary tract allowing adequate temperature management.

On the incorporation of iron into hexagonal barium titanate: II. Magnetic moment, electron paramagnetic resonance (EPR) and optical transmission.

Systematic measurements of the magnetic moment in dependence on temperature and magnetic field of hexagonal 6H-BaTiO3 + 0.04 BaO + x/2 Fe2O3 (0.005 ⩽ x ⩽ 0.05) ceramics were performed to study the influence of Fe ions on the magnetic properties. While the samples show Curie-Weiss paramagnetism for Fe concentrations ⩽1.0 mol%, antiferromagnetic interactions become manifest for 2.0 and 5.0 mol% iron. With increasing Fe content the antiferromagnetic interaction, which is assumed to be caused by a superexchange mechanism [Formula: see text], becomes stronger. At external magnetic fields smaller than 1 T a further, ferromagnetic interaction between Fe3+ ions is detected below 200 K. The interactions between Fe3+ ions in the samples with 2.0 and 5.0 mol% iron are also manifest in the EPR spectra by numerous lines with low intensity. Q-band EPR investigations of 5.0 mol% Fe doped single crystals confirm the existence of only one type of Fe3+-VO associates in the samples.

Drug-based perturbation screen uncovers synergistic drug combinations in Burkitt lymphoma.

Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma associated with MYC translocation. Here, we describe drug response profiling of 42 blood cancer cell lines including 17 BL to 32 drugs targeting key cancer pathways and provide a systematic study of drug combinations in BL cell lines. Based on drug response, we identified cell line specific sensitivities, i.e. to venetoclax driven by BCL2 overexpression and partitioned subsets of BL driven by response to kinase inhibitors. In the combination screen, including BET, BTK and PI3K inhibitors, we identified synergistic combinations of PI3K and BTK inhibition with drugs targeting Akt, mTOR, BET and doxorubicin. A detailed comparison of PI3K and BTKi combinations identified subtle differences, in line with convergent pathway activity. Most synergistic combinations were identified for the BET inhibitor OTX015, which showed synergistic effects for 41% of combinations including inhibitors of PI3K/AKT/mTOR signalling. The strongest synergy was observed for the combination of the CDK 2/7/9 inhibitor SNS032 and OTX015. Our data provide a landscape of drug combination effects in BL and suggest that targeting CDK and BET could provide a novel vulnerability of BL.

Green tea reduces body fat via upregulation of neprilysin.

BACKGROUND/OBJECTIVE: Consumption of green tea has become increasingly popular, particularly because of claimed reduction in body weight. We recently reported that animals with pharmacological inhibition (by candoxatril) or genetic absence of the endopeptidase neprilysin (NEP) develop an obese phenotype. We now investigated the effect of green tea extract (in drinking water) on body weight and body composition and the mediating role of NEP. SUBJECTS/METHODS: To elucidate the role of NEP in mediating the beneficial effects of green tea extract, 'Berlin fat mice' or NEP-deficient mice and their age- and gender-matched wild-type controls received the extract in two different doses (300 or 600 mg kg-1 body weight per day) in the drinking water. RESULTS: In 'Berlin fat mice', 51 days of green tea treatment did not only prevent fat accumulation (control: day 0: 30.5% fat, day 51: 33.1%; NS) but also reduced significant body fat (green tea: day 0: 27.8%, day 51: 20.9%, P<0.01) and body weight below the initial levels. Green tea reduced food intake. This was paralleled by a selective increase in peripheral (in kidney 17%, in intestine 92%), but not central NEP expression and activity, leading to downregulation of orexigens (like galanin and neuropeptide Y (NPY)) known to be physiological substrates of NEP. Consequently, in NEP-knockout mice, green tea extract failed to reduce body fat/weight. CONCLUSIONS: Our data generate experimental proof for the assumed effects of green tea on body weight and the key role for NEP in such process, and thus open a new avenue for the treatment of obesity.

Effects of the angiotensin-(1-7) receptor Mas on cell proliferation and on the population of doublecortin positive cells within the dentate gyrus and the piriform cortex.

Aside from the well-known biologically active angiotensin II, other biologically active angiotensins have been discovered, including angiotensin IV and angiotensin-(1-7). Some years ago, we and others discovered that the Mas proto-oncogene encodes a G protein-coupled receptor being essential for angiotensin-(1-7) signaling. Mas is not only expressed in the periphery but also within the brain, e.g. in the dentate gyrus (DG) and the piriform cortex (PC). Since the DG is capable of adult neurogenesis, we examined the impact of a deletion of Mas upon adult neurogenesis. Deletion of Mas did not alter cell proliferation in the adult DG (as monitored with phosphohistone H3) and did not alter cell death (as monitored with activated Caspase 3). However, Mas deficiency resulted in an increase in the number of doublecortin (DCX) positive cells, indicating that lack of Mas increases the number of this cell population. Concerning the PC, it is discussed whether adult neurogenesis occurs under physiological conditions in this area. We could demonstrate that Mas deficiency has an impact on cell division and on the population of DCX-positive cells within the PC. Since Mas is not expressed before birth within the brain, our data may suggest that adult hippocampal neurogenesis and neurogenesis occurring during prenatal development share several common mechanisms, but are, at least in part, differentially regulated. Moreover, since deficiency for Mas increases the numbers of DCX-positive young neurons, blockage of Mas might be beneficial in stimulating neurogenesis in adults.

[Transcatheter aortic valve implantation (TAVI)]. / Transkatheteraortenklappenersatz (TAVI).

Aortic valve stenosis (AS) is the most common acquired valve disease in the industrialized countries. Most patients--especially young and low-risk patients--can be safely and effectively operated with aortic valve replacement. Due to increasing life expectancy, however, the number of elderly patients with AS and various concomitant diseases will increase in the coming decades. For those elderly high-risk patients, transfemoral or transapical aortic valve implantation (TAVI) has evolved as a valuable alternative to conventional surgery. The TAVI approach has been shown to be superior to standard medical treatment in these high-risk patients. All patients considered for TAVI should be discussed in a consensus conference consisting of cardiac surgeons and cardiologists (heart team). Furthermore, for successful treatment with transcatheter techniques, sophisticated pre-interventional imaging is required to screen patients. Available data on TAVI from randomized trials and large-scale registries demonstrate that this method is very promising.

Aortic stenosis in high-risk patients. Surgical therapy.

Conventional aortic valve replacement is the standard approach for treating aortic stenosis, it is performed via a full or partial sternotomy, and is associated with low risks for patients and with excellent long-term outcomes. This also holds true for octogenarians, if they present without relevant comorbidities. After resection of the calcified native leaflets, biological prostheses with good functionality and durability are implanted. Elderly patients with an increasing risk profile, however, should be treated by a heart team using transcatheter approaches including cardiac surgery.

The German Aortic Valve Registry (GARY): a nationwide registry for patients undergoing invasive therapy for severe aortic valve stenosis.

Background The increasing prevalence of severe aortic valve defects correlates with the increase of life expectancy. For decades, surgical aortic valve replacement (AVR), under the use of extracorporeal circulation, has been the gold standard for treatment of severe aortic valve diseases. In Germany ~12,000 patients receive isolated aortic valve surgery per year. For some time, percutaneous balloon valvuloplasty has been used as a palliative therapeutic option for very few patients. Currently, alternatives for the established surgical procedures such as transcatheter aortic valve implantation (TAVI) have become available, but there are only limited data from randomized studies or low-volume registries concerning long-time outcome. In Germany, the implementation of this new technology into hospital care increased rapidly in the past few years. Therefore, the German Aortic Valve Registry (GARY) was founded in July 2010 including all available therapeutic options and providing data from a large quantity of patients.Methods The GARY is assembled as a complete survey for all invasive therapies in patients with relevant aortic valve diseases. It evaluates the new therapeutic options and compares them to surgical AVR. The model for data acquisition is based on three data sources: source I, the mandatory German database for external performance measurement; source II, a specific registry dataset; and source III, a follow-up data sheet (generated by phone interview). Various procedures will be compared concerning observed complications, mortality, and quality of life up to 5 years after the initial procedure. Furthermore, the registry will enable a compilation of evidence-based indication criteria and, in addition, also a comparison of all approved operative procedures, such as Ross or David procedures, and the use of different mechanical or biological aortic valve prostheses.Results Since the launch of data acquisition in July 2010, almost all institutions performing aortic valve procedures in Germany joined the registry. By now, 91 sites which perform TAVI in Germany participate and more than 15,000 datasets are already in the registry.Conclusion The implementation of new or innovative medical therapies needs supervision under the conditions of a well-structured scientific project. Up to now relevant data for implementation of TAVI and long-term results are missing. In contrast to randomized controlled trials, GARY is a prospective, controlled, 5-year observational multicenter registry, and a real world investigation with only one exclusion criterion, the absence of patients' written consent.

Immunohistochemical localization of the angiotensin-(1-7) receptor Mas in the murine forebrain.

Apart from the well-known biologically active angiotensin II, other biologically active angiotensins have been discovered, including angiotensin IV and angiotensin-(1-7). Some years ago, we and others discovered that the Mas proto-oncogene encodes a receptor that is essential for angiotensin-(1-7) signaling. Angiotensin-(1-7) is not only expressed in the periphery but also within the brain. Based on that, we examined the distribution of Mas within the murine brain, using an antibody directed against the 3(rd) cytoplasmic loop of the receptor protein. Strongest Mas protein expression was detected in the dentate gyrus of the hippocampus and within the piriform cortex. However, Mas protein expression is not restricted to these areas, since Mas immunopositive neurons were also seen in different parts of the cortex, hippocampus, amygdala, basal ganglia, thalamus and hypothalamus. Based on the expression of Mas protein in the cortex and the limbic system, angiotensin-(1-7) signaling may play a role in synaptic plasticity, learning, memory and emotion, as has been described for angiotensin II and IV.

Endurance and performance of two different concepts for left ventricular stimulation with bipolar epicardial leads in long-term follow-up.

BACKGROUND: Epicardial left ventricular (LV) leads represent an alternative for CRT therapy if transvenous lead implantation fails. Data on endurance, performance, the impact of the surgical approach (lateral minithoracotomy vs. median sternotomy simultaneously with other cardiac surgery), and the optimal technical concept (screw-in vs. suture-on) is limited. METHODS: Over a period of 48 months we evaluated 130 consecutive patients with comparable characteristics. A total of 54 screw-in (MyoDex™ 1084T, SJM) and 76 suture-on (Capture Epi 4968, Medtronic) bipolar epicardial steroid-eluting LV leads were implanted either via a left lateral or a median thoracotomy. Sensing, pacing threshold, impedance and NYHA class were recorded at defined time points. RESULTS: No surgery-related death or major complication was observed. At the time of implantation, the pacing threshold, sensing and NYHA class did not differ significantly between the two groups. The impedances of screw-in leads were significantly lower compared to those of suture-on leads. Suture-on leads showed a moderate initial drop in their pacing threshold but afterwards remained stable. Screw-in leads were characterized by a moderate but significant increase in the pacing threshold in the first year followed by a continuous decrease thereafter. Twenty-four months post-implantation no differences between both lead types could be detected. Sensing and NYHA class improved in both groups. The surgical approach had no significant impact on lead functionality. CONCLUSION: Our study showed that the implantation of epicardial leads was safe with very low complication rates. There was no superior technical epicardial lead concept (screw-in vs. suture-on leads) and all epicardial leads demonstrated an excellent long-term performance and durability. Therefore, it seems that epicardial leads represent a good alternative to transvenous leads and surgeons should be encouraged to implant epicardial leads during concomitant cardiac surgery when the indications for CRT are present.

Aortic valve prosthesis tracking for transapical aortic valve implantation.

PURPOSE: Transapical aortic valve implantation (TA-AVI) is a new minimally invasive surgical treatment of aortic stenosis for high-risk patients. The placement of aortic valve prosthesis (AVP) is performed under 2D X-ray fluoroscopic guidance. Difficult clinical complications can arise if the implanted valve is misplaced. Therefore, we present a method to track the AVP in 2D X-ray fluoroscopic images in order to improve the accuracy of the TA-AVI. METHODS: The proposed tracking method includes the template matching approach to estimate the position of AVP and a shape model of the prosthesis to extract the corner points of the AVP in each image of sequence. To start the AVP tracking procedure, an initialization step is performed by manually defining the corner points of the prosthesis in the first image of sequence to provide the required algorithm parameters such as the AVP model parameters. RESULTS: We evaluated the AVP tracking method on six 2D intra-operative fluoroscopic image sequences. The results of automatic AVP localization agree well with manually defined AVP positions. The maximum localization errors of tracked prosthesis are less than 1 mm and within the clinical accepted range. CONCLUSIONS: For assisting the TA-AVI, a method for tracking the AVP in 2D X-ray fluoroscopic image sequences has been developed. Our AVP tracking method is a first step toward automatic optimal placement of the AVP during the TA-AVI.

Receptor for AGEs (RAGE) blockade may exert its renoprotective effects in patients with diabetic nephropathy via induction of the angiotensin II type 2 (AT2) receptor.

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) contributes to the development and progression of diabetic nephropathy. In this study, we examined whether the protective effects of RAGE blockade are exerted via modulation of the renal angiotensin II type 2 (AT2) receptor. METHODS: Control and streptozotocin diabetic mice, wild-type or deficient in the AT2 receptor (At2 knockout [KO]) or RAGE (Rage KO), were studied for 24 weeks. Adenoviral overexpression of full-length Rage in primary rat mesangial cells was also used to determine the effects on AT2 production. RESULTS: With diabetes, Rage-deficient mice had less albuminuria, and an attenuation of hyperfiltration and glomerulosclerosis as compared with diabetic wild-type and At2 KO mice. Renal gene and protein expression of RAGE was elevated with diabetes. Diabetic Rage KO mice had a greater increase in renal AT2 receptor protein than was seen in diabetic wild-type mice. Diabetes-induced increases in renal cytosolic and mitochondrial superoxide generation were prevented in diabetic Rage KO mice, but enhanced in all At2 KO mice. Adenoviral overexpression of RAGE or AGE treatment decreased cell surface AT2 expression, in association with increasing superoxide generation; both were reversed using antioxidants N-acetylcysteine and apocynin, and soluble RAGE in primary mesangial cells. CONCLUSIONS/INTERPRETATION: RAGE appears to be a common and key modulator of AT2 receptor expression, a finding that would implicate a newly defined RAGE-AT2 axis in the development and progression of diabetic nephropathy.

Towards a new image guidance system for assisting transapical minimally invasive aortic valve implantation.

We propose a new image guidance system for assisting transapical minimally invasive aortic valve implantation. The goal is to define the exact positioning of aortic valve prosthesis, preventing the misplacement of the valve. The proposed system consists of two stand-alone modules. First, preoperative planning software uses DynaCT images with manual anatomical landmarks to calculate the size and optimal position of the prosthesis. Second, an intraoperative system is developed for tracking of the prosthesis and the coronary ostia in 2-D fluoroscopic images. Then the safe area of implantation is defined. The preliminary experimental results of preoperative planning and intraoperative tracking system are promising.

Postoperative development of aspirin resistance following coronary artery bypass.

BACKGROUND: Aspirin therapy is known to substantially reduce mortality and the rate of ischaemic complications after coronary artery bypass grafting (CABG). Rates of perioperative aspirin resistance cited in the literature are up to 50% and could be influenced by extracorporeal circulation. Thus, aspirin resistance after CABG may have a significant clinical relevance. MATERIALS AND METHODS: In 59 patients undergoing CABG (on-pump, off-pump and combined procedures) aspirin resistance was investigated by arachidonic acid induced platelet aggregometry. Clinical relevance was assessed with 12-month follow up. RESULTS: Two types of resistance were observed: A preoperative resistance (despite oral aspirin or in vitro addition) was present in 29% and a postoperative developing type was seen in 49% resulting in only 22% of patients with a 'normal' reaction to aspirin. If patients were already on oral aspirin at admission, the rate of resistance was significantly reduced. Off-pump surgery or pump-times exceeding 120 min had no significant impact on resistance. During the 12-month follow up (98.3%), there were three deaths (one stroke, one intestinal ischaemia, one mediastinitis after postoperative delirium) in patients with the perioperative resistance and none in other patients (P = 0.345). In none of those patients who presented with perioperative aspirin resistance, could this aspirin resistance be demonstrated when tested again after 12 months? CONCLUSIONS: Aspirin resistance is a transient phenomenon present in the majority of patients undergoing CABG. The three deaths in the resistant group may - although not statistically significant - indicate the possibility of a worse outcome for patients with aspirin resistance.

Ischemic and inflammatory lung impairment by extracorporeal circulation: effect of PARP-inhibition by INO1001.

OBJECTIVE: Among the complications after cardiac surgery the development of postoperative pulmonary distress is a serious problem. Typically, the patients leave the operating theatre with good blood gas values and O(2)-saturation, but develop their respiratory problems within the next hours/days. We investigated whether extracorporeal circulation may induce biochemical and histological changes in the lungs which may help to explain this development. METHODS: Piglets (6-10 kg) were anaesthetized using isoflurane and underwent extracorporeal circulation (ECC) with hypothermic (25-28 degrees C) cardioplegic arrest for 90 min followed by 3h reperfusion. An additional group received a poly(ADP-ribose) polymerase (PARP)-Inhibitor, INO1001. Cardiopulmonary monitoring was performed during the whole procedure. Finally, lungs were explanted and investigated by histomorphometry and immunohistology for heat shock protein HSP70 (indicator for cellular damage) and TNFalpha in comparison to normal piglets without ECC. RESULTS: Histologically we found significant swelling of the type I alveocytes (thickness increased from 2.4 to 3.2 microm), interstitial oedema, intra-alveolar erythrocyte (4.8 versus 0.4 erythrocytes/alveole) and granulocyte accumulation and fibrinous exudates. There was a significant up-regulation of TNFalpha and of the cellular repair enzyme HSP70, while in control piglets only minimal levels were observed. INO1001 significantly reduced ECC-induced elevation in TNFalpha and in HSP70. Despite the dramatic changes after heart-lung-machine (HLM), blood gases and gas transport were almost not affected at that time. CONCLUSIONS: ECC can lead to early significant histological and histochemical changes which have similarities with a beginning early stage shock lung, although - at 3h reperfusion - gas transport is still sufficient. INO1001 can partially antagonize these changes.

Catheter interventional treatment of Sano shunt obstruction in patients following modified Norwood palliation for hypoplastic left heart syndrome.

UNLABELLED: Shunts placed between the right ventricle and the pulmonary arteries, called Sano shunts, recently modified Norwood surgery for hypoplastic left heart syndrome. Patients with Sano shunts tend to be more stable thus reducing the interstage mortality of this still challenging complex cardiac anomaly. However, Sano shunt stenosis may develop and is a life threatening complication. We report on our experience in patients with Sano shunt obstruction. PATIENTS: Eight infants presenting with decreasing transcutaneous oxygen saturations (43-63%, median 58%) following modified Norwood procedures were shown to have relevant Sano shunt stenosis. None was suited for early stage two surgery (cavopulmonary Glenn anastomosis). Catheterization was performed at the age of 21 to 112 (median 85) days. Weight was 3.9 to 6.0 (median 4.8) kg. TECHNIQUE: Femoral 5F venous access. Long sheaths were not used. The shunt was entered with a 4F right Judkins catheter and a selective angiography was performed. The stenosis was localized proximal in 5, distal in 1 and proximal and distal in 2 patients. Ten coronary stents were implanted. RESULTS: There were no procedure related complications. Oxygen saturation increased immediately to 75-86% (median 80%) and remained above 70% during follow-up in all. Seven patients had successful stage two surgery 61-288 (median 134) days after stent implantation, one is awaiting this. CONCLUSIONS: Sano shunt obstruction can be treated safely and effectively by stent implantation. Early in-stent restenosis does not seem to be a problem.