Network-driven discovery yields new insight into Shox2-dependent cardiac rhythm control.

The homeodomain transcription factor SHOX2 is involved in the development and function of the heart's primary pacemaker, the sinoatrial node (SAN), and has been associated with cardiac conduction-related diseases such as atrial fibrillation and sinus node dysfunction. To shed light on Shox2-dependent genetic processes involved in these diseases, we established a murine embryonic stem cell (ESC) cardiac differentiation model to investigate Shox2 pathways in SAN-like cardiomyocytes. Differential RNA-seq-based expression profiling of Shox2+/+ and Shox2-/- ESCs revealed 94 dysregulated transcripts in Shox2-/- ESC-derived SAN-like cells. Of these, 15 putative Shox2 target genes were selected for further validation based on comparative expression analysis with SAN- and right atria-enriched genes. Network-based analyses, integrating data from the Mouse Organogenesis Cell Atlas and the Ingenuity pathways, as well as validation in mouse and zebrafish models confirmed a regulatory role for the novel identified Shox2 target genes including Cav1, Fkbp10, Igfbp5, Mcf2l and Nr2f2. Our results indicate that genetic networks involving SHOX2 may contribute to conduction traits through the regulation of these genes.

A zebrafish model for FHL1-opathy reveals loss-of-function effects of human FHL1 mutations.

Missense mutations in the four and a half LIM domain 1 (FHL1) gene were found to cause X-linked inherited myopathies of both skeletal and heart muscles. However, the mechanisms by which FHL1 mutations impact on FHL1 function and lead to alteration of muscle structure and function have not been deciphered yet. We generated here by Morpholino-modified antisense oligonucleotide-mediated gene knockdown fHL1-deficient zebrafish embryos. Similar to the human situation, fhl1a-morphants zebrafish displayed severe skeletal and heart muscle myopathy. Whereas ectopic expression of wild-type FHL1 (FHL1 wt) suppressed both skeletal and heart muscle myopathy in fhl1a-morphants zebrafish, overexpression of the FHL1-opathy associated human mutations FHL1-H123Y, FHL1-C132F or FHL1-C224W did not rescue skeletal and heart muscle myopathy in fhl1a-morphants. Overexpression of FHL1-H123Y, FHL1-C132F or FHL1-C224W in wild-type zebrafish did not induce myopathy in a dominant-negative mode. Altogether these results indicate that FHL1 mutations found to cause X-linked FHL1-opathies in humans consistently lead to severely impaired FHL1 function.

Comparison of video analysis and simulations of a drum coating process.

Tablet coating is a common unit operation in the pharmaceutical industry. To improve currently established processes, it is important to understand the influence of the process parameters on the coating quality. One of the critical parameters is the tablet velocity. In this work, numerical results are compared to results obtained experimentally. Tablet movement in the drums was simulated using the Discrete Element Method (DEM). The simulation parameters were adapted to fit the simulation to the experimental data. A comparison of the experimental and simulation results showed that the simulation correctly represents the real tablet velocity. A change in the velocity over time and its dependence on the rotation rates and the baffle position in the simulation were similar to the experimental results. In summary, simulations can improve the understanding of tablet coating processes and will thus provide insights into the underlying process mechanics, which cannot be obtained via ordinary experiments.

[Development and Periodicity of Human Exposures in Suicidal Intention Reported to the Centre Erfurt from 2004-2013]. / Entwicklung und Periodizität humaner Expositionen in suizidaler Absicht im Einzugsbereich des Giftnotrufs Erfurt von 2004­2013.

Aim of the Study: Exposures in suicide attempts are demanding for hospitals and poisons information centres (PICs). Therefore, the time characteristics of their frequency were studied. Methods: A retrospective analysis was undertaken of all human exposures reported to Centre Erfurt from the beginning of 2004 to the end of 2013 according to their frequency in the respective year, season, month, weekday, time of the weekday, circumstances of exposure, age and gender. Results: 59.7% of all exposures (n=137 104) were accidental, 23.4% occurred in suicide attempts and 3.3% in substance abuse. 0.3% of the suicide attempts resulted in death. Their number continuously increased from n=2 422 in 2004 to n=3 458 in 2013, but their relative frequency remained almost constant at 23.4%. Their highest numbers were reached in the spring and summer with maxima in July and August and minima in February and September. During the week, the most suicide attempts were observed between Sunday and Tuesday and the least on Friday. Highest rate of suicide attempts was seen at 10 pm and lowest at 6 am. The median of age was 39 years (first quartile 24 years, third quartile 50 years). The female proportion was almost twice as high as the male. Conclusions: Hospitals the Centre Erfurt is serving should be particularly prepared for exposures in suicide attempts in the spring and summer (especially in July and August), at the beginning of the week and shortly before midnight.

Prognostic factors associated with mortality in patients with septic arthritis: a descriptive cohort study.

OBJECTIVES: To evaluate the 30-day mortality rate of septic arthritis (SA) in adults in Funen, central Denmark, and to explore whether, at the time of SA presentation, risk factors for the 30-day mortality rate could be revealed. Our secondary objective was to describe the microbiological aetiologies, systemic signs of inflammation, and co-morbidity. METHOD: A descriptive study identifying patients with SA from central Denmark, during the period 2006-2013, by the use of joint fluid culture data retrieved from the electronic database at the Department of Clinical Microbiology, Odense University Hospital. Patients with a positive joint fluid culture were considered eligible and their medical records were examined. RESULTS: We identified 215 patients with SA, mean age 64.8 years. At presentation, mean C-reactive protein (CRP) was 204 mg/L, mean white blood cell count (WBC) 11.9 × 109/L, and mean body temperature 37.6°C. A total of 101 patients (47%) had a prosthetic joint, 46 (21%) had an inflammatory joint disease, and 24 (11%) had diabetes mellitus (DM). Staphylococcus aureus was the most common pathogen (104 patients, 48.4%). The 30-day mortality rate was 9.3% and the significant risk factor for death was liver disease at time of presentation [odds ratio (OR) 40.40, 95% confidence interval (CI) 5.38-303]. The other factors tested such as age > 65 years, elevated temperature, rheumatoid arthritis (RA), prostheses, and diabetes mellitus (DM) did not reach statistical significance. CONCLUSIONS: In our sample of patients with SA, we found a 30-day mortality rate in almost one in 10 adults. Among possible explanations, our study indicates that liver disease is a clinically relevant risk factor.

Simulation of a tablet coating process at different scales using DEM.

Spray coating of tablets is an important unit operation in the pharmaceutical industry and is mainly used for modified release, enteric protection, better appearance and brand recognition. It can also be used to apply an additional active pharmaceutical ingredient to the tablet core. Scale-up of such a process is an important step in commercialization. However, scale-up is not trivial and frequently, at manufacturing scales the required coating quality cannot be reached. Thus, we propose a method where laboratory experiments are carried out, yet scale-up is done via computational methods, i.e., by extrapolating results to larger scales. In the recent years, the Discrete Element Method (DEM) has widely been used to simulate tablet behavior in a laboratory scale drum coater. Due the increasing computational power and more sophisticated DEM algorithms, it has become possible to simulate millions of particles on regular PCs and model industrial scale tablet coating devices. In this work, simulations were performed on the laboratory, pilot and industrial scales and DEM was used to study how different scale-up rules influence the bed behavior on larger scales. The material parameters of the tablets were measured in the laboratory and a glued sphere approach was applied to model the tablet shape. The results include a vast amount of qualitative and quantitative data at the different scales. In conclusion, the evolution of the inter-tablet coating variation for the different scales and process parameters is presented. The results suggest that keeping the Froude number constant during the scale up process leads to faster processes as the cycle time is shorter and the spray residence time is more uniform when compared to keeping the circumferential velocity constant.

Analysis of large-scale tablet coating: Modeling, simulation and experiments.

This work concerns a tablet coating process in an industrial-scale drum coater. We set up a full-scale Design of Simulation Experiment (DoSE) using the Discrete Element Method (DEM) to investigate the influence of various process parameters (the spray rate, the number of nozzles, the rotation rate and the drum load) on the coefficient of inter-tablet coating variation (cv,inter). The coater was filled with up to 290kg of material, which is equivalent to 1,028,369 tablets. To mimic the tablet shape, the glued sphere approach was followed, and each modeled tablet consisted of eight spheres. We simulated the process via the eXtended Particle System (XPS), proving that it is possible to accurately simulate the tablet coating process on the industrial scale. The process time required to reach a uniform tablet coating was extrapolated based on the simulated data and was in good agreement with experimental results. The results are provided at various levels of details, from thorough investigation of the influence that the process parameters have on the cv,inter and the amount of tablets that visit the spray zone during the simulated 90s to the velocity in the spray zone and the spray and bed cycle time. It was found that increasing the number of nozzles and decreasing the spray rate had the highest influence on the cv,inter. Although increasing the drum load and the rotation rate increased the tablet velocity, it did not have a relevant influence on the cv,inter and the process time.

[Human Single Drug Exposures to Non-opioid Analgesics Reported to the Poisons Information Centre Erfurt from 2003 to 2012]. / Humane nichtopioide Analgetika-Monoexpositionen im Einzugsbereich des Giftnotrufes Erfurt von 2003-2012.

AIM OF THE STUDY: Because of their frequency, non-opioid analgesics (NOA) single drug exposures registered by Poisons Information Centre (PIC) Erfurt have been studied over a decade. METHODS: A retrospective analysis of frequencies, circumstances of exposure, symptom severity, and age groups in NOA single drug exposures received by the PIC Erfurt from the beginning of 2003 to the end of 2012 was undertaken. RESULTS: Of all 4749 NOA single drug exposures, the 10 most frequent were caused by paracetamol (n=1 686), ibuprofen (n=1 439), acetylsalicylic acid (n=456), dipyrone (n=274), diclofenac (n=267), flupirtine (n=138), naproxen (n=41), etoricoxib (n=36), indomethacin (n=24), and dexketoprofen (n=19). Paracetamol single drug exposures increased from 158 in 2003 to 216 in 2007 and fell afterwards to 133 in 2012. Ibuprofen single drug exposures continously rose from 57 in 2003 to 258 in 2012. Adults were more often involved in NOA (53.8%) and all single drug exposures (54.1%) than children (45.9% and 45.6%, respectively). Suicidal attempts were more frequent in NOA (43.1%) than in all single drug exposures (34.2%), whereas accidental exposures or exposures in abuse were less often (33.4 and 0.2%, 46.0 and 0.9% respectively). NOA single drug exposures resulted mostly in none to minor symptoms (77.0%) and rarely in moderate (2.1%) or severe symptoms (1.0%). One adult was found dead after probable ingestion of 32 g of acetylsalicylic acid in suicidal intention. CONCLUSIONS: Because many NOA are over-the-counter drugs, it is difficult to obtain data on their use. PIC data could provide information on the NOA use in the population.

Single test isolated lupus anticoagulant positivity is associated with increased plasma levels of inflammatory markers and dyslipidemia.

OBJECTIVE: To investigate whether a single positive test for lupus anticoagulant (LA) is associated with levels of inflammatory markers and traditional cardiovascular risk factors, independent of autoimmune disease, thrombophilia and occurrence of other antiphospholipid antibodies. METHODS: In a retrospective observational study we included persons referred for thrombophilia testing during 2011-2014. Persons with autoimmune disease, thrombophilia or presence of specific anti-phospholipid antibodies were excluded. Multivariate logistic regression analyses adjusted for age and sex was performed and odds ratios (ORs) with 95% confidence intervals (95% CI) calculated. RESULTS: Of 381 individuals tested, 271 fulfilled the criteria, of whom 22 (8%) were LA positive and 249 (92%) LA negative. LA positivity was associated with higher body mass index (BMI) (OR 1.12, 95% CI: 1.03-1.23, p = 0.01); C-reactive protein (OR 1.08 95% CI:1.04-1.11, p < 0.001); fibrinogen (OR 1.51 95% CI: 1.27-1.78, p < 0.001); coagulation factor VIII (FVIII) (OR 1.73 95% CI: 1.01-2.96, p = 0.046), low high density lipoprotein (HDL) (OR 0.03 95% CI: 0.00-0.19, p < 0.001) and high triglyceride (OR 1.81 95% CI: 1.12-2.92, p = 0.02) compared with LA negative individuals. CONCLUSION: This study shows that single test isolated LA positivity is associated with increased levels of inflammatory markers, low HDL cholesterol, elevated triglyceride and high BMI.

Level of Adherence to Prophylactic Osteoporosis Medication amongst Patients with Polymyalgia Rheumatica and Giant Cell Arteritis: A Cross-Sectional Study.

Objective. To estimate level of adherence to oral calcium and vitamin D supplementation as well as bisphosphonate amongst patients with PMR and GCA treated with glucocorticoids. Method. A total of 138 patients with the diagnosis of PMR and/or GCA registered in our department in December 2013. In this cross-sectional study we interviewed all the patients to measure level of adherence to calcium and vitamin D, as well as bisphosphonates. Results. Out of the 118 included patients, 88.9% of them were adherent to their prescription. Only 2 patients (1.7%) did not take calcium and vitamin D at all and 10 patients (8.5%) took their medication infrequently, 9 and 1 out of 10 patients took the medication 50-100% of the time and less than 50% of the prescribed dose, respectively. Sixty-one patients received additional treatment with bisphosphonate and 96.6% were adherent to this therapy. The remaining 3.4% of the patients did not take the medication at all. Forgetfulness, adverse side effects, and lack of understanding of treatment benefits were the most significant causes for nonadherence to calcium and vitamin D. Conclusions. Contrary to what we expected this study found that adherence to osteoporosis preventive medication in patients with PMR and GCA was high.

Electrophysiological characterization of spinal neurons in different models of diabetes type 1- and type 2-induced neuropathy in rats.

Diabetic polyneuropathy (DPN) is a devastating complication of diabetes. The underlying pathogenesis of DPN is still elusive and an effective treatment devoid of side effects presents a challenge. There is evidence that in type-1 and -2 diabetes, metabolic and morphological changes lead to peripheral nerve damage and altered central nociceptive transmission, which may contribute to neuropathic pain symptoms. We characterized the electrophysiological response properties of spinal wide dynamic range (WDR) neurons in three diabetic models. The streptozotocin (STZ) model was used as a drug-induced model of type-1 diabetes, and the BioBreeding/Worcester (BB/Wor) and Zucker diabetic fatty (ZDF) rat models were used for genetic DPN models. Data were compared to the respective control group (BB/Wor diabetic-resistant, Zucker lean (ZL) and saline-injected Wistar rat). Response properties of WDR neurons to mechanical stimulation and spontaneous activity were assessed. We found abnormal response properties of spinal WDR neurons in all diabetic rats but not controls. Profound differences between models were observed. In BB/Wor diabetic rats evoked responses were increased, while in ZDF rats spontaneous activity was increased and in STZ rats mainly after discharges were increased. The abnormal response properties of neurons might indicate differential pathological, diabetes-induced, changes in spinal neuronal transmission. This study shows for the first time that specific electrophysiological response properties are characteristic for certain models of DPN and that these might reflect the diverse and complex symptomatology of DPN in the clinic.

The bradykinin B1 receptor antagonist BI113823 reverses inflammatory hyperalgesia by desensitization of peripheral and spinal neurons.

BACKGROUND: Bradykinin is a neuropeptide released after tissue damage which plays an important role in inflammatory pain. The up-regulation of the bradykinin B1 receptor in response to inflammation makes it an attractive target for drug development. Aim was to investigate if the selective B1 receptor antagonist BI113823 reduces inflammation-induced mechanical hyperalgesia and if the effect is mediated via peripheral and/or spinal B1 receptor antagonism. METHODS: Electrophysiological recordings of peripheral afferents and spinal neurons were combined with behavioural experiments to better understand the underlying mechanisms of B1 receptor antagonism. Experiments were performed 24 h after injection of complete Freund's adjuvant (CFA) or saline into the paw of Wistar rats. A gene expression analysis for the B1 receptor was performed in different tissues. BI113823 was administered orally or intrathecally to assess effects on CFA-induced hyperalgesia. Peripheral afferents of the saphenous nerve as well as spinal wide dynamic range (WDR) and nociceptive-specific (NS) neurons were recorded, and mechanosensitivity was measured before and after BI113823 administration. RESULTS: BI113823 reduced CFA-induced mechanical hyperalgesia when administered orally or intrathecally. An increased B1 receptor gene expression was found in peripheral and spinal neural tissue. BI113823 significantly reduced mechanosensitivity of peripheral afferents and spinal NS neurons, but had no effect on WDR neurons. CONCLUSION: The selective bradykinin B1 receptor antagonist BI113823 reduces CFA-induced mechanical hyperalgesia which is mediated via antagonism of peripheral as well as spinal bradykinin B1 receptors. The selective modulation of CFA-sensitized spinal NS neurons by BI113823 could be a promising property for the treatment of inflammatory pain.

Which criteria demand additive stenting during catheter-directed thrombolysis?

Many factors are necessary for obtaining satisfactory results after catheter-directed thrombolysis (CDT) for iliofemoral deep venous thrombosis (DVT). Selections of patients, composition of the thrombolytic fluid, anticoagulation per- and post-procedural, recognition and treatment of persistent obstructive lesions of the iliac veins are the most important contributors. Stenting has been known for 15 to 20 years. The first publication on CDT in 1991 was combined with ballooning the iliac vein, an additive procedure which has been abandoned as an isolated procedure. This chapter will discuss selection, indication, such as an iliac compression syndrome, and outcome of iliac stenting in combination with CDT. The reported frequency of stenting used after CDT is very inconsistent, therefore this will be discussed in details. It is concluded that selection for stenting is of the greatest importance, when CDT is used for iliofemoral DVT, but strict criteria for stenting are not available in the existing literature. The potential value of intravascular ultrasound (IVUS) is also discussed.

[Human exposures to veterinary medicines reported to the Poisons Information Centre Erfurt from 2003 to 2012]. / Humane Tierarzneimittelexpositionen im Einzugsbereich des Giftnotrufes Erfurt 2003-2012.

AIM OF THE STUDY: The purpose of this study was to get information on all human exposures to veterinary medicines (HEVM) reported to the Poisons Information Centre (PIC) over a 10-year period. METHODS: A retrospective analysis of all HEVM was undertaken and a comparison was made to all human exposures (HE) registered by the PIC from the beginning of 2003 to the end of 2012 according to frequencies, circumstances of exposure, symptom severity, age groups, and substances involved in HEVM. RESULTS: In total, 389 cases of HEVM with 409 veterinary medicines were registered (0.30% of all HE, 360 monoexposures). The relative frequency of children and adults in HEVM (children: 52.4%, adults: 46.0%) and all HE (children: 48.7%, adults 48.7%) was the same with significant (p<0.05) differences in some age subgroups. The portion of accidental exposures was significantly (p<0.05) higher in HEVM (83.3%) than in all exposures (59.3%), whereas the portion of suicidal exposures was significantly (p<0.05) lower (HEVM: 6.4%, all exposures: 23.6%). Most frequent veterinary medicines (ATCvet) in HEVM were antiparasitic substances, insecticides and repellents (n=185), substances for the nervous system (n=48), substances for the cardiovascular system (n=35), and immunologicals (n=35). HEVM mostly resulted in no or mild symptoms (83.8%) and rarely in moderate (10/389, 2.6%) or even severe symptoms (5/389, 1.3%). In 4 of 5 cases of HEVM with severe symptoms, veterinary surgeons used products for animal euthanasia (n=3) or methadone (n=1). Once, self-medication with anthelmintics for several days by a goatherd resulted in transient blindness. CONCLUSIONS: In comparison to other HE, HEVM are rare. Most accidental HEVM in laymen result only in none to mild symptoms. If veterinary surgeons, however, swallow or inject products for animal euthanasia or opioids in suicidal intention, severe symptoms can be expected.

Functional genomics in zebrafish as a tool to identify novel antiarrhythmic targets.

In the face of an aging population and thereby an increasing number of patients suffering from heart rhythm diseases development of therapeutic agents is one of the major challenges in modern biomedical research. Antiarrhythmic drug discovery was mainly hindered by the limited knowledge of the molecular underpinnings of cardiac electrophysiology in health and disease. In recent years, the zebrafish has emerged as an effective model organism to dissect the pathology of human disorders in particular in the area of cardiovascular diseases. Especially, certain aspects of cardiac electrophysiology of the zebrafish such as action potential or heart rate are similar to that of humans. The zebrafish shares many features of human physiology and body plan but it develops extra-uterine and is initially transparent, allowing detailed and comprehensive characterization of cardiac development and function in vivo. Moreover, zebrafish are well amenable to large-scale forward and reverse functional genomics approaches, which has led to the identification of numerous novel genetic key-players and potential targets of cardiac disease. In this context, several zebrafish lines with mutations in defined ion channels have emerged as novel vertebrate models for human arrhythmia disorders such as long or short QT syndrome. In addition, due to its size and the high number of progeny, zebrafish are very suitable for rapid in vivo analysis of the bioactivity of small molecules and their therapeutic potential, especially in the context of cardiovascular diseases such as arrhythmias. In this review we highlight an assortment of established zebrafish models that enable the dissection of human heart rhythm disorders and the potential of this model system for the discovery of novel antiarrhythmic targets and drugs.

Indications for stenting during thrombolysis.

The most important vein segment to thrombolyse after deep venous thrombosis (DVT) is the outflow tract meaning the iliofemoral vein. Iliofemoral DVT is defined as DVT in the iliac vein and the common femoral vein. Spontaneous recanalization is less than 50%, particularly on the left side. The compression from adjacent structures, predominantly on the left side is known as the iliac vein compression syndrome. Therefore, it is essential that supplementary endovenous procedures have to be performed in case of persistent obstructive lesions following catheter-directed thrombolysis. Insertion of a stent in this position is the treatment of choice facilitating the venous flow into an unobstructed outflow tract either from the femoral vein or the deep femoral vein or both. The stent, made of stainless steel or nitinol, has to be self-expandable and flexible with radial force to overcome the challenges in this low-pressure system. The characteristics of the anatomy with external compression and often a curved vein segment with diameter difference make stent placement necessary. Ballooning alone has no place in this area. The proportion of inserted stents varies in the published materials with catheter-directed thrombolysis of iliofemoral deep venous thrombosis.

Long-term results using catheter-directed thrombolysis in 103 lower limbs with acute iliofemoral venous thrombosis.

OBJECTIVES: The long-term outcome of catheter-directed thrombolysis (CDT) in patients with acute iliofemoral venous thrombosis (IFVT) is evaluated in this study. MATERIAL AND METHODS: Patients presenting for treatment with IFVT between June 1999 and May 2007 were considered for treatment using CDT. The following inclusion criteria were used: first episode of IFVT, age below 60 years, age of thrombus <14 days and open distal popliteal vein. Ultrasonography (US) was used to verify the diagnosis. The popliteal vein was punctured under local anaesthesia using US guidance, and a multi-side-hole catheter with tip occlusion was placed in the thrombus. A solution of r-TPA was infused either continuously or using the pulse spray technique together with heparin. Any occlusion or residual stenosis in the iliac vein system was treated by stenting. Compression stockings and anticoagulation treatment were given for at least 12 months. Patients with severe thrombophilias were treated for longer periods. The patients were assessed by colour-duplex US for assessment of patency and valve function after 6 weeks, 3, 6 and 12 months and afterwards on a yearly basis. RESULTS: A total of 101 patients with 103 extremities affected by iliofemoral venous thrombosis were included (median age; 29 years, 78 women, and 79 had left-sided thrombosis). A stent was inserted in 57 limbs. The median follow-up time was 50 months (range 3 days-108 months). At 6 years, 82% of the limbs had patent veins with competent valves and without any skin changes or venous claudication. CONCLUSION: Treatment with CDT for IFVT achieves good patency and vein function after 6 years of follow-up in this highly selected group of patients. We suggest that results from future studies should be presented as Kaplan-Meier plots using venous patency without reflux as the main outcome, since it is an early indicator of the clinical outcome.