On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/µL, 1 GU/µL, and 5 × 10(3) GU/µL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

Acoustic monitoring of changes in well-defined hyaluronan layers exposed to chondrocytes.

The interaction of human-derived chondrocytes and thin hyaluronan layers was studied using the quartz crystal microbalance with dissipation (QCM-D) technique combined with light microscopy. This approach allowed unique real-time monitoring of the interface between the cells and the sensor surface. Our results suggest that the hyaluronan layer is rapidly degraded by chondrocytes.

[Pulmonary neuroendocrine neoplasms]. / Neuroendokrine Neoplasien der Lunge.

The pulmonary neuroendocrine neoplasms originate from the enterochromaffin cells which are diffusely distributed in the body. The incidence of these tumors has increased significantly in recent decades due to the available diagnostics. They make up about 1-2% of all lung tumors and 20-30% of all neuroendocrine neoplasms. The current WHO classification from 2004 divides them into typical carcinoids (TC), atypical carcinoids (AC), large cell neuroendocrine carcinomas (LCNEC) and small cell carcinomas (SCLC). The major neuroendocrine biomarkers are chromogranin A, synaptophysin and CD56. TC have a low mitotic rate of <2 mitoses/2mm(2) (10 HPF), whereas the mitotic rate of the AC is 2-10 mitoses/2 mm(2) (10 HPF). The Ki-67 staining is helpful to distinguish typical and atypical carcinoids from the highly malignant LCNEC and SCLC. Clinically, the patient presents usually with cough, hemoptysis or bronchial obstruction. The occurrence of a carcinoid or Cushing's syndrome and a tumor-associated acromegaly are rare. Surgical resection with radical lymph node dissection is the treatment of choice for achieving long-term survival. Endoscopic resection of the endobronchial tumor growth is a good alternative for inoperable endobronchially localized tumors. Peptide receptor radionuclide therapy (PRRT) is a promising treatment option for patients with metastatic or unresectable pulmonary neuroendocrine tumors. New targeted therapies using angiogenesis inhibitors, mTOR inhibitors, and tyrosine kinase inhibitors are being tested for their effectiveness in many previous studies. Typical carcinoid tumors metastasize less frequently than AC, the 5-year survival rate of patients with TC being over 90%. Patients with AC have a 5-year survival rate between 35% and 87%. The highly malignant LCNEC and SCLC, on the other hand, have a 5-year survival rate between 15% and 57%, and <5% respectively. The increasing number of therapeutic options and diagnostic procedures requires a multidisciplinary approach and decision-making in multidisciplinary tumor conferences to ensure a personalized treatment approach. Therefore patients with a neuroendocrine neoplasm of the lung should be treated in specialized centers.

[Hereditary Alzheimer's disease with amyloid angiopathy caused by amyloid precursor protein locus]. / Familiäre Alzheimer-Variante mit Amyloidangiopathie als Ausdruck einer APP-Gen-Duplikation.

We report a patient with early-onset autosomal dominant dementia. The CSF showed increased levels of tau protein and decreased amyloid beta (ratio 42:40) typical for Alzheimer's disease. Cerebral MRI revealed vascular lesions and white-matter changes around the posterior horns of the ventricles with only moderate atrophy of the brain. Susceptibility-weighted imaging detected multiple small hemorrhagic changes. Gene analysis revealed amyloid precursor protein (APP) locus duplication as the cause of hereditary Alzheimer's dementia. The co-occurrence of CSF changes typical for Alzheimer's disease and MRI findings of cerebral amyloid angiopathy is remarkable, as it is also described for APP locus duplication. In conjunction with a family history suggestive of hereditary dementia, such a constellation should lead to enhanced gene analysis.

Clinical and angiographic risk factors for stroke and death within 30 days after carotid endarterectomy and stent-protected angioplasty: a subanalysis of the SPACE study.

BACKGROUND: Carotid endarterectomy (CEA) and carotid artery stenting (CAS) are used to prevent ischaemic stroke in patients with stenosis of the internal carotid artery. Better knowledge of risk factors could improve assignment of patients to these procedures and reduce overall risk. We aimed to assess the risk of stroke or death associated with CEA and CAS in patients with different risk factors. METHODS: We analysed data from 1196 patients randomised to CAS or CEA in the Stent-Protected Angioplasty versus Carotid Endarterectomy in Symptomatic Patients (SPACE) trial. The primary outcome event was death or ipsilateral stroke (ischaemic or haemorrhagic) with symptoms that lasted more than 24 h between randomisation and 30 days after therapy. Six predefined variables were assessed as potential risk factors for this outcome: age, sex, type of qualifying event, side of intervention, degree of stenosis, and presence of high-grade contralateral stenosis or occlusion. The SPACE trial is registered at Current Controlled Trials, with the international standard randomised controlled trial number ISRCTN57874028. FINDINGS: Risk of ipsilateral stroke or death increased significantly with age in the CAS group (p=0.001) but not in the CEA group (p=0.534). Classification and regression tree analysis showed that the age that gave the greatest separation between high-risk and low-risk populations who had CAS was 68 years: the rate of primary outcome events was 2.7% (8/293) in patients who were 68 years old or younger and 10.8% (34/314) in older patients. Other variables did not differ between the CEA and CAS groups. INTERPRETATION: Of the predefined covariates, only age was significantly associated with the risk of stroke and death. The lower risk after CAS versus CEA in patients up to 68 years of age was not detectable in older patients. This finding should be interpreted with caution because of the drawbacks of post-hoc analyses.

[New strategies and materials in endoscopic vacuum therapy in the lower gastrointestinal tract]. / Neue Strategien und Materialien bei der endoskopischen Vakuumtherapie im unteren Gastrointestinaltrakt.

BACKGROUND: Endoscopic vacuum therapy is a widespread method in the postoperative treatment of lower and upper gastrointestinal (GI) tract leakage. OBJECTIVE: There is an absence of further technical development of the standardized material from 2007 for the lower GI tract. MATERIAL AND METHODS: New strategies and new materials for endoscopic vacuum therapy are presented. RESULTS: Alternative strategies in sponge placement, use of open-pore film drainage, use of a multiple sponge system, rinsing catheter, electronic pumps etc. enable the successful treatment of very complex pelvic defects. CONCLUSION: The wide variability of pelvic defects often necessitates a change in therapeutic strategies during the course of treatment for an optimized outcome.

30 day results from the SPACE trial of stent-protected angioplasty versus carotid endarterectomy in symptomatic patients: a randomised non-inferiority trial.

BACKGROUND: Carotid endarterectomy is effective in stroke prevention for patients with severe symptomatic carotid-artery stenosis, and carotid-artery stenting has been widely used as alternative treatment. Since equivalence or superiority has not been convincingly shown for either treatment, we aimed to compare the two. METHODS: 1200 patients with symptomatic carotid-artery stenosis were randomly assigned within 180 days of transient ischaemic attack or moderate stroke (modified Rankin scale score of < or =3) carotid-artery stenting (n=605) or carotid endarterectomy (n=595). The primary endpoint of this hospital-based study was ipsilateral ischaemic stroke or death from time of randomisation to 30 days after the procedure. The non-inferiority margin was defined as less than 2.5% on the basis of an expected event rate of 5%. Analyses were on an intention-to-treat basis. This trial is registered at Current Controlled Trials with the international standard randomised controlled trial number ISRCTN57874028. FINDINGS: 1183 patients were included in the analysis. The rate of death or ipsilateral ischaemic stroke from randomisation to 30 days after the procedure was 6.84% with carotid-artery stenting and 6.34% with carotid endarterectomy (absolute difference 0.51%, 90% CI -1.89% to 2.91%). The one-sided p value for non-inferiority is 0.09. INTERPRETATION: SPACE failed to prove non-inferiority of carotid-artery stenting compared with carotid endarterectomy for the periprocedural complication rate. The results of this trial do not justify the widespread use in the short-term of carotid-artery stenting for treatment of carotid-artery stenoses. Results at 6-24 months are awaited.

Metritis vaccination in Holstein dairy heifers using a herd-specific multivalent vaccine - Effects on uterine health and fertility in first lactation.

In cattle, acute puerperal metritis (APM) compromises animal welfare and has an economic impact to the farmer because of the detrimental effects on reproductive performance, milk production, and survivability. The aim of this randomized clinical study was to investigate the effects of a prepartum immunization using a herd-specific multivalent vaccine on incidence of APM, prevalence of clinical endometritis (CE), and selected measures of reproductive performance in primiparous Holstein cows. Pregnant nulliparous cows (230±4days of gestation) assigned to the vaccinated group (VG; n=142) received subcutaneously 5.0mL of a multivalent herd-specific vaccine containing inactivated whole bacterial cells of Trueperella pyogenes, Escherichia coli, Streptococcus uberis, Bacteroides species, and Peptostreptococcus species obtained from uterine swabs of primiparous cows suffering from APM at the time of enrolment and three weeks later. Heifers allocated to the control group (Co; n=144) remained unvaccinated. Rectal temperature in the first 10days in milk (DIM) did not differ between treatments, and no interaction between treatment and day was observed (interaction treatment*day: p=0.623). Incidence of APM (VG, 46.0%, Co, 48.9%, p=0.588), number of antibiotic doses per cow to treat APM (VG, 0.63±0.81, Co, 0.64±0.75, p=0.496), prevalence of CE at 35 DIM (VG, 24.6%, Co, 19.3%, p=0.350), first service pregnancy per artificial insemination (VG, 40.3%, Co, 45.5%, p=0.541), number of inseminations until 150 DIM (VG, 2.0±1.4, Co, 1.9±1.3, p=0.749), interval from calving to first service (median days, VG, 52, Co, 52, p=0.915), and interval from calving to pregnancy (median days, VG, 90, Co, 83, p=0.419) did not differ between VG and Co. Treatment did not affect activity of aspartate aminotransferase (AST) and creatine kinase (CK) or concentrations of beta-hydroxybutyrate (BHB) and haptoglobin in serum samples collected at 5 DIM. Furthermore, treatment did not influence yields of milk, fat or protein in the first 100 DIM. Moreover, no signs that vaccination provided an attenuation of severity of the APM cases were detected as the course of rectal body temperature and values of AST, CK, BHB, and haptoglobin were not influenced by treatment within the group of cows with diagnosed APM. Use of this herd-specific multivalent vaccine designed to reduce metritis was not efficacious at reducing the risk of uterine disease or influencing production and reproduction in dairy cows.

Development of a conjunctival tissue substitute on the basis of plastic compressed collagen.

Ocular surface disorders, such as pterygium, cicatricial pemphigoid and external disruptions, can cause severe inflammation, scarring, fornix shortening as well as ankyloblepharon. Current treatments do not resolve these conditions sufficiently. The aim of this study was to evaluate clinical applicability and suitability of plastic compressed collagen to serve as a substrate for the expansion of human conjunctival epithelial cells in order to develop an epithelialized conjunctival substitute for fornix reconstruction. Human conjunctival epithelial cells were expanded on plastic compressed collagen gels. Epithelial cell characteristics were evaluated by haematoxylin and eosin staining, electron microscopy and cytokeratin expression. The expression of putative epithelial progenitor cell markers p63α, ABCG2 and CK15 was assessed by immunostaining. The proliferative capacity and clonal growth of the cells was evaluated before (P0) and after expansion (P1) on the plastic compressed collagen gels by colony forming efficiency assay. The potential clinical applicability of this gel substitutes was evaluated by assessment of their biomechanical properties as well as their surgical handling. Human conjunctival epithelial cells cultured on plastic and plastic compressed collagen gels formed a confluent cell layer and expressed CK19. The cells showed expression of the putative epithelial progenitor cell markers p63α, ABCG2 and CK15 and sustained colony forming ability. The compressed collagen gels showed a high ultimate tensile strength and elasticity and the surgical handling of gels was comparable to amniotic membrane. An epithelialized conjunctival tissue construct on the basis of compressed collagen might therefore be a promising alternative bioartificial tissue substitute for conjunctival reconstruction. Copyright © 2015 John Wiley & Sons, Ltd.

Multiple reversible MR signal changes caused by Epstein-Barr virus encephalitis.

This report presents the imaging findings of an unusual case of Epstein-Barr virus (EBV) encephalitis. A young man presented with a short-lasting history of febrile infection, neuropsychologic deficits, ataxia, and seizures. MR imaging revealed fully reversible signal intensities (T2, diffusion-weighted imaging with a decreased apparent diffusion coefficient) in the splenium of the corpus callosum and both posterior hemispheres. EBV infection must be added to the list of differential diagnoses of (reversible) splenial lesions.

Monitoring of single and double lipid membrane formation with high spatiotemporal resolution using evanescent light scattering microscopy.

Formation and quality of single solid supported lipid membranes and double lipid membranes were investigated with single vesicle resolution using label-free evanescence light scattering microscopy (EvSM). For the formation of double lipid membranes we made use of electrostatic interaction between charged lipids and oppositely charged cations.

[Retinal bleeding and venous stasis in a 10-month-old infant after a fall?]. / Retinale Blutungen und venöse Stase bei einem 10 Monate alten Säugling nach Sturz?

This report describes the case of a 10-month-old infant, who was delivered to our hospital by the emergency physician intubated and in an unclear unconscious state. The father reported that the child had fallen from the couch to the ground. The consulted ophthalmologist reported venous stasis in both eyes including intraretinal and preretinal bleeding in all four quadrants, a diffuse vitreous hemorrhage in the right eye and temporal retinal wrinkling in both eyes. Based on these particular clinical findings a shaken baby syndrome was suspected. This report demonstrates the importance of recognizing and correctly interpreting the typical ophthalmological signs of physical child abuse in order to detect and prevent further mistreatment of children; moreover, the increasing importance of photographic documentation and histological work-up of the findings for forensic reasons are emphasized.

Localized reversible reduction of apparent diffusion coefficient in transient hypoglycemia-induced hemiparesis.

BACKGROUND AND PURPOSE: The pathophysiology of hypoglycemia shares a common mechanism with cerebral ischemia, but so far, little is known regarding MRI of humans with hypoglycemia. METHODS: We report a patient with left hemiparesis and dysarthria associated with a blood glucose level of 1.7 mmol/L. The patient recovered completely after glucose infusion. RESULTS: The initial diffusion-weighted imaging (DWI) showed increased signal intensities and a reduction of apparent diffusion coefficient (ADC) values localized in the corpus callosum (splenium) and asymmetrically in the corona radiata. After 48 hours, follow-up revealed complete recovery of DWI and ADC signal abnormalities. CONCLUSIONS: To our knowledge, this is the first presentation of a case with transient hypoglycemia-induced focal neurological deficits revealing completely reversible MRI changes in terms of disturbed DWI and ADC with a peculiar as yet undescribed topography.

Clinical significance of saccade analysis in early active Graves' ophthalmopathy.

PURPOSE: To assess whether saccadic eye movements show distinct changes in patients with early active Graves' ophthalmopathy (GO), which could serve as a diagnostic tool for early detection and treatment. METHODS: Each of two prospective studies included 10 patients with early acute GO and 10 age- and sex-matched control subjects. In the explorative study (ES) 15 dynamic parameters of saccades were analyzed. In the comparative study (CS) only those parameters were evaluated, which in ES had shown significant differences between patients and controls. Horizontal and vertical saccades of 10 degrees, 20 degrees, and 40 degrees including a fatigue test were recorded binocularly using the induction scleral search coil. RESULTS: The differences of saccadic dynamics between patients and controls were small, whereas intra- and interindividual standard deviations were large. In ES, 7.1% of the parameters showed significant differences at a level of P < or = 0.05. In CS, 2.1% of all parameters revealed repetitive significant differences. Despite statistical significance, individual data did not allow differentiation between patients and healthy individuals due to high standard deviations. CONCLUSIONS: In early active GO no clinically relevant saccadic changes were detected. These findings may be based on adaptation of the central saccadic generator. Inclusion of patients with fibrotic muscle changes due to long-standing disease could explain the contrasting results of previous studies. Consequently, analysis of saccades does not serve as a diagnostic tool during early active GO.

Application of model-predictive control based on artificial neural networks to optimize the fed-batch process for riboflavin production.

The fed-batch process for commercial production of riboflavin (vitamin B2) was optimized on-line using model-predictive control based on artificial neural networks (ANNs). The information required for process models was extracted from both historical data and heuristic rules. After each cultivation the process model was readapted off-line to include the most recent process data. The control signal (feed rate), however, was optimized on-line at each sampling interval. An optimizer simulated variations in the control signal and assessed the forecasted model outputs according to an objective function. The optimum feed profile for increasing the product yield (YB2/S) and the amount of riboflavin at the time of harvesting was adjusted continuously and applied to the process. In contrast to the control by set-point profiles, the novel ANN-control is able to react on-line to variations in the process and also to incorporate the new process information continuously. As a result, both the total amount of riboflavin produced and the product yield increased systematically by more than 10% and the reproducibility of seven subsequently optimized batches was enhanced.

[Severe course and contingent risk factors in optic neuropathy and myelopathy after tuberculostatics]. / Schwerwiegender Verlauf und belastende Faktoren bei Optikusneuropathie und Myelopathie durch Tuberkulostatika.

A male patient with tuberculous lymphadenopathy was treated with a four-fold therapy of ethambutol, isoniacide, rifampicin and pyracinamide. After 10 weeks the patient suffered from photophobia. Although ethambutol was discontinued vision decreased and visual field defects occurred as well as signs of myelopathy. Isoniacide was then discontinued and in the subsequent phase the vision was slowly restored over a period of 36 months. The combined toxicity of ethambutol and isoniacide seems to have been the main cause of the severe and protracted optic neuropathy.

[Indications for treating stenoses of the carotid artery]. / Indikationen zur Therapie von Stenosen der A. carotis.

The annual stroke risk for patients with asymptomatic stenoses of the carotid artery is around 1% in case of <70% stenosis (NASCET criteria) and 2-5% in patients with >70% stenosis. The risk of recurrent ischemic events for patients with symptomatic stenoses is much higher, around 15% during the first year. For more than 10 years, the efficacy of carotid surgery has been proven, and there is growing evidence to support surgery in case of asymptomatic stenosis. Patients with severe stenoses, male or elderly patients, and those with bilateral stenoses benefit more from surgery. Carotid artery stenting has not proven its safety or efficacy. Despite this lack of evidence, the method is used in many centers as an alternative to surgery. Especially symptomatic carotid artery stenosis should be used mainly in the setting of a randomized trial such as SPACE.

The Uromodulin C744G mutation causes MCKD2 and FJHN in children and adults and may be due to a possible founder effect.

Autosomal dominant medullary cystic kidney disease type 2 (MCKD2) is a tubulo-in terstitial nephropathy that causes renal salt wasting, hyperuricemia, gout, and end-stage renal failure in the fifth decade of life. This disorder was described to have an age of onset between the age of 20-30 years or even later. Mutations in the Uromodulin (UMOD) gene were published in patients with familial juvenile hyperuricemic nephropathy (FJHN) and MCKD2. Clinical data and blood samples of 16 affected individuals from 11 different kindreds were collected. Mutational analysis of the UMOD gene was performed by exon polymerase chain reaction (PCR) and direct sequencing. We found the heterozygous C744G (Cys248Trp) mutation, which was originally published by our group, in an additional four kindreds from Europe and Turkey. Age of onset ranged from 3 years to 39 years. The phenotype showed a variety of symptoms such as urinary concentration defect, vesicoureteral reflux, urinary tract infections, hyperuricemia, hypertension, proteinuria, and renal hypoplasia. Haplotype analysis showed cosegragation with the phenotype in all eight affected individuals indicating that the C744G mutation may be due to a founder effect. Moreover, we describe a novel T229G (Cys77Gly) mutation in two affecteds of one kindred. Three of the affected individuals were younger than 10 years at the onset of MCKD2/FJHN. Symptoms include recurrent urinary tract infections compatible with the published phenotype of the Umod knockout mouse model. This emphasizes that MCKD2 is not just a disease of the young adult but is also relevant for children.

Fate of the chromene encecalin in the interaction ofEncelia farinosa and its specialized herbivoreTrirhabda geminata.

Leaf beetles of the speciesTrirhabda geminata are specialized herbivores that are able to feed on the chemically well-protected foliage of the desert sunflowerEncelia farinosa, which contains the insecticidal chromene derivative encecalin. Chemical analysis of the beetles and their fecal excretions indicated that encecalin is present only in the alimentary canal and is not absorbed across the gut membrane, as previously shown for other herbivorous insects (e.g., the Egyptian armyworm,Spodoptera littoralis) that are susceptible to this chromene derivative. Further differences betweenT. geminata and nonadapted insects were observed with regard to the metabolism of encecalin. Whereas the encecalin-resistent leaf beetles metabolize encecalin mainly to encecalol by reduction of the acetyl group, susceptible insects, such as larvae ofS. littoralis, metabolize encecalin mainly by exoxidation of the 3,4 double bond, which creates a powerful alkylating agent and is responsible for the toxicity of encecalin. Reductive rather than oxidative metabolism of encecalin therefore seems important for the resistance ofT. geminata against the chemical defense of their host plantE. farinosa.