MyoRobot 2.0: An advanced biomechatronics platform for automated, environmentally controlled skeletal muscle single fiber biomechanics assessment employing inbuilt real-time optical imaging.

We present an enhanced version of our previously engineered MyoRobot system for reliable, versatile and automated investigations of skeletal muscle or linear polymer material (bio)mechanics. That previous version already replaced strenuous manual protocols to characterize muscle biomechanics properties and offered automated data analysis. Here, the system was further improved for precise control over experimental temperature and muscle single fiber sarcomere length. Moreover, it also now features the calculation of fiber cross-sectional area via on-the-fly optical diameter measurements using custom-engineered microscope optics. With this optical systems integration, the MyoRobot 2.0 allows to tailor a wealth of recordings for relevant physiological parameters to be sequentially executed in living single myofibers. Research questions include assessing temperature-dependent performance of active or passive biomechanics, or automated control over length-tension or length-velocity relations. The automatically obtained passive stress-strain relationships and elasticity modules are important parameters in (bio)material science. From the plethora of possible applications, we validated the improved MyoRobot 2.0 by assessing temperature-dependent myofibrillar Ca2+ sensitivity, passive axial compliance and Young's modulus. We report a Ca2+ desensitization and a narrowed dynamic range at higher temperatures in murine M. extensor digitorum longus single fibers. In addition, an increased axial mechanical compliance in single muscle fibers with Young's moduli between 40 - 60 kPa was found, compatible with reported physiological ranges. These applications demonstrate the robustness of our MyoRobot 2.0 for facilitated single muscle fiber biomechanics assessment.

The MyoRobot: A novel automated biomechatronics system to assess voltage/Ca2+ biosensors and active/passive biomechanics in muscle and biomaterials.

We engineered an automated biomechatronics system, MyoRobot, for robust objective and versatile assessment of muscle or polymer materials (bio-)mechanics. It covers multiple levels of muscle biosensor assessment, e.g. membrane voltage or contractile apparatus Ca2+ ion responses (force resolution 1µN, 0-10mN for the given sensor; [Ca2+] range ~ 100nM-25µM). It replaces previously tedious manual protocols to obtain exhaustive information on active/passive biomechanical properties across various morphological tissue levels. Deciphering mechanisms of muscle weakness requires sophisticated force protocols, dissecting contributions from altered Ca2+ homeostasis, electro-chemical, chemico-mechanical biosensors or visco-elastic components. From whole organ to single fibre levels, experimental demands and hardware requirements increase, limiting biomechanics research potential, as reflected by only few commercial biomechatronics systems that can address resolution, experimental versatility and mostly, automation of force recordings. Our MyoRobot combines optical force transducer technology with high precision 3D actuation (e.g. voice coil, 1µm encoder resolution; stepper motors, 4µm feed motion), and customized control software, enabling modular experimentation packages and automated data pre-analysis. In small bundles and single muscle fibres, we demonstrate automated recordings of (i) caffeine-induced-, (ii) electrical field stimulation (EFS)-induced force, (iii) pCa-force, (iv) slack-tests and (v) passive length-tension curves. The system easily reproduces results from manual systems (two times larger stiffness in slow over fast muscle) and provides novel insights into unloaded shortening velocities (declining with increasing slack lengths). The MyoRobot enables automated complex biomechanics assessment in muscle research. Applications also extend to material sciences, exemplarily shown here for spider silk and collagen biopolymers.

Adding dimension to cellular mechanotransduction: Advances in biomedical engineering of multiaxial cell-stretch systems and their application to cardiovascular biomechanics and mechano-signaling.

Hollow organs (e.g. heart) experience pressure-induced mechanical wall stress sensed by molecular mechano-biosensors, including mechanosensitive ion channels, to translate into intracellular signaling. For direct mechanistic studies, stretch devices to apply defined extensions to cells adhered to elastomeric membranes have stimulated mechanotransduction research. However, most engineered systems only exploit unilateral cellular stretch. In addition, it is often taken for granted that stretch applied by hardware translates 1:1 to the cell membrane. However, the latter crucially depends on the tightness of the cell-substrate junction by focal adhesion complexes and is often not calibrated for. In the heart, (increased) hemodynamic volume/pressure load is associated with (increased) multiaxial wall tension, stretching individual cardiomyocytes in multiple directions. To adequately study cellular models of chronic organ distension on a cellular level, biomedical engineering faces challenges to implement multiaxial cell stretch systems that allow observing cell reactions to stretch during live-cell imaging, and to calibrate for hardware-to-cell membrane stretch translation. Here, we review mechanotransduction, cell stretch technologies from uni-to multiaxial designs in cardio-vascular research, and the importance of the stretch substrate-cell membrane junction. We also present new results using our IsoStretcher to demonstrate mechanosensitivity of Piezo1 in HEK293 cells and stretch-induced Ca2+ entry in 3D-hydrogel-embedded cardiomyocytes.

Vegetarian diets in children: a systematic review.

PURPOSE: While the prevalence of children on vegetarian diets is assumed to be on the rise in industrialized countries, there are hardly any representative data available. In general, vegetarian diets are presumed to be healthy; nevertheless, there are concerns as to whether the dietary specifications required during infancy, childhood, and adolescence can be met. Therefore, the objective of this systematic review was to evaluate studies on the dietary intake and the nutritional or health status of vegetarian infants, children, and adolescents. METHODS: The database MEDLINE was used for literature search. In addition, references of reviews and expert opinions were considered. Inclusion criteria were (1) sufficient dietary information to define vegetarian type diet and (2) characteristics of nutritional or health status. Case reports and studies from non-industrialized countries were excluded. RESULTS: 24 publications from 16 studies published from 1988 to 2013 met our criteria. Study samples covered the age range from 0 to 18 years, and median sample size was 35. Five studies did not include a control group. With regard to biomarkers, anthropometry, and dietary or nutritional intake, the outcomes were diverse. Growth and body weight were generally found within the lower reference range. The intakes of folate, vitamin C, and dietary fiber were relatively high compared to reference values and/or control groups. Low status of vitamin B12 was reported in one study and low status of vitamin D in two studies. CONCLUSIONS: Due to the study heterogeneity, the small samples, the bias towards upper social classes, and the scarcity of recent studies, the existing data do not allow us to draw firm conclusions on health benefits or risks of present-day vegetarian type diets on the nutritional or health status of children and adolescents in industrialized countries.

Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells.

Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells.

The IsoStretcher: An isotropic cell stretch device to study mechanical biosensor pathways in living cells.

Mechanosensation in many organs (e.g. lungs, heart, gut) is mediated by biosensors (like mechanosensitive ion channels), which convert mechanical stimuli into electrical and/or biochemical signals. To study those pathways, technical devices are needed that apply strain profiles to cells, and ideally allow simultaneous live-cell microscopy analysis. Strain profiles in organs can be complex and multiaxial, e.g. in hollow organs. Most devices in mechanobiology apply longitudinal uniaxial stretch to adhered cells using elastomeric membranes to study mechanical biosensors. Recent approaches in biomedical engineering have employed intelligent systems to apply biaxial or multiaxial stretch to cells. Here, we present an isotropic cell stretch system (IsoStretcher) that overcomes some previous limitations. Our system uses a rotational swivel mechanism that translates into a radial displacement of hooks attached to small circular silicone membranes. Isotropicity and focus stability are demonstrated with fluorescent beads, and transmission efficiency of elastomer membrane stretch to cellular area change in HeLa/HEK cells. Applying our system to lamin-A overexpressing fibrosarcoma cells, we found a markedly reduced stretch of cell area, indicative of a stiffer cytoskeleton. We also investigated stretch-activated Ca(2+) entry into atrial HL-1 myocytes. 10% isotropic stretch induced robust oscillating increases in intracellular Fluo-4 Ca(2+) fluorescence. Store-operated Ca(2+) entry was not detected in these cells. The Isostretcher provides a useful versatile tool for mechanobiology.

Septic cardiomyopathy in rat LPS-induced endotoxemia: relative contribution of cellular diastolic Ca(2+) removal pathways, myofibrillar biomechanics properties and action of the cardiotonic drug levosimendan.

Cardiac dysfunction is a common complication in sepsis and is characterized by forward pump failure. Hallmarks of septic cardiomyopathy are decreased myofibrillar contractility and reduced Ca(2+) sensitivity but it is still not clear whether reduced pump efficiency is predominantly a diastolic impairment. Moreover, a comprehensive picture of upstream Ca(2+) handling mechanisms and downstream myosin biomechanical parameters is still missing. Ca(2+)-sensitizing agents in sepsis may be promising but mechanistic insights for drugs like levosimendan are scarce. Here, we used an endotoxemic LPS rat model to study mechanisms of sepsis on in vivo hemodynamics, multicellular myofibrillar Ca(2+) sensitivity, in vitro cellular Ca(2+) homeostasis and subcellular actomyosin interaction with intracardiac catheters, force transducers, confocal Fluo-4 Ca(2+) recordings in paced cardiomyocytes, and in vitro motility assay, respectively. Left ventricular ejection fraction and myofibrillar Ca(2+) sensitivity were depressed in LPS animals but restored by levosimendan. Diastolic Ca(2+) transient kinetics was slowed down by LPS but ameliorated by levosimendan. Selectively blocking intracellular and sarcolemmal Ca(2+) extrusion pathways revealed minor contribution of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) to Ca(2+) transient diastole in LPS-evoked sepsis but rather depressed Na(+)/Ca(2+) exchanger and plasmalemmal Ca(2+) ATPase. This was mostly compensated by levosimendan. Actin sliding velocities were depressed in myosin heart extracts from LPS rats. We conclude that endotoxemia specifically impairs sarcolemmal diastolic Ca(2+) extrusion pathways resulting in intracellular diastolic Ca(2+) overload. Levosimendan, apart from stabilizing Ca(2+)-troponin C complexes, potently improves cellular Ca(2+) extrusion in the septic heart.

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine.

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging.

Microarchitecture is severely compromised but motor protein function is preserved in dystrophic mdx skeletal muscle.

Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (approximately 1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities ("verniers") than wild-type fibers (approximately 130/1000 microm(3) vs. approximately 36/1000 microm(3)). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (approximately 38/1000 microm(3) counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies.

Cases of reduced cyathostomin egg-reappearance period and failure of Parascaris equorum egg count reduction following ivermectin treatment as well as survey on pyrantel efficacy on German horse farms.

In 2003 and 2004, on a total of 63 different German horse farms, a survey using the faecal egg count reduction (FECR) test was performed to investigate the efficacy of ivermectin (IVM, Ivomec) and pyrantel (PYR, Banminth) treatment against gastro-intestinal nematodes in a total of 767 horses. IVM treatment resulted in 100% reduction of the cyathostomin egg production 14 and 21 days post-treatment (d.p.t.) on 37 farms. On the remaining five farms, the mean faecal egg count reduction ranged between 97.7 and 99.9%. The mean cyathostomin FECR following PYR treatment ranged between 92.2 and 100% on the 25 farms tested. Therefore, based on the 90% FECR threshold suggested for detection of anthelmintic resistance in horses, neither IVM nor PYR anthelmintic resistance was detected. However, if the thresholds recommended for the detection of resistance in small ruminants were applied, on one and four farms signs of reduced IVM and PYR efficacy, respectively, were observed. In 2005, to further investigate these findings, the cyathostomin egg-reappearance period (ERP) following IVM treatment was examined on six selected farms, two of which were found to show less than 99.8% FECR in the previous survey. On these two latter farms, the ERP was less than 5 weeks, while on the other four it was at least 8 weeks. Earlier investigations described IVM cyathostomin ERP of at least 9 weeks. The efficacy of IVM to reduce Parascaris equorum egg excretion was also studied. On one farm in 2 consecutive years, IVM treatment did not lead to a significant reduction in P. equorum faecal egg counts in one and five young horses, respectively.

[Unique children -- unique headaches. Case reports of pediatric headache patients from an outpatient children's pain department]. / Individuelle Kinder -- individuelle Kopfschmerzen. Fallbeispiele kindlicher Kopfschmerzen aus einer multidisziplinären Kinderschmerzambulanz.

In the industrialized nations headache prevalence is increasing in children and adolescents. The nosologic classification determines the therapeutic strategy to follow. Three case reports illustrate the optimal cooperation of both a pediatric outpatient pain clinic and a pediatric psychosomatic pain clinic. We report on (1) a girl aged 2 years and 7 months with a 4-month history of headache episodes lasting about 15 min each with concomitant symptoms; (2) an 11-year-old boy with Schimmelpenning-Feuerstein-Mims syndrome, symptomatic focal epilepsy, psychomotor retardation, mild postinfectious internal hydrocephalus, and repeated heat-triggered episodes of right-sided headache beginning suddenly with a duration of 5-30 min and concomitant flush of his hemiface; and (3) a 12-year-old boy who for about 2 years has suffered from "migraine" 3 times a week, significantly impairing his quality of life. We discuss the patients' courses, diagnostic pitfalls, and therapeutic options. For the optimal treatment of children with headache not easily fitting into one of the categories, with significant comorbidity present, or if there is no adequate response to therapy conforming with guidelines, the help of an interdisciplinary pediatric pain clinic is invaluable.

Sterol regulatory element binding proteins (SREBP)-1a and SREBP-2 are linked to the MAP-kinase cascade.

The classic sterol regulatory cis element (sre-1) in the LDL receptor promoter mediates sterol regulatory element binding protein (SREBP)-binding and the effects of insulin and platelet derived growth factor (PDGF). To elucidate whether SREBP-1a and SREBP-2 play a direct role in insulin and PDGF action, stable cell lines of HepG2 deficient in either SREBP-1 or SREBP-2 were used. Transfection of these cells with the wild-type promoter fragment of the low density lipoprotein (LDL) receptor gene showed that the effects of insulin and PDGF were significantly reduced in both, SREBP-1- as well as SREBP-2-deficient cells. Insulin and PDGF action could be reconstituted again in these deficient cell lines by reintroducing SREBP-1a or SREBP-2. Preincubation of cells with either the phosphatidylinositol (PI)-3 kinase inhibitor wortmannin or the mitogen-activated protein (MAP) kinase cascade inhibitor PD 98059 showed that the latter abolished the stimulatory effects of insulin and PDGF on LDL receptor promoter activity completely, whereas wortmannin had no effect. Overexpression of upstream activators of the MAP kinases, like MEKK1 or MEK1, stimulated LDL receptor promoter activity several fold in an sre-1 related manner. These effects could be enhanced by coexpression of the transcriptional active N-terminal domains of SREBP-1a and SREBP-2. Using the heterologous Gal-4 system, we could show that intracellular activation of the MAP kinase cascade by ectopic expression of MEKK1 or MEK1 has a direct stimulatory effect on the transcriptional activity of SREBP-1a and SREBP-2. Experimental evidence for a direct link between MAP kinases and SREBPs was obtained due to the MAP kinases ERK1 and ERK2 phosphorylating recombinant GST-fusion proteins of SREBP-1a and SREBP-2, in vitro. We conclude that SREBP-1a and SREBP-2 mediate different regulatory effects converging at sre-1 and that they appear to be linked to the MAP kinase cascade, possibly being direct substrates of ERK1 and ERK2.

[Therapy program for children with hyperkinetic and oppositional problem behavior--organization and single case evaluation]. / Das Therapieprogramm für Kinder mit hyperkinetischem und oppositionellem Problemverhalten (THOP)-Aufbau und Einzelfall-Evaluation.

After a review of the research on the effectiveness of the different treatment modalities used in multimodal treatment of children with hyperkinetic disorders, a decision tree is described for planning multimodal treatment for school-aged children. Differences in treating preschool children are discussed. A mulitmodal treatment program for children with hyperkinetic and oppositional behavior problems is then described. It can be used for children aged 3 to 12 years old. The program has two parts, the parent-child program and the teacher-child program. The program contains intervention units that can be combined as necessary depending on the individual problem configuration. A case study is presented to illustrate the effects of this program in combination with stimulant medication.