Mesenchymal stem cells and myoblast differentiation under HGF and IGF-1 stimulation for 3D skeletal muscle tissue engineering.

BACKGROUND: Volumetric muscle loss caused by trauma or after tumour surgery exceeds the natural regeneration capacity of skeletal muscle. Hence, the future goal of tissue engineering (TE) is the replacement and repair of lost muscle tissue by newly generating skeletal muscle combining different cell sources, such as myoblasts and mesenchymal stem cells (MSCs), within a three-dimensional matrix. Latest research showed that seeding skeletal muscle cells on aligned constructs enhance the formation of myotubes as well as cell alignment and may provide a further step towards the clinical application of engineered skeletal muscle. In this study the myogenic differentiation potential of MSCs upon co-cultivation with myoblasts and under stimulation with hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) was evaluated. We further analysed the behaviour of MSC-myoblast co-cultures in different 3D matrices. RESULTS: Primary rat myoblasts and rat MSCs were mono- and co-cultivated for 2, 7 or 14 days. The effect of different concentrations of HGF and IGF-1 alone, as well as in combination, on myogenic differentiation was analysed using microscopy, multicolour flow cytometry and real-time PCR. Furthermore, the influence of different three-dimensional culture models, such as fibrin, fibrin-collagen-I gels and parallel aligned electrospun poly-ε-caprolacton collagen-I nanofibers, on myogenic differentiation was analysed. MSCs could be successfully differentiated into the myogenic lineage both in mono- and in co-cultures independent of HGF and IGF-1 stimulation by expressing desmin, myocyte enhancer factor 2, myosin heavy chain 2 and alpha-sarcomeric actinin. An increased expression of different myogenic key markers could be observed under HGF and IGF-1 stimulation. Even though, stimulation with HGF/IGF-1 does not seem essential for sufficient myogenic differentiation. Three-dimensional cultivation in fibrin-collagen-I gels induced higher levels of myogenic differentiation compared with two-dimensional experiments. Cultivation on poly-ε-caprolacton-collagen-I nanofibers induced parallel alignment of cells and positive expression of desmin. CONCLUSIONS: In this study, we were able to myogenically differentiate MSC upon mono- and co-cultivation with myoblasts. The addition of HGF/IGF-1 might not be essential for achieving successful myogenic differentiation. Furthermore, with the development of a biocompatible nanofiber scaffold we established the basis for further experiments aiming at the generation of functional muscle tissue.

Global assessment of the status of coral reef herbivorous fishes: evidence for fishing effects.

On coral reefs, herbivorous fishes consume benthic primary producers and regulate competition between fleshy algae and reef-building corals. Many of these species are also important fishery targets, yet little is known about their global status. Using a large-scale synthesis of peer-reviewed and unpublished data, we examine variability in abundance and biomass of herbivorous reef fishes and explore evidence for fishing impacts globally and within regions. We show that biomass is more than twice as high in locations not accessible to fisheries relative to fisheries-accessible locations. Although there are large biogeographic differences in total biomass, the effects of fishing are consistent in nearly all regions. We also show that exposure to fishing alters the structure of the herbivore community by disproportionately reducing biomass of large-bodied functional groups (scraper/excavators, browsers, grazer/detritivores), while increasing biomass and abundance of territorial algal-farming damselfishes (Pomacentridae). The browser functional group that consumes macroalgae and can help to prevent coral-macroalgal phase shifts appears to be most susceptible to fishing. This fishing down the herbivore guild probably alters the effectiveness of these fishes in regulating algal abundance on reefs. Finally, data from remote and unfished locations provide important baselines for setting management and conservation targets for this important group of fishes.

The Human Salivary Proteome Wiki: A Community-Driven Research Platform.

Saliva has become an attractive body fluid for on-site, remote, and real-time monitoring of oral and systemic health. At the same time, the scientific community needs a saliva-centered information platform that keeps pace with the rapid accumulation of new data and knowledge by annotating, refining, and updating the salivary proteome catalog. We developed the Human Salivary Proteome (HSP) Wiki as a public data platform for researching and retrieving custom-curated data and knowledge on the saliva proteome. The HSP Wiki is dynamically compiled and updated based on published saliva proteome studies and up-to-date protein reference records. It integrates a wide range of available information by funneling in data from established external protein, genome, transcriptome, and glycome databases. In addition, the HSP Wiki incorporates data from human disease-related studies. Users can explore the proteome of saliva simply by browsing the database, querying the available data, performing comparisons of data sets, and annotating existing protein entries using a simple, intuitive interface. The annotation process includes both user feedback and curator committee review to ensure the quality and validity of each entry. Here, we present the first overview of features and functions the HSP Wiki offers. As a saliva proteome-centric, publicly accessible database, the HSP Wiki will advance the knowledge of saliva composition and function in health and disease for users across a wide range of disciplines. As a community-based data- and knowledgebase, the HSP Wiki will serve as a worldwide platform to exchange salivary proteome information, inspire novel research ideas, and foster cross-discipline collaborations. The HSP Wiki will pave the way for harnessing the full potential of the salivary proteome for diagnosis, risk prediction, therapy of oral and systemic diseases, and preparedness for emerging infectious diseases.Database URL: https://salivaryproteome.nidcr.nih.gov/.

Comparison of calcium analysis, longitudinal microradiography and profilometry for the quantitative assessment of erosion in dentine.

Erosion of dentine causes mineral dissolution, while the organic compounds remain at the surface. Therefore, a determination of tissue loss is complicated. Established quantitative methods for the evaluation of enamel have also been used for dentine, but the suitability of these techniques in this field has not been systematically determined. Therefore, this study aimed to compare longitudinal microradiography (LMR), contacting (cPM) and non-contacting profilometry (ncPM), and analysis of dissolved calcium (Ca analysis) in the erosion solution. Results are discussed in the light of the histology of dentine erosion. Erosion was performed with 0.05 M citric acid (pH 2.5) for 30, 60, 90 or 120 min, and erosive loss was determined by each method. LMR, cPM and ncPM were performed before and after collagenase digestion of the demineralised organic surface layer, with an emphasis on moisture control. Scanning electron microscopy was performed on randomly selected specimens. All measurements were converted into micrometres. Profilometry was not suitable to adequately quantify mineral loss prior to collagenase digestion. After 120 min of erosion, values of 5.4 +/- 1.9 microm (ncPM) and 27.8 +/- 4.6 microm (cPM) were determined. Ca analysis revealed a mineral loss of 55.4 +/- 11.5 microm. The values for profilometry after matrix digestion were 43.0 +/- 5.5 microm (ncPM) and 46.9 +/- 6.2 (cPM). Relative and proportional biases were detected for all method comparisons. The mineral loss values were below the detection limit for LMR. The study revealed gross differences between methods, particularly when demineralised organic surface tissue was present. These results indicate that the choice of method is critical and depends on the parameter under study.

Effect of fluoride compounds on enamel erosion in vitro: a comparison of amine, sodium and stannous fluoride.

The aim of the study was to evaluate the relevance of cations in different fluoride compounds for their effectiveness as anti-erosive agents. Human enamel samples underwent a de- and re-mineralisation procedure for 10 days. Erosive demineralisation was performed with 0.05 M citric acid (pH 2.3) 6 x 2 min daily followed by immersion in the test solution 6 x 2 min each. Test solutions were: SnCl2 (815 ppm Sn; pH 2.6), NaF (250 ppm F; pH 3.5), SnF2 (250 ppm F, 809 ppm Sn; pH 3.5), amine fluoride (AmF, 250 ppm F; pH 3.5), AmF/NaF (250 ppm F; pH 4.3), and AmF/SnF2 (250 ppm F, 390 ppm Sn; pH 4.2). In the control group no fluoridation was performed. Mineral content was monitored by longitudinal microradiography. Finally, scanning electron microscopy was performed. The highest erosive mineral loss was found in the control group (48.0 +/- 17.1 microm). Mineral loss was nearly completely inhibited by AmF/SnF2 (5.7 +/- 25.1 microm; p < or = 0.001) and SnF2 (-3.8 +/- 14.4 microm; p < or = 0.001) treatments. Groups treated with SnCl2 (17.6 +/- 19.5 microm; p < or = 0.001) and NaF (13.2 +/- 21.7 microm; p < or = 0.001) showed a decrease in erosive mineral loss, AmF (41.6 +/- 16.0 microm) and AmF/NaF (27.7 +/- 28.4 microm) had no significant effect on erosion progression. The results indicate considerable differences between the fluoride compounds tested. Treatment with solutions containing SnF2 was most effective.

[Sporadic cutaneous lymphosarcoma of T-cell origin with involvement of lymph nodes and internal organs in a Holstein cow]. / Sporadische kutane T-Zell-Leukose mit Beteiligung von Lymphknoten und inneren Organen bei einer Holstein-Kuh.

Sporadic lymphosarcomas in adult cattle are rare entities with an unknown etiology. This case report describes the course of the disease in a 3.5-year-old cow of the breed German Holstein, which was presented to the veterinarian due to multifocal nodular skin lesions. Several superficial lymph nodes (Lymphonodi mandibulares, parotidei and mammariae) were enlarged, had a tight-elastic consistency and were freely movable. The histopathological and immunohistochemical examination of skin biopsies showed the presence of multifocal cutaneous T-cell lymphosarcomas consistent with a skin leukosis. Bovine leukemia virus infection was excluded by serological investigation of a milk sample and virological examination of a tissue sample, respectively. Seven weeks after the first clinical examination, the cow deteriorated rapidly and was euthanized. A post mortem examination revealed the presence of neoplastic cells within lymph nodes (all superficial lymph nodes of the carcass and Lymphonodi pulmonales), kidney and lungs as well as a liver rupture. Additionally, an overview of the case reports of sporadic bovine cutaneous lymphosarcomas published during the previous 15 years will be provided. The legal background for a further utilization of affected animals for milk and meat production will be discussed. This case report illustrates that sporadic bovine leukosis represents an important differential diagnosis for viral-, bacterial- and parasitic-induced skin lesions and enlargement of lymph nodes in adult cattle.

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells.

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.

Organization of a sensory neuropile in the auditory pathway of two groups of Orthoptera.

The anterior intermediate sensory neuropile (aISN) is a prominent neuropile in the ventral nerve cord of locusts and bushcrickets. Previous studies have shown that it receives its main sensory input from auditory receptors. In this paper we examine the structural and physiological relationship between tympanal receptor terminations and the dendrites of sound-sensitive interneurones in the homologous neuropile of locusts and bushcrickets. Each individual receptor fibre of the bushcricket terminates in a somewhat different target area of the neuropile. The ordering is with respect to the characteristic frequency of the fibres (tonotopic) in the anterior-posterior and dorsoventral axis. In the locust, representatives of the four tympanal receptor groups branch in different areas of the aISN. Most of the dorsal neuropilar region, and the anterior ventral region, do not receive input from tympanal receptors. The dendrites of identified sound-sensitive interneurones were examined in the context of this afferent projection. Local interneurones as well as intersegmental interneurones in bushcrickets have dendritic branches in the whole aISN or part of it and thus overlap with at least some receptors. By recording intracellularly from their main neurites, short-latency synaptic potentials were found in response to receptor spikes indicating monosynaptic input. The tuning of these neurones could be predicted by their dendritic morphology. In contrast, in the locust only local and bisegmental neurones are monosynaptically connected with tympanal receptors, but not the studied intersegmental neurones. This is consistent with the finding that most or all branches of intersegmental neurones lie in the dorsal area of neuropile where no receptors terminate. Anatomical and physiological evidence is presented for identified local neurones providing the excitatory and inhibitory synaptic input for such intersegmental neurones. The difference in the basic wiring diagram in the homologous neuropile of the two orthopteran groups is discussed with respect to the possible different roles that sound plays in their behaviour.

Distribution of synapses on two ascending interneurones carrying frequency-specific information in the auditory system of the cricket: evidence for GABAergic inputs.

Two identified cricket auditory interneurones, AN1 and AN2, were intracellularly labelled with horseradish peroxidase following physiological characterisation. The neurones, which have some structural similarities, have their somata in the prothoracic ganglion and axons that project to the brain. Although both carry auditory information, they have different response properties and participate in different types of phonotactic behaviour. Ultrathin sections from selected regions of their prothoracic arborisations were examined in the electron microscope after postembedding immunostaining for the inhibitory transmitter GABA. In the prothoracic ganglion AN1 branches only in the medial ventral association centre (mVAC) contralateral to the soma, and receives only iput synapses. Twenty-seven percent of these were made by processes immunoreactive for GABA. AN2 branches not only in mVAC on both sides of the ganglion but also in several other areas. It makes output synapses from large diameter neurites in mVAC on both sides of the ganglion as well as from neurites in more posterior regions of the neuropile. Most input synapses are received onto branches in the contralateral mVAC where about 19% were made from GABA-immunoreactive processes.

Distribution of input and output synapses on the central branches of bushcricket and cricket auditory afferent neurones: immunocytochemical evidence for GABA and glutamate in different populations of presynaptic boutons.

In order to investigate the synapses on the terminals of primary auditory afferents in the bushcricket and cricket, these were impaled with microelectrodes and after physiological characterisation, injected intracellularly with horseradish peroxidase. The tissue was prepared for electron microscopy, and immunocytochemistry for gamma-aminobutyric acid (GABA) and glutamate was carried out on ultrathin sections by using a post-embedding immunogold technique. The afferent terminals received many input synapses. Between 60-65% of these were made by processes immunoreactive for GABA and approximately 25% from processes immunoreactive for glutamate. The relative distribution of the different classes of input were analysed from serial section reconstruction of terminal afferent branches. Inputs from GABA and glutamate-immunoreactive processes appeared to be scattered at random over the terminal arborisation of the afferents both with respect to each other and to the architecture of the terminals. They were, however, always found close to the output synapses. The possible roles of presynaptic inhibition in the auditory afferents is discussed in the context of the auditory responses of the animals.

Effect of short-term topical corticosteroid treatment on mucosal enzyme systems in patients with distal inflammatory bowel disease.

BACKGROUND/AIMS: Glucocorticoids, even when administered topically, have a known early benefit on diarrheal symptoms in inflammatory bowel diseases which may not be explained exclusively by their anti-inflammatory effect. Therefore, we evaluated a possible early effect of topically administered glucocorticosteroids on the mucosal function of patients with distal inflammatory bowel disease in a prospective, controlled study, which was blinded for histological evaluation. METHODOLOGY: Eleven patients with distal ulcerative colitis or Crohn's disease, and 8 patients without intestinal inflammation were studied. A sigmoidoscopy with biopsy sampling (8-10) was performed before and 3 days after rectal administration of a hydrocortisone acetate foam preparation (100 mg b.i.d.). Prior to and after topical steroid treatment, basolateral (Na++K+)-ATPase activity (coupled optical assay), specific 3H ouabain binding (rapid filtration method), 5'-nucleotidase (microdetection method of phosphorus), and mucosal DNA levels (diphenylamine reaction) were determined from biopsy homogenates. Morphological and clinical characteristics were assessed according to established scores. RESULTS: Short-term topical GCS treatment significantly (p<0.05) stimulated (Na++K+)-ATPase activity (103%) as well as the number of active (Na++K+)-ATPase molecules (190%). In the healthy mucosa, only (Na++K+)-ATPase activity was stimulated (124%, p<0.05; specific 3H ouabain binding: 33%; p=0.09). As an unspecific GCS effect, apical 5'-nucleotidase was also stimulated (p<0.05; IBD: 50%; controls: 200%). As assessed by endoscopic and histological scores, as well as by mucosal DNA levels, morphological signs of intestinal inflammation remained unchanged during the study, whereas the daily stool frequency decreased significantly (p<0.05). CONCLUSIONS: In patients with distal inflammatory bowel disease, short-term treatment with topical GCS leads to a quick recovery from diarrheal symptoms, due to the early improvement of mucosal function prior to the occurrence of the well-known anti-inflammatory GCS effect.

A comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies.

Transmissible spongiform encephalopathies are associated with the accumulation of abnormal prion protein (PrP(Sc)) in the central nervous system which can be detected immunohistochemically. Using a monoclonal antibody (L42) to an epitope on the first alpha-helix of ruminant PrP, we compared previously reported immunohistochemical antigen unmasking and "visualization" systems. In addition, a variety of polyclonal and monoclonal antibodies to other epitopes on ruminant PrP were assessed. Antigen unmasking by hydrated autoclaving and proteinase K treatments, and antigen detection with L42 and an avidin-biotin complex system, enabled intra- and extra-neuronal PrP(Sc)to be demonstrated in scrapie-affected sheep carrying three different PrP alleles, as well as in cases of bovine spongiform encephalopathy.

Co-expression studies of the orphan carrier protein Slc10a4 and the vesicular carriers VAChT and VMAT2 in the rat central and peripheral nervous system.

The orphan carrier protein Slc10a4 represents a novel member of the so-called "sodium-bile acid co-transporter family," SLC10. Slc10a4 has a close phylogenetic relationship with the liver bile acid carrier Ntcp (Slc10a1), but has no transport activity for bile acids. In a previous study Slc10a4 proved to be predominantly expressed in the rat brain, where it was localized within cholinergic neurons. However, whether this cholinergic expression pattern was exclusive for Slc10a4 and whether this protein might also be expressed in the peripheral nervous system or other peripheral organs, remained unclear. Therefore, in the present study we analyzed the expression of Slc10a4 in neuronal and non-neuronal rat tissues more systematically, employing immunofluorescence co-localization studies of the vesicular acetylcholine transporter VAChT and the vesicular monoamine transporter VMAT2. The Slc10a4 protein was found to be widely expressed throughout structures of the CNS and peripheral nervous system. In addition to cholinergic neurons in the CNS, the retina, the neuromuscular junction and parasympathetic innervations, Slc10a4 was also localized in certain monoaminergic neurons and nerve fibers in the substantia nigra, the spinal cord and sympathetic innervations. Slc10a4 expression was also detected in granules of rat peritoneal and tissue mast cells using immunofluorescence and electron microscopy. Western blot and immunoprecipitation experiments with rat brain vesicle preparations revealed that the Slc10a4 protein was expressed in synaptic vesicles where it co-localized with synaptophysin, VAChT and VMAT2. This vesicular expression pattern was also shown in the rat adrenal pheochromocytoma cell line PC12 by immunofluorescence. Based on the findings of the present study we can speculate about the function of Slc10a4 as follows: (I) Slc10a4 could be a novel vesicular transporter for cholinergic and/or various monoaminergic neurotransmitters in the central and peripheral nervous system or (II) may be involved in the regulation of the synaptic vesicle sorting or exocytosis process.

Core then care: the nurse leader's role in "caring".

When discussing caring practices of health care professionals, often the nurse leader's role is not articulated, because the leader is not a direct caregiver. The leader, however, has the ability to potentiate or impede the level of practice that results in expert caring. In this article an argument is presented for the use of empowerment to promote the clinician-leader partnership model. Two theories--novice-to-expert and symphonology--are presented to assist leaders to create environments that foster expert level care.

Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale: preparative and analytical aspects as exemplified with paramecium cells.

We analyzed preparative and analytical aspects of the dynamic localization of Ca(2+) during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (approximately 100-nm thickness) or cilia (approximately 250-nm diameter). We used widely different standards to calibrate the ratio of CaK(alpha) net counts in relation to actual ¿Ca. Calibration curves showed a linear relationship and a detection limit of ¿Ca = 2 mM, while ¿Ca in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr(2+) can rapidly be substituted for Ca(2+) in the medium before and during stimulation, thus allowing one to determine Me(2+) fluxes. With our "model" cell, Paramecium, we showed that, upon stimulation (causing rapid Ca(2+) mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.

[A rare cause of upper gastrointestinal hemorrhage: large adenoma of Brunner's glands]. / Seltene Ursache einer oberen gastrointestinalen Blutung: Grosses Adenom der Brunner'schen Drüsen.

We report the rare case of a large adenoma of Brunner's glands as the cause of an upper gastrointestinal hemorrhage. Endoscopic resection is the therapy of choice in the appropriate setting. An unequivocal histological diagnosis by forceps biopsy often is not possible; there are no clear cut endoscopic criteria. The development of complications especially of severe hemorrhage increases with diameter of the adenoma. Therefore, the complete excision either endoscopically or by surgery should be attempted.

Distribution of synapses on two local auditory interneurones, ON1 and ON2, in the prothoracic ganglion of the cricket: relationships with GABA-immunoreactive neurones.

In the prothoracic ganglia of the cricket Gryllus bimaculatus two local auditory interneurones, ON1 and ON2, were labelled for electron microscopy by intracellular injection of horseradish peroxidase following physiological characterisation. The neurones branch in the median ventral association centre and the root of nerve 5 on both sides of the ganglion. As they are very similar in shape and position they may share a common embryological origin. Differences are found in the details of the fine branching pattern and in their physiology as ON1 is tuned particularly to low sound frequencies of 4-5 kHz whereas ON2 is more sensitive to frequencies above 8 kHz. Although the ON1 neurones inhibit each other and are involved in the inhibition of other auditory neurones they were not labelled by antibodies against the inhibitory transmitter GABA and their vesicles differ significantly from those in neurones that are. The same is true of the ON2 neurones whose vesicles also differ significantly from those in ON1 supporting light-microscope evidence that they may use different transmitters. The distribution of input and output synapses on the ipsilateral and contralateral branches of ON1 and ON2, and the proportion of the synapses made from and onto neuropilar processes immunoreactive for GABA was determined. In ON1 94% of the input synapses were received on the ipsilateral branches and 62% of the outputs made from the contralateral branches. This confirms previous physiological evidence that input is received ipsilaterally and output made contralaterally but the presence of some contralateral input and a significant ipsilateral output was unsuspected. Thirty percent of the input synapses on the ipsilateral side and 75% on the contralateral side were made from GABA-immunoreactive processes but processes postsynaptic to ON1 were rarely immunoreactive. The distribution of input synapses on ON2 was similar with 90% received on ipsilateral branches but a higher proportion of outputs (83%) was made from the contralateral side than in ON1. Thirty one percent of ipsilateral inputs were GABA-immunoreactive but only 14% on the contralateral side.