Spatio-temporal processing of surface EMG signals from the sternocleidomastoideus muscle to assess effects of radiotherapy on motor unit conduction velocity and firing rate—A pilot study

Radiation therapy causes both muscle and nerve tissue damage. However, the evolution and mechanisms of these damages are not fully understood. Information on the state of active muscle fibres and motoneurons can be obtained by measuring sEMG signals and calculating the conduction velocity (CV) and firing rate of individual motor units, respectively. The aim of this pilot study was to evaluate if the multi-channel surface EMG (sEMG) technique could be applied to the sternocleidomastoideus muscle (SCM) of radiotherapy patients, and to assess if the CV and firing rate are altered as a consequence of the radiation.

Surface EMG signals were recorded from the radiated and healthy SCM muscles of 10 subjects, while subjects performed isometric rotation of the head. CV and firing rate were calculated using two recently proposed methods based on spatio-temporal processing of the sEMG signals. The multi-channel sEMG technique was successfully applied to the SCM muscle and CV and firing rates were obtained. The measurements were fast and simple and comfortable for the patients. Sufficient data quality was obtained from both sides of seven and four subjects for the CV and firing rate analysis, respectively. No differences in CV or firing rate were found between the radiated and non-radiated sides (p = 0.13 and p = 0.20, respectively). Firing rate and CV were also obtained from a myokymic discharge pattern. It was found that the CV decreased significantly (p = 0.01) during the bursts.     

Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis

Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the physiology of mature biofilm in Bordetella pertussis, the agent responsible for whooping cough. Recent reports indicate that B. pertussis adopts a sessile biofilm as a strategy to persistently colonize the human host. However, since research in the past mainly focused on the planktonic lifestyle of B. pertussis, knowledge on biofilm formation of this important human pathogen is still limited. Comparative studies were carried out by combining 2-DE and Fourier transform infrared (FT-IR) spectroscopy with multivariate statistical methods. These complementary approaches demonstrated that biofilm development has a distinctive impact on B. pertussis physiology. Results from MALDI-TOF/MS identification of proteins together with results from FT-IR spectroscopy revealed the biosynthesis of a putative acidic-type polysaccharide polymer as the most distinctive trait of B. pertussis life in a biofilm. Additionally, expression of proteins known to be involved in cellular regulatory circuits, cell attachment and virulence was altered in sessile cells, which strongly suggests a significant impact of biofilm development on B. pertussis pathogenesis. In summary, our work showed that the combination of proteomics and FT-IR spectroscopy with multivariate statistical analysis provides a powerful tool to gain further insight into bacterial lifestyles.     

A New In Vitro Model to Study Cellular Responses after Thermomechanical Damage in Monolayer Cultures

Although electrosurgical instruments are widely used in surgery to cut tissue layers or to achieve hemostasis by coagulation (electrocautery), only little information is available concerning the inflammatory or immune response towards the debris generated. Given the elevated local temperatures required for successful electrocautery, the remaining debris is likely to contain a plethora of compounds entirely novel to the intracorporal setting. A very common in vitro method to study cell migration after mechanical damage is the scratch assay, however, there is no established model for thermomechanical damage to characterise cellular reactions. In this study, we established a new in vitro model to investigate exposure to high temperature in a carefully controlled cell culture system. Heatable thermostat-controlled aluminium stamps were developed to induce local damage in primary human umbilical vein endothelial cells (HUVEC). The thermomechanical damage invoked is reproducibly locally confined, therefore allowing studies, under the same experimental conditions, of cells affected to various degrees as well as of unaffected cells. We show that the unaffected cells surrounding the thermomechanical damage zone are able to migrate into the damaged area, resulting in a complete closure of the ‘wound’ within 48 h. Initial studies have shown that there are significant morphological and biological differences in endothelial cells after thermomechanical damage compared to the mechanical damage inflicted by using the unheated stamp as a control. Accordingly, after thermomechanical damage, cell death as well as cell protection programs were activated. Mononuclear cells adhered in the area adjacent to thermomechanical damage, but not to the zone of mechanical damage. Therefore, our model can help to understand the differences in wound healing during the early phase of regeneration after thermomechanical vs. mechanical damage. Furthermore, this model lends itself to study the response of other cells, thus broadening the range of thermal injuries that can be analysed.     

Species Differences in Developmental Toxicity of Epoxiconazole and Its Relevance to Humans

Epoxiconazole, a triazole‐based fungicide, was tested in toxicokinetic, prenatal and pre‐postnatal toxicity studies in guinea pigs, following oral (gavage) administration at several dose levels (high dose: 90 mg/kg body weight per day). Maternal toxicity was evidenced by slightly increased abortion rates and by histopathological changes in adrenal glands, suggesting maternal stress. No compound‐related increase in the incidence of malformations or variations was observed in the prenatal study. In the pre‐postnatal study, epoxiconazole did not adversely affect gestation length, parturition, or postnatal growth and development. Administration of epoxiconazole did not alter circulating estradiol levels. Histopathological examination of the placentas did not reveal compound‐related effects. The results in guinea pigs are strikingly different to those observed in pregnant rats, in which maternal estrogen depletion, pathological alteration of placentas, increased gestation length, late fetal death, and dystocia were observed after administration of epoxiconazole. In the studies reported here, analysis of maternal plasma concentrations and metabolism after administration of radiolabeled epoxiconazole demonstrated that the different results in rats and guinea pigs were not due to different exposures of the animals. A comprehensive comparison of hormonal regulation of pregnancy and birth in murid rodents and primates indicates that the effects on pregnancy and parturition observed in rats are not applicable to humans. In contrast, the pregnant guinea pig shares many similarities to pregnant humans regarding hormonal regulation and is therefore considered to be a suitable species for extrapolation of related effects to humans. Birth Defects Res (Part B) 98:230–246, 2013. © 2013 Wiley Periodicals, Inc.     

Pertussis toxin-sensitive G-proteins inhibit fibroblast growth factor-induced signaling in pancreatic acini

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C-γ, and phosphatidylinositol 3-kinase, whereas the role of G-proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G-proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C-γ1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF-2) and guanosine 5′-(γ-O-thio)triphosphate (GTPγS) caused an increase in inositol 1,4,5-trisphosphate (1,4,5-IP₃) production and amylase release. Combined stimulation of the acini with GTPγS and FGF-2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF-2 (1 nM) or vehicle (water) ADP-ribosylation of the α-subunit of G_i-type G-proteins by pertussis toxin was reduced in membranes prepared from FGF-2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with G_i-proteins. Pretreatment of acini with pertussis toxin which inhibits G_i-type G-proteins abolished the inhibitory effect of GTPγS on FGF-induced 1,4,5-IP₃ production and amylase release, whereas the stimulatory effects of FGF-2 and GTPγS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and G_i-type G-proteins and that G_i-type G-proteins exert an inhibitory influence on FGF-induced activation of phosphoinositide-specific phospholipase C in pancreatic acinar cells. © 1996 Wiley-Liss, Inc.     

A Voltage-Dependent Ca<Superscript>2+</Superscript> Influx Pathway Regulates the Ca<Superscript>2+</Superscript>-Dependent Cl<Superscript>−</Superscript> Conductance of Renal IMCD-3 Cells

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca²⁺-dependent Cl⁻ current (I_CLCA). Here we report that I_CLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al³⁺-sensitive, voltage-dependent, Ca²⁺ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (−60 mV), I_CLCA was found to be inactive and resting currents were predominantly K⁺ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of I_CLCA (T _0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in I_CLCA (T _0.5 ~ 100 s). The activation of I_CLCA by depolarization was reduced by lowering extracellular Ca²⁺ and completely inhibited by buffering cytosolic Ca²⁺ with EGTA, suggesting a role for Ca²⁺ influx in the activation of I_CLCA. However, raising bulk cytosolic Ca²⁺ at −60 mV did not produce sustained I_CLCA activity. Therefore I_CLCA is dependent on both an increase in intracellular Ca²⁺ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca²⁺ influx pathway that displays equal permeability to Ca²⁺ and Ba²⁺ ions and that is blocked by extracellular Al³⁺ and La³⁺. Furthermore, Al³⁺ completely and reversibly inhibited depolarization-induced activation of I_CLCA, thereby directly linking Ca²⁺ influx to activation of I_CLCA. We speculate that during sustained membrane depolarization, calcium influx activates I_CLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.     

Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E

The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T=1 icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.Flaviviruses form a genus in the family Flaviviridae (52) and comprise a number of important human pathogens such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis (TBE) viruses (30). They are small, enveloped viruses with only three structural proteins, designated C (capsid), M (membrane), and E (envelope). The E protein is oriented parallel to the viral membrane and forms a head-to-tail homodimeric complex (Fig. 1A and B). The structure of the E ectodomain (soluble E [sE])—consisting of about 400 amino acids and lacking the 100 C-terminal amino acids (including the so-called stem and two transmembrane helices)—has been determined by X-ray crystallography for several flaviviruses (Fig. ​(Fig.1A)1A) (25, 34, 36, 38, 44, 55). Both of the essential entry functions—receptor-binding and membrane fusion after uptake by receptor-mediated endocytosis—are mediated by E, which is therefore the primary target for virus-neutralizing antibodies (11, 42, 43, 45).Open in a separate windowFIG. 1.Structures and schematic representations of the TBE virus E protein, virions, and RSPs. In all panels, DI, DII, and DIII of the E protein are shown in red, yellow, and blue, respectively, and the fusion peptide (FP) is in orange. (A) Ribbon diagram of the sE dimer (top view). (B) Schematic of the full-length E dimer in a top view (upper panel) and side view (lower panel). The position of the two transmembrane helices of the membrane anchor and the two helices of the stem are based on Zhang et al. (54) and are shown in green and purple, respectively. (C) Pseudo-atomic structure of the virion based on cryo-EM reconstructions of dengue and West Nile viruses (27, 37, 54). One of the 30 rafts, each consisting of three parallel dimers, is highlighted. DIIIs of three monomers belonging to one icosahedral asymmetric unit are labeled by white stars. (D) Pseudo-atomic structure of RSP based on cryo-EM reconstructions (12).As revealed by cryo-electron microscopy (cryo-EM), mature infectious virions have smooth surfaces, comparable to a golf ball (27, 37). Their envelopes are icosahedrally symmetric and consist of a closed shell of 180 E monomers that are arranged in a herringbone-like pattern of 30 rafts of three dimers each (Fig. ​(Fig.1C)1C) (27). On the other hand, capsid-lacking subviral particles, which can be produced in recombinant form by the coexpression of prM and E, have a different symmetry, with 30 E dimers in a T=1 icosahedral structure (Fig. ​(Fig.1D)1D) (12, 49).The peculiar organization of E in virions is reminiscent of the tight packing of capsid proteins in nonenveloped viruses, for which it was shown that the native antigenic structure is strongly dependent on the intact capsid structure and not completely represented by isolated forms of capsid proteins (1, 41, 53). Such modulations of antigenic structure may be due to conformational changes in the course of packaging the capsid proteins into virions and/or to the fact that antibody binding sites at the virion surface are composed of residues that come together only through the juxtaposition of capsid proteins or neighboring protein subunits. Even in the case of spiky viral envelope proteins, the dependence of certain epitopes on the quaternary organization of the envelope glycoproteins has been described (8, 47).For flaviviruses, structural studies provide evidence for the considerable flexibility of E, especially at the junctions between the individual domains I, II, and III (DI, DII, and DIII) (7, 35, 55), suggesting that soluble forms may display differences in antigenic structure compared to those fixed in the closed envelope shell of whole virions. Furthermore, because of the tight packing of E at the virion surface, certain epitopes may be cryptic in the context of whole virus particles but accessible in soluble forms of E (40, 51).Studies on the antigenic structure of flaviviruses have used different antigen preparations including virions, recombinant subviral particles (RSPs), and soluble forms and subunits of E (10, 15-17, 32, 39, 40, 46, 49, 51), but so far no systematic comparative analysis of E in different physical forms and quaternary arrangements has been conducted. It was therefore the objective of our study, using TBE virus as a model, to investigate possible structural and/or antigenic differences between (i) soluble dimeric forms of E, including C-terminally truncated sE and detergent-solubilized full-length E (Fig. 1A and B); (ii) E in the context of whole virions (Fig. ​(Fig.1C);1C); and (iii) E in the context of RSPs (Fig. ​(Fig.1D).1D). For this purpose we used, and further characterized, a set of monoclonal antibodies (MAbs) directed to each of the three domains of E. All of these MAbs have neutralizing activity (17, 24) and therefore, by definition, react with infectious virions.Through these analyses, we demonstrate that the reactivity of several MAbs is significantly dependent on the quaternary arrangement of E and differs between virions, RSPs, and/or sE dimers. We thus provide evidence for previously unrecognized structural factors that have an impact on the antigenicity of the flavivirus E protein.     

Opposite effects of presynaptic 5-HT3 receptor activation on spontaneous and action potential-evoked GABA release at hippocampal synapses

5-HT(3) (serotonin type 3) receptors are targets of antiemetics, antipsychotics, and antidepressants and are believed to play a role in cognition. Nevertheless, contrasting results have been obtained with respect to their functions in the CNS and in the control of transmitter release. We used rat hippocampal neurons in single-neuron microcultures to identify the roles of presynaptic 5-HT(3) receptors at central synapses. 5-HT (10 microm) caused a transient > 10-fold increase in the frequency of miniature inhibitory postsynaptic currents without affecting amplitudes or kinetics. This effect was abolished by tropisetron (30 nm) and when Ca(2+) channels were blocked by 100 microm Cd(2+) it was mimicked and occluded when neurons were depolarized by 20 mm, but not 10 mm, K(+). Thus, activation of presynaptic 5-HT(3) receptors increased spontaneous GABA release by causing depolarization and opening of voltage-gated Ca(2+) channels. In microculture neurons, 5-HT transiently reduced action potential-evoked inhibitory autaptic currents by > 50%; this effect was blocked by tropisetron and mimicked by 20 mm, but not 10 mm, K(+). Miniature excitatory postsynaptic currents were not altered by 5-HT. Excitatory autaptic currents were tonically reduced, an effect attenuated by 5-HT(1A) antagonists. Thus, presynaptic 5-HT(3) receptors control GABA, but not glutamate, release and mediate opposite effects on spontaneous and action potential-dependent release.