In vivo myograph measurement of muscle contraction at optimal length

Background  

Current devices for measuring muscle contraction in vivo have limited accuracy in establishing and re-establishing the optimum muscle length. They are variable in the reproducibility to determine the muscle contraction at this length, and often do not maintain precise conditions during the examination. Consequently, for clinical testing only semi-quantitative methods have been used.     

Conservation of old individual trees and small populations is integral to maintain species' genetic diversity of a historically fragmented woody perennial

Historically fragmented and specialized habitats such as granite outcrops are understudied globally unique hot spots of plant evolution. In contrast to predictions based on mainstream population genetic theory, some granite outcrop plants appear to have persisted as very small populations despite prolonged geographic and genetic isolation. Eucalyptus caesia Benth. is a long‐lived lignotuberous tree endemic with a naturally fragmented distribution on granite outcrops in south‐western Australia. To quantify population to landscape‐level genetic structure, we employed microsatellite genotyping at 14 loci of all plants in 18 stands of E. caesia. Sampled stands were characterized by low levels of genetic diversity, small absolute population sizes, localized clonality and strong fine‐scale genetic subdivision. There was no significant relationship between population size and levels of heterozygosity. At the landscape scale, high levels of population genetic differentiation were most pronounced among representatives of the two subspecies in E. caesia as originally circumscribed. Past genetic interconnection was evident between some geographic neighbours separated by up to 20 km. Paradoxically, other pairs of neighbouring stands as little as 7 km apart were genetically distinct. There was no consistent pattern of isolation by distance across the 280 km range of E. caesia. Low levels of gene flow, together with strong drift within stands, provide some explanation of the patterns of genetic differentiation we observed. Individual genet longevity via the ability to repeatedly resprout and expand from a lignotuber may enhance the persistence of some woody perennial endemic plants despite small population size, minimal genetic interconnection and low heterozygosity.     

Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib

Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G-protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Because GRPR transactivates the epidermal growth factor receptor (EGFR), we investigated induction by GRP of Akt, an EGFR-activated signaling pathway, and examined effects of GRP on viability of non-small cell lung carcinoma (NSCLC) cells exposed to the EGFR tyrosine kinase inhibitor gefitinib. GRP induced Akt activation primarily through c-Src-mediated transactivation of EGFR. Transfection of dominant-negative c-Src abolished GRP-induced EGFR and Akt activation. GRP induced release of amphiregulin, and pre-incubation with human amphiregulin neutralizing antibody eliminated GRP-induced Akt phosphorylation. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely blocked GRP-initiated Akt phosphorylation. These results suggest that GRP stimulates Akt activation primarily via c-Src activation, followed by extracellular release of the EGFR ligand amphiregulin, leading to the activation of EGFR and PI3K. Pretreatment of NSCLC cells with GRP resulted in an increase in the IC(50) of gefitinib of up to 9-fold; this protective effect was mimicked by the pretreatment of cells with amphiregulin and reversed by Akt or PI3K inhibition. GRP appears to rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of the Akt pathway, suggesting GRPR and/or EGFR autocrine pathways in NSCLC cells may modulate therapeutic response to EGFR inhibitors.     

Myogenic stage, sarcomere length, and protease activity modulate localization of muscle-specific calpain

p94/calpain 3 is a Ca(2+)-binding intracellular protease predominantly expressed in skeletal muscles. p94 binds to the N2A and M-line regions of connectin/titin and localizes in the Z-bands. Genetic evidence showing that compromised p94 proteolytic activity leads to muscular dystrophy (limb-girdle muscular dystrophy type 2A) indicates the importance of p94 function in myofibrils. Here we show that a series of p94 splice variants is expressed immediately after muscle differentiation and differentially change localization during myofibrillogenesis. We found that the endogenous N-terminal (but not C-terminal) domain of p94 was not only localized in the Z-bands but also directly bound to sarcomeric alpha-actinin. These data suggest the incorporation of proteolytic N-terminal fragments of p94 into the Z-bands. In myofibrils localization of exogenously expressed p94 shifted from the M-line to N2A as the sarcomere lengthens beyond approximately 2.6 and 2.8 microm for wild-type and proteaseinactive p94, respectively. These data demonstrate for the first time that p94 proteolytic activity is involved in responses to muscle conditions, which may explain why p94 inactivation causes limb-girdle muscular dystrophy.     

Modified vaccinia virus Ankara induces Toll-like receptor-independent type I interferon responses

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus strain undergoing clinical evaluation as a replication-deficient vaccine vector against various infections and tumor diseases. To analyze the basis of its high immunogenicity, we investigated the mechanism of how MVA induces type I interferon (IFN) responses. MVA stimulation of bone marrow-derived dendritic cells (DC) showed that plasmacytoid DC were main alpha IFN (IFN-alpha) producers that were triggered independently of productive infection, viral replication, or intermediate and late viral gene expression. Increased IFN-alpha levels were induced upon treatment with mildly UV-irradiated MVA, suggesting that a virus-encoded immune modulator(s) interfered with the host cytokine response. Mice devoid of Toll-like receptor 9 (TLR9), the receptor for double-stranded DNA, mounted normal IFN-alpha responses upon MVA treatment. Furthermore, mice devoid of the adaptors of TLR signaling MyD88 and TRIF and mice deficient in protein kinase R (PKR) showed IFN-alpha responses that were only slightly reduced compared to those of wild-type mice. MVA-induced IFN-alpha responses were critically dependent on autocrine/paracrine triggering of the IFN-alpha/beta receptor and were independent of IFN-beta, thus involving "one-half" of a positive-feedback loop. In conclusion, MVA-mediated type I IFN secretion was primarily triggered by non-TLR molecules, was independent of virus propagation, and critically involved IFN feedback stimulation. These data provide the basis to further improve MVA as a vaccine vector.     

Influence of Sex on Suicidal Phenotypes in Affective Disorder Patients with Traumatic Childhood Experiences

Objectives

In the current study, we aimed to investigate the impact of childhood trauma on suicidal behaviour phenotypes in a group of patients with diagnosed affective disorder (unipolar or bipolar affective disorder).

Patients and Methods

Patients with and without a history of childhood abuse, measured by Childhood Trauma Questionnaire (CTQ), were assessed to explore risks for suicidal behaviour (including suicide attempt, self-harm and non-suicidal self-injury). The tested sample consisted of 258 patients (111 males and 147 females, in-patients and out-patients at the Department of Psychiatry and Psychotherapy, Medical University of Vienna and University Hospital Tulln, Lower Austria). Psychiatric diagnoses were derived from the SCAN (Schedules for Clinical Assessment in Neuropsychiatry) interview. In addition, patients were administered the Lifetime Parasuicidal Count (LPC), Suicidal Behaviour Questionnaire (SBQ-R), and Viennese Suicide Risk Assessment Scale (VISURIAS) questionnaires.

Results

In contrast to male suicide attempters, female suicide attempters showed both significantly higher total CTQ scores (p<0.001), and higher CTQ subscores (emotional, physical and sexual abuse, as well as emotional and physical neglect) in comparison to the non-suicidal control group. Besides, females with a history of self-harming behaviour (including suicidal intention) and Non-Suicidal-Self Injury (NSSI) had significantly higher CTQ total scores (p<0.001) than the control group.

Conclusion

These findings suggest gender differences in suicidal behaviour after being exposed to childhood trauma.     

Assessment of Model Based (Input) Impedance,Pulse Wave Velocity,and Wave Reflection in the Asklepios Cohort

Objectives

Arterial stiffness and wave reflection parameters assessed from both invasive and non-invasive pressure and flow readings are used as surrogates for ventricular and vascular load. They have been reported to predict adverse cardiovascular events, but clinical assessment is laborious and may limit widespread use. This study aims to investigate measures of arterial stiffness and central hemodynamics provided by arterial tonometry alone and in combination with aortic root flows derived by echocardiography against surrogates derived by a mathematical pressure and flow model in a healthy middle-aged cohort.

Methods

Measurements of carotid artery tonometry and echocardiography were performed on 2226 ASKLEPIOS study participants and parameters of systemic hemodynamics, arterial stiffness and wave reflection based on pressure and flow were measured. In a second step, the analysis was repeated but echocardiography derived flows were substituted by flows provided by a novel mathematical model. This was followed by a quantitative method comparison.

Results

All investigated parameters showed a significant association between the methods. Overall agreement was acceptable for all parameters (mean differences: -0.0102 (0.033 SD) mmHg*s/ml for characteristic impedance, 0.36 (4.21 SD) mmHg for forward pressure amplitude, 2.26 (3.51 SD) mmHg for backward pressure amplitude and 0.717 (1.25 SD) m/s for pulse wave velocity).

Conclusion

The results indicate that the use of model-based surrogates in a healthy middle aged cohort is feasible and deserves further attention.     

Proteomic and Metabolomic Analyses Reveal Contrasting Anti-Inflammatory Effects of an Extract of Mucor Racemosus Secondary Metabolites Compared to Dexamethasone

Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of Mucor racemosus (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424–102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac treatment on lipids were orthogonal to the effect of dexamethasone underlining differences in the overall mode of action.     

Diurnal changes in the xanthophyll cycle pigments of freshwater algae correlate with the environmental hydrogen peroxide concentration rather than non-photochemical quenching

Background and Aims In photosynthetic organisms exposure to high light induces the production of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), which in part is prevented by non-photochemical quenching (NPQ). As one of the most stable and longest-lived ROS, H₂O₂ is involved in key signalling pathways in development and stress responses, although in excess it can induce damage. A ubiquitous response to high light is the induction of the xanthophyll cycle, but its role in algae is unclear as it is not always associated with NPQ induction. The aim of this study was to reveal how diurnal changes in the level of H₂O₂ are regulated in a freshwater algal community.Methods A natural freshwater community of algae in a temporary rainwater pool was studied, comprising photosynthetic Euglena species, benthic Navicula diatoms, Chlamydomonas and Chlorella species. Diurnal measurements were made of photosynthetic performance, concentrations of photosynthetic pigments and H₂O₂. The frequently studied model organisms Chlamydomonas and Chlorella species were isolated to study photosynthesis-related H₂O₂ responses to high light.Key Results NPQ was shown to prevent H₂O₂ release in Chlamydomonas and Chlorella species under high light; in addition, dissolved organic carbon excited by UV-B radiation was probably responsible for a part of the H₂O₂ produced in the water column. Concentrations of H₂O₂ peaked at 2 µm at midday and algae rapidly scavenged H₂O₂ rather than releasing it. A vertical H₂O₂ gradient was observed that was lowest next to diatom-rich benthic algal mats. The diurnal changes in photosynthetic pigments included the violaxanthin and diadinoxanthin cycles; the former was induced prior to the latter, but neither was strictly correlated with NPQ.Conclusions The diurnal cycling of H₂O₂ was apparently modulated by the organisms in this freshwater algal community. Although the community showed flexibility in its levels of NPQ, the diurnal changes in xanthophylls correlated with H₂O₂ concentrations. Alternative NPQ mechanisms in algae involving proteins of the light-harvesting complex type and antioxidant protection of the thylakoid membrane by de-epoxidized carotenoids are discussed.