Review of Modelling Techniques for In Vivo

Background. Knowledge of the musculoskeletal loading conditions during strength training is essential for performance monitoring, injury prevention, rehabilitation, and training design. However, measuring muscle forces during exercise performance as a primary determinant of training efficacy and safety has remained challenging. Methods. In this paper we review existing computational techniques to determine muscle forces in the lower limbs during strength exercises in vivo and discuss their potential for uptake into sports training and rehabilitation. Results. Muscle forces during exercise performance have almost exclusively been analysed using so-called forward dynamics simulations, inverse dynamics techniques, or alternative methods. Musculoskeletal models based on forward dynamics analyses have led to considerable new insights into muscular coordination, strength, and power during dynamic ballistic movement activities, resulting in, for example, improved techniques for optimal performance of the squat jump, while quasi-static inverse dynamics optimisation and EMG-driven modelling have helped to provide an understanding of low-speed exercises. Conclusion. The present review introduces the different computational techniques and outlines their advantages and disadvantages for the informed usage by nonexperts. With sufficient validation and widespread application, muscle force calculations during strength exercises in vivo are expected to provide biomechanically based evidence for clinicians and therapists to evaluate and improve training guidelines

Dynamic circadian protein-protein interaction networks predict temporal organization of cellular functions.

Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (~24-hour) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes, PPI dynamics on this timescale are largely unknown. Here, we identified 109 novel PPIs among circadian clock proteins via a yeast-two-hybrid approach. Among them, the interaction of protein phosphatase 1 and CLOCK/BMAL1 was found to result in BMAL1 destabilization. We constructed a dynamic circadian PPI network predicting the PPI timing using circadian expression data. Systematic circadian phenotyping (RNAi and overexpression) suggests a crucial role for components involved in dynamic interactions. Systems analysis of a global dynamic network in liver revealed that interacting proteins are expressed at similar times likely to restrict regulatory interactions to specific phases. Moreover, we predict that circadian PPIs dynamically connect many important cellular processes (signal transduction, cell cycle, etc.) contributing to temporal organization of cellular physiology in an unprecedented manner

Tuning the Mammalian Circadian Clock: Robust Synergy of Two Loops

The circadian clock is accountable for the regulation of internal rhythms in most living organisms. It allows the anticipation of environmental changes during the day and a better adaptation of physiological processes. In mammals the main clock is located in the suprachiasmatic nucleus (SCN) and synchronizes secondary clocks throughout the body. Its molecular constituents form an intracellular network which dictates circadian time and regulates clock-controlled genes. These clock-controlled genes are involved in crucial biological processes including metabolism and cell cycle regulation. Its malfunction can lead to disruption of biological rhythms and cause severe damage to the organism. The detailed mechanisms that govern the circadian system are not yet completely understood. Mathematical models can be of great help to exploit the mechanism of the circadian circuitry. We built a mathematical model for the core clock system using available data on phases and amplitudes of clock components obtained from an extensive literature search. This model was used to answer complex questions for example: how does the degradation rate of Per affect the period of the system and what is the role of the ROR/Bmal/REV-ERB (RBR) loop? Our findings indicate that an increase in the RNA degradation rate of the clock gene Period (Per) can contribute to increase or decrease of the period - a consequence of a non-monotonic effect of Per transcript stability on the circadian period identified by our model. Furthermore, we provide theoretical evidence for a potential role of the RBR loop as an independent oscillator. We carried out overexpression experiments on members of the RBR loop which lead to loss of oscillations consistent with our predictions. These findings challenge the role of the RBR loop as a merely auxiliary loop and might change our view of the clock molecular circuitry and of the function of the nuclear receptors (REV-ERB and ROR) as a putative driving force of molecular oscillations

Eine Hochdurchsatz-Analyse zirkadianer Proteinstabilität

Circadian clocks are endogenous oscillations that drive 24-hour rhythms of physiology and behavior. Circadian rhythms exist in nearly all cells of the body with a gene-regulatory network as fundamental clockwork. These molecular clocks not only drive the rhythmic transcription of about ten percent of all genes but also seem to regulate rhythmic protein abundances by mechanisms beyond transcriptional control. Indeed, up to twenty percent of the mammalian proteome are circadian. The extent of post-transcriptional processes controlling circadian protein abundance, however, is hardly studied so far. Here, the contribution of timed degradation for the regulation of circadian protein rhythms is investigated. To this end, a human protein library was analyzed using a fluorescence based reporter system that measures alterations in protein abundance as readout of altered protein stability. In order to determine time-of-day dependent protein stability, the cellular clock was ’clamped’ at a specific circadian phase by the ectopic overexpression of CRY1, a strong negative inhibitor of molecular clock dynamics. Applying the fluorescence based method in cells with a ’clamped’ clock in comparison to unsynchronized cells enabled to perform a proteome-wide analysis of circadian stability. Revealed screen results represent a snapshot of the circadian proteome ’clamped’ at a phase of increased CRY1 level. About nine percent of analyzed proteins were identified as CAAPs (CRY1 mediated altered abundant proteins), representing proteins with potential circadian abundance as result of timed degradation. Indeed, rhythmically abundant proteins are enriched among CAAPs. Furthermore, in accordance with circadian proteome studies, CAAPs are overrepresented in processes related to vesicular trafficking and mitosis. Interestingly, CAAPs are underrepresented among modifiers of circadian dynamics. This postulates a role of circadian protein stability for the rhythmic fine-tuning of clock output functions rather than for regulatory mechanisms of molecular core clock dynamics. Together, the results of this study indicate first of all, an unexpected role of rhythmic protein degradation for the control of the circadian proteome, and secondly, suggest that rhythmic protein stability is essential as a timing signal for circadian clock output processes.Circadiane Uhren sind endogen getriebene Oszillationen, welche 24-Stunden Rhythmen der Physiologie und des Verhaltens steuern. Circadiane Rhythmen existieren in nahezu allen Zellen des Körpers und basieren auf einem genregulatorischen ’Uhrwerk’. Diese molekularen Uhren steuern die rhythmische Transkription von etwa zehn Prozent aller Gene. Darüber hinaus regulieren molekulare Uhren Proteinrhythmen, vermutlich durch zusätzliche Mechanismen neben der transkriptionellen Kontrolle. In der Tat liegen bis zu zwanzig Prozent des Säugetierproteoms circadian abundant vor. In welchem Umfang circadiane Proteinrhythmen durch post-transkriptionelle Prozesse kontrolliert werden, ist jedoch bisher wenig untersucht. In dieser Studie wurde der Beitrag des tageszeitspezifischen Abbaus zur Regulation circadianer Proteinrhythmen untersucht. Dazu wurde eine humane Proteinbibliothek unter Anwendung eines fluoreszenzbasierten Reportersystems untersucht. Das Reportersystem misst Unterschiede in der Proteinmenge als Kennzeichen verschiedener Proteinstabilitäten. Um tageszeitabhängige Proteinstabilität bestimmen zu können, wurde die zellulare Uhr in einer spezifischen Phase ’arretiert’. Dies erfolgte durch die ektopische Überexpression von CRY1, einem stark negativen Inhibitor der molekularen Uhr-Dynamik. Unter Anwendung der fluoreszenzbasierten Methode in Uhr-’arretierten’ Zellen im Vergleich zu unsynchronisierten Zellen, konnten circadiane Stabilitäten proteomweit analysiert werden. Die Ergebnisse der Untersuchung repräsentieren eine Momentaufnahme des circadianen Proteoms, ’arretiert’ in einer Phase erhöhter CRY1 Proteinmengen. Etwa neun Prozent aller untersuchten Proteine wurden als CAAPs (CRY1 mediated altered abundant proteins; deutsch: Proteine mit CRY1 induzierter, veränderter Abundanz) identifiziert. Diese stellen eine Gruppe von Proteinen mit vermutlich circadianer Abundanz als Ergebnis des tageszeitspezifischen Abbaus, dar. In der Tat sind rhythmisch abundante Proteine unter den CAAPs angereichert. In Übereinstimmung mit circadianen Proteomstudien, sind CAAPs in Prozessen des vesikulären Transports und der Mitose überrepräsentiert. Interessanterweise sind CAAPs unter molekularen Komponenten, welche die circadiane Uhr beeinflussen, unterrepräsentiert. Das deutet auf eine Funktion der circadianen Proteinstabilität in der rhythmischen Feinabstimmung Uhr-getriebener Prozesse hin, welche die molekulare Uhr selbst, nicht regulieren. Zusammenfassend weisen die Ergebnisse dieser Studie dem rhythmischen Proteinabbau eine unerwartete Rolle in der Regulation des circadianen Proteoms zu und deuten des Weiteren daraufhin, dass rhythmische Proteinstabilität als Übermittler von Zeitinformationen in Uhr-gesteuerten Prozessen essenziell ist

Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy

Wang Y, Schellenberg H, Walhorn V, Tönsing K, Anselmetti D. Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy. European Biophysics Journal. 2017;46(6):561-566

Systematic Interaction Mapping between 46 Circadian Clock Proteins and Associated Components.

<p>(A) Matrix based high-throughput yeast-two-hybrid interaction screen. (B) CLOCK interactors: Mating controls (top left); upon PPI reporter genes are activated (top middle: HIS, URA for growth selection, top right: lacZ for β-galactosidase activity). Bottom: Detected interactions with CLOCK; red lines: interactions previously discovered in yeast (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s001" target="_blank">Figure S1</a>). (C) Clock protein interaction matrix. Circles: interactions between two components not differentiating between bait and prey configuration. (D) Validation of new CLOCK and BMAL1 interactions in mammalian cells. HEK293 cells expressing CLOCK- or BMAL1-luciferase fusions were transfected with MYC-tagged components. Luciferase activity in anti-MYC co-immunoprecipitates is presented for one representative result of at least two independent experiments with similar results (for method and input controls also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s002" target="_blank">Figure S2</a>). MYC-β-galcactosidase fusions served as negative, MYC-BMAL1 and MYC-CRY1 as positive controls, respectively.</p

Network Neighborhood Contains Clock Modulating Components.

<p>Systematic RNAi-mediated downregulation of network neighborhood genes in dexamethasone-synchronized U2OS cells harboring a <i>Bmal1</i>-promoter luciferase reporter. Shown are altered oscillation dynamics (red dots with corresponding fit lines) for 16 genes achieved by individual RNAi constructs (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s009" target="_blank">Table S2</a>). For twelve genes, two RNAi constructs resulted in similar phenotypes, for nine genes only one construct was available in our laboratory library. Black dots with corresponding fit lines are controls representing the mean values of at least 80 irrelevant constructs. Period deviations from controls are shown.</p

Interaction Dynamics within the Liver Circadian Protein–Protein Network.

<p>(A) Interacting proteins are more likely to be co-expressed in time. Left: Co-expression of interacting proteins was calculated using the Pearson correlation coefficient (PCC) of circadian expression profiles in liver <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398-Hughes1" target="_blank">[6]</a> and compared to randomly selected protein pairs. Among interacting proteins co-expressed proteins (i.e. PCC>0.5) are significantly overrepresented (Chi squared test: p<10⁻¹⁵). 13% of interacting proteins have a PCC>0.5 compared to 4% for random pairs. Right: analogous analysis for the circadian PPI network. Co-expressed (PCC>0.5) interacting proteins are highly overrepresented (Chi squared test: p<10⁻¹⁵; 22% compared to 4% with PCC>0.5). (B) Left: heat map representing the predicted dynamics of protein–protein interaction based on their liver expression profiles. Interactions were classified as rhythmic if the product of their expression vectors shows highly significant periodic expression (FDR<10⁻⁵). Right: examples for interaction pairs and their predicted interaction phase. Red lines: products of individual transcript profiles of two interacting proteins. Dotted rectangles highlight predicted phase of interaction.</p

Protein Phosphatase 1 Modulates CLOCK/BMAL1 Function.

<p>(A) CLOCK and BMAL1 interactors identified in yeast and their paralogs were co-transfected with CLOCK/BMAL1 and an E-box containing luciferase reporter (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s007" target="_blank">Figure S7A</a>). Shown are means ± s.d. of CLOCK/BMAL1 modifiers (n = 3; *** p<0.001, t-test). (B) PPP1CA dose-dependently reduces CLOCK/BMAL1 transactivation (n = 3; means ± SD.). (C) PPP1CA is present in the CLOCK/BMAL1 complex. Murine livers were harvested at indicated times. Dashed lines: longer exposure. (LC: light chain; HC: heavy chain). (D) PPP1CA destabilizes BMAL1 protein. Left: Stability is reported by the change of EGFP to DsRed ratio <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398-Yen1" target="_blank">[30]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398-Yen2" target="_blank">[31]</a>. Right: PPP1CA co-expression with BMAL1, CLOCK or short-lived EGFP fusion proteins in U2OS cells reduces BMAL1 stability (mean ± s.d.; ***p<0.001; t-test; n = 3; (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s007" target="_blank">Figure S7B, S7C</a>). (E) Endogenous BMAL1 levels are reduced upon PPP1CA overexpression in U2OS cells. Depicted are two independent experiments. (F) PPP1CA reduces BMAL1 stability. U2OS cells stably expressing PPP1CA or GFP were harvested at the indicated time points after cycloheximide (CHX) application and protein levels were analyzed by Western blot. Shown is one representative of two independently performed experiments (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s007" target="_blank">Figure S7D</a>).</p