Covert sleep-related biological processes are revealed by probabilistic analysis in Drosophila

Sleep pressure and sleep depth are key regulators of wake and sleep. Current methods of measuring these parameters in Drosophila melanogaster have low temporal resolution and/or require disrupting sleep. Here we report analysis tools for high-resolution, noninvasive measurement of sleep pressure and depth from movement data. Probability of initiating activity, P(Wake), measures sleep depth while probability of ceasing activity, P(Doze), measures sleep pressure. In vivo and computational analyses show that P(Wake) and P(Doze) are largely independent and control the amount of total sleep. We also develop a Hidden Markov Model that allows visualization of distinct sleep/wake substates. These hidden states have a predictable relationship with P(Doze) and P(Wake), suggesting that the methods capture the same behaviors. Importantly, we demonstrate that both the Doze/Wake probabilities and the sleep/wake substates are tied to specific biological processes. These metrics provide greater mechanistic insight into behavior than measuring the amount of sleep alone.

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State-Switching Probability Reveals Sleep-Related Biological Drives inDrosophila

10.1101/263301 ◽

2018 ◽

Author(s):

Timothy D. Wiggin ◽

Patricia R. Goodwin ◽

Nathan C. Donelson ◽

Chang Liu ◽

Kien Trinh ◽

...

Keyword(s):

Analysis Tool ◽

Non Invasive ◽

Movement Data ◽

Environmental Perturbations ◽

Sleep Depth ◽

Computational Analyses ◽

And Control ◽

Insight Into ◽

Invasive Measurement

ABSTRACTSleep pressure and sleep depth are key regulators of wake and sleep. Current methods of measuring these parameters inDrosophila melanogasterhave low temporal resolution and/or require disrupting sleep. Here we report a novel analysis tool for high-resolution, non-invasive measurement of sleep pressure and depth from movement data. Probability of transitioning to an active state, P(Wake), measures sleep depth while probability of transitioning to an inactive state, P(Doze), measures sleep pressure.In vivoand computational analyses show that P(Wake) and P(Doze) are independent and control the amount of total sleep. Importantly, we demonstrate that these probabilities are tied to specific biological processes. Genetic and environmental perturbations demonstrate that a given amount of sleep can be produced by many combinations of underlying P(Wake) and P(Doze). We show that measuring sleep pressure and depth continuously, without disturbing on-going behavior, provides greater mechanistic insight into behavior than measuring the amount of sleep alone.

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SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

10.1101/2021.12.10.472112 ◽

2021 ◽

Author(s):

Scott B Biering ◽

Francielle Tramontini Gomes de Sousa ◽

Laurentia V. Tjang ◽

Felix Pahmeier ◽

Richard Ruan ◽

...

Keyword(s):

Transcriptional Response ◽

Barrier Dysfunction ◽

Vascular Leak ◽

Endothelial Barrier Dysfunction ◽

Mechanistic Insight ◽

Starting Point ◽

Signaling Axis ◽

Insight Into

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

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Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation

Nature Communications ◽

10.1038/s41467-019-12551-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Brianna J. Klein ◽

Suk Min Jang ◽

Catherine Lachance ◽

Wenyi Mi ◽

Jie Lyu ◽

...

Keyword(s):

Posttranslational Modification ◽

Memory Impairment ◽

Target Genes ◽

Epigenetic Mark ◽

Mechanistic Insight ◽

Genomic Analyses ◽

Insight Into

Abstract Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

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Mechanisms underlying sequence-dependent DNA hybridisation rates in the absence of secondary structure

10.1101/2021.12.17.473246 ◽

2021 ◽

Author(s):

Sophie Hertel ◽

Richard Spinney ◽

Stephanie Xu ◽

Thomas E Ouldridge ◽

Richard Morris ◽

...

Keyword(s):

Secondary Structure ◽

Dna Sequences ◽

Physical Factors ◽

Biological Processes ◽

Physical Mechanisms ◽

Mechanistic Insight ◽

Sequence Dependent ◽

Dna Hybridisation ◽

Kinetics Of ◽

Insight Into

The kinetics of DNA hybridisation are fundamental to biological processes and DNA-based technologies. However, the precise physical mechanisms that determine why different DNA sequences hybridise at different rates are not well understood. Secondary structure is one predictable factor that influences hybridisation rates but is not sufficient on its own to fully explain the observed sequence-dependent variance. Consequently, to achieve a good correlation with experimental data, current prediction algorithms require many parameters that provide little mechanistic insight into DNA hybridisation. In this context, we measured hybridisation rates of 43 different DNA sequences that are not predicted to form secondary structure and present a parsimonious physically justified model to quantify their hybridisation rates. Accounting only for the combinatorics of complementary nucleating interactions and their sequence-dependent stability, the model achieves good correlation with experiment with only two free parameters, thus providing new insight into the physical factors underpinning DNA hybridisation rates.

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Mycolactone toxin induces an inflammatory response by targeting the IL-1β pathway: Mechanistic insight into Buruli ulcer pathophysiology

PLoS Pathogens ◽

10.1371/journal.ppat.1009107 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009107

Author(s):

M. Foulon ◽

M. Robbe-Saule ◽

J. Manry ◽

L. Esnault ◽

Y. Boucaud ◽

...

Keyword(s):

Inflammatory Response ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Inflammatory Factors ◽

Dependent Manner ◽

Local Inflammatory Response ◽

Mechanistic Insight ◽

Insight Into

Mycolactone, a lipid-like toxin, is the major virulence factor of Mycobacterium ulcerans, the etiological agent of Buruli ulcer. Its involvement in lesion development has been widely described in early stages of the disease, through its cytotoxic and immunosuppressive activities, but less is known about later stages. Here, we revisit the role of mycolactone in disease outcome and provide the first demonstration of the pro-inflammatory potential of this toxin. We found that the mycolactone-containing mycobacterial extracellular vesicles produced by M. ulcerans induced the production of IL-1β, a potent pro-inflammatory cytokine, in a TLR2-dependent manner, targeting NLRP3/1 inflammasomes. We show our data to be relevant in a physiological context. The in vivo injection of these mycolactone-containing vesicles induced a strong local inflammatory response and tissue damage, which were prevented by corticosteroids. Finally, several soluble pro-inflammatory factors, including IL-1β, were detected in infected tissues from mice and Buruli ulcer patients. Our results revisit Buruli ulcer pathophysiology by providing new insight, thus paving the way for the development of new therapeutic strategies taking the pro-inflammatory potential of mycolactone into account.

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Seeing the Light: The Use of Zebrafish for Optogenetic Studies of the Heart

Frontiers in Physiology ◽

10.3389/fphys.2021.748570 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jonathan S. Baillie ◽

Matthew R. Stoyek ◽

T. Alexander Quinn

Keyword(s):

Experimental Model ◽

Optical Measurement ◽

Cardiac Activity ◽

Experimental Models ◽

Spatially Resolved ◽

And Control ◽

Spatiotemporal Control ◽

Insight Into

Optogenetics, involving the optical measurement and manipulation of cellular activity with genetically encoded light-sensitive proteins (“reporters” and “actuators”), is a powerful experimental technique for probing (patho-)physiological function. Originally developed as a tool for neuroscience, it has now been utilized in cardiac research for over a decade, providing novel insight into the electrophysiology of the healthy and diseased heart. Among the pioneering cardiac applications of optogenetic actuators were studies in zebrafish, which first demonstrated their use for precise spatiotemporal control of cardiac activity. Zebrafish were also adopted early as an experimental model for the use of optogenetic reporters, including genetically encoded voltage- and calcium-sensitive indicators. Beyond optogenetic studies, zebrafish are becoming an increasingly important tool for cardiac research, as they combine many of the advantages of integrative and reduced experimental models. The zebrafish has striking genetic and functional cardiac similarities to that of mammals, its genome is fully sequenced and can be modified using standard techniques, it has been used to recapitulate a variety of cardiac diseases, and it allows for high-throughput investigations. For optogenetic studies, zebrafish provide additional advantages, as the whole zebrafish heart can be visualized and interrogated in vivo in the transparent, externally developing embryo, and the relatively small adult heart allows for in situ cell-specific observation and control not possible in mammals. With the advent of increasingly sophisticated fluorescence imaging approaches and methods for spatially-resolved light stimulation in the heart, the zebrafish represents an experimental model with unrealized potential for cardiac optogenetic studies. In this review we summarize the use of zebrafish for optogenetic investigations in the heart, highlighting their specific advantages and limitations, and their potential for future cardiac research.

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In vivo quantitative analysis of Talin turnover in response to force

Molecular Biology of the Cell ◽

10.1091/mbc.e15-05-0304 ◽

2015 ◽

Vol 26 (22) ◽

pp. 4149-4162 ◽

Cited By ~ 11

Author(s):

Guðlaug Katrín Hákonardóttir ◽

Pablo López-Ceballos ◽

Alejandra Donají Herrera-Reyes ◽

Raibatak Das ◽

Daniel Coombs ◽

...

Keyword(s):

Fluorescence Recovery After Photobleaching ◽

Quantitative Imaging ◽

Three Dimensional ◽

Point Mutations ◽

Mechanical Forces ◽

Tissue Architecture ◽

Mechanistic Insight ◽

Adhesion Complex ◽

Insight Into

Cell adhesion to the extracellular matrix (ECM) allows cells to form and maintain three-dimensional tissue architecture. Cell–ECM adhesions are stabilized upon exposure to mechanical force. In this study, we used quantitative imaging and mathematical modeling to gain mechanistic insight into how integrin-based adhesions respond to increased and decreased mechanical forces. A critical means of regulating integrin-based adhesion is provided by modulating the turnover of integrin and its adhesion complex (integrin adhesion complex [IAC]). The turnover of the IAC component Talin, a known mechanosensor, was analyzed using fluorescence recovery after photobleaching. Experiments were carried out in live, intact flies in genetic backgrounds that increased or decreased the force applied on sites of adhesion. This analysis showed that when force is elevated, the rate of assembly of new adhesions increases such that cell–ECM adhesion is stabilized. Moreover, under conditions of decreased force, the overall rate of turnover, but not the proportion of adhesion complex components undergoing turnover, increases. Using point mutations, we identify the key functional domains of Talin that mediate its response to force. Finally, by fitting a mathematical model to the data, we uncover the mechanisms that mediate the stabilization of ECM-based adhesion during development.

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Mechanistic insight into the physiological relevance of helical blood flow in the human aorta: an in vivo study

Biomechanics and Modeling in Mechanobiology ◽

10.1007/s10237-010-0238-2 ◽

2010 ◽

Vol 10 (3) ◽

pp. 339-355 ◽

Cited By ~ 132

Author(s):

Umberto Morbiducci ◽

Raffaele Ponzini ◽

Giovanna Rizzo ◽

Marcello Cadioli ◽

Antonio Esposito ◽

...

Keyword(s):

Blood Flow ◽

In Vivo Study ◽

Human Aorta ◽

Mechanistic Insight ◽

Physiological Relevance ◽

Insight Into

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Combining theory, model and experiment to understand how theta rhythms are generated in the hippocampus

10.1101/115949 ◽

2017 ◽

Author(s):

Katie A. Ferguson ◽

Alexandra P. Chatzikalymniou ◽

Frances K. Skinner

Keyword(s):

Network Models ◽

Theory Model ◽

Network Activity ◽

Clear Understanding ◽

Cell Network ◽

Connection Probability ◽

Computational Analyses ◽

Insight Into

AbstractScientists have observed theta rhythms (3–12 Hz) in the hippocampus for decades, but we do not have a clear understanding of how they are generated. This is largely due to the complex, multi-scale and nonlinear nature of the brain. To obtain insight into mechanisms underlying the generation of theta rhythms, we develop cellular-based network models of the hippocampus based on a whole hippocampus in vitro preparation that spontaneously generates theta rhythms. Building on theoretical and computational analyses, we find that spike frequency adaptation and post-inhibitory rebound constitute a basis for theta generation in large, minimally connected CA1 pyramidal (PYR) cell network models with fast-firing parvalbumin-positive (PV+) inhibitory cells. The particular theta frequency is more controlled by PYR to PV+ cell interactions rather than PV+ to PYR cell ones. We identify two scenarios by which theta rhythms can emerge and they can be differentiated by the ratio of excitatory to inhibitory currents to PV+ cells, but not to PYR cells. Only one of the scenarios is consistent with data from the whole hippocampus preparation, which leads to the prediction that the connection probability from PV+ to PYR cells needs to be larger than from PYR to PV+ cells. Our models can serve as a platform on which to build and develop an understanding of in vivo theta generation, and of microcircuit dynamics in the hippocampus.SignificanceBrain rhythms have been linked to cognition and are disrupted in disease. This makes it essential to understand mechanisms underlying their generation. Theory and mathematical models help provide an understanding and generate hypotheses. Together with experiment they contribute a framework to dissect the cellular contributions to network activity. However, models are inherently biological approximations, and thus the specific experimental and theoretical context upon which they are built will shape their output. If the approximations and contexts are not taken into account, particularly when using previously constructed models, misinterpretations can arise. Here, we use both theory and microcircuit models derived from a specific experimental context to provide insight into cellular-based mechanisms involved in theta rhythm generation in the hippocampus.

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Specific miRNA-GPCR networks regulate Sox9a/Sox9b activities to promote gonadal renewal in zebrafish

10.1101/297820 ◽

2018 ◽

Author(s):

Xinlu Du ◽

Huiping Guo ◽

Ying Zhang ◽

Jiacheng Wu ◽

Minyou Li ◽

...

Keyword(s):

Regulatory Networks ◽

Sex Differentiation ◽

Gonadal Development ◽

Endocrine Function ◽

Mechanistic Insight ◽

Regulatory Circuits ◽

Primary Oocyte ◽

G Protein Coupled ◽

Insight Into

AbstractFertility and endocrine function rely on a tightly regulated synchronicity within the hypothalamic-pituitary gonadal (HPG) axis. FSH/cAMP/MAPK/ Sox9 axis signaling and its regulated specific miRNAs are thought to regulate vertebrate gonadal development and sex differentiation, and yet the regulatory networks are largely unknown. Here we construct small RNA and mRNA libraries from sexually matured ovary and testis of zebrafish to identify specific miRNA-target pairs. Integration of Targetscan prediction and in vivo induced gene expression highlight four specific miRNAs that conditionally target three G protein–coupled receptor (GPCR) x-Sox9 signaling genes, and implicate two regulatory circuits of miR430a-Sox9a in the testis and miR218a-Sox9b in the ovary. Co-injected Sox9a-miR430a mixture increases the proportion of spermatogonia but degenerates primary oocyte, while Sox9b-miR218a mixture induces renewal of ovarian follicles. Co-immunoprecipitation and mass-spectrometry analyses further reveal that miR430a and Sox9a synergistically activate testicular PKC/Rock1 signals while miR218a and Sox9b constrict ovary PKC/PI3K/Rock1 signaling. These results clarify specific miRNAs-GPCR regulatory networks of Sox9a/Sox9b switch, and also provide mechanistic insight into gonadal rejuvenation and plasticity.

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