Biology subjects

Jensen, M. H.

Publications and source records attributed to Jensen, M. H..

13 recordsLinked to original sources

bioRxiv2026

Development of a NanoBRET assay platform to detect intracellular target engagement in living cells for the Glycine Transporter 1 (SLC6A9)

Glycine transporter 1 (GlyT1) is a clinically investigated target whose conformational plasticity enables ligands to engage distinct extracellular and intracellular binding sites. However, methods to directly quantify intracellular binding remain limited and largely rely on radioligand-based approaches. Here, we report a NanoBRET-based binding assay to quantify intracellular GlyT1 target engagement in living cells using a fluorescent tracer derived from the GlyT1 inhibitor Cmpd1. With a structure-based approach followed by pharmacological characterization we identified a suitable meta-substituted exit vector for linker and fluorophore attachment while maintaining GlyT1 binding. A series of fluorescent probes was synthesized and evaluated, leading to the development of GlyT1-probe which displayed high-affinity binding and enabled robust NanoBRET measurements. Unlike the functional uptake assay, the NanoBRET assay distinguished intracellular from extracellular GlyT1 inhibitors and resolved structure-activity relationships at the intracellular binding site. This assay provides a quantitative approach to interrogate conformation-selective GlyT1 target engagement in living cells, facilitating the characterization of GlyT1 ligands targeting the intracellular site.

molecular biology↗

bioRxiv2024

Circadian Coupling Orchestrates Cell Growth

Single-cell circadian oscillators exchange extracellular information to sustain coherent circadian rhythms at the tissue level. Within cells, the circadian clock and the cell cycle couple, yet the mechanisms governing this interplay remain poorly elucidated. Here, we study the role of extracellular circadian communication in the intracellular coordination between the circadian clock and the cell cycle. We demonstrate that the loss of extracellular circadian synchronization disrupts circadian and cell cycle coordination within individual cells, impeding collective tissue growth. Coherent circadian rhythms yield oscillatory growth patterns, unveiling a global timing regulator of tissue dynamics. Knocking down core circadian elements abolishes observed effects, highlighting the central role of circadian clock regulation. Our research underscores the significance of tissue-level circadian disruption in regulating proliferation, thereby linking disrupted circadian clocks with oncogenic processes. These findings illuminate the intricate interplay between circadian rhythms, cellular signaling, and tissue physiology, enhancing our understanding of tissue homeostasis and growth regulation in both health and disease contexts.

cell biology↗

bioRxiv2024

Tipping-point transition from transient to persistent inflammation in pancreatic islets

Type 2 diabetes (T2D) is associated with a systemic increase in the pro-inflammatory cytokine IL-1{beta}. While transient exposure to low IL-1{beta} concentrations improves insulin secretion and {beta}-cell proliferation in pancreatic islets, prolonged exposure leads to impaired insulin secretion and collective {beta}-cell death. IL-1 is secreted locally by islet-resident macrophages and {beta}-cells; however it is unknown if and how the two opposing modes may emerge at single islet level. We investigated the duality of IL-1{beta} with a quantitative in-silico model of the IL-1 regulatory network in pancreatic islets. We find that the network can produce either transient or persistent IL-1 responses, when induced by pro-inflammatory and metabolic cues. This suggests that the duality of IL-1 may be regulated at the single islet level. We use two core feedbacks in the IL-1 regulation to explain both modes: First, a fast positive feedback in which IL-1 induces its own production through IL-1R/IKK/NF-{kappa}B pathway. Second, a slow negative feedback where NF{kappa}-{kappa}B upregulates inhibitors acting at different levels along the IL-1R/IKK/NF-{kappa}B pathway - IL-1 receptor antagonist and A20 among others. A transient response ensues when the two feedbacks are balanced. When positive feedback is dominating over the negative islets transit into the persistent inflammation mode. Consistent with several observations, where the size of islets was implicated in its inflammatory state, we find that large islets and islets with high density of IL-1{beta} amplifying cells are more prone to transit into persistent IL-1{beta} mode. Our results are likely not limited to IL-1{beta} but general for the combined effect of multiple pro-inflammatory cytokines and chemokines. Generalizing complex regulations in terms of two feedbacks of opposing nature and acting on different time scales provides a number of testable predictions, which call for dynamic monitoring of pro-inflammatory cytokines at the single islet level. AUTHOR SUMMARYDifferent expression or activity dynamics of the same proteins or signaling molecules can lead to opposing fates in cells and tissues. While it is known that brief and prolonged exposure to pro-inflammatory cytokine IL-1{beta} have opposing effects on the functionality and viability of pancreatic {beta}-cells, it is unclear if and how these differences in dynamics may arise at the single islet level. We use a mathematical model of the core feedback loops in the IL-1{beta} regulatory network to show that transient and persistent responses are the two characteristic dynamic modes of the IL-1{beta} response. The likelihood of each mode depends on systemic inflammation and elevated glycaemia and free fatty acids levels. We find that large islets are more prone to transit into the persistent mode, which may provide an explanation for why large islets are underrepresented in type 2 diabetes patients.

biophysics↗

bioRxiv2023

Local changes in potassium ions modulate dendritic integration

During neuronal activity the extracellular concentration of potassium ions ([K+]o) increases substantially above resting levels, but it remains unclear what role these [K+]o changes play in dendritic integration of synaptic inputs. We used mathematical formulations and biophysical modeling to explore the role of activity-dependent K+ changes near dendritic segments of a visual cortex pyramidal neuron, receiving synaptic inputs tuned to stimulus orientation. We found that the fine-scale spatial arrangement of inputs dictates the magnitude of [K+]o changes around the dendrites: Dendritic segments with similarly-tuned inputs can attain substantially higher [K+]o increases than segments with diversely-tuned inputs. These [K+]o elevations in turn increase dendritic excitability, leading to more robust and prolonged dendritic spikes. Ultimately, these local effects amplify the gain of neuronal input-output transformations, causing higher orientation-tuned somatic firing rates without compromising orientation selectivity. Our results suggest that local activity-dependent [K+]o changes around dendrites may act as a "volume knob" that determines the impact of synaptic inputs on feature-tuned neuronal firing.

neuroscience↗

bioRxiv2022

Homeostatic changes maintain the gain control of spinal motoneurones across the lifetime of C57BL/6J mice

Age-related changes in the excitability of spinal motoneurone have been observed in mouse models of neurodegenerative diseases affecting these neurones. How the excitability of spinal motoneurones change with healthy ageing in mice and how this compares with that seen in neurodegenerative diseases is unknown. Therefore, we performed in vivo intracellular recording from identified spinal motoneurones in C57BL/6 mice at three different ages (100, 300-400 and 600-750 days old). Behavioral tests confirmed a linear reduction in motor function across these ages (using the rotorod test). Significant differences were observed with respect to the features of individual somatic action potential with ageing including a decreased rate of rise and fall in aged mice. Surprisingly, the rate of rise of the action potential at the initial segment was altered in middle aged mice. Immunohistochemical labelling of the axon initial segment of the motoneurones confirmed structural changes occurring at middle age (decreased length and diameter) but returning to the earlier parameters in aged mice. To explore the effects on repetitive firing, this was tested across the age groups which showed surprising little difference as the mice aged, with a similar rheobase and I-f gain across all age groups (with the exception of a lower voltage threshold for action potential initiation in middle-aged mice). However, amplitudes of the after-hyperpolarization and the input resistance were both found to be significantly altered with age. We conclude that there are changes occurring in the intrinsic properties of spinal motoneurones that control their excitability over the lifetime of mice, although these do not develop in a linear fashion from young to old. We propose that these changes are homeostatic in nature and are able to compensate for one another to maintain a constant gain control across the lifetime.

neuroscience↗

bioRxiv2021

Kismet/CHD7/CHD8 affects gut biomechanics, the gut microbiome, and gut-brain axis in Drosophila melanogaster

The gut-brain axis may contribute to the pathophysiology of neurodevelopmental disorders, yet it is often unclear how risk genes associated with these disorders affect gut physiology in a manner that could impact microbial colonization. We addressed this question using Drosophila melanogaster with a null mutation in kismet, the ortholog of chromodomain helicase DNA-binding protein (CHD) family members CHD7 and CHD8. In humans, CHD7 and CHD8 are risk genes for neurodevelopmental disorders with co-occurring gastrointestinal symptoms. We found kismet mutant flies have a significant increase in gastrointestinal transit time, indicating functional homology of kismet with CHD7/CHD8 in vertebrates. To measure gut tissue mechanics, we used a high-precision force transducer and length controller, capable of measuring forces to micro-Newton precision, which revealed significant changes in the mechanics of kismet mutant guts, in terms of elasticity, strain stiffening, and tensile strength. Using 16S rRNA metagenomic sequencing, we also found kismet mutants have reduced diversity of gut microbiota at every taxonomic level and an increase in pathogenic taxa. To investigate the connection between the gut microbiome and behavior, we depleted gut microbiota in kismet mutant and control flies and measured courtship behavior. Depletion of gut microbiota rescued courtship defects of kismet mutant flies, indicating a connection between gut microbiota and behavior. In striking contrast, depletion of gut microbiome in the control strain reduced courtship activity. This result demonstrated that antibiotic treatment can have differential impacts on behavior that may depend on the status of microbial dysbiosis in the gut prior to depletion. We propose that Kismet influences multiple gastrointestinal phenotypes that contribute to the gut-brain axis to influence behavior. Based on our results, we also suggest that gut tissue mechanics should be considered as an element in the gut-brain communication loop, both influenced by and potentially influencing the gut microbiome and neuronal development.

developmental biology↗

bioRxiv2020

Spatiotemporal model of cellular mechanotransduction via Rho and YAP

How cells sense and respond to mechanical stimuli remains an open question. Recent advances have identified the translocation of Yes-associated protein (YAP) between nucleus and cytoplasm as a central mechanism for sensing mechanical forces and regulating mechanotransduction. We formulate a spatiotemporal model of the mechanotransduction signalling pathway that includes coupling of YAP with the cell force-generation machinery through the Rho family of GTPases. Considering the active and inactive forms of a single Rho protein (GTP/GDP-bound) and of YAP (non-phosphorylated/phosphorylated), we study the cross-talk between cell polarization due to active Rho and YAP activation through its nuclear localization. For fixed mechanical stimuli, our model predicts stationary nuclear-to-cytoplasmic YAP ratios consistent with experimental data at varying adhesive cell area. We further predict damped and even sustained oscillations in the YAP nuclear-to-cytoplasmic ratio by accounting for recently reported positive and negative YAP-Rho feedback. Extending the framework to time-varying mechanical stimuli that simulate cyclic stretching and compression, we show that the YAP nuclear-to-cytoplasmic ratios time dependence follows that of the cyclic mechanical stimulus. The model presents one of the first frameworks for understanding spatiotemporal YAP mechanotransduction, providing several predictions of possible YAP localization dynamics, and suggesting new directions for experimental and theoretical studies.

biophysics↗

bioRxiv2020

Theoretical analysis predicts an optimal therapeutic strategy in distinct parkinsonian landscapes of the striatum

Parkinsons disease (PD) results from a loss of dopaminergic neurons. The age of disease onset, its progression and symptoms vary significantly between patients, pointing to a complex relationship between neuron loss and PD etiology. Yet, our understanding of the clinical variability remains incomplete. Here, we use biophysical modelling to characterize the dopaminergic landscape in the healthy and denervated striatum. Based on currently proposed mechanisms causing PD, we model three distinct denervation patterns, and show notable differences in the dopaminergic network as denervation progresses. We find local and global differences in the activity of two types of striatal neurons depending on the denervation pattern. Finally, we identify an optimal cellular strategy for maintaining normal dopamine signaling when neurons degenerate stochastically within our model. Our results derive a conceptual framework in which the clinical variability of PD is rooted in distinct denervation patterns and forms testable predictions for future PD research.

biophysics↗

bioRxiv2020

mRNA secondary structure stability regulates bacterial translation insulation and re-initiation

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure precisely localized downstream of stop codons, which serves as the main factor governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that in 95% of 128 analyzed bacterial genomes representing all phyla, this structure is selectively depleted when re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious.

molecular biology↗

bioRxiv2019

Hierarchical domain model explains multifractal scaling of chromosome contact maps

Modern biological techniques such as Hi-C permit to measure probabilities that different chromosomal regions are close in space. These probabilities can be visualised as matrices called contact maps. In this paper, we introduce a multifractal analysis of chromosomal contact maps. Our analysis reveals that Hi-C maps are bifractal, i.e. complex geometrical objects characterized by two distinct fractal dimensions. To rationalize this observation, we introduce a model that describes chromosomes as a hierarchical set of nested domains and we solve it exactly. The predicted multifractal spectrum is in excellent quantitative agreement with experimental data. Moreover, we show that our theory yields to a more robust estimation of the scaling exponent of the contact probability than existing methods. By applying this method to experimental data, we detect subtle conformational changes among chromosomes during differentiation of human stem cells.

biophysics↗

bioRxiv2019

Constraints on somite formation in developing embryos

Segment formation in vertebrate embryos is a stunning example of biological self-organisation. Here, we present an idealized model of the presomitic mesoderm (PSM) as a one-dimensional line of oscillators. We use the model to derive constraints that connect the size of somites, and the timing of their formation, to the growth of the PSM and the gradient of the somitogenesis clock period across the PSM. Our analysis recapitulates the observations made recently in ex-vivo cultures of mouse PSM cells, and makes predictions for how perturbations, such as increased Wnt levels, would alter somite widths. Finally, our model makes testable predictions for the shape of the phase profile and somite widths at different stages of PSM growth. In particular, we show that the phase profile is robustly concave when the PSM length is steady and slightly convex in an important special case when it is decreasing exponentially. In both cases, the phase profile scales with the PSM length; in the latter case, it scales dynamically. This has important consequences for the velocity of the waves that traverse the PSM and trigger somite formation, as well as the effect of errors in phase measurement on somite widths.

developmental biology↗

bioRxiv2017

Tuning of recombinant protein expression in Escherichia coli by manipulating transcription, translation initiation rates and incorporation of non-canonical amino acids

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the E. coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. Based on the results, we developed a biophysical model, which suggests that the density of co-transcriptional translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

synthetic biology↗

bioRxiv2016

Impact of Zygosity in Bimodal Phenotype Distributions

Allele number, or zygosity, is a clear determinant of gene expression in diploid cells. But the relationship between the number of copies of a gene and its expression can be hard to anticipate, especially when the gene in question is embedded in a regulatory circuit that contains feedbacks. Here we study this question making use of the natural genetic variability of human populations, which allows us to compare the expression profiles of a receptor protein in natural killer cells between donors infected with human cytomegalovirus (HCMV) with one or two copies of the allele. Crucially, the distribution of gene expression in many of the donors is bimodal, indicative of the presence of a positive feedback somewhere in the regulatory environment of the gene. Three separate gene-circuit models differing in the location of the positive feedback with respect to the gene can all reproduce well the homozygous data. However, when the resulting fitted models are applied to the hemizygous donors, only one model (the one with the positive feedback located at the level of gene transcription) reproduces the experimentally observed gene-expression profile. In that way, our work shows that zygosity can help us relate structure and function of gene regulatory networks.\n\nAuthor SummaryNearly all mammalian cells, including human cells, have two copies of each chromosome, and thus possess two potentially different copies of each gene (which might be in some cases non-functional or even absent). Naiively one might expect that two identical copies of the gene would lead to the protein being expressed at twice the rate, but many factors can alter this simple calculation. One of these factors is the existence of feedback mechanisms affecting in one way or another the regulatory circuit in which our gene of interest is embedded. Here we study the relationship between the number of gene copies and the expression of a receptor protein that plays a crucial role in the recognition of pathogens by natural killer cells, which are important elements of the innate immune system. Experimental data of virus-infected donors reveals a bimodal expression profile of this receptor, typical of a positive feedback, and a clear difference between donors with one or two copies of the gene. Mathematical modeling allows us to find the likely location of the feedback loop within the genes regulatory circuit, by requiring the correct model to reproduce the expression profiles of both types of donors.

systems biology↗