In eukaryotes, the expression of specific genes is regulated by a combination of transcription factors (TFs) bound on regulatory regions of the genomic DNA (promoters and enhancers). Recent advances in genomic sequencing technology have enabled the measurements of TFs' footprints and binding affinities on DNA at the single-molecule level, facilitating the probing of binding cooperativity among adjacent TFs. This necessitates quantitative descriptions of TFs' binding cooperativity and understanding of its potential functional relevance. In this study, we show that the binding cooperativities between two adjacent TFs can be quantified by the [Formula: see text] coefficient, which can be experimentally determined. Under thermodynamic equilibrium, the binding affinities of two TFs can either increase together (positive cooperativity) or decrease together (negative cooperativity), but not in opposing directions (one increases while the other decreases). Within the framework of thermodynamics, we investigate the functional relevance of cooperativity. The functional relevance of positive cooperativity, which has been extensively discussed in the literature, is the sigmoidal binding curve around a TF concentration threshold (analogous to oxygen binding to hemoglobin), whereas the functional relevance of negative cooperativity is twofold. First, mutual exclusion of the two TFs enables bidirectional gene switching, akin to the CI-Cro system in phage [Formula: see text]. Second, while TFs often exhibit intranuclear concentration fluctuations, negative binding cooperativity assures fast TF dissociation from DNA and hence rapid response for gene expression regulation. Furthermore, the nonequilibrium steady states of living cells can lead to either positive or negative cooperativity, which can also be quantified by the [Formula: see text] coefficient.

Questions in cancer have engaged systems biologists for decades. During that time, the quantity of molecular data has exploded, but the need for abstractions, formal models, and simplifying insights has remained the same. This review brings together classic breakthroughs and recent findings in the field of cancer systems biology, focusing on cancer cell pathways for tumorigenesis and therapeutic response. Cancer cells mutate and transduce information from their environment to alter gene expression, metabolism, and phenotypic states. Understanding the molecular architectures that make each of these steps possible is a long-term goal of cancer systems biology pursued by iterating between quantitative models and experiments. We argue that such iteration is the best path to deploying targeted therapies intelligently so that each patient receives the maximum benefit for their cancer.

NF-κB is a crucial transcription factor in lymphocyte signaling. It is activated by environmental cues that drive lymphocyte differentiation to combat infections and cancer. As a key player in inflammation, NF-κB also significantly impacts autoimmunity and transplant rejection, making it an important therapeutic target. While the signaling molecules regulating this pathway are well-studied, the effect of changes in NF-κB signaling levels on T lymphocyte differentiation, fate, and function is not fully understood. Advances in computational biology and new NF-κB-inducible animal models are beginning to clarify these questions. In this review, we highlight recent findings related to T cells, focusing on how environmental cues affecting NF-κB signaling levels determine T cell fate and function.

Transcription factors relay information from the external environment to gene regulatory networks that control cell physiology. To confer signalling specificity, robustness and coordination, these signalling networks use temporal communication codes, such as the amplitude, duration or frequency of signals. Although much is known about how temporal information is encoded, a mechanistic understanding of how gene regulatory networks decode signalling dynamics is lacking. Recent advances in our understanding of phase separation of transcriptional condensates provide new biophysical frameworks for both temporal encoding and decoding mechanisms. In this Perspective, we summarize the mechanisms by which transcriptional condensates could enable temporal decoding through signal adaptation, memory and persistence. We further outline methods to probe and manipulate dynamic communication codes of transcription factors and condensates to rationally control gene activation.

As immune sentinel cells, macrophages are required to respond specifically to diverse immune threats and initiate appropriate immune responses. This stimulus-response specificity (SRS) is in part encoded in the signaling dynamics of the NFκB transcription factor. While experimental stimulus-response studies have typically focused on single defined ligands, in physiological contexts cells are exposed to multi-ligand mixtures. It remains unclear how macrophages combine multi-ligand information and whether they are able to maintain SRS in such complex exposure conditions. Here, we leveraged an established mathematical model that captures the heterogeneous single-cell NFκB responses of macrophage populations to extend experimental studies with systematic simulations of complex mixtures containing up to five ligands. Live-cell microscopy experiments for some conditions validated model predictions but revealed a discrepancy when TLR3 and TLR9 are stimulated. Refining the model suggested that the observed but unexpected ligand antagonism arises from a limited capacity for endosomal transport which is required for responses to CpG and pIC. With the updated model, we systematically analyzed SRS across all combinatorial-ligand conditions and employed three ways of quantifying SRS involving trajectory decomposition into informative trajectory features or machine learning. Our findings show that macrophages most effectively distinguish single-ligand stimuli, and distinguishability declines as more ligands are combined. However, even in complex combinatorial conditions, macrophages still maintain statistically significant distinguishability. These results indicate a robustness of innate immune response specificity: even in the context of complex exposure conditions, the NFκB temporal signaling code of macrophages can still classify immune threats to direct an appropriate response.

Macrophages sense diverse pathogens within complex environments and respond appropriately. Experimental studies have found that the NFκB pathway responds with stimulus-specific dynamics when macrophages are exposed to single ligand stimuli. However, it remains unclear complex contexts might erode this stimulus-specificity. Here we systematically studies NFκB responses using a mathematical model that provides simulations of the heterogeneous population of single cell responses. We show that although the model is parameterized to single ligand data it can predict the responses to multi-ligand mixtures. Indeed, model validation uncovered signaling antagonism between two ligands and the underlying mechanism. Importantly, we found that NFκB signaling dynamics distinguish ligands within multi-ligand mixtures indicating a robustness of the NFκB temporal code that was not previously appreciated.

Natural killer (NK) cells possess both innate and adaptive features. Here, we investigated NK cell activation across tissues during cytomegalovirus infection, which generates antigen-specific clonal expansion and long-lived memory responses. Longitudinal tracking and single-cell RNA sequencing of NK cells following infection revealed enhanced activation in the spleen, as well as early formation of a CD69lo precursor population that preferentially gave rise to adaptive NK cells. Splenic NK cells demonstrated heightened tumor necrosis factor alpha (TNF-α) signaling and increased expression of the receptor TNFR2, which coincided with elevated TNF-α production by splenic myeloid cells. TNFR2-deficient NK cells exhibited impaired interferon gamma (IFN-γ) production and expansion. TNFR2 signaling engaged two distinct nuclear factor κB (NF-κB) signaling arms-innate effector NK cell responses required canonical NF-κB signaling, whereas non-canonical NF-κB signaling enforced differentiation of CD69lo adaptive NK cell precursors. Thus, NK cell priming in the spleen during viral infection promotes an innate-to-adaptive transition, providing insight into avenues for generating adaptive NK cell immunity across diverse settings.

Postoperative cognitive dysfunction (POCD) impacts a significant number of patients annually, frequently impairing their cognitive abilities and resulting in unfavorable clinical outcomes. Aimed at addressing cognitive impairment, vagus nerve stimulation (VNS) is a therapeutic approach, which was used in many mental disordered diseases, through the modulation of vagus nerve activity. In POCD model, the enhancement of cognition function provided by VNS was shown, demonstrating VNS effect on cognition in POCD. In the present study, we primarily concentrates on elucidating the role of the VNS improving the cognitive function in POCD, via two potential mechanisms: the inflammatory microenvironment and epigenetics. This study provided a theoretical support for the feasibility that VNS can be a potential method to enhance cognition function in POCD.

A method to photomodulate dynamically transient DNA-based reaction circuits and networks is introduced. The method relies on the integration of photoresponsive o-nitrobenzyl-phosphate ester-caged DNA hairpin with a "mute" reaction module. Photodeprotection (λ=365 nm) of the hairpin structure separates a fuel strand triggering the dynamic, transient, operation of the DNA circuit/network. By temporal photocleavage of the hairpin within the course of transient operation of the circuit, photomodulation of the systems are demonstrated. The modulation amplitude and rhythms are controlled by the time-interval and cycle numbers of photo-deprotecting the hairpin structure. The method is applied to transiently photomodulate the catalytic activities of a DNAzyme, enabling the photomodulation of the transient assembly of a constitutional dynamic network (CDN) and the transient reconfiguration of the CDN framework. The different systems are supported by computational kinetic models allowing to predict, and experimentally validate, the behavior of the systems under variable auxiliary conditions. Moreover, the photomodulated transient CDNs are implemented as functional frameworks guiding the thrombin-catalyzed coagulation of fibrinogen to fibrin (fibrinogenesis) and photomodulated operation of a biocatalytic cascade.

Cells process dynamic signaling inputs to regulate fate decisions during development. While oscillations or waves in key developmental pathways, such as Wnt, have been widely observed, the principles governing how cells decode these signals remain unclear. By leveraging optogenetic control of the Wnt signaling pathway in both HEK293T cells and H9 human embryonic stem cells, we systematically map the relationship between signal frequency and downstream pathway activation. We find that cells exhibit a minimal response to Wnt at certain frequencies, a behavior we term anti-resonance. We developed both detailed biochemical and simplified hidden variable models that explain how anti-resonance emerges from the interplay between fast and slow pathway dynamics. Remarkably, we find that frequency directly influences cell fate decisions involved in human gastrulation; signals delivered at anti-resonant frequencies result in dramatically reduced mesoderm differentiation. Our work reveals a previously unknown mechanism of how cells decode dynamic signals and how anti-resonance may filter against spurious activation. These findings establish new insights into how cells decode dynamic signals with implications for tissue engineering, regenerative medicine, and cancer biology.

Decades of research have established that mammalian transcription factors (TFs) bind to each gene's regulatory regions and cooperatively control tissue specificity, timing, and intensity of gene transcription. Mapping the combination of TF binding sites genome wide is critically important for understanding functional genomics. Here, we report a technique to measure TFs' binding sites on the human genome with a near single-base resolution by footprinting with deaminase (FOODIE) on a single-molecule and single-cell basis. Single-molecule sequencing reads after enzymatic deamination allow detection of the TF binding fraction on a particular footprint and the binding cooperativity of any two adjacent TFs, which can be either positive or negative. As a newcomer of single-cell genomics, single-cell FOODIE enables the detection of cell-type-specific TF footprints in a pure cell population in a heterogeneous tissue, such as the brain. We found that genes carrying out a certain biological function together in a housing-keeping correlated gene module (CGM) or a tissues-specific CGM are coordinated by shared TFs in the gene's promoters and enhancers, respectively. Scalable and cost-effective, FOODIE allows us to create an open FOODIE database for cell lines, with applicability to human tissues and clinical samples.

Cellular processes are dynamic and often oscillatory, requiring precise coordination for optimal cell function.1,2,3,4,5,6,7 How distinct oscillatory processes can couple within a single cell remains an open question. Here, we use the cyanobacterial circadian clock8,9 as a model system to explore the coupling of oscillatory and pulsatile gene circuits. The cyanobacterial circadian clock generates 24-h oscillations in downstream targets10,11,12,13,14,15 to time processes across the day/night cycle.9,16,17,18,19,20,21,22 This timing is partly mediated by the clock's modulation of the activity of alternative sigma factors,14,23,24,25 which direct RNA polymerase to specific promoters.26 Using single-cell time-lapse microscopy and modeling, we find that the clock modulates the amplitude of expression pulses of the alternative sigma factor RpoD4, which occurs only at cell division. This pulse amplitude modulation (PAM), analogous to AM regulation in radio transmission,27 allows the clock to robustly generate a 24-h rhythm in rpoD4 expression despite rpoD4's pulsing frequency being non-circadian. By modulating cell division rates, we find that, as predicted by our model, PAM regulation generates the same 24-h period in rpoD4 pulse amplitude over a range of rpoD4 pulse frequencies. Furthermore, we identify a functional significance of rpoD4 expression levels: deletion of rpoD4 results in smaller cell sizes, whereas an increase in rpoD4 expression leads to larger cell sizes in a dose-dependent manner. Thus, our work reveals a link between the cell cycle, clock, and RpoD4 in cyanobacteria and suggests that PAM regulation can be a general mechanism for biological clocks to robustly modulate pulsatile downstream processes.

RNA translation to protein is paramount to creating life, yet RNA and protein correlations vary widely across tissues, cells, and species. To investigate these perplexing results, we utilize a time-series fixation method that combines static stimulation and a programmable formaldehyde perfusion to map pseudo-Signaling with Omics signatures (pSigOmics) of single-cell data from hundreds of thousands of cells. Using the widely studied nuclear factor kappa B (NFκB) mammalian signaling pathway in mouse fibroblasts, we discovered a novel asynchronous pseudotime regulation (APR) between RNA and protein levels in the quintessential NFκB p65 protein using single molecule spatial imaging. Prototypical NFκB dynamics are successfully confirmed by the rise and fall of NFκB response as well as A20 negative inhibitor activity by 90 min. The observed p65 translational APR is evident in both statically sampled timepoints and dynamic response gradients from programmable formaldehyde fixation, which successfully creates continuous response measurements. Finally, we implement a graph neural network model capable of predicting APR cell subpopulations from GAPDH RNA spatial expression, which is strongly correlated with p65 RNA signatures. Successful decision tree classifiers on Potential of Heat-diffusion for Affinity-based Trajectory Embedding embeddings of our data, which illustrate partitions of APR cell subpopulations in latent space, further confirm the APR patterns. Together, our data suggest an RNA-protein regulatory framework in which translation adapts to signaling events and illuminates how immune signaling is timed across various cell subpopulations.

Biological oscillators can specify time- and dose-dependent functions via dedicated control of their oscillatory dynamics. However, how biological oscillators, which recurrently activate noisy biochemical processes, achieve robust oscillations remains unclear. Here, we characterize the long-term oscillations of p53 and its negative feedback regulator Mdm2 in single cells after DNA damage. Whereas p53 oscillates regularly, Mdm2 from a single MDM2 allele exhibits random unresponsiveness to ∼9% of p53 pulses. Using allelic-specific imaging of MDM2 activity, we show that MDM2 alleles buffer each other to maintain p53 pulse amplitude. Removal of MDM2 allelic buffering cripples the robustness of p53 amplitude, thereby elevating p21 levels and cell-cycle arrest. In silico simulations support that allelic buffering enhances the robustness of biological oscillators and broadens their plausible biochemical space. Our findings show how allelic buffering ensures robust p53 oscillations, highlighting the potential importance of allelic buffering for the emergence of robust biological oscillators during evolution. A record of this paper's transparent peer review process is included in the supplemental information.

The tumor suppressor p53 responds to cellular stress and activates transcription programs critical for regulating cell fate. DNA damage triggers oscillations in p53 levels with a robust period. Guided by the theory of synchronization and entrainment, we developed a mathematical model and experimental system to test the ability of the p53 oscillator to entrain to external drug pulses of various periods and strengths. We found that the p53 oscillator can be locked and entrained to a wide range of entrainment modes. External periods far from p53's natural oscillations increased the heterogeneity between individual cells whereas stronger inputs reduced it. Single-cell measurements allowed deriving the phase response curves (PRCs) and multiple Arnold tongues of p53. In addition, multi-stability and non-linear behaviors were mathematically predicted and experimentally detected, including mode hopping, period doubling, and chaos. Our work revealed critical dynamical properties of the p53 oscillator and provided insights into understanding and controlling it. A record of this paper's transparent peer review process is included in the supplemental information.

Temporal properties of molecules within signaling networks, such as sub-cellular changes in protein abundance, encode information that mediate cellular responses to stimuli. How dynamic signals relay and process information is a critical gap in understanding cellular behaviors. In this work, we investigate transmission of information about changing extracellular cytokine concentrations from receptor-level supramolecular assemblies of IκB kinases (IKK) downstream to the nuclear factor κB (NF-κB) transcription factor (TF). In a custom robot-controlled microfluidic cell culture, we simultaneously measure input-output (I/O) encoding of IKK-NF-κB in dual fluorescent-reporter cells. When compared with single cytokine pulses, dose-conserving pulse trains prolong IKK assemblies and lead to disproportionately enhanced retention of nuclear NF-κB. Using particle swarm optimization, we demonstrate that a mechanistic model does not recapitulate this emergent property. By contrast, invoking mechanisms for NF-κB-dependent chromatin remodeling to the model recapitulates experiments, showing how temporal dosing that prolongs IKK assemblies facilitates switching to permissive chromatin that sequesters nuclear NF-κB. Remarkably, using simulations to resolve single-cell receptor data accurately predicts same-cell NF-κB time courses for more than 80% of our single cell trajectories. Our data and simulations therefore suggest that cell-to-cell heterogeneity in cytokine responses are predominantly due to mechanisms at the level receptor-associated protein complexes.

Most gene functions have been discovered through phenotypic observations under loss of function experiments that lack temporal control. However, cell signaling relies on limited transcriptional effectors, having to be re-used temporally and spatially within the organism. Despite that, the dynamic nature of signaling pathways have been overlooked due to the difficulty on their assessment, resulting in important bottlenecks. Here, we have utilized the rapid and synchronized developmental transitions occurring within the zebrafish embryo, in conjunction with custom NF-kB reporter embryos driving destabilized fluorophores that report signaling dynamics in real time. We reveal that NF-kB signaling works as a clock that controls the developmental progression of hematopoietic stem and progenitor cells (HSPCs) by two p65 activity waves that inhibit cell cycle. Temporal disruption of each wave results in contrasting phenotypic outcomes: loss of HSPCs due to impaired specification versus proliferative expansion and failure to delaminate from their niche. We also show functional conservation during human hematopoietic development using iPSC models. Our work identifies p65 as a previously unrecognized contributor to cell cycle regulation, revealing why and when pro-inflammatory signaling is required during HSPC development. It highlights the importance of considering and leveraging cell signaling as a temporally dynamic entity.

Insulin-like growth factor (IGF)-I mediates long-term activities that determine cell fate, including cell proliferation and differentiation. This study aimed to characterize the mechanisms by which IGF-I determines cell fate from the aspect of IGF-I signaling dynamics. In L6 myoblasts, myogenic differentiation proceeded under low IGF-I levels, whereas proliferation was enhanced under high levels. Mathematical and experimental analyses revealed that IGF-I signaling oscillated at low IGF-I levels but remained constant at high levels, suggesting that differences in IGF-I signaling dynamics determine cell fate. We previously reported that differential insulin receptor substrate (IRS)-1 levels generate a driving force for cell competition. Computational simulations and immunofluorescence analyses revealed that asynchronous IRS-1 protein oscillations were synchronized during myogenic processes through cell competition. Disturbances of cell competition impaired signaling synchronization and cell fusion, indicating that synchronization of IGF-I signaling oscillation is critical for myoblast cell fusion to form multinucleate myotubes.

Breast cancer (BC) is one of the primary causes of death in women worldwide. The challenges associated with adverse outcomes have increased significantly, and the identification of novel therapeutic targets has become increasingly urgent. Regulated cell death (RCD) refers to a type of cell death that can be regulated by several different biomacromolecules, which is distinctive from accidental cell death (ACD). In recent years, apoptosis, a representative RCD pathway, has gained significance as a target for BC medications. However, tumor cells exhibit avoidance of apoptosis and result in treatment resistance, which emphasizes further studies devoted to alternative cell death processes, namely necroptosis, pyroptosis, and ferroptosis. Here, in this review, we focus on summarizing the crucial signaling pathways of these RCD in BC. We further discuss the molecular mechanism and potentiality in clinical application of several prospective drugs, nanoparticles, and other small compounds targeting different RCD subroutines of BC. We also discuss the benefits of modulating RCD processes on drug resistance and the advantages of combining RCD modulators with conventional treatments in BC. This review will deepen our understanding of the relationship between RCD and BC, and shed new light on future directions to attack cancer vulnerabilities with RCD modulators for therapeutic purposes.

Kynurenic acid (KYNA) is an antioxidant degradation product of tryptophan that has been shown to have a variety of cytoprotective, neuroprotective and neuronal signalling properties. However, mammalian transporters and receptors display micromolar binding constants; these are consistent with its typically micromolar tissue concentrations but far above its serum/plasma concentration (normally tens of nanomolar), suggesting large gaps in our knowledge of its transport and mechanisms of action, in that the main influx transporters characterized to date are equilibrative, not concentrative. In addition, it is a substrate of a known anion efflux pump (ABCC4), whose in vivo activity is largely unknown. Exogeneous addition of L-tryptophan or L-kynurenine leads to the production of KYNA but also to that of many other co-metabolites (including some such as 3-hydroxy-L-kynurenine and quinolinic acid that may be toxic). With the exception of chestnut honey, KYNA exists at relatively low levels in natural foodstuffs. However, its bioavailability is reasonable, and as the terminal element of an irreversible reaction of most tryptophan degradation pathways, it might be added exogenously without disturbing upstream metabolism significantly. Many examples, which we review, show that it has valuable bioactivity. Given the above, we review its potential utility as a nutraceutical, finding it significantly worthy of further study and development.

The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in pancreatic ductal adenocardinoma (PDAC) tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single-cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single-cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC.

Tumor necrosis factor-α (TNFα) is a master cytokine which induces expression of chemokines and adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in endothelial cells to initiate the vascular inflammatory response. In this study, we identified neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, as a modulator of TNFα-induced inflammatory response of endothelial cells. NRP1 shRNA expression suppressed TNFα-stimulated leukocyte adhesion and expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs). Likewise, it reduced TNFα-induced phosphorylation of MAPK p38 but did not significantly affect other TNF-induced signaling pathways, such as the classical NFκB and the AKT pathway. Immunofluorescent staining demonstrated co-localization of NRP1 with the two receptors of TNF, TNFR1 and TNFR2. Co-immunoprecipitation further confirmed that NRP1 was in the same protein complex or membrane compartment as TNFR1 and TNFR2, respectively. Modulation of NRP1 expression, however, neither affected TNFR levels in the cell membrane nor the receptor binding affinities of TNFα. Although a direct interface between NRP1 and TNFα/TNFR1 appeared possible from a protein docking model, a direct interaction was not supported by binding assays in cell-free microplates and cultured cells. Furthermore, TNFα was shown to downregulate NRP1 in a time-dependent manner through TNFR1-NFκB pathway in HUVECs. Taken together, our study reveals a novel reciprocal crosstalk between NRP1 and TNFα in vascular endothelial cells.

The functional state of cells is dependent on their microenvironmental context. Prior studies described how polarizing cytokines alter macrophage transcriptomes and epigenomes. Here, we characterized the functional responses of 6 differentially polarized macrophage populations by measuring the dynamics of transcription factor nuclear factor κB (NF-κB) in response to 8 stimuli. The resulting dataset of single-cell NF-κB trajectories was analyzed by three approaches: (1) machine learning on time-series data revealed losses of stimulus distinguishability with polarization, reflecting canalized effector functions. (2) Informative trajectory features driving stimulus distinguishability ("signaling codons") were identified and used for mapping a cell state landscape that could then locate macrophages conditioned by an unrelated condition. (3) Kinetic parameters, inferred using a mechanistic NF-κB network model, provided an alternative mapping of cell states and correctly predicted biochemical findings. Together, this work demonstrates that a single analyte's dynamic trajectories may distinguish the functional states of single cells and molecular network states underlying them. A record of this paper's transparent peer review process is included in the supplemental information.

Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Guanylate-binding proteins (GBPs) are a family of interferon (IFN)-inducible GTPases and play a pivotal role in the host immune response to microbial infections. These are upregulated in immune cells after recognizing the lipopolysaccharides (LPS), the major membrane component of Gram-negative bacteria. In the present study, the expression pattern of GBP1-7 was initially mapped in phorbol 12-myristate 13-acetate-differentiated human monocytes THP-1 and mouse macrophages RAW 264.7 cell lines stimulated with LPS. A time-dependent significant expression of GBP1-7 was observed in these cells. Moreover, among the various GBPs, GBP1 has emerged as a central player in regulating innate immunity and inflammation. Therefore, to study the specific role of GBP1 in LPS-induced inflammation, knockdown of the Gbp1 gene was carried out in both cells using small interfering RNA interference. Altered levels of different cytokines (interleukin [IL]-4, IL-10, IL-12β, IFN-γ, tumor necrosis factor-α), inducible nitric oxide synthase, histocompatibility 2, class II antigen A, protein kinase R, and chemokines (chemokine (C-X-C motif) ligand 9 [CXCL9], CXCL10, and CXCL11) in GBP1 knockdown cells were reported compared to control cells. Interestingly, the extracellular-signal-regulated kinase 1/2 mitogen-activated protein (MAP) kinases and signal transducer and activator of transcription 1 (STAT1) transcription factor levels were considerably induced in knockdown cells compared to the control cells. However, no change in the level of phosphorylated nuclear factor-kB, c-Jun, and p38 transcription factors was observed in GBP1 knockdown cells compared to the control cells. This study concludes that GBP1 may alter the expression of cytokines, chemokines, and effector molecules mediated by MAP kinases and STAT1 transcription factors.

Circadian rhythms are generated by complex interactions among genes and proteins. Self-sustained ∼24 h oscillations require negative feedback loops and sufficiently strong nonlinearities that are the product of molecular and network switches. Here, we review common mechanisms to obtain switch-like behavior, including cooperativity, antagonistic enzymes, multisite phosphorylation, positive feedback, and sequestration. We discuss how network switches play a crucial role as essential components in cellular circadian clocks, serving as integral parts of transcription-translation feedback loops that form the basis of circadian rhythm generation. The design principles of network switches and circadian clocks are illustrated by representative mathematical models that include bistable systems and negative feedback loops combined with Hill functions. This work underscores the importance of negative feedback loops and network switches as essential design principles for biological oscillations, emphasizing how an understanding of theoretical concepts can provide insights into the mechanisms generating biological rhythms.

Glioblastoma Multiforme is a brain tumor distinguished by its aggressiveness. We suggested that this aggressiveness leads single-cell RNA-sequence data (scRNA-seq) to span a representative portion of the cancer attractors domain. This conjecture allowed us to interpret the scRNA-seq heterogeneity as reflecting a representative trajectory within the attractor's domain. We considered factors such as genomic instability to characterize the cancer dynamics through stochastic fixed points. The fixed points were derived from centroids obtained through various clustering methods to verify our method sensitivity. This methodological foundation is based upon sample and time average equivalence, assigning an interpretative value to the data cluster centroids and supporting parameters estimation. We used stochastic simulations to reproduce the dynamics, and our results showed an alignment between experimental and simulated dataset centroids. We also computed the Waddington landscape, which provided a visual framework for validating the centroids and standard deviations as characterizations of cancer attractors. Additionally, we examined the stability and transitions between attractors and revealed a potential interplay between subtypes. These transitions might be related to cancer recurrence and progression, connecting the molecular mechanisms of cancer heterogeneity with statistical properties of gene expression dynamics. Our work advances the modeling of gene expression dynamics and paves the way for personalized therapeutic interventions.

Macrophages are tissue resident innate phagocytic cells that take on contrasting phenotypes, or polarization states, in response to the changing combination of microbial and cytokine signals at sites of infection. During the opening stages of an infection, macrophages adopt the proinflammatory, highly antimicrobial M1 state, later shifting to an anti-inflammatory, pro-tissue repair M2 state as the infection resolves. The changes in gene expression underlying these transitions are primarily governed by nuclear factor kappaB (NF-κB), Janus kinase (JAK)/signal transducer and activation of transcription (STAT), and hypoxia-inducible factor 1 (HIF1) transcription factors, the activity of which must be carefully controlled to ensure an effective yet spatially and temporally restricted inflammatory response. While much of this control is provided by pathway-specific feedback loops, recent work has shown that the transcriptional co-regulators of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxy-terminal domain (CITED) family serve as common controllers for these pathways. In this review, we describe how CITED proteins regulate polarization-associated gene expression changes by controlling the ability of transcription factors to form chromatin complexes with the histone acetyltransferase, CBP/p300. We will also cover how differences in the interactions between CITED1 and 2 with CBP/p300 drive their contrasting effects on pro-inflammatory gene expression.

Individual cell sensing of external cues has evolved through the temporal patterns in signaling. Since nuclear factor κB (NF-κB) signaling dynamics have been examined using a single subunit, RelA, it remains unclear whether more information might be transmitted via other subunits. Using NF-κB double-knockin reporter mice, we monitored both canonical NF-κB subunits, RelA and c-Rel, simultaneously in single macrophages by quantitative live-cell imaging. We show that signaling features of RelA and c-Rel convey more information about the stimuli than those of either subunit alone. Machine learning is used to predict the ligand identity accurately based on RelA and c-Rel signaling features without considering the co-activated factors. Ligand discrimination is achieved through selective non-redundancy of RelA and c-Rel signaling dynamics, as well as their temporal coordination. These results suggest a potential role of c-Rel in fine-tuning immune responses and highlight the need for approaches that will elucidate the mechanisms regulating NF-κB subunit specificity.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 μM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1β, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

Cellular responses to TNF are inherently heterogeneous within an isogenic cell population and across different cell types. TNF promotes cell survival by activating pro-inflammatory NF-κB and MAPK signalling pathways but may also trigger apoptosis and necroptosis. Following TNF stimulation, the fate of individual cells is governed by the balance of pro-survival and pro-apoptotic signalling pathways. To elucidate the molecular mechanisms driving heterogenous responses to TNF, quantifying TNF/TNFR1 signalling at the single-cell level is crucial. Fluorescence live-cell imaging techniques offer real-time, dynamic insights into molecular processes in single cells, allowing for detection of rapid and transient changes, as well as identification of subpopulations, that are likely to be missed with traditional endpoint assays. Whilst fluorescence live-cell imaging has been employed extensively to investigate TNF-induced inflammation and TNF-induced cell death, it has been underutilised in studying the role of TNF/TNFR1 signalling pathway crosstalk in guiding cell-fate decisions in single cells. Here, we outline the various opportunities for pathway crosstalk during TNF/TNFR1 signalling and how these interactions may govern heterogenous responses to TNF. We also advocate for the use of live-cell imaging techniques to elucidate the molecular processes driving cell-to-cell variability in single cells. Understanding and overcoming cellular heterogeneity in response to TNF and modulators of the TNF/TNFR1 signalling pathway could lead to the development of targeted therapies for various diseases associated with aberrant TNF/TNFR1 signalling, such as rheumatoid arthritis, metabolic syndrome, and cancer.

Design patterns are generalized solutions to frequently recurring problems. They were initially developed by architects and computer scientists to create a higher level of abstraction for their designs. Here, we extend these concepts to cell biology to lend a new perspective on the evolved designs of cells' underlying reaction networks. We present a catalog of 21 design patterns divided into three categories: creational patterns describe processes that build the cell, structural patterns describe the layouts of reaction networks, and behavioral patterns describe reaction network function. Applying this pattern language to the E. coli central metabolic reaction network, the yeast pheromone response signaling network, and other examples lends new insights into these systems.

The NF-κB protein p65/RelA plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA regulates gene transcription and alternative splicing through promoter enrichment and genomic exon occupancy, respectively. The intricate ways in which p65/RelA simultaneously governs these functions across various genes remain to be fully elucidated. In this study, we employed the HTLV-1 Tax oncoprotein, a potent activator of NF-κB, to investigate its influence on the three-dimensional organization of the genome, a key factor in gene regulation. We discovered that Tax restructures the 3D genomic landscape, bringing together genes based on their regulation and splicing patterns. Notably, we found that the Tax-induced gene-gene contact between the two master genes NFKBIA and RELA is associated with their respective changes in gene expression and alternative splicing. Through dCas9-mediated approaches, we demonstrated that NFKBIA-RELA interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on RELA. Our findings shed light on new regulatory mechanisms upon HTLV-1 Tax and underscore the integral role of p65/RelA in coordinated regulation of NF-κB-responsive genes at both transcriptional and splicing levels in the context of the 3D genome.

Cancer remains the major cause of morbidity and mortality. The nuclear factor kappa-B (NF- κB) plays an indispensable role in cancer cell proliferation and drug resistance. The role of NF-κB is not only limited to tumor cell proliferation and suppression of apoptotic genes but it also induces EMT transition responsible for metastasis. Inhibition of the NF-κB pathway in cancer cells by herbal derivatives makes it a favorable yet promising target for cancer therapeutics.

The purpose of the study is to explore the inhibition potential of Nimbin and its analogs against NF-κB subunits p50 and p65.

In the present study, an herbal compound Nimbin and its derivative analogs were investigated to examine their impact on the p50 and p65 subunits of the NF-κB signaling pathway using in silico tools, namely molecular docking and simulation.

The molecular docking analysis revealed that Nimbin and its analogs may bind to p50 and p65 subunits with dG bind values ranging from -33.23 to -50.49 Kcal/mol. Interestingly, molecular dynamic simulation for the NO5-p65 complex displayed a stable conformation and convergence when compared to the NO4-p50 complex.

These results indicate that NO5 may have a potential inhibitory effect against NF-κB subunit p65, which needs to be further validated in in vitro and in vivo systems. Also, the results obtained emphasize and pave the way for exploring the Nimbin scaffold against NF-κB inhibition for cancer therapeutics.

Transcription factor dynamics is fundamental to determine the activation of accurate transcriptional programs and yet is heterogeneous at a single-cell level, even within homogeneous populations. We asked how such heterogeneity emerges for the nuclear factor κB (NF-κB). We found that clonal populations of immortalized fibroblasts derived from a single mouse embryo display robustly distinct NF-κB dynamics upon tumor necrosis factor ɑ (TNF-ɑ) stimulation including persistent, oscillatory, and weak activation, giving rise to differences in the transcription of its targets. By combining transcriptomics and simulations we show how less than two-fold differences in the expression levels of genes coding for key proteins of the signaling cascade and feedback system are predictive of the differences of the NF-κB dynamic response of the clones to TNF-ɑ and IL-1β. We propose that small transcriptional differences in the regulatory circuit of a transcription factor can lead to distinct signaling dynamics in cells within homogeneous cell populations and among different cell types.

Oscillatory behavior is critical to many life sustaining processes such as cell cycles, circadian rhythms, and notch signaling. Important biological functions depend on the characteristics of these oscillations (hereafter, oscillation characteristics or OCs): frequency (e.g., event timings), amplitude (e.g., signal strength), and phase (e.g., event sequencing). Numerous oscillating reaction networks have been documented or proposed. Some investigators claim that oscillations in reaction networks require nonlinear dynamics in that at least one rate law is a nonlinear function of species concentrations. No one has shown that oscillations can be produced for a reaction network with linear dynamics. Further, no one has obtained closed form solutions for the frequency, amplitude and phase of any oscillating reaction network. Finally, no one has published an algorithm for constructing oscillating reaction networks with desired OCs.

This is a theoretical study that analyzes reaction networks in terms of their representation as systems of ordinary differential equations. Our contributions are: (a) construction of an oscillating, two species reaction network [two species harmonic oscillator (2SHO)] that has no nonlinearity; (b) obtaining closed form formulas that calculate frequency, amplitude, and phase in terms of the parameters of the 2SHO reaction network, something that has not been done for any published oscillating reaction network; and (c) development of an algorithm that parameterizes the 2SHO to achieve desired oscillation, a capability that has not been produced for any published oscillating reaction network.

Our 2SHO demonstrates the feasibility of creating an oscillating reaction network whose dynamics are described by a system of linear differential equations. Because it is a linear system, we can derive closed form expressions for the frequency, amplitude, and phase of oscillations, something that has not been done for other published reaction networks. With these formulas, we can design 2SHO reaction networks to have desired oscillation characteristics. Finally, our sensitivity analysis suggests an approach to constructing a 2SHO for a biochemical system.

Cells at the site of an infection experience numerous biochemical signals that vary in amplitude, space, and time. Despite the diversity of dynamic signals produced by pathogens and sentinel cells, information-processing pathways converge on a limited number of central signaling nodes to ultimately control cellular responses. In particular, the NF-κB pathway responds to dozens of signals from pathogens and self, and plays a vital role in processing proinflammatory inputs. Studies addressing the influence of stimulus dynamics on NF-κB signaling are rare due to technical limitations with live-cell measurements. However, recent advances in microfluidics, automation, and image analysis have enabled investigations that yield high temporal resolution at the single-cell level. Here, we summarize the recent research which measures and models the NF-κB response to pulsatile and fluctuating stimulus concentrations, as well as different combinations and sequences of signaling molecules. Collectively, these studies show that the NF-κB network integrates external inflammatory signals and translates these into downstream transcriptional responses.

YAP is a transcriptional regulator that controls pluripotency, cell fate, and proliferation. How cells ensure the selective activation of YAP effector genes is unknown. This knowledge is essential to rationally control cellular decision-making. Here we leverage optogenetics, live-imaging of transcription, and cell fate analysis to understand and control gene activation and cell behavior. We reveal that cells decode the steady-state concentrations and timing of YAP activation to control proliferation, cell fate, and expression of the pluripotency regulators Oct4 and Nanog. While oscillatory YAP inputs induce Oct4 expression and proliferation optimally at frequencies that mimic native dynamics, cellular differentiation requires persistently low YAP levels. We identify the molecular logic of the Oct4 dynamic decoder, which acts through an adaptive change sensor. Our work reveals how YAP levels and dynamics enable multiplexing of information transmission for the regulation of developmental decision-making and establishes a platform for the rational control of these behaviors.

Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as a label-free imaging tool, capable of resolving cell types and organelles within 3D live samples, whilst monitoring their activity at tens of milliseconds resolution. Here, a D-FFOCT module design is presented which can be coupled to a commercial microscope with a stage top incubator, allowing non-invasive label-free longitudinal imaging over periods of minutes to weeks on the same sample. Long term volumetric imaging on human induced pluripotent stem cell-derived retinal organoids is demonstrated, highlighting tissue and cell organization processes such as rosette formation and mitosis as well as cell shape and motility. Imaging on retinal explants highlights single 3D cone and rod structures. An optimal workflow for data acquisition, postprocessing and saving is demonstrated, resulting in a time gain factor of 10 compared to prior state of the art. Finally, a method to increase D-FFOCT signal-to-noise ratio is demonstrated, allowing rapid organoid screening.