Search

Electrogenetics, the combination of electronics and genetics, is an emerging field of mammalian synthetic biology in which electrostimulation is used to remotely program user-designed genetic elements within designer cells to generate desired outputs. Here, we describe recent advances in electro-induced therapeutic gene expression and therapeutic protein secretion in engineered mammalian cells. We also review available tools and strategies to engineer electro-sensitive therapeutic designer cells that are able to sense electrical pulses and produce appropriate clinically relevant outputs in response. We highlight current limitations facing mammalian electrogenetics and suggest potential future directions for research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cell fate conversion by overexpressing defined factors is a powerful tool in regenerative medicine. However, identifying key factors for cell fate conversion requires laborious experimental efforts; thus, many of such conversions have not been achieved yet. Nevertheless, cell fate conversions found in many published studies were incomplete as the expression of important gene sets could not be manipulated thoroughly. Therefore, the identification of master transcription factors for complete and efficient conversion is crucial to render this technology more applicable clinically. In the past decade, systematic analyses on various single-cell and bulk OMICs data have uncovered numerous gene regulatory mechanisms, and made it possible to predict master gene regulators during cell fate conversion. By virtue of the sparse structure of master transcription factors and the group structure of their simultaneous regulatory effects on the cell fate conversion process, this study introduces a novel computational method predicting master transcription factors based on group sparse optimization technique integrating data from multi-OMICs levels, which can be applicable to both single-cell and bulk OMICs data with a high tolerance of data sparsity. When it is compared with current prediction methods by cross-referencing published and validated master transcription factors, it possesses superior performance. In short, this method facilitates fast identification of key regulators, give raise to the possibility of higher successful conversion rate and in the hope of reducing experimental cost., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

TLX (NR2E1), an orphan member of the nuclear receptor superfamily, is a transcription factor that has been described to be generally repressive in nature. It has been implicated in several aspects of physiology and disease. TLX is best known for its ability to regulate the proliferation of neural stem cells and retinal progenitor cells. Dysregulation, overexpression, or loss of TLX expression has been characterized in numerous studies focused on a diverse range of pathological conditions, including abnormal brain development, psychiatric disorders, retinopathies, metabolic disease, and malignant neoplasm. Despite the lack of an identified endogenous ligand, several studies have described putative synthetic and natural TLX ligands, suggesting that this receptor may serve as a therapeutic target. Therefore, this article aims to briefly review what is known about TLX structure and function in normal physiology, and provide an overview of TLX in regard to pathological conditions. Particular emphasis is placed on TLX and cancer, and the potential utility of this receptor as a therapeutic target., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phase separation of proteins regulates transcription. Here, we present a protocol to manipulate phase separation capacity of a protein. We use this protocol to disrupt phase separation by mutating residues at intrinsically disordered regions (IDRs). Further, we rescue the disabled phase separation by fusing an IDR known to drive phase separation. Phase separation promotes cell fate transitions, whereas disruption of phase attenuates the transitions. The major challenge is how to effectively predict mutation residues. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis., (© 2021 Perez Verdaguer et al.)

Podosomes and invadopodia, collectively termed invadosomes, are adhesive and degradative membrane structures formed in many types of cells and are well known for recruiting various proteases. However, another major class of degradative enzymes, deoxyribonuclease (DNase), remains unconfirmed and not studied in invadosomes. Here, using surface-immobilized nuclease sensor (SNS), we demonstrated that invadosomes recruit DNase to their core regions, which degrade extracellular double-stranded DNA. We further identified the DNase as GPI-anchored membrane-bound DNase X. DNase recruitment is ubiquitous and consistent in invadosomes of all tested cell types. DNase activity exhibits within a minute after actin nucleation, functioning concomitantly with protease in podosomes but preceding it in invadopodia. We further showed that macrophages form DNase-active podosome rosettes surrounding bacteria or micropatterned antigen islets, and the podosomes directly degrade bacterial DNA on a surface, exhibiting an apparent immunological function. Overall, this work reports DNase in invadosomes for the first time, suggesting a richer arsenal of degradative enzymes in invadosomes than known before., (© 2021 Pal et al.)

Comprehensively characterizing the cellular composition and organization of tissues has been a long-term scientific challenge that has limited our ability to study fundamental and clinical aspects of human physiology. The Human Cell Atlas (HCA) is a global collaborative effort to create a reference map of all human cells as a basis for both understanding human health and diagnosing, monitoring, and treating disease. Many aspects of the HCA are analogous to the Human Genome Project (HGP), whose completion presents a major milestone in modern biology. To commemorate the HGP's 20-year anniversary of completion, we discuss the launch of the HCA in light of the HGP, and highlight recent progress by the HCA consortium., Competing Interests: Declaration of Interests In the last 3 years, S.A.T. has consulted for Genentech and Roche, and is a member of SABs of Biogen, GlaxoSmithKline, and Foresite Labs. A.R. is a cofounder and equity holder of Celsius Therapeutics, an equity holder of Immunitas and was an SAB member of Neogene Therapeutics, Thermo Fisher Scientific, Asimov, and Syros Pharmaceuticals until July 31, 2020. Since August 1, 2020, A.R. is an employee of Genentech, a member of the Roche group. A.R. is an inventor on multiple patents to the Broad Institute in the area of single cell genomics. R.G.H.L. has no interests to declare., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In the mid-1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by a N -acetylglucosamine moiety ( O -GlcNAc) via an O -linkage overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O -GlcNAc modification does not lead to complex, branched glycan structures and is rapidly cycled on and off proteins by O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA), respectively. Since its discovery, O -GlcNAcylation has been shown to contribute to numerous cellular functions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O -GlcNAc cycling has been implicated in the progression of a wide range of diseases, such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O -GlcNAc turnover, the role of O -GlcNAcylation in regulating cellular physiology, and how dysregulation in O -GlcNAc cycling contributes to pathophysiological processes.

Bistable switches that produce all-or-none responses have been found to regulate a number of natural cellular decision making processes, and subsequently synthetic switches were designed to exploit their potential. However, an increasing number of studies, particularly in the context of cellular differentiation, highlight the existence of a mixed state that can be explained by tristable switches. The criterion for designing robust tristable switches still remains to be understood from the perspective of network topology. To address such a question, we calculated the robustness of several 2- and 3-component network motifs, connected via only two positive feedback loops, in generating tristable signal response curves. By calculating the effective potential landscape and following its modifications with the bifurcation parameter, we constructed one-parameter bifurcation diagrams of these models in a high-throughput manner for a large combinations of parameters. We report here that introduction of a self-activatory positive feedback loop, directly or indirectly, into a mutual inhibition loop leads to generating the most robust tristable response. The high-throughput approach of our method further allowed us to determine the robustness of four types of tristable responses that originate from the relative locations of four bifurcation points. Using the method, we also analyzed the role of additional mutual inhibition loops in stabilizing the mixed state.

Background: The complex composition of different cell types within a tissue can be estimated by deconvolution of bulk gene expression profiles or with various single-cell sequencing approaches. Alternatively, DNA methylation (DNAm) profiles have been used to establish an atlas for multiple human tissues and cell types. DNAm is particularly suitable for deconvolution of cell types because each CG dinucleotide (CpG site) has only two states per DNA strand-methylated or non-methylated-and these epigenetic modifications are very consistent during cellular differentiation. So far, deconvolution of DNAm profiles implies complex signatures of many CpGs that are often measured by genome-wide analysis with Illumina BeadChip microarrays. In this study, we investigated if the characterization of cell types in tissue is also feasible with individual cell type-specific CpG sites, which can be addressed by targeted analysis, such as pyrosequencing., Results: We compiled and curated 579 Illumina 450k BeadChip DNAm profiles of 14 different non-malignant human cell types. A training and validation strategy was applied to identify and test for cell type-specific CpGs. We initially focused on estimating the relative amount of fibroblasts using two CpGs that were either hypermethylated or hypomethylated in fibroblasts. The combination of these two DNAm levels into a "FibroScore" correlated with the state of fibrosis and was associated with overall survival in various types of cancer. Furthermore, we identified hypomethylated CpGs for leukocytes, endothelial cells, epithelial cells, hepatocytes, glia, neurons, fibroblasts, and induced pluripotent stem cells. The accuracy of this eight CpG signature was tested in additional BeadChip datasets of defined cell mixtures and the results were comparable to previously published signatures based on several thousand CpGs. Finally, we established and validated pyrosequencing assays for the relevant CpGs that can be utilized for classification and deconvolution of cell types., Conclusion: This proof of concept study demonstrates that DNAm analysis at individual CpGs reflects the cellular composition of cellular mixtures and different tissues. Targeted analysis of these genomic regions facilitates robust methods for application in basic research and clinical settings.

The regulation of gene expression is a fundamental process enabling cells to respond to internal and external stimuli or to execute developmental programs. Changes in gene expression are highly dynamic and depend on many intrinsic and extrinsic factors. In this review, we highlight the dynamic nature of transient gene expression changes to better understand cell physiology and development in general. We will start by comparing recent in vivo procedures to capture gene expression in real time. Intrinsic factors modulating gene expression dynamics will then be discussed, focusing on chromatin modifications. Furthermore, we will dissect how cell physiology or age impacts on dynamic gene regulation and especially discuss molecular insights into acquired transcriptional memory. Finally, this review will give an update on the mechanisms of heterogeneous gene expression among genetically identical individual cells. We will mainly focus on state-of-the-art developments in the yeast model but also cover higher eukaryotic systems.

Genetic programs operating in a history-dependent fashion are ubiquitous in nature and govern sophisticated processes such as development and differentiation. The ability to systematically and predictably encode such programs would advance the engineering of synthetic organisms and ecosystems with rich signal processing abilities. Here we implement robust, scalable history-dependent programs by distributing the computational labor across a cellular population. Our design is based on standardized recombinase-driven DNA scaffolds expressing different genes according to the order of occurrence of inputs. These multicellular computing systems are highly modular, do not require cell-cell communication channels, and any program can be built by differential composition of strains containing well-characterized logic scaffolds. We developed automated workflows that researchers can use to streamline program design and optimization. We anticipate that the history-dependent programs presented here will support many applications using cellular populations for material engineering, biomanufacturing and healthcare.

Myc and Ras are two of the most commonly activated oncogenes in tumorigenesis. Together and independently they regulate many cancer hallmarks including proliferation, apoptosis, and self-renewal. Recently, they were shown to cooperate to regulate host tumor microenvironment programs including host immune responses. But, is their partnership always cooperative or do they have distinguishable functions? Here, we provide one perspective that Myc and Ras cooperation depends on the genetic evolution of a particular cancer. This in turn, dictates when they cooperate via overlapping and identifiably distinct cellular- and host immune-dependent mechanisms that are cancer type specific., (©2020 American Association for Cancer Research.)

Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The cellular stress response (CSR) is pervasive to all domains of life. It has shaped the interaction between organisms and their environment since the origin of the first cell. Although the CSR has been subject to a myriad of nuanced modifications in the various branches of life present today, its core features remain preserved. The scientific literature covering the CSR is enormous and the broad scope of this brief overview was challenging. However, it is critical to conceptually understand how cells respond to stress in a holistic sense and to point out how fundamental aspects of the CSR framework are integrated. It was necessary to be extremely selective and not feasible to even mention many interesting and important developments in this expansive field. The purpose of this overview is to sketch out general and emerging CSR concepts with an emphasis on the initial cellular strain resulting from stress (macromolecular damage) and the evolutionarily most highly conserved elements of the CSR. Examples emphasize fish and aquatic invertebrates to highlight what is known in organisms beyond mammals, yeast, and other common models. Nonetheless, select pioneering studies using canonical models are also considered and the concepts discussed are applicable to all cells. More detail on important aspects of the CSR in aquatic animals is provided in the accompanying articles of this special issue., (© 2020 Wiley Periodicals, Inc.)

The mammalian circadian clock regulates a wide variety of physiological and behavioral processes. In turn, its disruption is associated with sleep deficiency, metabolic syndrome, neurological and psychiatric disorders, and cancer. At the turn of the century, the circadian clock was determined to be regulated by a transcriptional negative feedback mechanism composed of a dozen core clock genes. More recently, large-scale genomic studies have expanded the clock into a complex network composed of thousands of gene outputs and inputs. A major task of circadian research is to utilize systems biological approaches to uncover the governing principles underlying cellular oscillatory behavior and advance understanding of biological functions at the genomic level with spatiotemporal resolution. This review focuses on the genes and pathways that provide inputs to the circadian clock. Several emerging examples include AMP-activated protein kinase AMPK, nutrient/energy sensor mTOR, NAD + -dependent deacetylase SIRT1, hypoxia-inducible factor HIF1α, oxidative stress-inducible factor NRF2, and the proinflammatory factor NF-κB. Among others that continue to be revealed, these input pathways reflect the extensive interplay between the clock and cell physiology through the regulation of core clock genes and proteins. While the scope of this crosstalk is well-recognized, precise molecular links are scarce, and the underlying regulatory mechanisms are not well understood. Future research must leverage genetic and genomic tools and technologies, network analysis, and computational modeling to characterize additional modifiers and input pathways. This systems-based framework promises to advance understanding of the circadian timekeeping system and may enable the enhancement of circadian functions through related input pathways., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Gastrointestinal cancer, the most common solid tumor, has a poor prognosis. With the development of high-throughput sequencing and detection technology, recent studies have suggested that many chemical modifications of human RNA are involved in the development of human diseases, including cancer. m 6 A, the most abundant modification, was revealed to participate in a series of aspects of cancer progression. Recent evidence has shown that methyltransferase-like 3 (METTL3), the first identified and a critical methyltransferase, catalyzes m 6 A methylation on mRNA or non-coding RNA in mammals, affecting RNA metabolism. Abnormal m 6 A levels caused by METTL3 have been reported to be involved in different aspects of cancer development, including proliferation, apoptosis, and metastasis. In this review, we will shed light on recent findings regarding the biological function of METTL3 in gastrointestinal cancer and discuss future research directions and potential clinical applications of METTL3 for gastrointestinal cancer.

Protein degradation is tightly regulated inside cells because of its utmost importance for protein homeostasis (proteostasis). The two major intracellular proteolytic pathways are the ubiquitin-proteasome and the autophagy-lysosome systems which ensure the fate of proteins when modified by various members of the ubiquitin family. These pathways are tightly interconnected by receptors and cofactors that recognize distinct chain architectures to connect with either the proteasome or autophagy under distinct physiologic and pathologic situations. The degradation of proteasome by autophagy, known as proteaphagy, plays an important role in this crosstalk since it favours the activity of autophagy in the absence of fully active proteasomes. Recently described in several biological models, proteaphagy appears to help the cell to survive when proteostasis is broken by the absence of nutrients or the excess of proteins accumulated under various stress conditions. Emerging evidence indicates that proteaphagy could be permanently activated in some types of cancer or when chemoresistance is observed in patients.

Higher-order supramolecular complexes-dubbed signalosomes carry out key signaling and effector functions in innate immunity and inflammation. In this review, we present several recently discovered signalosomes that are formed either by stable protein-protein interactions or by dynamic liquid-liquid phase separation. Structural features of these signalosomes are highlighted to elucidate their functions and biological insights., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Autosomal dominant missense mutations that hyperactivate the leucine-rich repeat protein kinase-2 (LRRK2) are a common cause of inherited Parkinson's disease and therapeutic efficacy of LRRK2 inhibitors is being tested in clinical trials. In this review, we discuss the nuts and bolts of our current understanding of how the LRRK2 is misregulated by mutations and how pathway activity is affected by LRRK2 binding to membrane, microtubule filaments, and 14-3-3, as well as by upstream components such as Rab29 and VPS35. We discuss recent work that points toward a subset of Rab proteins comprising key physiological substrates that bind new sets of effectors, such as RILPL1/2, JIP3 and JIP4 after phosphorylation by LRRK2. We explore what is known about how LRRK2 regulates ciliogenesis, the endosomal-lysosomal system, immune responses and interplay with alpha-synuclein and tau and how this might be linked to Parkinson's' disease., Competing Interests: Conflicts of interest statement Nothing declared., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microglia polarization plays an important role in poststroke recovery. Inhibition of proinflammatory (M1) polarization and promotion of anti-inflammatory (M2) polarization of microglia are potential therapeutic strategies for inflammation reduction and neuronal recovery after stroke. Here, we evaluated the central nervous system (CNS)-targeted short interfering RNA (siRNA) delivery ability of functionalized curdlan nanoparticles (CMI) and investigated the nuclear factor-κB (NF-κB) p65 silencing efficiency of CMI-mediated siRNA in microglia, as well as the resulting neuroprotective effect of microglia polarization and neuroprotection in vitro and in vivo. The systemic delivery of NF-κB p65 siRNA (sip65) complexed to CMI nanoparticles in the mouse model of transient middle cerebral artery occlusion (tMCAO) resulted in the distribution of siRNA in microglia and significant silencing in NF-κB p65 in the peri-infarct region. Knockdown of NF-κB p65 resulted in M1 to M2 phenotypic transition of microglia, evidenced by the change in the expression pattern of signature cytokines as well as inducible nitric oxide synthase and CD206. Moreover, the CMI-mediated silencing of p65 increased the density of neurons and decreased pyknosis and edema in the peri-infarct region. Assessment of the neurological deficit score on the Bederson scale revealed a significantly reduced score in the mouse model of tMCAO treated with the sip65/CMI complex. Collectively, our data suggest that CMI nanoparticles are a promising CNS-targeting siRNA delivery system, and NF-κB p65 may be a potential therapeutic target for inflammation reduction and poststroke recovery.

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor ( FynSensor ) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate., Competing Interests: AM, RS, SU, SB, PR, SM, GG, SK, RD, BR, AG No competing interests declared

The process of cell competition results in the 'elimination of cells that are viable but less fit than surrounding cells'. Given the highly heterogeneous nature of our tissues, it seems increasingly likely that cells are engaged in a 'survival of the fittest' battle throughout life. The process has a myriad of positive roles in the organism: it selects against mutant cells in developing tissues, prevents the propagation of oncogenic cells and eliminates damaged cells during ageing. However, 'super-fit' cancer cells can exploit cell competition mechanisms to expand and spread. Here, we review the regulation, roles and risks of cell competition in organism development, ageing and disease., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The dynamics of the cellular proportion of mutant mtDNA molecules is crucial for mitochondrial diseases. Cellular populations of mitochondria are under homeostatic control, but the details of the control mechanisms involved remain elusive. Here, we use stochastic modelling to derive general results for the impact of cellular control on mtDNA populations, the cost to the cell of different mtDNA states, and the optimisation of therapeutic control of mtDNA populations. This formalism yields a wealth of biological results, including that an increasing mtDNA variance can increase the energetic cost of maintaining a tissue, that intermediate levels of heteroplasmy can be more detrimental than homoplasmy even for a dysfunctional mutant, that heteroplasmy distribution (not mean alone) is crucial for the success of gene therapies, and that long-term rather than short intense gene therapies are more likely to beneficially impact mtDNA populations., Competing Interests: The authors have declared that no competing interests exist.

Background: Modules of interacting components arranged in specific network topologies have evolved to perform a diverse array of cellular functions. For a network with a constant topological structure, its function within a cell may still be tuned by changing the number of instances of a particular component (e.g., gene copy number) or by modulating the intrinsic biochemical properties of a component (e.g., binding strength or catalytic efficiency). How such perturbations affect cellular response dynamics remains poorly understood. Here, we explored these effects in a common decision-making motif, cross-antagonism with autoregulation, by synthetically constructing this network in yeast., Results: We employed the engineering design strategy of reuse to build this topology with a single protein building block, TetR, creating necessary components through TetR mutations and fusion partners. We then studied the impact of several topology-preserving perturbations - strength of cross-antagonism, number of operator sites in a promoter, and gene dosage - on decision-making behavior. We found that reducing TetR repression strength, which hinders cross-antagonism, resulted in a loss of mutually exclusive cell responses. Unexpectedly, increasing the number of operator sites also impeded decision-making exclusivity, which may be a consequence of the averaging effect that arises when multiple transcriptional activators and repressors are accommodated at a given locus. Stochastic simulations of this topology revealed that, even for networks with high TetR repression strength and a low number of operator sites, increasing gene dosage can reduce exclusivity in response dynamics. We further demonstrated this result experimentally by quantifying gene copy numbers in selected yeast clones with differing phenotypic responses., Conclusions: Our study illustrates how parameters that do not change the topological structure of a decision-making network can nonetheless exert significant influence on its response dynamics. These findings should further inform the study of native motifs, including the effects of topology-preserving mutations, and the robust engineering of synthetic networks.

A diverse catalog of long noncoding RNAs (lncRNAs), which lack protein-coding potential, are transcribed from the mammalian genome. They are emerging as important regulators in gene expression networks by controlling nuclear architecture and transcription in the nucleus and by modulating mRNA stability, translation and post-translational modifications in the cytoplasm. In this Review, we highlight recent progress in cellular functions of lncRNAs at the molecular level in mammalian cells.

One of the most neglected aspects of chemotherapy are changes, and possible consequences of these changes, that occur in normal somatic cells. In this review, we summarize effects of selected drugs used to treat ovarian cancer (platin derivatives-cisplatin and carboplatin; and taxanes-paclitaxel and docetaxel) on cellular metabolism, acquisition of reactive stroma features, cellular senescence, inflammatory reactions, apoptosis, autophagy, mitophagy, oxidative stress, DNA damage, and angiogenesis in various types of normal cells, including fibroblasts, epithelial cells, endothelial cells, and neurons. The activity of these drugs against the normal cells is presented from a broader perspective of their desirable anti-tumoral effects.

Mitochondria play a central role in maintaining normal cellular homeostasis as well as contributing to the pathogenesis of numerous disease states. The advent of CRISPR-Cas9 screening technologies has greatly accelerated the study of mitochondrial biology. In this chapter, we review the various CRISPR-Cas9 screening platforms that are currently available and prior studies that leveraged this technology to identify genes involved in mitochondrial biology in both healthy and disease states. In addition, we discuss the challenges associated with current CRISPR-Cas9 platforms and potential solutions to further enhance this promising technology.

Cell morphological phenotypes, including shape, size, intensity, and texture of cellular compartments have been shown to change in response to perturbation with small molecule compounds. Image-based cell profiling or cell morphological profiling has been used to associate changes of cell morphological features with alterations in cellular function and to infer molecular mechanisms of action. Recently, the Library of Integrated Network-based Cellular Signatures (LINCS) Project has measured gene expression and performed image-based cell profiling on cell lines treated with 9515 unique compounds. These data provide an opportunity to study the interdependence between transcription and cell morphology. Previous methods to investigate cell phenotypes have focused on targeting candidate genes as components of known pathways, RNAi morphological profiling, and cataloging morphological defects; however, these methods do not provide an explicit model to link transcriptomic changes with corresponding alterations in morphology. To address this, we propose a cell morphology enrichment analysis to assess the association between transcriptomic alterations and changes in cell morphology. Additionally, for a new transcriptomic query, our approach can be used to predict associated changes in cellular morphology. We demonstrate the utility of our method by applying it to cell morphological changes in a human bone osteosarcoma cell line.

The high-order chromatin structure plays a non-negligible role in gene regulation. However, the mechanism, especially the sequence dependence for the formation of varied chromatin structures in different cells remains to be elucidated. As the nucleotide distributions in human and mouse genomes are highly uneven, we identified CGI (CpG island) forest and prairie genomic domains based on CGI densities of a species, dividing the genome into two sequentially, epigenetically, and transcriptionally distinct regions. These two megabase-sized domains also spatially segregate to different extents in different cell types. Forests and prairies show enhanced segregation from each other in development, differentiation, and senescence, meanwhile the multi-scale forest-prairie spatial intermingling is cell-type specific and increases in differentiation, helping to define cell identity. We propose that the phase separation of the 1D mosaic sequence in space serves as a potential driving force, and together with cell type specific epigenetic marks and transcription factors, shapes the chromatin structure in different cell types. The mosaicity in genome of different species in terms of forests and prairies could relate to observations in their biological processes like development and aging. In this way, we provide a bottoms-up theory to explain the chromatin structural and epigenetic changes in different processes.

Circular RNAs (circRNA) are non-coding RNAs characterized by a closed-loop structure providing increased stability and enhancing their ability to compete with messenger RNAs (mRNA) for microRNA (miRNA) binding. An important open question is if circRNA plays a physiological role in the cells regulating critical cellular functions. We address this question with a combination of theoretical modeling and experimental analysis. Theoretically, we investigated two possible scenarios, one in which circRNAs act as sponges for miRNAs but there is no other relation between the two RNAs and the other one where circRNAs are co-generated with their corresponding mRNAs. We thus compared the results with data reporting the level of circRNAs (ZEB1, CANCX, ABCC1) modulating specific miRNAs in 27 cell lines. Due to the high stability of circRNAs, we show that a miRNA mediated cross-talk between circRNA and mRNA appears for a broad range of physiological parameters. This confirms the relevance of circRNAs in cell regulation, suggesting that they could be used as biomarkers., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Voltage-gated ion channels were believed to be the only voltage-sensitive proteins in excitable (and some non-excitable) cells for a long time. Emerging evidence indicates that the voltage-operated model is shared by some other transmembrane proteins expressed in both excitable and non-excitable cells. In this review, we summarize current knowledge about voltage-operated proteins, which are not classic voltage-gated ion channels as well as the voltage-dependent processes in cells for which single voltage-sensitive proteins have yet to be identified. Particularly, we will focus on the following. (1) Voltage-sensitive phosphoinositide phosphatases (VSP) with four transmembrane segments homologous to the voltage sensor domain (VSD) of voltage-gated ion channels; VSPs are the first family of proteins, other than the voltage-gated ion channels, for which there is sufficient evidence for the existence of the VSD domain; (2) Voltage-gated proton channels comprising of a single voltage-sensing domain and lacking an identified pore domain; (3) G protein coupled receptors (GPCRs) that mediate the depolarization-evoked potentiation of Ca 2+ mobilization; (4) Plasma membrane (PM) depolarization-induced but Ca 2+ -independent exocytosis in neurons. (5) Voltage-dependent metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P 2 , PIP 2 ) in the PM. These recent discoveries expand our understanding of voltage-operated processes within cellular membranes., (© 2018 Wiley Periodicals, Inc.)

Various stress factors leading to protein damage induce the activation of an evolutionarily conserved cell protective mechanism, the heat shock response (HSR), to maintain protein homeostasis in virtually all eukaryotic cells. Heat shock factor 1 (HSF1) plays a central role in the HSR. HSF1 was initially known as a transcription factor that upregulates genes encoding heat shock proteins (HSPs), also called molecular chaperones, which assist in refolding or degrading injured intracellular proteins. However, recent accumulating evidence indicates multiple additional functions for HSF1 beyond the activation of HSPs. Here, we present a nearly comprehensive list of non-HSP-related target genes of HSF1 identified so far. Through controlling these targets, HSF1 acts in diverse stress-induced cellular processes and molecular mechanisms, including the endoplasmic reticulum unfolded protein response and ubiquitin-proteasome system, multidrug resistance, autophagy, apoptosis, immune response, cell growth arrest, differentiation underlying developmental diapause, chromatin remodelling, cancer development, and ageing. Hence, HSF1 emerges as a major orchestrator of cellular stress response pathways.

We report a microfluidic system that physically separates nuclear RNA (nucRNA) and cytoplasmic RNA (cytRNA) from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA, per cell, quantifies gene expression in the subcellular compartments, and combines them to create novel single-cell RNA-seq data. Leveraging SINC-seq, we discover distinct natures of correlation among cytRNA and nucRNA that reflect the transient physiological state of single cells. These data provide unique insights into the regulatory network of messenger RNA from the nucleus toward the cytoplasm at the single-cell level.

Fork head box O (FOXO) transcription factor is a key player in an evolutionarily conserved pathway. The mammalian FOXO family consists of FOXO1, 3, 4 and 6, are highly similar in their structure, function and regulation. To maintain optimum body function, the organisms have developed complex mechanisms for homeostasis. Importantly, it is well known that when these mechanisms dysregulate it results in the development of age-related disease. FOXO proteins are involved in a diverse cellular function and also have clinical significance including cell cycle arrest, cell differentiation, tumour suppression, DNA repair, longevity, diabetic complications, immunity, wound healing, regulation of metabolism and thus treatment of several types of diseases. By the combinations of post-translational modifications FOXO's serve as a 'molecular code' to sense external stimuli and recruit it as to specific regions of the genome and provide an integrated cellular response to changing physiological conditions. Akt/Protein kinase B a signaling pathway as a main regulator of FOXO to perform a diverse function in organisms. The present review summarizes the molecular and clinical aspects of FOXO transcription factor. And also elaborate the interaction of FOXO with the nucleosome remodelling complex to target genes, which is essential to cellular homeostasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Whereas the relationship between criticality of gene regulatory networks (GRNs) and dynamics of GRNs at a single-cell level has been vigorously studied, the relationship between the criticality of GRNs and system properties at a higher level has not been fully explored. Here we aim at revealing a potential role of criticality of GRNs in morphogenesis, which is hard to uncover through the single-cell-level studies, especially from an evolutionary viewpoint. Our model simulated the growth of a cell population from a single seed cell. All the cells were assumed to have identical intracellular GRNs. We induced genetic perturbations to the GRN of the seed cell by adding, deleting, or switching a regulatory link between a pair of genes. From numerical simulations, we found that the criticality of GRNs facilitated the formation of nontrivial morphologies when the GRNs were critical in the presence of the evolutionary perturbations. Moreover, the criticality of GRNs produced topologically homogeneous cell clusters by adjusting the spatial arrangements of cells, which led to the formation of nontrivial morphogenetic patterns. Our findings correspond to an epigenetic viewpoint that heterogeneous and complex features emerge from homogeneous and less complex components through the interactions among them. Thus, our results imply that highly structured tissues or organs in morphogenesis of multicellular organisms might stem from the criticality of GRNs.

Background: Small double-strand RNAs (dsRNAs) molecules are able to activate endogenous genes via an RNA-based promoter-targeting mechanism. Like RNA interference (RNAi), RNA activation (RNAa) is an evolutionarily conserved mechanism that is present in diverse eukaryotic organisms ranging from yeast to humans., Methods: The small activating RNAs (saRNAs) that are involved in RNAa have been successively used to activate gene expression in cultured cells. Thus, this emergent technique might allow us to develop biotechnological and therapeutic applications without the need to synthesize hazardous construct systems that harbor exogenous DNA sequences., Result and Conclusion: Accordingly, this article aims to provide insights into how RNAa cellular machinery can be manipulated to activate gene expression and for more effective clinical treatments of diverse diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Whole cell responses arise from coordinated interactions between diverse human gene products functioning within various pathways underlying sub-cellular processes (SCP). Lower level SCPs interact to form higher level SCPs, often in a context specific manner to give rise to whole cell function. We sought to determine if capturing such relationships enables us to describe the emergence of whole cell functions from interacting SCPs. We developed the Molecular Biology of the Cell Ontology based on standard cell biology and biochemistry textbooks and review articles. Currently, our ontology contains 5,384 genes, 753 SCPs and 19,180 expertly curated gene-SCP associations. Our algorithm to populate the SCPs with genes enables extension of the ontology on demand and the adaption of the ontology to the continuously growing cell biological knowledge. Since whole cell responses most often arise from the coordinated activity of multiple SCPs, we developed a dynamic enrichment algorithm that flexibly predicts SCP-SCP relationships beyond the current taxonomy. This algorithm enables us to identify interactions between SCPs as a basis for higher order function in a context dependent manner, allowing us to provide a detailed description of how SCPs together can give rise to whole cell functions. We conclude that this ontology can, from omics data sets, enable the development of detailed SCP networks for predictive modeling of emergent whole cell functions.

Ochratoxin A (OTA) is a mycotoxin produced by fungus belonging to Aspergillus and Penicillium genra. The aim of the present paper was to investigate if a low concentration OTA has toxic effect in pigs. Twelve piglets were fed with a control or an OTA (0.05 mg/kg feed) contaminated diet. After 30 days, animals were slaughtered and samples of blood and kidney were used for further analyses. The mycotoxin analyses showed a significant higher (6.25 times) concentration of OTA in the kidney of OTA intoxicated piglets than in control ones. While OTA has no effect on the urea and creatinine concentration, the microarray analysis of the effect of OTA on genome wide expression in the kidney of intoxicated piglets, revealed that approximately 105 different transcripts were significantly altered. As shown by the microarray results, 0.05 mg/kg of OTA can principally interfere with: i) canonical pathways (CD28 Signaling in T Helper Cells, Role of NFAT in Regulation of the Immune Response, Relaxin Signaling, IL-1 Signaling) ii) molecular and cellular function (cellular movement cellular function and maintenance, cellular growth and proliferation cellular assembly and organization, cell death and survival) etc. However, alteration of renal and urological system development and function and renal necrosis predicted through Ingenuity Pathway Analysis (IPA) were not supported by clinical pathological data. In conclusion, OTA toxicity was found even at low concentration of toxin, correlated with the activation of immune response pathways, oxidative stress response and early carcinogenic events. This effect need to be further investigated and analyzed in the context of human health., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although it was long thought that eukaryotic translation almost always initiates at an AUG start codon, recent advancements in ribosome footprint mapping have revealed that non-AUG start codons are used at an astonishing frequency. These non-AUG initiation events are not simply errors but instead are used to generate or regulate proteins with key cellular functions; for example, during development or stress. Misregulation of non-AUG initiation events contributes to multiple human diseases, including cancer and neurodegeneration, and modulation of non-AUG usage may represent a novel therapeutic strategy. It is thus becoming increasingly clear that start codon selection is regulated by many trans -acting initiation factors as well as sequence/structural elements within messenger RNAs and that non-AUG translation has a profound impact on cellular states., (© 2017 Kearse and Wilusz; Published by Cold Spring Harbor Laboratory Press.)

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.

Quorum sensing (QS) systems have been widely applied in biotechnology and synthetic biology that require coordinated, community-level behaviors. Meanwhile, the cell physiological state is another key parameter that affects metabolic pathway regulation. Here, we designed an autoinduced AND gate that responds to both microbial communities and the cell physiological state. A series of tunable QS systems in response to different cell densities were obtained through random mutagenesis of LuxR and optimization of the luxRI promoter; the corresponding suitable stationary phase sensing system was selected after monitoring the fluorescence process during cell growth. The application of the final synthetic device was demonstrated using the polyhydroxybutyrate (PHB) production system. The AND gate system increased PHB production by 1-2-fold in Escherichia coli. This synthetic logic gate is a tool for developing a general dynamic regulation system in metabolic engineering in response to complex signals, without using a specific sensor.

In the noisy cellular environment, gene products are subject to inherent random fluctuations in copy numbers over time. How cells ensure precision in the timing of key intracellular events despite such stochasticity is an intriguing fundamental problem. We formulate event timing as a first-passage time problem, where an event is triggered when the level of a protein crosses a critical threshold for the first time. Analytical calculations are performed for the first-passage time distribution in stochastic models of gene expression. Derivation of these formulas motivates an interesting question: Is there an optimal feedback strategy to regulate the synthesis of a protein to ensure that an event will occur at a precise time, while minimizing deviations or noise about the mean? Counterintuitively, results show that for a stable long-lived protein, the optimal strategy is to express the protein at a constant rate without any feedback regulation, and any form of feedback (positive, negative, or any combination of them) will always amplify noise in event timing. In contrast, a positive feedback mechanism provides the highest precision in timing for an unstable protein. These theoretical results explain recent experimental observations of single-cell lysis times in bacteriophage [Formula: see text] Here, lysis of an infected bacterial cell is orchestrated by the expression and accumulation of a stable [Formula: see text] protein up to a threshold, and precision in timing is achieved via feedforward rather than feedback control. Our results have broad implications for diverse cellular processes that rely on precise temporal triggering of events., Competing Interests: The authors declare no conflict of interest.

Genetic suppression occurs when the phenotypic defects caused by a mutation in a particular gene are rescued by a mutation in a second gene. To explore the principles of genetic suppression, we examined both literature-curated and unbiased experimental data, involving systematic genetic mapping and whole-genome sequencing, to generate a large-scale suppression network among yeast genes. Most suppression pairs identified novel relationships among functionally related genes, providing new insights into the functional wiring diagram of the cell. In addition to suppressor mutations, we identified frequent secondary mutations,in a subset of genes, that likely cause a delay in the onset of stationary phase, which appears to promote their enrichment within a propagating population. These findings allow us to formulate and quantify general mechanisms of genetic suppression., (Copyright © 2016, American Association for the Advancement of Science.)

Cells live in dynamic environments that necessitate perpetual adaptation. Since cells have limited resources to monitor external inputs, they are required to maximize the information content of perceived signals. This challenge is not unique to microscopic life: Animals use senses to perceive inputs and adequately respond. Research showed that sensory-perception is actively shaped by learning and expectation allowing internal cognitive models to "fill in the blanks" in face of limited information. We propose that cells employ analogous strategies and use internal models shaped through the long process of evolutionary adaptation. Given this perspective, we postulate that cells are prone to "misperceptions," analogous to visual illusions, leading them to incorrectly decode patterns of inputs that lie outside of their evolutionary experience. Mapping cellular misperception can serve as a fundamental approach for dissecting regulatory networks and could be harnessed to modulate cell behavior, a potentially new avenue for therapy., (© 2016 WILEY Periodicals, Inc.)