Cellular Systems Research Articles

Editorial on the Special Issue “Fluorescence Imaging and Analysis of Cellular Systems”

Fluorescence imaging has indeed become a cornerstone in modern cell biology due to its ability to offer highly sensitive, specific, and real-time visualization of cellular structures and dynamic processes [...]

Two dimensional computational model of calcium and IP3 interacting system dynamics in an obese hepatocyte cell

Abstract Calcium ion (Ca$^{2+}$) signaling is crucial in regulating numerous cellular processes vital for preserving structural integrity and functional equilibrium across diverse cell types. Both the calcium stores and mitochondria play significant roles in this signaling pathway. The calcium source may be in the form of a blip or a puff depending on the various conditions of the cellular systems. The one dimensional model of calcium dynamics with IP$_{3}$ gives crucial insight of feedback mechanisms influencing calcium homeostasis. In order to obtain deeper insights of local impacts of various mechanisms and feedbacks in hepatocyte cell, it is necessary to develop the models in higher dimensions. In order to get more deeper insights, two dimensional model is proposed assuming the phenomena to be uniform along z dimension. This research presents a two-dimensional computational model to analyse the interactive system dynamics of inositol 1,4,5-trisphosphate (IP$_{3}$) and Ca$^{2+}$, aiming to assess how these signaling patterns influence hepatocyte functionality which allows to incorporate puff type of calcium source under both obese and normal physiological states. It further examines the implications of calcium signaling on NADH synthesis, ATP production, and degradation rates. Numerical simulations are executed utilising the Crank-Nicolson method for temporal analysis and the Linear Finite Element Method for spatial analysis. Additionally, the study conducts a comparative analysis of calcium signaling between obese and normal hepatocyte. The findings offer enhanced insights into the interactive system dynamics of IP$_{3}$ and Ca$^{2+}$ in hepatocytes, elucidating the effects of various parameter alterations on cellular behaviour in both states.

Influence of breastfeeding on the ontogenesis of mucosal immunity in children

The role of breastfeeding in the development of the cellular immune system, antimicrobial strategies of neutrophils, and local cytokine levels was evaluated in a single-center, prospective, open-label, uncontrolled study of healthy infants who were either naturally or artificially breastfed. The main group included practically healthy children who were exclusively breastfed for the first year of life. Thirty-six children were examined during the first stage and 25 from this group during the second stage. The comparison group included children who received an adapted cow’s milk formula during the first half-year of their lives and a different formula during the second half-year. Thirty-one children were examined during stage one and 27 from this group were examined during stage two. A comprehensive study of mucosal immunity was conducted, assessing the cytokine profile and cellular components using the method of smear prints from the nasal mucosa. The results showed that the nature of feeding during the first year of life influences the formation of mucosal immunity and programming the postnatal development of the child’s immunological reactivity. In the absence of the immunomodulatory effects of breast milk, there is an imbalance in the cytokine profile, with a physiological deviation of the immune response linked to the predominance of Th2 profiles. In contrast, breastfeeding promotes the optimal cellular composition of the upper respiratory mucosa and a cytomorphological neutrophil profile with minimal destructive changes. Breastfeeding programs the optimal functional profile of neutrophils, including the level of receptor activity, intracellular biocidal activity, cytotoxicity, ability to complete phagocytosis. This natural infant feeding method has a long-term protective effect, preventing neutrophil destruction under the influence of environmental factors and respiratory viral agents, and ensuring optimal mucosal immunity development in the respiratory tract.

Skin Sensitization Potential of Sensitizers in the Presence of Metal Oxide Nanoparticles In Vitro

Silica (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) nanoparticles (NPs) are widely used in dermal products. Their skin sensitization potential, especially their effects in combination with known sensitizers, is poorly studied in vitro and their sensitization inconsistently reported in animal studies. In this study, cellular assays were used to identify different steps of sensitization, the activation of keratinocytes and dendritic cells, when cells were exposed to these NPs in the absence and presence of sensitizers. Cellular systems included HaCaT keratinocytes and U937 (U-SENS™) alone, as well as different co-culture systems of THP-1 cells with HaCaT cells (COCAT) and with primary keratinocytes. The effect of NPs differed between co-cultures and U-SENS™, whereas co-cultures with either primary keratinocytes or HaCaT cells responded similarly. Pre-exposure to ZnO NPs increased the U-SENS™ assay response to 2,4-dinitrochlorobenzene six-fold. The COCAT increase was maximally four-fold for the combination of SiO2 and trans cinnamaldehyde. When the THP-1 cells were separated from the keratinocytes by a membrane, the response of the co-culture system was more similar to U-SENS™. The direct contact with keratinocytes decreased the modulating effect of TiO2 and ZnO NPs but suggested an increase in response to sensitizers following dermal contact with SiO2 NPs.

A guide for blood-brain barrier models.

Understanding the intricate mechanisms underlying brain-related diseases hinges on unraveling the pivotal role of the blood-brain barrier (BBB), an essential dynamic interface crucial for maintaining brain equilibrium. This review offers a comprehensive analysis of BBB physiology, delving into its cellular and molecular components while exploring a wide range of in vivo and in vitro BBB models. Notably, recent advancements in 3D cell culture techniques are explicitly discussed, as they have significantly improved the fidelity of BBB modeling by enabling the replication of physiologically relevant environments under flow conditions. Special attention is given to the cellular aspects of in vitro BBB models, alongside discussions on advances in stem cell technologies, providing valuable insights into generating robust cellular systems for BBB modeling. The diverse array of cell types used in BBB modeling, depending on their sources, is meticulously examined in this comprehensive review, scrutinizing their respective derivation protocols and implications. By synthesizing diverse approaches, this review sheds light on the improvements of BBB models to capture physiological conditions, aiding in understanding BBB interactions in health and disease conditions to foster clinical developments.

THE ROLE OF INTRACELLULAR SIGNALING PATHWAYS IN THE DEVELOPMENT OF TROPHIC PATHOLOGIES OF THE LOWER EXTREMITIES AND THEIR REGENERATION UNDER TYPE 2 DIABETES (PART 1)

Introduction. This review article addresses the critical issue of the development and regeneration of chronic trophic ulcers in the context of type 2 diabetes. This pathological process is associated with inhibited cell proliferation, impaired differentiation of various cell types, and disrupted mechanisms that regulate cell death. An analysis of recent scientific literature also highlights the involvement of key intracellular signaling pathways in the development of chronic ulcerative pathologies of the lower extremities, as observed in both experimental animal models and patients with type II diabetes. Despite advancements, this issue remains insufficiently explored in both theory and practice, underscoring its ongoing relevance. The aim of this study is to identify the roles of key signaling pathways—transforming growth factor β (TGF-β), phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/Akt), and Wnt/β-catenin—in the inflammatory response, regenerative mechanisms, and healing processes of soft tissue damage and trophic ulcers in experimental animals and patients with type II diabetes. Materials and Methods. This study is based on an analysis of current scientific literature that addresses this topic. Results. It has been found out that changes in the content and activity of key molecules of signaling pathways lead to disruption of carbohydrate homeostasis and the occurrence of structural and functional dysfunction in damaged tissues against the background of type II diabetes. These include TGF-β, PI3K, Akt and β-catenin. Analysis of experimental data demonstrated that both under the conditions of type II diabetes development and in the occurrence of chronic ulcers of the lower extremities, against the background of this endocrine disease, there is an increase in the level of TGF-β. At the same time the activity of the PI3K/Akt signaling pathway in the above-mentioned studied groups was reduced. The relationship between the development of type II diabetes and the Wnt/β-catenin signaling pathway has been established. Suppression of its activity was accompanied by impaired regeneration of chronic trophic ulcers in type II diabetes. Conclusion. Thus, the mechanism of type II diabetes and chronic peptic ulcer disease, in the same pathology, is associated with a impaired activity of signaling cascades. This concerns the following cellular systems such as TGF-β, PI3K/Akt and Wnt/β-catenin. They can be considered as potential therapeutic targets for the development of newest methods for the treatment of chronic trophic ulcers in type II diabetes in order to accelerate the recovery process of volumetric tissue damage of the lower extremities.

Scalp condition improvement with botanical extracts possessing chemical and physical antioxidant activity.

Oxidative stress is implicated in scalp and hair health manifesting in several ways, including skin barrier, hair retention, healthy hair appearance and scalp sensation. We previously linked markers of oxidative damage to dandruff and skin barrier impairment and have linked key anti-dandruff technologies to the resolution of these issues. Here we expand the therapeutic space demonstrating many botanical extracts offer protective benefits against ROS stress via both chemical and biological antioxidant mechanisms. Chemical antioxidant activity of 10 botanical extracts was measured using oxygen radical absorbance capacity (ORAC) and biological antioxidant activity was measured using a Nrf-2 cell assay. A three-dimensional human keratinocyte skin equivalent model from MatTek was used to measure efficacy of reducing reactive oxygen species (ROS) from both the botanicals added as a solution and from a shampoo product. A 4-week consumer study with 56 panels was conducted with a leave-on treatment containing a botanical extract. Measurement of the oxidized lipids (9 and 13-Hydroxy-10E 12Z-octadecadienoic acid, HODE) extracted from scalp tape strips was made using gradient reversed-phase high-performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS). We demonstrated invitro that many botanical extracts exert antioxidant activity by quenching radicals in ORAC testing as well as activating biological antioxidant pathways via nrf-2 up-regulation in a cellular reporter system. Furthermore, we demonstrate these antioxidant activities for these botanicals in 3D skin models and ultimately show the reduction of lipid oxidation products in a consumer use context with a rosemary extract. Improved scalp condition can be achieved with incorporation of botanicals having the potential to exert antioxidant activity in hair/scalp care products.

Synthetic Biology in T-cell Engineering Research

Synthetic biology has emerged as a transformative discipline, enabling precise genetic and functional reprogramming of cellular systems. In T-cell engineering, it offers groundbreaking potential to revolutionize immunotherapy by endowing T cells with enhanced specificity, adaptability, and resilience against complex diseases such as cancer and autoimmune disorders. By integrating advanced genome-editing tools like CRISPR-Cas9 with modular synthetic constructs, researchers can design T cells with bespoke functionalities, such as tunable antigen recognition, controlled cytokine release, and resistance to immunosuppressive tumor microenvironments. This approach not only overcomes the limitations of conventional T-cell therapies but also facilitates the development of novel therapeutic paradigms, including "smart" cellular systems capable of sensing and responding to dynamic biological cues. Furthermore, synthetic circuits allow for the incorporation of logic-gated mechanisms to minimize off-target effects and enhance therapeutic precision. Despite these advancements, challenges remain in optimizing safety, scalability, and regulatory compliance. This research aims to explore the intersection of synthetic biology and T-cell engineering, highlighting cutting-edge methodologies, therapeutic applications, and emerging trends. By addressing current limitations and envisioning future possibilities, this work seeks to contribute to the growing body of knowledge driving synthetic biology toward clinical and industrial breakthroughs in cellular immunotherapy.

5G Analysis using Wi-Fi Analyzer Application

In the current cellular communication,5G connection is the most popular way to connect people together for real-time communication and data transmission. In order to improve the quality of service, researchers are studying the data generated from cellular communication systems to predict user. For the research analysis, Wi-Fi Analyzer applications that can measure parameters related to cellular communication and has been used as a measurement tool. The application shows the available signal networks in the nearby area together with the signal strength level. At the end of the research, it is concluded that the networks give good connection for the users

Systematizing cellular complexity: A Hilbertian approach to biological problems

Examining individual components of cellular systems has been successful in uncovering molecular reactions and interactions. However, the challenge lies in integrating these components into a comprehensive system-scale map. This difficulty arises due to factors such as missing links (unknown variables), overlooked nonlinearities in high-dimensional parameter space, downplayed natural noisiness and stochasticity, and a lack of focus on causal influence and temporal dynamics. Composite static and phenomenological descriptions, while appearing complicated, lack the essence of what makes the biological systems truly “complex.” The formalization of system-level problems is therefore important in constructing a meta-theory of biology. Addressing fundamental aspects of cellular regulation, adaptability, and noise management is vital for understanding the robustness and functionality of biological systems. These aspects encapsulate the challenges that cells face in maintaining stability, responding to environmental changes, and harnessing noise for functionality. This work examines these key problems that cells must solve, serving as a template for such formalization and as a step towards the axiomatization of biological investigations. Through a detailed exploration of cellular mechanisms, particularly homeostatic configuration, ion channels and harnessing noise, this paper aims to illustrate complex concepts and theories in a tangible context, providing a bridge between abstract theoretical frameworks and concrete biological phenomena.

Immune Cell-Derived Exosomes: A Cell-Free Cutting-Edge Tumor Immunotherapy.

Extracellular vesicles (EVs) are cellular communication molecules and are classified into three major subpopulations, such as microvesicles, apoptotic bodies, and exosomes. Among these, exosomes-based cancer research is a cutting-edge investigation approach to cancer understanding. During cancer progression , tumor-derived exosomes can reprogram the cellular system and promote cancer. Circulating exosomes in the body fluids such as blood, plasma, serum, saliva, CSF, sweat, and tears play a key role in identifying diagnostic and prognostic cancer biomarkers. Diverse therapeutic sources of exosomes including stem cells, plants, and immune cells, etc. exhibit significant cancer-healing properties. Although cancer-targeting immunotherapy is an effective strategy, it has limitations such as toxicity, and high costs. In comparison, immune cell-derived exosomes-based immunotherapy is a cell-free approach for cancer treatment and has advantages like less toxicity, biocompatibility, reduced immunogenicity, and efficient, target-specific cancer therapeutic development. This review highlights the therapeutic signature of immune cell-derived exosomes for cancer treatment.

Antioxidant, Apoptotic, and Wound Healing Effects of Xantolis Cambodiana Extracts in Normal Human Dermal Fibroblasts.

Xantolis cambodiana has demonstrated significant antioxidant properties; however, the mechanisms underlying its protective effects against oxidative stress in cellular systems remain unexplored. This work investigated the efficacy of methanolic extracts in exhibiting oxidative damage and examined their mechanisms. The methanolic extract had a high phenolic content (116.89±29.01 mg GAE/g FW) and exhibited scavenging of 2,2-diphenyl-1-picrylhydrazyl radicals with an IC50 value of 42.35±9.20 μg/ml. In addition, it had the highest antioxidant activity based on ferric-reducing antioxidant power (467.45±50.74 mg AA/100 g). Normal human dermal fibroblast (NHDF) cells were pretreated with the methanolic extract in a hydrogen peroxide (H2O2; 500 mM)-induced oxidative stress model, which resulted in a significant decrease in apoptosis and autophagy. Not only did the methanolic extract reduce mitochondrial membrane potential, it also stimulated NHDF cell migration and reduced reactive oxygen species production through mitochondrial dysfunction in the H2O2-induced stress model. These findings suggested that the methanolic extract (25 μg/ml) attenuated H2O2-induced oxidative stress in NHDF cells, significantly reducing apoptosis, autophagy, and mitochondrial dysfunction. Thus, this extract has the potential to support the wound healing process due to its antioxidant activity.

Clerodendrum trichotomum Extract Attenuates UV-B-Induced Skin Impairment in Hairless Mice by Inhibiting MAPK Signaling.

Exposure to solar ultraviolet-B (UV-B) radiation significantly accelerates skin aging by inducing the expression of matrix metalloproteinases (MMPs) such as MMP-1, leading to alterations in the extracellular matrix and consequent photoaging. Some plant components, renowned for their UV-absorbing and antioxidative properties, show potential for mitigating photoaging by reducing UV-B-induced MMP levels. In this context, we explored the inhibitory effects of Clerodendrum trichotomum extract (CTE) on UV-B-induced skin damage. The mechanism of CTE was predicted using network pharmacology approach. Also, antiaging efficacy was evaluated by mouse model and cellular system using human epidermal keratinocytes (HEKa), including its modulation of mitogen-activated protein kinase (MAPK) signaling pathways. CTE effectively counters UV-B-induced skin damage, as evidenced by the suppression of MMP-9 and MMP-1 expression in mice. We found that each fraction and chemical constituents of CTE suppressed UV-B-induced MMP-1 secretion in HEKa cells. CTE inhibits UV-B-induced skin aging by partially suppressing MMP-1 and MMP-9 secretion via the modulation of MAPK signaling pathways.

Stochastic geometry-based resource allocation scheme over cellular shotgun systems

Stochastic geometry-based resource allocation scheme over cellular shotgun systems

Research on the Correlation between Quantum Entanglement and Thinking Consciousness

The development of quantum technology has profoundly influenced research on consciousness. By analyzing the interaction of various microscopic particles in the universe, the article proposed that all things exhibit interconnected non-locality in both time and space, and that the interrelated connections of everything in the universe are based on quantum entanglement. Through analysis of the cellular and nervous system functions of organisms, as well as the influence of quantum science theories on consciousness and combined with research results in related fields, this paper presents the following viewpoints: first, life bodies exhibit quantum entanglement phenomena. Second, biological electricity phenomena are closely tied to consciousness. Third, both life bodies and non-life bodies are conscious, but different entities exhibit varying degrees of strong and weak consciousness. The strong and weak thinking consciousness influences the fluctuation of quantum and the size of quantum entanglement degree. Quantum fluctuations affect the consciousness of both life bodies and non-life bodies, and the entanglement quantum influences the degree of inductance between each other. Finally, the combination of quantum entanglement technology and thinking consciousness can realize the interconnection between human beings and the universe. Consciousness may affect the results measured in quantum mechanics when people observe. However, the macroscopic world mainly adopts the laws of classical physics to solve problems in life. Under certain conditions, the law of quantum physics and the law of classical physics can affect people’s lives together. No matter what angle we view the material world, it is real, and the universe is not illusory.

Comprehensive Analysis of Codon Usage Bias in Human Papillomavirus Type 51.

Human papillomavirus type 51 (HPV-51) is associated with various cancers, including cervical cancer. Examining the codon usage bias of the organism can offer valuable insights into its evolutionary patterns and its relationship with the host. This study comprehensively analyzed codon usage bias in HPV-51 by examining 64 complete genome sequences sourced from the NCBI GenBank database. Our analysis revealed no noteworthy preference for codon usage in HPV-51 overall. However, there was a noticeable bias towards A/T-ending codons, accompanied by GC3s below 32%. Dinucleotide frequency analysis revealed reduced frequencies for ApA, CpG, and TpC dinucleotides, while CpA and TpG dinucleotides were more frequent than others. Relative Synonymous Codon Usage analysis revealed 30 favored codons, primarily concluding with A/T nucleotides. Further analysis using Parity Rule 2, Effective Number of Codons plot, and neutrality plot indicated a balance between mutational pressure and natural selection, with natural selection being the primary force shaping codon usage bias. The Isoacceptor tRNA Pool analysis indicates that HPV-51 has a higher translation efficiency within the human cellular translational system. Moreover, the Codon Adaptation Index and Relative Codon Deoptimization Index analyses suggested a moderate adaptation of HPV-51 to human codon preferences. Our discoveries offer valuable perspectives on how HPV-51 evolves and uses genetic codes, contributing to a deeper comprehension of its endurance and disease-causing potential.

Zinc oxide nanoparticles at low dose mitigate lead toxicity in pea (Pisum sativum L.) seeds during germination by modulating metabolic and cellular defense systems

Addressing lead (Pb) pollution and contamination in soil and agriculture is urgent due to its widespread and long-lasting impacts on human health, food safety, and the environment. In the current study, we assessed the potential use of Zinc oxide nanoparticles (ZnONPs) in alleviating Pb toxicity in germinating seedlings. Pea (Pisum sativum L.) seedlings were exposed to Pb (83 mg/L) over a 7-day period, without and with ZnONPs amendment. Our results showed that the application of ZnONPs at 10 mg/L restored the morphological parameters affected by Pb, 34 %, 26 % and 23 %, for the length, fresh, and dry biomass of embryonic axis, respectively. ZnONPs decreased the activities of antioxidative enzymes of catalase (87.5 %), guaiacol peroxidase (60 %), and glutathione peroxidase (25 %), but increased the activities of ascorbate peroxidase (60 %) and glutathione reductase (25 %). These changes were associated with the increase (7 %) in the thiol groups, and reductions in the malondialdehyde (23 %), hydrogen peroxide (33 %), and carbonyl group levels (78 %), in addition to the stimulation of the activities of ROS-associated enzymes, including glycolate oxidase (40 %) and NADPH oxidase (by 89 %). Consequently, cell viability was increased by 66 %, while cellular death was reduced by 10 %, with ZnONPs, relative to the Pb-stressed seedlings. These findings highlight the mechanisms surrounding the ability of nanosized ZnO to mitigate the harmful effects of Pb on the growth and physiology of plants. Further investigation is needed to understand the mitigating effects of ZnONPs on Pb on the molecular and genomic levels in seedlings and plants, and ensure the sustainability of agricultural practices and food safety. Besides, site and source-specific integrated approaches must be practiced to formulate suitable remediation strategies.

Modulating TTN gene expression in human iPSC-CMs to investigate mechanisms and therapies in TTN-associated dilated cardiomyopathy

Abstract Background Familial dilated cardiomyopathy (DCM) is a common and harmful condition, and no available therapies target the underlying genetic mechanisms. Truncating variants in TTN are the most commonly identified genetic cause of DCM. However, it remains unclear how TTN variants drive disease. Contemporary thinking identifies haploinsufficiency, dominant negative effects, or a combination of both as likely driving mechanisms. There is competing evidence as to whether I-band and A-band mutations differ in severity or in the relative roles of haploinsufficiency and dominant-negative effects. Clarifying disease mechanisms will help pave the way to targeted genetic therapies. Purpose To modulate TTN gene expression in human-induced stem cell-derived cardiomyocyte (hiPSC-CM) models of TTN-associated DCM carrying I-band or A-band variants to provide insight into the comparative roles of haploinsufficiency and dominant negative mechanisms in DCM, and to identify novel therapeutic strategies. Methods We used CRISPR-Cas9 gene-editing to generate hiPSC lines with known disease-causing heterozygous truncating mutations in the I-band or A-band of TTN. These were screened for off-target mutations and pluripotency. hiPSCs were differentiated into hiPSC-CMs for phenotyping. We assayed TTN mRNA and titin protein levels. We trialled approaches for modulating TTN expression including CRISPR activation. Results Two hiPSC lines with heterozygous mutations in the I-band and A-band of TTN were generated using CRISPR/Cas-9 gene-editing. These hiPSC lines were successfully differentiated into hiPSC-CMs. Both cell lines demonstrated unchanged TTN mRNA expression compared to wildtype controls, but had reduced expression of full-length titin protein and identifiable truncated protein products. TTN mRNA expression was subsequently increased in these models using modulators of gene expression. Conclusions hiPSC-CM models of DCM with truncating mutations in the I-band or A-band of TTN were generated and phenotyped. These models were then used to trial gene expression modulation methods. This provides a human cellular system that can be used to better understand disease mechanisms of TTN-associated DCM, with the ultimate aim of establishing gene therapies for TTN-associated DCM.

Nucleotide-mediated modulation of chemoselective protein functionalization in a liquid-like condensed phase

Liquid-like protein condensates are ubiquitous in cellular system and are increasingly recognized for their roles in physiological processes. Condensed phase harbors distinctive chemical microenvironment, markedly different than dilute aqueous phase. Herein, we demonstrate chemoselective modification pattern of nucleophilic canonical amino acid sidechains (namely – cysteine, tyrosine and lysine) of the protein towards 4-chloro-7-nitrobenzofurazan in the dilute and condensed phase. We also delineate how the effect of nucleotides and their in situ enzymatic dissociation temporally modulate the protein condensate’s pH and the protein’s corresponding chemoselective modification. We have shown that the pH of the condensate decreases in the presence of nucleoside triphosphate, whereas it increases in the presence of nucleoside monophosphates or phosphate ion. For instance, we find lysine-specific modification gets inhibited in the presence of adenosine triphosphate (ATP), but significantly enhanced in the presence of monophosphates. This feature enables us to gain temporal control over dynamic change in protein functionalization via enzymatic ATP hydrolysis. Overall, this work substantiates the alteration in pH-responsiveness of Brønsted basicity of a protein’s ε-amine in the condensed phase. Furthermore, this environment sensitivity in chemoselective protein functionalization in condensed phase will be important in adaptable protein engineering to the chemical biology of protein phase separation.

Genome organization and stability in mammalian pre-implantation development

A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the de novo establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation.