Experimental Challenge Research Articles

Inferring the energy cost of resistance to parasitic infection and its link to a trade-off

BackgroundIn infected hosts, immune responses trigger a systemic energy reallocation away from energy storage and growth, to fuel a costly defense program. The exact energy costs of immune defense are however unknown in general. Life history theory predicts that such costs underpin trade-offs between host disease resistance and other fitness related traits, yet this has been seldom assessed. Here we investigate immune energy cost induced by infection, and their potential link to a trade-off between host resistance and fat storage that we previously exposed in sheep divergently selected for resistance to a pathogenic helminth.ResultsTo this purpose, we developed a mathematical model of host-parasite interaction featuring individual changes in energy allocation over the course of infection. The model was fitted to data from an experimental infectious challenge in sheep from genetically resistant and susceptible lines to infer the magnitude of immune energy costs. A relatively small and transient immune energy cost in early infection best explained within-individual changes in growth, energy storage and parasite burden. Among individuals, predicted responses assuming this positive energy cost conformed to the observed trade-off between resistance and storage, whereas a cost-free scenario incorrectly predicted no trade-off.ConclusionsOur mechanistic model fitting to experimental data provides novel insights into the link between energy costs and reallocation due to induced resistance within-individual, and trade-offs among individuals of selected lines. These will be useful to better understand the exact role of energy allocation in the evolution of host defenses, and for predicting the emergence of trade-offs in genetic selection.

Deep learning aided determination of the optimal number of detectors for photoacoustic tomography.

Photoacoustic tomography (PAT) is a non-destructive, non-ionizing, and rapidly expanding hybrid biomedical imaging technique, yet it faces challenges in obtaining clear images due to limited data from detectors or angles. As a result, the methodology suffers from significant streak artifacts and low-quality images. The integration of deep learning (DL), specifically convolutional neural networks (CNNs), has recently demonstrated powerful performance in various fields of PAT. This work introduces a post-processing-based CNN architecture named residual-dense UNet (RDUNet) to address the stride artifacts in reconstructed PA images. The framework adopts the benefits of residual and dense blocks to form high-resolution reconstructed images. The network is trained with two different types of datasets to learn the relationship between the reconstructed images and their corresponding ground truths (GTs). In the first protocol, RDUNet (identified as RDUNet I) underwent training on heterogeneous simulated images featuring three distinct phantom types. Subsequently, in the second protocol, RDUNet (referred to as RDUNet II) was trained on a heterogeneous composition of 81% simulated data and 19% experimental data. The motivation behind this is to allow the network to adapt to diverse experimental challenges. The RDUNet algorithm was validated by performing numerical and experimental studies involving single-disk, T-shape, and vasculature phantoms. The performance of this protocol was compared with the famous backprojection (BP) and the traditional UNet algorithms. This study shows that RDUNet can substantially reduce the number of detectors from 100 to 25 for simulated testing images and 30 for experimental scenarios.

Etiological characterization of multiple recombinant lineages of TJ-C6 porcine reproductive and respiratory syndrome virus in Tianjin, Northern China.

Recent outbreaks of PRRSV in live attenuated vaccine-immunized pig farms in Tianjin, China have raised questions about the etiological characteristics and pathogenicity of the PRRSV variant, which remains unknown. In this study, a multiple lineages recombinant PRRSV strain named TJ-C6, was isolated and identified. Phylogenetic trees and genome homology analyses revealed that TJ-C6 belonged to lineage 1.8 (NADC30-like) and with similar 131 discontinuous amino acid deletion pattern (111-aa + 1-a + 19-aa) in Nsp2-coding region, but it was classified in lineage 1.5 (NADC34-like) cluster based on ORF5 sequence. Furthermore, the recombination analyses revealed that TJ-C6 was a multiple recombinant virus among lineage 1.5(NADC34- like), lineage 1.8(NADC30- like), and lineage 3(GM2-Like) strains with four recombination breakpoints in Nsp9 (nt 7298/8111), ORF2 (nt 12213) and ORF6 (nt 14628), which was different from the previously prevalent PRRSV strain. Challenge experiments with 3-week-old piglets showed that TJ-C6 could cause piglets high fever, loss of appetite and severely histopathological lung lesions. Taken altogether, multiple co-circulating lineages of PRRSV strains in the swine population are accelerating the emergence of natural recombinant strains with variations in pathogenicity and highlight the importance of surveillance of newly emerging PRRSV strains in China.

Effects of Enterocytozoon hepatopenaei infection on intestinal immune defense and physiological processes of Penaeus vannamei

As the main pathogen causing growth retardation, EHP is considered to be mainly parasitic in the hepatopancreas of shrimp. However, the intestines of shrimp infected with EHP frequently exhibit syndromes such as jejunum and white midgut. Therefore, the challenge experiment was carried out in this study to compare the differences in intestinal histology, digestion and absorption, immune defense and oxidative stress of P. vannamei between the control group and EHP infection group. Histological analysis showed that EHP infection significantly damaged the intestine of the shrimp, including intestinal villus rupture and outer membrane impairment. Concurrently, EHP infection can trigger intestinal immune response, and the expression of key immune genes like Toll, myeloid differentiation factor, anti-lipopolysaccharide factor, and Relish was significantly enhanced, while the expression of IMD and alkaline phosphatase was suppressed. Additionally, antioxidant genes manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, and nuclear factor E2-related factor 2 were up-regulated to varying extents in EHP infection group, and the contents of lipid peroxides and malondialdehyde were heavily accumulated. Moreover, the expression levels of key genes involved in nutrient absorption, transport and synthesis, such as glucose transporter 1, Na+-K+ATPase, fatty acid synthase, acetyl-CoA carboxylase, rapamycin kinase, mTOR regulation-related protein, eukaryotic translation initiation factor 4E binding protein, ribosomal protein S6 kinase, were significantly up-regulated. However, the activities of amylase, lipase, and trypsin were inhibited in EHP infection group throughout the experiment. In summary, EHP infection damaged the intestine of P. vannamei, accompanied by immune response and oxidative stress. At the same time, nutrient transport and synthesis pathways were activated, while digestive enzyme activities were inhibited, indicating that in order to maintain survival, shrimps must accelerate material transport. Unfortunately, it remains in a state of nutrient deficiency that ultimately affects growth.

Study on the response mechanism of MicroRNA novel-13 and novel-44 to Vibrio parahaemolyticus infection in Pinctada fucata martensii

Pinctada fucata martensii (P. f. martensii) is one of the main pearl oysters cultured in artificial seawater in China. However, it is highly susceptible to pathogen infection under intensive cultivation near the coast. MicroRNAs (miRNAs), as an innovative and potent regulator of immune function, play a pivotal role in the immune response of pearl oysters to external stimuli and are a potent marker for the response of P. f. martensii to infection. This study identified two novel miRNAs, novel-13 and novel-44, from the whole transcriptome of the P. f. martensii hemocyte before and after infection with Vibrio parahaemolyticus. The dual luciferase results showed that novel-13 negatively regulated LAAO and novel-44 negatively regulated ILK. The activity of antioxidant-related enzymes increased significantly in the synthetic miRNA (novel-13 and novel-44) inhibitors and decreased significantly in the synthetic miRNA mimics. In the challenge experiment, injection with miRNA inhibitor increased the relative survival percentage by 10% compared with the control group. In conclusion, the overexpression of novel-13 and novel-44 can decrease the activity of immune and antioxidant-related enzymes, possibly affecting immune regulation in P. f. martensii by negatively regulating the LAAO and ILK target genes.

Theoretical Design for Thorium-Containing Two-Dimensional Materials.

Actinide elements are characterized by their unique electronic correlations, variable valence states, and localized 5f electrons, leading to unconventional electronic and topological properties in their compounds. The distinctive physical properties of actinide materials are maintained in low-dimensional forms, yet two-dimensional (2D) actinide materials remain largely unexplored due to their scarcity and the experimental challenges posed by their radioactivity. To fill the knowledge gap in 2D actinide materials, we theoretically designed a series of stable thorium-containing 2D materials, including MXenes, chalcogenides, halides, and other compounds with unique structures. These novel thorium-containing 2D materials show excellent thermodynamic, mechanical, and dynamical stability. Their electronic structures and potential applications were investigated. Considering the proper band edge positions, strong visible light absorption, and effective separation of photogenerated electron-hole pairs, ThPSe3 is proposed as a promising photocatalyst for water splitting. Our work significantly expands the 2D actinide materials family, and further opens new avenues for their experimental realization and applications.

Recombinant probiotic Escherichia coli delivers the polymeric protein of swine influenza virus for protection.

Swine influenza virus invades the host through the respiratory mucosa, which severely restricts the development of the pig breeding industry. To construct monomeric and trimeric vaccines, we developed recombinant Escherichia coli Nissle 1917 (EcN) strains that express the receptor binding site (RBS) of the hemagglutinin (HA) antigen from H1N1 swine influenza virus. After the mucosal immunization of mice, we found that probiotics activated CD40 and CD86 in DCs and increased the levels of IL-4 and IFN-γ secretion by T cells. Furthermore, the probiotics improved the function of the mucosal immune system, increased the level of SIgA, level of IgG and number of B220+IgA+, and activated germinal center B cells. The challenge experiment revealed that the probiotics alleviated weight loss, reduced pathological injury to the lungs, and protected the mice from virus infection. We also observed that the serum neutralizing antibodies of immunized piglets significantly increased, which reduced the shedding frequency of swine influenza virus in the nose of the piglets and reduced the pathological damage by activating the T cell immune response in infected piglets. Thus, the constructed probiotics are promising candidates for effective non-traditional swine influenza vaccines.

A Study on the Induction of Multi-Type Immune Responses in Mice via an mRNA Vaccine Based on Hemagglutinin and Neuraminidase Antigen.

The Influenza A virus (IAV), a pathogen affecting the respiratory system, represents a major risk to public health worldwide. Immunization remains the foremost strategy to control the transmission of IAV. The virus has two primary antigens: hemagglutinin (HA) and neuraminidase (NA). Our previous studies have demonstrated that an IAV NA mRNA vaccine can induce Th1-type immune responses in mice. This research examined the immune responses elicited by an mRNA vaccine targeting both HA and NA antigens in murine models. In this study, we used two dual-antigen immunization strategies: single-site immunization with an IAV HA+NA mRNA vaccine and multi-site immunization with an IAV HA mRNA vaccine and IAV NA mRNA vaccine. Hemagglutination-inhibiting antibody titer and neutralizing antibody titer in the sera of immunized mice were evaluated, and a viral challenge experiment was conducted. Additionally, the immune responses elicited by the two immunization strategies were characterized using flow cytometry and ELISA. Comparative analyses were performed with mice immunized individually with the IAV HA mRNA vaccine, IAV NA mRNA vaccine, and inactivated vaccine. The results showed that by using a multi-site immunization strategy, mice were able to generate higher levels of hemagglutination-inhibiting and neutralizing antibodies, and were protected in a viral challenge experiment. Moreover, the multi-site regimen also promoted the generation of cytotoxic T cells and maintained a balanced Th1/Th2 immune response. Using mRNA vaccine based on a HA and NA antigen with multi-site immunization strategy can induce higher levels of hemagglutination-inhibiting and neutralizing antibodies, and multi-type immune responses in mice, providing new theoretical and experimental support for advancing upcoming influenza vaccines.

What is the experimentalist challenge to the method of cases?

This paper puts forward a new formulation of the experimentalist challenge to the method of cases. Unlike previous attempts to articulate the challenge, the one proposed is based on a clear characterization of the targeted philosophical methodology. The method of cases is explicated as a form of thought experimentation aimed at testing philosophical hypotheses with a distinctive modal force. Given this explication, the empirical evidence gathered by experimental philosophers concerning the instability of case judgments is shown to constitute a stumbling block for the validation of the method as a rational form of hypothesis testing. The experimental challenge is presented as the challenge to compellingly indicate how this stumbling block can be removed, and the method of cases validated as a form of thought experimentation suitable for rationally testing philosophical hypotheses. The challenge, as presented in the text, is shown to be free from all the main criticisms leveled at previous versions of it and as one not yet met.

Comparison of bison and elk susceptibility to experimental challenge with Brucella abortus strain 2308

IntroductionBrucellosis is endemic in bison and elk in Yellowstone National Park and surrounding areas.MethodsA comparative study was conducted using data from naive (n = 82 and 67, respectively) and Brucella abortus strain RB51 (RB51) vaccinated (n-99 and 29, respectively) bison and elk experimentally challenged with virulent B. abortus strain during pregnancy.ResultsThe incidence of abortion, fetal infection, uterine or mammary infection, or infection in maternal tissues after experimental challenge was greater (p < 0.05) in naïve and vaccinated bison when compared to similar groups in elk. Vaccinated bison had lower (p < 0.002) abortion rates and recovery of Brucella from fetal or uterine/mammary tissues when compared to naïve bison. Vaccinated elk had reduced (p < 0.01) rates of maternal infection, but rates of abortion and fetal or uterine/mammary infection did not differ (p > 0.05) from naïve elk. Naïve and vaccinated bison had greater (p < 0.05) Brucella colonization in placentomes, and parotid and supramammary lymphatic tissues when compared to elk. In elk or bison that aborted, mean colonization in placentome tissues were typically more than 5 logs higher than in animals that did not abort.DiscussionThe results of our study suggest differences in disease pathogenesis between these two wildlife reservoirs of B. abortus.

A comparative evaluation of antibiotic and synbiotic supplementation on production performance and necrotic enteritis severity in broilers during an experimental necrotic enteritis challenge

The ban on antibiotics in the poultry diet resulted in re-emergence of several infectious diseases including necrotic enteritis (NE). These infectious diseases are leading to poor health and welfare as well as production and economic loss. Synbiotic could be a potential candidate to replace the antibiotics in poultry diet. Therefore, a 35-day study was conducted to compare the efficacy of synbiotic (PoultryStar®ME) and antibiotic (Stafac®50, Virginiamycin) supplementation during an experimentally induced necrotic enteritis infection. A total of 360 day-old chicks were randomly assigned to four treatment groups: Antibiotic, Challenge + Antibiotic, Synbiotic, and Challenge + Synbiotic, each with 6 replicates. The treatment groups referred as “Challenge + Antibiotic” and “Challenge + Synbiotic” were challenged, while their respective non-challenged treatment groups were “antibiotic” and “synbiotic”. NE in birds was induced by gavaging 1 × 104 oocysts of Eimeria maxima on day 14 (D14) and 1 × 108 CFU/mL of Clostridium perfringens on D19, 20, and 21. Both synbiotic and antibiotic supplementation during the NE challenge did not improve BW gain, feed intake, and feed conversion ratio at the end of the experiment (D0-35). However, antibiotic supplementation reduced mortality during the week of the challenge (D14-21) (P < 0.001). At D21, both synbiotic and antibiotic supplementation during the NE challenge did not decrease the intestinal lesion score (P < 0.001) compared to their respective non-challenged treatment groups. At D21, synbiotic supplementation during the NE challenge did not decrease intestinal permeability (P = 0.04) compared to the synbiotic group. At D21, antibiotic supplementation during the NE challenge increased the CD4+:CD8+ T cells (P < 0.001) in the cecal tonsil. It can be concluded that synbiotic supplementation elicited an immune response, decreasing the inflammatory response in the intestine and ameliorating the NE infection. Therefore, synbiotic could be a potential alternative to replace antibiotics in the poultry industry, but their efficacy needs to be improved through blending additional probiotics and prebiotics, and further exploration is required.

The Resurging of Hydrocarbon Gas as Early Sign of Battery Rollover Degradation.

Gas analysis offers real-time critical insights into the various processes occurring within batteries. However, monitoring battery degradation through gas formation remains relatively underexplored. Traditional coin cell setups pose challenges for long-cycle experiments and do not accurately reflect real-life battery usage. In this study, online electrochemical mass spectrometry (OEMS) was used to monitor gas evolution in a 4500 mAh NMC/graphite 21700 cylindrical battery throughout its cycling life. By tracking gas species and quantities, we identified distinct phases of gas evolution corresponding to different stages of degradation. Notably, the resurgence of hydrocarbon gases such as C2H4 emerged as an early indicator of rollover degradation. Furthermore, the continuous dissolution and reformation of the solid-electrolyte interphase (SEI) layer was identified as the primary driver of battery degradation. This study demonstrates the potential for integrating miniature gas sensors into battery systems for real-time health monitoring and early degradation detection.

Engineered Mycobacterium tuberculosis triple-kill-switch strain provides controlled tuberculosis infection in animal models.

Human challenge experiments could accelerate tuberculosis vaccine development. This requires a safe Mycobacterium tuberculosis (Mtb) strain that can both replicate in the host and be reliably cleared. Here we genetically engineered Mtb strains encoding up to three kill switches: two mycobacteriophage lysin operons negatively regulated by tetracycline and a degron domain-NadE fusion, which induces ClpC1-dependent degradation of the essential enzyme NadE, negatively regulated by trimethoprim. The triple-kill-switch (TKS) strain showed similar growth kinetics and antibiotic susceptibilities to wild-type Mtb under permissive conditions but was rapidly killed in vitro without trimethoprim and doxycycline. It established infection in mice receiving antibiotics but was rapidly cleared upon cessation of treatment, and no relapse was observed in infected severe combined immunodeficiency mice or Rag-/- mice. The TKS strain had an escape mutation rate of less than 10-10 per genome per generation. These findings suggest that the TKS strain could be a safe, effective candidate for a human challenge model.

Enhanced Reaction Kinetics in Stationary Two-Phase Flow through Porous Media.

Understanding the interaction between multiphase flow and reactive transport in porous media is critical for many environmental and industrial applications. When a nonwetting immiscible phase is present within the pore space, it can remain immobile, which we call unsaturated flow, or move, resulting in multiphase flow. Previous studies under unsaturated flow conditions have shown that, for a given flow rate, the product of a mixing-driven reaction increases as wetting phase saturation decreases. Conversely, the opposite effect is observed for a given Péclet number (i.e., the flow rate is adapted depending on the wetting phase saturation). However, the impact of multiphase flow dynamics on mixing-driven reactions is poorly understood due to experimental and numerical challenges. To assess the impact of multiphase flow conditions on product formation, we use an optimized chemiluminescence reaction and an experimental setup that allows the separate injection of reactants along with a stationary two-phase flow. In our experiments, the mass of the reaction product under stationary two-phase flow conditions increases faster than Fickian beyond the diffusive time. The global kinetics initially increase before experiencing a monotonic decrease with significant fluctuations caused by the displacement of the nonwetting phase. For a given flow rate of the wetting phase, product formation depends on the flow rate of the nonwetting immiscible phase.

Roadmap for Borophene Gas Sensors.

Borophene, a two-dimensional allotrope of boron, has emerged as a promising material for gas sensing because of its exceptional electronic properties and high surface reactivity. This review comprehensively overviews borophene synthesis methods, properties, and sensing applications. However, it is crucial to acknowledge the substantial gap between the abundance of theoretical literature and the limited experimental studies. While theoretical investigations have elucidated the stability and remarkable sensing capabilities of various borophene polymorphs across different gases, significant experimental challenges have hindered the translation of these theoretical predictions into practical devices. Consequently, this review carefully studies these challenges and shortcomings that are jeopardizing the practical implementation of borophene in real-world settings. Specifically, four key issues are thoroughly studied, such as superficial borophene oxidation upon exposure to the air, interference from relative humidity on gas molecule detection, lack of selectivity, and synthesis scalability. Finally, novel strategies are proposed to overcome these bottlenecks. By adopting these approaches, borophene can pave the way to drive the advancement of the next generation of sensing devices.

Gig Work at What Cost? Exploring Privacy Risks of Gig Work Platform Participation in the U.S.

In recent years, "gig work" platforms have gained popularity as a way for individuals to earn money; as of 2021, 16% of Americans have at some point earned money from such platforms. Despite their popularity and their history of unfair data collection practices and worker safety, little is known about the data collected from workers (and users) by gig platforms and about the privacy dark pattern designs present in their apps. This paper presents an empirical measurement of 16 gig work platforms' data practices in the U.S. We analyze what data is collected by these platforms, and how it is shared and used. Finally, we consider how these practices constitute privacy dark patterns. To that end, we develop a novel combination of methods to address gig-worker-specific challenges in experimentation and data collection, enabling the largest in-depth study of such platforms to date. We find extensive data collection and sharing with 60 third parties—including sharing reversible hashes of worker Social Security Numbers (SSNs)—along with dark patterns that subject workers to greater privacy risk and opportunistically use collected data to nag workers in off-platform messages. We conclude this paper with proposed interdisciplinary mitigations for improving gig worker privacy protections. After we disclosed our SSN-related findings to affected platforms, the platforms confirmed that the issue had been mitigated. This is consistent with our independent audit of the affected platforms. Analysis code and redacted datasets will be made available to those who wish to reproduce our findings.

Effect of microencapsulated Fiber2-displaying probiotics loaded with inulin nanoparticles on immunity against fowl adenovirus serotype 4 in chickens.

In this study, phthalate inulin nanoparticles (PINs) were chemically modified and characterized. The internalization of PINs into the probiotic E. faecalis, which delivering Fiber2 protein of fowl adenovirus serotype 4 (FAdV-4), was investigated. The expression of the Fiber2 protein in E. faecalis was detected using western blot analysis. To protect recombinant E. faecalis from degradation of in the gastric acid environment, sodium alginate was used to encapsulate the bacteria. The survival ratio and release of E. faecalis in simulated gastrointestinal fluid was assessed. Oral administration of microencapsulated E. faecalis loaded with PINs (Micro-E/Fiber2-PINs) or inulin (Micro-E/Fiber2-inulin) was conducted, followed by an experimental challenge with FAdV-4 in chickens to evaluate immune responses and protection. The results showed the internalization of PINs into the bacteria promoted bacteria growth, and significantly improved the expression level of Fiber2. After incubation in simulated gastric fluid, the number of viable bacteria from the Micro-E/Fiber2-PINs group was significantly higher than that from the E. faecalis/Fiber2 group. The release of bacteria from the microcapsules was completed within 30 min. Animal experiments demonstrated that oral immunization with Micro-E/Fiber2-PINs significantly enhanced humoral and cellular immune responses, relieved inflammatory injury in FAdV-targeted organs, and improved survival rate of challenged chickens. This study presents promising potential for developing oral vaccines against pathogen infection.

Studying the Function of tRNA Modifications: Experimental Challenges and Opportunities.

tRNAs are essential molecules in protein synthesis, responsible for translating the four-nucleotide genetic code into the corresponding amino acid sequence. RNA modifications play a crucial role in influencing tRNA folding, structure, and function. These modifications, ranging from simple methylations to complex hypermodified species, are distributed throughout the tRNA molecule. Depending on their type and position, they contribute to the accuracy and efficiency of decoding by participating in a complex network of interactions. The enzymatic processes introducing these modifications are equally intricate and diverse, adding further complexity. As a result, studying tRNA modifications faces limitations at multiple levels. This review addresses the challenges involved in manipulating and studying the function of tRNA modifications and discusses experimental strategies and possibilities to overcome these obstacles.

Measurement and modeling of excess pore-water pressure in warm saturated frozen soil based on dynamic loading effect

Measuring pore-water pressure (PWP) in frozen soils poses significant challenges in geotechnical testing experiments, and understanding PWP is crucial for unraveling the mechanism of frost heave generation in cold regions. This paper aims to clarify the development pattern of PWP in frozen soil through laboratory tests, specifically focusing on excess PWP generated under dynamic loading. Seven sets of triaxial tests were conducted to investigate the variations in excess PWP and deformation influenced by temperature, dynamic stress amplitude, and dry density. The results reveal that excess PWP in warm saturated frozen soil undergoes two stages: pore pressure increase and dissipation. The change of external factors mainly affects the peak value of excess PWP and the change rate of excess PWP. Unlike unfrozen soil, excess PWP has a small dissipation rate after the peak and may remain dynamically stable in the later stage of loading. In addition, two empirical models of excess PWP applicable to saturated frozen soils were proposed based on the developmental patterns of excess PWP in frozen soils, and the feasibility was validated using the results obtained from laboratory tests. The model is of great significance for predicting the development of excess PWP in frozen soil under dynamic load.

Recording Lineage History with Cellular Barcodes in the Mammary Epithelium and in Breast Cancer.

Lineage tracing methods have extensively advanced our understanding of physiological cell behaviour in vivo and in situ and have vastly contributed to decipher the phylogeny and cellular hierarchies during normal and tumour development. In recent years, increasingly complex systems have been developed to track thousands of cells within a given tissue or even entire organisms. Cellular barcoding comprises all techniques designed to genetically label single cells with unique DNA sequences or with a combination of fluorescent proteins, in order to trace their history and lineage production in space and time. We distinguish these two types of cellular barcoding as genetic or optical barcodes. Furthermore, transcribed cellular barcodes can integrate the lineage information with single-cell profiling of each barcoded cell. This enables the potential identification of specific markers or signalling pathways defining distinct stem cell states during development, but also signals promoting tumour growth and metastasis or conferring therapy resistance.In this chapter, we describe recent advances in cellular barcoding technologies and outline experimental and computational challenges. We discuss the biological questions that can be addressed using single-cell dynamic lineage tracing, with a focus on the study of cellular hierarchies in the mammary epithelium and in breast cancer.