Size Of Cell Population Research Articles

Sensory neurons regulate stimulus-dependent humoral immunity in mouse models of bacterial infection and asthma

Sensory neurons sense pathogenic infiltration to drive innate immune responses, but their role in humoral immunity is unclear. Here, using mouse models of Streptococcus pneumoniae infection and Alternaria alternata asthma, we show that sensory neurons are required for B cell recruitment and antibody production. In response to S. pneumoniae, sensory neuron depletion increases bacterial burden and reduces B cell numbers, IgG release, and neutrophil stimulation. Meanwhile, during A. alternata-induced airway inflammation, sensory neuron depletion decreases B cell population sizes, IgE levels, and asthmatic characteristics. Mechanistically, during bacterial infection, sensory neurons preferentially release vasoactive intestinal polypeptide (VIP). In response to asthma, sensory neurons release substance P. Administration of VIP into sensory neuron-depleted mice suppresses bacterial burden, while VIPR1 deficiency increases infection. Similarly, exogenous substance P delivery aggravates asthma in sensory neuron-depleted mice, while substance P deficiency ameliorates asthma. Our data, thus demonstrate that sensory neurons release select neuropeptides which target B cells dependent on the immunogen.

A somatic genetic clock for clonal species

Age and longevity are key parameters for demography and life-history evolution of organisms. In clonal species, a widespread life history among animals, plants, macroalgae and fungi, the sexually produced offspring (genet) grows indeterminately by producing iterative modules, or ramets, and so obscure their age. Here we present a novel molecular clock based on the accumulation of fixed somatic genetic variation that segregates among ramets. Using a stochastic model, we demonstrate that the accumulation of fixed somatic genetic variation will approach linearity after a lag phase, and is determined by the mitotic mutation rate, without direct dependence on asexual generation time. The lag phase decreased with lower stem cell population size, number of founder cells for the formation of new modules, and the ratio of symmetric versus asymmetric cell divisions. We calibrated the somatic genetic clock on cultivated eelgrass Zostera marina genets (4 and 17 years respectively). In a global data set of 20 eelgrass populations, genet ages were up to 1,403 years. The somatic genetic clock is applicable to any multicellular clonal species where the number of founder cells is small, opening novel research avenues to study longevity and, hence, demography and population dynamics of clonal species.

RIPK1 protects naive and regulatory T cells from TNFR1-induced apoptosis

The T cell population size is stringently controlled before, during, and after immune responses, as improper cell death regulation can result in autoimmunity and immunodeficiency. RIPK1 is an important regulator of peripheral T cell survival and homeostasis. However, whether different peripheral T cell subsets show a differential requirement for RIPK1 and which programmed cell death pathway they engage in vivo remains unclear. In this study, we demonstrate that conditional ablation of Ripk1 in conventional T cells (Ripk1ΔCD4) causes peripheral T cell lymphopenia, as witnessed by a profound loss of naive CD4+, naive CD8+, and FoxP3+ regulatory T cells. Interestingly, peripheral naive CD8+ T cells in Ripk1ΔCD4 mice appear to undergo a selective pressure to retain RIPK1 expression following activation. Mixed bone marrow chimeras revealed a competitive survival disadvantage for naive, effector, and memory T cells lacking RIPK1. Additionally, tamoxifen-induced deletion of RIPK1 in CD4-expressing cells in adult life confirmed the importance of RIPK1 in post-thymic survival of CD4+ T cells. Ripk1K45A mice showed no change in peripheral T cell subsets, demonstrating that the T cell lymphopenia was due to the scaffold function of RIPK1 rather than to its kinase activity. Enhanced numbers of Ripk1ΔCD4 naive T cells expressed the proliferation marker Ki-67+ despite the peripheral lymphopenia and single-cell RNA sequencing revealed T cell-specific transcriptomic alterations that were reverted by additional caspase-8 deficiency. Furthermore, Ripk1ΔCD4Casp8 ΔCD4 and Ripk1ΔCD4Tnfr1−/− double-knockout mice rescued the peripheral T cell lymphopenia, revealing that RIPK1-deficient naive CD4+ and CD8+ cells and FoxP3+ regulatory T cells specifically die from TNF- and caspase-8-mediated apoptosis in vivo. Altogether, our findings emphasize the essential role of RIPK1 as a scaffold in maintaining the peripheral T cell compartment and preventing TNFR1-induced apoptosis.

An Investigation of Size Distribution and Calcium Signaling in Human Platelets.

Background Platelets are thin disc-shaped blood cells that play a major role in hemostasis, maintenance of vascular integrity, and blood coagulation. Large platelets are more reactive and seen in patients with cardiovascular disease. This study aims to analyze the changes in platelet size of ex vivo activated platelets which phenotypically simulates that of a patient at risk of cardiovascular disease and elucidate the calcium signaling pathway responsible for this change. Methodology Platelets were isolated from adult human blood by differential centrifugation. Calcium was mobilized into platelets by treatment with calcium ionophore A23187 in the presence of Ca2+. Platelet size distribution was analyzed using Coulter Counter Multisizer 4. The following signaling parameters were studied: intracellular Ca2+ measurement (using Fura-2/AM by fluorescence spectrophotometry), Ca2+-dependent thiol protease calpain assay (using fluorogenic substrate t-butoxycarbonyl-Leu-metchloromethylcoumarin in fluorescence microplate reader), platelet-derived microparticles (using FACS Calibur flow cytometry), and cytoskeletal protein talin expression (by western immunoblotting). Results When adult platelets were treated with A23187 and Ca2+, two subcellular populations (<2 µm and between 2-4 µm) were noted. The mean size of the second cell population was significantly higher than that of resting platelets (2.94 ± 0.13 µm vs. 2.82 ± 0.15 µm, t = 4.605, p = 0.00). A23187 treatment led to elevated intracellular Ca2+, release of platelet-derived microparticles, increase in calpain activity, and cytoskeletal talin degradation. These events were inhibited by calpeptin (a specific calpain inhibitor). Conclusions Elevated calcium caused talin degradation by calpain activity. Breakdown of this cytoskeletal protein leads to relative swelling of cells reflected by the increase in platelet size.

On the dynamics and optimal control of a mathematical model of neuroblastoma and its treatment: Insights from a mathematical model

Celyvir is an advanced therapy medicine, consisting of mesenchymal stem cells (MSCs) containing the oncolytic virus ICOVIR 5. This paper sets out a dynamic system which attempts to capture the fundamental relationships between cancer, the immune system and adenoviruses. Two forms of treatment were studied: continuous and periodic, the second being closer to the real situation. In the analysis of the first model, in addition to identifying the critical points, their properties and bifurcation points, a number of numerical simulations were carried out. It has thus been shown that there are bistability regimes in which Celyvir can produce an equilibrium of tumor progression, or even freedom from tumor. A sensitivity analysis was also performed to determine which parameters are most important in the system. Subsequently, an optimal control problem with nonlinear objective functional has been formulated, where the therapeutic goal is not only to minimize the size of the tumor cell population and the total cost of treatment, but also to prevent the tumor from reaching a critical size. It has been shown that the optimal control is bang–bang. With the second model, a threshold value of viral load has been identified at which the success of the treatment could be ensured. It is clear in both models that a low viral load would lead to relapse of the disease. Finally, it is shown that a periodic bang–bang regime should be used to optimize treatment with Celyvir.

Coronin 1-dependent cell density sensing and regulation of the peripheral T cell population size.

The establishment and maintenance of peripheral T cells is important to ensure appropriate immunity. In mammals, T cells are produced in the thymus before seeding the periphery early in life, and thereafter progressive thymus involution impairs new T cell production. Yet, peripheral T cells are maintained lifelong at approximately similar cell numbers. The question thus arises: what are the mechanisms that enable the maintenance of the appropriate number of circulating T cells, ensuring that T cell numbers are neither too low nor too high? Here, we highlight recent research suggesting a key role for coronin 1, a member of the evolutionarily conserved family of coronin proteins, in both allowing T cells to reach as well as maintain their appropriate cell population size. This cell population size controlling pathway was found to be conserved in amoeba, mice and human. We propose that coronin 1 is an integral part of a cell-intrinsic pathway that couples cell density information with prosurvival signalling thereby regulating the appropriate number of peripheral T cells.

Border cell population size and oxidative stress in the root apex of Triticum aestivum seedlings exposed to fungicides.

Fungicides reduce the risk of mycopathologies and reduce the content of mycotoxins in commercial grain. The effect of fungicides on the structural and functional status of the root system of grain crops has not been studied enough. In this regard, we studied the phytocytotoxic effects tebuconazole (TEB) and epoxiconazole (EPO) and azoxystrobin (AZO) in the roots of Triticum aestivum seedlings in hydroponic culture. In the presence of EPO and AZO (but not TEB) inhibition of the root growth was accompanied by a dose-dependent increase in the content of malondialdehyde, carbonylated proteins, and proline in roots. TEB was characterized by a dose-dependent decrease in the total amount of border cells (BCs) and the protein content in root extracellular trap (RET). For EPO and AZO, the dose curves of changes in the total number of BCs were bell-shaped. AZO did not affect the protein content in RET. The protein content in RET significantly decreased by 3 times for an EPO concentration of 1µg/mL. The obtained results reveal that the BC-RET system is one of the functional targets of fungicides in the root system of wheat seedlings. Studied fungicides induce oxidative stress and structural and functional alterations in the BC-RET system that can affect their toxicity to the root system of crops.

The Multi-temporal and Multi-dimensional Global Urban Centre Database to Delineate and Analyse World Cities

Monitoring sustainable urban development requires comparable geospatial information on cities across several thematic domains. Here we present the first global database combining such information with city extents. The Global Human Settlement Urban Centre Database (GHS-UCDB) is produced by geospatial data integration to characterise more than 10,000 urban centres worldwide. The database is multi-dimensional and multi-temporal, containing 28 variables across five domains and having multitemporal attributes for one or more epochs when the UC are delineated (1975-1990-2000-2015). Delineation of urban centres for the year 2015 is performed via a logic of grid cell population density, population size, and grid cell contiguity defined by the Degree of Urbanisation method. Each of the urban centres has 160 attributes, including a validation assessment. The novel aspects of this database concern the thematic richness and temporal depth of the variables (across geography, socio-economic, environmental, disaster risk reduction, and sustainable development domains) and the type of geo-information provided (location and extent), featuring an overall consistency that allows comparative analyses across locations and time.

A guide to ERK dynamics, part 2: downstream decoding.

Signaling by the extracellular signal-regulated kinase (ERK) pathway controls many cellular processes, including cell division, death, and differentiation. In this second installment of a two-part review, we address the question of how the ERK pathway exerts distinct and context-specific effects on multiple processes. We discuss how the dynamics of ERK activity induce selective changes in gene expression programs, with insights from both experiments and computational models. With a focus on single-cell biosensor-based studies, we summarize four major functional modes for ERK signaling in tissues: adjusting the size of cell populations, gradient-based patterning, wave propagation of morphological changes, and diversification of cellular gene expression states. These modes of operation are disrupted in cancer and other related diseases and represent potential targets for therapeutic intervention. By understanding the dynamic mechanisms involved in ERK signaling, there is potential for pharmacological strategies that not only simply inhibit ERK, but also restore functional activity patterns and improve disease outcomes.

Clearing and replacing tissue-resident myeloid cells with an anti-CD45 antibody–drug conjugate

AbstractTissue-resident myeloid (TRM) cells in adults have highly variable lifespans, and may be derived from early embryonic yolk sac, fetal liver, or bone marrow. Some of these TRM cells are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplantation can replace long-lived brain TRM cells, resulting in clinical improvements in metabolic storage diseases. With the advent of antibody–drug conjugate (ADC)-targeted cell killing as a cell-selective means of transplant conditioning, we assessed the impact of anti-CD45–ADC on TRM cells in multiple tissues. Replacement of TRM cells ranged from 40% to 95% efficiencies in liver, lung, and skin tissues, after a single anti-CD45–ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM cells in tissues returned to pretreatment levels, suggesting a regulated control of TRM cell abundance. As expected, brain microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45–ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque-resident myeloid cells were efficiently replaced with anti-CD45–ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that the anti-CD45–ADC clears both hematopoietic stem and TRM cells from their niches, enabling cell replacement to achieve disease modification in a resident myeloid cell–driven disease.

BioNetGMMFit: estimating parameters of a BioNetGen model from time-stamped snapshots of single cells

Mechanistic models are commonly employed to describe signaling and gene regulatory kinetics in single cells and cell populations. Recent advances in single-cell technologies have produced multidimensional datasets where snapshots of copy numbers (or abundances) of a large number of proteins and mRNA are measured across time in single cells. The availability of such datasets presents an attractive scenario where mechanistic models are validated against experiments, and estimated model parameters enable quantitative predictions of signaling or gene regulatory kinetics. To empower the systems biology community to easily estimate parameters accurately from multidimensional single-cell data, we have merged a widely used rule-based modeling software package BioNetGen, which provides a user-friendly way to code for mechanistic models describing biochemical reactions, and the recently introduced CyGMM, that uses cell-to-cell differences to improve parameter estimation for such networks, into a single software package: BioNetGMMFit. BioNetGMMFit provides parameter estimates of the model, supplied by the user in the BioNetGen markup language (BNGL), which yield the best fit for the observed single-cell, time-stamped data of cellular components. Furthermore, for more precise estimates, our software generates confidence intervals around each model parameter. BioNetGMMFit is capable of fitting datasets of increasing cell population sizes for any mechanistic model specified in the BioNetGen markup language. By streamlining the process of developing mechanistic models for large single-cell datasets, BioNetGMMFit provides an easily-accessible modeling framework designed for scale and the broader biochemical signaling community.

CSF-1R+ Macrophages Control the Gut Microbiome-Enhanced Liver Invariant NKT Function through IL-18.

The gut microbiome is an important modulator of the host immune system. In this study, we found that altering the gut microbiome by oral vancomycin increases liver invariant NKT (iNKT) cell function. Enhanced iNKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both Ag-specific and Ag-independent invivo iNKT stimulations, with a more prominent effect in the liver than in the spleen. Fecal transplantation studies demonstrated that the iNKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of iNKT cell population size. Interestingly, when stimulated invitro, iNKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. iNKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing Ab or using genetically deficient mice attenuated the enhanced iNKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this iNKT activation. Using anti-CSF-1R depletion or LyzCrexCSF-1RLsL-DTR mice identified CSF-1R+ macrophages as a critical modulator of iNKT function. Vancomycin treatment had no effect on iNKT cell function invivo in IL-18 knockout macrophage reconstituted mice. Together, our results demonstrate that the gut microbiome controls liver iNKT function via regulating CSF-1R+ macrophages to produce IL-18.

Abstract 5810: The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer

Abstract Understanding how to therapeutically target colonic stem cells (SCs) is important because SC overpopulation drives CRC growth and development. Our goal is to therapeutically sensitize cancer SCs (CSCs) to differentiation-inducing effects of retinoid agents against CRC. We found that ALDH specifically marks normal and malignant colonic SCs and that retinoic acid (RA) signaling mainly occurs in colonic ALDH+ SCs. That RA signaling occurs through ALDH+ SCs was derived from finding that RA receptors and RA signaling components are selectively expressed in ALDH+ SCs. Indeed, All-transretinoic acid (ATRA) is a highly effective treatment for leukemia (APL) which has a RARA translocation. We conjecture that response of solid tumors to RA agents depends on RA pathway genotype. Our bioinformatics analysis shows mutations often occur in RA receptors in human CRCs. Therefore, we hypothesize that the ability of RA agents and RA metabolism blocking agents to induce differentiation of CSCs depends on RA pathway genotype. Methods: The ability of RA agents (ATRA, 13-cis retinoic acid, 9-cis retinoic acid) and RA metabolism blocking agents (Liarozole, Talarozole) to inhibit proliferation and induce differentiation of CRC cells was evaluated. Nanostring Profiling was used to measure effects of agents on mRNA expression of various markers. Results: Our results show that RA pathway genotype affects the response of RA agents in CRC cell lines. HCT116 and SW480 cells, which have RA receptor mutations, displayed resistance to ATRA. In contrast, HT29 cells that have wild-type RA receptors are sensitive to ATRA. All three cell lines however showed similar responses to the CYP26A1 inhibitor Liarozole. This finding indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism will increase intracellular RA levels. Nanostring profiling analysis shows ATRA treatment of HT29 cells increases expression of RARA, CYP26A1 (ATRA metabolizing enzyme), &amp; KRT20 (differentiation marker), and decreases expression of CSC markers LGR5 &amp; ALDH1A1. Moreover, ATRA treatment of both CRC cell lines decreases ALDH+ cell population size. These findings show upregulated RA signaling can increase differentiation and decrease CSC numbers in CRC. Discussion Our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Thus, therapeutically sensitizing CSCs to differentiation-inducing effects of RA agents should provide insight into design of new SC-targeted therapies for CRC. Citation Format: Victoria Oluwajuwon Hunsu, Caroline O. Facey, Bruce M. Boman. The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5810.

Abstract 642: CSF-1R+ macrophages control the gut microbiome-enhanced liver NKT function through IL-18

Abstract The gut microbiome is an important modulator of the host immune system. Here, we found that altering the gut microbiome by oral vancomycin increases liver NKT cell function. Enhanced NKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both antigen-specific and antigen-independent in vivo NKT stimulations, with a more prominent effect in liver as compared to the spleen. Fecal transplantation studies demonstrated that the NKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of NKT cell population size. Interestingly, when stimulated in vitro, NKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. NKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing antibody or using genetically deficient mice attenuated the enhanced NKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this NKT activation. Using anti-CSF-1R depletion or LyzCrexCSF-1RLsL-DTRmice identified CSF-1R+ macrophages as a critical modulator of NKT function. Together, our results demonstrate that the gut microbiome controls liver NKT function via regulating CSF-1R+ macrophages to produce IL-18. Citation Format: Chi Ma, Justin McCallen, John C. McVey, Qianfei Zhang, Benjamin Ruf, Kylynda Bauer, Sophie Wang, Chunwei Walter Lai, Giorgio Trinchieri, Jay A. Berzofsky, Firouzeh Korangy, Tim F. Greten. CSF-1R+ macrophages control the gut microbiome-enhanced liver NKT function through IL-18 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 642.

Redox metabolism maintains the leukemogenic capacity and drug resistance of AML cells

Rewiring of redox metabolism has a profound impact on tumor development, but how the cellular heterogeneity of redox balance affects leukemogenesis remains unknown. To precisely characterize the dynamic change in redox metabolism invivo, we developed a bright genetically encoded biosensor for H2O2 (named HyPerion) and tracked the redox state of leukemic cells insitu in a transgenic sensor mouse. A H2O2-low (HyPerion-low) subset of acute myeloid leukemia (AML) cells was enriched with leukemia-initiating cells, which were endowed with high colony-forming ability, potent drug resistance, endosteal rather than vascular localization, and short survival. Significantly high expression of malic enzymes, including ME1/3, accounted for nicotinamide adenine dinucleotide phosphate (NADPH) production and the subsequent low abundance of H2O2. Deletion of malic enzymes decreased the population size of leukemia-initiating cells and impaired their leukemogenic capacity and drug resistance. In summary, by establishing an invivo redox monitoring tool at single-cell resolution, this work reveals a critical role of redox metabolism in leukemogenesis and a potential therapeutic target.

Establishing a human bone marrow single cell reference atlas to study ageing and diseases.

Ageing in the human bone marrow is associated with immune function decline that results in the elderly being vulnerable to illnesses. A comprehensive healthy bone marrow consensus atlas can serve as a reference to study the immunological changes associated with ageing, and to identify and study abnormal cell states. We collected publicly available single cell transcriptomic data of 145 healthy samples encompassing a wide spectrum of ages ranging from 2 to 84 years old to construct our human bone marrow atlas. The final atlas has 673,750 cells and 54 annotated cell types. We first characterised the changes in cell population sizes with respect to age and the corresponding changes in gene expression and pathways. Overall, we found significant age-associated changes in the lymphoid lineage cells. The naïve CD8+ T cell population showed significant shrinkage with ageing while the effector/memory CD4+ T cells increased in proportion. We also found an age-correlated decline in the common lymphoid progenitor population, in line with the commonly observed myeloid skew in haematopoiesis among the elderly. We then employed our cell type-specific ageing gene signatures to develop a machine learning model that predicts the biological age of bone marrow samples, which we then applied to healthy individuals and those with blood diseases. Finally, we demonstrated how to identify abnormal cell states by mapping disease samples onto the atlas. We accurately identified abnormal plasma cells and erythroblasts in multiple myeloma samples, and abnormal cells in acute myeloid leukaemia samples. The bone marrow is the site of haematopoiesis, a highly important bodily process. We believe that our healthy bone marrow atlas is a valuable reference for studying bone marrow processes and bone marrow-related diseases. It can be mined for novel discoveries, as well as serve as a reference scaffold for mapping samples to identify and investigate abnormal cells.

Influence of abiotic factors on kinetics of viable populations of biocontrol agents in the phyllosphere of lettuce and strawberry leaves

AbstractThere is little information on the effect of temperature, relative humidity (RH) and vapour pressure deficit (VPD) on the viable populations of two commercial biocontrol strains, Bacillus subtilis QST 713 (recently classified as B. velezensis) and Gliocladium catenulatum J1446 (syn. Clonostachys rosea). The PMAxx‐qPCR molecular assay was used to quantify the viable biocontrol agent (BCA) populations on fully extended lettuce and strawberry leaves under a range of temperature and RH combinations. Overall, there was a small decline in the population size of viable cells for the two biocontrol organisms on strawberry and lettuce leaves within 10 days of application. However, for most experimental runs, which contained general UK agronomy climates, such a decline was not statistically significant. Moreover, for a few runs, the viable populations increased significantly in optimal BCA growth temperatures with high RH. Only temperature (ambient) and dew point significantly affected the rate of temporal changes in the viable biocontrol population size. Thus, an increasing temperature led to decreased daily mortality. It should be noted that much of the variability in the estimated daily mortality rates remains unaccounted for, thus unless under extreme conditions, the biocontrol efficacy in practice is less likely to be affected by the survival of biocontrol microbes but more likely by other factors that influence the density of biocontrol cells in the phyllosphere, especially dilution due to rapid host leaf expansion, spray coverage and rain wash‐off.

Simplified within-host and Dose–response Models of SARS-CoV-2

Understanding the mechanistic dynamics of transmission is key to designing more targeted and effective interventions to limit the spread of infectious diseases. A well-described within-host model allows explicit simulation of how infectiousness changes over time at an individual level. This can then be coupled with dose–response models to investigate the impact of timing on transmission. We collected and compared a range of within-host models used in previous studies and identified a minimally-complex model that provides suitable within-host dynamics while keeping a reduced number of parameters to allow inference and limit unidentifiability issues. Furthermore, non-dimensionalised models were developed to further overcome the uncertainty in estimates of the size of the susceptible cell population, a common problem in many of these approaches. We will discuss these models, and their fit to data from the human challenge study (see Killingley et al. (2022)) for SARS-CoV-2 and the model selection results, which has been performed using ABC-SMC. The parameter posteriors have then used to simulate viral-load based infectiousness profiles via a range of dose–response models, which illustrate the large variability of the periods of infection window observed for COVID-19.

Programmed death ligand 1 regulates epithelial-mesenchymal transition and cancer stem cell phenotypes in hepatocellular carcinoma through the serum and glucocorticoid kinase 2/β-catenin signaling pathway.

Programmed death ligand 1 (PD-L1) plays an important role in the occurrence of hepatocellular carcinoma (HCC). The present study indicated that epithelial-mesenchymal transition (EMT) and induction of cancer stem cell (CSC)-like properties contribute to metastasis of cancers. However, the molecular mechanisms underlying PD-L1 and EMT and CSC phenotypes in HCC remain to be elucidated. Here, we report that PD-L1 regulates not only EMT but also the stem-like transition in liver cancer cells. We observed high PD-L1 expression in CD133+ liver CSCs and CSC-enriched tumor spheres. Altering PD-L1 expression promoted liver CSC phenotypes by increasing the expression of stemness genes, the CD133+ cell population sizes, and the ability to form tumor spheres. Programmed death ligand 1 enhanced HCC cell tumorigenicity and invasion in nude mice. Additionally, PD-L1 overexpression in cells significantly increased cell motility and invasion, as well as the EMT process. Conversely, suppression of PD-L1 in cells had an opposite effect. Prolonged treatment of HCC cells with Akt inhibitor prefosine leads to activation of serum and glucocorticoid kinase 2 (SGK2) and rescued downregulation of PD-L1. Mechanistically, PD-L1 directly interacted with SGK2. Programmed death ligand 1 upregulated SGK2 and activated the SGK2/β-catenin signaling pathway, and promoted EMT and CSC expansion in liver cancer cells, highlighting the role of SGK2 in PD-L1-mediated EMT and CSC phenotypes in liver cancer cells. In conclusion, our findings suggest that PD-L1 activated the SGK2/β-catenin signaling pathway, to induce EMT and acquisition of a stem cell phenotype.

Clonal Hematopoiesis in Mice Is Common with Age and Accelerated By Microbial Exposure

Clonal hematopoiesis (CH) is a ubiquitous phenomenon of human aging - nearly all individuals older than 50 years have CH in a small but detectable fraction of their blood. Currently, the mechanisms driving clone evolution are poorly understood and experimental models recapitulating the emergent behavior of spontaneously-arising low-frequency clones (early CH) are lacking. To address this gap in knowledge, we endeavored to establish the mouse as a model for native clonal hematopoiesis. Laboratory mice live only 2-3 years but CH in humans develops over decades; thus, we considered whether natural CH would be observable outside of manipulated transplantation models. To address this question, we performed computational modeling incorporating published estimates of murine hematopoietic stem cell (HSC) mutation rate and population size. This analysis suggested that clonal expansions are likely to occur in mice, although at magnitudes-smaller clone sizes than in humans. Thus, we hypothesized that natural murine CH would be characterized by rare and small somatic clones. To test murine blood for CH, we employed targeted duplex consensus sequencing. In this strategy, each initial DNA molecule is uniquely barcoded such that reads derived from complementary starting strands are resolved to error-corrected sequences, enabling magnitudes-greater sensitivity relative to most other sequencing methods. We developed a panel to probe for rare variants in mouse homologs of human CH driver genes and queried unmanipulated, naturally-aged, laboratory mice across their lifespan, at ages spanning 4 to 37 months (roughly equivalent to humans between 20 to 100 years of age). Remarkably, we readily observed expanded variant clones in aged mice, with rare or absent clonal expansions in young mice (Figure 1). CH incidence increased with each year of age. Clones were most commonly driven by somatic mutations in known CH-associated genes, such as Dnmt3a, Tet2, and Bcor/Bcorl1. These variants occurred on the order of 1 mutant cell per 10,000 blood cells, often aligned with human hotspot loci, and were enriched for non-synonymous mutations above what is expected by genetic drift. Longitudinal sampling demonstrated the persistence of clones during aging and following transplantation. Thus, natural murine CH is broadly similar to that observed in humans, with the exception of the size of clones, as aligned with our predictions. Most laboratory mice are maintained throughout their life in highly controlled conditions with very low pathogen exposure, in stark contrast to the constant varied microbial exposures of humans. Because inflammation has been suggested to accelerate CH, we considered the possibility that a more natural microbial environment would augment murine CH. To test this, we employed a "normal microbial experience” (NME) model, in which laboratory mice are infected with common mouse microbes via exposure to fomite bedding (sourced from pet store mice), subjecting young and aged mice to short-term NME. Strikingly, we observe an increased number and size of expanded clones in NME-exposed aged mice relative to specific pathogen-free housed controls. Importantly, clones bearing mutations in Trp53 were particularly enriched in NME-exposed mice, as measured by clone count and clone magnitude (allele fraction). NME exposure transmits multiple types of pathogens; thus it is challenging to identify specific effects. Therefore, we performed targeted exposure to Mycobacterium avium, which we have previously shown can activate HSCs. We again observed an exacerbation of clonal expansion in aged, infected mice, with specific enrichment for Bcor/Bcorl1 mutant clones. In summary, we have unequivocally demonstrated that aged mice develop CH naturally within their captive lifespan. Genes and even mutational hotspots found in human CH are recapitulated, and the magnitude and number of clones are aligned with predictions based on mutation rate and HSC number. Moreover, native murine CH is significantly accelerated in the context of microbial exposure, revealing that driver genes offer context-specific advantages towards expansion. Our approach detects low-frequency clonal interplay and will be applicable for the development of therapeutic strategies to modulate CH by shifting the hematopoietic fitness landscape as a whole, rather than by targeting specific CH clones. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal