Proliferating Cell Population Research Articles

Apoptotic cell death in the mammalian central nervous system during development and in pathological conditions

Mammalian central nervous system (CNS) development involves genetically controlled, opposing processes, such as neuronal proliferation, migration, differentiation and death. The natural, developmental cell death is a ubiquitous phenomenon and is referred to as «programmed cell death» (PCD). Apoptosis, a type of PCD, is a central event in normal development of the CNS, playing an important role in the control of cell numbers and the establishment of neuronal circuitry. During embryogenesis, apoptosis takes place in proliferating cell populations and is involved in CNS morphogenesis. At later stages of development, apoptosis occurs in postmitotic neurons, because of the competition for limited supply of neurotrophic factors, that originally suppress the endogenous genetic death programme. Data concerning apoptotic cell death during normal CNS development of domestic mammals is lacking, therefore information about the developmental pattern of this phenomenon is restricted to rodents and rabbits. In these animals it has been suggested that apoptosis follows a mono- or biphasic time course and is completed duringan early, critical period of CNS development, that is characterized by morphological and functional neuronal maturation and synapse formation. Apart from its role in CNS development, apoptosis has also been implicated in neuronal loss accompanying neurodegenerative diseases and traumatic brain injuries in humans and animals. In the domestic canine brain, it has been shown that neurons die via apoptosis in Alzheimer's-like dementia, cerebellar abiotrophy, global and focal ischemia and virus-induced encephalopathies. In addition, cell death in ruminants with transmissible spongiform encephalopathy has been reported to be apoptotic in nature. A plethora of studies using animal models have been employed to elucidate the mechanisms than govern cell loss in neurological disorders. These studies provided strong evidence that experimental lesions of the connections between CNS areas and withdrawal of neurotrophic factors result in an increase of apoptosis, that is age-dependent. Specifically, developing neurons are more dependent on the integrity of their connections than mature ones. In addition, the response of neurons to apoptotic stimuli shows regional specificity. According to epidemiologic studies, CNS disorders are of major concern for animal and human public health, with a high socioeconomic impact. A major goal of neuroscientists is the development of therapeutic approaches for CNS repair. Contemporary strategies that are under trial include neurotrophic factor substitution and transplantation of stem cells. Investigation of the principles and mechanisms controlling cell loss in neurodegenerative diseases and traumatic brain injuries are universally considered of high priority and hopefully will lead to novel therapeutic approaches, with encouraging outcome. The present review summarises recent data on the molecular mechanisms and factors controlling neuronal apoptosis during development and in pathological conditions, describes popular animal models used in lesion studies and discusses therapeutic approaches aiming at preventing or restricting apoptotic cell death.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs).

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Multi-Cellular Modelling of Cellular Mechanisms Gives Insights on the Maintenance of Epidermal Tissue Structure

The inter-follicular epidermis (IFE) forms the outer-most layer of the skin. Many individual components fundamental to healthy IFE structure are known: proliferation occurs only in a basal layer; above this layer cells differentiate into keratinocytes forming further distinct layers before they are shed from the surface. However, a definitive understanding of how the balance between proliferation, differentiation, and cell shedding is maintained in IFE tissue during homeostasis does not yet exist. We have developed an agent-based multi-cellular computational model to simulate tissue homeostasis in the skin. Epidermal cells are represented as overlapping spheres, and cell divisions are represented as stochastic time-driven events. Cell movement is determined by adhesive attractive forces and repulsive forces between other cells and the basal membrane. The magnitude of these forces depends upon the types of the interacting bodies, and can vary depending on factors such as cell age and location. Using this model we have analysed the impact of different cell mechanisms and behaviours on the tissue in order to investigate alternate hypotheses around maintenance of tissue structure. Mechanisms investigated include cell division, cell adhesion and stiffness, and cell death. Results of this study provide insights on important considerations in modelling this system. In particular, both choice of biological assumptions and the mathematical representation of cell division impact the spatial distribution of proliferative cell populations. We find that the cell mechanisms and behaviours critical to maintenance of the tissue width are proliferation rate, spatial adhesion variation, and cell loss.

Dental Tissue-Derived Mesenchymal Stem Cells: Applications in Tissue Engineering.

In recent years, mesenchymal stem cells (MSCs) derived from dental tissue have gained in popularity for tissue-engineering and regenerative medicine applications. The highly proliferative and self-renewing population of dental stem cells has the neural crest as their origin. This expands their applicability for regeneration of tissues from both ectochyme and mesenchymal origin. Ease of tissue harvest, high initial yield of cells, low population-doubling time, plasticity, multipotential capabilities, and immunomodulatory properties make them a suitable candidate for various therapeutic strategies. Furthermore, dental tissue-derived cells can be transformed into induced pluripotent stem cells to customize cell-based regenerative approaches. However, there is currently a lack of exhaustive comparative profiles of these dental tissues and their regenerative applications. We thereby present a comprehensive compilation of morphofunctional analyses and tissue-engineering applications of MSCs that are derived from tooth germ, exfoliated deciduous teeth, periodontal ligament, gingiva, dental pulp, alveolar bone, dental follicle, and apical papilla. Immunoregulatory properties of dental stem cells provide potential for both autologous and allogenic tissue-engineering approaches. In vitro and animal studies show promise for using dental stem cells in regenerative medicine. Eventually, the orchestration of clinical trials will require systematic monitoring of spontaneous in vitro transformations and complications associated with graft versus host response as well as a thorough understanding of underlying anabolic mechanisms.

Cellular Proliferative Domains: Barriers to Migration

An important aspect of the analysis of the proliferation of cell populations as composed of a set of intercommunicating logistic domains is the nature of the barriers between adjacent domains and the regulation of cellular migration between them. The nature of these inter-domain boundaries is discussed in relation to the central role of L-type calcium channels in the control of cell motility and it is proposed that domain boundaries are determined by proteoglycans which act as calcium chelators and hence regulate the local extracellular Ca⁺⁺ concentration. The selective migratory advantage gained by overexpression of calcium channels is briefly alluded to.

IL-17A-Induced PLET1 Expression Contributes to Tissue Repair and Colon Tumorigenesis.

This study identifies a novel mechanism linking IL-17A with colon tissue repair and tumor development. Abrogation of IL-17A signaling in mice attenuated tissue repair of dextran sulfate sodium (DSS)-induced damage in colon epithelium and markedly reduced tumor development in an azoxymethane/DSS model of colitis-associated cancer. A novel IL-17A target gene, PLET1 (a progenitor cell marker involved in wound healing), was highly induced in DSS-treated colon tissues and tumors in an IL-17RC-dependent manner. PLET1 expression was induced in LGR5+ colon epithelial cells after DSS treatment. LGR5+PLET1+ marks a highly proliferative cell population with enhanced expression of IL-17A target genes. PLET1 deficiency impaired tissue repair of DSS-induced damage in colon epithelium and reduced tumor formation in an azoxymethane/DSS model of colitis-associated cancer. Our results suggest that IL-17A-induced PLET1 expression contributes to tissue repair and colon tumorigenesis.

Screening and identification of host proteins interacting with Theileria annulata cysteine proteinase (TaCP) by yeast-two-hybrid system

BackgroundTheileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs) are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP) is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells.MethodsThe cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid) for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP) and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING.ResultsTwo host proteins, CRBN (Bos taurus cereblon transcript variant X2) and Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit) were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs.ConclusionsThis study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are involved in many cellular processes, such as ubiquitylation regulating, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. The interaction with CRBN and Ppp4C indicate that TaCP possibly is involved in regulating signaling pathways and cell proliferation, which is helpful for understanding the interaction between T. annulata and host cells.

Two States of Myeloma Stem Cells.

The identity of so-called malignant stem cells in multiple myeloma has long been controversial. However, it is now appreciated that a small population of myeloma cells has a stem-like capacity for self-renewal and resides within the population of recognizable myeloma plasma cells, although whether these cells are quiescent or proliferative remains unresolved. It is also now accepted that subpopulations of myeloma plasma cells alternate bidirectionally and dynamically between immature and mature phenotypes, and both subpopulations are capable of clonogenicity invitro and in animal models. Mounting evidence suggests that in multiple myeloma, small populations of quiescent myeloma stem cells behave as tumor-initiating cells as a result of their interaction with the endosteal bone marrow niche. However, a different, small population of proliferative myeloma stem cells produces the tumor bulk, and this population is likely responsible for the emergence of evolved subclones. A cellular-based plasticity model in which 2 states of stem-like cell compartments-quiescent and proliferative-exist simultaneously and are regulated dynamically by the microenvironment, epigenetics, and genetic aberrations may explain various clinical scenarios and the lack of consensus among some reported studies.

Tbx3-dependent amplifying stem cell progeny drives interfollicular epidermal expansion during pregnancy and regeneration

The skin surface area varies flexibly in response to body shape changes. Skin homeostasis is maintained by stem cells residing in the basal layer of the interfollicular epidermis. However, how the interfollicular epidermal stem cells response to physiological body shape changes remains elusive. Here, we identify a highly proliferative interfollicular epidermal basal cell population in the rapidly expanding abdominal skin of pregnant mice. These cells express Tbx3 that is necessary for their propagation to drive skin expansion. The Tbx3+ basal cells are generated from Axin2+ interfollicular epidermal stem cells through planar-oriented asymmetric or symmetric cell divisions, and express transit-amplifying cell marker CD71. This biased division of Axin2+ interfollicular epidermal stem cells is induced by Sfrp1 and Igfbp2 proteins secreted from dermal cells. The Tbx3+ basal cells promote wound repair, which is enhanced by Sfrp1 and Igfbp2. This study elucidates the interfollicular epidermal stem cell/progeny organisation during pregnancy and suggests its application in regenerative medicine.

Integrating Biological and Mathematical Models to Explain and Overcome Drug Resistance in Cancer, Part 2: from Theoretical Biology to Mathematical Models

Despite the promise of targeted cancer therapy (i.e., drugs targeted towards specific signaling pathways supposed to be essential to the survival and proliferation of cancer cells), unexpected treatment failures in the clinic are common. Tumor cell heterogeneity, which can result from the plasticity of individual cancer cells and/or evolutionary dynamics, has revealed the importance to study cancer at the level of the entire cell population. Here, we explore mathematical models that describe tumor dynamics under pressure of anticancer drugs by integrating cancer cell population heterogeneity and evolutionary behavior. We further explore mathematical modeling as a theoretical tool to analyze and predict the behavior of cancer cell populations as a whole, and not only of individual cells, which may reveal new clues to therapy failure and ways to overcome it. An evolutionary perspective that relies on the “atavistic theory of cancer” together with the so-called “cold genes”, and the involvement of “bet hedging” in tumors, has recently changed our vision of cell plasticity in cancer. These new perspectives provide a sound theoretical basis to the emergence of resistance in cancer cell populations and to its possible reversibility. Continuous mathematical models of the evolutionary dynamics of proliferative cell populations already exist, that take into account the heterogeneity of cancer cell populations, allowing to study the evolutionary potential of cancer cell populations and predict their behavior. Those models, in turn, can be used to probe population growth control by incorporating functions (in the mathematical model) that represent the action of drugs on the mechanisms driving proliferation, and furthermore to suggest new therapeutic strategies in the clinic of cancers. In this second part of our review, that can be taken independently of Part I, we focus on the level of cell populations, the only one amenable to completely take into account phenotype plasticity in its observable consequences on the evolution of proliferative diseases, and on heterogeneity, that makes sense only in the context of cell populations, together with the fundamental evolutionary potential of cancer cell populations (and not only of single cancer cells). These are modern views that come from the transposition from ecology of species to the biology of cancer (for further reading on evolution and cancer, the reader is referred to the recent comprehensive book on the subject Ujvari et al. 2017). Finally, we present a brief review of mathematical models, with their features and their use in cell population studies, to account for phenomena found in cancer, focusing on drug resistance. We contend that a good understanding of what mathematical models can do to tackle the question of drug resistance in cancer can shed light on the mechanisms of resistance and means to control them and help design principles for biological experiments to be performed at the lab and therapeutical strategies to be applied in the clinic of cancers.

Role of the TGF-β pathway in dedifferentiation of human mature adipocytes.

Dedifferentiation of adipocytes contributes to the generation of a proliferative cell population that could be useful in cellular therapy or tissue engineering. Adipocytes can dedifferentiate into precursor cells to acquire a fibroblast‐like phenotype using ceiling culture, in which the buoyancy of fat cells is exploited to allow them to adhere to the inner surface of a container. Ceiling culture is usually performed in flasks, which limits the ability to test various culture conditions. Using a new six‐well plate ceiling culture approach, we examined the relevance of TGF‐β signaling during dedifferentiation. Adipose tissue samples from patients undergoing bariatric surgery were digested with collagenase, and cell suspensions were used for ceiling cultures. Using the six‐well plate approach, cells were treated with SB431542 (an inhibitor of TGF‐β receptor ALK5) or human TGF‐β1 during dedifferentiation. Gene expression was measured in these cultures and in whole adipose tissue, the stromal–vascular fraction (SVF), mature adipocytes, and dedifferentiated fat (DFAT) cells. TGF‐β1 and collagen type I alpha 1 (COL1A1) gene expression was significantly higher in DFAT cells compared to whole adipose tissue samples and SVF cells. TGF‐β1, COL1A1, and COL6A3 gene expression was significantly higher at day 12 of dedifferentiation compared to day 0. In the six‐well plate model, treatment with TGF‐β1 or SB431542, respectively, stimulated and inhibited the TGF‐β pathway as shown by increased TGF‐β1, TGF‐β2, COL1A1, and COL6A3 gene expression and decreased expression of TGF‐β1, COL1A1, COL1A2, and COL6A3, respectively. Treatment of DFAT cells with TGF‐β1 increased the phosphorylation level of SMAD 2 and SMAD 3. Thus, a new six‐well plate model for ceiling culture allowed us to demonstrate a role for TGF‐β in modulating collagen gene expression during dedifferentiation of mature adipocytes.

Hypothermia after cranial irradiation protects neural progenitor cells in the subventricular zone but not in the hippocampus

Purpose: To explore if hypothermia can reduce the harmful effects of ionizing radiation on the neurogenic regions of the brain in young rats.Materials and methods: Postnatal day 9 rats were randomized into two treatment groups, hypo- and normothermia, or a control group. Treatment groups were placed in chambers submerged in temperature-controlled water baths (30 °C and 36 °C) for 8 h, after receiving a single fraction of 8 Gy to the left hemisphere. Seven days’ post-irradiation, we measured the sizes of the subventricular zone (SVZ) and the granule cell layer (GCL) of the hippocampus, and counted the number of proliferating (phospho-histone H3+) cells and microglia (Iba1 + cells).Results: Irradiation caused a 53% reduction in SVZ size in the normothermia group compared to controls, as well as a reduction of proliferating cell numbers by >50%. These effects were abrogated in the hypothermia group. Irradiation reduced the number of microglia in both treatment groups, but resulted in a lower cell density of Iba1 + cells in the SVZs of the hypothermia group. In the GCL, irradiation decreased both GCL size and the proliferating cell numbers, but with no difference between the treatment groups. The number of microglia in the GCL did not change.Conclusions: Hypothermia immediately after irradiation protects the SVZ and its proliferative cell population but the GCL is not protected, one week post-irradiation.

Planarian Epidermal Stem Cells Respond to Positional Cues to Promote Cell-Type Diversity

Successful regeneration requires that progenitors of different lineages form the appropriate missing cell types. However, simply generating lineages is not enough. Cells produced by a particular lineage often have distinct functions depending on their position within the organism. How this occurs in regeneration is largely unexplored. In planarian regeneration, new cells arise from a proliferative cell population (neoblasts). We used the planarian epidermal lineage to study how the location of adult progenitor cells results in their acquisition of distinct functional identities. Single-cell RNA sequencing of epidermal progenitors revealed the emergence of distinct spatial identities as early in the lineage as the epidermal neoblasts, with further pre-patterning occurring in their post-mitotic migratory progeny. Establishment of dorsal-ventral epidermal identities and functions, inresponse to BMP signaling, required neoblasts. Our work identified positional signals that activate regionalized transcriptional programs in the stem cell population and subsequently promote cell-type diversity in the epidermis.

A High-Throughput Biology Approach to Identify Novel Therapies Specifically Targeting AML Blasts and Leukemic Stem Cells

IntroductionWhile the majority of patients diagnosed with acute myeloid leukemia (AML) will respond to standard induction chemotherapy, recurrent and refractory disease rates are high. Much needed new therapies for AML are currently in development; however, many drugs indiscriminately target rapidly proliferating cells and are ineffective against indolent, more quiescent leukemic stem cells. To identify novel therapies specific for AML blasts and leukemic stem cells (LSC), we developed a high-throughput, multi-parametric flowcytometry(FC) based method that simultaneously distinguishes specific cell populations and their differential drug responses. Using this method, we could distinguish healthy cells from leukemic stem cells and assess the effects of several drugs on these cell populations as well as the immune profile of individual patients. In addition, we identified drugs that could target both blasts and LSC and could potentially be repositioned for the treatment of AML.MethodsMononuclear cells were enriched from bone marrow (BM) aspirates collected from 10 patients by density gradient centrifugation and seeded to pre-drugged 96-well plates at 100,000 cells/well. The drugs were tested in a 10,000-fold concentration range and included standard chemotherapy agents (cytarabineandidarubicin), corticoid steroid dexamethasone,rapalogtemosirolimus, PI3K/mTORinhibitoromipalisib, JAK1/2 inhibitorruxolitinib,Srckinase inhibitordasatiniband Bcl-2 inhibitorvenetoclax. The cells were incubated with the drugs for 3 days in RPMI medium supplemented with conditioned medium from the human BM stromal cell line HS-5. The cells were stained using six different antibodies to identify blasts (CD45+, CD34+/CD33+, CD123+), LSC (CD45+, CD34+, CD38-, CD96+), healthy stem cells (HSC) (CD45+, CD34+ CD38-, CD96-), normal BM cells (CD45+, CD34-) and lymphocytes (CD45+ high, CD34-/CD33-, SSH low). We usedAnnexinV and 7-AAD to detect and remove dead and apoptotic cells. The stained cells were measured within the 96-well plates using theiQueScreener PLUS instrument (Intellicyt) in 20 minutes and at least 50,000 events acquired per well. Live cell numbers for different cell populations were calculated and dose response curves generated for each population and drug. For comparison, the viability of the samples was also assessed using theCellTiterGlo(CTG) assay.ResultsAssay set up and plate reading were accomplished within a relatively short time (staining 1 h, plate reading 20 min) with more than 5 million cell events acquired from each plate. Dose response curves generated from the live cell counts of the FC assay and viability readouts of the CTG assay were highly similar for cytotoxic drugs.(Fig. 1A).However, for targeted therapies, CTG overestimated the effect of JAK andmTORinhibitors by approximately 20% compared to the FC readouts (Fig. 1B). High variation in drug responses was observed between patient samples indicating patient and sample heterogeneity. In addition, cell populations within the same patient samples showed very different responses to the tested drugs. For example lymphocytes were sensitive tovenetoclax, but unresponsive to the other tested drugs. In contrast, blast cells were sensitive to most drugs and especially tovenetoclaxandruxolitinibwhen compared to other cell populations, suggesting that these drugs are particularly effective against this rapidly proliferating cell population.(Fig. 1C).Interestingly,ruxolitinibwas also effective at targeting LSCs, although HSCs were equally sensitive to this drug.ConclusionBy applying a high-throughput FC based method, we could easily assess the effects of several drugs on AML patient samples and distinguish the functional impact on different cell populations. The method differentiated leukemic and healthy stem cells and identified drugs that could target LSC, which are believed to be a main cause of relapses in AML. Interestingly, JAK1/2 inhibitorruxolitinib, approved for the treatment ofmyelofibrosisand polycythemiavera, was effective at targeting AML blasts and LSC. In addition, this method accounts for inter- and intra-patient variability, and may be useful for a precision medicine based approach to identify optimal, patient-specific treatment strategies. [Display omitted] DisclosuresHeckman:Celgene: Research Funding; Pfizer: Research Funding.

Genome-Wide Association Study for Identifying Loci that Affect Fillet Yield, Carcass, and Body Weight Traits in Rainbow Trout (Oncorhynchus mykiss).

Fillet yield (FY, %) is an economically-important trait in rainbow trout aquaculture that affects production efficiency. Despite that, FY has received little attention in breeding programs because it is difficult to measure on a large number of fish and cannot be directly measured on breeding candidates. The recent development of a high-density SNP array for rainbow trout has provided the needed tool for studying the underlying genetic architecture of this trait. A genome-wide association study (GWAS) was conducted for FY, body weight at 10 (BW10) and 13 (BW13) months post-hatching, head-off carcass weight (CAR), and fillet weight (FW) in a pedigreed rainbow trout population selectively bred for improved growth performance. The GWAS analysis was performed using the weighted single-step GBLUP method (wssGWAS). Phenotypic records of 1447 fish (1.5 kg at harvest) from 299 full-sib families in three successive generations, of which 875 fish from 196 full-sib families were genotyped, were used in the GWAS analysis. A total of 38,107 polymorphic SNPs were analyzed in a univariate model with hatch year and harvest group as fixed effects, harvest weight as a continuous covariate, and animal and common environment as random effects. A new linkage map was developed to create windows of 20 adjacent SNPs for use in the GWAS. The two windows with largest effect for FY and FW were located on chromosome Omy9 and explained only 1.0–1.5% of genetic variance, thus suggesting a polygenic architecture affected by multiple loci with small effects in this population. One window on Omy5 explained 1.4 and 1.0% of the genetic variance for BW10 and BW13, respectively. Three windows located on Omy27, Omy17, and Omy9 (same window detected for FY) explained 1.7, 1.7, and 1.0%, respectively, of genetic variance for CAR. Among the detected 100 SNPs, 55% were located directly in genes (intron and exons). Nucleotide sequences of intragenic SNPs were blasted to the Mus musculus genome to create a putative gene network. The network suggests that differences in the ability to maintain a proliferative and renewable population of myogenic precursor cells may affect variation in growth and fillet yield in rainbow trout.

Analysis of Cell Lineage Trees by Exact Bayesian Inference Identifies Negative Autoregulation of Nanog in Mouse Embryonic Stem Cells

Many cellular effectors of pluripotency are dynamically regulated. In principle, regulatory mechanisms can be inferred from single-cell observations of effector activity across time. However, rigorous inference techniques suitable for noisy, incomplete, and heterogeneous data are lacking. Here, we introduce stochastic inference on lineage trees (STILT), an algorithm capable of identifying stochastic models that accurately describe the quantitative behavior of cell fate markers observed using time-lapse microscopy data collected from proliferating cell populations. STILT performs exact Bayesian parameter inference and stochastic model selection using a particle-filter-based algorithm. We use STILT to investigate theautoregulation of Nanog, a heterogeneously expressed core pluripotency factor, in mouse embryonic stem cells. STILT rejects the possibility of positiveNanog autoregulation with high confidence; instead, model predictions indicate weak negative feedback. We use STILT for rational experimental design andvalidate model predictions using novel experimental data. STILT is available for download as an open source framework from http://www.imsb.ethz.ch/research/claassen/Software/stilt---stochastic-inference-on-lineage-trees.html.

Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution

AbstractChanges in the oxygenation state of microenvironments within solid tumors are associated with the development of aggressive cancer phenotypes. Factors that influence cellular hypoxia have been characterized; however, methods for measuring the dynamics of oxygenation at a cellular level in vivo have been elusive. We report a series of tellurium‐containing isotopologous probes for cellular hypoxia compatible with mass cytometry (MC)—technology that allows for highly parametric interrogation of single cells based on atomic mass spectrometry. Sequential labeling with the isotopologous probes (SLIP) in pancreatic tumor xenograft models revealed changes in cellular oxygenation over time which correlated with the distance from vasculature, the proliferation of cell populations, and proximity to necrosis. SLIP allows for capture of spatial and temporal dynamics in vivo using enzyme activated probes.

Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution.

Changes in the oxygenation state of microenvironments within solid tumors are associated with the development of aggressive cancer phenotypes. Factors that influence cellular hypoxia have been characterized; however, methods for measuring the dynamics of oxygenation at a cellular level in vivo have been elusive. We report a series of tellurium-containing isotopologous probes for cellular hypoxia compatible with mass cytometry (MC)-technology that allows for highly parametric interrogation of single cells based on atomic mass spectrometry. Sequential labeling with the isotopologous probes (SLIP) in pancreatic tumor xenograft models revealed changes in cellular oxygenation over time which correlated with the distance from vasculature, the proliferation of cell populations, and proximity to necrosis. SLIP allows for capture of spatial and temporal dynamics in vivo using enzyme activated probes.

ZSCAN5B and primate-specific paralogs bind RNA polymerase III genes and extra-TFIIIC (ETC) sites to modulate mitotic progression.

Mammalian genomes contain hundreds of genes transcribed by RNA Polymerase III (Pol III), encoding noncoding RNAs and especially the tRNAs specialized to carry specific amino acids to the ribosome for protein synthesis. In addition to this well-known function, tRNAs and their genes (tDNAs) serve a variety of other critical cellular functions. For example, tRNAs and other Pol III transcripts can be cleaved to yield small RNAs with potent regulatory activities. Furthermore, from yeast to mammals, active tDNAs and related “extra-TFIIIC” (ETC) loci provide the DNA scaffolds for the most ancient known mechanism of three-dimensional chromatin architecture. Here we identify the ZSCAN5 TF family - including mammalian ZSCAN5B and its primate-specific paralogs - as proteins that occupy mammalian Pol III promoters and ETC sites. We show that ZSCAN5B binds with high specificity to a conserved subset of Pol III genes in human and mouse. Furthermore, primate-specific ZSCAN5A and ZSCAN5D also bind Pol III genes, although ZSCAN5D preferentially localizes to MIR SINE- and LINE2-associated ETC sites. ZSCAN5 genes are expressed in proliferating cell populations and are cell-cycle regulated, and siRNA knockdown experiments suggested a cooperative role in regulation of mitotic progression. Consistent with this prediction, ZSCAN5A knockdown led to increasing numbers of cells in mitosis and the appearance of cells. Together, these data implicate the role of ZSCAN5 genes in regulation of Pol III genes and nearby Pol II loci, ultimately influencing cell cycle progression and differentiation in a variety of tissues.

The impact of cell proliferation and radionuclide uptake rate in radio-immunotherapy

Purpose The instantaneous-uptake model of O’Donoghue [”The impact of tumor cell proliferation in radio-immunotherapy”, Cancer 73, 974–980 (1994)] for a proliferating cell population irradiated by an exponentially decreasing dose-rate is being extended to arbitrary uptake rates of the radiopharmaceutical in tumor cells. Methods The time derivative of the survival curve is examined and an expression for the minimum of the surviving fraction is deduced along with a new expression for the biological effective dose (BED). Surviving fractions are calculated over a clinically relevant parameter range to establish general trends. Results Results are presented for the therapy radionuclides Y-90, I-131, and P-32, assuming uptake half-times 1–24 h, extrapolated initial dose-rates 0.5–1 Gy h-1 and a biological clearance half-life of 7 days. Cell doubling time equals 2 days. The exponential-uptake rate of the radiopharmaceutical by the targeted cells appears to have a considerable effect on the survival of a proliferating cell population (even for uptake half-times of only a few hours) and thence might need to be considered in the radiobiological models of tumor cell-kill in radio-immunotherapy. The differences between the exponential-uptake model and the instantaneous-uptake model become larger for high peak dose-rates, slow uptakes, and (slightly) for radionuclides with higher half-life. Moreover, the sensitivity of the cell survival on the uptake was found to be higher for the tumor cells with higher radio-sensitivity. Conclusion Neglecting an uptake phase may result in a considerable overestimation of cell kill.