Episome partitioning and symmetric cell divisions: Quantifying the role of random events in the persistence of HPV infections.
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ABSTRACT:
SUBMITTER: Beneteau T
PROVIDER: S-EPMC8448377 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hematopoietic stem cells (HSCs) are somatic stem cells that continuously generate lifelong supply of blood cells through a balance of symmetric and asymmetric divisions. It is well established that the HSC pool increases with age. However, not much is known about the underlying cause for these observed changes. Here, using a novel method combining single-cell ex vivo HSC expansion with mathematical modeling, we quantify HSC division types (stem cell-stem cell (S-S) division, stem cell-progenitor cell (S-P) division, and progenitor cell-progenitor cell (P-P) division) as a function of the aging process. Our time-series experiments reveal how changes in these three modes of division can explain the increase in HSC numbers with age. Contrary to the popular notion that HSCs divide predominantly through S-P divisions, we show that S-S divisions are predominant throughout the lifespan of the animal, thereby expanding the HSC pool. We, therefore, provide a novel mathematical model-based experimental validation for reflecting HSC dynamics in vivo.
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Project description:The ASPM (abnormal spindle-like microcephaly-associated) protein has previously been implicated in the determination of human cerebral cortical size, but the cell biological basis of this regulation has not been studied. Here we investigate the role of Aspm in mouse embryonic neuroepithelial (NE) cells, the primary stem and progenitor cells of the mammalian brain. Aspm was found to be concentrated at mitotic spindle poles of NE cells and to be down-regulated with their switch from proliferative to neurogenic divisions. Upon RNA interference in telencephalic NE cells, Aspm mRNA is reduced, mitotic spindle poles lack Aspm protein, and the cleavage plane of NE cells is less frequently oriented perpendicular to the ventricular surface of the neuroepithelium. The alteration in the cleavage plane orientation of NE cells increases the probability that these highly polarized cells undergo asymmetric division, i.e., that apical plasma membrane is inherited by only one of the daughter cells. Concomitant with the resulting increase in abventricular cells in the ventricular zone, a larger proportion of NE cell progeny is found in the neuronal layer, implying a reduction in the number of NE progenitor cells upon Aspm knock-down relative to control. Our results demonstrate that Aspm is crucial for maintaining a cleavage plane orientation that allows symmetric, proliferative divisions of NE cells during brain development. These data provide a cell biological explanation of the primary microcephaly observed in humans with mutations in ASPM, which also has implications for the evolution of mammalian brains.
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Project description:Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), four polyamides were identified that possessed apparent IC(50)s?150nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA1, PA25, and related polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our polyamides, is accompanied by significant cellular toxicity.
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Project description:Successful maintenance of cellular lineages critically depends on the fate decision dynamics of stem cells (SCs) upon division. There are three possible strategies with respect to SC fate decision symmetry: (a) asymmetric mode, when each and every SC division produces one SC and one non-SC progeny; (b) symmetric mode, when 50% of all divisions produce two SCs and another 50%-two non-SC progeny; (c) mixed mode, when both the asymmetric and two types of symmetric SC divisions co-exist and are partitioned so that long-term net balance of the lineage output stays constant. Theoretically, either of these strategies can achieve lineage homeostasis. However, it remains unclear which strategy(s) are more advantageous and under what specific circumstances, and what minimal control mechanisms are required to operate them. Here we used stochastic modeling to analyze and quantify the ability of different types of divisions to maintain long-term lineage homeostasis, in the context of different control networks. Using the example of a two-component lineage, consisting of SCs and one type of non-SC progeny, we show that its tight homeostatic control is not necessarily associated with purely asymmetric divisions. Through stochastic analysis and simulations we show that asymmetric divisions can either stabilize or destabilize the lineage system, depending on the underlying control network. We further apply our computational model to biological observations in the context of a two-component lineage of mouse epidermis, where autonomous lineage control has been proposed and notable regional differences, in terms of symmetric division ratio, have been noted-higher in thickened epidermis of the paw skin as compared to ear and tail skin. By using our model we propose a possible explanation for the regional differences in epidermal lineage control strategies. We demonstrate how symmetric divisions can work to stabilize paw epidermis lineage, which experiences high level of micro-injuries and a lack of hair follicles as a back-up source of SCs.
| S-EPMC4689538 | biostudies-literature
Project description:Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell divisions. When we offset the metaphase plate position by creating an asymmetric centriole distribution on each pole, we find that metaphase plates relocate to the middle of the spindle before anaphase. The spindle assembly checkpoint enables this centering mechanism by providing cells enough time to correct metaphase plate position. The checkpoint responds to unstable kinetochore-microtubule attachments resulting from an imbalance in microtubule stability between the two half-spindles in cells with an asymmetric centriole distribution. Inactivation of the checkpoint prior to metaphase plate centering leads to asymmetric cell divisions and daughter cells of unequal size; in contrast, if the checkpoint is inactivated after the metaphase plate has centered its position, symmetric cell divisions ensue. This indicates that the equatorial position of the metaphase plate is essential for symmetric cell divisions.
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Project description:The DNA damage response (DDR) of host cells is utilized by a number of viruses to establish and propagate their genomes in the infected cells. We examined the expression of the DDR genes during Kaposi's sarcoma-associated herpesvirus (KSHV) infection of human peripheral blood mononuclear cells (PBMCs). The genes were mostly downregulated, except H2AX, which was upregulated during infection. H2AX is important for gammaherpesvirus infectivity, and its phosphorylation at serine 139 is crucial for maintenance of latency during mouse gamma-herpesvirus 68 (MHV-68) infection. We now also observed phosphorylation of H2AX at serine 139 during KSHV infection. H2AX is a histone H2A isoform shown to interact with the latency-associated nuclear antigen (LANA) encoded by KSHV. Here, we show that LANA directly interacted with H2AX through domains at both its N and C termini. The phosphorylated form of H2AX (?H2AX) was shown to colocalize with LANA. Chromatin immunoprecipitation (ChIP) assays showed that a reduction in H2AX levels resulted in reduced binding of LANA with KSHV terminal repeats (TRs). Binding preferences of H2AX and ?H2AX along the KSHV episome were examined by whole-episome ChIP analysis. We showed that ?H2AX had a higher relative binding activity along the TR regions than that of the long unique region (LUR), which highlighted the importance of H2AX phosphorylation during KSHV infection. Furthermore, knockdown of H2AX resulted in decreased KSHV episome copy number. Notably, the C terminus of LANA contributed to phosphorylation of H2AX. However, phosphorylation was not dependent on the ability of LANA to drive KSHV-infected cells into S-phase. Thus, H2AX contributes to association of LANA with the TRs, and phosphorylation of H2AX is likely important for its increased density at the TRs.
| S-EPMC3624323 | biostudies-literature
Project description:Many RNAs, proteins, and organelles are present in such low numbers per cell that random segregation of individual copies causes large "partitioning errors" at cell division. Even symmetrically dividing cells can then by chance produce daughters with very different composition. The size of the errors depends on the segregation mechanism: Control systems can reduce low-abundance errors, but the segregation process can also be subject to upstream sources of randomness or spatial heterogeneities that create large errors despite high abundances. Here we mathematically demonstrate how partitioning errors arise for different types of segregation mechanisms and how errors can be greatly increased by upstream heterogeneity but remarkably hard to avoid through controlled partitioning. We also show that seemingly straightforward experiments cannot be straightforwardly interpreted because very different mechanisms produce identical fits and present an approach to deal with this problem by adding binomial counting noise and testing for convexity or concavity in the partitioning error as a function of the binomial thinning parameter. The results lay a conceptual groundwork for more effective studies of heterogeneity among growing and dividing cells, whether in microbes or in differentiating tissues.
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Project description:BackgroundAll quantifications of mortality, morbidity, and other health measures involve numerous sources of error. The routine quantification of random sampling error makes it easy to forget that other sources of error can and should be quantified. When a quantification does not involve sampling, error is almost never quantified and results are often reported in ways that dramatically overstate their precision.DiscussionWe argue that the precision implicit in typical reporting is problematic and sketch methods for quantifying the various sources of error, building up from simple examples that can be solved analytically to more complex cases. There are straightforward ways to partially quantify the uncertainty surrounding a parameter that is not characterized by random sampling, such as limiting reported significant figures. We present simple methods for doing such quantifications, and for incorporating them into calculations. More complicated methods become necessary when multiple sources of uncertainty must be combined. We demonstrate that Monte Carlo simulation, using available software, can estimate the uncertainty resulting from complicated calculations with many sources of uncertainty. We apply the method to the current estimate of the annual incidence of foodborne illness in the United States.SummaryQuantifying uncertainty from systematic errors is practical. Reporting this uncertainty would more honestly represent study results, help show the probability that estimated values fall within some critical range, and facilitate better targeting of further research.
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Project description:BackgroundCircumcision and lower human papillomavirus (HPV) viral loads in men are possibly associated with a reduced risk of HPV transmission to women. However, the association between male circumcision and HPV viral load remains unclear.MethodsSwab specimens from the glans and shaft of the penis were collected from men enrolled in a circumcision trial in Kisumu, Kenya. GP5+/6+ polymerase chain reaction (PCR) was used to identify HPV DNA types. HPV-16 and HPV-18 loads were measured with a LightCycler real-time PCR and classified as high (>250 copies/scrape) or low (≤250 copies/scrape).ResultsA total of 1159 men were randomly assigned to undergo immediate circumcision, and 1140 men were randomly assigned to the control arm (these individuals were asked to remain uncircumcised until the study ended). The hazard of acquisition of high-viral load infections in the glans was lower in the circumcision arm, compared with the control arm, for HPV-16 (hazard ratio [HR], 0.32 [95% confidence interval {CI}, .20-.49]) and HPV-18 (HR, 0.34 [95% CI, .21-.54]). The 6-month risk of HPV persistence among men with high-viral load infections in the glans at baseline was lower in the circumcision arm, compared with the control arm, for HPV-16 (risk ratio [RR], 0.36 [95% CI, .18-.72]) and HPV-18 (RR 0.34 [95% CI, .13-.86]). Weaker and less precise results were obtained for shaft samples.ConclusionsMale circumcision could potentially reduce the risk of HPV transmission to women by reducing the hazard of acquisition, and the risk of persistence of high-HPV viral load infections in the glans in men.
| S-EPMC4402376 | biostudies-literature
Project description:Proper functioning of sensory systems requires the generation of appropriate numbers and proportions of neuronal subtypes that encode distinct information. Perception of color relies on signals from multiple cone photoreceptor types. In cone-dominated retinas, each cone expresses a single opsin type with peak sensitivity to UV, long (L) (red), medium (M) (green), or short (S) (blue) wavelengths. The modes of cell division generating distinct cone types are unknown. We report here a mechanism whereby zebrafish cone photoreceptors of the same type are produced by symmetric division of dedicated precursors. Transgenic fish in which the thyroid hormone receptor β2 (trβ2) promoter drives fluorescent protein expression before L-cone precursors themselves are produced permitted tracking of their division in vivo. Every L cone in a local region resulted from the terminal division of an L-cone precursor, suggesting that such divisions contribute significantly to L-cone production. Analysis of the fate of isolated pairs of cones and time-lapse observations suggest that other cone types can also arise by symmetric terminal divisions. Such divisions of dedicated precursors may help to rapidly attain the final numbers and proportions of cone types (L > M, UV > S) in zebrafish larvae. Loss- and gain-of-function experiments show that L-opsin expression requires trβ2 activity before cone differentiation. Ectopic expression of trβ2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trβ2 expression could explain why some species have mixed, and others have pure, cone types.
| S-EPMC3773785 | biostudies-literature