Stochastic modeling of stress erythropoiesis using a two-type age-dependent branching process with immigration.

The erythroid lineage is a particularly sensitive target of radiation injury. We model the dynamics of immature (BFU-E) and mature (CFU-E) erythroid progenitors, which have markedly different kinetics of recovery, following sublethal total body irradiation using a two-type reducible age-dependent branching process with immigration. Properties of the expectation and variance of the frequencies of both types of progenitors are presented. Their explicit expressions are derived when the process is Markovian, and their asymptotic behavior is identified in the age-dependent (non-Markovian) case. Analysis of experimental data on the kinetics of BFU-E and CFU-E reveals that the probability of self-renewal increases transiently for both cell types following sublethal irradiation. In addition, the probability of self-renewal increased more for CFU-E than for BFU-E. The strategy adopted by the erythroid lineage ensures replenishment of the BFU-E compartment while optimizing the rate of CFU-E recovery. Finally, our analysis also indicates that radiation exposure causes a delay in BFU-E recovery consistent with injury to the hematopoietic stem/progenitor cell compartment that give rise to BFU-E. Erythroid progenitor self-renewal is thus an integral component of the recovery of the erythron in response to stress.

Phase II study of gemcitabine, oxaliplatin in combination with panitumumab in KRAS wild-type unresectable or metastatic biliary tract and gallbladder cancer.

BACKGROUND: Current data suggest that platinum-based combination therapy is the standard first-line treatment for biliary tract cancer. EGFR inhibition has proven beneficial across a number of gastrointestinal malignancies; and has shown specific advantages among KRAS wild-type genetic subtypes of colon cancer. We report the combination of panitumumab with gemcitabine (GEM) and oxaliplatin (OX) as first-line therapy for KRAS wild-type biliary tract cancer. METHODS: Patients with histologically confirmed, previously untreated, unresectable or metastatic KRAS wild-type biliary tract or gallbladder adenocarcinoma with ECOG performance status 0-2 were treated with panitumumab 6 mg kg(-1), GEM 1000 mg m(-2) (10 mg m(-2) min(-1)) and OX 85 mg m(-2) on days 1 and 15 of each 28-day cycle. The primary objective was to determine the objective response rate by RECIST criteria v.1.1. Secondary objectives were to evaluate toxicity, progression-free survival (PFS), and overall survival. RESULTS: Thirty-one patients received at least one cycle of treatment across three institutions, 28 had measurable disease. Response rate was 45% and disease control rate was 90%. Median PFS was 10.6 months (95% CI 5-24 months) and median overall survival 20.3 months (95% CI 9-25 months). The most common grade 3/4 adverse events were anaemia 26%, leukopenia 23%, fatigue 23%, neuropathy 16% and rash 10%. CONCLUSIONS: The combination of gemcitabine, oxaliplatin and panitumumab in KRAS wild type metastatic biliary tract cancer showed encouraging efficacy, additional efforts of genetic stratification and targeted therapy is warranted in biliary tract cancer.

Saddlepoint approximations to the moments of multitype age-dependent branching processes, with applications.

This article proposes saddlepoint approximations to the expectation and variance-covariance function of multitype age-dependent branching processes. The proposed approximations are found accurate, easy to implement, and much faster to compute than by simulating the process. Multiple applications are presented, including the analyses of clonal data on the generation of oligodendrocytes from their immediate progenitor cells, and on the proliferation of Hela cells. New estimators are also constructed to analyze clonal data. The proposed methods are finally used to approximate the distribution of the generation, which has recently found several applications in cell biology.

Transition in specification of embryonic metazoan DNA replication origins.

In early Xenopus embryos, in which ribosomal RNA genes (rDNA) are not transcribed, rDNA replication initiates and terminates at 9- to 12-kilobase pair intervals, with no detectable dependence on specific DNA sequences. Resumption of ribosomal RNA (rRNA) synthesis at late blastula and early gastrula is accompanied by a specific repression of replication initiation within transcription units; the frequency of initiation within intergenic spacers remains as high as in early blastula. These results demonstrate that for rRNA genes, circumscribed zones of replication initiation emerge in intergenic DNA during the time in metazoan development when the chromatin is remodeled to allow gene transcription.

Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito.

An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

Segregation and rearrangement of coamplified genes in different lineages of mutant cells that overproduce adenylate deaminase.

Four genes encoding proteins designated as W, X, Y1, and Y2 were found previously to be amplified at different levels in a Chinese hamster fibroblast mutant line selected for overproduction of adenylate deaminase. To gain information on the molecular mechanisms responsible, we studied the levels of amplification and the structures of these four genes in several lineages of mutant cells with comparable activities of adenylate deaminase, the selected enzyme. Only the W gene was amplified in all the lines. In one line, the X, Y1, and Y2 genes were coamplified, while in others either the Y1 gene or the pair X and Y2 were coamplified. The results were consistent with linkage of all the genes--in a particular order--in an amplifiable sequence with variable endpoints. Novel joints with a nonrandom distribution were observed. We frequently detected rearranged copies of the W gene, but very few novel joints were present in the other three genes in the six highly amplified lines examined. Some of the novel joints in gene W were highly amplified; they were generated by reamplification of a rearrangement that appeared at an early selection step. In some lines, reamplification was accompanied by deletion or mass correction of preexisting units. We discuss mechanisms which might account for these observations.

Hemicatenanes form upon inhibition of DNA replication.

Plasmid DNA incubated in interphase Xenopus egg extracts is normally assembled into chromatin and then into synthetic nuclei which undergo one round of regulated replication. During a study of restriction endonuclease cut plasmid replication intermediates (RIs) by the Brewer-Fangman 2D gel electrophoresis technique, we have observed the formation of a strong spike of X-shaped DNA molecules in extracts that otherwise yield very little or no RIs. Formation of these joint molecules is also efficiently induced in replication-competent extracts upon inhibition of replication fork progression by aphidicolin. Although their electrophoretic properties are quite similar to those of Holliday junctions, 2D gels of doubly cut plasmids show that these junctions can link two plasmid molecules at any pair of DNA sequences, with no regard for sequence homology at the branch points. Neutral-neutral-alkaline 3D gels show that the junctions only contain single strands of parental size and no recombinant strands. A hemicatenane, in which one strand of a duplex is wound around one strand of another duplex, is the most likely structure to account for these observations. The mechanism of formation of these novel joint DNA molecules and their biological implications are discussed.

Bi-directional replication and random termination.

Two-dimensional (2D) agarose gel electrophoresis was used to study termination of DNA replication in a shuttle vector, YRp7', when it replicated in Escherichia coli, Saccharomyces cerevisiae and Xenopus egg extracts. In E. coli, the 2D gel patterns obtained were consistent with uni-directional replication initiated at a specific site, the ColE1 origin. In consequence, termination also occurred precisely at the ColE1 origin. In Xenopus egg extracts, the particular shape of the bubble arc as well as the triangular smear detected to the left of the simple-Y pattern indicated random initiation and termination. In S.cerevisiae, initiation occurred at the ARS1 origin and replication proceeded in a bi-directional manner. However, termination did not always occur at a specific site 180 degrees across from the origin, but almost all along the south hemisphere of the plasmid. Inversion, deletion or replacement of DNA sequences located throughout this hemisphere did not eliminate random termination. Analysis of the replication intermediates of another yeast plasmid bearing a different origin, ARS305, also exhibited random termination. We propose that the random termination events observed in S.cerevisiae could be due to an asynchronous departure of both forks from the bi-directional origin in addition to differences in the rate of fork progression. These observations could be extended to all bi-directional origins.

Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations.

We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin's fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

Developmental regulation of replication fork pausing in Xenopus laevis ribosomal RNA genes.

In early Xenopus embryos, replication forks move along the rRNA genes (rDNA) at a uniform rate and terminate at multiple, apparently random sites. In contrast, a polar replication fork barrier (RFB) is found at the 3' end of the rRNA genes in Xenopus cultured cells. We have now analysed the replication intermediates of Xenopus rDNA from a wide range of developmental stages by 2D gel electrophoresis. Surprisingly, up to 15 different replication fork pausing sites (RFPs) simultaneously appear in the rDNA at the midgastrula stage, when rRNA transcription abruptly increases. They disappear during the neurula stage, except for a polar RFP at the 3' end of Xthe transcription unit, which persists to the tadpole stage. The latter RFP is found at the same location as the RFB in cultured cells; however the arrest of replication forks at this RFP is not absolute, since termination occurs at multiple positions throughout the rDNA repeat. The efficiency of fork arrest at this RFP remains constant from midgastrula to early tadpole, and decreases around hatching. The transient appearance of multiple RFPs at midgastrula may reflect some chromatin remodeling associated with developmental activation of rRNA transcription.

Replication fork density increases during DNA synthesis in X. laevis egg extracts.

Duplication of the eukaryotic genome depends on the temporal and spatial organization of DNA replication during the cell cycle. To investigate the genomic organization of DNA replication in a higher eukaryote, multiple origins of replication must be simultaneously analyzed over large regions of the genome as DNA synthesis progresses through S phase of the cell cycle. We have employed a novel technique that allows for the quantitative analysis of DNA replication on a genome wide basis. The technique involves stretching and aligning individual DNA molecules on a glass surface. As a model system, Xenopus laevis egg extract was used to differentially label sperm chromatin at successive time points after the start of DNA synthesis. The differentially labeled DNA allows earlier and later replicating sequences to be distinguished, and hence the sites of DNA synthesis at any given time can be directly visualized. Genomic DNA was extracted, and measurements made on the linearized molecules provided a comprehensive analysis of the spatial and temporal organization of DNA replication in the X. laevis in vitro replication system. It was found that: (i) DNA synthesis initiates asynchronously at irregular intervals but continuously as DNA replication advances; and (ii) that the frequency of initiation (the number of activated origins per kilobase) increases as DNA synthesis nears completion. The implications of these findings for the regulation of DNA replication in early embryos is discussed.

Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos.

Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.3-42.2 kbp) in Xenopus egg extracts by two-dimensional gel electrophoresis and electron microscopy. We confirm that replication initiates without sequence specificity on all plasmids. We demonstrate for the first time that multiple initiation events occur on large plasmids, but not on small (<10 kb) plasmids, at average intervals of approximately 10 kb. Origin interference may prevent multiple initiation events on small plasmids. Multiple initiation events are neither synchronous nor regularly spaced. Bubble density is higher on later than on earlier replication intermediates, showing that initiation frequency increases throughout S phase, speeding up replication of late intermediates. We suggest that potential origins are abundant and randomly distributed, but that the increase of initiation frequency during S phase, and possibly origin interference, regulate origin activation to ensure rapid completion of replication.

Mechanisms and consequences of replication fork arrest.

Chromosome replication is not a uniform and continuous process. Replication forks can be slowed down or arrested by DNA secondary structures, specific protein-DNA complexes, specific DNA-RNA hybrids, or interactions between the replication and transcription machineries. Replication arrest has important implications for the topology of replication intermediates and can trigger homologous and illegitimate recombination. Thus, replication arrest may be a key factor in genome instability. Several examples of these phenomena are reviewed here.

Role of nuclear architecture in the initiation of eukaryotic DNA replication.

The eukaryotic genome is compacted in the cell nucleus, in a way that allows its faithful and ordered replication each cell cycle. Chromatin is organized into topologically constrained loops that are anchored to the nuclear matrix by specific attachment regions (SARs). Chromatin loops were proposed to correspond to replication units. In particular, it has been suggested that replication origins coincide with SARs. Critical examination of these hypotheses has long been hampered by the elusive nature of higher eukaryotic DNA replication origins and termini. In recent years, however, a number of loci have been mapped for both SARs and replication units, and studies on the nuclear localization of replicating DNA and replication proteins have begun. We review these data and argue that they question this model. We then try to delineate other aspects of chromosome compartmentalization and cell-cycle remodeling which might be responsible for the specification and activation of metazoan DNA replication origins.

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.

Stochastic modeling in toxicokinetics. Application to the in vivo micronucleus assay.

A stochastic model for the in vivo micronucleus assay is presented. This model describes the kinetic of the rate of micronucleated polychromatic erythrocytes induced by the administration of a mutagenic compound. For this, biological assumptions are made both on the erythropoietic system and on the mechanisms of action of the compound. Its pharmacokinetic profile is also taken into account and it is linked to the induced toxicological effect. This model has been evaluated by analyzing the induction of micronuclei is mice bone marrow by a mutagenic compound, 6-mercaptopurine (6-mp). This analysis enabled to make interesting remarks about the induction of micronuclei by 6-mp and to put to light an unsuspected wavy kinetic by optimizing the experimental design of the in vivo micronucleus assay.

Clinical factors affecting engraftment and transfusion needs in SCT: a single-center retrospective analysis.

Successful utilization of SCT modalities often requires utilization of both red cell and platelet transfusions. In this retrospective evaluation of clinical factors affecting transplant engraftment and transfusion utilization at a single transplant center in 505 patients from 2005 through 2009, we found that graft type, donor type and the conditioning regimen intensity significantly affected both the neutrophil engraftment time (P<0.001) and the platelet engraftment time (P<0.001). SCT patients required an average of 6.2 red cell units, and 7.9 platelet transfusions in the first 100 days with a wide s.d. Among auto-SCT patients, 5% required neither RBC nor platelet transfusions. Some reduced-intensity transplants were also associated with no transfusion need, and in allogeneic transplants, conditioning regimen intensity was positively correlated with platelet transfusion events as assessed by multivariate analysis. Other patient characteristics such as gender, graft type, donor type, underlying disease and use of TBI were all independently associated with transfusion needs in SCT patients. Further studies are required to understand the means to minimize transfusions and potential related complications in SCT patients.

Electrocardiographic method for identifying drug-induced repolarization abnormalities associated with a reduction of the rapidly activating delayed rectifier potassium current.

Several important non-cardiac drugs have been removed from the market after revealing harmful effect that was not identified during prior safety-assessment studies. We developed a new technique for the measurements of repolarization abnormalities from surface ECGs; this method improves sensitivity and specificity of the current technique used to identify the presence of abnormal ion current kinetics in the myocardial cells namely a prolongation of the QT interval on the surface ECG signal. We described in this paper the method and preliminary results, revealing the superiority of our technique that may play a role in the future of drug-safety assessment.

Large inverted duplications in amplified DNA of mammalian cells form hairpins in vitro upon DNA extraction but not in vivo.

I have analysed the duplex-to-hairpin transition of large inverted duplications with a short asymmetric center which are found in the amplified DNA of two mammalian cell lines resistant to cytotoxic drugs. Psoralen crosslinking experiments establish that this transition does not occur in vivo, but takes place in a significant portion of the palindromes during genomic DNA purification, at the phenol-chloroform extraction step. The introduction of single strand nicks in the DNA by gamma irradiation prior to its purification does not prevent hairpin formation but instead facilitates it. These results show that the rate-limiting step of the duplex-to-hairpin transition does not require negative supercoiling, and that transient melting of large segments of cellular DNA occurs during phenol-chloroform extraction. I also show, and discuss the fact, that only cellular DNA, and not cloned palindromic DNA, is able to undergo hairpin formation by this mechanism. These results bear practical implications for the study of inverted repeated DNA sequences in eukaryotic cells.

Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos.

We have analysed the replication of the chromosomal ribosomal DNA (rDNA) cluster in Xenopus embryos before the midblastula transition. Two-dimensional gel analysis showed that replication forks are associated with the nuclear matrix, as in differentiated cells, and gave no evidence for single-stranded replication intermediates (RIs). Bubbles, simple forks and double Ys were found in each restriction fragment analysed, showing that replication initiates and terminates without detectable sequence specificity. Quantification of the results and mathematical analysis showed that the average rDNA replicon replicates in 7.5 min and is 9-12 kbp in length. This time is close to the total S phase duration, and this replicon size is close to the maximum length of DNA which can be replicated from a single origin within this short S phase. We therefore infer that (i) most rDNA origins must be synchronously activated soon in S phase and (ii) origins must be evenly spaced, in order that no stretch of chromosomal DNA is left unreplicated at the end of S phase. Since origins are not specific sequences, it is suggested that this spatially and temporally concerted pattern of initiation matches some periodic chromatin folding, which itself need not rely on DNA sequence.